Effects of the antiandrogen flutamide on the expression of protein kinase C isoenzymes in LNCaP and PC3 human prostate cancer cells.

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.

Altered SMRT levels disrupt vitamin D3 receptor signalling in prostate cancer cells.

We hypothesized that key antiproliferative target genes for the vitamin D receptor (VDR) were repressed by an epigenetic mechanism in prostate cancer cells resulting in apparent hormonal insensitivity. To explore this possibility, we examined nuclear receptor corepressor expression in a panel of nonmalignant and malignant cell lines and primary cultures, and found frequently elevated SMRT corepressor mRNA expression often associated with reduced sensitivity to 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)2D3). For example, PC-3 and DU-145 prostate cancer cell lines had 1.8-fold and twofold increases in SMRT mRNA relative to normal PrEC cells (P<0.05). Similarly, 10/15 primary tumour cultures (including three matched to normal cells from the same donors) had elevated SMRT mRNA levels; generally NCoR1 and Alien were not as commonly elevated. Corepressor proteins often have associated histone deacetylases (HDAC) and reflectively the antiproliferative action of 1alpha,25(OH)2D3 can be 'restored' by cotreatment with low doses of HDAC inhibitors such as trichostatin A (TSA, 15 nM) to induce apoptosis in prostate cancer cell lines. To decipher the transcriptional events that lead to these cellular responses, we undertook gene expression studies in PC-3 cells after cotreatment of 1alpha,25(OH)2D3 plus TSA after 6 h. Examination of known VDR target genes and cDNA microarray analyses revealed cotreatment of 1alpha,25(OH)2D3 plus TSA cooperatively upregulated eight (out of 1176) genes, including MAPK-APK2 and GADD45alpha. MRNA and protein time courses and inhibitor studies confirmed these patterns of regulation. Subsequently, we knocked down SMRT levels in PC-3 cells using a small interfering RNA (siRNA) approach and found that GADD45alpha induction by 1alpha,25(OH)2D3 alone became very significantly enhanced. The same distortion of gene responsiveness, with repressed induction of GADD45alpha was found in primary tumour cultures compared and to matched peripheral zone (normal) cultures from the same donor. These data demonstrate that elevated SMRT levels are common in prostate cancer cells, resulting in suppression of target genes associated with antiproliferative action and apparent 1alpha,25(OH)2D3-insensitivity. This can be targeted therapeutically by combination treatments with HDAC inhibitors.

Regulation of the expression of protein kinase C isoenzymes in rat ventral prostate: effects of age, castration and flutamide treatment.

Protein kinase C (PKC) isoenzymes are involved in cell function, growth, apoptosis and neoplastic transformation in the prostate gland. We detected by means of Western blot the expression of the classical alpha and beta1, the novel epsilon and the atypical zeta isoforms of PKC in ventral prostates from rats with different extents of plasma testosterone levels and/or androgen imprinting on the gland. The expression of the four isoforms decreased in 5-day castrated rats showing apoptotical regression of the gland and a drastic reduction of circulating testosterone. However, the expression of PKC isoenzymes (alpha, beta1, epsilon ) increased in prostates from pubertal (35-days old) rats that are characterized by relatively low but extremely bioactive testosterone plasma levels. Treatment of adult rats for 14 days with flutamide (daily s.c. injection of 15 mg/Kg B.W.) resulted in increased expression of the four isoenzymes; it occurred in the presence of increased (normal rats) or drastically reduced (rats castrated after 9 days of flutamide administration) levels of plasma testosterone conceivably through a direct effect of this nonsteroidal antiandrogen on prostate cells. Measurements of PKC(alpha) activity were in agreement with the observations on protein expression and showed that flutamide (that is extensively used in the treatment of advanced prostate cancer) elicits some impairment in the mechanisms of translocation of this isoform from the cytosol to the membrane. Thus, in addition to the possibility of direct effects of flutamide upon the rat prostate, we present evidence that the levels of circulating androgens and/or their bioactivity in the gland regulate the expression of various important PKC isoforms.