QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peehl, D. M. Articles by Feldman, D. Search for Related Content PubMed PubMed Citation Articles by Peehl, D. M. Articles by Feldman, D.

---

Supplement: Nutritional Genomics and Proteomics in Cancer Prevention

Pathways Mediating the Growth-Inhibitory Actions of Vitamin D in Prostate Cancer¹

Donna M. Peehl^*, Aruna V. Krishnan and David Feldman^,2

Departments of ^* Urology and Medicine, Stanford University School of Medicine, Stanford, CA 94305

² To whom correspondence should be addressed. E-mail: feldman{at}cmgm.stanford.edu.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
Vitamin D is emerging as an important dietary factor that affects the incidence and progression of many malignancies including prostate cancer. The active form of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃], inhibits the growth and stimulates the differentiation of prostate cancer cells. We have studied primary cultures of normal and cancer-derived prostatic epithelial cells as well as established human prostate cancer cell lines to elucidate the molecular pathways of 1,25(OH)₂D₃ actions. These pathways are varied and appear to be cell specific. In LNCaP cells, 1,25(OH)₂D₃ mainly causes growth arrest through the induction of insulin-like growth factor binding protein-3 and also stimulates apoptosis to a much smaller extent. We have used cDNA-microarray analyses to identify additional genes that are regulated by 1,25(OH)₂D₃ and to raise novel therapeutic targets for use in the chemoprevention or treatment of prostate cancer. Less calcemic analogs of 1,25(OH)₂D₃ that have more antiproliferative activity are being developed that will be more useful clinically. In target cells, 1,25(OH)₂D₃ induces 24-hydroxylase, the enzyme that catalyzes its self inactivation. Cotreatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of 1,25(OH)₂D₃. The combination of other anticancer agents such as retinoids with vitamin D offers another promising therapeutic approach. A small clinical trial has shown that 1,25(OH)₂D₃ can slow the rate of prostate-specific antigen increase in prostate cancer patients, which demonstrates proof of the concept that vitamin D or its analogs are clinically effective. Our research is directed at understanding the mechanisms of vitamin D action in prostate cells with the goal of developing chemoprevention and treatment strategies to improve prostate cancer therapy.

KEY WORDS: • 1,25-dihydroxVycholecalciferol • vitamin D analogs • vitamin D receptor • 24-hydroxylase • target genes

Background: hormonal actions of vitamin D in prostate

Vitamin D and its metabolites are candidates for prevention and therapy of prostate cancer. This concept is based on a large body of evidence from epidemiological, cell culture, animal and clinical studies that shows antitumor activity of vitamin D in prostate cells. The vitamin D axis involves a complex series of events, and the impact of vitamin D on the prostate occurs at several levels and is mediated by many different pathways.

Vitamin D metabolism and sunlight

The first step in the synthesis of the active form of vitamin D begins in the skin ( 1). Ultraviolet (UV)³ radiation converts the precursor 7-dehydrocholesterol to cholecalciferol (vitamin D-3). Epidemiological studies reveal a correlation between exposure to UV radiation and prostate cancer risk. Schwartz and Hulka ( 2) demonstrated that mortality rates from prostate cancer in the U.S.A. are inversely correlated with UV radiation, although this was an indirect link with vitamin D. Also, it is clear that age is the strongest risk factor for prostate cancer, and the elderly are frequently vitamin D deficient due to several factors that include less exposure to UV radiation ( 3). Race also is a risk factor for prostate cancer, with the clinical incidence in American blacks twice that of Caucasians. This also may be related to vitamin D deficiency in that high melanin levels in darkly pigmented skin block UV radiation and inhibit the formation of vitamin D-3 ( 4).

    Diet. Dietary forms of vitamin D include calciferol in supplemented milk, ergocalciferol (vitamin D-2) in plants and vitamin D-3 in animal products. Diet is proposed to be a risk factor for prostate cancer, and the low risk of prostate cancer for indigenous Japanese is postulated to be related to their traditional diet. This diet, among other attributes, is rich in oily fish, which are an important dietary source of vitamin D-3.

    Metabolism of provitamin D to active vitamin D. The active form of vitamin D, 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃], is formed through two sequential hydroxylation steps ( 5, 6). In the first step, hepatic hydroxylation of vitamin D-3 by vitamin D 25-hydroxylase generates 25-hydroxycholecalciferol [25(OH)D₃], which is then hydroxylated (mainly in the kidneys) to form 1,25(OH)₂D₃ by the enzyme 25(OH)D₃ 1-hydroxylase (1-hydroxylase). Age has an impact at this point on the vitamin D axis as well, because activity of these enzymes declines with age ( 7). Thus, the elderly are frequently deficient in vitamin D not only due to less exposure to UV radiation, but also owing to a decline with increasing age in the ability to synthesize 1,25(OH)₂D₃.

    Vitamin D and prostate cancer risk. Evidence for vitamin D deficiency as a risk factor for prostate cancer first came from a prospective case-control study of vitamin D metabolite levels in sera collected in the San Francisco Bay area between 1964 and 1971 that were matched for age, race and day of storage. In this study ( 8), mean levels of serum 1,25(OH)₂D₃ were 1.81pg/mL lower in cases diagnosed with prostate cancer than in controls (P = 0.002). This finding remains controversial, because other studies fail to confirm an association between serum levels of vitamin D metabolites and prostate cancer risk. Interestingly, another epidemiological study published recently ( 9) reports that high levels of dietary calcium are a significant risk factor for prostate cancer. This may be relevant to the relationship of vitamin D and prostate cancer, because high levels of serum calcium suppress parathyroid hormone and reduce the renal production of 1,25(OH)₂D₃.

    Prostate as a target of 1,25(OH)₂D₃. Classically, 1,25(OH)₂D₃ is considered to interact exclusively with bone, intestine, kidney and parathyroid glands to regulate calcium and phosphate homeostasis ( 10). Now we recognize that 1,25(OH)₂D₃ is a secosteroid hormone that regulates growth and differentiation in a wide variety of cells by binding to the vitamin D receptor (VDR), which is a member of the steroid hormone receptor gene superfamily ( 11, 12). Evidence that VDR are present in human prostatic tissues dates back to 1988 ( 13), and epithelial cells are found ( 14) to be the primary target of 1,25(OH)₂D₃ in the prostate.

Circulating 1,25(OH)₂D₃ binds to VDR in prostatic epithelial cells and sets in motion a series of events that leads to several biological actions ( 11, 12). VDR functions as a ligand-dependent transcription factor that binds to specific DNA sequences on target genes. Vitamin D response elements (VDRE) have been identified in several target genes. VDR bind to VDRE as heterodimers with the retinoid X receptor (RXR). Transcriptional activation by nuclear hormone receptors such as VDR requires their interaction with coactivators and corepressors.

Many factors influence the transcription program initiated by ligand binding to VDR and the end effect on cellular phenotype. Genetic polymorphisms may affect VDR function. Several molecular epidemiological studies link VDR polymorphisms with prostate cancer risk and/or progression ( 15). Many coactivators and corepressors modulate activity of VDR, and these also may have polymorphisms that affect individual response or may carry mutations in their genes in premalignant or cancer cells to make VDR more or less active. Also, cell-type specific responses to 1,25(OH)₂D₃ are believed to some extent be due to cell-type specific expression of different sets of coactivators and/or corepressors ( 16). The number of VDR per cell also can be regulated in malignant cells by autocrine, paracrine or endocrine growth factors or hormones ( 17).

Strong evidence supports a role for 1,25(OH)₂D₃ as a critical regulator of growth and differentiation in the prostate ( 14, 18– 21). Application of 1,25(OH)₂D₃ to animal models of prostate cancer demonstrates efficacy as an antitumor agent in vivo. Finally, a pilot clinical study ( 22) shows that 1,25(OH)₂D₃ can slow the rate of tumor doubling in patients with early recurrent prostate cancer after radical prostatectomy or radiation therapy. The combination of all of these observations at many different levels of investigation provides a strong rationale for additional studies on the development of vitamin D as a preventive and therapeutic agent in prostate cancer ( 14, 18– 21).

