FK228阻断细胞生存信号通路诱导前列腺癌细胞凋亡

目的：研究组蛋白去乙酰化酶（HDAC）抑制剂FK228诱导前列腺癌细胞系DU145凋亡的作用机制。方法：MTT比色法测定FK228抑制DUl45细胞增殖及其对细胞的杀伤效应；瑞氏-姬姆萨染色观察细胞形态的变化；流式细胞术分析细胞周期的改变；蛋白印迹实验检测细胞内蛋白表达水平的变化。结果：FK228明显抑制DU145细胞体外增殖，并介导细胞死亡；12．5ng／ml FK228作用细胞48h后，细胞存活率降至63．7％；伴有细胞形态改变及细胞周期阻滞于G0／G1期；细胞内多种重要的激酶蛋白，包括EGFR、Her2、Raf-1、Src、Cdk4、Akt及凋亡抑制蛋白Survivin均发生了不同程度的降解，细胞内两条重要的生存信号通路Raf-MEK—ERK及P13K／Akt被阻断，细胞发生凋亡。结论：FK228可以通过清除细胞内重要信号蛋白、阻断细胞生存信号通路来诱导DU145细胞发生凋亡。     

曲古菌素A诱导前列腺癌DU-145细胞有丝分裂的灾变

目的：研究组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor,HDACi）曲古菌素（trichostatina A,TSA）对前列腺癌DU145细胞有丝分裂的影响,探讨HDACi杀伤肿瘤细胞的新机制。方法：将前列腺癌DC-145细胞分成不加药对照组和不同剂量（100、200、300、400nmol/L）TSA加药组,药物作用一定时间后,MTT法检测TSA对DU145细胞的杀伤效应,瑞氏-姬姆萨染色观察细胞形态的变化,流式细胞术分析细胞周期的改变,免疫荧光染色观察DU145细胞异常的有丝分裂现象,Western blotting检测TSA处理对DU145细胞某些调控蛋白表达的影响。结果：TSA处理诱导DU145细胞发生有丝分裂,TSA处理24h后多核细胞数目由0.24%增加至1.21%。细胞周期计数结果显示,TSA处理后有丝分裂各期细胞比例发生明显改变,表现为有丝分裂前中期细胞所占比例增加,后末期细胞所占比例减少。免疫荧光染色显示,细胞出现多极纺锤体、染色体分离滞后等异常有丝分裂现象。TSA作用于DU145细胞后,能明显抑制Survivin蛋白的表达,增强细胞骨架蛋白Tubulin的乙酰化,并诱导P21蛋白高表达。结论：TSA能够诱使DU145细胞发生有丝分裂灾变,其机制可能与TSA降低Survivin蛋白的表达以及增强微管蛋白的乙酰化有关。     

Downregulation of c-FLIP sensitizes DU145 prostate cancer cells to Fas-mediated apoptosis

Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasL(TET)) [Hyer et al., Molecular Therapy, 2000; 2:348-58 (ref.12)]. The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasL(TET) to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasL(TET) is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasL(TET) treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.     

Transforming growth factor ß1 induces apoptosis by suppressing FLICE‐like inhibitory protein in DU145 prostate epithelial cells

Transforming growth factor ß (TGFß) is a paracrine mediator of prostate epithelial cell apoptosis. In rodents, castration induces production of TGFβ by stromal cells, which leads to apoptosis of epithelial cells. To identify potential mediators of this cell death pathway, we developed a model using DU145 cells, a tumorigenic human prostate epithelial cell line. We discovered that at low density, in low mitogen media, DU145 cells apoptose when treated with TGFβ1. Prior to the onset of death, TGFβ1 treatment downregulated the expression of the caspase inhibitor FLICE‐like inhibitory protein (FLIP), at both the mRNA and protein level, suggesting a causal role between FLIP downregulation and cell death. To confirm the importance of FLIP in TGFβ1‐induced apoptosis, we employed small interfering RNA (siRNA) to silence FLIP expression. Doing so led to apoptosis, which is consistent with the hypothesis that FLIP prevents death in these cells. Furthermore, inhibition of caspase‐8 by siRNA knockdown partially rescued the apoptotic effects of TGFβ1, suggesting a role for death receptor signaling components in TGFß‐mediated death of prostate epithelial cells. © 2008 Wiley‐Liss, Inc.     

