紫龙金对前列腺癌细胞系DU-145的体外作用

目的　探讨中药紫龙金 (ZLJ)对雄激素非依赖型人前列腺癌细胞系DU 14 5的体外作用及其机制。方法　应用四甲基偶氮唑蓝 (MTT)比色法、软琼脂集落生长试验和流式细胞术测定ZLJ对DU 14 5的增殖抑制、集落生长抑制、周期阻滞和诱导凋亡作用 ,应用逆转录 聚合酶链反应 (RT PCR)和Westernblot方法检测对凋亡相关基因 /蛋白bcl 2和bax ,抑癌基因 /蛋白p16表达的影响。结果　ZLJ具有时间和剂量依赖性增殖抑制、G0 /G1期阻滞和诱导凋亡作用 ,作用 72hIC50 为 0 .42g/L ,ZLJ 0 .1g/L作用 14d对DU 14 5细胞集落生长抑制率为 87.9% ,ZLJ 0 .5 g/L作用 2dG0 /G1期细胞比例从 41.3 0 %增高至 76.70 %。ZLJ可以下调bcl 2基因 /蛋白 ,上调bax和p16基因 /蛋白的表达。 结论　ZLJ可能通过抑制DU 14 5细胞的集落生长、增殖抑制、G0 /G1期阻滞、诱导凋亡、下调bcl 2基因 /蛋白 ,上调bax和 p16基因 /蛋白表达等作用机制发挥抗肿瘤作用。     

人抑癌基因PTEN对前列腺癌细胞系生物学行为的影响

目的　探讨外源性人抑癌基因PTEN表达对前列腺癌细胞系LNCaP、DU 14 5细胞增殖和侵袭转移能力的影响。　方法　利用携带人PTEN基因的可调控性腺病毒 (Ad PTEN ) ,体外转染人前列腺癌细胞系LNCaP、DU 14 5,RT PCR、Westernblot检测目的基因不同水平的表达 ,通过细胞生长试验、流式细胞仪分析技术以及Boyden小室法检测LNCaP、DU 14 5转染前后细胞增殖、细胞周期、细胞凋亡和细胞体外侵袭力的变化。　结果　转染Ad PTEN后LNCaP、DU 14 5细胞的PTEN表达由阴性转为阳性 ,转染后对LNCaP、DU 14 5细胞生长有抑制作用 ,阻滞于G0 ～G1期 ,早期细胞凋亡率增加 ,LNCaP细胞 (6.89± 0 .51) % ;DU14 5细胞 (5.44± 1.13 ) % ,与对照组相比差异有显著性 ,Boyden小室法检测转染后体外侵袭力受到明显抑制。 　结论　重组腺病毒介导的人PTEN基因在体外对前列腺癌细胞系LNCaP、DU 14 5的细胞增殖和体外侵袭力有明显抑制作用 ,可望作为前列腺癌基因治疗的有效工具     

熊果酸对前列腺癌细胞雄激素非依赖性的逆转作用

目的 探讨中药成分熊果酸对雄激素非依赖性前列腺癌(AIPC)的治疗作用及其机制.方法 应用熊果酸处理体外培养的人雄激素依赖性前列腺癌(ADPC)细胞株LNCaP和AIPC细胞株DU145,噻唑蓝(MTT)比色法检测细胞活性及对人工合成雄激素R1881的反应性,免疫细胞化学检测熊果酸对雄激素受体(AR)、糖皮质激素受体(GR)、前列腺特异性抗原(PSA)及成活因子HSP90和白细胞介素(IL)-6表达的影响,逆转录.聚合酶链反应(RT-PCR)检测熊果酸对DU145细胞AR mRNA表达的影响.结果 熊果酸对不同浓度雄激素下的LNCaP细胞均呈浓度和时间依赖性生长抑制,20 mg/L的熊果酸作用96 h对LNCaP细胞的抑制率近50%.0.1 nmoL/L的R1881为最适生长浓度,熊果酸作用后,LNCaP细胞生长的最适雄激素浓度上升了10倍;熊果酸对DU145细胞的生长有浓度和时间依赖性抑制效应,DU145细胞对AR阻断剂羟氟他胺缺乏反应,熊果酸作用同时再应用氟他胺比单纯熊果酸的作用更明显,对细胞抑制率明显上升.熊果酸作用后,LNCaP和DUl45细胞IL-6、HSF90表达均明显下降(P＜0.05),DU145细胞GR表达明显降低(P＜0.01),AR和PSA蛋白及AR mRNA出现再表达.结论 熊果酸能改善前列腺癌细胞对雄激素的反应性,使LNCaP细胞对雄激素的依赖性加强,并诱发了DU145细胞对雄激素的反应性,其部分机制是降低了GR、HSP90、IL-6的表达并促进AR再表达.     

