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Vitamin D and Its Role in Cancer and Immunity: A Prescription for Sunlight

^* Integrative GI Nutrition Services, Capsule Endoscopy, Division of Gastroenterology and Liver Disease, Johns Hopkins Hospital, Baltimore, Maryland

Correspondence: Correspondence: Gerard E. Mullin, MD, MHS, FACP, CNSP, FACN, AGAF, Director of Integrative GI Nutrition Services, Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21205. Electronic mail may be sent to gmullin1{at}jhmi.edu.

Vitamin D has been recognized for more than a century as essential for the normal development and mineralization of a healthy skeleton. More extensive roles for vitamin D were suggested by the discovery of the vitamin D receptor (VDR) in tissues that are not involved in calcium and phosphate metabolism. VDR has been discovered in most tissues and cells in the body and is able to elicit a wide variety of biologic responses. These observations have been the impetus for a reevaluation of the physiologic and pharmacologic actions of vitamin D. Here, we review the role of vitamin D in regulation of the immune system and its possible role in the prevention and treatment of cancer and immune-mediated diseases.

	Vitamin D Overview

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
Most humans depend on sun exposure to satisfy their requirements for vitamin D. Brief exposure to direct sunlight on a bright summer day is all the body needs to produce sufficient cholecalciferol (or vitamin D₃).¹ Skin exposure to ultraviolet B light (wavelength 290–315 nm) initiates the photochemical conversion of 7-dehydrocholesterol to previtamin D₃ in the epidermis and dermis. Body heat then catalyzes the rapid isomerization of previtamin D₃ to vitamin D₃ (cholecalciferol), which binds to the vitamin-D-binding protein (VDBP) in the extracellular space.² Vitamin D₃ is transported to the liver, which uses 25-hydroxylase to produce 25-hydroxyvitamin D₃, or 25(OH)D₃.

Although endogenous production of vitamin D typically provides most of the vitamin D requirement, vitamin D can be ingested orally as either vitamin D₃ (cholecalciferol) or D₂ (ergocalciferol, derived from the irradiation of plant sterols). Very few foods naturally contain vitamin D: oily fish and fish products such as cod-liver oil, salmon, mackerel, and herring contain approximately 300–500 international units (IU) of vitamin D per serving.³ Foods in the United States that are fortified with vitamin D include milk, orange juice, and some cereals, breads, yogurts, and cheeses. Most European countries permit margarine and some cereals to be fortified with vitamin D, and a few countries, including Sweden, also permit milk to be fortified with this vitamin.

25-Hydroxyvitamin D [25(OH)D] derived from endogenous production or exogenous sources is the major vitamin D metabolite in the human body's circulation system and is the best known indicator of vitamin D status. There is debate regarding cutoffs for vitamin D deficiency and insufficiency using 25(OH)D concentrations. The current thinking is that the traditional cutoff for deficiency, 10 ng/mL of 25(OH)D (above which protection from rickets or osteomalacia is conferred), is too low to indicate vitamin D adequacy. A low serum 25(OH)D concentration, often referred to as hypovitaminosis D, is not simply a biochemical abnormality. It is associated with physiologic, pathologic, and clinical evidence of vitamin D deficiency, including increased parathyroid hormone secretion, increased bone turnover, osteoporosis and mild osteomalacia, and an increased risk of hip and other fractures. To prevent more subtle or long-term effects of vitamin D insufficiency, circulating concentrations of at least 30 ng/mL 25(OH)D may be warranted.³

In the kidney, 25(OH)D is further hydroxylated to 1,25-dihydroxyvitamin D [1,25(OH)₂D₃, calcitriol], the most biologically active form of the vitamin, by renal 1-hydroxylase under the control of parathyroid hormone. 1,25(OH)₂D₃ principally targets the intestine, kidney, and bone to regulate calcium and phosphate homeostasis. Calcitriol is a lipid-soluble hormone that interacts with its vitamin D receptor (VDR) in the small intestine to increase the expression of an epithelial calcium channel, calcium-binding protein, and a variety of other proteins to help in transport of calcium from the intestinal lumen into circulation. Calcitriol also interacts with its VDR in the osteoblasts, which results in an increase in the mobilization of osteoclast precursors to become mature osteoclasts. The end result is mobilization of calcium stores from the skeleton to maintain calcium homeostasis.^1–6 These well-defined, calcemic functions of vitamin D are illustrated in Figure 1. It should be noted that 1,25(OH)₂D₃ circulates at concentrations 1000-fold less than that of 25(OH)D, and that its half-life is hours rather than weeks, as is the case for 25(OH)D.

View larger version (29K): [in this window] [in a new window]   Figure 1. Calcemic and noncalcemic roles of vitamin D and their potential health consensequences.

More recently, VDRs were found in cells of tissues not involved in calcium homeostasis, and extrarenal tissues were found to produce 1,25(OH)₂D₃. These pathways are also indicated in Figure 1 and supported the hypothesis that vitamin D plays additional roles in cellular differentiation and the control of proliferation in a variety of cell types. These results have bolstered at a biochemical level what epidemiologists have been witnessing in observational studies: that vitamin D status may protect against certain cancers, as well as some autoimmune conditions.

	Vitamin D and Cancer Prevention

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
Epidemiologic Studies
Geography, sunlight, and cancer risk. Most of the information about vitamin D and cancer prevention has been based on observational, case-control, or cohort studies linking sunlight exposure to cancer incidence or survival. The first of these studies was documented nearly a half century ago, when it was reported that individuals living in the northeast regions of the United States had an approximately 2-fold higher risk of dying of cancer than those living in southern regions.⁷ Recently, several more studies found a similar association between latitude and the risk of developing prostate, breast, and colon cancer in the United States and Europe.^8–10 Colon cancer mortality was subsequently found to be higher among people from the Northeastern United States compared with people living in southern states.¹¹ It is now well established that the risk of developing and dying of prostate, breast, colon, ovarian, esophageal, non-Hodgkin's lymphoma, and a number of other lethal cancers correlated with living at higher latitudes.^11–24

Several investigators have confirmed the reciprocal relationship of sunlight exposure and cancer. Most recently, Grant^24,25 reported that increased sun exposure decreases mortality from common cancers in both white men and women.⁷ Grant¹⁷ suggested that 25% of breast cancer mortality in Europe was related to living at a higher latitude and being vitamin D deficient.

