Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway

The identification and development of novel nontoxic phytochemicals that target androgen and androgen receptor (AR) signaling remains a priority for prostate cancer (PCA) control. In the present study, we assessed the antiandrogenic efficacy of isosilybin B employing human PCA LNCaP (mutated AR), 22Rv1 (mutated AR) and LAPC4 (wild-type AR) cells. Isosilybin B (10-90 microM) treatment decreased the AR and prostate specific antigen (PSA) levels in LNCaP, 22Rv1 and LAPC4 cells, but not in non-neoplastic human prostate epithelial PWR-1E cells. Isosilybin B treatment also inhibited synthetic androgen R1881-induced nuclear localization of AR, PSA expression and cell growth, and caused G(1) arrest. In mechanistic studies identifying AR degradation, isosilybin B caused increased phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which was linked with AR degradation as pretreatment with PI3K inhibitor (LY294002)-restored AR level. Further, overexpression of kinase-dead Akt largely reversed isosilybin B mediated-AR degradation suggesting a critical role of Akt in AR degradation. Antibody pull-down results also indicated that isosilybin B treatment enhances the formation of complex between Akt, Mdm2 and AR, which promotes phosphorylation-dependent AR ubiquitination and its degradation by proteasome. Together, present findings identify a novel mechanism for isosilybin B-mediated anticancer effects in human PCA cells.     

Short hairpin RNA knockdown of the androgen receptor attenuates ligand-independent activation and delays tumor progression

Progression to androgen independence is the lethal end stage of prostate cancer. We used expression of androgen receptor (AR)-targeted short hairpin RNAs (shRNA) to directly test the requirement for AR in ligand-independent activation of androgen-regulated genes and hormone-independent tumor progression. Transient transfection of LNCaP human prostate cancer cells showed that AR shRNA decreased R1881 induction of the prostate-specific antigen (PSA)-luciferase reporter by 96%, whereas activation by forskolin, interleukin-6, or epidermal growth factor was inhibited 48% to 75%. Whereas the antiandrogen bicalutamide provided no further suppression, treatment with the mitogen-activated protein kinase (MAPK) inhibitor U0126 completely abrogated the residual activity, indicating a MAPK-dependent, AR-independent pathway for regulating the PSA promoter. Expression of doxycycline-inducible AR shRNA expression in LNCaP cells resulted in decreased levels of AR and PSA as well as reduced proliferation in vitro. When these cells were grown as xenografts in immunocompromised mice, induction of AR shRNA decreased serum PSA to below castration nadir levels and significantly retarded tumor growth over the entire 55-day experimental period. This is the first demonstration that, by inducibly suppressing AR expression in vivo, there is an extensive delay in progression to androgen independence as well as a dramatic inhibition of tumor growth and decrease in serum PSA, which exceeds that seen with castration alone. Based on these findings, we propose that suppressing AR expression may provide superior therapeutic benefit in reducing tumor growth rate than castration and may additionally be very effective in delaying progression to androgen independence.     

Progression to androgen-independent LNCaP human prostate tumors: cellular and molecular alterations

Lethal phenotypes of human prostate cancer are characterized by progression to androgen-independence and metastasis. For want of a clinically relevant animal model, mechanisms behind this progression remain unclear. Our study used an in vivo model of androgen-sensitive LNCaP human prostate cancer cell xenografts in male SCID mice to study the cellular and molecular biology of tumor progression. Primary tumors were established orthotopically, and the mice were then surgically castrated to withdraw androgens. Five generations of androgen-independent tumors were developed using castrated host mice. Tumor samples were used to determine expressions of cellular and molecular markers. Androgen-independent tumors had increased proliferation and decreased apoptosis compared to androgen-sensitive tumors, outcomes associated with elevated expression of p53, p21/waf1, bcl-2, bax and the bcl-2/bax ratio. Blood vessel growth in androgen-independent tumor was associated with increased expression of vascular endothelial growth factor. Overexpression of androgen receptor mRNA and reduced expression of androgen receptor protein in androgen-independent tumors suggest that the androgen receptor signaling pathway may play an important role in the progression of human prostate cancer to androgen-independence. The in vivo orthotopic LNCaP tumor model described in our study mimics the clinical course of human prostate cancer progression. As such, it can be used as a model for defining the molecular mechanisms of prostate cancer progression to androgen-independence and for evaluating the effect of preventive or therapeutic regimens for androgen-independent human prostate cancer.     

