Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1.

Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.

Proliferative response of human prostate cancer cell to hormone inhibited by androgen receptor antisense RNA.

BACKGROUND: The failure of endocrine treatment for advanced prostate cancer might be related to aberrant activation of androgen receptor (AR). Prostate cancer cell line LNCaP contains AR that can be activated by androgen, estrogen and progesterone. This study was set to investigate the effects of antisense AR RNA on growth of LNCaP cultured in medium containing varied concentrations of R1881, 17beta-estradiol, and progesterone, respectively. METHODS: LNCaP cells transfected with antisense AR RNA retroviral vector pL-AR-SN were designated as LNCaPas-AR. LNCaP cells containing empty vector pLXSN served as LNCaPNeo. LNCaP and LNCaPNeo were taken as controls. In vitro cell growth assay, proliferative cells of LNCaP and tranfected LNCaPs were counted by typan staining when they cultured with synthetic androgen R1881, 17beta-estradiol, and progesterone, respectively. RESULTS: Growth of LNCaPas-AR was inhibited significantly (P < 0.05) compared with that of LNCaP and LNCaPNeo at 1 nmol/L R1881, 10 nmol/L 17beta-estradiol, and 1 nmol/L progesterone, respectively. No difference was seen between LNCaP and LNCaPNeo (P > 0.05). Microscopic observation showed that LNCaP and LNCaPNeo cells grew well, but only few LNCaPas-AR cells were alive. CONCLUSIONS: Our observations indicate that antisense AR RNA retroviral vector pL-AR-SN could change androgen-independent characteristics of LNCaP cells, which might shed some novel insights into the treatment of androgen-independent prostate cancer.

Differential modulation of androgen receptor transcriptional activity by the nuclear receptor co-repressor (N-CoR).

Antiandrogens are widely used agents in the treatment of prostate cancer, as inhibitors of AR (androgen receptor) action. Although the precise mechanism of antiandrogen action is not yet elucidated, recent studies indicate the involvement of nuclear receptor co-repressors. In the present study, the regulation of AR transcriptional activity by N-CoR (nuclear receptor co-repressor), in the presence of different ligands, has been investigated. Increasing levels of N-CoR differentially affected the transcriptional activity of AR occupied with either agonistic or antagonistic ligands. Small amounts of co-transfected N-CoR repressed CPA (cyproterone acetate)- and mifepristone (RU486)-mediated AR activity, but did not affect agonist (R1881)-induced AR activity. Larger amounts of co-transfected N-CoR repressed AR activity for all ligands, and converted the partial agonists CPA and RU486 into strong AR antagonists. In the presence of the agonist R1881, co-expression of the p160 co-activator TIF2 (transcriptional intermediary factor 2) relieved N-CoR repression up to control levels. However, in the presence of RU486 and CPA, TIF2 did not functionally compete with N-CoR, suggesting that antagonist-bound AR has a preference for N-CoR. The AR mutation T877A (Thr877-->Ala), which is frequently found in prostate cancer and affects the ligand-induced conformational change of the AR, considerably reduced the repressive action of N-CoR. The agonistic activities of CPA- and hydroxyflutamide-occupied T877A-AR were hardly affected by N-CoR, whereas TIF2 strongly enhanced their activities. These results indicate that lack of N-CoR action allows these antiandrogens to act as strong agonists on the mutant AR.

Mechanistic studies of the effects of the retinoid N-(4-hydroxyphenyl)retinamide on prostate cancer cell growth and apoptosis.

To explore the mechanisms underlying the chemopreventive effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in prostate cancer, we evaluated the anti-proliferative and apoptosis-inducing effects of 4-HPR in the androgen-sensitive human prostate cancer cell line LNCaP. 4-HPR decreased the number of viable LNCaP cells (as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) in a dose-dependent manner. Although 4-HPR exerted a modest G1 cell-cycle block (as determined by flow cytometry), its effect on reduced cell number appeared to result primarily from induction of apoptosis (as measured by an enzyme-linked immunosorbent assay and flow-cytometric assays). The mitogenic effects of R1881, a non-metabolizable androgen that potently induces LNCaP cell proliferation, was completely blocked by greater than 0.5 microM 4-HPR. Furthermore, increasing the R1881 concentration in the presence of 2.0 microM 4-HPR increased apoptotic cell death. 4-HPR decreased prostate-specific antigen (PSA) protein levels in conditioned medium and decreased PSA mRNA expression. 4-HPR also decreased the ratio of bcl-2 to bax mRNA expression in LNCaP cells by approximately 45%, indicating that the apoptotic effects of 4-HPR may be mediated, at least in part, by alterations in the bcl-2/bax-regulated apoptotic pathway. N-acetylcysteine (4 mM) completely blocked the anti-proliferative and apoptotic-inducing effects of 4-HPR, suggesting that an oxidative mechanism may be involved. We concluded that (i) 4-HPR exerts growth-suppressive and apoptotic effects on LNCaP cells, (ii) 4-HPR can interact with androgen to suppress proliferation and induce apoptosis, (iii) the apoptotic effects of 4-HPR may be mediated in part by the bcl-2/bax pathway, and (iv) a pro-oxidant mechanism may contribute to the anti-proliferative and apoptotic-inducing effects of 4-HPR.

Androgen and glucocorticoid regulation of androgen receptor cDNA expression.