These observations are also supported by a report of men who had little sun exposure and developed prostate cancer 3–5 years earlier than men who had optimal sun exposure.²⁶ Vitamin D deficiency was reported to increase the risk for prostate cancer, and sunlight exposure is inversely proportional to prostate cancer mortality. Colon cancer incidence was evaluated in California because the state has a wide range of latitudes. Living in San Diego was reported to significantly decrease the risk of developing colon cancer compared with living in San Francisco or further north.²⁷

There has been speculation that subjects living further north synthesized less vitamin D and that therefore vitamin D seems to be protective against tumor development or growth. The sunlight effect therefore seems to attenuate the risk of numerous cancers, such as prostate, breast, colon, ovarian, non-Hodgkin's lymphoma, esophageal, stomach, pancreatic, rectal, kidney, corpus uteri, lung, and bladder. These studies, which controlled for other important variables such as smoking and lifestyle, provide indirect evidence of a possible causal relation between low vitamin D status and cancer risk.

View larger version (27K): [in this window] [in a new window]   Figure 2. 1,25(OH)2D3 regulates prostate cell growth. Vitamin D can influence the regulation of key genetic elements of cell differentiation and proliferation by either 1,25(OH)2D3 entering the nucleus of the prostate cell or via conversion of 25 (OH)D3 by 1,25-OHase in the mitochondrion. Adapted from Progress in Biophysics & Molecular Biology Volume 62, Holick MF. Vitamin D: its role in cancer prevention and treatment, pp 49–59, © 2006 with permission from Elsevier. AR, androgen receptor; BAG 1L, Bcl-2 associated athanogene-1; BCL-2/BCL-XL, B-cell leukemia/lymphoma family; CDK2, cyclin-dependent kinase 2; cIAP1 and cIAP2, cIAP1 and cIAP2 are members of a protein family; Mc1-1, Myeloid Cell Leukemia 1; p21, p27, and p53, tumor proteins 21, 27, and 53; PSA, prostate specific antigen; RXR, retinoic X receptors; VDR, vitamin D receptor; XIAP, X-linked mammalian inhibitor of apoptosis protein.

Putative mechanisms for vitamin D protection against cancer. The original hypothesis for why exposure to sunlight decreased the risk of common cancers was that increased production of vitamin D₃ in the skin resulted in higher circulating levels of 25(OH)D₃, which could be metabolized by the kidneys to 1,25(OH)₂D₃. The discovery that VDRs existed in tissues that were not involved in calcium metabolism (ie, prostate, breast) reinforced the possibility that enhanced production of renal 1,25(OH)₂D₃ could influence noncalcemic tissues. Studies demonstrated that 1,25(OH)₂D₃ markedly inhibited a variety of genes responsible for cellular proliferation, including p21 and p27, and was also responsible for enhancing apoptotic activities and a variety of genes that regulate cellular differentiation.²⁹

However, because the kidney's production of 1,25(OH)₂D₃ is exquisitely regulated, it could not serve as the source of the active 1,25(OH)₂D₃ responsible for the newly recognized cellular functions. Thus, another hypothesis to explain the observations that sunlight provided a protective effect via vitamin D was needed.^1–4 Another possibility was that the "target" cells of interest were themselves converting 25(OH)D to 1,25(OH)₂D₃. Along these lines, cultured keratinocytes and prostate cells obtained from nondiseased prostate biopsies converted 25(OH)D to 1,25(OH)₂D₃^18,27,30 (Figure 2). Following these observations, it has been noted that the colon, lung, breast, and other tissues all express the 25(OH)D-1-hydroxylase (1-OHase; cyp 27 B1).³¹ Thus, it has been suggested that raising blood levels of 25(OH)D provides an adequate substrate for the prostate, colon, and breast to produce their own local 1,25(OH)₂D₃, which, in turn, is capable of regulating a variety of cellular processes that help control cellular growth and prevent malignancy.

Animal models. Although the epidemiologic evidence linking sun exposure to cancer risk seems strong, whether vitamin D deficiency per se promotes tumor growth remained unclear. To this end, a study evaluated the growth of a mouse colon cancer cell line MC-26 in Balb/c mice that were either vitamin D deficient or vitamin D sufficient.³² The tumors grew much more rapidly in the vitamin D–deficient mice. By the end of the study on day 19, tumors in the vitamin D–deficient mice were on average 80% larger than in the vitamin D–sufficient mice. The 25(OH)D levels in the vitamin D–deficient mice at the end of the study were <5 ng/mL, whereas the vitamin D–sufficient mice maintained a 25(OH)D level of 35 ng/mL. This observation provides strong corroborating data supporting the concept that vitamin D sufficiency is important for reducing tumor cell growth. There is now emerging data that vitamin D may play a role in altering colon cancer cell growth. Spina et al³³ performed studies using murine models of colon cancer to demonstrate that those mice who were sufficient in vitamin D had a significantly lower (p < .05) tumor burden compared with vitamin D–deficient mice. Furthermore, using Gemini analog A in the same mouse model of colon cancer, these investigators demonstrated that the tumor burden was decreased by 50% in the treatment group when compared with placebo or 1,25(OH)₂D₃ (p < .05; Figure 3).

View larger version (34K): [in this window] [in a new window]   Figure 3. 1,25(OH)2D3 and a novel vitamin D analog regulate colon cancer cell growth in mice. A novel Gemini analog was shown to diminish tumor volume when administered while a dose response reduction effect is shown for 1,25(OH)2D3. Adapted with permission from Spina CS, Tangpricha V, Uskokovic M, Adorinic L, Maehr H, Holick MF. Vitamin D and cancer. Anticancer Res. 2006;26:2515–2524.

