Molecular activity of 1,25-dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] exerts anti-proliferative, differentiating and apoptotic effects on prostatic cells. These activities, in addition to epidemiologic findings that link Vitamin D to prostate cancer risk, support the use of 1,25(OH)(2)D(3) for prevention or therapy of prostate cancer. The molecular mechanisms by which 1,25(OH)(2)D(3) exerts antitumor effects on prostatic cells are not well-defined. In addition, there is heterogeneity among the responses of various prostate cell lines and primary cultures to 1,25(OH)(2)D(3) with regard to growth inhibition, differentiation and apoptosis. To understand the basis of these differential responses and to develop a better model of Vitamin D action in the prostate, we performed cDNA microarray analyses of primary cultures of normal and malignant human prostatic epithelial cells, treated with 50 nM of 1,25(OH)(2)D(3) for 6 and 24 h. CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) was the most highly upregulated gene. Significant and early upregulation of dual specificity phosphatase 10 (DUSP10), validated in five additional primary cultures, points to inhibition of members of the mitogen-activated protein kinase (MAPK) superfamily as a key event mediating activity of 1,25(OH)(2)D(3) in prostatic epithelial cells. The functions of other regulated genes suggest protection by 1,25(OH)(2)D(3) from oxidative stress. Overall, these results provide new insights into the molecular basis of antitumor activities of Vitamin D in prostate cells.     

1alpha,25-Dihydroxyvitamin D3-3beta-(2)-bromoacetate,an affinity labeling derivative of 1alpha,25-dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells

In this report we describe that 1,25(OH)(2)D(3)-3-BE, a VDR-affinity labeling analog of 1,25(OH)(2)D(3), showed strong and dose-dependent growth-inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF-7 breast cancer, PC-3, and LNCaP prostate cancer and PZ-HPV-7 immortalized normal prostate cell-lines. Furthermore, 10(-6) M of 1,25(OH)(2)D(3)-3-BE induced apoptosis specifically in LNCaP and PC-3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)(2)D(3)-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)(2)D(3)-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)(2)D(3) and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)(2)D(3) and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)(2)D(3)-3-BE displayed similar growth-inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen-independent PC-3 cell-lines. Therefore, these results raise the possibility that 1,25(OH)(2)D(3)-3-BE or similar VDR-cross linking analogs of 1,25(OH)(2)D(3) might be considered for further development as potential candidates for prostate cancer.     

Vitamin D and multiple sclerosis

Vitamin D is a principal regulator of calcium homeostasis. However, recent evidence has indicated that vitamin D can have numerous other physiological functions including inhibition of proliferation of a number of malignant cells including breast and prostate cancer cells and protection against certain immune mediated disorders including multiple sclerosis (MS). The geographic incidence of MS indicates an increase in MS with a decrease in sunlight exposure. Since vitamin D is produced in the skin by solar or UV irradiation and high serum levels of 25-hydroxyvitamin D (25(OH)D) have been reported to correlate with a reduced risk of MS, a protective role of vitamin D is suggested. Mechanisms whereby the active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) may act to mediate this protective effect are reviewed. Due to its immunosuppressive actions, it has been suggested that 1,25(OH)(2)D(3) may prevent the induction of MS.     

PTHrP contributes to the anti-proliferative and integrin alpha6beta4-regulating effects of 1,25-dihydroxyvitamin D(3)

Parathyroid hormone-related protein (PTHrP) increases the growth and metastatic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] suppresses PTHrP expression and exerts an anti-proliferative effect in prostate carcinoma cells. We used the human prostate cancer cell line C4-2 as a model system to ask whether down-regulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the anti-proliferative effects of 1,25(OH)(2)D(3). Since PTHrP increases the expression of the pro-invasive integrin alpha6beta4, we also asked whether 1,25(OH)(2)D(3) decreases integrin alpha6beta4 expression in C4-2 cells, and whether modulation of PTHrP expression by 1,25(OH)(2)D(3) plays a role in the effects of 1,25(OH)(2)D(3) on integrin alpha6beta4 expression. Two strategies were utilized to modulate PTHrP levels: overexpression of PTHrP (-36 to +139) and suppression of endogenous PTHrP expression using siRNAs. We report a direct correlation between PTHrP expression, C4-2 cell proliferation and integrin alpha6beta4 expression at the mRNA and cell surface protein level. Treatment of parental C4-2 cells with 1,25(OH)(2)D(3) decreased cell proliferation and integrin alpha6 and beta4 expression. These 1,25(OH)(2)D(3) effects were significantly attenuated in cells with suppressed PTHrP expression. 1,25(OH)(2)D(3) regulates PTHrP expression via a negative vitamin D response element (nVDRE) within the noncoding region of the PTHrP gene. The effects of 1,25(OH)(2)D(3) on cell proliferation and integrin alpha6beta4 expression were significantly attenuated in cells overexpressing PTHrP (-36 to +139), which lacks the nVDRE. These findings suggest that one of the pathways via which 1,25(OH)(2)D(3) exerts its anti-proliferative effects is through down-regulation of PTHrP expression.     