Interleukin-4 enhances prostate-specific antigen expression by activation of the androgen receptor and Akt pathway

Androgen receptor (AR) plays an important role in the development and progression of prostate cancer upon the action of androgen through the binding of the androgen-responsive elements (AREs) on the target genes. Abnormal activation of the AR by nonandrogen has been implicated in the progression of androgen-independent prostate cancer. The levels of interleukin-4 (IL-4) are significantly elevated in sera of patients with hormone refractory prostate cancer. The potential role of IL-4 on the activation of AR was investigated in prostate cancer cells. IL-4 enhances AR-mediated prostate-specific antigen (PSA) expression and ARE-containing gene activity through activation of the AR in the androgen ablation condition in human prostate cancer cells. The AR can also be sensitized by IL-4 and activated by significantly lower levels of androgen (10 pM of R1881) in prostate cancer cells. IL-4 enhances nuclear translocation of AR and increases binding of the AR to the ARE in LNCaP prostate cancer cells. Blocking of the Akt pathway by an Akt-specific inhibitor LY294002 abrogates IL-4-induced PSA expression and AR signaling. These results demonstrate that IL-4 enhances PSA expression through activation of the AR and Akt signaling pathways in LNCaP prostate cancer cells. Understanding IL-4-induced signaling leading to abnormal activation of AR will provide insights into the molecular mechanisms of androgen-independent progression of prostate cancer cells.     

Genistein affects androgen-responsive genes through both androgen- and estrogen-induced signaling pathways

This study examined the mechanisms by which the prostate cancer chemopreventive agent genistein modulates gene expression in LNCaP human prostate cancer cells. Expression of androgen- and estrogen-regulated genes was measured in LNCaP cells cultured in the presence or absence of hormonal stimulation and the presence or absence of genistein. Genistein strongly suppressed basal expression of androgen-responsive gene (ARG) mRNAs, including prostate-specific antigen (PSA) and Ste20-related proline-alanine-rich kinase (SPAK). However, genistein had little or no effect on basal expression of two other ARGs, beta2-microglobulin (B2M) or selenoprotein P (SEPP1). Culturing LNCaP cells in the presence of the synthetic androgen R1881-induced increases in PSA, SPAK, B2M, and SEPP1 mRNA levels. The R1881-induced expression of these genes was uniformly blocked by genistein. For PSA and SPAK, genistein also blocked or downregulated 17beta-estradiol-induced increases in mRNA expression. These results indicate that genistein selectively alters expression of ARG mRNAs in LNCaP cells through modulation of both androgen- and estrogen-induced signaling pathways.     

Androgen receptor-dependent PSA expression in androgen-independent prostate cancer cells does not involve androgen receptor occupancy of the PSA locus

It is widely suspected that androgen-independent prostate cancer growth depends on androgen receptor signaling via ill-defined mechanisms. Prostate-specific antigen (PSA) expression is often used to measure androgen receptor activity in cells and prostate cancer progression in patients. In the present study, we have compared androgen receptor activity using PSA and human male germ cell-associated kinase (hMAK), as read-outs in androgen-dependent LNCaP and androgen-independent C4-2B cells. As expected, very little PSA and hMAK expression were detected in LNCaP cells in the absence of androgens, whereas substantial expression of PSA was observed only in C4-2B cells under the same conditions. The addition of dihydrotestosterone to the culture medium increased the expression of both genes in both cell types. Comprehensive chromatin immunoprecipitation analysis of the entire PSA locus and an androgen-response element in hMAK unexpectedly revealed that androgen receptor was not occupying any site in the absence of dihydrotestosterone in either cell type. In line with the expression data, and in the absence of dihydrotestosterone, histone acetylation and RNA polymerase II occupancy was substantial at the PSA locus in C4-2B but not in LNCaP cells. In the presence of dihydrotestosterone, androgen receptor was found to occupy mainly the enhancer region of PSA in both cell types, accompanied with increases in histone acetylation and RNA polymerase II occupancy. Although the androgen receptor was not directly involved in the androgen-independent expression of PSA in C4-2B cells, small interfering RNA knock-down of androgen receptor significantly reduced PSA expression in both the presence and absence of dihydrotestosterone. In contrast, hMAK expression was decreased only in the presence of dihydrotestosterone after androgen receptor knock-down. We conclude that androgen-independent expression of PSA in C4-2B cells does not rely on the direct occupancy of the androgen receptor at the PSA locus, but is nevertheless affected indirectly via unknown androgen receptor-dependent mechanism(s) that influence the expression from some but not all androgen receptor target genes.     