靶向TRPV6的siRNA对前列腺癌LNCaP细胞抑制作用的体外研究

目的:运用RNA干扰(RNAi)技术阻断前列腺癌LNCaP细胞中瞬时感受器电位离子通道蛋白V亚家族6(TRPV6)基因的表达,探讨TRPV6基因沉默对LNCaP细胞增殖、细胞周期及凋亡的影响。方法:针对TR-PV6基因,构建2条小干扰RNA(siRNA)序列;在脂质体介导下转染前列腺癌LNCaP细胞,用RT-PCR测定TRPV6mRNA的表达;MTT法和流式细胞技术测定siRNA对LNCaP细胞增殖、细胞周期及凋亡的影响。结果:两条siRNA序列均能有效地阻断LNCaP细胞中TRPV6基因在mRNA水平上的表达(P<0.01),且mRNA表达随着转染时间的延长而减少,转染72h后TRPV6mRNA的表达减少至对照组的27%和23%;siRNA转染能显著抑制LNCaP细胞增殖,转染48h细胞增殖抑制率最高,分别为34.53%和29.32%,与转染24h和72h比较有统计学意义(P<0.05);转染48h后,siRNA转染组G0～G1期细胞增多,S期细胞数量明显减少,与空白对照组及阴性对照组相比有统计学差异(P<0.01);siRNA转染组细胞的凋亡率分别为14.45%和12.73%,显著高于空白对照组及阴性对照组3.78%和5.22%(P<0.05)。结论:针对TRPV6的siRNA在体外能有效地抑制LNCaP细胞TRPV6mRNA的转录,同时能够抑制前列腺癌LNCaP细胞增殖,使细胞周期出现G0/G1期阻滞,细胞凋亡率显著增加。     

苦参碱对前列腺癌细胞增殖及雄激素受体功能的抑制作用

目的:探讨苦参碱(matrine)对雄激素依赖性前列腺癌细胞株(LNCaP)的增殖及雄激素受体(androgen re-ceptor,AR)表达的抑制作用。方法:分别用0.5、1.0、1.5、2.0、3.0g/L浓度的苦参碱作用于LNCaP细胞12、24、36h后MTT法检测细胞生长活性;台盼蓝拒染法测定细胞生长曲线;24h后流式细胞仪测定细胞周期变化;24h后Western印迹法检测细胞内AR的表达。结果:苦参碱能抑制LNCaP细胞的生长,呈剂量与时间依赖性,不同浓度苦参碱组之间与不同作用时间组之间的差异均有显著性意义(P<0.01)。苦参碱诱导LNCaP细胞出现剂量依赖性G2/M期阻滞(P<0.01);细胞内AR的表达随苦参碱剂量依赖性减少(P<0.01)。结论:苦参碱通过下调细胞内AR表达和阻滞细胞周期进展来抑制LNCaP细胞的体外生长。     