Dietary intake of vitamin D and cancer risk. Aside from sun exposure, others sources of vitamin D are in the form of diet and supplements. There are several epidemiologic studies of the association between vitamin D and breast cancer risk. Studies reporting dietary and supplemental intake demonstrate inconsistent results (Table 1).^{20,35–38,41} Three case-controlled studies demonstrated no association between dietary vitamin D intake and breast cancer risk^35,39,40; however, small sample size and selection bias may have skewed the results. On the other hand, the Nurses Health Study demonstrated an inverse association between vitamin D intake and breast cancer risk among premenopausal women but no association among postmenopausal women.⁴¹ Along these lines, the Cancer Prevention Study II Nutrition Cohort found no correlation between dietary intake of vitamin D and development of breast cancer in postmenopausal women,³⁸ and 2 additional studies determined that the dietary intake of vitamin D in the female adolescent had no bearing in determining future breast cancer risk.^36,37 However, researchers provided additional preliminary evidence demonstrating a potential role of vitamin D in decreasing breast cancer risk. In a population-based, case-controlled study in Ontario, women with breast cancer (identified through the Ontario Cancer Registry) and controls were interviewed about their past and present diets and sun exposure.

The investigators' preliminary analysis suggests that earlier exposures to vitamin D during breast development in adolescence may be more significant for reducing breast cancer risk than recent exposures.^42,43 A recent study seeking to estimate the amount of vitamin D required to reduce the incidence of breast cancer was performed via a meta-analysis of 2 large previously published studies that measured vitamin D concentrations in the blood [as serum 25(OH)D] and subsequent breast cancer development. The results demonstrated that women who consumed 1000 IU a day of vitamin D in addition to the normal background amount had a 10% reduced risk of breast cancer.⁴³ Garland and colleagues⁴⁴ plotted a dose-response gradient and found that as quintiles of serum 25(OH)D (0–11 ng/mL, 12–25 ng/mL, 26–31 ng/mL, 32–42 ng/mL, and >42 ng/mL) increased, the risk of breast cancer significantly decreased. A serum 25(OH)D concentration exceeding 52 ng/mL (which would require intake of >2700 IU/d of vitamin D₃ in an individual weighing 70 kg) was associated with 50% lower risk of breast cancer compared with a serum concentration of <12 ng/mL. The authors noted that the US median intake of 320 IU/d of vitamin D is only about one-tenth of the amount found to be associated with a 50% reduction of breast cancer incidence. They concluded that increasing daily intake of vitamin D, perhaps by fortification of foods, should be considered. Studies analyzing endogenous circulating vitamin D levels and breast cancer risk are shown in Table 2.^{12,19,34,45,46}

Biochemical indicators of vitamin D status and cancer risk. There has been considerable debate recently about concentrations of circulating 25(OH)D that can be viewed as optimal for preventing vitamin D deficiency and, in particular, promoting health. Prospective and retrospective studies have shown that circulating levels of 25(OH)D above 50 nmol/L (20 ng/mL) were associated with a 30%–50% decreased risk of developing prostate, breast, and colon cancer.⁴⁷

A recent study by Dr Walter Willett's group from Harvard University found that higher serum vitamin D levels correlated with reduced risk for certain forms of cancers in a population of middle-aged to elderly men in a retrospective analysis of data from a major study of health professionals.⁴⁸ Giovannucci and colleagues⁴⁸ analyzed prospective data from Harvard's Health Professionals Follow-up Study, which includes >50,000 men aged 40–75 years. Higher serum levels of vitamin D were associated with a significantly lower incidence of colorectal, pancreatic, esophageal, and oral/pharyngeal cancers. An increase of 25 nmol/L in serum vitamin D level was associated with a 17% decline in overall cancer incidence and a 29% drop in total cancer deaths. For cancers involving the digestive system, a 25 nmol/L increase in vitamin D reduced cancer incidence by 43% and mortality by 45%. This study was the first to examine total cancer incidence and mortality by using a comprehensive assessment of factors that determine 25(OH)D levels. The authors estimated that cancer mortality for US men could be reduced by 29% if serum vitamin D levels were increased by 25 nmol/L throughout the male population. The findings from this cohort study are the latest of several^49–52 linking vitamin D status with reduced cancer risk, and are some of the most compelling yet. The results, with lower risks of most (but not all) forms of cancer, are also some of the most broad-based, and they indicate that vitamin D may have a role in most human tumors. Factors associated with higher serum levels of vitamin D were white race, residence in the southern United States, higher intakes of vitamin D, body mass index <22 kg/m², and more physical activity. Vitamin D supplements (as opposed to dietary vitamin D) had only a slight effect on serum vitamin D. The investigators noted that increasing one's serum vitamin D by 25 nmol/L (the basis of their relative-risk calculation) might mean adding at least 1500 IU/d of vitamin D, far exceeding the US Department of Agriculture's recommended dietary allowance (RDA) of vitamin D currently set at 200–400 IU for people aged 1–70 years. A glass of milk contains only 100 IU and would increase serum vitamin D by only 2–3 nmol/L, they noted, but "a fair-skinned individual can produce 20,000 IU of vitamin D in the skin through 20–30 minutes of sun exposure." A recent editorial also noted that "the amount of sun needed to produce adequate levels of vitamin D, at least for bone health, is modest and can be obtained in a light-skinned person by a brief summertime afternoon stroll."⁵³ Other previous reports support a role for vitamin D in the prevention of colorectal cancer.^15,22,54 Giovannucci⁵⁵ has recently published a review of the data on colon cancer risk and vitamin D.

