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Abstract: We found previously that long-chain fatty-acid-CoA ligase 3 (FACL3), a critical enzyme for activation of long-chain fatty acids, was upregulated by 1α, 25(OH)2D3 at an mRNA and enzyme activity levels in prostate cancer cells. Our further study indicated that the FACL3 mediated 1α,25(OH)2D3 inhibition of fatty acid synthase (FAS), which is associated with many cancers, including prostate cancer. In the current study, we investigated an FACL3 protein expression and its regulation by 1α, 25(OH)2D3 and its synthetic analogs EB1089 and CB1093 in prostate cancer cells. The results showed that the expression of an FACL3 protein was upregulated by 1α, 25(OH)2D3, EB1089 and CB1093 in LNCaP cells, consistent with their upregulation of an FACL3 mRNA expression. In addition, the FACL3 expression was found to be markedly low at both mRNA and protein levels in more transformed prostate cancer PC-3 and DU145 cells compared with less transformed LNCaP cells. The data suggest that decreased FACL3 expression might be associated with a more malignant phenotype of prostate cancer. [ABSTRACT FROM AUTHOR]

Vitamin D receptor (VDR) and liver X receptor (LXR) are nuclear receptors, which regulate gene transcription upon binding of their specific ligands. VDR seems to play a role in the regulation of prostate cancer cell proliferation. ATPbinding cassette transporter A1 (ABCA1) is known to be a target gene of LXR and it has been reported to be inhibited by androgen and to be involved in the regulation of LNCaP proliferation. We find that calcitriol (1α,25(OH)2D3) inhibits both basal and a LXR agonist, TO-901317, induced ABCA1 mRNA expression but has no effect on the mRNA expression of ATP-binding cassette transporter G1 (ABCG1), LXRα nor LXRβ. TO-901317 increases both basal and calcitriol induced 25-hydroxyvitamin D3-24-hydroxylase (CYP24) mRNA expression and it slightly but significantly inhibits VDR mRNA expression. The inhibition of ABCA1 by calcitriol appears to be androgen-independent. Cell growth assay shows that when each of calcitriol and 5α-dihydrotestosterone (DHT) was co-treated with ABCA1 blocker, glybenclamide, cell-growth is significantly decreased compared to their own treatments respectively. Our study suggests a possible interaction between calcitriol and TO-901317 in LNCaP cells. Alike DHT, the inhibition of ABCA1 by calcitriol may be involved in its regulation of LNCaP growth. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin D seems to be involved in the control of prostate cancer cell growth. 17β-Hydroxysteroid dehydrogenases type 2, type 4 and type 5 are enzymes which regulate intracellular concentration of active sex steroid hormones, which in turn, regulate the development, growth, and function of the prostate and play a role in the development and progression of prostate cancer. Using quantitative real-time PCR we find that calcitriol up-regulates HSD17B type 2, type 4 and type 5 in human prostate cancer LNCaP and PC3 cells but not in stromal cells. LXR agonist, TO-901317, suppresses the expression of HSD17B2 mRNA and inhibits calcitriol induced HSD17B2 expression. TO-901317 up-regulates the expression of HSD17B5 but not that of HSD17B4. 5α-Dihydrotestosterone up-regulates the expression of HSD17B2 and HSD17B4 but it significantly inhibits HSD17B5 expression by 70%. Calcitriol has no effect on DHT mediated expression of the three genes. The regulation of HSD17B2, HSD17B4 and HSD17B5 by ligands of LXR and VDR as well as AR in prostate cancer cells suggests a complex interaction of these signaling systems in the prostate. [Copyright &y& Elsevier]

Abstract: 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase, CYP24) is an important inactivating enzyme controlling the concentrations of both active metabolites 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. In this paper, we demonstrate that 25-hydroxyvitamin D3 at 500nM significantly increases the expression of 24-hydroxylase mRNA and the increase is significantly decreased by 5α-dihydrotestosterone (DHT) at concentrations of 1–100nM in androgen-sensitive prostate cancer cells LNCaP. 25-Hydroxyvitamin D3 at 500nM and 1α,25-dihydroxyvitamin D3 at 10nM inhibit LNCaP cell growth, and the growth inhibition is enhanced by 1nM DHT. Neither 25-hydroxyvitamin D3 nor 1α,25-dihydroxyvitamin D3 at physiological concentrations has growth effect. However, in the presence of 1nM DHT, both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 at physiological concentrations are clearly antiproliferative. These data demonstrate that DHT enhances the antiproliferative activity of Vitamin D3 hormones by inhibiting their inactivating enzyme. Most previous studies on Vitamin D3 action in cell cultures have used pharmacological concentrations of 1α,25-dihydroxyvitamin D3, the present results demonstrate, for the first time, that both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 at physiological concentrations are active in the presence of physiological concentration of androgen. The combined use of androgen and Vitamin D3 metabolites could be a promising treatment for prostate cancer. [Copyright &y& Elsevier]

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1α,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells. [Copyright &y& Elsevier]

The antiproliferative effect of 1α,25(OH)2D3 on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D3 and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1α,25(OH)2D3 and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96 h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1α,25(OH)2D3 had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48 h by around twofold. The upregulation of FACL3/ACS3 expression by 1α,25(OH)2D3 was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a 14C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1α,25(OH)2D3 on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D3 failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D3 was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D3 regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D3 is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D3 antiproliferative effect in prostate cancer LNCaP cells. [Copyright &y& Elsevier]

