Bcl-2 antagonizes the combined apoptotic effect of transforming growth factor-beta and dihydrotestosterone in prostate cancer cells

BACKGROUND: We previously demonstrated that dihydrotestosterone (DHT) enhances transforming growth factor-beta (TGF-beta) -induced apoptosis in human prostate cancer cells (Endocrinology 2001;142:2419-2426). METHODS: In this study, the ability of the apoptosis suppressor bcl-2 to directly antagonize the combined apoptotic effect of TGF-beta and DHT in the androgen-sensitive LNCaP TbetaRII prostate cancer cells was examined. The previously cloned TGF-RbetaII receptor LNCaP cells, responsive to both TGF-beta and androgens, were engineered to overexpress the bcl-2 oncoprotein and the profile of apoptosis induction was analyzed in response to TGF-beta alone (5.0 ng/ml) or in combination with DHT (1 nM). RESULTS: Biological characterization of cloned LNCaP TbetaRII hygromycin/bcl-2 transfectants demonstrated that bcl-2 overexpression resulted in a significant inhibition of the combined TGF-beta and DHT apoptotic effect in prostate cancer cells (P < 0.01). Furthermore, molecular analysis indicated that this antagonistic effect of bcl-2 on apoptosis was due to partial suppression of TGF-beta and DHT-mediated induction of caspase-1 expression and activation in LNCaP TbetaRII-hygro/bcl-2 transfectants. These results support a potential bcl-2 interference with the TGF-beta and androgen apoptotic signaling in prostate cancer cells by means of an antagonistic effect on caspase-1 activation. CONCLUSION: This evidence may have mechanistic significance in understanding the contribution of bcl-2 overexpression in the development of androgen-independent prostate cancer by means of conferring resistance to TGF-beta-mediated apoptosis.     

Expression of human AR cDNA driven by its own promoter results in mild promotion, but not suppression, of growth in human prostate cancer PC-3 cells

Aim： To examine the physiological role of the androgen receptor （AR） in the PC-3 cell line by transfecting full-length functional AR cDNA driven by its natural human AR promoter. Methods： We generated an AR-expressing PC-3（AR）9 stable clone that expresses AR under the control of the natural human AR promoter and compared its proliferation to that of the PC-3（AR）2 （stable clone that expresses AR under the control of the cytomegalovirus （CMV） promoter, established by Heisler et al.） after androgen treatment. Results： We found that dihydrotestosterone （DHT） from 0.001 nmol/L to 10 nmol/L induces cell cycle arrest or inhibits proliferation of PC-3（AR）2 compared with its vector control, PC-3（plRES）. In contrast, PC-3（AR）9 cell growth slightly increased or did not change when treated with physiological concentrations of 1 nmol/L DHT. Conclusion： These data suggest that intracellular control of AR expression levels through the natural AR promoter might be needed for determining AR function in androgen-independent prostate cancer （AIPC） PC-3 cells. Unlike previous publications that showed DHT mediated suppression of PC-3 growth after transfection of viral promoter-driven AR overexpression, we report here that DHT-mediated PC-3 proliferation is slightly induced or does not change compared with its baseline after reintroducing AR expression driven by its own natural promoter, as shown in PC-3（AR）9 prostate cancer cells.     

前列腺雄激素调节基因:一个新的雄激素非依赖性前列腺癌治疗的潜在靶点(英文)

