The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth

The antiproliferative effect of 1alpha,25(OH)(2)D(3) 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 D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) 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 D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) 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 D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.     

Expression and vitamin D3 regulation of long-chain fatty-acid-CoA ligase 3 in human prostate cancer cells

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)(2)D(3) at an mRNA and enzyme activity levels in prostate cancer cells. Our further study indicated that the FACL3 mediated 1α,25(OH)(2)D(3) 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)(2)D(3) 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)(2)D(3), 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.     

Prostate cancer cell type-specific involvement of the VDR and RXR in regulation of the human PTHrP gene via a negative VDRE

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDREhPTHrP) within the human PTHrP gene and involves an interaction between nVDREhPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRalpha forms part of the nuclear protein complex that interacts with nVDREhPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9-cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9-cis-retinoic acid enhances the 1,25(OH)2D3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9-cis-retinoic acid. Promoter activity driven by nVDREhPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH)2D3 and EB1089 in both cell lines. Co-treatment with these compounds and 9-cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH)2D3 in a cell type-specific manner in prostate cancer cells.     

Expression and secretion of N-acylethanolamine-hydrolysing acid amidase in human prostate cancer cells

N-acylethanolamines (NAEs) are a class of bioactive lipid molecules in animal tissues, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. Enzymatic hydrolysis of NAEs is considered to be an important step to regulate their endogenous levels. Lysosomal NAE-hydrolysing acid amidase (NAAA) as well as fatty acid amide hydrolase (FAAH) is responsible for this reaction. Here, we report relatively high expression of NAAA in human prostate cancer cells (PC-3, DU-145 and LNCaP) and prostate epithelial cells (PrEC), with the highest mRNA level in LNCaP cells. FAAH and the NAE-forming enzyme N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) were also detected in these cells. NAAA activity in LNCaP cells could be distinguished from coexisting FAAH activity, based on their different pH dependency profiles and specific inhibition of FAAH activity by URB597. These results showed that both the enzymes were functionally active. We also found that NAAA was partly secreted from LNCaP cells, which underlined possible usefulness of this enzyme as a biomarker of prostate cancer.     

Androgen receptor auto-regulates its expression by a negative feedback loop through upregulation of IFI16 protein

Expression of androgen receptor (AR) in prostate epithelial cells is thought to regulate cell proliferation, differentiation, and survival. However, the molecular mechanisms remain unclear. We report that re-expression of AR in PC-3 human prostate cancer cell line resulted in upregulation of IFI16 protein, a negative regulator of cell growth. We found that the IFI16 protein bound to AR in a ligand-dependent manner and the DNA-binding domain (DBD) of the AR was sufficient to bind IFI16. Furthermore, re-expression of IFI16 protein in LNCaP prostate cancer cells, which do not express IFI16 protein, resulted in downregulation of AR expression and an inhibition of the expression of AR target genes. Our observations identify a role for IFI16 protein in AR-mediated functions.     

Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells

Glial cells play a pivotal role in brain fatty acid metabolism and membrane biogenesis. However, the potential regulation of lipogenesis and cholesterologenesis by fatty acids in glial cells has been barely investigated. Here, we show that physiologically relevant concentrations of various saturated, monounsaturated, and polyunsaturated fatty acids significantly reduce [1-(14)C]acetate incorporation into fatty acids and cholesterol in C6 cells. Oleic acid was the most effective at depressing lipogenesis and cholesterologenesis; a decreased label incorporation into cellular palmitic, stearic, and oleic acids was detected, suggesting that an enzymatic step(s) of de novo fatty acid biosynthesis was affected. To clarify this issue, the activities of acetyl-coenzyme A carboxylase (ACC) and FAS were determined with an in situ digitonin-permeabilized cell assay after incubation of C6 cells with fatty acids. ACC activity was strongly reduced ( approximately 80%) by oleic acid, whereas no significant change in FAS activity was observed. Oleic acid also reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). The inhibition of ACC and HMGCR activities is corroborated by the decreases in ACC and HMGCR mRNA abundance and protein levels. The downregulation of ACC and HMGCR activities and expression by oleic acid could contribute to the reduced lipogenesis and cholesterologenesis.     

Regulation of aromatase and 5alpha-reductase by 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3), dexamethasone and progesterone in prostate cancer cells

Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5alpha-dihydrotestosterone are produced from testosterone by aromatase and 5alpha-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 D(3) (25OHD(3)), 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)], dexamethasone, and progesterone regulate the expression of aromatase and 5alpha-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD(3), 1alpha,25-(OH)(2)D(3), dexamethasone, or progesterone. Aromatase and 5alpha-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [(3)H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10nM dexamethasone, 1-100 nM 1alpha,25-(OH)(2)D(3) and 100 nM-10 microM 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 5alpha-reductase type I mRNA was not regulated by 1alpha,25-(OH)(2)D(3) and no expression of 5alpha-reductase type II was detected in LNCaP.     

Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte

Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC₅₀) of 0.14 μM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARγ during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.     

Differential expression of galectins in normal, benign and malignant prostate epithelial cells: silencing of galectin-3 expression in prostate cancer by its promoter methylation

Galectins (gal), a family of soluble beta-galactoside-binding proteins present at the cell surface, are involved in cancer progression and metastasis. Here we investigated the expression of several galectins in normal (PrEC), benign (BPH-1), and malignant (LNCaP) prostate epithelial cells and found that all galectins, except gal1 are differentially expressed. The gal3, 7, and 9 are highly expressed in PrEC, but not in LNCaP cells. Out of seven isoforms of gal8, the proto isoform gal8e and our newly discovered proto isoform gal8g were upregulated in LNCaP cells compared to PrEC, whereas the two tandem-repeat isoforms gal8a and gal8b were equally expressed in these cells. To determine if the silencing of gal3 in LNCaP cells was due to promoter methylation, LNCaP cells were treated with azacytidine. Azacytidine treatment induced the expression of gal3 in LNCaP cells, indicating that the gal3 gene was silenced by methylation of its promoter. To examine further, we evaluated cytosine methylation in gal3 promoter in LNCaP, normal prostate and placenta DNA and observed that it is highly methylated in LNCaP but not in normal cells and azacytidine completely abolished this methylation in LNCaP cells. Similar to prostate cancer cells, gal3 promoter was highly methylated in human prostate cancer tissue but not in normal tissue. To our knowledge, this is the first report indicating that gal3 expression is regulated by promoter methylation in LNCaP cells and prostate tumors. The methylation of gal3 promoter may constitute a powerful tool for early diagnosis of prostate cancer.     

Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro

Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)₂D₃ on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)₂D₃ (10⁻⁶, 10⁻⁷, 10⁻¹⁰ M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)₂D₃, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)₂D₃ in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.     

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

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

Inhibition of Proliferation and Induction of Autophagy by Atorvastatin in PC3 Prostate Cancer Cells Correlate with Downregulation of Bcl2 and Upregulation of miR-182 and p21

The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in a dose- and time-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells.     

14-3-3sigma is down-regulated in human prostate cancer

The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.