Whole Body Health Sale

Abstracts

Vitamin D: 266 Research Abstracts

Cancer

1. Nutr Rev. 2003 Jul;61(7):227-38. Vitamin D and vitamin D analogs as cancer chemopreventive agents. Guyton KZ, Kensler TW, Posner GH. CCS Associates, 2005 Landings Drive, Mountain View, CA 94043, USA.

Epidemiologic studies have associated vitamin D, attained through nutrition and sun exposure, with reduced cancer risk. Although dose-limiting hypercalcemia has limited the use of natural vitamin D in cancer prevention, several promising new synthetic vitamin D analogs (deltanoids) are under development. Examples are KH-1060, EB-1089, 1alpha-hydroxyvitamin D5, vitamin D2, and QW-1624F2-2. Clinical targets for deltanoids include colon, prostate, and breast. Studies to elucidate the molecular mechanisms underlying the observed efficacy of deltanoids are ongoing. The vitamin D receptor, a steroid/thyroid receptor superfamily member, appears to control most deltanoid effects on proliferation, apoptosis, differentiation, and angiogenesis.

2. Circ Res. 2000 Aug 4;87(3):214-20. 1 alpha,25-dihydroxyvitamin D(3) inhibits angiogenesis in vitro and in vivo. Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfield AE. Wellcome Trust Centre for Cell Matrix Research, Department of Medicine University of Manchester, Manchester, UK.

Modulation of angiogenesis is now a recognized strategy for the prevention and treatment of pathologies categorized by their reliance on a vascular supply. The purpose of this study was to evaluate the effect of 1 alpha,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)], the active metabolite of vitamin D(3), on angiogenesis by using well-characterized in vitro and in vivo model systems. 1,25(OH)(2)D(3) (1 x 10(-9) to 1 x 10(-7) mol/L) significantly inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting and elongation in vitro in a dose-dependent manner and had a small, but significant, inhibitory effect on VEGF-induced endothelial cell proliferation. 1, 25(OH)(2)D(3) also inhibited the formation of networks of elongated endothelial cells within 3D collagen gels. The addition of 1, 25(OH)(2)D(3) to endothelial cell cultures containing sprouting elongated cells induced the regression of these cells, in the absence of any effect on cells present in the cobblestone monolayer. Analysis of nuclear morphology, DNA integrity, and enzymatic in situ labeling of apoptosis-induced strand breaks demonstrated that this regression was due to the induction of apoptosis specifically within the sprouting cell population. The effect of 1,25(OH)(2)D(3) on angiogenesis in vivo was investigated by using a model in which MCF-7 breast carcinoma cells, which had been induced to overexpress VEGF, were xenografted subcutaneously together with MDA-435S breast carcinoma cells into nude mice. Treatment with 1,25(OH)(2)D(3) (12.5 pmol/d for 8 weeks) produced tumors that were less well vascularized than tumors formed in mice treated with vehicle alone. These results highlight the potential use of 1,25(OH)(2)D(3) in both the prevention and regression of conditions characterized by pathological angiogenesis.

3. Carcinogenesis. 2000 Jul;21(7):1341-5.

Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis.

Kensler TW, Dolan PM, Gange SJ, Lee JK, Wang Q, Posner GH.

Department of Environmental Health Sciences and Department of Epidemiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA. tkensler@jhsph.edu

Development of vitamin D analogs (deltanoids) as chemopreventive agents requires separation of desirable antiproliferative and pro-differentiating activities from the undesirable calcemic activity also found in the hormone calcitriol (1 alpha, 25-dihydroxyvitamin D(3)). Therefore, several conceptually new deltanoids were synthesized with modifications to the 1alpha- and/or 25-hydroxyl groups, positions traditionally considered essential for stimulating biological responses. In this study, 1 beta-hydroxymethyl-3-epi-25-hydroxyvitamin D(3), a non-calcemic CH(2) homolog of the natural hormone with antiproliferative activity in vitro, was ineffective as an inhibitor of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced induction of ornithine decarboxylase activity in mouse epidermis. However, a hybrid analog incorporating not only the calcemia-ablating 1 beta-hydroxymethyl alteration, but potentiating C,D ring 16-unsaturation and side chain 24,24-fluorination and 26, 27-homologation was found to be as effective as calcitriol. Several non-calcemic 24- or 25-t-butyl sulfones, some containing side chain fluorination but all lacking the 25-hydroxyl group, were also shown to be active in this assay. Three sulfones and the 1 beta-hydroxymethyl hybrid were evaluated as inhibitors of multistage carcinogenesis in mouse skin. Female CD-1 mice were initiated with a single dose of 7,12-dimethylbenz[a]anthracene and then promoted twice weekly for 20 weeks with TPA. Deltanoids were applied topically 30 min before TPA. Unlike calcitriol, none of the atypical deltanoids affected body weight gain in these animals. Minimal effects on urinary calcium excretion were observed following chronic treatment with these analogs. All deltanoids inhibited the incidence and multiplicity of papilloma formation, with the hybrid analog showing the greatest efficacy. With this deltanoid, tumor incidence was significantly reduced by 28% and tumor multiplicity by 63%. These results, coupled with the rich chemical diversity available in side chain sulfur-containing deltanoids, particularly when combined with A ring modifications such as 1 beta-hydroxylalkyl groups, provide important new advances in the fundamental understanding of chemical structure-biological activity relationships as well as more potent and safe vitamin D analogs for cancer chemoprevention and other medicinal uses.

Colon Cancer

4. Nat Rev Cancer. 2003 Aug;3(8):601-14.

Chemoprevention of colon cancer by calcium, vitamin D and folate: molecular mechanisms.

Lamprecht SA, Lipkin M.

Strang Cancer Prevention Center and Strang Cancer Research Laboratory at The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. lampres@mail.rockefeller.edu

Recent findings have indicated that dietary calcium, vitamin D and folate can modulate and inhibit colon carcinogenesis. Supporting evidence has been obtained from a wide variety of preclinical experimental studies, epidemiological findings and a few human clinical trials. Important molecular events and cellular actions of these micronutrients that contribute to their tumour-modulating effects are discussed. They include a complex series of signalling events that affect the structural and functional organization of colon cells. Biochem Biophys Res Commun. 2002 Dec 20;299(5):730-8.

5. Liganded VDR induces CYP3A4 in small intestinal and colon cancer cells via DR3 and ER6 vitamin D responsive elements.

Thompson PD, Jurutka PW, Whitfield GK, Myskowski SM, Eichhorst KR, Dominguez CE, Haussler CA, Haussler MR.

Department of Biochemistry and Molecular Biophysics, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.

The nuclear vitamin D receptor (VDR) mediates the effects of 1,25-dihydroxyvitamin D(3) (1,25D(3)) to alter intestinal gene transcription and promote calcium absorption. Because 1,25D(3) also exerts anti-cancer effects, we examined the efficacy of 1,25D(3) to induce cytochrome P450 (CYP) enzymes. Exposure of human colorectal adenocarcinoma cells (HT-29) to 10(-8)M 1,25D(3) resulted in >/=3-fold induction of CYP3A4 mRNA and protein as assessed by RT-PCR and Western blotting, respectively. Six vitamin D responsive element (VDRE)-like sequences in the promoter region of the CYP3A4 gene were then individually tested for their ability to enhance transcription. A canonical DR3-type element in the distal region of the promoter (-7719-GGGTCAgcaAGTTCA-7733), and a proximal, non-classical everted repeat with a spacer of 6 bp (ER6; -169-TGAACTcaaaggAGGTCA-152) were identified as functional VDREs in this CYP gene. These data suggest that 1,25D(3)-dependent, VDR-mediated induction of CYP3A4 may constitute a chemoprotective mechanism for detoxification of enteric xenobiotics and carcinogens.

6. Mol Cell Endocrinol. 2001 Oct 25;183(1-2):141-9.

Synthetic low-calcaemic vitamin D(3) analogues inhibit secretion of insulin-like growth factor II and stimulate production of insulin-like growth factor-binding protein-6 in conjunction with growth suppression of HT-29 colon cancer cells.

Oh YS, Kim EJ, Schaffer BS, Kang YH, Binderup L, MacDonald RG, Park JH.

Division of Life Sciences and Institute of Environment and Life Science, Hallym University, 1 Okchon Dong, Chunchon, 200-702, South Korea.

The aims of the present study were to compare the ability of various synthetic analogues of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25-(OH)(2)D(3)] to inhibit proliferation of HT-29 cells, a human colon adenocarcinoma cell line. HT-29 cells were incubated for 144 h with various concentrations (0-100 nM) of 1 alpha,25-(OH)(2)D(3), or the analogues EB1089, CB1093 or 1 beta,25-(OH)(2)D(3). All these analogues except 1 beta,25-(OH)(2)D(3) inhibited cell proliferation, but relative potencies and efficacies of EB1089 and CB1093 were much greater than that of the native vitamin. Cells grew in serum-free medium, reaching a plateau density at day 10 of culture, and addition of 10 nM 1 alpha,25-(OH)(2)D(3) or 1 beta,25-(OH)(2)D(3) did not alter the long-term growth characteristics of HT-29 cells. However, cells treated with 10 nM EB1089 or CB1093 grew at a rate slower than control and reached final densities that were 53+/-1 and 36+/-2% lower than control, respectively. Immunoblot analysis of serum-free conditioned medium using a monoclonal anti-insulin-like growth factor-(IGF)-II antibody showed that both 10 nM EB1089 and CB1093 markedly inhibited secretion of both mature 7500 M(r) and higher M(r) forms of IGF-II. Ligand blot and immunoblot analyses of conditioned media revealed the presence of IGFBPs of M(r) 24,000 (IGFBP-4), 30,000 (glycosylated IGFBP-4), 35,000 (IGFBP-2) and 32,000-34,000 (IGFBP-6). The level of IGFBP-2 was decreased by 42+/-8 and 49+/-7% by 10 nM EB 1089 and CB1093, respectively, compared to controls. IGFBP-6 was increased approximately twofold by EB1089 and CB1093, and exogenously added IGFBP-6 inhibited HT-29 cell proliferation. These results suggest that inhibition of HT-29 cell proliferation by EB1089 and CB1093 may be attributed, at least in part, to the decreased secretion of IGF-II. The increase in IGFBP-6 concentration coupled with its high affinity for IGF-II may also contribute to decreased cellular proliferation by an indirect mechanism involving sequestration of endogenously produced IGF-II.

7. Cancer Causes Control. 2000 May;11(5):459-66.

Calcium, vitamin D, sunshine exposure, dairy products and colon cancer risk (United States).

Kampman E, Slattery ML, Caan B, Potter JD.

Fred Hutchinson Cancer Research Center, Cancer Prevention Research Program, Seattle, WA 98109-1024, USA.

OBJECTIVE: Epidemiologic studies on calcium, vitamin D and colon cancer are inconsistent, whereas experimental studies more regularly show a protective effect. To evaluate potential sources of inconsistencies, data from a large case-control study were analyzed, stratifying on potential effect modifiers. METHODS: Data were collected by certified interviewers in Northern California, Utah and Minnesota. Analyses included 1993 incident colon cancer cases and 2410 population-based controls. Multivariate logistic regression models included age, sex, BMI, family history, physical activity, intake of energy, dietary fiber, aspirin and NSAIDs. RESULTS: Dietary calcium was inversely associated with colon cancer risk in men (OR highest vs lowest quintile = 0.6, 95% CI = 0.5-0.9) and women (OR = 0.6, 95% CI = 0.4-0.9). No statistically significant associations were observed for dietary vitamin D or sunshine exposure. Consumption of total low-fat dairy products was associated with a statistically significantly decreased risk in men and women (ORs highest vs lowest category of intake = 0.8 and 0.7 respectively). Calcium supplement use was inversely associated with risk in both sexes (ORs use vs non-use = 0.8). Vitamin D supplements were inversely associated with risk in men (OR = 0.5) and women (OR = 0.6) but confidence limits included 1.0. CONCLUSIONS: These data provide additional support of an inverse association between high levels of calcium intake and colon cancer risk.

8. Int J Oncol. 1999 May;14(5):979-85.

Novel 19-nor-hexafluoride vitamin D3 analog (Ro 25-6760) inhibits human colon cancer in vitro via apoptosis.

Evans SR, Soldatenkov V, Shchepotin EB, Bogrash E, Shchepotin IB.

Department of Surgery, George Washington University, Washington, DC 20037, USA.

Our previously performed experiments clearly showed a significant VDR-mediated growth inhibitory effect of 1,25-dihydroxyvitamin D3 and its synthetic analogs in a variety of human cancer cells including human colon and breast cancer, soft tissue sarcoma, and malignant melanoma cell lines. The mechanisms by which 1, 25-dihydroxyvitamin D3 and its synthetic analogs growth inhibit human cancer cells is poorly elucidated. The exposure of human colon cancer cells HT-29 to 1,25-dihydroxyvitamin D3 or its analog, 1alpha, 25-dihydroxy-16-ene-23yne-26,27-hexafluoro-19-nor-choleca lci ferol (Ro 25-6760), at the 10(-6) M concentration resulted in significant growth inhibition with induction of the apoptotic process after three days of treatment detected by TUNEL assay and agarose gel electrophoresis of DNA. As a logical link with DNA fragmentation analyses and TUNEL assay, cleavage of the 116 kDa PARP protein was accompanied by the appearance of a characteristic 85 kDa fragment of PARP in a population of floating cells after both treatments. The results of cell cycle analysis showed a G0/G1 phase block after three days of administration of either compound when compared with untreated cells. On day 4, G0/G1 cell cycle arrest remained on the same level in comparison with control. Paralleling the G0/G1 phase block, was a notable decrease in the number of cells in the S phase which also became significant after three days of treatment. The results of these experiments show that the newly developed 19-nor synthetic vitamin D3 analog, Ro 25-6760, as well as 1, 25-dihydroxyvitamin D3, induced the expression of p21waf1, resulted in a significant G1/G0 cell cycle arrest leading to impressive growth inhibition and induction of apoptosis associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) showing a possible involvement of apoptosis-specific activation of the ICE/CED-3 proteolitic pathway.

9. Dis Colon Rectum. 1997 Mar;40(3):317-21. (Animal Study) Vitamin D3 analog, EB1089, inhibits growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model.

Akhter J, Chen X, Bowrey P, Bolton EJ, Morris DL.

University of New South Wales, Department of Surgery, Sydney, Australia.

PURPOSE: In this study, we investigated the effect of the vitamin D3 analog, EB1089, on the growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model. METHODS: BALB/c Nu/Nu nude mice were inoculated subcutaneously with 10(6) LoVo cells. EB1089 dissolved in isopropanol was administered intraperitoneally and orally on alternate days at doses of 0.1, 0.5, and 2.5 microg/kg/day. Control animals received isopropanol alone. Tumor volumes estimated using the formula 0.5 X length X (width)2. The tumor kinetic index was determined by immunohistochemical detection of proliferating cell nuclear antigen. RESULTS: Significant dose-dependent inhibition of tumor growth was seen. After 20 days of treatment with 0.1 microg/kg/day EB1089, mean tumor volume in treated mice was 41 to 49 percent less than that in control animals (P < 0.01). Significant inhibition of tumor growth was also seen with 0.5 microg/kg/day EB1089 after 22 days of treatment (51 percent of control P < 0.01). Treatment with 2.5 microg/kg/day resulted in weight loss that required termination of this group; these mice were subsequently found to be hypercalcemic. The tumor kinetic index was significantly lower in tumors treated with 0.1 microg/kg/day EB1089 compared with that for control tumors (8 vs. 30 percent in controls). CONCLUSION: These findings suggest that the vitamin D3 analog, EB1089, is a potent antiproliferative agent for some human colon cancers.

10. Anticancer Res. 1996 Jul-Aug;16(4B):2333-7.

Vitamin D receptor and cytokeratin expression may be progression indicators in human colon cancer.

Cross HS, Bajna E, Bises G, Genser D, Kallay E, Potzi R, Wenzl E, Wrba F, Roka R, Peterlik M.

Department of General and Experimental Pathology, University Hospital (AKH), Vienna, Austria.

Epidemiological data suggest the protective role of vitamin D against the development of colorectal carcinoma in man. This could be due to the anti-mitogenic effect of the steroid hormone on human colon carcinoma cells which is mediated by a specific nuclear vitamin D receptor (VDR). Western blot analysis showed that VDR expression increases during the transition from normal mucosa to polyps and later to pT3 tumors. In later stages, however, VDR is dramatically reduced. Cytokeratin 20, which was monitored as a differentiation marker, decreases in parallel with advancing proliferation and disappears from "normal" mucosa adjacent to later stage carcinoma. Interestingly, VDR density was conspicuously higher in all tumors tested when compared to adjacent "normal" tissue. This suggest that, up to a certain degree of dedifferentiation, malignant colonocytes can upregulate the VDR, probably as a counteractive measure in response to tumor cell growth, but that this ability is finally lost in highly undifferentiated carcinoma cells.

11. Am J Epidemiol. 1996 May 1;143(9):907-17.

Calcium, vitamin D, and dairy foods and the occurrence of colon cancer in men.

Kearney J, Giovannucci E, Rimm EB, Ascherio A, Stampfer MJ, Colditz GA, Wing A, Kampman E, Willett WC.

Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, USA.

To examine the associations between intakes of calcium, Vitamin D, and dairy foods and the risk of colon cancer, the authors analyzed data from a prospective study of 47,935 US male professionals, 40-75 years of age and free of cancer in 1986. Within this cohort, 203 new cases of colon cancer were documented between 1986 and 1992. After adjusting for age and total energy intake, the authors found that the intake of calcium from foods and supplements was inversely associated with colon cancer risk (relative risk (RR) = 0.58, 95% confidence interval (CI) 0.39-087 between high and low intakes of calcium). However, after adjusting for confounding variables, they found that the trend was no longer statistically significant (p = 0.22), and the relative risk for the highest quintile group of intake was attenuated: 0.75 (95% CI 0.48-1.15). Similar results were observed for total vitamin D intake; the age- and energy-adjusted relative risk was 0.54% (95% CI 0/34-0/85) for the highest versus lowest quintile group, and this was attenuated in the multivariate model (RR = 0.66, 95% CI 0.42-1.05). The inverse association was weaker for dietary vitamin D (RR highest vs. lowest quintile = 0.88. 95% CI 0.54-1.42) and strongest for vitamin D arising from vitamin supplements (RR = 0.48, 95% CI 0.22-1.02). Thus, it is possible that other components of multivitamin use rather than vitamin D accounted for the reduction in risk. Consumption of milk and fermented dairy products was not significantly associated with the risk of colon cancer; individuals consuming two or more glasses of "whole" or skim milk per day had a relative risk of 1.09 (95% CI 0.69-1.72), compared with those who consumed "whole or skim milk less than once a month. These prospective data do not support the hypothesis that calcium intake is strongly protective against colon cancer risk, although a modest association cannot be excluded.

12. Cancer Res. 1996 Feb 1;56(3):623-32.

Antiproliferative responses to two human colon cancer cell lines to vitamin D3 are differently modified by 9-cis-retinoic acid.

Kane KF, Langman MJ, Williams GR.

Department of Medicine, Queen Elizabeth Hospital, University of Birmingham, Edgbaston, United Kingdom.

1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] exerts antiproliferative actions in colorectal cancer, but their underlying molecular mechanisms have not been determined. 1,25(OH)2D3 regulates target gene transcription via a specific nuclear vitamin D receptor (VDR), which mediates hormone action preferentially as a heterodimer with 9-cis-retinoic acid receptors (RXRs). We investigated the actions of 1,25(OH)2D3 and 9-cis-retinoic acid (RA) in two human colon cancer cell lines, HT-29 and Caco-2. Both expressed mRNAs encoding VDR, RXR alpha, and RXR gamma, and VDR was regulated posttranscriptionally in Caco-2 cells. There was an antiproliferative response of both cell lines to 1,25(OH)2D3. 9-cis-RA exerted antiproliferative effects on Caco-2 cells but blocked 1,25(OH)2D3 actions in HT-29 cells. The 1,25(OH)2D3-responsive gene 25-hydroxyvitamin D3 24-hydroxylase was induced in both cell lines b 1,25(OH)2D3 but in only HT-29 cells by 9-cis-RA. 1,25(OH)2D3 and 9-cis-RA cotreatment enhanced 24-hydroxylase expression in HT-29 cells only. The 24-hydroxylase enzyme is known to result in catabolism of 1,25(OH)2D3 and attenuation of its actions. Increased 24-hydroxylase activity in HT-29 cells, but not in Caco-2 cells, in response to 9-cis-RA may account for some of the complex cell-specific responses demonstrated in these studies.

13. Am J Epidemiol. 1993 Jun 15;137(12):1302-17.

Relation of calcium, vitamin D, and dairy food intake to incidence of colon cancer among older women. The Iowa Women's Health Study.

Bostick RM, Potter JD, Sellers TA, McKenzie DR, Kushi LH, Folsom AR.

Department of Family Practice and Community Health, Medical School, University of Minnesota, Minneapolis 55454.

To investigate whether a high intake of calcium, vitamin D, or dairy products may protect against colon cancer, the authors analyzed data from a prospective cohort study of 35,216 Iowa women aged 55-69 years without a history of cancer who completed a dietary questionnaire in 1986. Through 1990, 212 incident cases of colon cancer were documented. Adjusted for age, intakes of calcium and vitamin D were significantly inversely associated with the risk of colon cancer. The relative risks for the highest quintile of intake as compared with the lowest were 0.52 (95% confidence interval (CI) 0.33-0.82) for calcium and 0.54 (95% CI 0.35-0.84) for vitamin D. After multivariate adjustment, the trends were no longer statistically significant and the relative risks for the highest versus the lowest quintiles of calcium and vitamin D intakes were attenuated: 0.68 (95% CI 0.41-1.11) for calcium and 0.73 (95% CI 0.45-1.18) for vitamin D. Although the multivariate-adjusted findings did not reach statistical significance at p < or = 0.05, when considered in the context of the whole body of literature on this subject, they are consistent with a possible role for calcium or vitamin D in modestly reducing colon cancer risk.

14. Endocrinology. 1993 Apr;132(4):1808-14.

Regulation of vitamin D receptor abundance and responsiveness during differentiation of HT-29 human colon cancer cells.

Zhao X, Feldman D.

Division of Endocrinology, Stanford University School of Medicine, California 94305.

We have studied the effects of 1,25-dihydroxyvitamin D [1,25-(OH)2D3] on cellular differentiation in the HT-29 human colon cancer cell line. Our aim was to evaluate the regulation of 1,25-dihydroxyvitamin D receptor (VDR) abundance and hormone responsiveness during the transition of rapidly proliferating to differentiated cells. Differentiation was induced by three means: cells were cultured in galactose-supplemented medium without glucose (GAL), grown on Matrigel-coated surfaces (MTG), or treated with 1,25(OH)2D3. Cell proliferation, assessed by [3H]thymidine incorporation, was equivalently inhibited by treatment with 1,25(OH)2D3, GAL or MTG. Differentiation was assessed by the induction of amino-oligo peptidase activity which was low in the proliferating cells. Following treatment with 1,25(OH)2D3, or growth in GAL or on MTG, amino-oligo peptidase activity increased 8- to 9-fold. The abundance of VDR measured by [3H]1,25(OH)2D3 binding, decreased to half without significant change in affinity, in cells differentiated by all three means compared to proliferating cells. Northern blot analyses of differentiated cells showed decreased steady-state levels of VDR messenger RNA (mRNA), indicating that all three treatments similarly decreased the abundance of VDR, at least in part, at the mRNA level. When exposed to 1,25(OH)2D3, the proliferating cells exhibited homologous up-regulation of VDR as well as the induction of 24-hydroxylase mRNA; the differentiated cells failed to exhibit both of these biological responses. Our findings demonstrate that 1,25(OH)2D3, GAL and MTG treatment all inhibit HT-29 cell proliferation and stimulate differentiation. Postproliferative differentiation achieved by the three approaches was associated with decreased VDR abundance, loss of VDR homologous up-regulation, and development of hormone unresponsiveness to 1,25(OH)2D3.

15. Gut. 1992 Dec;33(12):1660-3.

Vitamin D and its metabolites inhibit cell proliferation in human rectal mucosa and a colon cancer cell line.

Thomas MG, Tebbutt S, Williamson RC.

Department of Surgery, Royal Postgraduate Medical School, Hammersmith Hospital, London.