目的:初步研究前列腺雄激素调节基因(PAR)与雄激素—雄激素受体信号转导通路的关系及其在前列腺癌细胞恶性转化过程中的作用,探讨通过抑制 PAR 基因治疗雄激素非依赖性前列腺癌的可能性。方法:用 RT-PCR 检测 LNCaP、PC3细胞中 PAR 基因 mRNA 表达水平的差异。分别用 RT-PCR 检测双氢睾酮对LNCaP、PC3及稳定转染了 pcDNA3-AR 的 PC3细胞株 PC3-AR 的 PAR 基因 mRNA 表达的调节作用,并观察这一调节作用是否可被雄激素受体拮抗剂氟他胺阻断。进一步用 RNA 干扰技术下调 PC3细胞 PAR 的表达,用细胞计数、软琼脂克隆形成实验、流式细胞术研究 PAR 基囚表达下调对 PC3细胞生长的抑制作用。结果:PC3细胞 PAR 基因 mRNA 的表达是 LNCaP 细胞的3倍;双氢睾酮可调节 LNCaP 和 PC3-AR 细胞株PAR 基因 mRNA 表达水平,此种对 PAR 表达的调节作用可被氟他胺阻断:双氢睾酮对 PC3细胞 PAR 基因mRNA 表达无明显影响。RNA 干扰可抑制 PC3细胞 PAR 基因表达,使细胞增殖受抑制,细胞周期阻滞于G_2-M 期,凋亡增加。结论:PAR 可能是雄激素—雄激素受体信号转导通路下游的与雄激素非依赖性前列腺癌恶性表型密切相关的癌基因,有望成为其基冈和药物治疗的靶点。     

昆布多糖硫酸酯对PC-3细胞PTEN和P27kip1基因表达的影响

目的:研究昆布多糖硫酸酯(LAMS)对体外培养的非激素依赖型人前列腺癌细胞株PC-3的作用,以及对PC-3细胞PTEN和P27kip1基因表达的影响,并探讨其抗肿瘤的作用机制。方法:分别用0、50、100、200μg/ml浓度的LAMS作用于PC-3细胞,用WST-8法检测其对细胞生长的抑制作用,利用流式细胞术(FCM)分析细胞周期和凋亡的变化,用荧光显微镜观察PC-3细胞形态的变化,RT-PCR和Western印迹检测细胞增殖、凋亡相关基因PTEN和P27kip1mRNA及蛋白的表达。结果:LAMS能抑制PC-3细胞的增殖,呈时间-剂量效应;不同浓度LAMS诱导PC-3细胞出现剂量依赖性S期阻滞;LAMS作用于PC-3细胞后凋亡率增加,并出现凋亡的形态学改变,相关基因PTEN和P27kip1mRNA及蛋白的表达呈剂量依赖性增加。结论:LAMS能抑制体外PC-3细胞的生长,并促进其S期阻滞和凋亡,PTEN和P27kip1mRNA及蛋白的表达明显增加,可能是其抑制前列腺癌生长的机制之一。     

Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells.

BACKGROUND: Prostate cancer is a worldwide significant health care problem, due to its high incidence and mortality. In particular, androgen-independent tumors have the worst prognosis, because they are refractory to almost all kinds of available therapy. Hence, there is the need of new treatment opportunities targeting androgen-independent, growth factor-mediated, tumor signaling. One of these new promising opportunities is vitamin D3 and its related analogues. METHODS: We investigated the effect of a vitamin D3 analogue, analogue (V), on proliferation of several human prostate cancer cells in basal condition and after treatment with KGF, one of the intraprostatic growth factors that might participate in the progression of prostate cancer. In addition, in the androgen-independent cell line DU 145, we also studied the effect of analogue (V), KGF, and their mutual interaction on protein tyrosine phosphorylation, bcl-2 expression and apoptosis. RESULTS: Overall, we found that analogue (V) dose-dependently decreased basal and KGF-induced prostate cancer cell growth, although to a different extent. Maximal effect was obtained in DU 145 cells. In these cells, KGF stimulated tyrosine phosphorylation of a protein corresponding to its receptor, induced bcl-2 expression, and prolonged cell survival. Analogue (V) not only counteracted all these KGF-mediated events, but also decreased basal bcl-2 expression, therefore, allowing DU 145 cells to undergo an apoptotic program. CONCLUSIONS: Our results indicated that in prostate cancer cells analogue (V) decreased basal and KGF-induced cell proliferation. This effect, at least in DU 145 cells, is in part mediated by negative interactions with cell survival and KGF signaling.     