Growth inhibitory actions of vitamin D in prostate cancer cells

    Vitamin D activity in prostate cancer cell lines. Miller et al. ( 23) were the first to show that LNCaP human prostate cancer cells express VDR and are responsive to 1,25(OH)₂D₃. We extended their observations by characterizing VDR in a variety of prostate cancer cell lines, and we were the first to show antiproliferative activity of 1,25(OH)₂D₃ and its analogs in prostate cancer cells ( 24, 25). Studies from our laboratory ( 26) provide evidence that the upregulation of insulin-like growth factor binding protein-3 (IGFBP-3) expression by 1,25(OH)₂D₃ is a necessary component of 1,25(OH)₂D₃-mediated inhibition of LNCaP cell growth. Treatment with 1,25(OH)₂D₃ increases IGFBP-3 mRNA levels. Importantly, addition of IGFBP-3 antisense oligonucleotides abrogates 1,25(OH)₂D₃-mediated growth inhibition ( Fig. 1), which suggests that in LNCaP cells, the growth-inhibitory action of 1,25(OH)₂D₃ depends on IGFBP-3 upregulation. Our data together with that of other investigators ( 27– 36) show that human prostate cancer cell lines vary in their expression of VDR, sensitivity to growth regulation by 1,25(OH)₂D₃ or analogs and induction of apoptosis by 1,25(OH)₂D₃. The majority of the cell lines express VDR and exhibit growth inhibition in response to 1,25(OH)₂D₃. A notable exception is the DU 145 cell line, which expresses functional VDR but is resistant to growth inhibition. The LNCaP cells also are unique in comparison with other prostate cancer cell lines in that 1,25(OH)₂D₃ causes these cells to undergo apoptosis in addition to cell-cycle arrest. Blutt et al. ( 37) show evidence of apoptosis in LNCaP cells treated with 1,25(OH)₂D₃ for 6 d and also show the downregulation of the proapoptotic proteins Bcl-2 and Bcl-X_L.

View larger version (13K): [in this window] [in a new window]   FIGURE 1  Abrogation of 1,25-dihydroxycholecalciferol [1,25(OH)2D3]-mediated growth inhibition of LNCaP cells by insulin-like growth factor binding protein-3 (IGFBP-3) antisense oligonucleotides. Cells were seeded in 96-well plates and grown in serum-free growth medium for 4 d with 10 nmol/L of 1,25(OH)2D3 or ethanol vehicle (+ and -, respectively) along with 8 µg of antisense or sense IGFBP-3 oligonucleotides/mL. No oligonucleotides were added to the control group. DNA concentrations were determined at the end of the experiment, and values in cells treated with vehicle for each group were defined as 100%. Values are shown as means ± SE of three experiments. [Reproduced with permission from Boyle et al. (2001) J. Urol. 165: 1319–1324.]

Vitamin D also modulates other phenotypic traits of prostate cancer cells in a manner that is consistent with tumor-suppressive activity. Schwartz et al. ( 41) found that 1,25(OH)₂D₃ inhibits the invasiveness of prostate cancer cells, which is presumably related to decreased expression of the matrix metalloproteinases MMP-2 and MMP-9 in 1,25(OH)₂D₃-treated cells. We showed ( 42) that 1,25(OH)₂D₃ decreases the expression of ₆ and ß₄ integrins in prostate cancer cells, which results in reduced cellular invasion, adhesion and migration to the basement-membrane protein laminin. These effects of 1,25(OH)₂D₃ are considered to be related to the ability of 1,25(OH)₂D₃ to inhibit metastases, which was demonstrated in a rat model of prostate cancer ( 43).

    Vitamin D activity in primary cultures of prostatic cells. In 1994, we were the first to show ( 44) that primary cultures of epithelial cells from normal, benign prostatic hyperplasia (BPH) and malignant human prostate tissues express VDR and that 1,25(OH)₂D₃ exhibits potent antiproliferative effects. In this initial study, we also noted that prostatic stromal cells have considerably fewer VDR than epithelial cells and are much less sensitive to growth suppression by 1,25(OH)₂D₃ than epithelial cells. Fresh, uncultured prostatic tissues also bind 1,25(OH)₂D₃, which demonstrates the presence of VDR in vivo. From these studies, we conclude that epithelial cells are the main target of vitamin D action in the prostate, and that primary cultures would provide a valuable model system to further investigate the role of vitamin D in the normal biology of the prostate as well as an alternative to established cancer cell lines for investigation of vitamin D activity. We noted that primary cultures differ in at least one significant way from cell lines in their response to 1,25(OH)₂D₃: the growth inhibition of cell lines is generally reversible even after rather lengthy 1,25(OH)₂D₃ exposure ( 45), whereas primary cultures (normal as well as malignant) become irreversibly growth suppressed after as little as 2 h of 1,25(OH)₂D₃ exposure ( 44).

    Vitamin D activity in androgen-responsive prostate cells. Although the predominant effect of 1,25(OH)₂D₃ on prostatic epithelial cells is growth inhibition, Miller et al. reported ( 23) that growth of LNCaP cells is modestly stimulated by 1,25(OH)₂D₃ in medium that contains charcoal-stripped (androgen-depleted) serum instead of whole serum. We evaluated the response of primary cultures to 1,25(OH)₂D₃ in the presence of charcoal-stripped serum, but found that this did not make 1,25(OH)₂D₃ growth stimulatory ( 44). In subsequent studies from our laboratory and others, it was shown that this effect is uniquely related to the androgen-responsive nature of LNCaP cells. The actions of 1,25(OH)₂D₃ in LNCaP cells are in fact androgen dependent and are mediated in part by upregulation of the androgen receptor (AR) by 1,25(OH)₂D₃ ( 45– 48). In another androgen-responsive pair of prostate cancer cell lines, MDA-PCa 2a and 2b ( 49) with unique mutations in the AR gene ( 50, 51), we found that growth inhibition by 1,25(OH)₂D₃ occurs through androgen-dependent as well as androgen-independent mechanisms ( 52). Similar cross talk between the androgen- and vitamin D–signaling pathways is reported for ovarian ( 53) and breast cancer ( 54) cells in which VDR and AR are upregulated by their cognate ligands.

Activity of vitamin D in patients with prostate cancer

In 1995, Osborn et al. ( 55) published the results of a phase II clinical trial in which patients with hormone-refractory prostate cancer were treated with 1,25(OH)₂D₃. Although no objective responses were seen, serum levels of PSA declined in several of the patients. In 1998, we reported the benefit of 1,25(OH)₂D₃ in a pilot clinical trial of early recurrent prostate cancer after radical prostatectomy or radiation therapy ( 22). As shown in Table 1, all of the seven subjects showed a statistically significant prolongation of the serum PSA doubling time, thereby establishing proof of principle that 1,25(OH)₂D₃ has beneficial effects when used to treat men with prostate cancer. Recently, we developed a rationale for combining ketoconazole and 1,25(OH)₂D₃ or vitamin D analogs to treat prostate cancer ( 56). Ketoconazole inhibits cytochrome P450 enzymes such as those that synthesize androgens ( 57) and is used as a second-line androgen-ablation therapy to treat advanced prostate cancer. However, ketoconazole also inhibits the enzyme 1-hydroxylase that synthesizes 1,25(OH)₂D₃, and therefore, men on ketoconazole therapy are likely to be vitamin D deficient. Van Veldhuizen et al. ( 58) showed that treating vitamin D deficiency in patients with metastatic prostate cancer may improve bone pain and muscle strength. We reasoned that combination therapy with ketoconazole and 1,25(OH)₂D₃ might alleviate the side effects of vitamin D deficiency caused by ketoconazole as well as provide additional therapeutic activity through antitumor effects of 1,25(OH)₂D₃. Indeed, our studies on primary prostatic epithelial cultures reveal enhanced antiproliferative activity of the combined drugs ( 59).

Factors that modulate activity of 1,25(OH)₂D₃ on prostatic epithelial cells

    Combination therapy. In addition to searching for less-calcemic vitamin D analogs that could be used more efficaciously for therapy than 1,25(OH)₂D₃, investigators have tested a variety of agents for additive or synergistic antiproliferative activity for use in combination with 1,25(OH)₂D₃. Clinically relevant agents that enhance the antiproliferative activity of 1,25(OH)₂D₃ on prostatic epithelial cells include platinum drugs ( 62) and paclitaxel ( 63). Using primary cultures of prostate cancer cells, we found enhanced activity of 1,25(OH)₂D₃ when combined with suramin ( 64). Suramin is a polysulfonated napthylurea compound that is used with some efficacy to treat patients with advanced prostate cancer. For the most part, the mechanism of additive or synergistic activity of 1,25(OH)₂D₃ with these drugs is not understood.