Androgen receptor (AR) levels are regulated by androgens, other steroids and non-steroidal hormones via complex, tissue-specific processes. Since alterations in receptor levels may influence cellular sensitivity to androgens, understanding AR regulation is of fundamental and potentially therapeutic significance. In most target tissues and AR-containing cell lines, AR mRNA is down-regulated in response to androgens. We have reconstituted this androgen-mediated down-regulation of AR mRNA in COS 1 cells transfected with a human AR cDNA under the control of the cytomegalovirus (CMV) promoter. The sequences mediating receptor mRNA down-regulation are represented within the AR cDNA and not within the CMV promoter. Androgenic down-regulation of AR cDNA expression was time- and dose-dependent, resembling native AR mRNA down-regulation. In addition, androgenic regulation of the receptor cDNA was not dependent on protein synthesis suggesting that AR and/or another pre-existing protein(s) is involved in this process. In COS 1 cells co-transfected with androgen and glucocorticoid receptor cDNAs, dexamethasone mimicked the action of androgen in down-regulating AR mRNA. This response depended on glucocorticoid receptors. Androgen had little effect on steady-state levels of AR protein consistent with reports that androgen down-regulates AR mRNA but increases AR protein half-life (Kemppainen et al. (1992) J. Biol. Chem. 267, 968-974; Zhou et al. (1995) Mol. Endocrinol. 9, 208-218). However, glucocorticoids decreased AR protein levels in cells that co-expressed androgen and glucocorticoid receptors. These results indicate that sequences represented in the AR cDNA mediate AR mRNA down-regulation by both androgens and glucocorticoids. Inhibition of AR mRNA and protein by glucocorticoids suggests that these steroids may modulate androgen action in tissues, such as mammary gland and prostate, which express both androgen and glucocorticoid receptors.

Androgens inhibit the proliferation of a variant of the human prostate cancer cell line LNCaP.

UNLABELLED: The paradoxical androgen response of R2, a subline of the human prostate cancer cell line LNCaP, is described here. Two androgens (DHT and R1881) decreased, in a dose-dependent manner, R2 cell proliferation and [3H]thymidine incorporation. These ligand and cell specific effects were accompanied by an increase in the metabolism of the vital dye MTT and in cell protein content. Both androgens increased the doubling time and the percentage of G0-G1 cells. No evidence of androgen-induced apoptosis was found. Cloning allowed the selection of two cell populations on the basis of the response to 10 nM of R1881. Long term culture of uncloned R2 cells with R1881 modified reversibly the pattern of androgen response. R2 was compared to the androgen-stimulated LNCaP-FGC subline to investigate the causes of their different androgen responsiveness. The androgen receptor (number, affinity for hormones and antihormones, sedimentation constant and molecular weight) and androgen receptor genes (exon size and exon 8 sequence) were found to be identical in the two sublines. EGF stimulated LNCaP-FGC but not R2. Both cells were slightly stimulated by basic FGF but were insensitive to IGF-I and TGF beta 1. IN CONCLUSION: (1) androgens inhibit the proliferation of R2 cells possibly by introducing a G0-G1 block; (2) this inhibition is incomplete because, at least in part, the R2 cell population is heterogeneous; (3) chronic androgen treatment induces reversible cell adaptation; and (4) there is no evidence that the loss of the classical stimulatory effect of androgen on cell proliferation and the gain of inhibitory effect are due to androgen receptor alteration or to a specific action of one of the four growth factors tested.

Androgenic and antiandrogenic control on epidermal growth factor, epidermal growth factor receptor, and androgen receptor expression in human prostate cancer cell line LNCaP.

Both androgen and antiandrogen treatments enhance the proliferation rate of the hormone-dependent prostate cancer cell line LNCaP, expressing a mutated androgen receptor (AR). We studied the modification of the expression of epidermal growth factor (EGF), of its receptor (EGF-R), and of androgen receptor (AR) in the LNCaP cell line, under basal conditions and during androgen (R1881) and antiandrogen hydroxy-flutamide (OH-FLU) treatment. After prolonged R1881 administration, a marked increase of EGF release was observed, completely blocked by the addition of OH-FLU. The Scatchard plot analysis of EGF-R binding revealed two classes of binding sites with high and low affinity. The administration of OH-FLU alone or combined with R1881 did not modify the affinity constants, while the low-affinity component disappeared after androgen administration. Both androgen and antiandrogen administration led to a significant increase of the EGF-R high-affinity component. AR mRNA and protein levels were downregulated by R1881 treatment. Following OH-FLU administration, AR mRNA was slightly downregulated, and there was not a strict parallelism between AR mRNA levels and AR binding capacity. When combined with R1881, OH-FLU partially counteracted the androgen-induced AR downregulation. Our data show that EGF-R binding capacity is the only parameter constantly raised in cell proliferation with respect to quiescent cells, and highlights the nonunivocal action of OH-FLU on androgen-induced effects.

Antiproliferative effects of suramin on androgen responsive tumour cells.

UNLABELLED: The effect of the polyanionic drug suramin on two androgen responsive tumour cell lines was studied. Human prostate tumour (LNCaP) cell growth is stimulated two- to three-fold by the synthetic androgen R1881 (0.1 nM) or EGF (1 ng/ml). Suramin (0.01-1.0 mM) inhibited the growth of LNCaP cells in a dose dependent way, both in the presence and absence of androgen or EGF. Growth was arrested in the G0/G1 phase of the cell cycle, but was resumed after removal of suramin. DDT-1 hamster ductus deferens tumour cells are stimulated by PDGF (25 ng/ml), b-FGF (10 ng/ml) and testosterone (10 nM). Suramin inhibited PDGF and b-FGF stimulated cell growth. However in the presence of testosterone, suramin showed a biphasic effect: stimulatory at low dose (0.01 mM) and inhibitory above 0.01 mM. Suramin decreased the apparent affinity of EGF binding sites on LNCaP cells with a two- to eight-fold increase in Kd at 0.1 and 1.0 mM suramin, respectively. IN CONCLUSION: suramin counteracts the growth stimulatory effects of both androgens and growth factors on androgen sensitive tumour cells. The effects are reversible after withdrawal of suramin.