Phosphorylation of mitogen-activated protein kinase is inhibited by calcitonin in DU145 prostate cancer cells

One of the causes of insensitivity to androgen ablation therapy in prostate cancer is thought to be attributable to elevated neuropeptides secreted by neuroendocrine cells in the tumor mass. Calcitonin (CT), one of these neuropeptides, is reported to be associated with the growth of prostate cancer. There is an increase in mitogen-activated protein (MAP) kinase activation as prostate cancer progresses to a more advanced and androgen-independent disease. We examined the effect of CT on signal transduction and the relation between CT and early-response genes in the human androgen-insensitive prostate cancer cell line, DU145. The basal phosphorylation level of extracellular signal-regulated kinase 1/2, which is a key kinase in the mediation of growth factor-induced mitogenesis in prostate cancer cells, was constitutively up-regulated. N-[2-(4-bromocinnamyl) aminoethyl]-5-isoquinoline-sulfonamide (H89), a specific inhibitor of protein kinase A, potentiated the effects of more increased phosphorylation of extracellular signal-regulated kinase 1/2. CT induced the inhibition of this MAP kinase phosphorylation, and this effect was completely abolished by pretreatment with H89. Our findings demonstrate that CT caused the inhibition of constitutive MAP kinase phosphorylation in a protein kinase A-dependent manner in DU145. The transient increase of c-fos expression was detected after CT treatment, whereas expression of c-jun RNA was down-regulated after CT treatment. These results suggest that CT may regulate early-response genes, c-fos and c-jun, via a MAP kinase cascade. In conclusion, these findings suggest that DU145 might be a useful model as a therapeutic approach of neuropeptides in androgen-independent prostatic carcinoma.     

环王巴明诱导DU145细胞凋亡的机制研究

目的:研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制.方法:不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化.流式细胞术观察细胞凋亡率变化,RT-PCR检测5、10μmol/L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2 mRNA表达水平差异.结果:当环王巴明浓度大于5μmol/L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变.环王巴明5μmol/L组Gli-1、Bcl-2mRNA表达减弱,但同对照组差异无统计学意义(P>0.05),与空白及DMSO对照组相比10μmol/L环王巴明可以显著下调Gli-1、Bcl-2 mRNA表达(P<0.01).结论:环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2mRNA表达,活化细胞凋亡的线粒体途径有关.     

环王巴明诱导DU145细胞凋亡的机制研究

目的：研究环王巴明诱导DU145细胞凋亡作用及其对Gli-1和Bcl-2 mRNA表达的影响,探讨环王巴明诱导DU145细胞凋亡的机制。方法：不同浓度环王巴明处理DU145细胞后,吖啶橙染色,荧光显微镜下观察细胞形态变化。流式细胞术观察细胞凋亡率变化,RT—PCR检测5、10μmol／L环王巴明作用72h后的实验组和对照组Gli-1、Bcl-2mRNA表达水平差异。结果：当环王巴明浓度大于5μmol／L作用72h后,荧光显微镜下可见细胞核固缩或碎裂,核仁变形等典型的凋亡形态学改变。环王巴明5μmol／L组Gli-1、Bcl-2 mRNA表达减弱,但同对照组差异无统计学意义（P〉0．05）,与空白及DMSO对照组相比10μmol／L环王巴明可以显著下调Gli—1、Bcl-2 mRNA表达（P〈0．01）。结论：环王巴明可诱导人前列腺癌DU145细胞株凋亡,其作用机制可能与下调Gli-1和Bcl-2 mRNA表达,活化细胞凋亡的线粒体途径有关。     