Other cancers where vitamin D plays a protective role. Polesel et al⁵⁶ reported that vitamin D also serves a protective role against the development of non-Hodgkin's lymphoma. Vitamin D (chiefly contained in fish) provided protection against non-Hodgkin's lymphoma that was stronger in women, whereas no differences emerged according to age (odds ratio [OR] = 0.4; 95% confidence interval [CI], 0.2–0.9). Vitamin D also seems to play a protective role in pancreatic cancer.⁵⁷ In 2 US cohorts, increased intakes of vitamin D were associated with reduced risks for pancreatic cancer, suggesting a potential role for vitamin D in the pathogenesis and prevention of this malignancy.

Melanoma, on the other hand, has been linked to sun exposure. Thus, the risk of excessive sun exposure needs to be weighed against the benefit of sun-derived vitamin D. Li et al⁵⁸ reported that genetic variants (ie, TaqI t protective allele and FokI f risk allele) in VDR may alter risk of cutaneous melanoma. Thus, genetic alternations in VDRs may in part determine the risk for developing cutaneous melanoma from sun exposure.

	Mechanisms Involved in the Anticancer Effects of Vitamin D

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
Over the past quarter century, evidence for the anticancer properties of vitamin D and its analogs has been emerging in the literature. The volume of data supports a multipronged attack that involves growth arrest at the G1 phase of the cell cycle, apoptosis, tumor-cell differentiation, disruption of growth-factor-mediated cell survival signals, and inhibition of angiogenesis and cell adhesion (Figure 2). Processes involved in the tumor suppressive activity of vitamin D analogues include inhibition of cell proliferation, induction of apoptosis, inhibition of cell adhesion, G₁-phase cell-cycle arrest, promotion of cell differentiation, inhibition of angiogenesis, alteration of growth factors, and inhibition of metastasis.^59,60 Thus, VDR-mediated pathways constitute potential therapeutic targets for cancer prevention and treatment.

Proposed Cellular Mechanisms
Cell cycle regulators. Direct regulation of the cell cycle by 1,25(OH)₂D₃ has been studied predominantly in in vitro systems. The 1,25(OH)₂D₃-VDR system arrests the cancerous cell cycle at the G₀-G₁ transition through multiple mechanisms.⁵⁹ In the monomyelocytic cell line U937, 1,25(OH)₂D₃-activated VDR directly binds to the promoter of p21 and induces its expression. Furthermore, 1,25(OH)₂D₃ increases the expression of p27 as well as p15, p16, and p18, which effectively inhibits the G1-to-S phase transition (Figure 4).⁶⁰ Furthermore, in the MCF-7 breast cancer cell line, treatment with 1,25(OH)₂D₃ inhibits cellular proliferation, and microarray analysis demonstrated an up-regulation of a number of cell cycle regulatory genes, including p21-activated kinase 1 and p53.⁶¹ Thus, vitamin D is a potent regulator of cellular differentiation and proliferation through direct regulation of cell cycle proteins.

View larger version (14K): [in this window] [in a new window]   Figure 4. Nutrient regulation of cell cycle proteins. Vitamin D has broad effects in regulating the cell cycle by regulating the expression of p53, Cip/waf p21, p27, p57, p15, p16, and p18, which effectively inhibits the G1-to-S phase transition. Adapted with permission from Bohnsack BL, Hirschi KK. Red light, green light: signals that control endothelial cell proliferation during embryonic vascular development. Cell Cycle. 2004;3:1506–1511.

	Role of 1,25(OH)2D3 in Regulating Cell Growth and Terminal Differentiation

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
1,25(OH)₂D₃ regulates proliferation and differentiation of different kinds of cells, including keratinocytes, osteoblasts, and hematopoietic cells.² 1,25(OH)₂D₃ has been shown to inhibit cellular growth and induced differentiation of M-1 leukemic cells and HL-60 leukemic cells expressing VDR.⁶² 1,25(OH)₂D₃ inhibits proliferation and induces differentiated function in osteoblasts, keratinocytes, and hematopoietic cells.⁶³ 1,25(OH)₂D₃ is generally associated with inhibiting proliferation and inducing differentiation. The growth inhibition of cancer cells by 1,25(OH)₂D₃ is associated with growth factor signaling through transforming growth factor-β (TGF-β), which is a potent inhibitor of proliferation of many cell types and is involved in cell cycle control and apoptosis. Vitamin D analogs induce an autocrine TGF-β activity through increasing expression of TGF-β isoforms or TGF-β receptors in nonmalignant and malignant cells.⁶⁴ Insulin-like growth factor (IGF)-binding protein 3 induction by 1,25(OH)₂D₃ seems to contribute to its antiproliferative and proapoptotic actions in primary cancer cells.^65–67 Recent microarray studies of gene expression profiles in cancer cells have further highlighted the capacity of 1,25(OH)₂D₃ analogs to drive malignant cells to a more differentiated state.⁶⁸

	Role of 1,25(OH)2D3 in Apoptosis

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
1,25(OH)₂D₃-induced apoptosis is an important contributor to its growth-suppressing and anticancer properties. 1,25(OH)₂D₃ analogs have been shown to induce apoptosis in cancer cells by modulating B-cell leukemia/lymphoma-2 genes (Bcl-2) and Bax proteins (which is a proapoptotic member of the Bcl-2 protein family), tumor necrosis factor (TNF)-, and caspase-dependent and independent mechanisms.⁶⁹

	Role of 1,25(OH)2D3 in Controlling Tumor Invasion and Metastasis

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
Aside from growth inhibition, vitamin D and its analogs decrease the invasiveness of several cell lines in vitro, and they inhibit angiogenesis and metastasis in xenograft and transgenic mouse models in vivo.⁷⁰ In cultured malignant cells, 1,25(OH)₂D₃ and its analogs down-regulate cell-invasion-associated proteases, including matrix metalloproteinases 2 and 9 and serine proteinases.⁷¹ In prostate and colon cells, 1,25(OH)₂D₃ and its analogs increase the expression of E-cadherin, a tumor suppressor associated with the metastatic potential of cells, and inhibit the oncogenic β-catenin signaling.⁷² 1,25(OH)₂D₃ and its analogs inhibit the proliferation of some tumor-derived endothelial cells, inhibit the expression of vascular endothelial cell growth factor that induces angiogenesis in tumors, and suppress tenascin-C, which promotes growth, invasion, and angiogenesis during tumorigenesis.⁷³

Breast, colon, prostate, skin, lung, and a variety of other cell lines, when exposed to 1,25(OH)₂D₃, showed marked inhibition of cellular growth and induction of terminal differentiation.⁷⁴ Although vitamin D is not currently used as a chemotherapeutic agent, it shows promise for drug development in the treatment of certain cancers. However, at this point, it is far from a certainty.