Like calcium, vitamin D may protect against colorectal neoplasia as it reduces epithelial cell proliferation and induces differentiation. Although its therapeutic use is limited by its effects on calcium metabolism, analogues such as calcipotriol produce little hypercalcaemia. Stathmokinetic and immunohistochemical techniques were used to study the effect of 1,25 (OH)2 D3 and its analogues on cell proliferation in human rectal mucosa and a colon cancer cell line. Paired sigmoidoscopic biopsy specimens were obtained from 17 control patients and five patients with familial adenomatous polyposis. Explants were established in organ culture, with or without the addition of vitamin D. Proliferation was assessed using (1) metaphase arrest to determine the crypt cell production rate (CCPR) and (2) Ki-67 monoclonal antibody directed against an antigen present in proliferating cells. 1,25 (OH)2 D3 in concentrations of 1 microM-100 pM (10(-6)-10(-10) M) reduced the CCPR (cells/crypt/hour) from 4.74 to 2.15-2.67 (p < 0.001), and the Ki-67 labelling index from 7.28-3.74 (p < 0.01). Likewise, vitamin D2, 10 nM (10(-8) M) reduced the CCPR from 4.74-2.74 (p < 0.05) and calcipotriol from 4.86-2.38 (p < 0.05). In familial adenomatous polyposis patients 1,25 (OH)2 D3 100 pM (10(-10) M) halved the CCPR from 8.75-4.22. Calcipotriol (10(-5) M to 10(-9) M) produced a clearcut dose response inhibition of HT-29 cell growth. Thus, vitamin D and its metabolites inhibit proliferation in normal and premalignant rectal epithelium and suppress growth in a colorectal cancer cell line.

16. Am J Clin Nutr. 1991 Jul;54(1 Suppl):193S-201S.

Can colon cancer incidence and death rates be reduced with calcium and vitamin D?

Garland CF, Garland FC, Gorham ED.

Department of Community and Family Medicine, University of California, San Diego, La Jolla 92093-0607.

It was proposed in 1980 that vitamin D and calcium could reduce the risk of colon cancer. This assertion was based on the decreasing gradient of mortality rates from north to south, suggesting a mechanism related to a favorable influence of ultraviolet-induced vitamin D metabolites on metabolism of calcium. A 19-y prospective study of 1954 Chicago men found that a dietary intake of greater than 3.75 micrograms vitamin D/d was associated with a 50% reduction in the incidence of colorectal cancer, whereas an intake of greater than or equal to 1200 mg Ca/d was associated with a 75% reduction. Clinical and laboratory studies further support these findings. A nested case-control study based on serum drawn from a cohort of 25,620 individuals reported that moderately elevated concentrations of 25-hydroxyvitamin D, in the range 65-100 nmol/L, were associated with large reductions (P less than 0.05) in the incidence of colorectal cancer.

17. Anticancer Res. 1987 Jul-Aug;7(4B):817-21.

The role of vitamin D3 in the proliferation of a human colon cancer cell line in vitro.

Lointier P, Wargovich MJ, Saez S, Levin B, Wildrick DM, Boman BM.

M. D. Anderson Hospital and Tumor Institute, Section of Gastrointestinal Oncology and Digestive Diseases, Houston, Texas 77030.

LoVo, a cultured colon cancer cell line, is shown to possess a receptor for 1,25-dihydroxy vitamin D3 (1 alpha,25(OH)2D3) with a low capacity (28 fmol/mg protein) and high affinity (Kd: 1.9 x 10(-21)0M). When these cells were grown in monolayer culture in a chemically defined serum-free medium, a significant inhibition of proliferation was seen in the presence of 10 nM to 1 microM of 1 alpha,25(OH)2D3 (p less than 0.005. Furthermore, 1 alpha,25(OH)2D3 delayed early attachment of cells. After 8 days of treatment, aggregated cuboidal cells showed a marked change to an apparently spindle like morphology. The 1 alpha,25(OH)2D3 growth-inhibitory effect was modulated by verapamil (1 microM), a calcium channel blocker, hydrocortisone (1 microM), and moxestrol (1 mM), an estrogen analogue, and 2% charcoal-treated fetal bovine serum. This study represents the first demonstration of 1 alpha,25(OH)2D3 modulation of growth of human colon cells.

18. Int J Epidemiol. 1980 Sep;9(3):227-31.

Do sunlight and vitamin D reduce the likelihood of colon cancer?

Garland CF, Garland FC.

It is proposed that vitamin D is a protective factor against colon cancer. This hypothesis arose from inspection of the geographic distribution of colon cancer deaths in the U.S., which revealed that colon cancer mortality rates were highest in places where populations were exposed to the least amounts of natural light--major cities, and rural areas in high latitudes. The hypothesis is supported by a comparison of colon cancer mortality rates in areas that vary in mean daily solar radiation penetrating the atmosphere. A mechanism involving cholecalciferol (vitamin D3) is suggested. The possibility that an ecological fallacy or other indirect association explains the findings is explored.

Melanoma

19. Br J Dermatol. 2002 Aug;147(2):197-213.

Vitamin D and systemic cancer: is this relevant to malignant melanoma?

Osborne JE, Hutchinson PE.

Department of Dermatology, Leicester Royal Infirmary, Leicester LE1 5WW, UK. joyos@doctors.org.uk

1,25-dihydroxyvitamin D3[1,25(OH)2D3] is a well-known potent regulator of cell growth and differentiation and there is recent evidence of an effect on cell death, tumour invasion and angiogenesis, which makes it a candidate agent for cancer regulation. The classical synthetic pathway of 1,25(OH)2D3 involves 25- and 1 alpha-hydroxylation of vitamin D3, in the liver and kidney, respectively, of absorbed or skin-synthesized vitamin D3. There is recent focus on the importance in growth control of local metabolism of 1,25(OH)2D3, which is a function of local tissue synthetic hydroxylases and particularly the principal catabolizing enzyme, 24-hydroxylase. The classical signalling pathway of 1,25(OH)2D3 employs the vitamin D nuclear receptor (VDR), which is a transcription factor for 1,25(OH)2D3 target genes. Effects of this pathway include inhibition of cellular growth and invasion. Cytoplasmic signalling pathways are increasingly being recognized, which similarly may regulate growth and differentiation but also apoptosis. 1,25(OH)2D3 has a major inhibitory effect on the G1/S checkpoint of the cell cycle by upregulating the cyclin dependent kinase inhibitors p27 and p21, and by inhibiting cyclin D1. Indirect mechanisms include upregulation of transforming growth factor-beta and downregulation of the epidermal growth factor receptor. 1,25(OH)2D3 may induce apoptosis either indirectly through effects on the insulin-like growth receptor and tumour necrosis factor-alpha or more directly via the Bcl-2 family system, the ceramide pathway, the death receptors (e.g. Fas) and the stress-activated protein kinase pathways (Jun N terminal kinase and p38). Inhibition of tumour invasion and metastasis potential has been demonstrated and mechanisms include inhibition of serine proteinases, metalloproteinases and angiogenesis. The lines of evidence for an effect of vitamin D3 in systemic cancer are the laboratory demonstration of relevant effects on cellular growth, differentiation, apoptosis, malignant cell invasion and metastasis; epidemiological findings of an association of the occurrence and outcome of cancers with derangements of vitamin D3/1,25(OH)2D3 and the association of functional polymorphisms of the VDR with the occurrence of certain cancers. In addition, vitamin D3 analogues are being developed as cancer chemotherapy agents. There is accumulating evidence that the vitamin D3/1,25(OH)2D3/VDR axis is similarly important in malignant melanoma (MM). MM cells express the VDR, and the antiproliferative and prodifferentiation effects of 1,25(OH)2D3 have been shown in cultured melanocytes, MM cells and MM xenografts. Recently, an inhibitory effect on the spread of MM cells has been demonstrated, low serum levels of 1,25(OH)2D3 have been reported in MM patients and the VDR polymorphisms have been shown to be associated with both the occurrence and outcome of MM. The relationship between solar irradiation and MM is more complex than for the systemic cancers. As in other cancers, there is evidence of a protective effect of vitamin D3 in MM, but ultraviolet radiation, which is a principal source of vitamin D3, is mutagenic. Further work is necessary on the influence of serum vitamin D3 levels on the occurrence and prognosis of MM, the effects of sun protection measures on serum vitamin D3 levels in temperate climates and epidemiological studies on geographical factors and skin type on the prognosis of MM. Meanwhile, it would seem mandatory to ensure an adequate vitamin D3 status if sun exposure were seriously curtailed, certainly in relation to carcinoma of breast, prostate and colon and probably also MM.

20. Clin Cancer Res. 2000 Feb;6(2):498-504.

Vitamin D receptor polymorphisms are associated with altered prognosis in patients with malignant melanoma.

Hutchinson PE, Osborne JE, Lear JT, Smith AG, Bowers PW, Morris PN, Jones PW, York C, Strange RC, Fryer AA.

Department of Dermatology, Leicester Royal Infirmary, United Kingdom.

Calcitriol [1,25(OH)2D3], the hormonal derivative of vitamin D3, is an antiproliferative and prodifferentiation factor for several cell types, including cultured melanocytes and malignant melanoma (MM) cells. Several polymorphisms of the vitamin D receptor (VDR) gene have been described including a FokI RFLP in exon 2, BsmI, and ApaI polymorphisms in intron 8 and an adjacent TaqI RFLP in exon 9. Alterations in vitamin D/1,25(OH)2D3 levels and polymorphisms of the VDR have been shown to be associated with several systemic malignancies. We hypothesize that polymorphism in this gene may be associated with altered susceptibility and outcome in patients with MM. A hospital-based case-control study, using 316 MM cases and 108 controls, was used to assess associations with MM susceptibility. Breslow thickness, the most important single prognostic factor in MM, was used as the outcome measure. Polymorphisms at the FokI and TaqI restriction sites were determined using PCR-based methods. Polymorphism at the FokI, but not TaqI, RFLP was associated with an altered risk of MM (P = 0.014). More importantly, variant alleles were associated with increased Breslow thickness. Thus, homozygosity for variant alleles at both RFLP (ttff genotype combination) was significantly associated with thicker tumors. (> or = 3.5 mm; P = 0.001; odds ratio = 31.5). Thus, polymorphisms of the VDR gene, which would be expected to result in impaired function, are associated with susceptibility and prognosis in MM. These data suggest that 1,25(OH)2D3, the ligand of the VDR, may have a protective influence in MM, as has been proposed for other malignancies.

21. Med Hypotheses. 1997 Apr;48(4):351-4.

A role for photoproducts of vitamin D in the etiology of cutaneous melanoma?

Braun MM, Tucker MA.

Epidemiology and Biostatistics Program, National Cancer Institute, National Institutes of Health, Rockville, MD 20852, USA.

Several clinical and epidemiological aspects of cutaneous melanoma seem anomalous because they contrast with other sunlight-associated skin cancers. For example, persons with the greatest risk of melanoma are not those with the greatest cumulative solar exposure, the anatomic areas that receive the most solar exposure are not preferentially affected, and the incidence of the disease is seasonal, with more cases reported in summer than winter. This article discusses the synthesis and biologic effects of vitamin D photoproducts and suggests that sun-related local skin effects, mediated by vitamin D photoproducts, on melanocytes previously damaged by excessive solar exposure may help explain the seemingly anomalous aspects of melanoma.

22. J Surg Res. 1996 Feb 15;61(1):127-33.

Vitamin D receptor and growth inhibition by 1,25-dihydroxyvitamin D3 in human malignant melanoma cell lines.

Evans SR, Houghton AM, Schumaker L, Brenner RV, Buras RR, Davoodi F, Nauta RJ, Shabahang M.

Department of Surgery, Lombardi Cancer Center, Georgetown University Hospital, Washington, DC 20007, USA.

The expression of vitamin D receptors (VDR) and growth inhibition induced by 1,25-dihydroxyvitamin D3 have been noted in certain human malignant melanoma cell lines. In this study, widely disparate levels of VDR mRNA expression were demonstrated in a panel of eight human malignant melanoma cell lines. Quantitation of receptor level by ligand binding assay showed a similar pattern. Proliferation and growth curve analysis was performed in two cell lines: RPMI 7951 (high VDR) and SK-MEL-28 (low VDR). Significant growth inhibition was noted in RPMI 7951 cells at 10(-9) M 1,25-dihydroxyvitamin D3. SK-MEL-28 cells, which express much lower levels of VDR, did not show any growth inhibition except at extremely high concentrations of 1,25-dihydroxyvitamin D3, namely 10(-5) M. These findings suggest a receptor-mediated mechanism of growth inhibition for 1,25-dihydroxyvitamin D3 and a role for this hormone in the growth of malignant melanoma cells.

23. J Clin Endocrinol Metab. 1983 Sep;57(3):627-31.

The synthesis of vitamin D metabolites by human melanoma cells.

Frankel TL, Mason RS, Hersey P, Murray E, Posen S.

Two melanin-producing human melanoma cell lines originally established from fresh surgical specimens were incubated with 25 hydroxyvitamin D3 (25 OHD3). Both cell lines produced material comigrating with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3) and 24,25 dihydroxyvitamin D3 (24,25(OH)2D3) in straight and reverse phase high performance liquid chromatography systems and displacing the relevant labeled ligands in competitive binding assays. The material designated 1,25(OH)2D3 was found almost entirely within the cells, whereas 24,25(OH)2D3 was evenly distributed between cells and medium. The synthesis of dihydroxylated materials was time dependent and was not observed if the cells were boiled before incubation with 25 OHD3. Preincubation with 1,25(OH)2D3 caused an increase in the synthesis of 24,25(OH)2D3 and a decrease in the synthesis of 1,25(OH)2D3. Michaelis-Menten constant (Km) values were 1.4 X 10(-9) mol/liter 25 OHD3 for the 1-alpha-hydroxylase enzyme and 72 X 10(-9) mol/liter for 24-hydroxylase. These studies constitute further evidence for the extrarenal synthesis of 1,25(OH)2D3. The suppressibility of 1 alpha-hydroxylase by preincubation with 1,25(OH)2D3 suggests a regulatory function for this system in the skin.

Breast Cancer

24. Int J Cancer. 2003 Aug 20;106(2):178-86.

Vitamin D enhances caspase-dependent and -independent TNFalpha-induced breast cancer cell death: The role of reactive oxygen species and mitochondria.

Weitsman GE, Ravid A, Liberman UA, Koren R.

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Calcitriol, the hormonal form of vitamin D, potentiates the activity of some common anticancer drugs and agents of the anticancer immune system, including tumor necrosis factor alpha (TNFalpha). TNFalpha-induced cytotoxicity is due to both caspase-dependent and -independent pathways. Cotreatment with calcitriol enhanced both modes of TNFalpha-induced death in MCF-7 breast cancer cells. It increased caspase-3-like activity as assayed by the cleavage of poly-(ADP-ribose)polymerase and of the fluorogenic substrate ac-DEVD-AMC. It also enhanced TNFalpha-induced caspase-independent cytotoxicity in the presence of the pan-caspase inhibitor zD-2,6-dichlorobenzoyloxymethylketone. The antioxidants N-acetylcysteine, reduced glutathione, lipoic acid and ascorbic acid markedly reduced the enhancing effect of the hormone on TNFalpha-induced caspase activation. N-acetylcysteine and reduced glutathione also decreased caspase-independent cytotoxicity in the presence or absence of calcitriol, indicating that reactive oxygen species (ROS) have a key role in the cross talk between TNFalpha and calcitriol. Mitochondrial damage is common to both TNFalpha-induced caspase-dependent and -independent pathways and may underlie excessive production of ROS. Mitochondrial membrane potential (DeltaPsi) was assessed by the specific potential-sensitive fluorescent probe JC-1. The hormone augmented the drop in DeltaPsi and release of cytochrome c from mitochondria, induced by TNFalpha. The effect of calcitriol on DeltaPsi was mimicked by rotenone, which increased both the drop in DeltaPsi and caspase activation induced by TNFalpha. It is possible that the interaction of TNFalpha and calcitriol on the level of the mitochondria is the underlying mechanism responsible for the enhancement of TNFalpha-induced, ROS-mediated caspase-dependent and -independent cell death. Copyright 2003 Wiley-Liss, Inc.

25. J Steroid Biochem Mol Biol. 2003 Feb;84(2-3):181-92.

Efficacy of Vitamin D compounds to modulate estrogen receptor negative breast cancer growth and invasion.

Flanagan L, Packman K, Juba B, O'Neill S, Tenniswood M, Welsh J.

Department of Biological Sciences, University of Notre Dame, 126 Galvin Life Sciences Building, IN 46556, USA.

In estrogen receptor (ER) positive breast cancer cells such as MCF-7 cells, the anti-tumor effects of 1,25(OH)(2)D(3) (1,25D(3)) may be secondary to disruption of estrogen mediated survival signals. If so, then sensitivity to 1,25D(3) mediated growth arrest could be reduced in estrogen independent breast cancer cells. The aim of these studies was to determine the effects of 1,25D(3) and EB1089 on the ER negative, invasive human breast cancer cell line SUM-159PT. 1,25D(3) and EB1089 reduced SUM-159PT cell growth subsequent to elevation of p27 and p21 levels. 1,25D(3) mediated apoptosis of SUM-159PT cells was associated with an enrichment of membrane bound bax, a redistribution of cytochome c from the mitochondria to the cytosol and PARP cleavage. 1,25D(3) and EB1089 also inhibited SUM-159PT cell invasion through an 8 microM Matrigel membrane. In pre-clinical studies, EB1089 dramatically reduced the growth of SUM-159PT xenografts in nude mice. The decreased size of tumors from EB1089 treated mice was associated with decreased proliferation and increased DNA fragmentation. Our data support the concept that Vitamin D(3) compounds trigger apoptosis by mechanisms independent of estrogen signaling. These studies indicate that Vitamin D(3) based therapeutics may be beneficial, alone or in conjunction with other agents, for the treatment of estrogen independent breast cancer.

26. Histochem J. 2002 Jan-Feb;34(1-2):35-40.

Analysis of vitamin D-receptor (VDR) and retinoid X-receptor alpha in breast cancer.

Friedrich M, Axt-Fliedner R, Villena-Heinsen C, Tilgen W, Schmidt W, Reichrath J.

Department of Gynecology and Obstetrics, University Hospital of Saarland, Homburg/Saar, Germany.

The expression of vitamin D-receptor (VDR) and retinoid X-receptor alpha (RXR-alpha) has been analysed immunohistochemically in benign (n = 62 and n = 5 respectively) and malignant (n = 228 and n = 15 respectively) breast tissue samples using a monoclonal antibody 9A7gamma against VDR and a polyclonal antibody against RXR-alpha. A recently developed immunoreactive scoring method (IRS) was employed. The expression of VDR was detected at the RNA-level using the reverse transcriptase-polymerase chain reaction. A statistically significant higher expression of VDR at the protein level was seen in breast cancer compared with benign breast tissue, whereas at the mRNA level no visible differences in the expression of VDR were found. A higher expression of RXR-alpha was seen in breast cancer compared with benign breast tissue. Our findings indicate that breast tissue may be a new target organ for therapeutically applied vitamin D and retinoid analogues. VDR and RXR-alpha are upregulated at the protein level in breast carcinomas as compared to normal breast tissue, indicating a possibly increased sensitivity to therapeutically applied vitamin D analogues. New vitamin D analogues exerting less calcemic side effects may be promising new drugs for the treatment or chemoprevention of breast carcinomas as well as of precancerous breast lesions. Combination therapies of vitamin D and retinoid analogues with fewer side effects seen promising for the treatment of breast cancer.

27. J Natl Cancer Inst. 2002 Sep 4;94(17):1301-11.

Intake of dairy products, calcium, and vitamin d and risk of breast cancer.

Shin MH, Holmes MD, Hankinson SE, Wu K, Colditz GA, Willett WC.

Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, USA.

BACKGROUND: Laboratory data suggest that calcium and vitamin D, found at high levels in dairy products, might reduce breast carcinogenesis. However, epidemiologic studies regarding dairy products and breast cancer have yielded inconsistent results. We examined data from a large, long-term cohort study to evaluate whether high intake of dairy products, calcium, or vitamin D is associated with reduced risk of breast cancer. METHODS: We followed 88 691 women in the Nurses' Health Study cohort from the date of return of their food-frequency questionnaire in 1980 until May 31, 1996. Dietary information was collected in 1980 and updated in 1984, 1986, 1990, and 1994. We identified 3482 women (premenopausal = 827, postmenopausal = 2345, and uncertain menopausal status = 310) with incident invasive breast cancer. We used pooled logistic regression to estimate multivariable relative risks (RRs) using 2-year time increments. The RRs and 95% confidence intervals (CIs) were calculated for each category of intake compared with the lowest intake group. All statistical tests were two-sided. RESULTS: Intakes of dairy products, calcium, or vitamin D were not statistically significantly associated with breast cancer risk in postmenopausal women. In premenopausal women, however, consumption of dairy products, especially of low-fat dairy foods and skim/low-fat milk, was inversely associated with risk of breast cancer. The multivariable RRs comparing highest (>1 serving/day) and lowest (<or=3 servings/month) intake categories were 0.68 (95% CI = 0.55 to 0.86) for low-fat dairy foods and 0.72 (95% CI = 0.56 to 0.91) for skim/low-fat milk. Dairy calcium (>800 mg/day versus <or=200 mg/day; RR = 0.69, 95% CI = 0.48 to 0.98), total vitamin D (>500 IU/day versus <or=150 IU/day; RR = 0.72, 95% CI = 0.55 to 0.94), and lactose (quintile 5 versus quintile 1; RR = 0.68, 95% CI = 0.54 to 0.86] also had inverse associations with premenopausal breast cancer risk. By taking into account supplemental calcium and vitamin D intake, we found that association with calcium was due mainly to dairy sources whereas the association with vitamin D may be independent of dairy intake. CONCLUSIONS: We found no association between intake of dairy products and breast cancer in postmenopausal women. Among premenopausal women, high intake of low-fat dairy foods, especially skim/low-fat milk, was associated with reduced risk of breast cancer. Similar inverse associations were seen with components (calcium and vitamin D) of dairy foods, but their independent associations with breast cancer are difficult to distinguish.

28. J Biol Chem. 2002 Aug 23;277(34):30738-45. Epub 2002 Jun 18.

Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells.

Mathiasen IS, Sergeev IN, Bastholm L, Elling F, Norman AW, Jaattela M.

Apoptosis Laboratory, Danish Cancer Society, Strandboulevarden 49, DK 2100 Copenhagen O, Denmark.

The active form of vitamin D(3) (1,25(OH)(2)D(3)) induces an increase in the intracellular free calcium ([Ca(2+)](i)) and caspase-independent cell death in human breast cancer cells. Here we show that the treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) or its chemotherapeutic analog, EB 1089, releases Ca(2+) from the endoplasmic reticulum. The increase in [Ca(2+)](i) was associated with the activation of a calcium-dependent cysteine protease, mu-calpain. Interestingly, ectopic expression of a calcium-binding protein, calbindin-D(28k), in MCF-7 cells not only attenuated the elevation in [Ca(2+)](i) and calpain activation, but also reduced death triggered by vitamin D compounds. Similarly, the inhibition of calpain activity by structurally unrelated chemical inhibitors increased the survival of the cells and reduces the amount of annexin V-positive cells. Despite the complete absence of effector caspase activation, transmission electron microscopy of MCF-7 cells treated with 1,25(OH)(2)D(3) or EB 1089 revealed apoptosis-like morphology characterized by the condensed cytoplasm, nuclei, and chromatin. Overall, these results suggest that calpain may take over the role of the major execution protease in apoptosis-like death induced by vitamin D compounds. Thus, these compounds may prove useful in the treatment of tumors resistant to therapeutic agents dependent on the classical caspase cascade.

29. J Biol Chem. 2002 Jul 19;277(29):25884-92. Epub 2002 Apr 30.

The p38 and JNK pathways cooperate to trans-activate vitamin D receptor via c-Jun/AP-1 and sensitize human breast cancer cells to vitamin D(3)-induced growth inhibition.

Qi X, Pramanik R, Wang J, Schultz RM, Maitra RK, Han J, DeLuca HF, Chen G.

Department of Radiation Oncology, Loyola University of Chicago, Maywood, Illinois 60153, USA.

The signaling connection between mitogen-activated protein kinases(MAPKs) and nuclear steroid receptors is complex and remains mostly unexplored. Here we report that stress-activated protein kinases p38 and JNK trans-activate nuclear steroid vitamin D receptor (VDR) gene and increase vitamin D(3)-dependent growth inhibition in human breast cancer cells. Activation of p38 and JNK by an active MAPK kinase 6 stimulates VDR promoter activity independently of the ligand vitamin D(3) and estrogen receptor expression. Moreover, stimulation of the endogenous stress pathways by adenovirus-mediated delivery of recombinant MAPK kinase 6 also activates VDR and sensitizes MCF-7 cells to vitamin D(3)-dependent growth inhibition. Both the p38 and JNK MAPK pathways and the downstream transcription factor c-Jun/AP-1 are required for the VDR stimulation, as revealed by application of their dominant negatives, the specific p38 inhibitor SB203580, and site-directed mutagenesis of the AP-1 element in the VDR promoter. The essential role of the p38 and JNK stress pathways in up-regulation of VDR expression is further confirmed by using the chemical stimulator arsenite. These results establish a signaling connection between the stress MAPK pathways and steroid hormone receptor VDR expression and thereby offer new insights into regulation of cell growth by the MAPK pathways through regulation of vitamin D(3)/VDR activity.