Androgen receptor expression in relation to apoptosis and the expression of cell cycle related proteins in prostate cancer

The expression of several genes involved in the regulation of cell cycle and apoptosis may be regulated via the androgen receptor (AR) in the prostate. AR may have a role in the prognosis of prostatic carcinoma. The aim was to examine AR expression status and its relationship with markers of proliferation, apoptosis and cell cycle control in prostate cancer. Expression of AR, bcl-2, bax, Ki-67 and p53 was examined in paraffin-embedded tissues from 50 cases of prostate carcinoma by immunohistochemistry and evaluated using an index of staining. Detection of apoptotic cells was performed by TUNEL method. Correlation between AR expression and apoptosis, proliferation index, bcl-2, bax and p53 and also clinicopathological parameters including stage, pathological grade and Gleason score were determined. AR expression was observed in all cases with mean expression of 81%±15 and mean staining index of 141±65. No correlation was found between AR expression and apoptosis detected in patients. The mean AR staining index was 170±72 in bcl-2 positive tumors versus 120±53 in bcl-2 negative tumors showing a significant association between AR and bcl-2 expression (p=0.015). AR expression also showed a significant association with bcl-2/bax ratio (r=0.321, p=0.023) and Ki-67 proliferation staining index (r=0.396, p=0.004). Although a significant correlation between Ki-67 and p53 with differentiation status of the tumors was observed (p<0.004) no correlation was found with AR. AR expression showed no prognostic value regarding its correlation with stage and differentiation status of the prostate carcinoma. However, its significant correlation with Ki-67 and bcl-2 that are markers of cell survival suggest its contribution to tumor cell progression.     

PKC-mediated secretion of death factors in LNCaP prostate cancer cells is regulated by androgens

Activation of PKCdelta in androgen-dependent LNCaP prostate cancer cells leads to apoptosis via the activation of p38 MAPK and JNK cascades. We have recently shown that treatment of LNCaP cells with phorbol 12-myristate 13-acetate (PMA) leads to a PKCdelta-mediated autocrine release of death factors, including the cytokines TNFalpha and TRAIL, and that conditioned medium (CM) collected from PMA-treated LNCaP cells promotes the activation of the extrinsic apoptotic cascade. Interfering with this autocrine loop either at the level of factor release or death receptor activation/signaling markedly impaired the PMA apoptotic response. In the present study we show that this PKCdelta-dependent autocrine mechanism is greatly influenced by androgens. Indeed, upon androgen depletion, which down-regulates PKCdelta expression, TNFalpha and TRAIL mRNA induction and release by PMA are significantly diminished, resulting in a reduced apoptogenic activity of the CM and an impaired ability of the CM to activate p38 MAPK and JNK. These effects can be rescued by addition of the synthetic androgen R1881. Furthermore, RNAi depletion of the androgen-receptor (AR) from LNCaP cells equally impaired PMA responses, suggesting that PKC-mediated induction of death factor secretion and apoptosis in LNCaP prostate cancer cells are highly sensitive to hormonal control.     

Effect of calcitriol on prostate-specific antigen in vitro and in humans.