Interactions between vitamin D and retinoids have been recognized for some time and are believed to occur through the RXR, with which both VDR and the retinoic acid (RA) receptor dimerize ( 65). When we tested a variety of factors for the ability to enhance the response of primary cultures of normal and malignant prostatic epithelial cells to vitamin D, we found synergistic activity between all-trans RA and 1,25(OH)₂D₃ ( 64). We found similar synergistic growth-inhibitory activity of 9-cis RA and 1,25(OH)₂D₃ on LNCaP cells ( 48) as noted by others ( 45, 66). Various other retinoids sensitize prostate cancer cell lines to 1,25(OH)₂D₃ or analogs ( 67– 69). Retinoids have been tested in clinical trials for prostate cancer, but as with 1,25(OH)₂D₃, therapeutic efficacy is limited by toxicity.

Another clinically relevant observation of ours is that hydrocortisone at 3 µmol/L dramatically reduces the growth-inhibitory action of 1,25(OH)₂D₃ on primary cultures of prostate cancer cells. In the absence of hydrocortisone, 1,25(OH)₂D₃ is at least 10-fold more potent as an antiproliferative agent ( 64). Our observation that hydrocortisone mutes the antiproliferative activity of 1,25(OH)₂D₃ is noteworthy, because a clinical trial combining 1,25(OH)₂D₃ and glucocorticoid therapy is in progress at the University of Pittsburgh. The rationale for this trial is based on enhanced activity of 1,25(OH)₂D₃ with glucocorticoids in other experimental systems ( 70) and the expectation that glucocorticoid action on the intestine will reduce the tendency toward hypercalcemia. However, the outcome of combining vitamin D and glucocorticoids is cell-type dependent ( 71, 72), and our finding suggests that this approach may be contraindicated for prostate cancer. Glucocorticoids are shown to regulate VDR levels in a number of experimental systems ( 17, 71). However, we did not find changes in VDR levels in primary cultures of prostatic cancer cells in response to hydrocortisone ( 64), which suggests that the downregulation of VDR is apparently not the mechanism by which hydrocortisone blocks the antiproliferative activity of 1,25(OH)₂D₃ in these cells.

    Vitamin D and genistein. Vitamin D and its analogs are believed to have the potential to prevent as well as treat prostate cancer ( 73). The antiproliferative and differentiating effects of 1,25(OH)₂D₃ on normal epithelial cells in the rat prostate ( 74) in conjunction with in vitro studies of cultured cells support this concept. The vitamin D analog Ro24-5531 shows chemopreventive activity in the androgen-promoted carcinoma model of the Lobund-Wistar rat seminal vesicle and prostate ( 75). Therefore, in addition to considering combinations of vitamin D and other agents that might have additive or synergistic therapeutic activity, investigators have begun to search for combinations that could have enhanced chemopreventive properties. One such factor that is emerging is genistein. This compound is the most-abundant isoflavone in soy, and the association between decreased risk of certain cancers, including prostate cancer, and soy consumption is attributed to genistein. A recent study demonstrates that genistein potentiates the antiproliferative effect of vitamin D analogs on HL-60 leukemia cells ( 76), and a similar enhancement of activity of 1,25(OH)₂D₃ by genistein is found in prostate cells ( 77). The mechanism is unclear, but several recent abstracts ( 78, 79) suggest that genistein may potentiate activity of vitamin D by decreasing enzymatic activity of 24-hydroxylase, which metabolizes 1,25(OH)₂D₃ to less-active products.

    Resistance of some prostate cancer cells to growth suppression by vitamin D. Although many of the established prostate cancer cell lines or primary cultures of adenocarcinoma-derived cells respond to vitamin D, there is abundant evidence that prostate cancer cells may become resistant to antiproliferative activity of 1,25(OH)₂D₃. This resistance can develop at several levels such as through the loss or decreased expression of VDR or RXR, through VDR polymorphisms that diminish its function, through elevated expression of VDR corepressors, by increased expression of enzymes that metabolize 1,25(OH)₂D₃ or by other means. An example of loss of response to vitamin D through loss of VDR is shown by the cancer cell line JCA-1 (formerly believed to be prostatic but now of questionable origin) ( 80). VDR is not detectable in these cells, and they do not respond to 1,25(OH)₂D₃. Stable transfection of the cells with VDR cDNA makes these cells sensitive to growth inhibition by vitamin D ( 81). We ( 24) and others noted that the growth of DU 145 cells is not inhibited by 1,25(OH)₂D₃ despite the presence of a functional VDR with apparently normal binding affinity for 1,25(OH)₂D₃ in these cells. It seems that several factors can explain the insensitivity of DU 145 cells to growth inhibition by 1,25(OH)₂D₃. VDR abundance to some degree determines the magnitude of response to 1,25(OH)₂D₃. Like JCA-1 and PC-3 cells, DU 145 cells have relatively low numbers of VDR. Stable transfection of VDR into these cells results in increased levels of receptors and increased growth inhibition by 1,25(OH)₂D₃ ( 28). Therefore, increased levels of VDR are one mechanism by which cells may become more responsive to vitamin D.

The three cell lines mentioned as well as another, TSU-Pr1 (also of questionable origin), have the highest levels of 1,25(OH)₂D₃-inducible 24-hydroxylase of all of the prostate cancer cell lines studied so far. This enzyme initiates the catabolism of 1,25(OH)₂D₃ to inactive metabolites. Studies from our laboratory ( 82) show that inhibition of 24-hydroxylase by liarozole, a compound that blocks P450 enzymes including 24-hydroxylase, permits growth inhibition of DU 145 cells by physiological concentrations of 1,25(OH)₂D₃ ( Fig. 2). Clinical application of drugs such as liarozole or ketoconazole with similar anti-P450 activity ( 83) may improve the efficacy of vitamin D therapy. The greater response of DU 145 cells to certain vitamin D analogs than to 1,25(OH)₂D₃ also may relate to lesser metabolism of the analogs by 24-hydroxylase or to other interactions of the analogs with receptors or vitamin D–binding protein ( 31, 84). Thus, overexpression of 24-hydroxylase is a mechanism that can limit growth-suppressive activity of 1,25(OH)₂D₃ on prostate cancer cells.

View larger version (12K): [in this window] [in a new window]   FIGURE 2  Effects of 1,25(OH)2D3 and liarozole on DU 145 cell growth. Effects of 10 nmol 1,25(OH)2D3/L, 1 µmol liarozole/L or a combination of both on DU 145 cell growth were measured over a time course of 6 d with fresh medium (that contains the agents) replenished every 2 d. Cell proliferation was estimated using the MTS assay. Data are expressed as means ± SD of three experiments. *P < 0.05, compared with vehicle-treated control group. [Reproduced with permission from Ly et al. (1999) Endocrinology 140: 2071–2076. Copyright owner: The Endocrine Society.]

One important concept to emerge from these studies is that different signaling pathways are used by VDR to regulate genes involved in growth control versus genes involved in other functions. This is apparent in the ability of cells resistant to growth inhibition by 1,25(OH)₂D₃ to still exhibit VDR upregulation and/or 24-hydroxylase induction in response to 1,25(OH)₂D₃ ( 24). The presence of analogs with less-calcemic but equal or enhanced growth-inhibitory effects also suggests that different signaling pathways may be involved in these responses.

    1-hydroxylase activity in prostatic cells. Schwartz et al. ( 88) initially reported that normal human prostatic epithelial cells have 1-hydroxylase activity and synthesize 1,25(OH)₂D₃ from 25(OH)D₃. 1-hydroxylase activity is present at low levels in PC-3 and DU 145 cells and is absent in LNCaP cells. We confirmed these results and extended the studies to primary cultures derived from cancer ( 89). Whereas normal cells indeed had significant 1-hydroxylase activity, 14 of 15 cancer-derived cell strains had significantly reduced activity (5- to 10-fold lower than normal cells). The biological significance of this observation is evident from the data shown in Figure 3. Because of the very low activity of 1-hydroxylase in primary cancer strains, 25(OH)D₃ is unable to inhibit the growth of these cancer cells due to lack of conversion to the active hormone 1,25(OH)₂D₃. Normal cells, on the other hand, are inhibited by 25(OH)D₃ through conversion to 1,25(OH)₂D₃ by 1-hydroxylase activity ( 89). We did not detect 1-hydroxylase mRNA in prostatic stromal cells, which indicates that expression of this enzyme is specific to epithelial cells of the prostate. The prostate joins the list of extrarenal sites of 1-hydroxylase production, and the significance of local production of 1,25(OH)₂D₃ is a subject of considerable interest. If reduced activity of 1-hydroxylase is confirmed in prostate cancer tissues, then loss of local production of 1,25(OH)₂D₃ may be an important mechanism by which cancer escapes the antitumor effects of dietary vitamin D or circulating 1,25(OH)₂D₃.