30. Endocr Relat Cancer. 2002 Mar;9(1):45-59.

Mechanisms implicated in the growth regulatory effects of vitamin D in breast cancer.

Colston KW, Hansen CM.

Department of Oncology, Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, London SW17 0RE, UK. k.colston@sghms.ac.uk

It is now well established that, in addition to its central role in the maintenance of extracellular calcium levels and bone mineralization, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, also acts as a modulator of cell growth and differentiation in a number of cell types, including breast cancer cells. The anti-proliferative effects of 1,25(OH)(2)D(3) have been linked to suppression of growth stimulatory signals and potentiation of growth inhibitory signals, which lead to changes in cell cycle regulators such as p21(WAF-1/CIP1) and p27(kip1), cyclins and retinoblastoma protein as well as induction of apoptosis. Such studies have led to interest in the potential use of 1,25(OH)(2)D(3) in the treatment or prevention of certain cancers. Since this approach is limited by the tendency of 1,25(OH)(2)D(3) to cause hypercalcaemia, synthetic vitamin D analogues have been developed which display separation of the growth regulating effects from calcium mobilizing actions. This review examines mechanisms by which 1,25(OH)(2)D(3) and its active analogues exert both anti-proliferative and pro-apoptotic effects and describes some of the synthetic analogues that have been shown to be of particular interest in relation to breast cancer.

31. Cancer Res. 2001 Feb 15;61(4):1439-44.

Vitamin D is a prooxidant in breast cancer cells.

Koren R, Hadari-Naor I, Zuck E, Rotem C, Liberman UA, Ravid A.

Basil and Gerald Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva, Israel. rkoren@post.tau.ac.il

The anticancer activity of the hormonal form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D], is associated with inhibition of cell cycle progression, induction of differentiation, and apoptosis. In addition, 1,25(OH)2D3 augments the activity of anticancer agents that induce excessive reactive oxygen species generation in their target cells. This study aimed to find out whether 1,25(OH)2D3, acting as a single agent, is a prooxidant in cancer cells. The ratio between oxidized and reduced glulathione and the oxidation-dependent inactivation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) are considered independent markers of cellular reactive oxygen species homeostasis and redox state. Treatment of MCF-7 breast cancer cells with 1,25(OH)2D3 (10-100 nM for 24-48 h) brought about a maximal increase of 41+/-13% (mean +/- SE) in the oxidized/reduced glutathione ratio without affecting total glutathione levels. The in situ activity of glutathione peroxidase and catalase were not affected by 1,25(OH)2D3, as assessed by the rate of H2O2 degradation by MCF-7 cell cultures. Neither did treatment with 1,25(OH)2D3 affect the levels of glutathione reductase or glutathione S-transferase as assayed in cell extracts. The hormone did not affect overall glutathione consumption and efflux as reflected in the rate of decline of total cellular glutathione after inhibition of its synthesis by buthionine sulfoximine. The extent of reversible oxidation-dependent inactivation of GAPDH in situ was determined by comparing the enzyme activity before and after reduction of cell extracts with DTT. The oxidized fraction was 0.13+/-0.02 of total GAPDH in control cultures and increased by 56+/-5.3% after treatment with 1,25(OH)2D3, which did not affect the total reduced enzyme activity. Treatment with 1,25(OH)2D3 resulted in a approximately 40% increase in glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the generation of NADPH. This enzyme is induced in response to various modes of oxidative challenge in mammalian cells. Taken together, these findings indicate that 1,25(OH)2D3 causes an increase in the overall cellular redox potential that could translate into modulation of redox-sensitive enzymes and transcription factors that regulate cell cycle progression, differentiation, and apoptosis.

32. Br J Cancer. 2001 Mar 2;84(5):686-90.

Anti-oestrogen resistant human breast cancer cell lines are more sensitive towards treatment with the vitamin D analogue EB1089 than parent MCF-7 cells.

Larsen SS, Heiberg I, Lykkesfeldt AE.

Department of Tumor Endocrinology, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, Copenhagen, DK-2100 O, Denmark.

Most breast cancer patients treated with anti-oestrogens will eventually develop resistance towards treatment. Therefore it is important to find new therapeutic agents effective for treatment of patients relapsing on anti-oestrogen. The vitamin D analogue EB1089 (Seocalcitol(TM)) is a promising new agent for treatment of breast cancer patients with advanced disease, and in this study we show that two different anti-oestrogen-resistant human breast cancer cell lines are more sensitive towards treatment with EB1089, than the parent MCF-7 cell line. The two resistant cell lines both express a lower content of the anti-apoptotic protein Bcl-2, and we suggest that this may explain the higher sensitivity towards EB1089. The importance of Bcl-2 for response to EB1089 is supported by our observation that oestradiol abrogates the effect of EB1089 in cell lines which increase Bcl-2 in response to oestradiol treatment. Overall these results indicate that treatment with Seocalcitol(TM)may prove effective when patients become refractory to anti-oestrogen therapy, and that Bcl-2 may be used as a predictive marker. Copyright 2001 Cancer Research Campaign.

33. Steroids. 2001 Mar-May;66(3-5):309-18.

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells.

Pirianov G, Colston KW.

Department of Oncology, Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, Cranmer Terrace, SW17 0RE, London, UK.

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.

34. Mol Cell Endocrinol. 2001 Feb 14;172(1-2):69-78.

Interactions of vitamin D analogue CB1093, TNFalpha and ceramide on breast cancer cell apoptosis.

Pirianov G, Colston KW.

Department of Oncology, Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, Cranmer Terrace, Tooting, SW17 ORE, London, UK.

Mechanisms by which vitamin D analogues promote apoptosis in tumour cells are unclear. In this study we have examined possible interactions between the synthetic vitamin D analogue CB1093 and two other known mediators of apoptosis, TNFalpha and ceramide, in MCF-7, T47D and Hs578T breast cancer cells. These studies indicated that cytosolic phospholipase A(2) (cPLA(2)) is involved in CB1093 as well as TNFalpha-mediated cell death. CB1093 promoted both TNFalpha and ceramide-induced c-PLA(2) activation, which was inversely related to loss of cell viability in MCF-7 and Hs578T cells. TNFalpha alone (5-20 ng/ml) failed to induce cytotoxicity and activation of cPLA(2) in T47D cells. However, pretreatment of these cells with CB1093 potentiated C(2)-ceramide-induced cPLA(2) activation and cell death. Treatment with CB1093 alone induced loss of cell viability and DNA fragmentation in all three cell lines by 5 days and these effects were accompanied by activation of cPLA(2). Furthermore, co-treatment with the cPLA(2) inhibitor AACOCF(3) led to partial protection against loss of cell viability induced by CB1093 in Hs578T and T47D cells as well as MCF-7 cells. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not C(2)-ceramide and CB1093-mediated release of arachidonic acid and cell death in MCF-7 cells. These results indicate that CB1093 potentiates responsiveness of breast cancer cells to TNFalpha and suggest that ceramide and/or cPLA(2) might be involved as downstream effectors in vitamin D-mediated caspase-independent cell death.

35. J Biol Chem. 2001 Mar 23;276(12):9101-7. Epub 2000 Oct 26.

Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells.

Narvaez CJ, Welsh J.

Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.

36. Cancer Res. 2000 Aug 15;60(16):4412-8. (Animal Study)

The vitamin D analogue EB 1089 prevents skeletal metastasis and prolongs survival time in nude mice transplanted with human breast cancer cells.

El Abdaimi K, Dion N, Papavasiliou V, Cardinal PE, Binderup L, Goltzman D, Ste-Marie LG, Kremer R.

Department of Medicine, McGill University and Royal Victoria Hospital, Montreal, Quebec, Canada.

1,25-Dihydroxyvitamin D has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of EB 1089, a low calcemic analogue of vitamin D, on the development of osteolytic bone metastases after intracardiac injection of the human breast cancer cell line MDA-MB-231 in nude mice. Animals injected with tumor cells were implanted simultaneously with osmotic minipumps containing either EB 1089 or vehicle. Both groups remained normocalcemic for the duration of the experiment. The total number of bone metastases, the mean surface area of osteolytic lesions, and tumor burden within bone per animal were markedly decreased in EB1089-treated mice. Furthermore, longitudinal analysis revealed that mice treated with EB1089 displayed a marked increase in survival and developed fewer bone lesions and less hind limb paralysis over time as compared with untreated animals. These results suggest that EB1089 may be beneficial in the prevention of metastatic bone lesions associated with human breast cancer.

Biochem Biophys Res Commun. 2000 Jul 5;273(2):675-80.

37. Vitamin D induced up-regulation of keratinocyte growth factor (FGF-7/KGF) in MCF-7 human breast cancer cells.

Lyakhovich A, Aksenov N, Pennanen P, Miettinen S, Ahonen MH, Syvala H, Ylikomi T, Tuohimaa P.

Tampere University Medical School, Tampere, 33101, Finland. alex.lyakhovich@uta.fi

Keratinocyte growth factor (FGF-7/KGF) is a secreted member of the fibroblast growth factor family, which functions primarily as an important paracrine mediator of cell growth and differentiation. Inhibitory pathways of vitamin D may also involve participation of some growth factors. To determine whether vitamin D may play a role in the expression of FGF-7, we investigated FGF-7 expression in human breast cancer cells treated with 1,25-dihydroxyvitamin D3, which inhibited the growth of the cells. By means of cDNA microarray, RT-PCR, and Western blot analysis, we have shown an increase in expression of FGF-7 on both mRNA and protein levels after vitamin D exposure. This is the first demonstration of vitamin D regulation of FGF-7 expression and its possible involvement in mediating growth and differentiation by vitamin D. Copyright 2000 Academic Press.

38. Eur J Cancer. 2000 Apr;36(6):780-6.

Induction of differentiation by 1alpha-hydroxyvitamin D(5) in T47D human breast cancer cells and its interaction with vitamin D receptors.

Lazzaro G, Agadir A, Qing W, Poria M, Mehta RR, Moriarty RM, Das Gupta TK, Zhang XK, Mehta RG.

Department of Surgical Oncology, University of Illinois College of Medicine, 840 S. Wood St (M/C 820), Chicago 60612, USA.

The role of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in cell differentiation is well established. However, its use as a differentiating agent in a clinical setting is precluded due to its hypercalcaemic activity. Recently, we synthesised a relatively non-calcaemic analogue of vitamin D(5), 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)), which inhibited the development of carcinogen-induced mammary lesions in culture and suppressed the incidence of chemically induced mammary carcinogmas in rats. In the present study, we determined the differentiating effects of 1alpha-(OH)D(5) in T47D human breast cancer cells and compared its effects with 1,25(OH)(2)D(3). Cells incubated with either 10 or 100 nM of the analogues inhibited cell proliferation in a dose-dependent manner, as measured by the dimethylthiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Similar growth-inhibitory effects were also observed for MCF10(neo) cells. Both vitamin D analogues induced cell differentiation, as determined by induction of casein expression and lipid production. However, MCF10(neo) cells failed to respond to either vitamin D analogue and did not undergo cell differentiation. Since the cell differentiating effect of vitamin D is considered to be mediated via the vitamin D receptor (VDR), we examined the induction of VDR using reverse transcriptase-polymerase chain reaction (RT-PCR) in both cells. The results showed that, in T47D cells, both 1,25(OH)(2)D(3) and 1alpha(OH)D(5) induced VDR in a dose-dependent manner. Moreover, both analogues of vitamin D upregulated the expression of vitamin D response element-chloramphenicol acetyl transferase (VDRE-CAT). These results collectively indicate that 1alpha-(OH)D(5) may mediate its cell-differentiating action via VDR in a manner similar to that of 1,25(OH)(2)D(3).

39. Oncol Res. 1999;11(6):265-71.

Influence of static magnetic field on the antiproliferative effects of vitamin D on human breast cancer cells.

Pacini S, Aterini S, Pacini P, Ruggiero C, Gulisano M, Ruggiero M.

Department of Anatomy, Histology and Forensic Medicine, University of Firenze at the Careggi General Hospital, Italy.

We describe the effect of a 0.2 tesla (T) static magnetic field generated by a magnetic resonance tomograph and of vitamin D treatment on a human breast cancer cell line (MCF-7). Cell damage and proliferation were monitored by measuring the incorporation of [3H]thymidine in duplicating DNA and by the clonogenic assay. [3H]Thymidine incorporation in MCF-7 was stimulated by vitamin D at low doses (10(-12)-10(-10) M), whereas it was inhibited at higher concentrations (10(-9)-10(-6) M). Magnetic field treatment (0.2 T) decreased [3H]thymidine incorporation in human breast cancer cells, eliminating the proproliferative effect of low doses of vitamin D, and enhanced the vitamin D antiproliferative effect, further reducing [3H]thymidine incorporation, from -12.5% (P < 0.05) to -66.7% (P < 0.001), over the range of 10(-9) to 10(-6) M. In the clonogenic assay, ability of MCF-7 to form colonies was inhibited by vitamin D 10(-9) M and above, whereas 3-h exposure to 0.2 T magnetic field had no effect on the number of cell colonies formed. In conclusion, vitamin D treatment yields a permanent antiproliferative effect, while magnetic field exposure only temporarily slows down cellular growth. These findings suggest that therapy with vitamin D may prove beneficial for chemoprevention or treatment of breast cancer. Static magnetic field, alone or in combination, does not appear to represent an effective candidate for breast cancer therapy, at least at the intensity used in the present study.

40. Eur J Cancer. 1999 Nov;35(12):1717-23.

Vitamin D analogues suppress IGF-I signalling and promote apoptosis in breast cancer cells.

Xie SP, Pirianov G, Colston KW.

Department of Oncology, Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, London, U.K.

Survival factors are known to promote cell viability, and factor deprivation can be a potent apoptotic signal. Insulin-like growth factors are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cells with insulin-like growth factor-I (IGF-I) being the most effective in many breast cancer cell lines. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues inhibit IGF-I-stimulated growth of MCF-7 human breast cancer cells. The aim of this study was to determine the relationship between inhibition of IGF-I responsiveness and induction of apoptosis by vitamin D analogues in breast cancer cells. Vitamin D analogues EB1089 and CB1093 inhibited autonomous and IGF-I-stimulated growth of MCF-7 and T47D cells and autonomous growth of IGF-I-insensitive Hs578T cells. In MCF-7 cells, IGF-I alone (4 nM) protected against apoptosis mediated by serum deprivation. Co-treatment with vitamin D analogues prevented the anti-apoptotic effects of IGF-I. In T47D cells, IGF-I treatment provided only partial protection against apoptosis induced by serum deprivation and co-incubation of serum-deprived cells with 100 nM CB1093 and IGF-I abrogated this partial protection. In Hs578T cells, addition of IGF-I did not prevent apoptosis induced by serum deprivation. However, treatment with CB1093 attenuated the protective effect of the serum in these cells. Our findings suggest that vitamin D analogues inhibit IGF-I signalling pathways to promote apoptosis in breast cancer cells.

41. Ann N Y Acad Sci. 1999;889:107-19.

Calcium and vitamin D. Their potential roles in colon and breast cancer prevention.

Garland CF, Garland FC, Gorham ED.

Department of Family and Preventive Medicine, University of California, San Diego 92093, USA. cgarland@ucsd.edu

The geographic distribution of colon cancer is similar to the historical geographic distribution of rickets. The highest death rates from colon cancer occur in areas that had high prevalence rates of rickets--regions with winter ultraviolet radiation deficiency, generally due to a combination of high or moderately high latitude, high-sulfur content air pollution (acid haze), higher than average stratospheric ozone thickness, and persistently thick winter cloud cover. The geographic distribution of colon cancer mortality rates reveals significantly low death rates at low latitudes in the United States and significantly high rates in the industrialized Northeast. The Northeast has a combination of latitude, climate, and air pollution that prevents any synthesis of vitamin D during a five-month vitamin D winter. Breast cancer death rates in white women also rise with distance from the equator and are highest in areas with long vitamin D winters. Colon cancer incidence rates also have been shown to be inversely proportional to intake of calcium. These findings, which are consistent with laboratory results, indicate that most cases of colon cancer may be prevented with regular intake of calcium in the range of 1,800 mg per day, in a dietary context that includes 800 IU per day (20 micrograms) of vitamin D3. (In women, an intake of approximately 1,000 mg of calcium per 1,000 kcal of energy with 800 IU of vitamin D would be sufficient.) In observational studies, the source of approximately 90% of the calcium intake was vitamin D-fortified milk. Vitamin D may also be obtained from fatty fish. In addition to reduction of incidence and mortality rates from colon cancer, epidemiological data suggest that intake of 800 IU/day of vitamin D may be associated with enhanced survival rates among breast cancer cases.

42. Int J Cancer. 1999 Dec 10;83(6):723-6.

Association of A vitamin D receptor polymorphism with sporadic breast cancer development.

Curran JE, Vaughan T, Lea RA, Weinstein SR, Morrison NA, Griffiths LR.

Genomics Research Centre, Griffith University Gold Coast, Southport, Queensland, Australia.

Breast cancer is the leading cause of cancer death among Australian women and its incidence is annually increasing. Genetic factors are involved in the complex etiology of breast cancer. The seco-steroid hormone, 1.25 dihydroxy vitamin D3 can influence breast cancer cell growth in vitro. A number of studies have reported correlations between vitamin D receptor (VDR) gene polymorphisms and several diseases including prostate cancer and osteoporosis. In breast cancer, low vitamin D levels in serum are correlated with disease progression and bone metastases, a situation also noted in prostate cancer and suggesting the involvement of the VDR. In our study, 2 restriction fragment length polymorphisms (RFLP) in the 3' region (detected by Apa1 and Taq1) and an initiation codon variant in the 5' end of the VDR gene (detected by Fok1) were tested for association with breast cancer risk in 135 females with sporadic breast cancer and 110 cancer-free female controls. Allele frequencies of the 3' Apa1 polymorphism showed a significant association (p = 0.016; OR = 1.56, 95% CI = 1.09-2.24) while the Taq1 RFLP showed a similar trend (p = 0.053; OR = 1.45, 95% CI = 1.00-2.00). Allele frequencies of the Fok1 polymorphism were not significantly different (p = 0.97; OR = 0.99, 95% CI = 0.69-1.43) in the study population. Our results suggest that specific alleles of the VDR gene located near the 3' region may identify an increased risk for breast cancer and justify further investigation of the role of VDR in breast cancer.

43. Cancer Res. 1999 Oct 1;59(19):4848-56.

Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by Bcl-2 but does not involve known caspases or p53.

Mathiasen IS, Lademann U, Jaattela M.

Apoptosis Laboratory, Institute of Cancer Biology, Danish Cancer Society, Copenhagen.

The hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, and its two analogues, EB 1089 and CB 1093, are novel putative anticancer agents with an interesting profile of induction of growth inhibition, differentiation, and apoptosis in tumor cells. To study the signaling pathways mediating these events, we used two human breast cancer cell lines: MCF-7 cells, expressing a wild-type p53 tumor suppressor protein, and T47D cells, lacking a functional p53. Vitamin D compounds induced a growth arrest followed by apoptosis in both cell lines at concentrations ranging from 1 to 100 nM, indicating that p53 is not necessary for growth-inhibitory effects induced by vitamin D compounds. Surprisingly, apoptosis induced by these compounds occurred also independently of known caspases. Inhibition of caspase activation by overexpression of a cowpox-derived caspase inhibitor CrmA or by addition of inhibitory peptides acetyl-Asp-Glu-Val-Asp-aldehyde (200 microM), acetyl-Ile-Glu-Thr-Asp-aldehyde (50 microM), and Z-Val-Ala-D,L-Asp-fluoromethylketone (1 microM) showed no effect on the induction of growth arrest or apoptosis by vitamin D compounds under assay conditions in which apoptosis induced by TNF or staurosporine was effectively inhibited. Moreover, overexpression of caspase-3 in MCF-7 cells had no sensitizing effect to vitamin D compounds, and neither caspase-3-like protease activity nor cleavage of a caspase substrate poly(ADP)ribose polymerase was detected in lysates from apoptotic cells following the treatment with these compounds. Contrary to CrmA, overexpression of an antiapoptotic protein Bcl-2 in MCF-7 cells conferred a nearly complete protection from apoptosis induced by vitamin D compounds. Taken together, these data indicate that vitamin D compounds induce apoptosis via a novel caspase- and p53-independent pathway that can be inhibited by Bcl-2. This may prove useful in the treatment of tumors that are resistant to therapeutic agents that are dependent on the activation of p53 and/or caspases.

44. Int J Oncol. 1999 Sep;15(3):589-94.

Inhibition of insulin-like growth factor I receptor signaling by the vitamin D analogue EB1089 in MCF-7 breast cancer cells: A role for insulin-like growth factor binding proteins.

Rozen F, Pollak M.

Lady Davis Institute for Medical Research of the Jewish General Hospital and Departments of Medicine and Oncology, McGill University, Montreal, Quebec H3T 1E2, Canada.

Insulin-like growth factors I and II (IGF-I and IGF-II) are potent mitogens involved in growth regulation of breast epithelial cells and are implicated in the pathophysiology of breast cancer. Their bioactivity is enhanced or inhibited by specific IGF-binding proteins (IGFBPs). Vitamin D-related compounds (VDRCs) have been shown to inhibit proliferation and induce apoptosis of MCF-7 breast carcinoma cells. We have previously demonstrated that VDRCs antagonize the growth-promoting activity of IGF-I by stimulating autocrine production of IGFBP-5 in MCF-7 cells, but the effect of VDRCs on IGF-I receptor (IGF-IR) intracellular signaling has not been elucidated. We report here that the vitamin D analogue EB1089 interferes with the IGF-IR signaling pathway by attenuating IGF-I-induced tyrosine phosphorylation of IRS-1, and to a lesser extent, IRS-2. It does not affect protein levels of IRS-1, IRS-2 or IGF-IR. However, EB1089 does not inhibit tyrosine phosphorylation of IRS-1 induced by des(1-3) IGF-I, an IGF-I analogue with greatly reduced affinity for IGFBPs. Furthermore, we demonstrate that an antisense IGFBP-5 oligodeoxynucleotide attenuates EB1089-induced inhibition of IGF-I-stimulated tyrosine phosphorylation of IRS-1 and EB1089-induced IGFBP-5 accumulation. These data strongly suggest that IGFBP-5 plays a functional role in the interfering action of EB1089 with the IGF-IR signal transduction pathway.

45. Cancer Epidemiol Biomarkers Prev. 1999 May;8(5):399-406.

Vitamin D and breast cancer risk: the NHANES I Epidemiologic follow-up study, 1971-1975 to 1992. National Health and Nutrition Examination Survey.

John EM, Schwartz GG, Dreon DM, Koo J.

Northern California Cancer Center, Union City 94587, USA. ejohn@nccc.org

We analyzed data from the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study to test the hypothesis that vitamin D from sunlight exposure, diet, and supplements reduces the risk of breast cancer. We identified 190 women with incident breast cancer from a cohort of 5009 white women who completed the dermatological examination and 24-h dietary recall conducted from 1971-1974 and who were followed up to 1992. Using Cox proportional hazards regression, we estimated relative risks (RRs) for breast cancer and 95% confidence intervals, adjusting for age, education, age at menarche, age at menopause, body mass index, alcohol consumption, and physical activity. Several measures of sunlight exposure and dietary vitamin D intake were associated with reduced risk of breast cancer, with RRs ranging from 0.67-0.85. The associations with vitamin D exposures, however, varied by region of residence. The risk reductions were highest for women who lived in United States regions of high solar radiation, with RRs ranging from 0.35-0.75. No reductions in risk were found for women who lived in regions of low solar radiation. Although limited by the relatively small size of the case population, the protective effects of vitamin D observed in this prospective study are consistent for several independent measures of vitamin D. These data support the hypothesis that sunlight and dietary vitamin D reduce the risk of breast cancer.

46. Br J Pharmacol. 1998 Nov;125(5):953-62.

EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro.

James SY, Mercer E, Brady M, Binderup L, Colston KW.

Division of Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School London.

1. Effects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea-induced rat mammary tumours in vivo were investigated. 2. At a dose of 0.5 microg kg(-1) body weight, EB1089 caused significant inhibition of tumour progression over the 28 day treatment period in the absence of a significant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 microg kg(-1)) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death. 3. Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3' DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 microg kg(-1)) for 14 days. 4. Effects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF-7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089. 5. The effects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl-2 and bax were examined by Western analysis. In MCF-7 cell cultures treated with 1,25(OH)2D3 or EB1089 (1 x 10(-8) M), bcl-2 protein levels were decreased in a time-dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl-2/bax ratio favouring increased susceptibility of MCF-7 cells to undergo apoptosis. 6. These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death.