	Vitamin D Analogs for Cancer Treatment

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
As discussed earlier, the nuclear VDR has been isolated from a variety of target cells and tissues (Table 3), suggesting that vitamin D compounds may have therapeutic potential throughout several body systems. The major drawback of 1,25(OH)₂D₃, however, is its effect on calcium metabolism, which results in hypocalcaemia and hypercalciuria. Newly developed vitamin D analogs with lower calcemic activity have been shown to retain many therapeutic properties of 1,25(OH)₂D₃.^75–106 MC 903 is a vitamin D analog with low (2/0.05) ratio of cell growth inhibition to calcemic activity. In contrast, CB 966 (6/0.2), CB 1093 (160/0.27) and KH 1060 (1000/1.3) have much higher ratios making them potentially effective therapeutic agents without the potential risk of toxicity.⁷⁵ More than 2000 synthetic analogs of the biologic form of vitamin D (1,25(OH)₂D₃) are presently known. These analogs interfere with the molecular switch of nuclear 1,25(OH)₂D₃ signaling.

Most 1,25(OH)₂D₃ analogs have been identified as agonists, a few are antagonists (ie, ZK 159222), and only Gemini analogs (analogs having additional side-chains attached) and some of its derivatives act under restricted conditions as nonagonists.

Five vitamin D analogs have been approved for clinical use in a variety of disorders: calcipotriol (Dovonex; Leo Pharmaceuticals, Copenhagen, Denmark) for the treatment of psoriasis, 19-nor-1,25(OH)₂D₂ (Zemplar; Abbott Laboratories, Abbott Park, IL) for secondary hyperparathyroidism, doxercalciferol (Hectorol; Bone Care Int, Madison, WI) for reduction of elevated parathyroid hormone levels, 22-oxacalcitriol (Maxacalcitol; Chugai Pharmaceuticals, Tokyo, Japan), and alfacalcidol. Thus, these synthetic analogs which maintain antiproliferative effects but do not have strong calcemic activity have been developed and show promising results (Table 4). Studies using the human osteosarcoma cell line MG-63 demonstrated that 3 of these analogs (KH1060, EB1089, and CB1093), have a greater antiproliferative effect than 1,25(OH)₂D₃. Treatment with these analogs increases p27 protein levels by increasing expression and decreasing degradation. The analogs cause decreased levels of cyclin E, decreased CDK2 kinase activity and hypophosphorylation of Rb, and inhibition of the G1-to-S phase transition.¹⁰⁵ Similar results have been seen in neuroblastoma cell lines treated with a 20-epi-1,25(OH)₂D₃ analog,¹⁰⁶ suggesting that in the future, these 1,25(OH)₂D₃ analogs may be useful in chemotherapeutic regimens.

View this table: [in this window] [in a new window]   Table 4 Clinical development of 1,25(OH)2D3 and its analogues for cancer treatment

Several other analogs are currently being tested in preclinical and clinical trials for the treatment of various types of cancer and osteoporosis, as well as immunosuppression.⁸⁰ Vitamin D analogs are effective treatments and are widely used as drugs for hyperproliferative skin disorders such as psoriasis, and in suppression of secondary hyperparathyroidism and parathyroid hyperplasia resulting from chronic renal insufficiency.^107–109 For instance, Gemini analogs have been recently found to be 100–1000 times more potent in their antiproliferative activity than the natural hormone. Studies in mice suggest that they may be useful in the treatment of some cancers, including colon cancer.²⁷ Understanding how analogs exert their selective actions may allow for the design of more effective and safer vitamin D compounds for the treatment of a wide range of clinical disorders. The promising vitamin D analogs under development for the treatment of cancer are shown in Table 4^76–88; the in vivo effects of vitamin D analogs in animal models for breast cancer, prostate cancer, and colon cancer are shown in Table 5^89–106; and the molecular targets for vitamin D compounds in cancer are shown in Table 6. Optimal administration of vitamin D analogs is only being realized, with high-dose intermittent administration overcoming bioavailability and hypercalcemic problems. Combination therapy with cytotoxic agents (taxols and cisplatins), anti-resportive agents (biphosphonates), agents blocking vitamin D metabolism (ketoconazole), ionizing radiation, antiproliferative agents (retinoic acids), antihypercalcemic agents (dexamethasone), histone deacetylase inhibitors, or cytochrome P450 inhibitors may achieve better results through synergy, as shown in vitro.^110–115 Understanding how analogs exert their tissue-specific selective actions may allow for the design of more effective and safer vitamin D compounds for the treatment of hyperproliferative disorders, including cancer.

	Vitamin D and the Immune System

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
T Cells as the Main Target
1,25(OH)₂D₃ seems to modulate immunity principally via regulating T-cell function. VDR has been found to be expressed on virtually every type of cell involved in immunity (Table 3).^110–113 The immunomodulatory actions of vitamin D are elicited through its direct action on T-cell and antigen-presenting cell (APC) functions. Thus, 1,25(OH)₂D₃ may have an important physiologic role in immunoregulation and therapeutic target in immune-mediated diseases.