米非司酮诱导雄激素非依赖性前列腺癌细胞凋亡的实验研究

目的探讨米非司酮在体外诱导雄激素非依赖性前列腺癌DU-145、PC-3细胞凋亡的作用。方法采用四甲基偶氮唑蓝法检测1,10,50和100μmol／L米非司酮作用于前列腺癌DU-145、PC-3细胞24～120h的吸光度（A）值,用流式细胞仪检测10μmol／L米非司酮作用24和48h后DU．145、PC-3细胞凋亡率的变化;采用免疫组化法检测米非司酮作用DU-145、PC．3细胞后bax、bcl-2、血管内皮生长因子（VEGF）蛋白表达的变化。结果1μmol／L米非司酮组的A值与对照组相比,差异无统计学意义（P〉0．05）;10,50和100μmol／L米非司酮组的A值与对照组比较,差异有统计学意义（P〈0．01）;米非司酮对前列腺癌DU-145、PC-3细胞的抑制作用呈时间-剂量依赖性。10μmol／L米非司酮作用前列腺癌DU-145细胞24和48h的凋亡率分别为15．3％和30．4％,PC-3细胞的凋亡率分别为22．2％和32．0％。经10μmol／L米非司酮作用后,DU-145、PC-3细胞中VEGF和bcl-2蛋白的表达明显减少,而bax的表达显著增加（P〈0．05）。结论米非司酮以时间．剂量依赖性方式抑制激素非依赖性前列腺癌DU-145和Pc-3细胞的增殖,其作用可能是通过降低VEGF蛋白的表达。从而下调bcl-2、激活bax蛋白的表达来实现。     

Differential mechanisms of bicalutamide-induced apoptosis in prostate cell lines

Bicalutamide is a non-steroidal antiandrogen used in the treatment of prostate cancer. Although widely accepted as an androgen receptor antagonist, the mechanism by which it induces apoptosis remains unclear. Defining exact pathways by which bicalutamide induces its apoptotic effects would help to advance its clinical applications. We aimed to (a) examine the apoptotic effects of bicalutamide at 24 h and (b) comment on the role of the caspases and calpains in mediating bicalutamide-induced apoptosis in androgen-dependent and androgen-independent cells. PWR-1E, PC-3 and DU-145 cells were treated with bicalutamide and assessed for apoptosis by flow cytometry at 24 h. DU-145 cells were used to compare differences between two different metastatic receptor-negative cells and to verify apoptotic induction at 48 h. To delineate a specific pathway of action for bicalutamide, PC-3 and PWR-1E cells were pretreated with specific inhibitors of caspase-dependent (zVAD-FMK) and caspase-independent pathways (calpain 2 inhibitor). Bicalutamide induced apoptosis in androgen-dependent PWR-1E cells via a caspase-dependent and calpain-independent mechanism. In androgen-independent PC-3 cells, bicalutamide also induced apoptosis by mechanisms that were partially inhibited by pan-caspase inhibition but were partially calpain dependent. Understanding into how bicalutamide exerts its effects in androgen-independent cells will yield further insights into the treatment of hormone-refractory disease.     

Mitogenic signaling in androgen sensitive and insensitive prostate cancer cell lines

PURPOSE: To investigate the role of a specific mitogen activated protein kinase, extracellular signal-regulated kinase (ERK), in regulating cell proliferation induced by three potentially important prostate cancer mitogens that signal via different classes of receptors. MATERIALS AND METHODS: Androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cell lines were used in these studies. Epidermal growth factor (EGF), lysophosphatidic acid (LPA), and dihydrotestosterone (DHT) were the mitogenic stimulants and AG1478, a receptor tyrosine kinase inhibitor, and PD98059, an inhibitor of MEK, were the chemical inhibitors used in this study. Cell proliferation was measured using the WST-1 assay and ERK expression and activation was determined by immunoblotting for phospho- and total ERK. RESULTS: In androgen-sensitive LNCaP cells, epidermal growth factor (EGF) and dihydrotestosterone (DHT) both enhanced cell proliferation. EGF-stimulation dramatically increased ERK phosphorylation while DHT did not. In the androgen-insensitive cell line, PC-3, EGF- and LPA-induced ERK phosphorylation and cell proliferation. Inhibition of EGF- and LPA- induced ERK activation with the EGF receptor inhibitor, AG1478, or the MEK inhibitor, PD98059, attenuated their proliferative effects. Neither inhibitor had an effect on DHT stimulated cell proliferation. CONCLUSIONS: These data demonstrate heterogeneity of mitogenic signaling in prostate cancer cells, and support the hypothesis that androgens and growth factors utilize divergent signaling pathways in prostate cancer to induce proliferation.     