47. J Mol Endocrinol. 1998 Feb;20(1):157-62.

Growth inhibition of both MCF-7 and Hs578T human breast cancer cell lines by vitamin D analogues is associated with increased expression of insulin-like growth factor binding protein-3.

Colston KW, Perks CM, Xie SP, Holly JM.

Division of Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, London, UK.

The effects of two vitamin D analogues, EB1089 and CB1093, on insulin-like growth factor binding protein (IGFBP) expression have been examined in MCF-7 and Hs578T human breast cancer cell lines. Both vitamin D analogues inhibited IGF-1 stimulated growth of MCF-7 cells and enhanced the production of IGFBP-3 as determined by Western-ligand blotting. Recombinant human IGFBP-3 inhibited the growth of MCF-7 cells over the concentration range 1-235 ng/ml. Hs578T cells were unresponsive to the mitogenic effects of IGF-1 but growth was inhibited by the two vitamin D analogues. Treatment of Hs578T cells with EB1089 and CB1093 (10 nM) as well as 100 nM 9-cis retinoic acid (9-cis RA) or all-trans retinoic acid (ATRA) was associated with increased accumulation of IGFBP-3 in conditioned medium. Furthermore, cotreatment of Hs578T cells with EB1089 and 9-cis RA led to augmented effects on both inhibition of cell growth and IGFBP-3 accumulation in conditioned medium as assessed by Western ligand blotting and radioimmunoassay. These findings suggest a role for IGFBP-3 in the growth inhibitory effects of vitamin D analogues.

48. J Endocrinol. 1997 Sep;154(3):495-504.

Vitamin D derivatives inhibit the mitogenic effects of IGF-I on MCF-7 human breast cancer cells.

Xie SP, James SY, Colston KW.

Division of Gastroenterology, Endocrinology and Metabolism, St George's Hospital Medical School, London, UK.

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.

49. J Steroid Biochem Mol Biol. 1995 Aug;54(3-4):147-53.

Modulation of vitamin D receptor and estrogen receptor by 1,25(OH)2-vitamin D3 in T-47D human breast cancer cells.

Davoodi F, Brenner RV, Evans SR, Schumaker LM, Shabahang M, Nauta RJ, Buras RR.

Department of Surgery, Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, DC, USA.

1,25(OH)2-Vitamin D3 inhibits breast cancer cell proliferation through interaction with the vitamin D receptor (VDR). Regulation of VDR is under the influence of several factors which include the functional ligand for this receptor (1,25(OH)2-vitamin D3) as well as heterologous steroid hormones. We evaluated the nature of homologous regulation in T-47D human breast cancer cells with a radiolabelled ligand binding assay and a ribonuclease protection assay for VDR. Significant VDR up-regulation, as measured by hormone binding assays, occurred with pre-incubations with 10(-9)M through 10(-6)M 1,25(OH)2-vitamin D3 (P < 0.05). A 7-fold VDR up-regulation with 10(-8)M 1,25(OH)2-vitamin D3 occurred at 4 h treatment and was not associated with an increase in VDR mRNA expression on ribonuclease protection assay. This supports the hypothesis that up-regulation of VDR is probably the result of ligand-induced stabilization of pre-existing receptor. All-trans-retinoic acid, the progesterone analog R-5020, and prednisone were found to induce heterologous up-regulation of the VDR. We then determined with ligand binding assays whether 1,25(OH)2-vitamin D3 could influence receptor levels for another hormone in a manner analogous to the heterologous regulation of VDR. Regulation of estrogen receptor (ER) by 1,25(OH)2-vitamin D3 was studied in T-47D and MDA-MB-231 breast cancer cells. Incubation of T-47D cells, which are ER (+), with 10(-8)M 1,25(OH)2-vitamin D3 did not result in up-regulation of ER. Yet estrogen binding was significantly up-regulated in a cell line that is ER(-), MDA-MB-231. The increased estrogen binding was associated with a shift in binding affinity and ribonuclease protection assay showed absence of ER mRNA in these cells, suggesting an up-regulation of estrogen binding proteins and not of the ER itself.

50. J Mol Endocrinol. 1995 Jun;14(3):391-4.

Vitamin D derivatives in combination with 9-cis retinoic acid promote active cell death in breast cancer cells.

James SY, Mackay AG, Colston KW.

Department of Clinical Biochemistry, St George's Hospital Medical School, London.

The effects of the novel vitamin D analogue, EB1089 alone, or in combination with the retinoid, 9-cis retinoic acid (9-cis RA) on indices of apoptosis in MCF-7 breast cancer cells have been examined. EB1089 was capable of reducing bcl-2 protein, a suppressor of apoptosis, and increasing p53 protein levels in MCF-7 cell cultures following 96h treatment. In the presence of 9-cis RA, EB1089 acted to further enhance the down-regulation and up-regulation of bcl-2 and p53 respectively. Furthermore, EB1089 induces DNA fragmentation in MCF-7 cells, a key feature of apoptosis, alone and in combination with 9-cis RA in situ. The observation that EB1089 and 9-cis RA act in a cooperative manner to enhance induction of apoptosis in these cells may have therapeutic implications.

51. Cancer Lett. 1995 May 25;92(1):77-82.

The antiproliferative effect of vitamin D analogs on MCF-7 human breast cancer cells.

Brenner RV, Shabahang M, Schumaker LM, Nauta RJ, Uskokovic MR, Evans SR, Buras RR.

Department of Surgery, Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, DC 20007-2197, USA.

We analyzed the antiproliferative effect of 1,25-dihydroxyvitamin D3 and four vitamin D analogs on MCF-7, a human breast cancer cell line known to express the vitamin D receptor. Growth curve studies and [3H]thymidine incorporation assays were used to assess the antiproliferative effect of 1,25-dihydroxyvitamin D3 (vitamin D), Ro 23-7553, Ro 24-5531, Ro 25-5317, and Ro 24-5583. Growth of MCF-7 cells was significantly inhibited by 1,25-dihydroxyvitamin D3 and all four analogs at 10(-8) M (P < 0.05). MCF-7 cells treated with analog had significantly less [3H]thymidine incorporation than cells treated with 1,25-dihydroxyvitamin D3 (P < 0.05). The affinity of the analogs for the vitamin D receptor was similar to that of 1,25-dihydroxyvitamin D3. These results demonstrate that analogs of 1,25-dihydroxyvitamin D3 are potent antiproliferative agents on human breast cancer cells and that this activity is likely mediated through the vitamin D receptor.

52. J Endocrinol. 1994 Jun;141(3):555-63.

Effects of a new synthetic vitamin D analogue, EB1089, on the oestrogen-responsive growth of human breast cancer cells.

James SY, Mackay AG, Binderup L, Colston KW.

Department of Clinical Biochemistry, St George's Hospital Medical School, London, UK.

The anti-proliferative effects of the novel vitamin D analogue, EB1089, were assessed in the hormone-dependent breast cancer cell line, MCF-7, in vitro. In the present study, EB1089 was shown to be at least an order of magnitude more potent at inhibiting MCF-7 cell proliferation than the native hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Treatment of MCF-7 cell cultures with combinations of oestradiol and EB1089 ranging from 5 x 10(-11) M to 5 x 10(-9) M revealed the ability of EB1089 to suppress the mitogenic effects of oestradiol in these cells dose-dependently, as determined by [3H]thymidine incorporation and cell counts. EB1089 also exhibited a significant time- and dose-dependent decrease in MCF-7 oestrogen receptor (ER) concentration, as assessed by ligand binding assay. A fourfold reduction of ER levels by 5 x 10(-9) M EB1089 relative to control ER levels was observed, whilst 5 x 10(-9) M 1,25(OH)2D3 produced a significant but less dramatic decrease in ER levels. In addition, reduction of ER protein in EB1089-treated cell cultures was also demonstrated using an oestrogen receptor enzyme immunoassay. The interaction of EB1089 and anti-oestrogens on the oestradiol-stimulated growth of MCF-7 cells was investigated. The treatment of cell cultures with 5 x 10(-10) M EB1089 in combination with the pure anti-oestrogen, ICI 182,780 (5 x 10(-8) M), and in the presence of between 5 x 10(-10) M and 5 x 10(-9) M oestradiol, produced an augmented inhibition of MCF-7 cell proliferation compared with the actions of either compound alone.(ABSTRACT TRUNCATED AT 250 WORDS)

53. Cancer Res. 1994 Apr 1;54(7):1653-6. (Animal Study)

1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531), a new deltanoid (vitamin D analogue) for prevention of breast cancer in the rat.

Anzano MA, Smith JM, Uskokovic MR, Peer CW, Mullen LT, Letterio JJ, Welsh MC, Shrader MW, Logsdon DL, Driver CL, et al.

Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892.

We have used the vitamin D analogue, 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531), for inhibition of mammary carcinogenesis induced by N-nitroso-N-methylurea (NMU) in Sprague-Dawley rats. Rats were first treated with a single dose of either 15 or 50 mg/kg body weight NMU and then fed Ro24-5531 (2.5 or 1.25 nmol/kg of diet) for 5-7 months. Ro24-5531 significantly extended tumor latency and lessened tumor incidence as well as tumor number in rats treated with the lower dose of NMU. In rats treated with the higher dose of NMU, Ro24-5531 was fed in combination with tamoxifen; in these experiments, Ro24-5531 significantly enhanced the ability of tamoxifen to reduce total tumor burden, as well as to increase the probability that an animal would be tumor free at the end of the experiment. In vitro, Ro24-5531 was 10-100 times more potent than 1,25-dihydroxyvitamin D3 for inhibition of proliferation of human breast cancer cell lines as well as primary cultures of cells from 2 patients with acute myelogenous leukemia. When fed chronically, Ro24-5531 did not elevate serum calcium in the present studies. We propose the new term, "deltanoids," for the set of molecules composed of vitamin D and its synthetic analogues, in a manner similar to the naming of "retinoids" for the corresponding set of molecules related to vitamin A.

54. Breast Cancer Res Treat. 1994;31(2-3):191-202.

Vitamin D receptors in breast cancer cells.

Buras RR, Schumaker LM, Davoodi F, Brenner RV, Shabahang M, Nauta RJ, Evans SR.

Department of Surgery, Georgetown University, Washington DC.

1,25-(OH)2-Vitamin D3, the active metabolite of vitamin D, is a secosteroid hormone with known differentiating activity in leukemic cells. Studies have demonstrated the presence of vitamin D receptors (VDR) in a wide range of tissues and cell types. Antiproliferative activity of 1,25-(OH)2-vitamin D3 has been documented in osteosarcoma, melanoma, colon carcinoma, and breast carcinoma cells. This study was designed to analyze vitamin D receptor level in breast cancer cells as a marker of differentiation and as a predictor of growth inhibition by 1,25-(OH)2-vitamin D3. VDR messenger RNA was found to be present in relatively high levels in well-differentiated cells and in low levels in poorly differentiated cells. All cell lines had detectable VDR mRNA. Radiolabeled ligand binding assay showed a similar pattern. MCF-7 and T47D cells, which express VDR at moderate levels, showed significant growth inhibition by 10(-9) M1,25-(OH)2-vitamin D3 (p < 0.05). MDA-MB-231 cells, which have very low levels of VDR, demonstrated no growth inhibition by 1,25-(OH)2-vitamin D3 at concentrations up to 10(-6) M. Based on these results it can be stated that VDR expression is lost with de-differentiation and that receptor is essential for the antiproliferative response to 1,25-(OH)2-vitamin D3.

55. Adv Exp Med Biol. 1994;364:109-14.

Vitamin D adequacy: a possible relationship to breast cancer.

Newmark HL.

Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

(1) Low levels of dietary calcium and vitamin D, biochemically interrelated, increase the promoting action of high dietary fat on chemically induced mammary carcinogenesis in animal studies. (2) High dietary fat increases mammary epithelial cell proliferation, particularly the "hormonally driven" hyperproliferation during breast growth and development in young animals. Increased dietary calcium (and probably vitamin D) lessens the increase of proliferation induced by high fat. These data, although limited, suggest that the maximum effect of diet (high fat increase, as well as calcium and vitamin D modulation) on eventual breast cancer may be during puberty, and adolescence, when the mammary gland is actively growing and developing. (3) An inverse epidemiological correlation has been developed between sunlight availability as a source of vitamin D and the risk of breast cancer in the U.S. and Canada. (4) Current vitamin D and calcium dietary intake in the U.S. is far below the RDA in all female age groups, particularly for the elderly. (5) Reduction of breast cancer risk, and simultaneously osteoporosis, might be achieved by increasing dietary intake of calcium and vitamin D to RDA levels. This may be particularly applicable to females during puberty and adolescence.

56. Biochem Pharmacol. 1992 Dec 15;44(12):2273-80. (Animal Study)

EB1089: a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro.

Colston KW, Mackay AG, James SY, Binderup L, Chander S, Coombes RC.

Department of Clinical Biochemistry, St Georges Hospital Medical School, Tooting, London, U.K.

EB1089 is a novel vitamin D analogue which has been tested for its effects on breast cancer cell growth in vitro, using the established human breast cancer cell line MCF-7, and in vivo on the growth of established rat mammary tumours. Both EB1089 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited MCF-7 cell proliferation with the synthetic analogue being at least an order of magnitude more potent than the native hormone. In vivo anti-tumour effects were investigated using the N-methyl-nitrosourea-induced rat mammary tumour model. Oral treatment with EB1089 was tested at three doses. With the lower dose, significant inhibition of tumour growth was seen in the absence of a rise in serum calcium. The same dose of 1,25-(OH)2D3 had no effect on tumour growth but caused hypercalcaemia. With the higher dose of EB1089, striking tumour regression was seen although serum calcium rose. This report demonstrates that EB1089 possess enhanced anti-tumour activity coupled with reduced calcaemic effects relative to 1,25-(OH)2D3 and thus may have therapeutic potential as an anti-tumour agent.

57. Biochem Pharmacol. 1992 Aug 18;44(4):693-702.

Effects of synthetic vitamin D analogues on breast cancer cell proliferation in vivo and in vitro.

Colston KW, Chander SK, Mackay AG, Coombes RC.

Department of Clinical Biochemistry, St George's Hospital Medical School, London, U.K.

Calcipotriol (MC903) is a novel vitamin D analogue which effects cellular differentiation and proliferation in vitro and has reduced effects on calcium metabolism in vivo. In the present study its in vitro activity was evaluated using the MCF-7 breast cancer cell line, and its effects on calcium metabolism and mammary tumour growth were measured in vivo in adult female rats. Calcipotriol was compared to the natural metabolite of vitamin D3, 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3] and its synthetic analogue 1 alpha hydroxycholecalciferol [1 alpha(OH)D3]. Both calcipotriol and 1,25(OH)2D3 produced significant inhibition of MCF-7 cell proliferation at a concentration of 5 x 10(-11) M. Intraperitoneal administration of calcipotriol to normal female rats showed that the analogue was 100-200 times less active than 1,25(OH)2D3 in raising serum calcium concentration and urinary calcium excretion. Anti-tumour activity of the vitamin D analogues was investigated in vivo using the nitrosomethylurea-induced rat mammary tumor model. Rats, maintained on a low calcium diet, were treated with 1 alpha(OH)D3 (0.25 and 1.25 micrograms/kg). Both doses produced a response rate of 25% but hypercalcaemia developed. Treatment with calcipotriol (50 micrograms/kg) of rats maintained on a normal laboratory diet caused inhibition of tumour progression (response rate 17%) without the development of severe hypercalcaemia. This study supports the concept that vitamin D derivatives may inhibit breast cancer cell proliferation in vivo.

58. Lancet. 1989 Jan 28;1(8631):188-91.

Possible role for vitamin D in controlling breast cancer cell proliferation.

Colston KW, Berger U, Coombes RC.

Department of Chemical Pathology, St George's Hospital Medical School, London.

By means of an immunocytochemical method the 1.25-dihydroxyvitamin D [1.25(OH)2D] receptor status of tumours from 136 patients with primary carcinoma of the breast was determined. Patients with receptor-positive tumours had significantly longer disease-free survival than those with receptor-negative tumours (Chi2 = 4.01, p less than 0.05). 1.25(OH)2D3 inhibits the proliferation of several established human breast cancer cell lines in vitro. Effects of 1.25(OH)2D3 on breast tumour growth in vitro were assessed by means of the nitrosomethylurea-induced rat mammary tumour model of hormone-responsive breast cancer. Treatment of tumour-bearing animals with 0.1 microgram of the synthetic analogue, 1 alpha-hydroxyvitamin D3, three times weekly produced significant inhibition of tumour progression. Taken together, these studies suggest that the levels of 1.25(OH)2D occurring in vivo may exert an inhibitory effect on receptor-positive tumours. Further studies are required to evaluate the role of vitamin D metabolites in the treatment of human malignant disease.

Prostate Cancer

59. Endocr Relat Cancer. 2003 Jun;10(2):131-40.

The role of vitamin D and retinoids in controlling prostate cancer progression.

Peehl DM, Feldman D.

Department of Urology, Stanford University School of Medicine, Stanford, California, USA. dpeehl@stanford.edu

Prostate cancer is a leading cause of cancer-related deaths in many countries. Premalignant lesions and invasive cancer occur more frequently in the prostate than in any organ other than the skin. Yet, the incidence of clinically detected prostate cancer is much lower than the histopathological incidence. The slow growth of prostate cancer and the low incidence of clinically manifest disease in some geographical locations or racial/ethnic groups suggest that prostate cancer can be controlled, perhaps by dietary factors. Vitamin D and retinoids have emerged as leading candidates both to prevent and to treat prostate cancer. Many of the activities of these compounds, established from epidemiological studies, research with cell culture and animal models, and clinical trials, are consistent with tumor suppressor effects. However, retinoids may have additional tumor enhancer properties that balance or negate anti-cancer activity. This perhaps explains the overall lack of protective effects of vitamin A compounds against prostate cancer found in epidemiological studies, and the minimal efficacy of retinoids in clinical trials to treat prostate cancer. While current efforts focus on developing strategies to use vitamin D compounds to control prostate cancer, the possibility exists that prostate cancer cells may become resistant to tumor suppressor effects of vitamin D. Analyses of experimental model systems show that prostate cancer cells become less sensitive to vitamin D through loss of receptors or signaling molecules that mediate vitamin D's actions, or through changes in metabolic enzymes that synthesize or degrade vitamin D compounds. The potential promise of exploiting vitamin D to control prostate cancer is tempered by the possibility that prostate cancer, perhaps even at early stages, may develop mechanisms to escape tumor suppressor activities of vitamin D and/or retinoids.

60. Int J Urol. 2003 May;10(5):261-6.

Vitamin D receptor gene polymorphism in familial prostate cancer in a Japanese population.

Suzuki K, Matsui H, Ohtake N, Nakata S, Takei T, Koike H, Nakazato H, Okugi H, Hasumi M, Fukabori Y, Kurokawa K, Yamanaka H.

Department of Urology, Gunma University School of Medicine, Gunma, Japan. kazu@showa.gunma-u.ac.jp

AIM: Vitamin D acts as an antiproliferative agent against prostate cells. Epidemiological study has shown that a low level of serum vitamin D concentration is a risk factor for prostate cancer. Vitamin D acts via vitamin D receptor (VDR), and an association of genetic polymorphisms of the VDR gene has been reported. In the current study, we examined the association of VDR gene polymorphisms with familial prostate cancer in a Japanese population. METHODS: We performed a case-control study consisting of 81 familial prostate cancer cases and 105 normal control subjects. Three genetic polymorphisms (BsmI, ApaI and TaqI) in the VDR gene were examined by the restriction fragment restriction length polymorphism method. RESULTS: Overall, there was no significant association of the VDR gene polymorphisms with familial prostate cancer risk in the cases and control subjects. However, a weak association between BsmI or TaqI genotypes and cancer risk was observed in subjects under 70 years of age. Stratification of cases by clinical stage or pathological grade did not show significant association between the VDR gene polymorphisms and prostate cancer risk. CONCLUSION: In the present study, we could not confirm any significant association between VDR gene polymorphisms with familial prostate cancer risk in a Japanese population. Further large-scale case-control studies are warranted to confirm the importance of VDR gene polymorphisms in familial prostate cancer.

61. J Cell Biochem. 2003 Feb 1;88(2):363-71.

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression.

Krishnan AV, Peehl DM, Feldman D.

Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA.

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued. Copyright 2002 Wiley-Liss, Inc.

62. Cancer Metastasis Rev. 2002;21(2):147-58.

Vitamin D-related therapies in prostate cancer.

Johnson CS, Hershberger PA, Trump DL.

candace.johnson@roswellpark.org

Calcitriol or 1,25-dihydroxycholecalciferol (vitamin D) is classically known for its effects on bone and mineral metabolism. Epidemiological data suggest that low vitamin D levels increase the risk and mortality from prostate cancer. Calcitriol is also a potent anti-proliferative agent in a wide variety of malignant cell types including prostate cancer cells. In prostate model systems (PC-3, LNCaP, DU145, MLL) calcitriol has significant anti-tumor activity in vitro and in vivo. Calcitriol's effects are associated with an increase in cell cycle arrest, apoptosis, differentiation and in the modulation of growth factor receptors. Calcitriol induces a significant G0/G1 arrest and modulates p21(Waf/Cip1) and p27(Kip1), the cyclin dependent kinase inhibitors. Calcitriol induces PARP cleavage, increases the bax/bcl-2 ratio, reduces levels of phosphorylated mitogen-activated protein kinases (P-MAPKs, P-Erk-1/2) and phosphorylated Akt (P-Akt), induces caspase-dependent MEK cleavage and up-regulation of MEKK-1, all potential markers of the apoptotic pathway. Glucocorticoids potentiate the anti-tumor effect of calcitriol and decrease calcitriol-induced hypercalcemia. In combination with calcitriol, dexamethasone results in a significant time- and dose-dependent increase in VDR protein and an enhanced apoptotic response as compared to calcitriol alone. Calcitriol can also significantly increase cytotoxic drug-mediated anti-tumor efficacy. As a result, phase I and II trials of calcitriol either alone or in combination with the carboplatin, paclitaxel, or dexamethasone have been initiated in patients with androgen-dependent and -independent prostate cancer and advanced cancer. Patients were evaluated for toxicity, maximum tolerated dose (MTD), schedule effects, and PSA response. Data from these studies indicate that high-dose calcitriol is feasible on an intermittent schedule, the MTD is still being delineated and dexamethasone or paclitaxel appear to ameliorate toxicity. Studies continue to define the MTD of calcitriol whichcan be safely administered on this intermittent schedule either alone or with other agents and to evaluate the mechanisms of calcitriol effects in prostate cancer.

63. BJU Int. 2002 Oct;90(6):607-16. (Animal Study)

Vitamin D receptor-dependent antitumour effects of 1,25-dihydroxyvitamin D3 and two synthetic analogues in three in vivo models of prostate cancer.

Oades GM, Dredge K, Kirby RS, Colston KW.

Department of Urology, St. George's Hospital and Medical School, London, UK. gmoades@baus.org.uk

OBJECTIVE: To determine the in vitro and in vivo effects of 1,25-dihydroxyvitamin D3 (calcitriol) and two newer less hypercalcaemic analogues, EB1089 and CB1093 (as the use of calcitriol as a therapeutic agent in humans has been limited by hypercalcaemia) in three rodent models of prostate cancer. MATERIALS AND METHODS: The highly metastatic MAT LyLu Dunning prostate model, PAIII tumours in Lobund-Wistar rats and LNCaP xenografts in nude mice were used. Vitamin D receptor (VDR) expression and binding were assessed in all cell lines. The effects of calcitriol, EB1089 and CB1093 on tumour growth, cell cycle and angiogenesis in vitro, and growth and serum calcium levels in vivo, were assessed. RESULTS: The growth of prostate adenocarcinoma was inhibited by calcitriol, EB1089 and CB1093 in the Dunning prostate model. Although both analogues increased serum calcium levels, the levels were significantly less than in rats treated with calcitriol. Tumour growth was also inhibited in male athymic nu/nu mice with LNCaP tumour xenografts. PAIII cells failed to express functional VDR and were insensitive to calcitriol and its analogues, either in vitro or in vivo. The analogues of calcitriol did not inhibit angiogenesis in a rat aorta assay. CONCLUSION: This is the first report comparing the actions of calcitriol and its analogues in different in vivo models. The results suggest that the newer less hypercalcaemic analogues of calcitriol may offer a novel therapeutic option for treating prostate cancer. VDR-dependent growth inhibition and not the inhibition of angiogenesis is the main mechanism of action of these compounds in vivo. J Urol. 2002 Oct;168(4 Pt 1):1583-8.

64. Preclinical activity of ketoconazole in combination with calcitriol or the vitamin D analogue EB 1089 in prostate cancer cells.

Peehl DM, Seto E, Hsu JY, Feldman D.

Department of Urology, Stanford University School of Medicine, California 94305-5118, USA.