Molecular mechanisms mediating the anti-proliferative effects of Vitamin D in prostate cancer

Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth and stimulates the differentiation of prostate cancer (PCa) cells. The effects of calcitriol are varied, appear to be cell-specific and result in growth arrest and stimulation of apoptosis. Our goal was to define the genes involved in the multiple pathways mediating the anti-proliferative effects of calcitriol in PCa. We used cDNA microarray analysis to identify calcitriol target genes involved in these pathways in both LNCaP human PCa cells and primary prostatic epithelial cells. Interestingly, two of the target genes that we identified play key roles in the metabolism of prostaglandins (PGs), which are known stimulators of PCa cell growth and progression. The expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene was significantly decreased by calcitriol, while that of PG inactivating 15-prostaglandin dehydrogenase gene (15-PGDH) was increased. We postulate that this dual action of calcitriol would reduce the levels of biologically active PGs in PCa cells decreasing their proliferative stimulus and contribute to the growth inhibitory actions of calcitriol. In addition, we propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs that inhibit COX activity, as a potential therapeutic strategy to improve the potency and efficacy of both drugs in the treatment of PCa.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

1,25-Dihydroxyvitamin D3 inhibits tumor necrosis factor-alpha-induced adhesion molecule expression in endothelial cells

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.     

Activation of rapid signaling pathways does not contribute to 1α,25‐dihydroxyvitamin D3‐induced growth inhibition of mouse prostate epithelial progenitor cells

The active form of vitamin D, 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D) inhibits the growth of prostate epithelial cells, however the underlying mechanisms have not been clearly delineated. In the current study, the impact of 1,25(OH)₂D on the rapid activation of extracellular‐regulated kinase (ERK) 1/2 and protein kinase C α (PKCα), and the role of these pathways in growth inhibition was examined in immortalized mouse prostate epithelial cells, MPEC3, that exhibit stem/progenitor cell characteristics. 1,25(OH)₂D treatment suppressed the growth of MPEC3 in a dose and time dependent manner (e.g., 21% reduction at three days with 100 nM 1,25(OH)₂D treatment). However, ERK1/2 activity was not altered by 100 nM 1,25(OH)₂D treatment for time points from 1 min to 1 h in either serum‐containing or serum‐free medium. Similarly, PKCα activation (translocation onto the plasma membrane) was not regulated by short‐term treatment of 100 nM 1,25(OH)₂D. In conclusion, 1,25(OH)₂D did not mediate rapid activation of ERK1/2 or PKCα in MPEC3 and therefore the growth inhibitory effect of 1,25(OH)₂D is independent of rapid activation of these signaling pathways in this cell type. J. Cell. Biochem. 107: 1031–1036, 2009. © 2009 Wiley‐Liss, Inc. This article was published online 2 June 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 15 June 2009.     

Vitamin D regulated keratinocyte differentiation

The epidermis is the largest organ in the body. It is comprised primarily of keratinocytes which are arranged in layers that recapitulates their programmed life cycle. Proliferating keratinocytes are on the bottom-the stratum basale. As keratinocytes leave the stratum basale they begin to differentiate, culminating in the enucleated stratum corneum which has the major role of permeability barrier. Calcium and the active metabolite of vitamin D, 1,25(OH)(2)D(3), play important roles in this differentiation process. The epidermis has a gradient of calcium with lowest concentrations in the stratum basale, and highest concentrations in the stratum granulosum where proteins critical for barrier function are produced. Vitamin D is made in different layers of the epidermis, but 1,25(OH)(2)D(3) is made primarily in the stratum basale. Together calcium and 1,25(OH)(2)D(3) regulate the ordered differentiation process by the sequential turning on and off the genes producing the elements required for differentiation as well as activating those enzymes involved in differentiation. Animal models in which the sensing mechanism for calcium, the receptor for 1,25(OH)(2)D(3), or the enzyme producing 1,25(OH)(2)D(3) have been rendered inoperative demonstrate the importance of these mechanisms for the differentiation process, although each animal model has its own phenotype. This review will examine the mechanisms by which calcium and 1,25(OH)(2)D(3) interact to control epidermal differentiation.     

Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.     

Vitamin D compounds in leukemia

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3,] possess in vitro multiple anti-cancer activities including growth arrest, induction of apoptosis and differentiation of a variety of different types of malignant cells. However, its use as a therapeutic agent is hindered by its calcemic effects. Analogs of 1,25(OH)(2)D3 have enhanced anti-tumor activity, with reduced calcemic effects. However, limited clinical studies using vitamin D compounds have not yet achieved major clinical success. Nevertheless, pre-clinical studies suggest that the combination of either 1,25(OH)(2)D3 or its analogs with other agents can have additive or synergistic anti-cancer activities, suggesting future clinical studies.     

Nucleo-cytoplasmic cycling of the vitamin D receptor in the enterocyte-like cell line, Caco-2

We examined the effects of 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on the distribution and mobility of the vitamin D receptor (VDR) in the enterocyte-like Caco-2 cell. Confocal microscopy showed that a green fluorescent protein-vitamin D receptor (GFP-VDR) fusion protein is predominantly nuclear (58%) and it does not associate with the apical or basolateral membrane of proliferating or polarized, differentiated cells. In contrast to the previously studied cell types, neither endogenous VDR nor GFP-VDR levels accumulate in the nucleus following 1,25(OH)(2)D(3) treatment (100 nM, 30 min). However, in nuclear photobleaching experiments nuclear GFP-VDR import was significantly increased by 1,25(OH)(2)D(3) during both an early (0-5 min) and later (30-35 min) period (20% per 5 min). Compared to the natural ligand, nuclear import of GFP-VDR was 60% lower in cells treated with the 1,25(OH)(2)D(3) analog, 1-alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228, 5 min, 100 nM). Downstream events like ligand-induced association of VDR with chromatin at 1 h and the accumulation of CYP24 mRNA were significantly lower in Ro-26-9228 treated cells compared to 1,25(OH)(2)D(3) (60 and 95% lower, respectively). Collectively our data are consistent with a role for ligand-induced nuclear VDR import in receptor activation. In addition, ligand-dependent VDR nuclear import appears to be balanced by export, thus accounting for the lack of nuclear VDR accumulation even when VDR import is significantly elevated.