Lysosomotropic acid ceramidase inhibitor induces apoptosis in prostate cancer cells

Purpose Alterations in ceramide metabolism have been reported in prostate cancer (PCa), resulting in escape of cancer cells from ceramide-induced apoptosis. Specifically, increased expression of lysosomal acid ceramidase (AC) has been shown in some primary PCa tissues and in several PCa cell lines. To determine if this represents a novel therapeutic target, we designed and synthesized LCL204, a lysosomotropic analog of B13, a previously reported inhibitor of AC Methods Prostate cancer cell lines were treated with LCL204 for varying times and concentrations. Effects of treatment on cytotoxicity, sphingolipid content, and apoptotic markers were assessed. Results Treatment of DU145 PCa cells resulted in increased ceramide and decreased sphingosine levels. Interestingly, LCL204 caused degradation of AC in a cathepsin-dependent manner. We also observed rapid destabilization of lysosomes and the release of lysosomal proteases into the cytosol following treatment with LCL204. Combined, these events resulted in mitochondria depolarization and executioner caspase activation, ultimately ending in apoptosis Conclusions These results provide evidence that treatment with molecules such as LCL204, which restore ceramide levels in PCa cells may serve as a new viable treatment option for PCa.     

Characterization of prostate cancer DU145 cells expressing the recombinant androgen receptor

Prostate cancer (PC) develops as a consequence of abnormal androgenic stimulation. Unfortunately, most of the PC cell lines are androgen independent (like DU145), or express mutated forms of androgen receptor (AR). We have produced and characterized a new stably transfected PC line expressing the AR (DU145-AR). Untreated DU145-AR cells showed a lower proliferation rate than mock transfected cells, but responded to testosterone treatment. PSA mRNA, undetectable in mock DU145 cells, was present and upregulated by testosterone in DU145-AR. About 5% of DU 145-AR cells showed modification of morphology and enriched of f-actin after testosterone treatment. Moreover, in DU145-AR plasminogen activator (PA) activity and secreted urokinase type plasminogen activator (uPA) protein were lower than in AR negative cells; again testosterone induced PA activity and uPA protein only in DU145-AR. These results indicate that, in general, the effects of unactivated AR is to suppress function(s) in DU145 cells and the addition of testosterone restores the normal properties associated with the untransfected cells. Some of the effects described may thus be mediated by a ligand-independent activation of AR in DU145 cells.     

Anisomycin activates JNK and sensitises DU 145 prostate carcinoma cells to Fas mediated apoptosis

Treatment of the hormone refractory prostate cancer cell line DU 145 with sublethal concentrations of chemotherapeutic drugs has been reported to sensitise these cells to Fas mediated apoptosis. However, the mechanism by which this occurs has not been determined. Our group has shown that inhibition of JNK activity completely abrogates the effects of chemotherapeutic drugs. Using anisomycin, a potent JNK agonist, we have demonstrated a role for JNK in Fas mediated apoptosis in DU 145 cells. Inhibition of Caspase 8 and Caspase 9 completely inhibits this process which suggests that DU 145 cells require mitochondrial amplification of the Fas apoptotic signal. Furthermore, we have shown that inhibition of Fas mediated apoptosis is an early event in DU 145 cells, occurring upstream of Caspase 8 cleavage. It is hoped that identifying the target of JNK will allow novel therapies to be developed for the treatment of hormone refractory prostate cancer. Such therapies are especially important because no single or combined treatment to date has significantly prolonged survival in patients with hormone refractory prostate cancer.     