PURPOSE: Ketoconazole is a general inhibitor of P450 enzymes, of which some are necessary for androgen biosynthesis and the metabolism of vitamin D compounds. We tested the growth inhibitory activity of ketoconazole combined with 1,25-dihydroxyvitamin D3 (calcitriol) and with the vitamin D analogue EB 1089 in a preclinical model of prostate cancer. MATERIALS AND METHODS: Clonal assays with primary cultures of human prostatic cancer cells were performed to test anti-proliferative effects of ketoconazole alone or in combination with calcitriol or EB 1089. Enzyme substrate reactions were done to determine whether the ability of ketoconazole to potentiate the activity of calcitriol or EB 1089 was due to the inhibition of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the enzyme that initiates conversion of active vitamin D compounds to inactive products. RESULTS: Ketoconazole, calcitriol and EB 1089 each inhibited the growth of prostatic cancer cells. In combination 0.1 microg./ml. ketoconazole potentiated growth inhibitory activity of calcitriol 50-fold and EB 1089 10-fold. Induction of 24-hydroxylase by calcitriol or EB 1089 was partially blocked by this level of ketoconazole. CONCLUSIONS: Combination therapy with ketoconazole and calcitriol or EB 1089 may enhance antitumor activities of vitamin D compounds for prostate cancer and alleviate side effects of vitamin D deficiency that are likely associated with ketoconazole therapy.

65. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 25;777(1-2):261-8.

Isoflavonoids inhibit catabolism of vitamin D in prostate cancer cells.

Farhan H, Wahala K, Adlercreutz H, Cross HS.

Institute of Pathophysiology, University of Vienna Medical School, AKH, Wahringergurtel 18-20 A-1090, Vienna, Austria.

The high ingestion of soybean products in Asian countries has been suggested to be responsible for a reduced incidence of prostate cancer. The mechanism of action, however, is unknown. Our data demonstrate that genistein and some isoflavone metabolites reduce the activity of 25-D3-24-hydroxylase (CYP24) in the human prostate cancer-derived cell line DU-145. CYP24 is also responsible for degradation of the active vitamin D metabolite 1,25-dihydroxyvitamin D3 which is known to be antimitotic and prodifferentiating in prostate cancer cells. High levels of CYP24 frequently found in prostate cancer cells may thus degrade the active metabolite. This could be prevented by ingestion of genistein-containing food such as soybeans.

66. Urol Clin North Am. 2002 Feb;29(1):95-106, ix.

Vitamin D and prostate cancer.

Konety BR, Getzenberg RH.

Departments of Urology, Pathology, and Pharmacology, University of Pittsburgh, 5200 Centre Avenue, G-40, Pittsburgh, PA 15232, USA.

Current approaches to the management of prostate cancer include surgery, radiation therapy or hormonal manipulation either individually or in combination. Diet is increasingly being recognized as playing a role in many cancers including that of the prostate. There is now considerable evidence suggesting a role for vitamin D in prostate cancer. In this article, we have reviewed the current evidence supporting the use of vitamin D in the prevention and treatment of prostate cancer.

67. Cancer Epidemiol Biomarkers Prev. 2002 Jun;11(6):555-63. (Animal Study)

The G gamma / T-15 transgenic mouse model of androgen-independent prostate cancer: target cells of carcinogenesis and the effect of the vitamin D analogue EB 1089.

Perez-Stable CM, Schwartz GG, Farinas A, Finegold M, Binderup L, Howard GA, Roos BA.

Geriatric Research, Education, and Clinical Center and Research Service, Veterans Affairs Medical Center, Miami, Florida 33125, USA. cperez@med.miami.edu

Transgenic mouse models of prostate cancer provide unique opportunities to understand the molecular events in prostate carcinogenesis and for the preclinical testing of new therapies. We studied the G gamma T-15 transgenic mouse line, which contains the human fetal globin promoter linked to SV40 T antigen (Tag) and which develops androgen-independent prostate cancer. Using the immunohistochemistry of normal mouse prostates before tumor formation, we showed that the target cells of carcinogenesis in G gamma T-15 mice are located in the basal epithelial layer. We tested the efficacy of the 1,25(OH)(2)D(3) analogue, EB 1089, to chemoprevent prostate cancer in these transgenic mice. Compared with treatment with placebo, treatment with EB 1089 at three different time points before the onset of prostate tumors in mice did not prevent or delay tumor onset. However, EB 1089 significantly inhibited prostate tumor growth. At the highest dose, EB 1089 inhibited prostate tumor growth by 60% (P = 0.0003) and the growth in the number of metastases, although this dose also caused significant hypercalcemia and weight loss. We conducted several in vitro experiments to explore why EB 1089 did not prevent the occurrence of the primary tumors. EB 1089 significantly inhibited the growth of a Tag-expressing human prostate epithelial cell line, BPH-1, and an androgen-insensitive subline of LNCaP cells [which was not inhibited by 1,25(OH)(2)D(3)]. Thus, neither Tag expression nor androgen insensitivity explain the absence of chemopreventive effect. Conversely, neither 1,25(OH)(2)D(3) nor EB 1089 inhibited the growth of the normal rat prostate basal epithelial cell line NRP-152. It is likely that EB 1089 was not effective in delaying the growth of the primary tumor in G gamma T-15 transgenic mice because the target cells of carcinogenesis in these mice are located in the basal epithelial layer. We conclude that G gamma T-15 transgenic mice are a useful model for testing vitamin D-based therapies in androgen-insensitive prostate cancer but are not suitable for studies of vitamin D-based chemoprevention. The superiority of EB 1089 over 1,25(OH)(2)D(3) in the growth suppression of androgen-insensitive prostate cancer cells supports the use of EB 1089 in androgen-insensitive prostate cancer.

68. Cancer Res. 2002 Jun 1;62(11):3084-92.

A novel targeting modality to enhance adenoviral replication by vitamin D(3) in androgen-independent human prostate cancer cells and tumors.

Hsieh CL, Yang L, Miao L, Yeung F, Kao C, Yang H, Zhau HE, Chung LW.

Department of Urology, Molecular Urology and Therapeutics Program, Emory University School of Medicine, Atlanta, GA 30322, USA. chsieh2@emory.edu

We report the development of a novel replication-competent adenoviral vector, Ad-hOC-E1, containing a single bidirectional human osteocalcin (hOC) promoter to drive both the early viral E1A and E1B gene. This vector selectively replicated in OC-expressing but not non-OC-expressing cells, with viral replication enhanced at least 10-fold on vitamin D(3) exposure. Both the artificial TATA-box and hOC promoter element in this bidirectional promoter construct were controlled by a common OC regulatory element which selectively activated OC expression in cells. The expression ofE1A and E1B gene by Ad-hOC-E1 can be markedly induced by vitamin D(3). Unlike Ad-sPSA-E1, an adenoviral vector with viral replication controlled by a strong super prostate-specific antigen (sPSA) promoter which only replicates in PSA-expressing cells with androgen receptor (AR), Ad-hOC-E1 retarded the growth of both androgen-dependent and androgen-independent prostate cancer cells irrespective of their basal level of AR and PSA expression. A single i.v. administration of 2 x 10(9) plaque-forming units of Ad-hOC-E1 inhibited the growth of previously established s.c. DU145 tumors (an AR- and PSA-negative cell line). Viral replication is highly enhanced by i.p. administration of vitamin D(3). Ultimately, enhancing Ad-hOC-E1 viral replication by vitamin D(3) may be used clinically to treat localized and osseous metastatic prostate cancer in men.

69. Mol Cell Endocrinol. 2002 Apr 25;190(1-2):115-24.

Regulation of PTH-related protein gene expression by vitamin D in PC-3 prostate cancer cells.

Tovar Sepulveda VA, Falzon M.

Department of Pharmacology and Toxicology and Sealy Center for Molecular Science, University of Texas Medical Branch, 10th and Market Streets, , Galveston 775550 1031, USA.

Parathyroid hormone-related protein (PTHrP) is expressed by prostate cancer cells. Since PTHrP increases prostate cancer cell growth and enhances the osteolytic effects of prostate cancer cells, it is important to control PTHrP expression in prostate cancer. Vitamin D exerts a protective effect against prostate cancer through its antiproliferative actions. We investigated whether this steroid also downregulates PTHrP gene transcription, using the human prostate cancer cell line PC-3 as a model system. We report that PTHrP mRNA and secreted protein levels are downregulated by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via a transcriptional mechanism. We also show that PTHrP gene expression is upregulated, also via a transcriptional mechanism, by epidermal growth factor (EGF), which is normally secreted by prostate cancer cells. 1,25(OH)(2)D(3) reversed the EGF-induced PTHrP upregulation at both the mRNA and protein levels. Since PTHrP enhances prostate cancer cell growth, this study demonstrates the importance of maintaining adequate levels of 1,25(OH)(2)D(3).

70. Mol Cell Endocrinol. 2002 Jan 15;186(1):69-79.

Vitamin D-mediated growth inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer.

Yang ES, Maiorino CA, Roos BA, Knight SR, Burnstein KL.

Department of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, PO Box 016189 (R-189), Miami, FL 33136, USA.

1,25-(OH)(2) vitamin D(3) (1,25-(OH)(2) D), the active metabolite of vitamin D, exerts antiproliferative effects on a variety of tumor cells including prostate. This inhibition requires vitamin D receptors (VDRs) as well as downstream effects on the G1 to S phase checkpoint of the cell cycle. Recent data raise the possibility that androgen plays a role in the antiproliferative effects of 1,25-(OH)(2) D in prostate cancer cells; however, this hypothesis has been difficult to test rigorously as the majority of prostate cancer cell lines (unlike human prostate tumors) lack androgen receptors (ARs). We utilized two different models of androgen-independent prostate cancer that express functional ARs and VDRs to evaluate a possible role of androgen in 1,25-(OH)(2) D mediated growth inhibition. We stably introduced the AR cDNA into the human prostate cancer cell line ALVA 31, which expresses functional VDR but is relatively resistant to growth inhibition by 1,25-(OH)(2) D. Neither ALVA-AR nor the control cells, ALVA-NEO, exhibited substantial growth inhibition by 1,25-(OH)(2) D in the presence or absence of androgen. This observation suggests that the basis for the resistance of ALVA 31 to 1,25-(OH)(2) D-mediated growth inhibition is not the lack of AR. The second model was LNCaP-104R1, an AR-expressing androgen independent prostate cancer cell line derived from androgen dependent LNCaP. 1,25-(OH)(2) D inhibited the growth of LNCaP-104R1 cells in the absence of androgen and this effect was not blocked by the antiandrogen Casodex. As was observed in the parental LNCaP cells, this effect was correlated with G1 phase cell cycle accumulation and upregulation of the cyclin dependent kinase inhibitor (CKI) p27, as well as increased association of p27 with cyclin dependent kinase 2. These findings suggest that the antiproliferative effects of 1,25-(OH)(2) D do not require androgen-activated AR but do involve 1,25-(OH)(2) D induction of CKIs required for G1 cell cycle checkpoint control.

71. J Steroid Biochem Mol Biol. 2001 Jan-Mar;76(1-5):125-34.

Vitamin D and prostate cancer.

Tuohimaa P, Lyakhovich A, Aksenov N, Pennanen P, Syvala H, Lou YR, Ahonen M, Hasan T, Pasanen P, Blauer M, Manninen T, Miettinen S, Vilja P, Ylikomi T.

Medical School, University of Tampere, 33014, Tampere, Finland. pentti.tuohimaa@uta.fi

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(2000) (847-852)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had 25OH-vitamin D (25-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum 25-VD, whereas low serum 25-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest 25-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high 25-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum 25-VD concentrations. After 1-month exposure, there was no significant increase in the serum 25-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that 25-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and 24-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-2, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since keratinocyte growth factor (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

72. Endocrinology. 2000 Jul;141(7):2567-73.

Hepatocyte growth factor and vitamin D cooperatively inhibit androgen-unresponsive prostate cancer cell lines.

Qadan LR, Perez-Stable CM, Schwall RH, Burnstein KL, Ostenson RC, Howard GA, Roos BA.

Geriatric Research, Education, and Clinical Center and Research Service, Veterans Affairs Medical Center, Department of Medicine, University of Miami School of Medicine, Florida 33101, USA.

Expression of MET, the receptor for hepatocyte growth factor (HGF), has been associated with androgen-insensitive prostate cancer. In this study we evaluated MET activation by HGF and HGF action in prostate cancer cell lines. HGF causes phosphorylation (activation) of the MET receptor in three androgen-unresponsive cell lines (DU 145, PC-3, and ALVA-31) together with morphological change. Although HGF is known to stimulate the growth of normal epithelial cells, including those from prostate, we found that HGF inhibited ALVA-31 and DU 145 (hormone-refractory) cell lines. Moreover, HGF and vitamin D additively inhibited growth in each androgen-unresponsive cell line, with the greatest growth inhibition in ALVA-31 cells. Further studies in ALVA-31 cells revealed distinct cooperative actions of HGF and vitamin D. In contrast to the accumulation of cells in G1 seen during vitamin D inhibition of androgen-responsive cells (LNCaP), growth inhibition of the androgen-unresponsive ALVA-31 cell line with the HGF and vitamin D combination decreased, rather than increased, the fraction of cells in G1, with a corresponding increase in the later cell cycle phases. This cell cycle redistribution suggests that in androgen-unresponsive prostate cancer cells, HGF and vitamin D act together to slow cell cycle progression via control at sites beyond the G1/S checkpoint, the major regulatory locus of growth control in androgen-sensitive prostate cells.

73. Cancer Res. 2000 Feb 15;60(4):779-82. (Animal Study)

Comment in: Cancer Res. 2001 May 15;61(10):4294.

A calcitriol analogue, EB1089, inhibits the growth of LNCaP tumors in nude mice.

Blutt SE, Polek TC, Stewart LV, Kattan MW, Weigel NL.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Limited options for the treatment of prostate cancer have spurred the search for new therapies. One innovative approach is the use of 1alpha,25-dihydroxyvitamin D3 (calcitriol) analogues to inhibit cancer growth. We demonstrate here that the calcitriol analogue, EB1089, extensively inhibits the growth of LNCaP prostate cancer cells in culture and causes the cells to both accumulate in G0-G1 and undergo apoptosis. Importantly, we found that EB1089 inhibits the growth of LNCaP tumor xenografts in nude mice. Because of these antiproliferative properties in vivo, EB1089 is a potential new therapeutic agent for the treatment of prostate cancer.

74. J Urol. 2000 Jan;163(1):187-90.

Treatment of vitamin D deficiency in patients with metastatic prostate cancer may improve bone pain and muscle strength.

Van Veldhuizen PJ, Taylor SA, Williamson S, Drees BM.

Department of Internal Medicine, Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA.

PURPOSE: We performed a phase II study to determine whether pain associated with prostate cancer bone metastasis would respond to vitamin D replacement and parameters of muscle strength would be improved by vitamin D replacement therapy. MATERIALS AND METHODS: After a 4-week placebo period, eligible patients received orally 2,000 units vitamin D daily for 12 weeks. Pain questionnaires and measurements of muscle strength were competed at study enrollment and every 4 weeks thereafter. Serum calcium and vitamin D were measured at each clinic visit. RESULTS: A total of 16 patients with advanced hormone refractory prostate cancer were enrolled in this phase II study, of whom 7 (44%) had decreased baseline vitamin D. With vitamin D treatment, 4 patients (25%) had improvement in pain scores and 6 (37%) had improvement in muscle strength measurements. Improvement in pain scores correlated with improvement in subjective symptoms but did not result in a significant decrease in regular scheduled analgesic requirements. CONCLUSIONS: Vitamin D deficiency develops in a significant percent of patients with advanced hormone refractory prostate cancer. Supplementation with vitamin D may be a useful adjunct for improving pain, muscle strength and quality of life in this patient population.

75. Eur Urol. 1999;35(5-6):392-4.

Vitamin D and prostate cancer risk.

Peehl DM.

Department of Urology, Stanford University School of Medicine, Stanford, Calif. 94305, USA. dpeehl@leland.stanford.edu

Prostate cancer is a progressive, multistep disease which presents many stages for intervention. Microscopic cancer is found in the prostate beginning by age 30 in about 20% of men, and the incidence increases steadily so that by the time a man is 90 years old, he has almost a 100% chance of having cancer in his prostate. Independent, multiple foci of cancer are present in the majority of prostate specimens, and the incidence of premalignant lesions is even higher than that of cancer. Yet, despite the high incidence of microscopic cancer, only 8% of men in the US present with clinically significant disease during their lifetime. Furthermore, only 3% of men in the US die of prostate cancer. In no other human cancer is there such disparity between the high incidence of microscopic malignancy and the relatively low death rate. Thus, there are many windows of opportunity for control of prostate cancer. Evidence from diverse areas of study - epidemiologic, molecular, genetic, cellular, animal models, and clinical trials - suggests that vitamin D may be an effective preventive agent against prostate cancer.

76. Proc Soc Exp Biol Med. 1999 Jun;221(2):89-98.

Vitamin D and prostate cancer.

Blutt SE, Weigel NL.

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Classically, the actions of vitamin D have been associated with bone and mineral metabolism. More recent studies have shown that vitamin D metabolites induce differentiation and/or inhibit cell proliferation of a number of malignant and nonmalignant cell types including prostate cancer cells. Epidemiological studies show correlations between the risk factors for prostate cancer and conditions that can result in decreased vitamin D levels. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol), inhibits growth of both primary cultures of human prostate cancer cells and cancer cell lines, but the mechanism by which the cells are growth-inhibited has not been clearly defined. Initial studies suggest that calcitriol alters cell cycle progression and may also initiate apoptosis. One of the disadvantages of using vitamin D in vivo is side-effects such as hypercalcemia at doses above physiological levels. Analogs of calcitriol have been developed that have comparable or more potent antiproliferative effects but are less calcemic. Further research into the mechanisms of vitamin D action in prostate and identification of suitable analogs for use in vivo may lead to its use in the treatment or prevention of prostate cancer.

77. Cancer Causes Control. 1998 Dec;9(6):559-66.

Comment in: Cancer Causes Control. 1998 Dec;9(6):541-3.

Dairy products, calcium, phosphorous, vitamin D, and risk of prostate cancer (Sweden)

Chan JM, Giovannucci E, Andersson SO, Yuen J, Adami HO, Wolk A.

Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115, USA.

OBJECTIVES: Dairy products consistently have been associated with an increased risk of prostate cancer, yet the mechanism of this relationship remains unknown. Recent hypotheses propose that 1,25 dihydroxyvitamin D (1,25 D) is protective for prostate cancer. One study in the United States found that calcium consumption, which can lower circulating 1,25 D, was associated with higher risk of advanced prostate cancer, and we sought to address this hypothesis in a distinct population. METHODS: We analyzed data from a population-based case-control study of prostate cancer conducted in Orebro, Sweden, with 526 cases and 536 controls. Using unconditional logistic regression models, we examined the relationship of dairy products, dietary calcium, phosphorous, and vitamin D with risk of total, extraprostatic, and metastatic prostate cancer. RESULTS: Calcium intake was an independent predictor of prostate cancer (relative risk (RR) = 1.91, 95 percent confidence interval (CI) 1.23-2.97 for intake > or = 1183 vs. < 825 mg/day), especially for metastatic tumors (RR = 2.64, 95 percent CI 1.24-5.61), controlling for age, family history of prostate cancer, smoking, and total energy and phosphorous intakes. High consumption of dairy products was associated with a 50 percent increased risk of prostate cancer. CONCLUSIONS: Our results support the hypothesis that high calcium intake may increase risk of prostate cancer, and this relation may underlie previously observed associations between dairy products and prostate cancer.

78. Clin Cancer Res. 1997 Aug;3(8):1331-8.

Three synthetic vitamin D analogues induce prostate-specific acid phosphatase and prostate-specific antigen while inhibiting the growth of human prostate cancer cells in a vitamin D receptor-dependent fashion.

Hedlund TE, Moffatt KA, Uskokovic MR, Miller GJ.

Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

Numerous studies have indicated that the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 protects against the development of clinical prostate cancer (PC). Whether this hormone also has therapeutic potential for patients with advanced PC has not yet been evaluated. Several synthetic vitamin D analogues are now available that have reduced hypercalcemic effects and yet effectively induce differentiation in some cell types. For these reasons, these analogues may be safer and more effective for cancer therapy than the natural hormone. In the current study, 13 such analogues were screened for their abilities to inhibit the growth of PC cell lines. Three of the most consistently effective analogues (Ro 23-7553, Ro 24-5531, and Ro 25-6760) were then chosen for further analysis. Growth studies using clones of the JCA-1 cell line that were transfected with the vitamin D receptor cDNA indicate that the antiproliferative effects of these analogues require vitamin D receptor expression. Furthermore, these three analogues induce the secretion of prostate-specific acid phosphatase and prostate-specific antigen (two markers of the differentiated prostatic phenotype) in the cell line LNCaP. These in vitro studies suggest that Ro 23-7553, Ro 24-5531, and Ro 25-6760 should be further evaluated as therapeutic agents for the treatment of PC.

79. Int J Oncol. 1998 Jul;13(1):137-43.

Regulation of insulin-like growth factor (IGF) II and IGF binding protein 3 autocrine loop in human PC-3 prostate cancer cells by vitamin D metabolite 1,25(OH)2D3 and its analog EB1089.

Huynh H, Pollak M, Zhang JC.

Lady Davis Research Institute of the Jewish General Hospital and Departments of Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada.

Prostate cancer and benign prostate hyperplasia (BPH) are major public health problems. Prostate epithelial cell proliferation is regulated by insulin-like growth factor I (IGF-I) which is mitogenic and anti-apoptotic, and IGF binding protein 3 (IGFBP-3) which is an apoptotic agent in these cells. We demonstrate that the 1,25(OH)2D3 and its analog EB1089-induced growth inhibition was associated with increased IGFBP-3 mRNA abundance, IGFBP-3 mRNA stability, IGFBP-3 protein accumulation, and decreased IGF-II gene expression. Anti-IGF-II antibody and exogenous recombinant human IGFBP-3 inhibit PC-3 cell proliferation. The results document the inhibitory effects of 1,25(OH)2D3 and EB1089 on the IGF system of mitogens in prostate cancer cells, and suggest a potential therapeutic use of EB1089 in treatment of BPH and prostate cancer.

80. Cancer Epidemiol Biomarkers Prev. 1996 Feb;5(2):121-6.

Circulating vitamin D metabolites in relation to subsequent development of prostate cancer.

Gann PH, Ma J, Hennekens CH, Hollis BW, Haddad JG, Stampfer MJ.

Division of Preventive Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

An emerging hypothesis suggests that vitamin D metabolites suppress the development of prostate cancer. In a recent epidemiological study, elevated levels of 1,25-dihydroxyvitamin D (1,25-D) in blood were associated with a greatly reduced risk, particularly in older men. We conducted a nested case-control study to evaluate the relationship between plasma levels of the two major vitamin D metabolites, 1,25-D and 25-hydroxyvitamin D (25-D), and subsequent diagnosis of prostate cancer. We also measured vitamin D-binding protein to investigate the influence of free metabolite levels on risk. Plasma samples from 14,916 participants in the Physicians' Health Study were collected and frozen in 1982-1983. This analysis included 232 cases diagnosed up to 1992 and 414 age-matched control participants. Vitamin D metabolite and vitamin D-binding protein assays were conducted without knowledge of case-control status. Median levels of 25-D, 1,25-D, and vitamin D-binding protein were indistinguishable between cases and controls. Analysis of risk for increasing quartiles of total or free metabolites did not reveal a pattern of decreasing risk. For 1,25-D, men in the highest quartile had an odds ratio of 0.88 (95% confidence interval = 0.53-1.45) compared to those in the lowest quartile. Significant reductions in risk were not seen in analyses restricted to older men, to cases occurring > 3 years from blood collection, or to cases presenting as aggressive prostate cancer. Nonsignificant inverse associations for 1,25-D appeared for some groups according to 25-D level, particularly when the cutoff for defining low 25-D was reduced. These results do not support the hypothesis that high circulating levels of vitamin D metabolites reduce prostate cancer risk, although small to moderate effects cannot be excluded.

81. Cancer Epidemiol Biomarkers Prev. 1995 Sep;4(6):655-9.

Seasonal variation in vitamin D, vitamin D-binding protein, and dehydroepiandrosterone: risk of prostate cancer in black and white men.

Corder EH, Friedman GD, Vogelman JH, Orentreich N.

Center for Demographic Studies, Duke University, Durham, North Carolina 27708, USA.