C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells

BACKGROUND: Adenocarcinoma of the prostate is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths among men in the United States. The most successful therapies to date for this tumor have involved some form of androgen ablation. However, these therapies become ineffective as the tumor evolves to an androgen-insensitive state. Ceramide is a lipid second messenger that has been shown to mediate growth arrest or cell death when added exogenously to prostate cancer cells. As a first step toward understanding the events that lead to the transition of prostate cancer cells to an androgen-independent state, we considered investigating the effect of androgen ablation on endogenous ceramide levels in androgen-sensitive and androgen-insensitive prostate cancer cells. METHODS: To investigate the mechanisms of growth arrest/apoptosis in androgen-sensitive (LNCaP) and insensitive (DU-145, PC-3) cells, we used various methods including nonyl acridine orange (NAO) staining, propidium iodide (PI) staining/cell-cycle analysis, lipid analysis, and Western blotting assays. RESULTS: In this study, we demonstrate that androgen ablation drives G(0)/G(1)-phase cell-cycle arrest followed by progressive apoptosis in vitro, in LNCaP cells. Lipid analysis indicated an increase in C16 ceramide, which was generated via the de novo pathway as revealed by blockade of ceramide synthase by fumonisin B1. The addition of 5alpha-dihydrotestosterone (DHT) or fumonisin B1 rescued LNCaP cells from apoptosis induced by androgen ablation, and decreased levels of intracellular C16 ceramide. Neither apoptosis nor an increase in C16 ceramide was observed in androgen-independent cell lines following androgen ablation.     

Dutasteride, the dual 5alpha-reductase inhibitor, inhibits androgen action and promotes cell death in the LNCaP prostate cancer cell line

BACKGROUND: Reduction of T to DHT by 5alphaR in the prostate enhances androgenic activity for most targets. Inhibition of 5alphaR activity with finasteride attenuates androgen action in men and animal models. The objective of this study was to compare and contrast the effects of a potent new 5alphaR inhibitor, dutasteride, with finasteride in the LNCaP prostate cancer cell line. METHODS: LNCaP cells were incubated for varying times with T or DHT in steroid-free medium in the absence or presence of increasing doses of dutasteride or finasteride and the effects on 5alphaR activity, PSA accumulation in the medium, and on cell proliferation were determined. Drug effects on apoptosis were investigated using Annexin V staining and a cell death ELISA assay. Effects of the drugs on AR ligand-binding activity and on AR protein levels were determined. RESULTS: Dutasteride inhibited (3)H-T conversion to (3)H-DHT and, as anticipated, inhibited T-induced secretion of PSA and proliferation. However the drug also inhibited DHT-induced PSA secretion and cell proliferation (IC(50) approximately 1 microM). Finasteride also inhibited DHT action but was less potent than dutasteride. Dutasteride competed for binding the LNCaP cell AR with an IC(50) approximately 1.5 microM. High concentrations of dutasteride (10-50 microM), but not finasteride, in steroid-free medium, resulted in enhanced cell death, possibly by apoptosis. This was accompanied by loss of AR protein and decreased AR ligand-binding activity. Occupation of AR by R1881 partly protected against cell death and loss of AR protein. PC-3 prostate cancer cells, which do not contain AR, also were killed by high concentrations of dutasteride, as well as by 50 microM finasteride. CONCLUSIONS: Dutasteride exhibited some inhibitory actions in LNCaP cells possibly related to 5alphaR inhibition but also had antiandrogenic effects at relatively low concentrations and cell death-promoting effects at higher concentrations. Finasteride also was antiandrogenic, but less than dutasteride. The antiandrogenic effects may be mediated by the mutant LNCaP cell AR. Promotion of cell death by dutasteride can be blocked, but only in part, by androgens.     