BACKGROUND: Calcitriol, the natural ligand for the vitamin D receptor, has significant potential in prostate cancer treatment. Measurement of its antineoplastic activity in prostate cancer clinical trials may be complicated by effects of calcitriol on prostate-specific antigen (PSA) production. We examined the effects of calcitriol at similar concentration on cell proliferation, androgen receptor (AR) expression, and PSA production in vitro and on PSA concentrations in prostate cancer patients. EXPERIMENTAL DESIGN: LNCaP prostate cancer cell proliferation was examined by cell counts 6 days after exposure to a range of concentrations of calcitriol. AR and PSA protein was quantified in LNCaP cells over 96 hours after exposure to 1 nmol/L calcitriol. Serum PSA and free PSA was serially measured by immunoassay over a period of 8 days in patients with hormone-na?ve prostate cancer after a single dose of 0.5 microg/kg calcitriol. RESULTS: Calcitriol treatment resulted in dose-dependent growth inhibition of LNCaP with approximately 50% growth inhibition at the clinically achievable concentration of 1 nmol/L. Time-dependent up-regulation of AR expression and of PSA production in LNCaP cells was shown at the same concentration. No significant change in serum PSA or free PSA over 8 days was seen in eight subjects treated with a single dose of 0.5 microg/kg calcitriol. The analysis was powered to detect a 1.23-fold change between the baseline and day 8 serum PSA. CONCLUSIONS: At clinically achievable concentrations, calcitriol inhibits growth and induces AR and PSA expression in LNCaP cells. We did not detect similar changes in serum PSA or free PSA in patients exposed to similar concentrations of calcitriol. Thus, a PSA flare, predicted by preclinical systems, is unlikely to occur in patients and therefore unlikely to complicate interpretation of clinical trial outcomes.     

The impact of bcl-2 expression and bax deficiency on prostate homeostasis in vivo

Prostatic glandular epithelial cells undergo apoptosis in response to androgen-deprivation. The molecular determinants of androgen-responsiveness in these cells are incompletely understood. Recent evidence suggests that bcl-2 gene family members may be important in this context. We used the probasin promoter to target a human bcl-2 transgene specifically to the prostate in order to assess its impact on conferring resistance to androgen withdrawal in, otherwise sensitive, prostatic glandular epithelial cells in vivo. We examined the contribution of bax to mediating androgen-responsiveness in prostatic glandular epithelial cells using bax knockout mice. The histologic appearance of the prostates from probasin-bcl-2 transgenic mice or bax-/- mice did not differ from those of control littermates. There was no evidence of hyperplastic or neoplastic growth. There was no difference between probasin bcl-2 transgenic mice, bax-/- mice, and control littermates in steady-state levels of apoptosis. Following castration our findings suggest that both bax and bcl-2 may each contribute to the androgen-responsiveness of prostatic glandular epithelial cells. It is apparent from these results, however, that bax is not required to mediate cell death in prostatic glandular epithelial cells following castration. A comparison between the apoptotic indices in the ventral prostate from the probasin-bcl-2 and bax-/- mice following castration suggests that the presence of bcl-2 may be a more important indicator of androgen-sensitivity than a deficiency of bax.     

Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate‐specific antigen expression and secretion

Vacuolar ATPases (V‐ATPases) comprise specialized and ubiquitously distributed pumps that acidify intracellular compartments and energize membranes. To gain new insights into the roles of V‐ATPases in prostate cancer (PCa), we studied the effects of inhibiting V‐ATPase pumps in androgen‐dependent (LNCaP) and androgen‐independent (C4‐2B) cells of a human PCa progression model. Treatment with nanomolar concentrations of the V‐ATPase inhibitors bafilomycin A or concanamycin A reduced the in vitro invasion in both cell types by 80%, regardless that V‐ATPase was prominent at the plasma membrane of C4‐2B cells and only traces were detected in the low‐metastatic LNCaP parental cells. In both cell types, intracellular V‐ATPase was excessive and co‐localized with prostate‐specific antigen (PSA) in the Golgi compartment. V‐ATPase inhibitors reversibly excluded PSA from the Golgi and led to the accumulation of largely dispersed PSA‐loaded vesicles of lysosomal composition. Inhibition of acridine orange staining and transferrin receptor recycling suggested defective endosomal and lysosomal acidification. The inhibitors, additionally, interfered with the AR‐PSA axis under conditions that reduced invasion. Bafilomycin A significantly reduced steady‐state and R1881‐induced PSA mRNA expression and secretion in the LNCaP cells which are androgen‐dependent, but not in the C4‐2B cells which are androgen ablation‐resistant. In the C4‐2B cells, an increased susceptibility to V‐ATPase inhibitors was detected after longer treatments, as proliferation was reduced and reversibility of bafilomycin‐induced responses impaired. These findings make V‐ATPases attractive targets against early and advanced PCa tumors.