人前列腺癌细胞系DU 145中肿瘤干细胞样侧群细胞的分离

目的:分离人前列腺癌细胞系DU 145中的侧群(side population,SP)细胞,并初步分析其生物学特性。方法:采用荧光激活细胞分类(fluorescence activated cell sorting,FACS)技术,从DU 145细胞中分离出侧群细胞,并检测其比例;继而培养于无血清培养基中,观察其生长特性。采用反转录聚合酶链反应(RT-PCR)技术检测侧群细胞中ABCG2的表达水平。结果:DU 1 4 5细胞中存在含量极少的侧群细胞,比例约1.1%;培养于无血清培养基中成簇生长。和对应的母系DU 145细胞相比,DU 145侧群细胞的ABCG2表达增高。结论:人前列腺癌细胞系DU 145中存在具有肿瘤干细胞特性的侧群细胞。     

UVB-activated indole-3-acetic acid induces apoptosis of PC-3 prostate cancer cells

Indole-3-acetic acid (IAA) has recently shown anticancer activity in combination with horseradish peroxidase. The current study demonstrated that IAA irradiated with ultraviolet B (IAA(UVB)) is able to generate free radicals and induce cell death in a time-dependent fashion in PC-3 prostate cancer cells, while PC-3 cells treated with IAA alone exhibited no toxic responses. It was also found through Western blot analysis that the cytotoxic effect of IAA(UVB) resulted from apoptosis. Treatment with IAA(UVB) for 24 hours showed a significant increase in phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, the stress signaling proteins. Furthermore, pro-caspases (-3, -8, and -9) were clearly down-regulated and poly(ADP-ribose) polymerase cleavages were demonstrated in the group treated with IAA(UVB). Flow cytometric analysis also demonstrated the induction of apoptosis by IAA(UVB) in PC-3 cells. In conclusion, this study demonstrated that IAA induced cell death in combination with UVB irradiation by increasing apoptosis in PC-3 cells.     

Defects in death-inducing signalling complex formation prevent JNK activation and Fas-mediated apoptosis in DU 145 prostate carcinoma cells

Androgen-independent prostate carcinomas are resistant to chemotherapy and cell lines derived from androgen-independent prostate carcinomas such as DU 145 cells are highly resistant to Fas-mediated apoptosis. The incubation of DU 145 cells with anti-Fas IgM agonistic antibody of Fas receptor fails to activate JNK, a stress kinase involved in regulating apoptosis. We have previously shown that JNK activation is sufficient and necessary to promote Fas-mediated apoptosis in DU 145 cells. We investigate the mechanisms by which JNK activation and apoptosis are abrogated. HSP27 is overexpressed in DU 145 cells and has previously been reported to sequester DAXX and prevent JNK activation in cells treated with anti-Fas IgM. However, we find no evidence that HSP27 interacts with DAXX in DU 145 cells. Instead, we find that FADD does not interact with caspase-8 and this results in defective death-inducing signalling complex formation following Fas receptor activation.     

Endogenous secretion of epidermal growth factor peptides stimulates growth of DU145 prostate cancer cells

The growth rate of DU145 prostate cancer cells in vitro is slowed considerably by changing the growth medium every 24 h, suggesting dependence upon endogenously-secreted growth factors. Because previous studies have identified epidermal growth factor (EGF) in the conditioned medium from DU145 cells, [35S]labeled EGF was selectively immunoprecipitated from the culture medium at 24-h intervals for quantitation. Under the culture conditions used, there was an initial phase of slow growth, the EGF level secreted per cell was highest on day 3 after plating, and an increase in cell number was most evident between days 3 and 4. Finally, growth was assayed under culture conditions where the medium was replaced every 24 h with fresh medium in the absence or presence of 10 ng/ml added EGF. The EGF was able to increase the cell growth up to the levels seen in cultures where the medium was unchanged during the entire period. We interpret these results as evidence that endogenously secreted EGF-like growth factors participate in an autocrine growth stimulation of DU145 cells.     