View larger version (21K): [in this window] [in a new window]   FIGURE 3  Growth inhibition by 25-hydroxycholecalciferol [25(OH)D3; triangles] in comparison with 1,25(OH)2D3 (circles) in prostate epithelial cells. (A) In normal prostatic epithelial cells (E-CZ-2, E-PZ-8 and E-PZ-12), 25(OH)D3 and 1,25(OH)2D3 are equally growth inhibitory at concentrations between 0.01 and 10 nmol/L. (B) In primary cultures of cancer cells (E-CA-6, E-CA-10, E-CA-12), 1,25(OH)2D3 induces 20–40% more growth inhibition than 25(OH)D3. (C) Prostate cancer cell line LNCaP, although fully responsive to 1,25(OH)2D3, is resistant to treatment with 25(OH)D3. [Reproduced with permission from Hsu et al. (2001) Cancer Res. 61: 2852–2856.]

Identification of new target genes by cDNA microarray

    Transactivation of target genes by 1,25(OH)₂D₃. Molecular mechanisms of action of 1,25(OH)₂D₃ are beginning to be resolved ( 11). It is known that 1,25(OH)₂D₃ elicits biological responses through the regulation of transcription of various target genes that are involved in cell growth, apoptosis and cell differentiation ( 14, 18– 21). Genes regulated by 1,25(OH)₂D₃ range from those involved in bone mineralization and immune modulation to those implicated in signaling pathways that regulate cell cycle, proliferation and expression of structural proteins. However, it is clear that the effects of 1,25(OH)₂D₃ on cellular phenotype are cell and tissue specific, and data show that these specific effects have their basis in differential gene expression profiles induced by 1,25(OH)₂D₃ in different tissues.

    Gene expression profiles. To understand the basis of the differential phenotypic responses to 1,25(OH)₂D₃ among primary cultures and various cell lines and to begin to identify its molecular targets, we performed cDNA-microarray analyses of primary cell cultures (one strain of normal cells and one strain derived from an adenocarcinoma of Gleason grade 3/3) and LNCaP cells. Cells were exposed to fresh medium 2 d before vehicle or 50 nmol of 1,25(OH)₂D₃/L were added, and mRNA was isolated at 6 and 24 h after treatment. Microarray analyses were conducted with chips carrying 20,000 genes. When comparing our results with the published profiles of genes that are regulated by 1,25(OH)₂D₃ in squamous carcinoma cells ( 99), we found no commonly regulated genes. We found a very limited number of genes commonly regulated between prostate cells and breast cells after microarray analyses of 1,25(OH)₂D₃-treated breast cancer cell lines MCF-7 and MDA-MB 231 (unpublished data). As additional findings from genetic profiling become available, it appears that the spectrum of genes induced by any given factor differs markedly among different cell types ( 100). Prostate-specific effects of 1,25(OH)₂D₃ are therefore expected and can be used to develop molecular markers that reflect activity of vitamin D in the prostate.

Several noteworthy observations emerged from microarray analyses. First, we found the highest fold increase in the expression of 24-hydroxylase in both normal and cancer-derived primary cell cultures. We had previously seen upregulation of this gene by 1,25(OH)₂D₃ at both the RNA and enzymatic level in primary cultures, which confirms the microarray technique. The promoter of this gene contains VDRE, and its regulation by 1,25(OH)₂D₃ in the kidney is very important for maintaining vitamin D homeostasis in the body. In contrast, we did not detect any increase in 24-hydroxylase RNA in our microarray analysis of LNCaP cells, which confirms our previously reported results that were based on Northern blot analyses ( 24).

There was overlap in the profiles of genes regulated by 1,25(OH)₂D₃ in normal and cancer-derived primary cultures, but there also were striking differences. Of the early-response genes (i.e., upregulated twofold or more at 6 h), dual-specificity phosphatase 10 was the most highly upregulated gene in both normal and cancer-derived cultures. Dual phosphatase 10 inactivates mitogen-activated protein kinase (MAPK), which suggests that an important feature of the growth-inhibitory activity of 1,25(OH)₂D₃ may be the inhibition of the growth-promoting activity of MAPK. Early upregulation of protein kinase A anchor protein (gravin) 12 by 1,25(OH)₂D₃ in both normal and cancer cells also is of interest and is perhaps related to the recently reported 1,25(OH)₂D₃-regulated packaging of protein kinase C in chondrocytes ( 101).

Metallothionein genes are particularly noteworthy for their preferential induction by 1,25(OH)₂D₃ in normal but not cancer-derived primary epithelial cells. In fact, several metallothionein genes are significantly downregulated by 1,25(OH)₂D₃ in cancer cells. Metallothioneins constitute the majority of intracellular protein thiols and as such are considered to act as cell-survival factors. Upregulation of metallothioneins in normal prostatic epithelial cells is consistent with the lack of apoptotic activity of 1,25(OH)₂D₃ in primary prostatic epithelial cells. Certain metallothioneins are reportedly overexpressed in prostate cancer ( 102), and the downregulation of metallothioneins in cancer cells by 1,25(OH)₂D₃, as we noted in this study, might be beneficial for therapy. Vitamin D is believed to participate in the antioxidant machinery ( 103), and our microarray results attest to this type of activity in prostate cells. Thioredoxin reductase 1, which is involved in maintaining redox balance, is an early-response gene in both normal and cancer-derived primary cultures. Glutathione-S-transferase-1-like-1 and superoxide dismutase 2, late-response genes induced by 1,25(OH)₂D₃ in normal and cancer-derived primary cultures, respectively, also are linked to protection from oxidative damage. These genes are unlikely to be directly involved in growth suppression but undoubtedly are key elements in mediating the chemopreventive activity of 1,25(OH)₂D₃ by preventing DNA damage caused by reactive oxygen species.

Our results also are noteworthy for the absence of regulation of certain genes by 1,25(OH)₂D₃. More than 50 genes are described as being responsive to vitamin D including at least 26 genes that contain promoters in which VDRE are identified ( 104, 105). Calbindin, osteocalcin and osteopontin are not regulated in primary cultures of prostatic cells. The human prostate cancer cell line PC-3 expresses osteocalcin, and an osteocalcin promoter–luciferase construct exhibits strong vitamin D–induced activity in these cells ( 106). The cell-cycle inhibitor p21 also is not induced by 1,25(OH)₂D₃ in our primary prostate cell strains. Reports regarding the role of p21 in mediating vitamin D signaling are variable. Although the promoter of p21 has a VDRE ( 107), p21 is not consistently regulated by vitamin D in different types of cells. The p27 gene, which is also implicated in growth inhibition by vitamin D, is not regulated in primary cultures of prostatic cells. However, vitamin D is believed to upregulate p27 by increasing the rate of mRNA translation and extending the half-life of the p27 protein rather than by altering the level of p27 mRNA ( 108).

In our microarray study using LNCaP cells, the expression of the IGFBP-3 gene shows the highest fold increase after 1,25(OH)₂D₃ treatment (33-fold induction at 24 h), yet this gene does not appear to be regulated by 1,25(OH)₂D₃ in our analysis of primary cultures. We previously implicated IGFBP-3 ( 26) as a key mediator of vitamin D activity in LNCaP cells (see Fig. 1). In contrast, we do not see regulation of IGFBP-3 by 1,25(OH)₂D₃ in primary cultures, and in fact, primary cultures do not express IGFBP-3. Clearly, IGFBP-3 is not mandatory for growth inhibition of all prostatic epithelial cells. IGFBP-3 may be linked to apoptosis induced by 1,25(OH)₂D₃ ( 109). In LNCaP cells, however, 1,25(OH)₂D₃ induces IGFBP-3, which in turn increases p21 expression and results in growth arrest ( 26). Vitamin D does not induce apoptosis in primary cultures, and the lack of regulation of IGFBP-3 as well as Bax or Bcl-2 are consistent with this response. In contrast, 1,25(OH)₂D₃ alters the ratio of expression of Bcl-2/Bax in LNCaP cells, which undergo apoptosis in response to vitamin D ( 37).