Our previous study provided evidence that higher serum levels of the active form of vitamin D, 1,25-dihydroxyvitamin D (1, 25-D), might possibly slow the progression of subclinical to clinically significant prostate cancer in both black and white men, especially after age 57. This paper extends the prior study by contrasting seasonal variation in 1,25-D and its precursor, 25-hydroxyvitamin D (25-D), in case and control subjects. In addition, the risk of prostate cancer is related to serum levels of vitamin D-binding protein (VDBP) and total dehydroepiandrosterone and to polymorphic variation in VDBP. The expected elevated summer levels of 25-D were seen in case and control subjects and, as expected, 1,25-D did not vary throughout the year in the control subjects. Unexpectedly, lower case levels of 1,25-D were limited largely to the summer months (P = 0.01) in both black and white cases and to cases greater than or equal to the median age of 57 years. Levels of VDBP and dehydroepiandrosterone and the frequencies of VDBP polymorphisms were similar in case and control subjects, although striking differences were seen in allelic frequencies in black and white men. These observations provide additional evidence that vitamin D metabolism may impact the risk of prostate cancer.

82. Anticancer Res. 1994 May-Jun;14(3A):1077-81.

Human prostate cancer cells: inhibition of proliferation by vitamin D analogs.

Schwartz GG, Oeler TA, Uskokovic MR, Bahnson RR.

Department of Clinical Epidemiology and Family Medicine, University of Pittsburgh Medical Center, PA.

1,25-Dihydroxyvitamin D [1,25(OH)2D3, calcitriol] can inhibit the proliferation of some human prostate cancer cells but its clinical use is limited by hypercalcemia. We therefore explored the bioactivity of less calcemic vitamin D analogs. We studied the effects of calcitriol and 3 synthetic analogs at concentrations of 10(-6) to 10(-12) M on the in vitro proliferation of 3 human prostate carcinoma cell lines: DU 145, PC-3, and LNCaP. Calcitriol and analogs showed significant antiproliferative activity on PC-3 and LNCaP cells. DU 145 cells were inhibited by the analogs only. We conclude that vitamin D analogs warrant further investigation as therapeutic agents in prostate cancer.

83. Endocrinology. 1993 May;132(5):1952-60.

Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines.

Skowronski RJ, Peehl DM, Feldman D.

Department of Medicine, Stanford University School of Medicine, California 94305.

It has been suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. In this study three human prostate carcinoma cell lines, LNCaP, DU-145, and PC-3, were examined both for the presence of specific 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] receptors (VDRs) and also employed to study the effects of hormone on cell proliferation and differentiation. Ligand binding experiments demonstrated classical VDR in all three cell lines examined with an apparent dissociation constant of 7.5, 5.4, and 6.3 x 10(-11) M for LNCaP, DU-145, and PC-3 cells, respectively. Corresponding binding capacity for the three prostate carcinoma cell lines were 27, 31, and 78 fmol/mg protein, respectively. The presence of VDR in the three cell lines was also confirmed by immunocytochemistry. In addition, one major 4.6-kilobase messenger RNA transcript hybridizing with a specific human VDR complementary DNA probe was identified in all three cell lines. Interestingly, both DU-145 and PC-3 but not LNCaP cell lines exhibited 1,25(OH)2D3-stimulated induction of 24-hydroxylase messenger RNA employed as a marker of 1,25(OH)2D3 action. Physiological levels of 1,25(OH)2D3 dramatically inhibited proliferation of the LNCaP and PC-3 cell lines. However, in spite of the presence of high affinity VDR, proliferation of DU-145 cells was not inhibited by 1,25(OH)2D3 at the doses tested. Treatment with 1,25(OH)2D3 caused a dose-dependent stimulation of prostate-specific antigen secretion by LNCaP cells. In conclusion, these results demonstrate that these three human prostate carcinoma cell lines all possess specific VDR and that 1,25(OH)2D3 treatment can elicit both an antiproliferative and a differentiating action on these cancer cells. The findings lend support to the hypothesis that vitamin D might exert beneficial actions on prostate cancer risk.

84. Anticancer Res. 1990 Sep-Oct;10(5A):1307-11.

Is vitamin D deficiency a risk factor for prostate cancer? (Hypothesis).

Schwartz GG, Hulka BS.

Department of Epidemiology, University of North Carolina, School of Public Health, Chapel Hill 27599.

Prostate cancer is a major cause of cancer death among males, yet little is known about its etiology. We hypothesize that Vitamin (Hormone) D deficiency may underlie the major risks for prostate cancer, including age, Black race, and northern latitudes. These factors all are associated with decreased synthesis of Vitamin D. Mortality rates from prostate cancer in the U.S. are inversely correlated with ultraviolet radiation, the principal source of Vitamin D. This hypothesis is consistent with known antitumor properties of Vitamin D, and may suggest new avenues for research in prostate cancer.

Ovarian Cancer

85. Int J Cancer. 2000 Apr 1;86(1):40-6.

Androgen receptor and vitamin D receptor in human ovarian cancer: growth stimulation and inhibition by ligands.

Ahonen MH, Zhuang YH, Aine R, Ylikomi T, Tuohimaa P.

Department of Anatomy, Medical School, University of Tampere, Finland.

The data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and androgens are essential for regulation of growth and differentiation in, e.g., human reproductive tissues. We investigated the possible cross-talk between 1,25(OH)2D3 and androgens in the human ovarian cancer cell line OVCAR-3. Our data demonstrate that 1,25(OH)2D3 and androgen (dihydrotestosterone, DHT) regulate the growth of OVCAR-3 cells. Nine days' treatment of OVCAR-3 cells with 100 nM DHT resulted in 48% stimulation of growth, whereas growth inhibition (73%) was observed after treatment with 100 nM 1,25(OH)2D3. The combination of 1,25(OH)2D3 and DHT showed that 1,25(OH)2D3 clearly reduces the growth-stimulatory effect of DHT on OVCAR-3 cells. Moreover, Western blot analysis revealed that these cells contain receptors for 1,25(OH)2D3 (VDR) and androgen (AR). Expression of VDR and AR was up-regulated by their cognate ligands. Up-regulation of AR by 1,25(OH)2D3 and of VDR by DHT provides evidence of cross-talk between 2 signaling pathways in OVCAR-3 cells. We also studied the immuno-histochemical distribution of VDRs and ARs in rat ovaries and human ovarian cancer cases. In rat ovaries, VDRs were observed mainly in granulosa and theca cells and ARs in granulosa cells and surface epithelium. In the human ovarian cancer cases studied, 43% were VDR-positive and 64% AR-positive. Combining the results suggests that the growth of ovarian tissue might be regulated by 1,25(OH)2D3 and androgen.

86. Int J Epidemiol. 1994 Dec;23(6):1133-6.

Sunlight, vitamin D, and ovarian cancer mortality rates in US women.

Lefkowitz ES, Garland CF.

Department of Family and Preventive Medicine, University of California, San Diego, La Jolla 92093-0620, USA.

BACKGROUND. In general, ovarian cancer incidence and mortality is higher in northern than southern latitudes. This ecologic study tests the hypothesis that vitamin D produced in the skin from sunlight exposure may be associated with a protective action in ovarian cancer mortality. METHODS. The association between average annual sunlight energy and age-specific ovarian cancer mortality rates in counties containing the 100 largest US cities was evaluated for 1979-1988. Simple linear regression was performed by decade using sunlight and ozone as independent variables and ovarian cancer rates as the dependent variable. Multiple regression was used to adjust for ozone and sulphur dioxide, since these atmospheric components may absorb ultraviolet light. RESULTS. Fatal ovarian cancer in these areas was inversely proportional to mean annual intensity of local sunlight in a univariate analysis (P = 0.0001), and in a regression adjusted for air pollution (P = 0.04). The association was also seen when restricted to 27 major urban areas of the US; however, probably due to a small sample size, this statistic did not reach significance. CONCLUSIONS. This ecologic study supports the hypothesis that sunlight may be a protective factor for ovarian cancer mortality.

Psoriasis

87. Cutis. 2002 Nov;70(5 Suppl):21-4.

Vitamin D and scalp psoriasis.

Koo J.

Department of Dermatology, University of California, San Francisco, USA.

Calcipotriene has been shown to be safe and effective for the treatment of psoriasis. For scalp psoriasis, the safety advantage of this nonsteroid agent is as important as its efficacy. Even though monotherapy with calcipotriene solution may not always be efficacious for severe scalp psoriasis, many patients are managed effectively with a sequential therapy regimen consisting of 3 phases. In phase 1 (clearing), patients apply clobetasol solution or gel in the morning and calcipotriene solution in the evening daily for 2 weeks. After the scalp psoriasis improves, clobetasol is reduced to weekends and calcipotriene solution is applied on weekdays (phase 2, transitional). Phase 3 is maintenance on calcipotriene solution alone to prevent recurrence. For patients with recalcitrant scalp psoriasis-where only a clobetasol-strength, superpotent topical corticosteroid is effective-a flip-flop therapy regimen has been proposed that allows for the safe, prolonged use of clobetasol solution by limiting its treatment to twice a day for 2-week periods with the use of calcipotriene solution twice a day for a minimum of 2 weeks during the corticosteroid-free in-between periods.

88. BMJ. 2000 Apr 8;320(7240):963-7.

Comment in: BMJ. 2000 Aug 12;321(7258):452.

Systematic review of comparative efficacy and tolerability of calcipotriol in treating chronic plaque psoriasis.

Ashcroft DM, Po AL, Williams HC, Griffiths CE.

Centre for Evidence-Based Pharmacotherapy, School of Life and Health Sciences, Aston University, Birmingham B4 7ET.

OBJECTIVES: To evaluate the comparative efficacy and tolerability of topical calcipotriol in the treatment of mild to moderate chronic plaque psoriasis. DESIGN: Quantitative systematic review of randomised controlled trials. SUBJECTS: 6038 patients with plaque psoriasis reported in 37 trials. MAIN OUTCOME MEASURES: Mean difference in percentage change in scores on psoriasis area and severity index, and response rate ratios for both patients' and investigators' overall assessments of marked improvement or better. Adverse effects were estimated with the rate ratio, rate difference, and number needed to treat. RESULTS: Calcipotriol was at least as effective as potent topical corticosteroids, calcitriol, short contact dithranol, tacalcitol, coal tar, and combined coal tar 5%, allantoin 2%, and hydrocortisone 0.5%. Calcipotriol caused significantly more skin irritation than potent topical corticosteroids (number needed to treat to harm for irritation 10, 95% confidence interval 6 to 34). Calcipotriol monotherapy also caused more irritation than calcipotriol combined with a potent topical corticosteroid (6, 4 to 8). However, the number needed to treat for dithranol to produce lesional or perilesional irritation was 4 (3 to 5). On average, treating 23 patients with short contact dithranol led to one more patient dropping out of treatment owing to adverse effects than if they were treated with calcipotriol. CONCLUSIONS: Calcipotriol is an effective treatment for mild to moderate chronic plaque psoriasis, more so than calcitriol, tacalcitol, coal tar, and short contact dithranol. Only potent topical corticosteroids seem to have comparable efficacy at eight weeks. Although calcipotriol caused more skin irritation than topical corticosteroids this has to be balanced against the potential long term effects of corticosteroids. Skin irritation rarely led to withdrawal of calcipotriol treatment. Longer term comparative trials of calcipotriol versus dithranol and topical corticosteroids are needed to see whether these short term benefits are mirrored by long term outcomes such as duration of remission and improvement in quality of life.

89. J Med Assoc Thai. 1999 Oct;82(10):974-7.

Treatment of psoriasis vulgaris with topical vitamin D analogue (calcipotriol): open multicenter study.

Kullavanijaya P, Gritiyarangsan P, Huiprasert P, Leenutaphong V.

Department of Medical Services, Ministry of Public Health, Bangkok, Thailand.

Sixty-one psoriasis patients, 46 males and 15 females (mean age: 40 years, range: 20-70 years) with baseline PASI score of 7.16 (+/- 3.66 SD) were enrolled in the study. All subjects were advised to apply calcipotriol ointment twice daily for 6 weeks. Six patients dropped out, five after 2 weeks and one after 4 weeks of treatment. PASI scores of fifty five patients were reduced to 2.16 per cent, 46.78 per cent and 55.55 per cent by 2 weeks, 4 weeks and 6 weeks respectively versus the baseline. Overall clinical assessment showed remission in 7.27 per cent marked improvement 74.54 per cent and slight improvement 18.18 per cent. Mild erythema were observed in fourteen patients (22.95%) that were mostly transient except for one patient. Serum creatinine, calcium and phosphate were normal throughout the study.

90. J Am Acad Dermatol. 1996 Nov;35(5 Pt 1):690-5.

Effect of UVB phototherapy and oral calcitriol (1,25-dihydroxyvitamin D3) on vitamin D photosynthesis in patients with psoriasis.

Prystowsky JH, Muzio PJ, Sevran S, Clemens TL.

Irving Center for Clinical Research, New York, USA.

BACKGROUND: Phototherapy and activated froms of vitamin D help clear psoriasis. OBJECTIVE: The influence of UVB phototherapy and oral calcitriol (1,25-dihydroxyvitamin D3) on vitamin D photosynthesis was assessed in 16 patients. METHODS: Patients were randomly selected to receive orally either placebo or calcitriol (0.5 to 2 micrograms daily) for the duration of the 8-week study; all patients received approximately 21 UVB treatments. Before and after treatment, serum levels of 25-hydroxyvitamin D and calcitriol were measured by high-pressure liquid chromatography. RESULTS: Although calcitriol had no additive effect on phototherapy as a treatment modality, a significant increase in serum 25-hydroxyvitamin D levels occurred in both groups; in three patients extraordinarily high levels developed (> 120 ng/ml). Oral calcitriol significantly increased calcitriol serum levels. Increased serum calcitriol did not inhibit cutaneous synthesis of vitamin D or its hepatic conversion to serum 25-hydroxyvitamin D. CONCLUSION: UVB induces high levels of vitamin D photosynthesis. Because oral or topical calcitriol alone helps clear psoriasis, studies to explore the possible influence of UVB phototherapy on its production should be considered. If UVB phototherapy induces cutaneous calcitriol synthesis this could explain the lack of added benefit to treatment when oral calcitriol is administered with phototherapy.

91. Br J Dermatol. 1996 Sep;135(3):347-54.

Vitamin D analogues in psoriasis: effects on systemic calcium homeostasis.

Bourke JF, Iqbal SJ, Hutchinson PE.

Department of Dermatology, Leicester Royal Infirmary, UK.

Vitamin D and its analogues are effective in the treatment of psoriasis. The principal concern about the use of these agents is the possibility of adverse effects on systemic calcium homeostasis. We review the effects of vitamin D and its analogues on systemic calcium homeostasis and discuss the implications for patients with psoriasis.

92. Clin Sci (Lond). 1994 May;86(5):627-32.

Cyclosporin A and vitamin D metabolism: studies in patients with psoriasis and in rats.

Shaw AJ, Hayes ME, Davies M, Edwards BD, Ballardie FW, Chalmers RJ, Mawer EB.

University of Manchester Bone Disease Research Centre, Department of Medicine, U.K.

1. Cyclosporin A, an immunosuppressive drug used to treat psoriasis, stimulates renal synthesis of 1,25-dihydroxyvitamin D in rats. 1,25-Dihydroxyvitamin D can also reduce the activity of psoriasis, and in the present study we have examined the possibility that cyclosporin A mediates some of its actions in psoriasis by renal or extra-renal production of 1,25-dihydroxyvitamin D. 2. Treatment of 12 psoriatic patients with cyclosporin A (5 mg day-1 kg-1) for 3 months significantly improved the psoriasis activity and severity index and reduced glomerular filtration rate, but serum 1,25-dihydroxyvitamin D levels were not changed. However, 1-3 months after stopping cyclosporin A treatment, an increase in the psoriasis activity and severity index score was accompanied by a small, but significant, increase in serum 1,25-dihydroxyvitamin D concentration. Plasma 1,25-dihydroxyvitamin D levels in rats gavaged with cyclosporin A (15 mg day-1 kg-1 for 2 weeks) were significantly increased compared with controls, but a lower dose of cyclosporin A (2.4 mg day-1 kg-1) had no effect. Renal 25-hydroxyvitamin D-24-hydroxylase activity in rat kidney homogenates was not different between control and cyclosporin A-treated rats. Renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity was not detectable in these homogenates. Extra-renal production of 1,25-dihydroxyvitamin D by activated macrophages isolated from the synovial fluid of patients with inflammatory arthritis was reduced after incubation with cyclosporin A (0.1-10 mumol/l) for 30 h or 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

93. Tidsskr Nor Laegeforen. 1993 Nov 30;113(29):3580-1.

[New treatment of psoriasis with the vitamin D analogue calcipotriol]

[Article in Norwegian]

Austad J.

Rikshospitalet, Oslo.

A new topical treatment for psoriasis was introduced in 1992 when the vitamin D analogue calcipotriol was registered in Norway. This is a new therapeutic principle for psoriasis. Calcipotriol induces differentiation and inhibits proliferation of the keratinocytes. Application twice daily for 6-8 weeks gives a 60-70% improvement in plaque type psoriasis. No serious side effects have been reported when using up to 100 grams of the ointment weekly.

94. J Am Acad Dermatol. 1992 Dec;27(6 Pt 1):1001-8.

Treatment of psoriasis with calcipotriol and other vitamin D analogues.

Kragballe K.

Department of Dermatology, Marselisborg Hospital, University of Aarhus, Denmark.

The discovery of a high-affinity receptor for the bioactive form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25[OH]D3), in most skin cells has led to the finding of previously unknown effects of vitamin D on epidermal growth and on the skin immune system. 1,25(OH)2D3 inhibits epidermal proliferation and promotes epidermal differentiation. These properties provided the rationale for introducing 1,25(OH)2D3 in the treatment of psoriasis vulgaris. In addition to 1,25(OH)2D3, the synthetic vitamin D3 analogues 1 alpha(OH)D3, 1,24(OH)2D3, and calcipotriol have undergone clinical evaluation. Calcipotriol has been studied most extensively. Compared with 1,25(OH)2D3, calcipotriol is about 200 times less potent in its effects on calcium metabolism, although similar in receptor affinity. In double-blind, placebo-controlled, randomized studies, topical calcipotriol (50 micrograms/gm, up to 100 gm weekly) has been shown to be efficacious and safe for the treatment of psoriasis. A similar therapeutic profile has been seen in long-term studies. In comparative studies topical calcipotriol is slightly more efficacious than betamethasone 17-valerate and dithranol. The mode of action of calcipotriol and other vitamin D3 analogues in psoriasis is not known. Although vitamin D3 analogues affect epidermal growth, their immunosuppressive properties may be equally important for their antipsoriatic effect.

95. Br J Dermatol. 1992 Aug;127(2):71-8.

Vitamin D analogues and psoriasis.

Berth-Jones J, Hutchinson PE.

Department of Dermatology, Leicester Royal Infirmary, U.K.

Topical vitamin D analogues offer a new, effective, more convenient and generally well-tolerated option for the treatment of psoriasis. Only psoriasis vulgaris has been intensively studied, but other forms of the disease may also respond. Both calcitriol and calcipotriol have been shown to be effective in numerous clinical trials, and the latter has compared well with betamethasone valerate and short-contact dithranol in controlled studies. Their mechanism of action is not yet fully understood and may prove complex. The most important effect may be a direct regulation of keratinocyte proliferation and differentiation. However, these compounds also have potent immunological properties, and may act by inhibition of cytokine production by keratinocytes or lymphocytes. Topical application of vitamin D analogues appears generally to be remarkably safe, but hypercalcaemia and hypercalciuria may develop if large quantities are used.

96. Nutr Rev. 1992 May;50(5):138-42.

Vitamin D and psoriasis.

Lowe KE, Norman AW.

Department of Biochemistry, University of California, Riverside 92521.

Skin can serve as the source of vitamin D when exposed to sunlight so that cutaneous 7-dehydrocholesterol can be converted to the vitamin. Skin is also a target organ for the hormone form of vitamin D: 1,25-(OH)2D3. Both skin keratinocytes grown in tissue culture and samples of human skin have the nuclear receptor for 1,25(OH)2D3. New results suggest that this hormone or its analogs may be effective in treating some forms of psoriasis.

97. DICP. 1991 Jul-Aug;25(7-8):835-9.

Vitamin D therapy in psoriasis.

Araugo OE, Flowers FP, Brown K.

Department of Pharmacy Practice, College of Pharmacy, University of Florida, Gainesville.

The use of vitamin D3 in the treatment of psoriasis is discussed with emphasis on positive and negative results of many clinical trials. Investigations indicate the treatment with topical vitamin D3 provides consistently more rapid clinical improvement than its oral counterpart, with no reported adverse effects. Studies have shown that 68 of 83 patients exhibited significant improvement of their psoriatic lesions with the topical application of vitamin D3 analogs, including 1,24-dihydroxycholecalciferol, calcitriol, and MC 903. Clinical trials involving 35 patients treated with oral vitamin D3 analogs resulted in moderate improvement in 24 of the patients. Adverse effects can be minimized by bedtime dosing and possibly the use of new noncalciotropic analogs. Vitamin D3 analogs appear to provide one more promising treatment option for psoriasis.

98. Acta Derm Venereol. 1990;70(4):351-4.

Vitamin D metabolism in psoriasis before and after phototherapy.

Guilhou JJ, Colette C, Monpoint S, Lancrenon E, Guillot B, Monnier L.

Department of Dermatology and Phlebology, Hopital Saint Charles, Montpellier, France.

Epidermis plays a major role in vitamin D synthesis and is a target tissue for 1,25 (OH)2 vitamin D, which could be involved in abnormal proliferation and differentiation of psoriatic keratinocytes. We investigated plasma calcium, phosphorus, alkaline phosphatases, parathyroid hormone, 25 (OH) D, 24,25 (OH)2 D and 1,25 (OH)2 D in 15 control subjects and 20 psoriatic patients before and after 3 weeks of phototherapy (UVB or PUVA). Before irradiation, all parameters were similar in psoriatics and controls, except for serum phosphorus (lower in psoriasis p less than 0.01). After phototherapy, P rose to normal values in psoriatic patients; 25 (OH) D and 24,25 (OH)2 D were dramatically increased by UVB (but not by PUVA) in psoriatic patients as well as in controls; 1,25 (OH)2 D was unmodified in controls but was significantly increased in psoriasis. Since 1,25 (OH)2 D has been reported to be an effective treatment for psoriasis, the UV-induced increase in 1,25 (OH)2 D could account for the beneficial effect of phototherapy in psoriasis.

99. Acta Derm Venereol. 1989;69(2):147-50.

Efficacy of topical treatment in psoriasis with MC903, a new vitamin D analogue.

Staberg B, Roed-Petersen J, Menne T.

Department of Dermatology, Gentofte Hospital, Copenhagen, Denmark.

In 10 in-patients with chronic plaque psoriasis, the antipsoriatic effect of MC903, a new synthetic analogue of vitamin D was evaluated. In each patient two symmetrical located psoriatic plaques were selected for the study. Topical treatment with MC903 cream (containing 1.2 mg MC903 per g cream) was compared with placebo cream in a double-blind, controlled, left-right, randomized way during 6 weeks of therapy. Compared with baseline, the clinical (erythema, scaling and infiltration) improvement was significant after 1 week of therapy with MC903 cream, while lateral comparison showed MC903 cream significantly better than cream base after 4 weeks of therapy (p less than 0.05). Measurements of skin blood flow by the laser Doppler technique in evaluating the disease activity was not superior to the clinical assessments. In 3 patients the psoriatic lesions treated with MC903 cream cleared completely during 6 weeks of therapy. No essential adverse reactions were observed. MC903 has a potent effect on cell proliferation and cell differentiation, but has minimal effect on calcium metabolism. It is concluded that this synthetic vitamin D analogue is potentially useful in the treatment of psoriasis.

100. Acta Derm Venereol. 1988;68(5):436-9.

Is the effect of phototherapy in psoriasis partly due to an impact on vitamin D metabolism?

Staberg B, Oxholm A, Klemp P, Hartwell D.

Department of Dermatology, Gentofte Hospital, Copenhagen, Denmark.

To elucidate the effect of phototherapy on vitamin D metabolism in psoriatics, the serum concentrations of the major vitamin D metabolites (25-hydroxy-vitamin D (25(OH)D), 1,25-dihydroxy-vitamin D (1,25(OH)2D), and 24,25-dihydroxy-vitamin D (24,25(OH)2D)) were studied in 10 patients with disseminated psoriasis, both before and after phototherapy. Some 3-4 weeks of Goeckerman therapy induced significantly increased serum levels of 25(OH)D (mean: 24.6 ng/ml versus 54.4 ng/ml; (p less than 0.001] and 24,25(OH)2D (mean: 2.01 ng/ml versus 3.49 ng/ml; (p less than 0.001)). After phototherapy the mean serum level of 1,25(OH)2D increased nearly to the level found in healthy controls (mean: 23.8 vs. 32.2 pg/ml). However, this increase was not significant. It is shown that conventional phototherapy does have an impact on vitamin D metabolism in psoriatics. Since previous investigations have indicated an abnormal vitamin D metabolism in patients with psoriasis, it is possible that the beneficial effect of phototherapy in this disease might be due partly to an impact on vitamin D metabolism.