蛋白酶体抑制剂通过内质网应激影响前列腺癌DU145细胞的生长

目的:探讨蛋白酶体抑制剂(epoxomicin,EPO）可否诱导前列腺癌DU145细胞产生内质网应激以及内质网应激阻断剂是否可阻断这种作用。方法:不同浓度EPO处理DU145不同时间,MTS检测细胞生长情况,流式细胞仪检测细胞凋亡情况,Real-time PCR检测内质网应激相关分子mRNA表达情况;以及内质网应激阻断剂4-PBA阻断内质网应激后的情况。结果:EPO抑制DU145细胞生长,并呈现浓度梯度依赖;EPO处理组细胞凋亡率明显高于正常组,且EPO 20nmol组凋亡率高于10nmol组;CHOP、XBP-1s、GRP78 mRNA明显升高,72h最为明显;XBP-1及XBP-1s基因转录水平在EPO处理后都升高,而XBP-1s随着处理时间逐渐增加,XBP-1随着时间基因转录水平变化不明显;4-PBA可阻断CHOP mRNA表达,阻断EPO对DU145细胞数量的减少。结论:EPO抑制DU145细胞生长,并促进凋亡,其机制可能与激活内质网应激并激发CHOP、XBP-1s、XBP-1、GRP78分子表达等有关;4-PBA可阻断EPO对细胞生长抑制的影响。     

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.     

Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells

Development of effective agents for treatment of hormone-refractory prostate cancer has become a national medical priority. We have reported recently that apigenin (4',5,7-trihydroxyflavone), found in many common fruits and vegetables, has shown remarkable effects in inhibiting cell growth and inducing apoptosis in many human prostate carcinoma cells. Here we demonstrate the molecular mechanism of inhibitory action of apigenin on androgen-refractory human prostate carcinoma DU145 cells that have mutations in the tumor suppressor gene p53 and pRb. Treatment of cells with apigenin resulted in a dose- and time-dependent inhibition of growth, colony formation, and G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk)2, 4, and 6, with concomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. The induction of WAF1/p21 and its growth inhibitory effects by apigenin appears to be independent of p53 and pRb status of these cells. Apigenin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in a significant increase in cleaved fragments of caspase-9, -3, and poly(ADP-ribose) polymerase (PARP). Further, apigenin treatment resulted in downmodulation of the constitutive expression of nuclear factor-kappaB (NF-kappaB)/p65 and NF-kappaB/p50 in the nuclear fraction that correlated with an increase in the expression of IkappaB-alpha (IkappaBalpha) in the cytosol. Taken together, we concluded that molecular mechanisms during apigenin-mediated growth inhibition and induction of apoptosis in DU145 cells was due to (1) modulation in cell-cycle machinery, (2) disruption of mitochondrial function, and (3) NF-kappaB inhibition.     

Pancratistatin induces apoptosis and autophagy in metastatic prostate cancer cells

The Amaryllidaceae alkaloid pancratistatin has been proven to selectively induce apoptotic cell death in a variety of human cancer cells with an insignificant effect on non-cancerous cells. In this study we report, for the first time, the effects of pancratistatin (PST) on models of metastatic prostate cancer. The effects of pancratistatin on prostate cancer DU145 and LNCaP cell lines was assessed by microscopy, enzymatic activity assays and Western blotting. Apoptosis was determined by nuclear condensation and caspase activation, and autophagy was observed by MDC staining and LC3 expression levels. Human prostate xenografts were used to test the potential therapeutic efficacy of intra-tumor administration of pancratistatin in vivo. Pancratistatin treatment reduced cell viability and induced apoptosis in androgen-responsive (LNCaP) and androgen-refractory (DU145) prostate cancer cell lines in a dose- and time-dependent manner, but with an insignificant effect on normal human fibroblast (NHF) cells at the effective dose. Increased reactive oxygen species production and collapse of mitochondrial membrane potential resulted from treatment with pancratistatin in both cancer cell lines. This study presents the novel finding that pancratistatin treatment caused decreased migration capacity and increased autophagy levels in metastatic prostate cancer cells. Importantly, in this proof-of-concept study, pancratistatin reduced the volume of xenograft tumors compared to control-treated animals, and was well-tolerated. Our results highlight the potential of pancratistatin for clinical development as a selective therapeutic for treatment of metastatic prostate cancer.