In conclusion, the microarray studies described reveal many biologically relevant molecular targets of 1,25(OH)₂D₃ in human prostatic epithelial cells and provide a starting point for additional investigations to more fully elucidate the mechanism of vitamin D action in prostate. The identification of genes primarily responsible for the anticancer effects of 1,25(OH)₂D₃ could lead to a tissue or serum marker that shows the status of vitamin D–mediated tumor-suppressor activity in the prostate. Such markers could be used to identify individuals who need supplemental vitamin D treatment for prostate cancer prevention or therapy. Less-calcemic analogs of 1,25(OH)₂D₃ with more-potent antiproliferative activity will be more useful clinically. Cotreatment with 24-hydroxylase inhibitors enhances the antiproliferative activity of 1,25(OH)₂D₃. The combination of other anticancer agents such as retinoids with vitamin D is another promising therapeutic approach. In the future, new therapeutic targets may become apparent when the mechanism for vitamin D–induced anticancer activity is more completely understood.

	FOOTNOTES

 
¹ Published in a supplement to The Journal of Nutrition. Presented at the "Nutritional Genomics and Proteomics in Cancer Prevention Conference" held September 5–6, 2002, in Bethesda, MD. This meeting was sponsored by the Center for Cancer Research, National Cancer Institute; Division of Cancer Prevention, National Cancer Institute; National Center for Complementary and Alternative Medicine, National Institutes of Health; Office of Dietary Supplements, National Institutes of Health; Office of Rare Diseases, National Institutes of Health; and the American Society for Nutritional Sciences. Guest editors for the supplement were Young S. Kim and John A. Milner, Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.

³ Abbreviations used: 1,25(OH)₂D₃, 1,25-dihydroxycholecalciferol; 25(OH)D₃, 25-hydroxycholecalciferol; BPH, benign prostatic hyperplasia; HPV, human papillomavirus; IGFBP-3, insulin-like growth factor binding protein-3; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PSA, prostate-specific antigen; RA, retinoic acid; RXR, retinoid X receptor; UV, ultraviolet; VDR, vitamin D receptor; VDRE, vitamin D response element.

	LITERATURE CITED

TOP ABSTRACT LITERATURE CITED

 

1. Holick, M. F. (1997) Photobiology of vitamin D. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 33–39. Academic Press, San Diego, CA.

2. Schwartz, G. G. & Hulka, B. S. (1990) Is vitamin D deficiency a risk factor for prostate cancer? (Hypothesis). Anticancer Res. 10: 1307–1311.[Medline]

3. Chapuy, M.-C. & Meunier, P. J. (1997) Vitamin D insufficiency in adults and the elderly. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 679–693. Academic Press, San Diego, CA.

4. Mitra, D. & Bell, N. H. (1997) Racial, geographic, genetic, and body habitus effects on vitamin D metabolism. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 521–532. Academic Press, San Diego, CA.

5. Gascon-Barre, M. (1997) The vitamin D 25-hydroxylase. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 41–55. Academic Press, San Diego, CA.

6. Henry, H. L. (1997) The 25-hydroxyvitamin D 1a-hydroxylase. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 57–68. Academic Press, San Diego, CA.

7. Halloran, B. P. & Portale, A. A. (1997) Vitamin D metabolism: the effects of aging. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 541–554. Academic Press, San Diego, CA.

8. Corder, E. H., Guess, H. A., Hulka, B. S., Friedman, G. D., Sadler, M., Vollmer, R. T., Lobaugh, B., Drezner, M. K., Vogelman, J. H. & Orentreich, N. (1993) Vitamin D and prostate cancer: a prediagnostic study with stored sera. Cancer Epidemiol. Biomarkers Prev. 2: 467–472.[Abstract]

9. Giovannucci, E., Rimm, E. B., Wolk, A., Ascherio, A., Stampfer, M. J., Colditz, G. A. & Willett, W. C. (1998) Calcium and fructose intake in relation to risk of prostate cancer. Cancer Res. 58: 442–447.[Abstract/Free Full Text]

10. Feldman, D., Malloy, P. J. & Gross, C. (2001) Vitamin D: biology, action and clinical implications. In: Osteoporosis (Marcus, R., Feldman, D. & Kelsey, J., eds.), vol. 1, pp. 257–303. Academic Press, San Diego, CA.

11. Haussler, M. R., Whitfield, G. K., Haussler, C. A., Hsieh, J. C., Thompson, P. D., Selznick, S. H., Dominguez, C. E. & Jurutka, P. W. (1998) The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J. Bone Miner. Res. 13: 325–349.[Medline]

12. Malloy, P. J., Pike, J. W. & Feldman, D. (1999) The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocr. Rev. 20: 156–188.[Abstract/Free Full Text]

13. Berger, U., Wilson, P., MCClelland, R. A., Colston, K., Haussler, M. R., Pike, J. W. & Coombes, R. C. (1988) Immunocytochemical detection of 1,25-dihydroxyvitamin D receptors in normal human tissues. J. Clin. Endocrinol. Metab. 67: 607–613.[Abstract/Free Full Text]

14. Gross, C., Peehl, D. M. & Feldman, D. (1997) Vitamin D and prostate cancer. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 1125–1139. Academic Press, San Diego, CA.

15. Feldman, D. (1997) Androgen and vitamin D receptor gene polymorphisms: the long and short of prostate cancer risk. J. Natl. Cancer Inst. 89: 109–111.[Free Full Text]

16. MCKenna, N. J. & O'Malley, B. W. (2002) Minireview: nuclear receptor coactivators—an update. Endocrinology 143: 2461–2465.[Abstract/Free Full Text]

17. Krishnan, A. & Feldman, D. (1997) Regulation of vitamin D receptor abundance. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 179–200. Academic Press, San Diego, CA.

18. Blutt, S. E. & Weigel, N. L. (1999) Vitamin D and prostate cancer. Proc. Soc. Exp. Biol. Med. 221: 89–98.[Medline]

19. Konety, B. R., Johnson, C. S., Trump, D. L. & Getzenberg, R. H. (1999) Vitamin D in the prevention and treatment of prostate cancer. Semin. Urol. Oncol. 17: 77–84.[Medline]

20. Miller, G. (1999) Vitamin D and prostate cancer: biologic interactions and clinical potentials. Cancer Metastasis Rev. 17: 353–360.

21. Feldman, D., Zhao, X. Y. & Krishnan, A. V. (2000) Vitamin D and prostate cancer. Endocrinology 141: 5–9.[Free Full Text]

22. Gross, C., Stamey, T., Hancock, S. & Feldman, D. (1998) Treatment of early recurrent prostate cancer with 1,25-dihydroxyvitamin D3 (calcitriol). J. Urol. 159: 2035–2040. [Published erratum: J. Urol. (1998) 160: 840.][Medline]

23. Miller, G. J., Stapleton, G. E., Ferrara, J. A., Lucia, M. S., Pfister, S., Hedlund, T. E. & Upadhya, P. (1992) The human prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1 alpha,25-dihydroxyvitamin D3. Cancer Res. 52: 515–520.[Abstract/Free Full Text]

24. Skowronski, R. J., Peehl, D. M. & Feldman, D. (1993) Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines. Endocrinology 132: 1952–1960.[Abstract/Free Full Text]

25. Skowronski, R. J., Peehl, D. M. & Feldman, D. (1995) Actions of vitamin D3, analogs on human prostate cancer cell lines: comparison with 1,25-dihydroxyvitamin D3. Endocrinology 136: 20–26.[Abstract]

26. Boyle, B. J., Zhao, X. Y., Cohen, P. & Feldman, D. (2001) Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1. J. Urol. 165: 1319–1324.[Medline]

27. Schwartz, G. G., Oeler, T. A., Uskokovic, M. R. & Bahnson, R. R. (1994) Human prostate cancer cells: inhibition of proliferation by vitamin D analogs. Anticancer Res. 14: 1077–1081.[Medline]

28. Zhuang, S. H., Schwartz, G. G., Cameron, D. & Burnstein, K. L. (1997) Vitamin D receptor content and transcriptional activity do not fully predict antiproliferative effects of vitamin D in human prostate cancer cell lines. Mol. Cell. Endocrinol. 126: 83–90.[Medline]