101. Arch Dermatol. 1987 Dec;123(12):1677-1683a.

Skin as the site of vitamin D synthesis and target tissue for 1,25-dihydroxyvitamin D3. Use of calcitriol (1,25-dihydroxyvitamin D3) for treatment of psoriasis.

Holick MF, Smith E, Pincus S.

US Department of Agriculture/Human Nutrition Research Center, Tufts University, Boston, MA.

Vitamin D is a hormone, not a vitamin. The skin is responsible for producing vitamin D. During exposure to sunlight, ultraviolet radiation penetrates into the epidermis and photolyzes provitamin D3 to previtamin D3. Previtamin D3 can either isomerize to vitamin D3 or be photolyzed to lymisterol and tachysterol. Vitamin D is also sensitive to sunlight and is photolyzed to 5,6-transvitamin D3, suprasterol I, and suprasterol II. In Boston, solar irradiation only produces previtamin D3 in the skin between the months of March and October. Aging, sunscreens, and melanin all diminish the capacity of the skin to produce previtamin D3. Once formed, vitamin D3 enters the circulation and is sequentially metabolized to 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3). The epidermis possesses receptors for 1,25-(OH)2-D3. 1,25-(OH)2-D3 inhibits the proliferation of cultured keratinocytes and induces them to terminally differentiate. The topical or oral administration of 1,25-(OH)2-D3 has proved to be effective for the treatment of psoriasis. Therefore, the skin is the site for the synthesis of vitamin D and a target tissue for its active metabolite. The successful use of 1,25-(OH)2-D3 for the treatment of psoriasis heralds a new approach for the treatment of this enigmatic disorder.

102. Acta Derm Venereol. 1987;67(1):65-8.

Abnormal vitamin D metabolism in patients with psoriasis.

Staberg B, Oxholm A, Klemp P, Christiansen C.

To elucidate if psoriatic skin involvement induces changes in vitamin D metabolism, the serum concentrations of the major vitamin D metabolites (25-hydroxy-vitamin D(2+3) (25OHD), 1,25-dihydroxyvitamin D(2+3) (1,25(OH)2D), and 24,25-dihydroxyvitamin D(2+3) (24,25(OH)2D)) were studied in a group of patients with psoriasis, who had not been exposed to ultraviolet radiation at least three months before the investigation. Serum concentrations of 1,25(OH)2D were significantly reduced in 17 patients with disseminated psoriasis compared to healthy age and sex matched controls (22.3 pg/ml versus 35.0 pg/ml (p less than 0.001)) and compared to 15 patients with moderate extended psoriasis (22.3 pg/ml versus 38.3 pg/ml (p less than 0.005)). Serum concentrations of the two other metabolites were not significantly decreased. In patients with moderate psoriatic skin manifestations, the values of the three vitamin D metabolites were normal. It is concluded that patients with disseminated psoriasis demonstrate decreased serum concentrations of the vitamin D metabolite 1,25(OH)2D. Since 1,25(OH)2D plays a role in differentiation and proliferation of epidermal cells, the abnormal low serum level of 1,25(OH)2D might be of importance for the abnormalities in cell maturation and proliferation found in psoriatic skin.

Diabetes

103. J Clin Endocrinol Metab. 2003 Jul;88(7):3137-40.

Vitamin D receptor gene polymorphism affects onset pattern of type 1 diabetes.

Motohashi Y, Yamada S, Yanagawa T, Maruyama T, Suzuki R, Niino M, Fukazawa T, Kasuga A, Hirose H, Matsubara K, Shimada A, Saruta T.

Department of Internal Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan. asmd@sc.itc.keio.ac.jp

Type 1 diabetes mellitus is recognized as a T-cell-mediated autoimmune disease. Vitamin D compounds are known to suppress T-cell activation by binding to the vitamin D receptor (VDR); and thus, VDR gene polymorphisms may be related to T-cell-mediated autoimmune diseases. We, therefore, investigated a VDR gene polymorphism in type 1 diabetes. We examined the VDR gene Bsm I polymorphism in 203 type 1 diabetic patients and 222 controls, and the association between the VDR gene polymorphism and type 1 diabetes and their onset pattern. We found a significantly higher frequency of B allele in type 1 diabetics overall, compared with controls (P = 0.0010). Moreover, there was a significant difference in B-allele frequency between acute-onset type 1 diabetics and controls (P = 0.0002), whereas this difference was not observed between slow-onset type 1 diabetics and controls. Regardless of the existence of islet-associated autoantibody, we found a significant difference in B-allele frequency between acute-onset type 1 diabetics and controls. In conclusion, we found an association between a VDR gene polymorphism and acute-onset type 1 diabetes. Assessment of this VDR gene polymorphism may contribute to prediction of the onset pattern in individuals with a high risk of type 1 diabetes.

104. Lancet. 2001 Nov 3;358(9292):1500-3.

Comment in: Lancet. 2001 Nov 3;358(9292):1476-8. Lancet. 2002 Apr 6;359(9313):1246-7; discussion 1247-8. Lancet. 2002 Apr 6;359(9313):1246; discussion 1247-8. Lancet. 2002 Apr 6;359(9313):1247; discussion 1247-8. Lancet. 2002 Apr 6;359(9313):1248.

Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study.

Hypponen E, Laara E, Reunanen A, Jarvelin MR, Virtanen SM.

Department of Paediatric Epidemiology and Biostatistics, Institute of Child Health, WC1N 1EH, London, UK. e.hypponen@ich.ucl.ac.uk

BACKGROUND: Dietary vitamin D supplementation is associated with reduced risk of type 1 diabetes in animals. Our aim was to ascertain whether or not vitamin D supplementation or deficiency in infancy could affect development of type 1 diabetes. METHODS: A birth-cohort study was done, in which all pregnant women (n=12055) in Oulu and Lapland, northern Finland, who were due to give birth in 1966 were enrolled. Data was collected in the first year of life about frequency and dose of vitamin D supplementation and presence of suspected rickets. Our primary outcome measure was diagnosis of type 1 diabetes by end of December, 1997. FINDINGS: 12058 of 12231 represented live births, and 10821 (91% of those alive) children were followed-up at age 1 year. Of the 10366 children included in analyses, 81 were diagnosed with diabetes during the study. Vitamin D supplementation was associated with a decreased frequency of type 1 diabetes when adjusted for neonatal, anthropometric, and social characteristics (rate ratio [RR] for regular vs no supplementation 0.12, 95% CI 0.03-0.51, and irregular vs no supplementation 0.16, 0.04-0.74. Children who regularly took the recommended dose of vitamin D (2000 IU daily) had a RR of 0.22 (0.05-0.89) compared with those who regularly received less than the recommended amount. Children suspected of having rickets during the first year of life had a RR of 3.0 (1.0-9.0) compared with those without such a suspicion. INTERPRETATION: Dietary vitamin D supplementation is associated with reduced risk of type 1 diabetes. Ensuring adequate vitamin D supplementation for infants could help to reverse the increasing trend in the incidence of type 1 diabetes.

105. Diabetologia. 1999 Jan;42(1):51-4.

Vitamin D supplement in early childhood and risk for Type I (insulin-dependent) diabetes mellitus. The EURODIAB Substudy 2 Study Group.

[No authors listed]

The initiation of the immunopathogenetic process that can lead to Type I (insulin-dependent) diabetes mellitus in childhood probably occurs early in life. Studies in vitro have shown that vitamin D3 is immunosuppressive or immunomodulating and studies in experimental models of autoimmunity, including one for autoimmune diabetes, have shown vitamin D to be protective. Seven centres in Europe with access to population-based and validated case registers of insulin-dependent diabetes patients participated in a case-control study focusing on early exposures and risk of Type I diabetes. Altogether data from 820 patients and 2335 control subjects corresponding to 85% of eligible patients and 76% of eligible control subjects were analysed. Questions focused on perinatal events and early eating habits including vitamin D supplementation. The frequency of vitamin D supplementation in different countries varied from 47 to 97% among control subjects. Vitamin D supplementation was associated with a decreased risk of Type I diabetes without indication of heterogeneity. The Mantel-Haenszel combined odds ratio was 0.67 (95% confidence limits: 0.53, 0.86). Adjustment for the possible confounders: a low birth weight, a short duration of breast feeding, old maternal age and study centre in logistic regression analysis did not affect the significant protective effect of vitamin D. In conclusion, this large multicentre trial covering many different European settings consistently showed a protective effect of vitamin D supplementation in infancy. The findings indicate that activated vitamin D might contribute to immune modulation and thereby protect or arrest an ongoing immune process initiated in susceptible people by early environmental exposures.

106. J Nutr Sci Vitaminol (Tokyo). 1985 Dec;31 Suppl:S27-32.

Osteopenia and circulating levels of vitamin D metabolites in diabetes mellitus.

Imura H, Seino Y, Ishida H.

The degree of diabetic osteopenia and serum vitamin D metabolite levels were measured in 14 type 1 (insulin-dependent) and 168 type 2 (non-insulin-dependent) diabetic patients. Based on six indices obtained by microdensitometry, we found the bone mass in 28.6% of type 1 and 26.2% of type 2 diabetic patients to be decreased and in 14.3% and 11.9%, respectively, the decrease was severe. Our method of analysis of bone mass has shown that diabetic osteopenia differs from typical osteoporosis in character. In addition, serum 24,25-dihydroxyvitamin D was significantly decreased both in type 1 and in type 2 diabetes (p less than 0.01), but 1,25-dihydroxyvitamin D was significantly decreased only in type 1 diabetes (p less than 0.01) compared to the controls, being lower than that in type 2 diabetes (p less than 0.05). On the other hand, 25-hydroxyvitamin D was similar to that of the controls, in both types of diabetes.

Pregnancy

107. J Pediatr. 2003 Feb;142(2):169-73.

Hypovitaminosis D and vitamin D deficiency in exclusively breast-feeding infants and their mothers in summer: a justification for vitamin D supplementation of breast-feeding infants.

Dawodu A, Agarwal M, Hossain M, Kochiyil J, Zayed R.

Department of Paediatrics, Faculty of Medicine and Health Sciences, UAE University, United Arab Emirates.

OBJECTIVE: To determine the prevalence of hypovitaminosis D in exclusively breast-feeding infants and their mothers in a community where maternal sunshine exposure is low. STUDY DESIGN: Serum levels of calcium, phosphate, alkaline phosphatase, 25-hydroxy vitamin D (25-OHD), and intact parathyroid hormone were measured in 90 unsupplemented healthy term breast-feeding Arab/South Asian infants and their mothers in summer. Maternal dietary vitamin D intake was also estimated. RESULTS: The median age of infants was 6 weeks. The median serum 25-OHD concentrations in mothers (8.6 ng/mL) and infants (4.6 ng/mL) were low, and 61% of the mothers and 82% of the 78 infants tested had hypovitaminosis D (serum 25-OHD <10 ng/mL). The infants with hypovitaminosis D had elevated serum alkaline phosphatase and a tendency to higher serum intact parathyroid hormone levels. The average daily maternal vitamin D intake from commercial milk was 88 IU. CONCLUSIONS: Hypovitaminosis D is common in summer in exclusively breast-feeding infants and their mothers. The results provide justification for vitamin D supplementation of breast-feeding infants and mothers in the United Arab Emirates. Low vitamin D intake probably contributed to low maternal vitamin D status.

108. Calcif Tissue Int. 2002 Oct;71(4):364-75. Epub 2002 Aug 29. (Animal Study)

Vitamin D deficiency in guinea pigs: exacerbation of bone phenotype during pregnancy and disturbed fetal mineralization, with recovery by 1,25(OH)2D3 infusion or dietary calcium-phosphate supplementation.

Rummens K, van Bree R, Van Herck E, Zaman Z, Bouillon R, Van Assche FA, Verhaeghe J.

Department of Obstetrics and Gynecology, Katholieke Universiteit Leuven, 3000 Leuven, Belgium. katrien.rummens@uz.kuleuven.ac.be

Vitamin D (D) deficiency during human pregnancy appears to disturb fetal growth and mineralization, but fetal development is normal in D-deficient rats and vitamin D receptor gene-ablated mice. We used the guinea pig model to investigate maternal and fetal effects of D deficiency. Pregnant (Pr) and nonpregnant (NPr) animals were fed a D-replete (+D) or D-deficient diet (-D) for 8 weeks. We further studied whether the effects of a -D diet are reversed by continuous 1,25(OH)2D3 infusion (-D+1,25) and/or by a lactose-, Ca- and P-enriched D-deficient diet (-D+Ca/P). Bone analyses included histomorphometry of the proximal tibiae, dual-energy X-ray absorptiometry (DXA), and quantitative computed tomography (QCT) of the femora. Depletion of 25(OH)D3 and 1,25(OH)2D3 levels and the D-deficiency syndrome were more severe in pregnant animals. Indeed, Pr/-D but not NPr/-D guinea pigs were hypophosphatemic, and showed robust increases in growth plate width and osteoid surface and thickness; in addition, bone mineral density on DXA was lower in Pr/-D animals only, which was exclusively in cortical bone on QCT. Bone phenotype was partly normalized in Pr/-D+1,25 and Pr/-D+Ca/P animals. Compared with +D fetuses, -D fetuses had very low or undetectable 25(OH)D3 and 1,25(OH)2D3, were hypercalcemic and hypophosphatemic, and had lower osteocalcin levels. In addition, body weight and total body bone mineral content were 10-15% lower; histomorphometry showed hypertrophic chondrocyte zone expansion and hyperosteoidosis. 1,25(OH)2D3 levels were restored in -D+1,25 fetuses, and the phenotype was partially corrected. Similarly, the fetal +D phenotype was rescued in large part in -D+Ca/P fetuses, despite undetectable circulating 25(OH)D3 and 1,25(OH)2D3. We conclude that pregnancy markedly exacerbates D deficiency, and that augmenting Ca and P intake overrides the deleterious effects of D deficiency on fetal development.

109. J Gynecol Obstet Biol Reprod (Paris). 2001 Dec;30(8):761-7.

[Winter supplementation in the 3rd trimester of pregnancy by a dose of 80,000 IU of vitamin D]

[Article in French]

Madelenat P, Bastian H, Menn S.

Hopital Bichat Claude-Bernard, 46, rue Henri-Huchard, 75877 Paris.

A non-comparative study was conducted to examine the effects of 80,000 IU vitamin D given in a single dose to 59 pregnant women from northern or southern France between their 27th and 32nd week of gestation during the winter season. Serum levels of 25 hydroxy-vitamin D (25 OH D), intact Parathyroid Hormone (iPTH), calcium, phosphates, proteins were measured at the inclusion, at delivery (mother and arterial cord) and in the newborn between the 3rd and the 5th day of life. The mothers' sun exposure and their vitamin D dietary intakes were evaluated with scores at the inclusion and at delivery. Before vitamin D supplementation, 34% of the women had a 25 OH D concentration below 10 ng/ml and 32% had hypocalcemia. At delivery, only one woman had a low 25 OH D concentration, whereas 15% of the women showed hypocalcemia. No neonatal hypocalcemia was observed and no vitamin D overdose was recorded in this study. The mothers' vitamin D dietary intakes were quite high; the lack of sun exposure during last summer appeared as a major vitamin D deficiency risk. A single dose of 80,000 IU vitamin D, taken between the 27th and the 32nd amenorrhoea weeks in winter, seems to be a good compromise between efficacy and tolerance.

110. Cochrane Database Syst Rev. 2000;(2):CD000228.

Vitamin D supplementation in pregnancy.

Mahomed K, Gulmezoglu AM.

Department of Obstetrics and Gynaecology, University of Zimbabwe, PO Box A178, Avondale, Harare, Zimbabwe. kmahomed@healthnet.zw

BACKGROUND: Vitamin D deficiency can occur in people whose diet is relatively low in the vitamin and those who are not exposed to much sunlight. OBJECTIVES: The objective of this review was to assess the effects of vitamin D supplementation on pregnancy outcome. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group trials register and the Cochrane Controlled Trials Register (October 1998). SELECTION CRITERIA: Acceptably controlled trials of vitamin D supplementation during pregnancy. DATA COLLECTION AND ANALYSIS: One reviewer assessed trial quality and extracted data. MAIN RESULTS: Two trials involving 232 women were included. In one trial the mothers had higher mean daily weight gain and lower number of low birthweight infants. In the other trial the supplemented group had lower birthweights. REVIEWER'S CONCLUSIONS: There is not enough evidence to evaluate the effects of vitamin D supplementation during pregnancy.

111. J Clin Endocrinol Metab. 1999 Dec;84(12):4541-4.

Vitamin D supplementation during infancy is associated with higher bone mineral mass in prepubertal girls.

Zamora SA, Rizzoli R, Belli DC, Slosman DO, Bonjour JP.

Department of Pediatrics, University Hospital, Geneva, Switzerland. samuel.zamora@hcuge.ch

The objective of this study was to determine whether vitamin D supplementation of breast-fed infants during the first year of life is associated with greater bone mineral content and/or areal bone mineral density (aBMD) in later childhood. The design was a retrospective cohort study. One hundred and six healthy prepubertal Caucasian girls (median age, 8 yr; range, 7-9 yr) were classified as vitamin D supplemented or unsupplemented during the first year of life on the basis of a questionnaire sent to participating families and their pediatricians. Bone area (square centimeters) and bone mineral content (grams) were determined by dual energy x-ray absorptiometry at six skeletal sites. Vitamin D receptor (VDR) 3'-gene polymorphisms (BsmI) were also determined. The supplemented (n = 91) and unsupplemented (n = 15) groups were similar in terms of season of birth, growth in the first year of life, age, anthropometric parameters, and calcium intake at time of dual energy x-ray absorptiometry. The supplemented group had higher aBMD at the level of radial metaphysis (mean +/- SEM, 0.301+/-0.003 vs. 0.283+/-0.008; P = 0.03), femoral neck (0.638+/-0.007 vs. 0.584+/-0.021; P = 0.01), and femoral trochanter (0.508+/-0.006 vs. 0.474+/-0.016; P = 0.04). At the lumbar spine level aBMD values were similar (0.626+/-0.006 vs. 0.598+/-0.019; P = 0.1). In a multiple regression model taking into account the effects of vitamin D supplementation, height, and VDR genotype on aBMD (dependent variable), femoral neck aBMD remained higher by 0.045 g/cm2 in the supplemented group (P = 0.02). Vitamin D supplementation in infancy was found to be associated with increased aBMD at specific skeletal sites later in childhood in prepubertal Caucasian girls.

112. Calcif Tissue Int. 1999 Jul;65(1):23-8.

Bone mineral density of the spine and femur in healthy Saudi females: relation to vitamin D status, pregnancy, and lactation.

Ghannam NN, Hammami MM, Bakheet SM, Khan BA.

Department of Medicine (MBC-46), King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.

Bone mineral density (BMD) measurements of the anterio-posterior lumbar spine and the proximal femur using dual-energy x-ray absorptiometry, as well as relevant clinical and biochemical parameters, were determined in 321 healthy Saudi females in order to establish reference values and to study the effects of physical and lifestyle factors on BMD. Mean +/- SD of age, body mass index (BMI), number of pregnancies, and total duration of lactation were 35.4 +/- 11.3 years, 26.5 +/- 5.2 kg/m2, 3.1 +/- 3.1, and 23.7 +/- 42.4 months, respectively. Mean +/- SD of serum calcium, 25-hydroxyvitamin D (25OHD), and PTH levels were 2.37 +/- 0.09 mmol/liter, 24.5 +/- 17.2 nmol/liter, and 52.0 +/- 30.8 pg/ml, respectively. Peak BMD values were observed around age 35 years at the spine and earlier at the femur. Compared with USA females, Saudi females had lower weight-matched Z scores at the spine (-0.126 +/- 1. 078, P = 0.04), femoral neck (-0.234 +/- 0.846, P < 0.0001), and Ward's triangle (-0.269 +/- 1.015, P < 0.0001). Further, the prevalence of osteopenia and osteoporosis in subjects >/=31 years old were 18-41% and 0-7%, respectively, depending on the site examined. Severe hypovitaminosis D (25OHD level </=20 nmol/liter) was present in 52% of the subjects. However, there was no correlation between 25OHD level and BMD at any site. Parathyroid hormone (PTH) levels correlated significantly with 25OHD levels (r = -0.28, P < 0.0001) and with weight-matched BMD Z scores at the spine (r = -0.17, P = 0.005), femoral neck (r = -0.16, P = 0.007), and Ward's triangle (r = -0.2, P = 0.0008), suggesting that the distribution of 25OHD levels in the cohort is below the threshold needed for maintaining normal BMD. On the other hand, number of pregnancies and total duration of lactation correlated with weight-matched BMD Z scores at the spine (r = -0.17, P = 0.003; r = -0.1, P = 0.08, respectively). We conclude that BMD in healthy Saudi females is significantly lower than in their USA counterparts. This may be due in part to increased number of pregnancies and longer duration of lactation together with prevalent vitamin D deficiency. http://link.springer-ny.com/link/service/journals/00223/bibs /65n1p23. html

PIP: This paper examines the relationship between bone mineral density (BMD) of the spine and femur and vitamin D status, pregnancy, and lactation among women in Saudi Arabia. The aims of the study are the following: 1) establish normative data for BMD at the anterio-posterior lumbar spine and femur using dual x-ray absorptiometry; 2) compare the BMD of Saudi females and their US counterparts; and 3) examine the relation of BMD to vitamin D status, pregnancy, and lactation. Samples included 321 healthy Saudi females recruited from the city of Riyadh, Saudi Arabia. Results suggest that the mean standard deviation (SD) of age, body mass index, number of pregnancies, and total duration of lactation were, respectively, 35.4 +or- 11.3 years, 26.5 +or- 5.2 kg/sq. m, 3.1 +or- 3.1, and 23.7 +or- 42.4 months. Mean +or- SD of serum calcium, 25-hydroxyvitamin D (25OHD), and PTH levels were 2.37 +or- 0.09 mmol/liter, 24.5 +or- 17.2 nmol/liter, and 52.0 +or- 30.8 pg/ml, respectively. Peak BMD values were observed around age 35 years at the spine and earlier at the femur. Compared with US females, Saudi females had lower weight-matched Z scores at the spine, femoral neck, and Ward's triangle. On the other hand, the number of pregnancies and total duration of lactation correlated with weight-matched BMD Z scores at the spine. This made the BMD in healthy Saudi females significantly lower than their US counterparts. This may due to the increase number of pregnancies and longer duration of lactation together with prevalent vitamin D deficiency.

113. Arch Pediatr. 1995 Apr;2(4):373-6.

[Vitamin D supplementation in pregnancy: a necessity. Committee for Nutrition]

[Article in French]

[No authors listed]

In septentrional countries without vitamin D supplementation of milk and dairy products, many pregnant women are vitamin D deficient. Consequently vitamin D deficiency is present in many newborn infants, which may lead to neonatal hypocalcemia and vitamin D deficient rickets. A prevention of vitamin D deficiency is therefore a necessity in pregnant women through a vitamin D supplementation. This can be done either by a daily supplementation of 400 IU during all pregnancy, or a daily supplementation of 1,000 IU during the third trimester, or by giving a unique dose of 100,000 to 200,000 IU during the seventh month of pregnancy.

114. Am J Clin Nutr. 1994 Feb;59(2 Suppl):484S-490S; discussion 490S-491S.

Do North American women need supplemental vitamin D during pregnancy or lactation?

Specker BL.

Department of Pediatrics, University of Cincinnati Medical Center, OH 45267-0541.

Studies in European and other countries have shown that vitamin D deficiency during pregnancy may adversely affect fetal growth, bone ossification, tooth enamel formation, and neonatal calcium homeostasis. Whether effects of vitamin D deficiency on pregnant or lactating mothers differ from effects observed in nonpregnant or nonlactating women is not clear. Poor maternal vitamin D status during lactation results in low breast-milk vitamin D. However, human milk usually contains small vitamin D amounts and, under normal circumstances, the sunshine exposure of human-milk--fed infants is the major factor affecting their vitamin D status. Mothers at risk of vitamin D deficiency are those who avoid dairy products, which are routinely vitamin D fortified, and live in more northern latitudes. Dark-skinned women also are theoretically at risk of vitamin D deficiency. Sunshine exposure is a major vitamin D source, and given adequate exposure, supplemental vitamin D is not necessary. However, defining adequate sunshine exposure is difficult.

115. Ann Nutr Metab. 1991;35(4):208-12. (Animal Study)

Effect of vitamin D supplementation during pregnancy on the neonatal skeletal growth in the rat.

Marya RK, Saini AS, Jaswal TS.

Department of Physiology, Medical College, Rohtak, India.

In rats on normal intakes of calcium, phosphorus and vitamin D3, 3,000 and 7,500 IU of vitamin D3 were injected on the 10th day of pregnancy and the pups were investigated for the skeletal growth on 28th day of age. Compared to controls, the pups in the supplemented groups showed significantly greater dry weight and ash weight of the tibiae. However, the ash weight/dry bone weight ratios in the supplemented groups were not different from controls. Histological examination of the upper ends of decalcified tibiae and plasma calcium estimations revealed no abnormality in any group. The results suggest that vitamin D3 supplementation in pregnancy enhances the skeletal growth of the pups which involves both the organic and inorganic components.