Abstract: 1α,25–dihydroxyvitamin D3 (1α,25(OH)2D3 or calcitriol) is an active hormone that regulates cellular proliferation and induces apoptosis in cancer cells. Here we report on a new calcitriol target gene in prostate cancer cells, tumour necrosis factor α (TNF–α). Calcitriol and its analogue CB1093 up–regulate TNF–α mRNA expression in LNCaP and PC–3 cells. The stimulation is dose–dependent in both of these cell lines, demonstrated by the quantitative real–time polymerase chain reaction. Calcitriol and CB1093 act synergistically with human recombinant TNF–α in activation of TNF–α mRNA expression in LNCaP but not in PC–3 cells. Transcriptional activation of TNF–α gene by calcitriol or CB1093 does not lead to TNF–α protein secretion, however calcitriol and CB1093 enhance TPA–stimulated TNF–α production in LNCaP cells. We did not observe any significant effect of calcitriol on regulation of TNFR1 at the level of gene expression. Nor does calcitriol affect transcriptional regulation of cytokine (IL–1, IL–6) and cytokine receptor genes in LNCaP and PC–3 prostate cancer cell lines. Calcitriol and its analogue CB1093 at 10 nM concentration induce programmed cell death in LNCaP cells. Combined addition of human recombinant TNF–α with calcitriol or CB1093 cause enhanced effect in induction of apoptosis. We conclude that under physiological conditions vitamin D activates only the transcription of TNF–α gene, for TNF–α protein synthesis additional cofactors are required. Therefore a cooperation of vitamin D and TNF–α may play an important role in the control of cell growth in prostate cancer. [Copyright &y& Elsevier]

Abstract: Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5α-dihydrotestosterone are produced from testosterone by aromatase and 5α-reductase, respectively. Metabolites of vitamin D have shown to inhibit the growth of prostate cancer cells. The aim of the present study was to verify whether 25-hydroxyvitamin D3 (25OHD3), 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], dexamethasone, and progesterone regulate the expression of aromatase and 5α-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD3, 1α,25-(OH)2D3, dexamethasone, or progesterone. Aromatase and 5α-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [3H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10nM dexamethasone, 1–100nM 1α,25-(OH)2D3 and 100nM–10μM progesterone. The induction was enhanced when hormones were used synergistically. Real-time RT-PCR analysis showed no regulation of the expression of aromatase mRNA by any steroids tested in either LNCaP or PC3 cells. The expression of 5α-reductase type I mRNA was not regulated by 1α,25-(OH)2D3 and no expression of 5α-reductase type II was detected in LNCaP. [Copyright &y& Elsevier]

Abstract: A20 is a TNF-inducible primary response gene and its product, a zinc finger protein, has antiapoptotic function in several cancer cells. We studied A20 gene expression in the Vitamin D- and TNF-sensitive LNCaP cell line and in the Vitamin D- and TNF-resistant PC-3 cell line. The results of the quantitative real-time RT-PCR analyses demonstrated that the basal level of A20 mRNA production in PC-3 cells was considerably higher than in LNCaP cells that is associated with the resistance of PC-3 cells. TNF induced A20 gene expression in both cell lines, but with different effect. A20 mRNA expression was down-regulated by 10nM calcitriol within 3–9h after treatment and up-regulated by androgen reaching maximal values by 6h after stimulation in LNCaP cells. We conclude that A20 may be involved in the regulation of cell proliferation by TNF, Vitamin D, and androgen in prostate cancer. [Copyright &y& Elsevier]

Abstract: 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) is an important inactivating enzyme and its expression is induced by 25-hydroxyvitamin D3 (25OHD3) and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) through action of heterodimers of vitamin D receptor (VDR) and retinoid X receptor (RXR). RXRs also act as heterodimer partners for retinoic acid receptors (RARs), mediating the action of all-trans-retinoic acid (ATRA). Prostate stroma plays a crucial role in prostate cancer development and benign prostatic hyperplasia. We demonstrate here that ATRA markedly reduced the expression of 24-hydroxylase mRNA induced by 25OHD3 and 1α,25-(OH)2D3 in human prostatic stromal cells P29SN and P32S but not in epithelial cells PrEC or cancer cells LNCaP. By using transfection and RAR-selective ligands, we found that the inhibitory effect of ATRA on 24-hydroxylase expression in stromal cells was mediated by RARα but not by RARβ. Moreover, the ATRA-induced expression of RARβ was also mediated by RARα. The combined treatment of 1α,25-(OH)2D3 and RARα agonist Am80 at 10nM exhibited strong growth-inhibitory effect whereas either alone had no effect. Our data suggest that ATRA suppresses 24-hydroxylase expression through RARα-dependent signaling pathway and can enhance vitamin D action in suppression of cell growth. [Copyright &y& Elsevier]

Abstract: Calcitriol, a hormonal form of Vitamin D, regulates growth of normal and cancer cells of various origins by modulation of peptide growth factors signaling. Platelet-Derived Growth Factor (PDGF) signaling pathway is involved in prostate cancer progression. We studied the expression of PDGF receptors in human prostate primary stromal cells and cancer epithelial cell lines and growth response to PDGF-BB isoform. We found that the expression of PDGF receptors and PDGF-BB-mediated cell growth are regulated by calcitriol in prostate cells. Quantitative RT-PCR analysis revealed a lower level of mRNA for PDGF receptors in LNCaP and PC-3 cells than in primary stromal cells. Western blotting showed a high amount of PDGFRα and β proteins in primary stromal cells that could not be detected in LNCaP, which may explain the resistance of LNCaP cells to growth-promoting effect of PDGF-BB. Addition of Epidermal Growth Factor (EGF) to the culture medium induces the expression of PDGFRβ and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRβ expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRα and PDGF-B mRNA expression. We suggest that inhibition of PDGFRβ expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB. [Copyright &y& Elsevier]