Effects on T Cells and T Helper 1 Cytokine Profiles: Relevance to Autoimmunity
In autoimmune patients, T cells target tissues such as the central nervous system (multiple sclerosis [MS]), the gut (Crohn's disease, inflammatory bowel diseases [IBD]), the joints (rheumatoid arthritis) and the pancreas (type-1 diabetes). The common denominator between these diseases is that T-helper 1 (Th1) cells secrete a proinflammatory profile of cytokines (TNF-, interferon [INF], IFN) at the site of pathology, which drives the disease process. In general, if a treatment for Th1-mediated autoimmunity works, it suppresses the number or activity of Th1 cells or antagonizes the cytokines (TNF- in particular) that they produce. In addition, treatments that work for one Th1-driven disease are likely to suppress other Th1-driven autoimmune diseases (eg, infliximab used to treat Crohn's disease and rheumatoid arthritis).

Vitamin D regulates T cells both directly and indirectly via APCs.^120,121 When vitamin D is deficient or signals through the VDR are weakened, Th1 cell actions are intensified, whereas regulatory T cells and Th2 cells are diminished, thus favoring an autoimmune Th1 response.^122,123 1,25(OH)₂D₃ increases regulatory T cells and Th2 cells (anti-inflammatory cytokines) while suppressing Th1 cell activities.¹²⁴ VDRs are required to maintain a physiologic balance of Th1 and Th2 cell responses, and furthermore in the absence of the VDR, Th2 cell functions are diminished.¹²⁵ 1,25(OH)₂D₃ suppresses production of the Th1 proinflammatory cytokines interleukin-2 (IL-2) by binding to the distal nuclear factor of activated T cells (NF-AT) binding site in the promoter of the human IL-2 gene and IFN through the interaction of VDRs, with a vitamin D response element (VDRE) in the promoter region of the IFN gene.^126,127

Because vitamin D deficiency favors a proinflammatory Th1 immune response, does supplementation rebalance immunity? In vitro addition of 1,25(OH)₂D₃ has recently been shown to enhance and promote differentiation of type 2 T-helper (Th2) lymphocytes through a direct effect on naïve CD4⁺ T cells. 1,25(OH)₂D₃ has been shown in vitro to inhibit the development of Th1 cells while promoting the development of Th2 cells.^121,128,129 Thus, 1,25(OH)₂D₃ seems to modulate T-cell differentiation, driving cells toward the Th2 phenotype and inhibiting Th1 development.

Thus, the action of 1,25(OH)₂D₃ in antagonizing Th1 cytokine production while promoting Th2 function may have an important therapeutic role in diseases whereby Th1 cytokine responses drive the immunopathology.

Effects on APCs
The secretion of cytokines by APCs such as macrophages and dendritic cells (DCs) is crucial for the recruitment and activation of T lymphocytes. The actions of APCs on T cells are also influenced by 1,25(OH)₂D₃. Interleukin-12 (IL-12) is the principal APC-derived cytokine that determines the direction (Th1 vs Th2) of the immune response. Thus, Th1-stimulating cytokines are inhibited by 1,25(OH)₂D₃ in DCs, as well as in other APCs.¹²⁴ IL-12 stimulates the development of CD4⁺ Th1 lymphocytes and inhibits the development of CD4⁺ Th2 lymphocytes. By inhibiting IL-12, 1,25(OH)₂D₃ shifts the immune response away from a Th1 and toward a Th2 profile. Finally, 1,25(OH)₂D₃ stimulates DC production of the immunosuppressive cytokine IL-10, which antagonizes the Th1 driving effects of IL-12.¹³⁰ These observations provide a rationale for use of vitamin D and its analogs in the prevention and treatment of autoimmune disease.

Autoimmune Disease
Autoimmune diseases occur because of an inappropriate immune-mediated attack against self-tissue, resulting in tissue injury and disease. Both environmental and genetic factors play a vital role in disease development. Vitamin D availability via sun exposure or diet may contribute to the development of both MS and IBD. In support of this view, vitamin D and signaling through the VDR have been shown in mice to dictate the outcome of experimental MS and IBD. Vitamin D seems to regulate T-cell development and function, which may influence the outcome of the immune response either toward or away from autoimmunity.^131,132 For example, in the absence of vitamin D and signals delivered through the VDR, autoreactive T cells develop, whereas in the presence of active 1,25(OH)₂D₃ and a functional VDR, the balance in the T-cell response is restored and autoimmunity avoided.¹³³

Vitamin D from sunlight exposure is lower in areas where IBD occurs most often, as IBD is most prevalent in northern climates such as North America and Northern Europe.^134,135 Vitamin D deficiency is common in patients with IBD even when the disease is in remission.^136,137 Why vitamin D deficiency occurs more frequently in IBD is unclear. It is probably due to the combined effects of low vitamin D intake, malabsorption of many nutrients including vitamin D, and decreased outdoor activities in climates that are not optimal for vitamin D synthesis in the skin.

The strongest evidence that the VDR and its ligand have important roles in the pathogenesis of IBD comes from studies in mouse models. IL-10 knockout mice, model for the Crohn's disease form of IBD, spontaneously develop enterocolitis within 5–8 weeks of birth due to an uncontrolled immune response to resident intestinal flora in conventional animal facilities.¹³⁸ Approximately 30% of mice die subsequent to the development of severe anemia and weight loss. By contrast, IL-10 knockout mice raised in pathogen-free facilities develop a milder form of enterocolitis that does not result in death. Experimental IBD is induced by TNF-- and IFN-secreting Th1 cells. Th2 or T regulatory cells inhibit both the development and function (cytokine secretion) of Th1 cells. Cantorna¹²³ was the first to establish an experimental link between vitamin D status and IBD. The author showed that vitamin D deficiency exacerbates the symptoms of enterocolitis and increases morbidity and mortality in IL-10 knockout mice, whereas supplementation with vitamin D ameliorates IBD symptoms, reduces inflammation, and improves histologic scores and mortality.¹²³ They noted that 100% of the vitamin D–deficient IL-10 KO mice expressed symptoms of IBD (diarrhea, rectal bleeding) and 60% died before 9 weeks of age from complications of severe IBD. In contrast, vitamin D–sufficient mice showed no outward symptoms of IBD at the same time point.¹³⁴ Supporting the vitamin D deficiency data, mice that are both IL-10 and VDR deficient (double VDR/IL-10 KO) develop a fulminating form of experimental IBD that leads to 100% mortality by 7 weeks of age.¹²³ Interestingly, the severity of IBD in the VDR/IL-10 knockout mice is the same regardless of whether or not disease-causing microorganisms are present in the colony. Both vitamin D deficiency and VDR deficiency render experimental IBD more severe. These observations provide strong evidence that establishes vitamin D and VDR as a physiologic regulator of intestinal inflammation in IBD. The accumulating evidence for the immunomodulatory effects of VDR ligands certainly provides a rationale for further investigation of their potential in the treatment of IBD. A schematic depicting the potential role of vitamin D in Crohn's disease is shown in Figure 5.