Resveratrol sensitizes androgen independent prostate cancer cells to death-receptor mediated apoptosis through multiple mechanisms

BACKGROUND: A critical factor in prostate cancer development and progression is the altered expression of apoptotic regulatory proteins which renders cells resistant to both hormone- and chemo-therapies. Resveratrol, a dietary component with chemopreventive properties has been reported to resensitize a variety of cancer cell types to apoptosis. In the current study, the ability of resveratrol pre-treatment to sensitize hormone refractory prostate cancer cell lines (PC-3 and DU145) to apoptosis and the mechanisms involved were investigated. METHODS: Apoptosis was assessed using several established parameters and protein expression was analyzed by Western blot and flow cytometry. IAP knockdown was achieved using RNAi while inhibition of Akt phosphorylation was achieved by pre-incubation with the PI3-kinase inhibitor LY294002. RESULTS: Pre-treatment with resveratrol sensitized PC-3 and DU145 cells to agents that specifically target death receptors (TRAIL, Fas, TNFalpha) but not agents that initiate apoptosis through other mechanisms (Etoposide, Paclitaxel, Tunicamycin, Thapsigargin). Resveratrol pre-treatment altered the expression of IAPs and Bax, and decreased Akt phosphorylation in PC-3 cells, leading to increased caspase activation and apoptosis. While knockdown of IAPs using siRNA did not mimic the effects of resveratrol, inhibition of Akt phosphorylation using LY294002 sensitized PC-3 cells to TRAIL induced apoptosis but not to etoposide or tunicamycin. CONCLUSION: Altering apoptotic susceptibility in advanced androgen independent disease requires manipulation of a broad signaling pathway. Use of resveratrol or inhibition of Akt phosphorylation may represent an important therapeutic approach in combination with conventional therapies for the treatment of prostate cancer.     

Downregulation of androgen receptor expression by luteolin causes inhibition of cell proliferation and induction of apoptosis in human prostate cancer cells and xenografts

BACKGROUND: Androgen receptor (ARs) play a crucial role in the development and progression of prostate cancer. Recent studies have suggested that prostate cancer cell proliferation is inhibited by AR downregulation. Our aim was to investigate how luteolin, a natural flavonoid, affects cell growth and AR expression in prostate cancer cells and xenografts. METHODS: We assessed prostate cancer cell (LNCaP, DU145, and PC-3) proliferation and apoptosis by MTT assay, flow cytometric analysis, and Western analysis. AR function was measured by evaluating the AR target molecule, prostate-specific antigen (PSA), by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. We determined the mechanism of AR downregulation with cycloheximide chase assays, proteasome inhibitor, and coimmunoprecipitation experiments. The effects of luteolin on growth inhibition in vivo were examined by LNCaP xenografts in SCID mice. RESULTS: Luteolin significantly repressed prostate cancer cell proliferation and induced apoptosis in LNCaP cells. PC-3 and DU145 cells were less susceptible to luteolin-mediated growth inhibition. Luteolin simultaneously suppressed intracellular and secreted PSA levels and repressed AR mRNA and protein expression in a dose- and time-dependent manner. Luteolin reduced the association between AR and heat-shock protein 90, causing AR degradation through a proteasome-mediated pathway in a ligand-independent manner. Luteolin also suppressed LNCaP xenograft tumor growth in SCID mice. CONCLUSION: Luteolin-mediated AR downregulation contributes to the inhibition of cell proliferation and the induction of apoptosis in LNCaP human prostate cancer cells, suggesting that AR is a molecular target for luteolin-mediated anticancer activity. Luteolin may act as a chemopreventive or chemotherapeutic agent for prostate cancer.