29. Miller, G. J., Stapelton, G. E., Hedlund, T. E. & Moffatt, K. A. (1995) Vitamin D receptor expression, 24-hydroxylase activity, and inhibition of growth by 1a,25-dihydroxyvitamin D₃ in seven human prostatic carcinoma cell lines. Clin. Cancer Res. 1: 997–1003.[Abstract]

30. Wang, X., Chen, X., Akhter, J. & Morris, D. L. (1997) The in vitro effect of vitamin D3 analogue EB-1089 on a human prostate cancer cell line (PC-3). Br. J. Urol. 80: 260–262.[Medline]

31. de Vos, S., Holden, S., Heber, D., Elstner, E., Binderup, L., Uskokovic, M., Rude, B., Chen, D. L., Le, J., Cho, S. K. & Koeffler, H. P. (1997) Effects of potent vitamin D3 analogs on clonal proliferation of human prostate cancer cell lines. Prostate 31: 77–83.[Medline]

32. Hedlund, T. E., Moffatt, K. A., Uskokovic, M. R. & Miller, G. J. (1997) 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. Clin. Cancer Res. 3: 1331–1338.[Abstract]

33. Kubota, T., Koshizuka, K., Koike, M., Uskokovic, M., Miyoshi, I. & Koeffler, H. P. (1998) 19-Nor-26,27-bishomo-vitamin D3 analogs: a unique class of potent inhibitors of proliferation of prostate, breast, and hematopoietic cancer cells. Cancer Res. 58: 3370–3375.[Abstract/Free Full Text]

34. Koike, M., Koshizuka, K., Kawabata, H., Yang, R., Taub, H. E., Said, J., Uskokovic, M., Tsuruoka, N. & Koeffler, H. P. (1999) 20-Cyclopropyl-cholecalciferol vitamin D3 analogs: a unique class of potent inhibitors of proliferation of human prostate, breast and myeloid leukemia cell lines. Anticancer Res. 19: 1689–1697.[Medline]

35. Hisatake, J., Kubota, T., Hisatake, Y., Uskokovic, M., Tomoyasu, S. & Koeffler, H. P. (1999) 5,6-Trans-16-ene-vitamin D3: a new class of potent inhibitors of proliferation of prostate, breast, and myeloid leukemic cells. Cancer Res. 59: 4023–4029.[Abstract/Free Full Text]

36. Chen, T. C., Schwartz, G. G., Burnstein, K. L., Lokeshwar, B. L. & Holick, M. F. (2000) The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as therapeutic agents for prostate cancer. Clin. Cancer Res. 6: 901–908.[Abstract/Free Full Text]

37. Blutt, S. E., MCDonnell, T. J., Polek, T. C. & Weigel, N. L. (2000) Calcitriol-induced apoptosis in LNCaP cells is blocked by overexpression of Bcl-2. Endocrinology 141: 10–17.[Abstract/Free Full Text]

38. Schwartz, G. G., Hill, C. C., Oeler, T. A., Becich, M. J. & Bahnson, R. R. (1995) 1,25-Dihydroxy-16-ene-23-yne-vitamin D3 and prostate cancer cell proliferation in vivo. Urology 46: 365–369.[Medline]

39. Blutt, S. E., Polek, T. C., Stewart, L. V., Kattan, M. W. & Weigel, N. L. (2000) A calcitriol analogue, EB1089, inhibits the growth of LNCaP tumors in nude mice. Cancer Res. 60: 779–782.[Abstract/Free Full Text]

40. Polek, T. C., Murthy, S., Blutt, S. E., Boehm, M. F., Zou, A., Weigel, N. L. & Allegretto, E. A. (2001) Novel nonsecosteroidal vitamin D receptor modulator inhibits the growth of LNCaP xenograft tumors in athymic mice without increased serum calcium. Prostate 49: 224–233.[Medline]

41. Schwartz, G. G., Wang, M. H., Zang, M., Singh, R. K. & Siegal, G. P. (1997) 1 alpha,25-Dihydroxyvitamin D (calcitriol) inhibits the invasiveness of human prostate cancer cells. Cancer Epidemiol. Biomarkers Prev. 6: 727–732.[Abstract/Free Full Text]

42. Sung, V. & Feldman, D. (2000) 1,25-Dihydroxyvitamin D3 decreases human prostate cancer cell adhesion and migration. Mol. Cell. Endocrinol. 164: 133–143.[Medline]

43. Lokeshwar, B. L., Schwartz, G. G., Selzer, M. G., Burnstein, K. L., Zhuang, S. H., Block, N. L. & Binderup, L. (1999) Inhibition of prostate cancer metastasis in vivo: a comparison of 1,23-dihydroxyvitamin D (calcitriol) and EB1089. Cancer Epidemiol. Biomarkers Prev. 8: 241–248.[Abstract/Free Full Text]

44. Peehl, D. M., Skowronski, R. J., Leung, G. K., Wong, S. T., Stamey, T. A. & Feldman, D. (1994) Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells. Cancer Res. 54: 805–810.[Abstract/Free Full Text]

45. Esquenet, M., Swinnen, J. V., Heyns, W. & Verhoeven, G. (1996) Control of LNCaP proliferation and differentiation: actions and interactions of androgens, 1alpha,25-dihydroxycholecalciferol, all-trans retinoic acid, 9-cis retinoic acid, and phenylacetate. Prostate 28: 182–194.[Medline]

46. Hsieh, T. Y., Ng, C. Y., Mallouh, C., Tazaki, H. & Wu, J. M. (1996) Regulation of growth, PSA/PAP and androgen receptor expression by 1 alpha,25-dihydroxyvitamin D3 in the androgen-dependent LNCaP cells. Biochem. Biophys. Res. Commun. 223: 141–146.[Medline]

47. Hsieh, T. & Wu, J. M. (1997) Induction of apoptosis and altered nuclear/cytoplasmic distribution of the androgen receptor and prostate-specific antigen by 1alpha,25-dihydroxyvitamin D3 in androgen-responsive LNCaP cells. Biochem. Biophys. Res. Commun. 235: 539–544.[Medline]

48. Zhao, X. Y., Ly, L. H., Peehl, D. M. & Feldman, D. (1999) Induction of androgen receptor by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells. Endocrinology 140: 1205–1212.[Abstract/Free Full Text]

49. Navone, N. M., Olive, M., Ozen, M., Davis, R., Troncoso, P., Tu, S. M., Johnston, D., Pollack, A., Pathak, S., von Eschenbach, A. C. & Logothetis, C. J. (1997) Establishment of two human prostate cancer cell lines derived from a single bone metastasis. Clin. Cancer Res. 3: 2493–2500.[Abstract/Free Full Text]

50. Zhao, X. Y., Boyle, B., Krishnan, A. V., Navone, N. M., Peehl, D. M. & Feldman, D. (1999) Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a. J. Urol. 162: 2192–2199.[Medline]

51. Zhao, X. Y., Malloy, P. J., Krishnan, A. V., Swami, S., Navone, N. M., Peehl, D. M. & Feldman, D. (2000) Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor. Nat. Med. 6: 703–706.[Medline]

52. Zhao, X. Y., Peehl, D. M., Navone, N. M. & Feldman, D. (2000) 1alpha,25-Dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms. Endocrinology 141: 2548–2556.[Abstract/Free Full Text]

53. Ahonen, M. H., Zhuang, Y. H., Aine, R., Ylikomi, T. & Tuohimaa, P. (2000) Androgen receptor and vitamin D receptor in human ovarian cancer: growth stimulation and inhibition by ligands. Int. J. Cancer 86: 40–46.[Medline]

54. Escaleira, M. T., Sonohara, S. & Brentani, M. M. (1993) Sex steroids induced up-regulation of 1,25-(OH)2 vitamin D3 receptors in T 47D breast cancer cells. J. Steroid Biochem. Mol. Biol. 45: 257–263.[Medline]

55. Osborn, J. L., Schwartz, G. G., Smith, D. C., Bahnson, R., Day, R. & Trump, D. L. (1995) Phase II trial of oral 1,25- dihydroxyvitamin D (calcitriol) in hormone refractory prostate cancer. Urol. Oncol. 1: 195–198.