View larger version (31K): [in this window] [in a new window]   Figure 5. Potential role of vitamin D in Crohn's disease. In Crohn's disease, bacterial antigens drive antigen-presenting cells (DCs) to produce cytokines such as interleukin-12 (IL-12) which drive a T-helper 1 (Th1) proinflammatory response to induce macrophages, which produce TNF and neutrophil chemoattractive agents, ultimately resulting in the production of noxious agents and tissue injury. The damaged intestinal tissue is more permeable to antigens that drive the vicious cycle of antigen-presentation, local immune activation, and tissue injury. Anti-inflammatory cytokines such as interleukin-10 (IL-10), made by regulatory T cells (T regs), anatagonize Th1 proinflammatory processes by stimulating T-helper 2 function. Vitamin D anatagonizes Th1 proinflammatory responses by interfering with antigen presentation and Th1 activation, up-regulating Th2 cytokines and down-regulating NFB in macrophages. Ag, antigen; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

A large epidemiologic study showed an inverse relationship between vitamin D status and the development of MS. The study showed that women with the highest vitamin D intakes (including supplements) had a 40% reduction in the risk of developing disease.¹³⁹ Like IBD, vitamin D deficiency is common in patients with MS.¹²² The cause of low vitamin D levels in MS patients is also likely to be due to a combination of low vitamin intakes and decreased outdoor activities in climates that are not optimal for vitamin D synthesis in the skin. In vivo, the immune targets of vitamin D have been defined primarily in Th1-driven autoimmune diseases. Vitamin D deficiency accelerates the development of experimental MS and type-1 diabetes.^139–142 Conversely, 1,25(OH)₂D₃ treatment suppressed the development of these Th1-mediated autoimmune diseases.¹⁴³ In addition, 1,25(OH)₂D₃ treatment of mice with ongoing MS symptoms halted the disease progression in these mice, showing that vitamin D altered the immune response even after the disease had been established. 1,25(OH)₂D₃ has been shown to inhibit Th1-driven responses in a number of different models.¹⁴²

Vitamin D may also play a role in preventing type I diabetes mellitus. Autoimmune type 1 diabetes can be prevented in nonobese diabetic mice by 1,25(OH)₂D₃ and its analogs.^144,145 Treatment of NOD mice from weaning until old age not only prevented clinical diabetes, it also prevented the histologic lesion insulitis.¹⁴⁶ In this model of autoimmune diabetes, up-regulation of regulatory immune cells and a shift from Th1 toward Th2 lymphocytes locally in the pancreases of treated mice can be observed. This protective Th2 population is induced not only at the site of the β cell attack but also in the peripheral immune system.¹⁴⁷ After immunization of 1,25(OH)₂D₃-treated NOD mice with a diabetes-specific autoantigen (a peptide of GAD65), lymphocytes of the draining lymph nodes showed an increased IL-4 and decreased IFN production in vitro and in vivo. Strikingly, this immune deviation induced by 1,25(OH)₂D₃ is limited to pancreatic autoantigens and could not be seen after immunization with the β cell-irrelevant protein ovalbumin.

Other effects on the immune system of NOD mice have been described, the most important being a restoration of the defective apoptosis sensitivity of lymphocytes, leading to a more efficient elimination of potentially dangerous autoimmune effector cells.¹⁴⁸ This increased apoptosis induced by 1,25(OH)₂D₃ and its analogs in DCs and T lymphocytes of NOD mice has been described after treatment with different apoptosis-inducing signals, such as corticosteroids, and could help to explain why an early short-term treatment with these agents, before onset of autoimmunity, confers long-term protection and promotes tolerance restoration.

Besides preventing the onset of autoimmune diseases, 1,25(OH)₂D₃ and its analogs are also able to treat ongoing autoimmune diseases. Treatment of NOD mice with analogs of 1,25(OH)₂D₃ can prevent the progression of an initial β cell attack (reflected by the presence of insulitis) to clinical overt diabetes.¹⁴⁹ Interestingly, in this model of ongoing autoimmune destruction, no induction of suppressor cells by 1,25(OH)₂D₃ could be demonstrated. Nevertheless, within the pancreases of protected mice, again a shift from Th1 toward Th2 cytokines is noted. Further, analogs of 1,25(OH)₂D₃ are able to inhibit the recurrence of autoimmune diabetes after syngeneic islet transplantation in NOD mice.^150,151

Development of noncalcemic vitamin D analogs could permit sustained systemic administration without causing significant hypercalcemia, thus allowing wider clinical applications in the future. Vitamin D supplementation, which is an inexpensive and efficient way to prevent vitamin D deficiency, might also help reduce the risk of autoimmune disease.