116. Gynecol Obstet Invest. 1988;25(2):99-105.

Alterations in vitamin D metabolites and minerals in diabetic pregnancy.

Kuoppala T.

Department of Obstetrics and Gynecology, University Central Hospital of Tampere, Finland.

Vitamin D metabolites and minerals involved in bone metabolism were studied in 68 control mothers, 14 gestational diabetics and 68 insulin-dependent diabetics during pregnancy and at delivery. 25(OH)D and 1,25(OH)2D concentrations were significantly (p less than 0.001) lower in insulin-dependent diabetics than in the control or gestational diabetic groups. A similar difference was also observed between infants. 24,25(OH)2D, phosphorus and magnesium values were similar in all groups. Corrected calcium values were significantly lower in both mothers (p less than 0.001) and infants (p less than 0.05) in the insulin-dependent group than in the other two groups. Postpartum, 10% of infants of diabetic mothers received calcium therapy. Our results show alterations in vitamin D and mineral metabolism in pregnant insulin-dependent diabetics and their newborn infants and indicate observation during pregnancy and after delivery.

117. Obstet Gynecol. 1986 Sep;68(3):300-4.

Vitamin D supplementation in pregnancy: a controlled trial of two methods.

Mallet E, Gugi B, Brunelle P, Henocq A, Basuyau JP, Lemeur H.

A randomized study was conducted to evaluate the effects of single-dose and daily vitamin D supplementation in pregnant women during the last trimester of a winter pregnancy in the Northwest of France. The women were divided into three randomized groups: one (N = 21) was given a vitamin D2 supplement of 1000 IU/day during the last three months of pregnancy, one (N = 27) was given a single oral dose of 5 mg at the seventh month of pregnancy, and one (N = 29) acted as a control. Venous plasma samples were obtained at delivery from the women and from cord blood, and levels of calcium, 25-OHD, and 1,25(OH)2D were determined. No significant difference in plasma calcium concentration was found among the three groups, but within each group plasma calcium concentrations were higher in the cord samples than in the respective maternal samples. The levels of the two metabolites measured were consistently lower in the cord samples than in the respective maternal samples. Cord 25-OHD concentrations correlated with those of maternal plasma. No significant modification of maternal calciuria or of the birth weight of term infants was observed. 25-OHD concentrations were greater in maternal and cord plasma from treated mothers, but only a slight difference was observed between the supplemented groups. 1,25(OH)2D concentrations were not significantly different in the three groups. A single 5-mg dose of vitamin D given orally at the seventh month of pregnancy provides effective prophylaxis in the authors' region.

118. J Pediatr. 1986 Aug;109(2):328-34.

Vitamin D supplementation during pregnancy: effect on neonatal calcium homeostasis.

Delvin EE, Salle BL, Glorieux FH, Adeleine P, David LS.

We assessed whether modification of vitamin D nutritional status during the last trimester of pregnancy affects maternal and neonatal calcium homeostasis. At the end of the first trimester, 40 pregnant women were randomly assigned to either of two groups, and blood taken to assess the basal values of Ca, Pi, Mg, iPTH, 25-OHD, and 1,25(OH)2D. From the sixth month on, group 1 (+D) received 1000 IU vitamin D3 daily; group 2 (-D) served as control. At the time of delivery, maternal serum 25-OHD was higher in the +D group (P less than 0.0005). Ca, Pi, iPTH, and 1,25(OH)2D were not affected. At term, venous cord 25-OHD levels were also higher in the +D group (P less than 0.0005), and 1,25(OH)2D levels slightly lower (P less than 0.05), but neither Ca, Pi, nor iPTH differed between the two groups. Serum CaT dropped significantly (P less than 0.002) at 4 days of age in the infants from both groups, although to a lesser extent in these from the +D group (P less than 0.05). Circulating iPTH increased in both groups. Serum 25-OHD remained low in the -D group, and dropped slightly in the +D group; 1,25(OH)2D remained stable during the first 4 days of life in the -D group, and increased in the +D group (P less than 0.001). Our data demonstrate the importance of providing adequate maternal vitamin D stores to ensure better perinatal handling of calcium. This is of particular importance for populations at risk for hypovitaminosis D.

119. Hum Nutr Clin Nutr. 1986 Jul;40(4):287-93.

Serum levels of vitamin D metabolites, calcium, phosphorus, magnesium and alkaline phosphatase in Finnish women throughout pregnancy and in cord serum at delivery.

Kuoppala T, Tuimala R, Parviainen M, Koskinen T, Ala-Houhala M.

Serum concentrations of 25(OH)D, 24,25(OH)2D, 1,25(OH)2D, total calcium, protein, phosphorus, magnesium and alkaline phosphatase were measured in two groups of Finnish women throughout pregnancy and in cord serum at delivery. The autumn group delivered in August-September and the spring group in February-March. There was strong seasonal variation in the 25(OH)D concentrations in both groups. Maternal values (mean +/- s.d.) at delivery were 44.3 +/- 20.8 nmol/l in autumn and 26.0 +/- 13.0 nmol/l in spring. Fetal concentrations were 28.8 +/- 14.3 and 18.3 +/- 11.3 nmol/l, respectively. In both mothers and infants low 25(OH)D values were measured in winter. In the autumn group 7 out of 21 mothers (33 per cent) and in the spring group 17 out of 36 mothers (47 per cent) had values below 17 nmol/l, which is the lowest winter reference value recorded in our laboratory. No significant seasonal variation was observed in dihydroxylated vitamin D metabolites, although 24,25(OH)2D values were a little higher in summer than in winter. Concentrations of 1,25(OH)2D tended to rise towards delivery. Corrected calcium, magnesium and phosphorus concentrations did not change during pregnancy. Fetal calcium and phosphorus concentrations were significantly (P less than 0.001) higher than maternal ones. The data indicate that many mothers and infants have poor vitamin D status in the latitude of Finland. Our results support the concept that vitamin D supplementation should be considered in Finland for pregnant women at least in winter.

120. Rev Fr Gynecol Obstet. 1986 Jun-Jul;81(6-7):365-7.

[Vitamin D and pregnancy: value of a single dose in the third trimester. Comparative study of 100 cases]

[Article in French]

Rideau F, Allisy C, Girard O, Michel F, Sommier M, Wipff J.

A comparative study (100 treated subjects, 100 controls) was carried out at the Centre Hospitalier at Meaux during the winter of 1984-85 in which pregnant women were given single doses of vitamin D to be taken at the seventh month of pregnancy. The results obtained for neonatal blood calcium levels clearly demonstrated the value of vitamin D supplements in pregnant women. This study was original in that a single dose of vitamin D was provided; this led to almost perfect therapeutic observance (99%).

121. Acta Paediatr Scand. 1983 Nov;72(6):817-21.

Effect of season and vitamin D supplementation on plasma concentrations of 25-hydroxyvitamin D in Norwegian infants.

Markestad T.

Plasma concentrations of 25-hydroxyvitamin D (25OHD) were determined in 81 vitamin D supplemented or unsupplemented infants at the end of winter. The values were compared with maternal levels and with concentrations found in 22 unsupplemented infants at the end of summer. The 25OHD levels of the neonates were lower, but closely related to maternal values (r = 0.95, p less than 0.0005). Unsupplemented breast-fed infants had lower 25OHD levels at 6 weeks than at 4 days (16 +/- 7 vs. 32 +/- 15 nmol/l, mean +/- 1 SD, p less than 0.0005). The mean 25OHD level of vitamin D supplemented 6-12 months old infants was intermediate between those of the unsupplemented nursed groups and the unsupplemented children studied during summer (53 +/- 28 vs. 85 +/- 28 nmol/l, p less than 0.0005). Six weeks old infants who had received a milk formula containing 400 IU vitamin D3 per liter had levels similar to the latter group (92 +/- 21 nmol/l). The data suggest that the vitamin D stores acquired during fetal life, or from ultraviolet light exposure during the summer, may be inadequate to maintain safe levels of 25OHD throughout the winter, but that a daily supplement of 400 IU is adequate to establish concentrations in the summer range.

122. Br J Obstet Gynaecol. 1983 Oct;90(10):971-6.

Vitamin D metabolism in normal and hypoparathyroid pregnancy and lactation. Case report.

Markestad T, Ulstein M, Bassoe HH, Aksnes L, Aarskog D.

Plasma concentrations of vitamin D metabolites in 17 non-pregnant women, 22 pregnant women at delivery, and in eight lactating women 3 and 16 days after delivery, were compared with those in a postpartum hypoparathyroid patient treated with 1 alpha-hydroxyvitamin D (1 alpha-OHD). The mean concentration of 1,25-dihydroxyvitamin D [1,25-(OH)2 D] was 203 (SD 61) pmol/l in the pregnant, and 86 (SD 27) pmol/l in the non-pregnant women (P less than 0.0005). The levels 3 and 16 days after delivery were similar [57 (11) compared with 62 (19) pmol/l], and lower than the non-pregnant value (P less than 0.01). The 25-hydroxyvitamin D (25-OHD) concentration remained unchanged between the 3rd and 16th days after delivery, whereas the 24,25-dihydroxyvitamin D [24,25-(OH)2D] level increased from 2.7 (SD 1.8) to 3.7 (SD 2.3) nmol/l (P less than 0.025). The patient temporarily required an increased supplement of l alpha-OHD during pregnancy, but a dose which was appropriate before pregnancy resulted in marked hypercalcaemia and a rise of 1,25-(OH)2D concentration within 16 days of delivery despite lactation. The results suggest that the metabolic need for the active vitamin D metabolite 1,25-(OH)2D is increased during pregnancy and rapidly reduced during early lactation in healthy and hypoparathyroid women.

123. Gynecol Obstet Invest. 1981;12(3):155-61.

Effects of vitamin D supplementation in pregnancy.

Marya RK, Rathee S, Lata V, Mudgil S.

Serum calcium, inorganic phosphate and heat-labile alkaline phosphatase (HLAP) have been estimated in maternal and cord sera of 120 pregnant women at labour. 75 women who did not take any vitamin D supplements during pregnancy showed statistically significant hypocalcaemia, hypophosphataemia and elevation of HLAP. Hypocalcaemia and hypophosphataemia were present in cord blood, too. 25 women who had received 1,200 U vitamin D/day throughout the 3rd trimester, showed significantly lower HLAP levels and increased fetal birth weight but there was no other improvement in maternal or cord blood chemistry. Administration of vitamin D in two large doses of 600,000 U each in the 7th and 8th months of pregnancy in 20 women proved more efficacious. Statistically significant improvement was observed in all the three biochemical parameters in maternal as well as cord sera. Fetal birth weight was also significantly greater with this mode of therapy.

124. Lancet. 1977 Jan 29;1(8005):222-5.

Altered vitamin-D metabolism in pregnancy.

Turton CW, Stanley P, Stamp TC, Maxwell JD.

Low levels of plasma-25-hydroxy-vitamin-D (25-OHD) (less than 16 nmol/1) were found in 33% of a group of pregnant women studied in South London. Non-White women in the last 10 weeks of pregnancy had significantly reduced levels compared with non-pregnant controls (P less than 0.02). Plasma-25-OHD was unrelated to dietary vitamin-D intake, age, parity, social class, plasma-calcium, and plasma-albumin. Reduction of plasma-25-OHD could contribute to the fall in plasma-calcium during pregnancy, and may result from enhanced maternal metabolism or increased utilisation of vitamin D by the fetus.

Tuberculosis

125. J Assoc Physicians India. 2002 Apr;50:554-8.

Comment in: J Assoc Physicians India. 2003 Mar;51:325-6; author reply 327.

Tuberculosis and vitamin D deficiency.

Sasidharan PK, Rajeev E, Vijayakumari V.

Department of Medicine, Calicut Medical College, Kerala.

OBJECTIVES: a) To find out the normal level of 25 hydroxy vitamin D in healthy individuals b) To look for evidence of vitamin D deficiency in patients with active tuberculosis. METHODS: There were 35 cases of pulmonary and extra-pulmonary tuberculosis and 16 controls, whose clinical characteristics, dietary intake of vitamin D and biochemical characteristics including serum vitamin D levels were compared. Exclusion criteria: malabsorption, liver or renal disorders, intake of drugs, which can reduce vitamin D levels, HIV infection, diabetes, immunosuppressive treatment, and severe protein energy malnutrition. RESULTS: There was a statistically significant difference (p < 0.005) in mean vitamin D levels between controls (19.5 ng/ml) and study subjects (10.7 ng/ml). Sixteen patients out of 35 had values well below the lower limit of normal (9 ng/ml). No one in the control group had vitamin D level less than 9 ng/ml. However the mean vitamin D level in the control group was less than the mean value quoted in the literature from the West. Sunlight exposure was adequate in those with deficiency but there was reduced dietary intake of vitamin D. CONCLUSIONS: Serum 25 hydroxy vitamin D levels less than 9 ng/ml indicates deficiency. Vitamin D deficiency exists in patients with tuberculosis and it is possibly a cause rather than effect of the disease; deficiency is due to decreased dietary intake. Vitamin D deficiency can occur without any symptoms. If symptoms are present, it indicates severe deficiency. Serum calcium and phosphorus values do not often predict the existence of deficiency.

126. Calcif Tissue Int. 2000 Jun;66(6):476-8.

Comment on: Calcif Tissue Int. 1997 Jan;60(1):91-3.

Vitamin D deficiency and susceptibility to tuberculosis.

Chan TY.

Division of Clinical Pharmacology, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.

Vitamin D, a modulator of macrophage function, can activate human anti-mycobacterial activity. Vitamin D deficiency is therefore associated with a higher risk of tuberculosis (TB) infection, as indicated by several observations. First, TB tends to occur during the colder seasons when cutaneous synthesis of vitamin D from sun exposure is reduced and serum vitamin D levels are lower. Second, patients with untreated TB, particularly those from a temperate climate, have lower serum vitamin D levels than healthy subjects. Third, the incidence of TB is higher among subjects with relatively low serum vitamin D levels, such as the elderly, uremic patients, and Asian immigrants in the U.K.

127. Lancet. 2000 Feb 19;355(9204):618-21.

Comment in: Lancet. 2000 Feb 19;355(9204):588-9. Lancet. 2000 Jul 1;356(9223):73-4; discussion 74-5. Lancet. 2000 Jul 1;356(9223):74; discussion 74-5. Lancet. 2000 Jul 1;356(9223):74; discussion 74-5. Lancet. 2001 Mar 24;357(9260):961.

Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study.

Wilkinson RJ, Llewelyn M, Toossi Z, Patel P, Pasvol G, Lalvani A, Wright D, Latif M, Davidson RN.

Division of Infectious Diseases, Case Western Reserve University, Cleveland, OH, USA.

BACKGROUND: Susceptibility to disease after infection by Mycobacterium tuberculosis is influenced by environmental and host genetic factors. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of M. tuberculosis. This effect may be influenced by polymorphisms at three sites in the vitamin D receptor (VDR) gene. We investigated the interaction between serum vitamin D (25-hydroxycholecalciferol) concentrations and VDR genotype on susceptibility to tuberculosis. METHODS: This study was a hospital-based case-control analysis of Asians of Gujarati origin, a mainly vegetarian immigrant population with a high rate of tuberculosis. We typed three VDR polymorphisms (defined by the presence of restriction endonuclease sites for Taq1, Bsm1, and Fok1) in 91 of 126 untreated patients with tuberculosis and 116 healthy contacts who had been sensitised to tuberculosis. Serum 25-hydroxycholecalciferol was recorded in 42 contacts and 103 patients. FINDINGS: 25-hydroxycholecalciferol deficiency was associated with active tuberculosis (odds ratio 2.9 [95% CI 1.3-6.5], p=0.008), and undetectable serum 25-hydroxycholecalciferol (<7 nmol/L) carried a higher risk of tuberculosis (9.9 [1.3-76.2], p=0.009). Although there was no significant independent association between VDR genotype and tuberculosis, the combination of genotype TT/Tt and 25-hydroxycholecalciferol deficiency was associated with disease (2.8 [1.2-6.5]) and the presence of genotype ff or undetectable serum 25-hydroxycholecalciferol was strongly associated with disease (5.1 [1.4-18.4]). INTERPRETATION: 25-hydroxycholecalciferol deficiency may contribute to the high occurrence of tuberculosis in this population. Polymorphisms in the VDR gene also contribute to susceptibility when considered in combination with 25-hydroxycholecalciferol deficiency.

128. Ethn Health. 1998 Nov;3(4):247-53.

Does vitamin D deficiency account for ethnic differences in tuberculosis seasonality in the UK?

Douglas AS, Ali S, Bakhshi SS.

Birmingham Communicable Disease Unit, UK.

OBJECTIVES: Notifications of tuberculosis in England and Wales are reported to peak in the summer season. The purpose of this study was to confirm that finding and to determine to what extent patients of Indian Subcontinent (ISC) ethnic origin contributed to the seasonality. The clinical presentation of the disease is presumed to occur some months following reactivation of the endogenous latent focus of tuberculosis infection. There arises the possibility of vitamin D deficiency producing immunological inadequacy at the end of winter and beginning of spring. PATIENTS AND METHODS: Monthly (or 4-weekly) aggregated data over 7 years were collected from the three countries of mainland Britain, England, Wales, Scotland and from the city of Birmingham in England. The notifications from Birmingham were divided into those of ISC ethnic origin and 'whites'. The presence or absence of seasonality was determined by fitting a sinusoidal curve by the technique called 'cosinor analysis'. In this method amplitude gives a measure of the extent of the seasonal variation. RESULTS: The summer peak of clinical diagnosis was confirmed in the UK series from England, Wales and Scotland. In England and Wales without Scotland a larger seasonal variation was present. Scotland, with a lower proportion of population of ISC ethnic origin, was examined separately and the results in Scotland alone failed to confirm seasonality. In the data from Birmingham, seasonality was confirmed with a greater amplitude, particularly in those over 60 years of age. The finding was influenced by those of ISC ethnic origin, seasonality not being present in the 'white' population. CONCLUSION: The results from Birmingham are very striking, but there were almost three times as many patients in the ISC ethnic group as in indigenous 'white' patients. A series with larger numbers of 'white' patients would be necessary to confirm the absence of seasonality in the 'white' population. The discussion reviews the evidence that vitamin D may have an important hormonal role in immunological defence in the prevention of tuberculosis.

129. Calcif Tissue Int. 1997 Jan;60(1):91-3.

Comment in: Calcif Tissue Int. 2000 Jun;66(6):476-8.

Differences in vitamin D status and calcium intake: possible explanations for the regional variations in the prevalence of hypercalcemia in tuberculosis.

Chan TY.

Department of Clinical Pharmacology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.

The prevalence of hypercalcemia in patients with untreated tuberculosis (TB) varies widely between countries. Since the vitamin D status and calcium intake are important determinants of hypercalcemia in TB, these two factors were compared among four populations (U.K., Hong Kong, Malaysia, Thailand) with a low prevalence (<3%) and two populations (Sweden, Australia) with a high prevalence (>25%). In the three Asian countries, the circulating vitamin D levels are abundant, but the calcium intakes are low. Subjects from the U.K. have the lowest circulating vitamin D level of all, although their calcium intake is high. In Sweden and Australia, both the circulating vitamin D levels and calcium intakes are high. Since serum 1,25(OH)2D concentration will only be raised if its substance for extrarenal conversion, 25(OH)D, is plentiful and the effect of a given serum 1,25 (OH)2D concentration on serum calcium is determined by the calcium intake, it is postulated that the regional variation in the prevalence of hypercalcemia in TB may be due to differences in the circulating vitamin D levels and calcium intakes in these populations.

130. Eur Respir J. 1994 Jun;7(6):1103-10.

Vitamin D metabolism by alveolar immune cells in tuberculosis: correlation with calcium metabolism and clinical manifestations.

Cadranel JL, Garabedian M, Milleron B, Guillozzo H, Valeyre D, Paillard F, Akoun G, Hance AJ.

INSERM U.82, Faculte de Medecine Xavier Bichat, Paris, France.

The aim of this study was to investigate the relationship between the pulmonary vitamin D metabolism in tuberculosis and the calcium metabolism abnormalities and other clinical characteristics of the disease. The metabolism of 25-hydroxyvitamin D3 (25(OH)D3) by alveolar immune cells recovered by bronchoalveolar lavage (BAL) was evaluated in parallel to the results of calcium metabolism, 25(OH) D and 1,25 dihydroxyvitamin D (1,25(OH)2D) plasma levels and other clinical parameters obtained in 14 tuberculosis patients. Whilst predominant metabolites produced by lavage cells in patients and controls were 5(E)--and 5(Z) -19-nor-10-oxo-25(OH)D3, 1,25(OH)2D3 was produced by cells from all tuberculosis patients but not by cells from controls. Calcium metabolism abnormalities were observed in only some patients, but the production of 1,25 (OH)2D3 by lavage cells was found to correlate both with 1,25(OH)2D levels (r = 0.67) and post-load urinary calcium excretion (r = 0.59). 1,25(OH)2D3 production by lavage cells was increased in patients of black origin, and those presenting with hilar adenopathy without pulmonary infiltrates, and was correlated with the number of lymphocytes recovered by lavage (r = 0.87). We conclude that 1,25(OH)2D3 production by alveolar immune cells makes a major contribution to the abnormalities in calcium metabolism seen in tuberculosis patients, and may be partly dependent on the clinical characteristics evaluated here.

131. Tubercle. 1985 Sep;66(3):187-91.

A study of vitamin D levels in Indonesian patients with untreated pulmonary tuberculosis.

Grange JM, Davies PD, Brown RC, Woodhead JS, Kardjito T.

The levels of serum vitamin D, measured as 25-hydroxycholecalciferol (25-OHD3), among 40 Indonesian patients with pulmonary tuberculosis and 38 healthy controls were very similar. In both groups the distribution of the serum 25-OHD3 levels were bimodal with about a quarter of the individuals belonging to the group with higher levels. There was a tendency for controls in this group to be tuberculin negative and for patients in this group to have less extensive active pulmonary disease. Although it is uncertain whether such associations result from a direct effect of vitamin D on protective immune reactions, the use of this vitamin as an adjunct to antituberculosis therapy merits consideration.

Leukemia

132. Blood. 2001 Dec 1;98(12):3290-300.

The acute promyelocytic leukemia-associated protein, promyelocytic leukemia zinc finger, regulates 1,25-dihydroxyvitamin D(3)-induced monocytic differentiation of U937 cells through a physical interaction with vitamin D(3) receptor.

Ward JO, McConnell MJ, Carlile GW, Pandolfi PP, Licht JD, Freedman LP.

Programs of Cell Biology and Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

Monocyte differentiation induced by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor alpha (RARalpha) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D(3) receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)(2)D(3) activation from both a minimal VDR responsive reporter and the promoter of p21(WAF1/CIP1), a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)(2)D(3)-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)(2)D(3) to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)(2)D(3) induces monocytic differentiation in hematopoietic cells.

133. Cancer Lett. 2000 Mar 13;150(1):1-13.

Anticlastogenic potential of 1alpha,25-dihydroxyvitamin D3 in murine lymphoma.

Sarkar A, Saha BK, Basak R, Mukhopadhyay I, Karmakar R, Chatterjee M.

Department of Pharmaceutical Technology, Jadavpur University, Calcutta, India.

Vitamin D3, having gained scientific interest for so long because of its role in mineral homeostasis, has now received great importance as a possible antitumor agent. This study was undertaken in an attempt to visualize the possible anticlastogenic potential of the vitamin in an ascitic mouse lymphoma model namely, Dalton's lymphoma. Frequencies of structural type chromosomal aberrations, sister chromatid exchanges and micronucleus assays have been chosen as the genotoxic endpoints in the proposed investigation. All these cytogenetic markers have been found to be markedly elevated during the progression of lymphoma in bone marrow cells. Vitamin D3 effectively suppressed the frequencies of chromosomal aberrations and sister chromatid exchanges in the lymphoma-bearing mice during the entire phase of tumor growth that significantly coupled with almost two-fold increase in survival time (37 +/- 2 and 68 +/- 2 days in lymphoma controls and vitamin D3-treated lymphoma-bearing mice, respectively), thus substantiating the antineoplastic efficacy of this secosteroid. The outcome of this study also is clearly reflected in the depletion of circulating (serum) vitamin D3 levels in the lymphoma control mice compared with normal (vehicle) controls while a still higher level was maintained in the VD3-treated lymphoma mice. This anticlastogenic property of the vitamin has so far been neglected and this is the first attempt to unravel the vitamin D3's effect in combating tumor development in vivo by limiting the frequencies of chromosomal aberrations, sister chromatid exchanges and micronuclei at least in transplantable murine model studied herein.