56. Peehl, D. M., Seto, E. & Feldman, D. (2001) Rationale for combination ketoconazole/vitamin D treatment of prostate cancer. Urology 58: 123–126.[Medline]

57. Feldman, D. (1986) Ketoconazole and other imidazole derivatives as inhibitors of steroidogenesis. Endocr. Rev. 7: 409–420.[Abstract/Free Full Text]

58. Van Veldhuizen, P. J., Taylor, S. A., Williamson, S. & Drees, B. M. (2000) Treatment of vitamin D deficiency in patients with metastatic prostate cancer may improve bone pain and muscle strength. J. Urol. 163: 187–190.[Medline]

59. Peehl, D. M., Seto, E., Hsu, J. Y. & Feldman, D. (2002) Preclinical activity of ketoconazole in combination with calcitriol or the vitamin D analog EB 1089 in prostate cancer cells. J. Urol. 168: 1583–1588.[Medline]

60. Bouillon, R., Okamura, W. H. & Norman, A. W. (1995) Structure-function relationships in the vitamin D endocrine system. Endocr. Rev. 16: 200–257.[Abstract/Free Full Text]

61. Zhao, X. Y., Eccleshall, T. R., Krishnan, A. V., Gross, C. & Feldman, D. (1997) Analysis of vitamin D analog-induced heterodimerization of vitamin D receptor with retinoid X receptor using the yeast two-hybrid system. Mol. Endocrinol. 11: 366–378.[Abstract/Free Full Text]

62. Moffatt, K. A., Johannes, W. U. & Miller, G. J. (1999) 1alpha,25Dihydroxyvitamin D3 and platinum drugs act synergistically to inhibit the growth of prostate cancer cell lines. Clin. Cancer Res. 5: 695–703.[Abstract/Free Full Text]

63. Hershberger, P. A., Yu, W. D., Modzelewski, R. A., Rueger, R. M., Johnson, C. S. & Trump, D. L. (2001) Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis. Clin. Cancer Res. 7: 1043–1051.[Abstract/Free Full Text]

64. Peehl, D. M., Wong, S. T., Cramer, S. D., Gross, C. & Feldman, D. (1995) Suramin, hydrocortisone and retinoic acid modify inhibitory effects of 1,25-dihydroxyvitamin D₃ on prostatic epithelial cells. Urol. Oncol. 1: 188–194.

65. Kliewer, S. A., Umesono, K., Mangelsdorf, D. J. & Evans, R. M. (1992) Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355: 446–449.[Medline]

66. Blutt, S. E., Allegretto, E. A., Pike, J. W. & Weigel, N. L. (1997) 1,25-Dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cells in G1. Endocrinology 138: 1491–1497.[Abstract/Free Full Text]

67. Campbell, M. J., Park, S., Uskokovic, M. R., Dawson, M. I. & Koeffler, H. P. (1998) Expression of retinoic acid receptor-beta sensitizes prostate cancer cells to growth inhibition mediated by combinations of retinoids and a 19-nor hexafluoride vitamin D3 analog. Endocrinology 139: 1972–1980.[Abstract/Free Full Text]

68. Campbell, M. J., Park, S., Uskokovic, M. R., Dawson, M. I., Jong, L. & Koeffler, H. P. (1999) Synergistic inhibition of prostate cancer cell lines by a 19-nor hexafluoride vitamin D3 analogue and anti-activator protein 1 retinoid. Br. J. Cancer 79: 101–107.[Medline]

69. Elstner, E., Campbell, M. J., Munker, R., Shintaku, P., Binderup, L., Heber, D., Said, J. & Koeffler, H. P. (1999) Novel 20-epi-vitamin D3 analog combined with 9-cis-retinoic acid markedly inhibits colony growth of prostate cancer cells. Prostate 40: 141–149.[Medline]

70. Yu, W. D., MCElwain, M. C., Modzelewski, R. A., Russell, D. M., Smith, D. C., Trump, D. L. & Johnson, C. S. (1998) Enhancement of 1,25-dihydroxyvitamin D3-mediated antitumor activity with dexamethasone. J. Natl. Cancer Inst. 90: 134–141.[Abstract/Free Full Text]

71. Hirst, M. & Feldman, D. (1982) Glucocorticoid regulation of 1,25(OH)₂vitamin D₃ receptors: divergent effects on mouse and rat intestine. Endocrinology 111: 1400–1402.[Abstract/Free Full Text]

72. Raisz, L. G. & Lukert, B. P. (1997) Glucocorticoid and vitamin D interactions. In: Vitamin D (Feldman, D., Glorieux, F. H. & Pike, J. W., eds.), pp. 789–796. Academic Press, San Diego, CA.

73. Lieberman, R., Bermejo, C., Akaza, H., Greenwald, P., Fair, W. & Thompson, I. (2001) Progress in prostate cancer chemoprevention: modulators of promotion and progression. Urology 58: 835–842.[Medline]

74. Konety, B. R., Schwartz, G. G., Acierno, J. S., JR., Becich, M. J. & Getzenberg, R. H. (1996) The role of vitamin D in normal prostate growth and differentiation. Cell Growth Differ. 7: 1563–1570.[Abstract]

75. Lucia, M. S., Anzano, M. A., Slayter, M. V., Anver, M. R., Green, D. M., Shrader, M. W., Logsdon, D. L., Driver, C. L., Brown, C. C. & Peer, C. W. (1995) Chemopreventive activity of tamoxifen, N-(4-hydroxyphenyl)retinamide, and the vitamin D analogue Ro24-5531 for androgen-promoted carcinomas of the rat seminal vesicle and prostate. Cancer Res. 55: 5621–5627.[Abstract/Free Full Text]

76. Siwinska, A., Opolski, A., Chrobak, A., Wietrzyk, J., Wojdat, E., Kutner, A., Szelejewski, W. & Radzikowski, C. (2001) Potentiation of the antiproliferative effect in vitro of doxorubicin, cisplatin and genistein by new analogues of vitamin D. Anticancer Res. 21: 1925–1929.[Medline]

77. Rao, A., Woodruff, R. D., Wade, W. N., Kute, T. E. & Cramer, S. D. (2002) Genistein and vitamin D synergistically inhibit human prostate epithelial cell growth. J. Nutr. 132: 3191–3194.[Abstract/Free Full Text]

78. Farhan, H. & Cross, H. S. (2002) Effects of estrogenic and antiestrogenic substances on vitamin D metabolism in prostate cancer cells. Proceedings of the American Association for Cancer Research 93rd Annual Meeting, San Francisco, CA.

79. Lechner, D. & Cross, H. S. (2002) Phytoestrogens influence vitamin D metabolism—relevance for colon cancer prevention. Proceedings of the First International Symposium on Vitamin D Analogs in Cancer Prevention Therapy, Hamburg, Germany.

80. van Bokhoven, A., Varella-Garcia, M., Korch, C., Hessels, D. & Miller, G. J. (2001) Widely used prostate carcinoma cell lines share common origins. Prostate 47: 36–51.[Medline]

81. Hedlund, T. E., Moffatt, K. A. & Miller, G. J. (1996) Stable expression of the nuclear vitamin D receptor in the human prostatic carcinoma cell line JCA-1: evidence that the antiproliferative effects of 1 alpha, 25-dihydroxyvitamin D3 are mediated exclusively through the genomic signaling pathway. Endocrinology 137: 1554–1561.[Abstract]

82. Ly, L. H., Zhao, X. Y., Holloway, L. & Feldman, D. (1999) Liarozole acts synergistically with 1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activity. Endocrinology 140: 2071–2076.[Abstract/Free Full Text]

83. Loose, D. S., Kan, P. B., Hirst, M. A., Marcus, R. A. & Feldman, D. (1983) Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes. J. Clin. Invest. 71: 1495–1499.

84. Campbell, M. J., Reddy, G. S. & Koeffler, H. P. (1997) Vitamin D3 analogs and their 24-oxo metabolites equally inhibit clonal proliferation of a variety of cancer cells but have differing molecular effects. J. Cell. Biochem. 66: 413–425.[Medline]

85. Peehl, D. M., Wong, S. T. & Rhim, J. S. (1996) Transformation-altered growth factor responses of human prostatic epithelial cells. Rad. Oncol. Investig. 3: 330–332.

86. Gross, C., Skowronski, R. J., Plymate, S. R., Rhim, J. S., Peehl, D. M. & Feldman, D. (1996) Simian virus 40-, but not human papillomavirus-, transformation of prostatic epithelial cells results in loss of growth-inhibition by 1,25-dihydroxyvitamin D₃. Int. J. Oncol. 8: 41–47.