	VDR Polymorphisms and Disease

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
Most of the biologic activities of 1,25(OH)₂D₃ are mediated by a high-affinity receptor that acts as a ligand-activated transcription factor. The major steps involved in the control of gene transcription by the VDR include ligand binding, heterodimerization with retinoid X receptor (RXR), binding of the heterodimer to VDREs, and recruitment of other nuclear proteins into the transcriptional preinitiation complex. Thus, genetic alterations of the VDR gene could lead to important defects on gene activation, affecting calcium metabolism, cell proliferation, and immune function. For example, VDR gene mutations cause vitamin D–resistant rickets, a rare monogenetic disease.¹⁵² A polymorphism is a genetic variant that appears in at least 1% of the population. These changes can occur in noncoding parts of the gene (introns), so they would not be seen in the protein product. Changes in these regulatory parts of the gene would then affect the degree of expression of the gene and thus the levels of the protein. The discovery of genetic variants linked with susceptibility of diseases can be the key to advances in preventive medicine. In general, association studies can be used to test whether a polymorphism occurs more frequently in the cases studied than in the controls. If a relationship with the disease emerges from association studies, this finding would strongly support the idea that the candidate gene is in some way involved in the disease. The diseases associated with VDR polymorphisms are summarized in Table 7.¹⁵³

Carling et al¹⁵⁴ reported a relationship between the BsmI polymorphism and primary hyperparathyroidism. Recently, 2 meta-analyses performed by Thakkinstian et al^155,156 demonstrated a positive association between the b allele and bone mass density. Furthermore, it was shown that the haplotypes Bat and BAt were significantly associated with osteoporosis.

An association has been described between VDR polymorphisms and susceptibility to and outcome of some cancers, like breast, prostate, and colon cancers. In 1997, Ingles et al¹⁵⁷ published one of the first reports finding a relationship between the polyA polymorphism of the VDR gene and prostate cancer in the US population. Taylor et al¹⁵⁸ showed the relationship between the TaqI polymorphism and an increased risk of prostate cancer, whereas the restriction site (tt) was associated with a lower risk with higher levels of 1,25(OH)₂D₃.¹⁵⁹ However, other investigators found no association between TaqI or polyA and prostate cancer.^160–162 In the case of other VDR polymorphisms (FokI, BsmI), conflicting results can be found as well.^{159,162–165} In addition, a recent meta-analysis of 28 different studies was performed, and no relationship was found between any of the former VDR polymorphisms and prostate cancer susceptibility.¹⁶⁶

As with prostate cancer, conflicting results have been reported regarding the possible relationship between breast cancer and VDR polymorphisms. The majority of the reports present in the literature found no relationship between the risk of breast cancer and TaqI polymorphism, but some of them presented a link between TaqI polymorphism and risk of metastases.^167–171

The BsmI shows an opposite pattern, with a relationship detected in 3 reports.^172–174 In the case of FokI the consensus is higher, and most of the reports showed no association with increased risk of breast cancer.^171,173,174 To date, studies investigating the relationship between polyA or ApaI^170,171 and the risk of breast cancer showed a link between these VDR polymorphisms and the possibility of presenting a tumor. The number of papers analyzing VDR polymorphisms in other cancer types is significantly lower. Regarding colon carcinoma, there are reports showing an association with BsmI polymorphism,^175,176 whereas conflicting results are found regarding FokI.^177,178

VDR polymorphisms have been described in a number of autoimmune diseases. A positive relationship between the B allele of the BsmI polymorphism and a lack of a relationship between the FokI polymorphism and the incidence of systemic lupus erythematosus has been observed.^179–181 In the case of Crohn's disease, a link has been suggested between the TaqI, ApaI and FokI polymorphisms and disease susceptibility.¹⁸² Furthermore, a link between BsmI and FokI with primary biliary cirrhosis and autoimmune hepatitis has also been found.^183,184 In MS patients, a higher presence of the bA haplotype has been detected,^185,186 whereas TaqI¹⁸⁷ and FokI¹⁸⁸ polymorphisms were observed.

In summary, a vast amount of information has been collected through the years regarding the association of vitamin D polymorphisms with susceptibility to contract different diseases. Unfortunately, the results obtained so far are conflicting, and the role of VDR polymorphisms remains obscure. Therefore, the use of VDR polymorphisms as diagnostic tools, or even as markers for a higher propensity toward some diseases, is still a matter of debate.

	Recommendations and Conclusion

Top Vitamin D Overview Vitamin D and Cancer... Mechanisms Involved in the... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Role of 1{alpha},25(OH)2D3 in... Vitamin D Analogs for... Vitamin D and the... VDR Polymorphisms and Disease Recommendations and Conclusion

 
Vitamin D deficiency is a common clinical problem in the United States.^189–194 Because most patients with mild deficiency are asymptomatic, physicians should have a high index of suspicion in populations at highest risk for deficiency. Identification and treatment of patients with vitamin D deficiency are important for optimal bone development and muscle strength. The major source of vitamin D for both children and adults comes from reasonable sun exposure. In the absence of sun exposure, most experts now agree that 1000 IU of vitamin D is needed daily in the absence of sun exposure to maintain a healthy blood level of 25(OH)D of between 75 and 125 nmol/L (30–50 ng/mL).^194–200 However, vitamin D supplementation is not widely practiced and most supplements only contain 400 IU of vitamin D. As reviewed in this manuscript, in addition to bone health, there is mounting scientific evidence that implicates vitamin D deficiency with an increased risk of autoimmune disease and many common deadly cancers. Children over the age of 1 year and all adults should receive 1000 IU of vitamin D per day, or have judicious sun exposure to satisfy their vitamin D requirement. Measurement of 25(OH)D should be encouraged. The risks and potential benefits of vitamin D supplementation should be further studied for use in patients who either have or are at high risk for breast, colon, and prostate cancer and autoimmune diseases.

The authors acknowledge Kerry Schulze, PhD, Johns Hopkins School of Public Health, Department of Human Nutrition, and Susan Kreimer, MS, a freelance medical writer in New York, for their editorial contributions. The work was supported in part by grant number AT00437 from the National Center for Complementary & Alternative Medicine of the National Institutes of Health (Adrian S. Dobs, MD, MHS, principal investigator).

1 Holick MF. Vitamin D: the underappreciated D-lightful hormone that is important for skeletal and cellular health. Curr Opin Endocrinol Diabetes. 2002;9:87 –98.[CrossRef]

Nutrition in Clinical Practice, Vol. 22, No. 3, 305-322 (2007)
DOI: 10.1177/0115426507022003305


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