87. Agadir, A., Lazzaro, G., Zheng, Y., Zhang, X. K. & Mehta, R. (1999) Resistance of HBL100 human breast epithelial cells to vitamin D action. Carcinogenesis 20: 577–582.[Abstract/Free Full Text]

88. Schwartz, G. G., Whitlatch, L. W., Chen, T. C., Lokeshwar, B. L. & Holick, M. F. (1998) Human prostate cells synthesize 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Cancer Epidemiol. Biomarkers Prev. 7: 391–395.[Abstract/Free Full Text]

89. Hsu, J. Y., Feldman, D., MCNeal, J. E. & Peehl, D. M. (2001) Reduced 1alpha-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25-hydroxyvitamin D3-induced growth inhibition. Cancer Res. 61: 2852–2856.[Abstract/Free Full Text]

90. Morrison, N. A., Qi, J. C., Tokita, A., Kelly, P. J., Crofts, L., Nguyen, T. V., Sambrook, P. N. & Eisman, J. A. (1994) Prediction of bone density from vitamin D receptor alleles. Nature 367: 284–287.[Medline]

91. Ingles, S. A., Coetzee, G. A., Ross, R. K., Henderson, B. E., Kolonel, L. N., Crocitto, L., Wang, W. & Haile, R. W. (1998) Association of prostate cancer with vitamin D receptor haplotypes in African-Americans. Cancer Res. 58: 1620–1623.[Abstract/Free Full Text]

92. Gross, C., Eccleshall, T. R., Malloy, P. J., Villa, M. L., Marcus, R. & Feldman, D. (1996) The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women. J. Bone Miner. Res. 11: 1850–1855.[Medline]

93. Xu, Y., Shibata, A., MCNeal, J. E., Stamey, T. A., Feldman, D. & Peehl, D. M. (2003) Vitamin D receptor start codon polymorphism (Fok1) and prostate cancer progression. Cancer Epidemiol. Biomarkers Prev. 12: 23–27.[Abstract/Free Full Text]

94. Stamey, T. A., MCNeal, J. E., Yemoto, C. M., Sigal, B. M. & Johnstone, I. M. (1999) Biological determinants of cancer progression in men with prostate cancer. J. Am. Med. Assoc. 281: 1395–1400.[Abstract/Free Full Text]

95. Arai, H., Miyamoto, K., Taketani, Y., Yamamoto, H., Iemori, Y., Morita, K., Tonai, T., Nishisho, T., Mori, S. & Takeda, E. (1997) A vitamin D receptor gene polymorphism in the translation initiation codon: effect on protein activity and relation to bone mineral density in Japanese women. J. Bone Miner. Res. 12: 915–921.[Medline]

96. Gross, C., Krishnan, A. V., Malloy, P. J., Eccleshall, T. R., Zhao, X. Y. & Feldman, D. (1998) The vitamin D receptor gene start codon polymorphism: a functional analysis of FokI variants. J. Bone Miner. Res. 13: 1691–1699.[Medline]

97. Jurutka, P. W., Remus, L. S., Whitfield, G. K., Thompson, P. D., Hsieh, J. C., Zitzer, H., Tavakkoli, P., Galligan, M. A., Dang, H. T., Haussler, C. A. & Haussler, M. R. (2000) The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB. Mol. Endocrinol. 14: 401–420.[Abstract/Free Full Text]

98. Whitfield, G. K., Remus, L. S., Jurutka, P. W., Zitzer, H., Oza, A. K., Dang, H. T., Haussler, C. A., Galligan, M. A., Thatcher, M. L., Encinas Dominguez, C. & Haussler, M. R. (2001) Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. Mol. Cell. Endocrinol. 177: 145–159.[Medline]

99. Akutsu, N., Lin, R., Bastien, Y., Bestawros, A., Enepekides, D. J., Black, M. J. & White, J. H. (2001) Regulation of gene expression by 1alpha,25-dihydroxyvitamin D3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma cells. Mol. Endocrinol. 15: 1127–1139.[Abstract/Free Full Text]

100. Schulze, A., Lehmann, K., Jefferies, H. B., MCMahon, M. & Downward, J. (2001) Analysis of the transcriptional program induced by Raf in epithelial cells. Genes Dev. 15: 981–994.[Abstract/Free Full Text]

101. Schwartz, Z., Sylvia, V. L., Larsson, D., Nemere, I., Casasola, D., Dean, D. D. & Boyan, B. D. (2002) 1alpha,25(OH)₂D₃ regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis. J. Biol. Chem. 277: 11828–11837.[Abstract/Free Full Text]

102. Zhang, X. H., Jin, L., Sakamoto, H. & Takenaka, I. (1996) Immunohistochemical localization of metallothionein in human prostate cancer. J. Urol. 156: 1679–1681.[Medline]

103. Garcion, E., Sindji, L., Leblondel, G., Brachet, P. & Darcy, F. (1999) 1,25-Dihydroxyvitamin D3 regulates the synthesis of gamma-glutamyl transpeptidase and glutathione levels in rat primary astrocytes. J. Neurochem. 73: 859–866.[Medline]

104. Hannah, S. S. & Norman, A. W. (1994) 1 alpha,25(OH)₂ vitamin D₃-regulated expression of the eukaryotic genome. Nutr. Rev. 52: 376–382.[Medline]

105. Carlberg, C. & Polly, P. (1998) Gene regulation by vitamin D3. Crit. Rev. Eukaryot. Gene Expr. 8: 19–42.[Medline]

106. Yeung, F., Law, W. K., Yeh, C. H., Westendorf, J. J., Zhang, Y., Wang, R., Kao, C. & Chung, L. W. (2002) Regulation of human osteocalcin promoter in hormone-independent human prostate cancer cells. J. Biol. Chem. 277: 2468–2476.[Abstract/Free Full Text]

107. Liu, M., Lee, M. H., Cohen, M., Bommakanti, M. & Freedman, L. P. (1996) Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes Dev. 10: 142–153.[Abstract/Free Full Text]

108. Wang, Q. M., Jones, J. B. & Studzinski, G. P. (1996) Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells. Cancer Res. 56: 264–267.[Abstract/Free Full Text]

109. Nickerson, T. & Huynh, H. (1999) Vitamin D analogue EB1089-induced prostate regression is associated with increased gene expression of insulin-like growth factor binding proteins. J. Endocrinol. 160: 223–229.[Abstract]

This article has been cited by other articles:

				M. THILL, D. FISCHER, S. BECKER, T. CORDES, C. DITTMER, K. DIEDRICH, D. SALEHIN, and M. FRIEDRICH Prostaglandin Metabolizing Enzymes in Correlation with Vitamin D Receptor in Benign and Malignant Breast Cell Lines Anticancer Res, September 1, 2009; 29(9): 3619 - 3625. [Abstract] [Full Text] [PDF]

				G. E. Mullin and A. Dobs Vitamin D and Its Role in Cancer and Immunity: A Prescription for Sunlight Nutr Clin Pract, June 1, 2007; 22(3): 305 - 322. [Abstract] [Full Text] [PDF]

				S. Swami, A. V. Krishnan, J. Moreno, R. B. Bhattacharyya, D. M. Peehl, and D. Feldman Calcitriol and Genistein Actions to Inhibit the Prostaglandin Pathway: Potential Combination Therapy to Treat Prostate Cancer J. Nutr., January 1, 2007; 137(1): 205S - 210S. [Abstract] [Full Text] [PDF]

				S. S. Harris Vitamin D and African Americans J. Nutr., April 1, 2006; 136(4): 1126 - 1129. [Abstract] [Full Text] [PDF]

				D. L. Bemis, J. L. Capodice, M. Desai, R. Buttyan, and A. E. Katz A Concentrated Aglycone Isoflavone Preparation (GCP) That Demonstrates Potent Anti-Prostate Cancer Activity In vitro and In vivo Clin. Cancer Res., August 1, 2004; 10(15): 5282 - 5292. [Abstract] [Full Text] [PDF]

				H. S. Cross, E. Kallay, D. Lechner, W. Gerdenitsch, H. Adlercreutz, and H. J. Armbrecht Phytoestrogens and Vitamin D Metabolism: A New Concept for the Prevention and Therapy of Colorectal, Prostate, and Mammary Carcinomas J. Nutr., May 1, 2004; 134(5): 1207S - 1212S. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peehl, D. M. Articles by Feldman, D. Search for Related Content PubMed PubMed Citation Articles by Peehl, D. M. Articles by Feldman, D.