左旋棉酚体外诱导人前列腺癌LNCaP细胞凋亡的研究

目的研究左旋棉酚对人前列腺癌LNCaP细胞体外增殖和凋亡的影响。方法 MTT法检测左旋棉酚对LNCaP细胞增殖的影响,透射电镜观察细胞超微结构的改变,TUNEL染色观察细胞凋亡,流式细胞术检测细胞周期的改变,半定量RT-PCR检测Bcl-2及BakmRNA的表达水平。结果左旋棉酚在体外能显著抑制LNCaP细胞增殖,呈浓度-时间依赖性。左旋棉酚可以诱导LNCaP细胞发生典型的凋亡形态学改变和G0/G1期阻滞。RT-PCR显示Bcl-2mRNA表达水平降低,BakmRNA表达水平升高。结论左旋棉酚在体外能诱导前列腺癌细胞凋亡,其主要原因可能与BakmRNA的表达升高及Bcl-2mRNA的表达下调有关。     

苦参碱对雄激素依赖性前列腺癌细胞的影响

目的：探讨苦参碱（Matrine）对雄激素依赖性前列腺癌细胞株（LNCaP）凋亡及前列腺特异抗原（prostate specific antigen,PSA）表达的影响。方法：分别用0.5g／L、1.0g／L、1.5g／L、2.0g／L浓度的苦参碱作用于LNCaP细胞12h、24h、36h后MTT法检测细胞生长活性;24h后流式细胞仪测定细胞凋亡的变化;24h后Western印迹法检测细胞内Bel-2和Bax的表达;12h、24h、36h后化学发光法检测LNCaP细胞培养液中PSA的变化。结果：苦参碱能抑制LNCaP细胞的生长,呈剂量与时间依赖性,不同浓度苦参碱组之间与不同作用时间组之间的差异均有统计学意义（P〈0.05）。苦参碱诱导LNCaP细胞凋亡,各浓度组凋亡细胞比例均显著高于对照组,差异有统计学意义（P〈0.05）;LNCaP细胞内Bcl-2含量呈浓度依赖性下降,Bax含量呈浓度依赖性升高（P〈0.01）;LNCaP细胞培养液中PSA的表达显著下降（P均〈0.05）。结论：苦参碱能显著抑制LNCaP细胞的体外生长,诱导其凋亡,并抑制PSA的表达。     

前列腺癌细胞凋亡通路基因表达与凋亡反应性的关系

目的　探讨凋亡通路基因表达改变与前列腺癌细胞凋亡反应性的关系。　方法　凋亡诱导剂鬼臼乙叉甙分别作用前列腺癌激素敏感与不敏感细胞LNCaP和PC 3,Hoechst 332 5 8染色检测凋亡发生率。收集LNCaP和PC 3细胞后提取总RNA ,合成cDNA探针并以生物素标记 ,在凋亡通路特异基因寡核苷酸片段cDNA膜上进行杂交反应 ,检测基因mRNA表达。　结果　鬼臼乙叉甙能诱导两种细胞凋亡 ,但PC 3细胞的凋亡反应性小于LNCaP细胞。与LNCaP细胞比较 ,PC 3细胞显著下调的基因为Bcl1 0、CIDE A、GADD4 5a和RIP2 ,以及Caspase 4、5和 6 ,显著上调的基因为TRAF4。两种细胞均强烈表达Caspase 1 4和TNFR2。　结论　凋亡通路基因表达改变与前列腺癌细胞凋亡反应性不同有关 ,在前列腺癌激素敏感性转化中可能有重要作用     

RBBP4对前列腺癌细胞生长和侵袭能力的影响

目的：研究视网膜母细胞瘤结合蛋白4(retinoblastoma binding protein4,RBBP4)对前列腺癌细胞侵袭、迁移、增殖及肿瘤生长等生物学行为的影响.方法构建 RBBP4过表达慢病毒载体转染及未转染 LNCaP、DU145细胞株,分别通过 Transwell 实验、Wound healing 实验、CCK8及流式细胞技术检测前列腺癌细胞的侵袭、迁移、增殖和凋亡,异体肿瘤种植模型研究RBBP4对前列腺癌细胞成瘤能力的影响.结果 RBBP4上调表达明显促进前列腺癌细胞的迁移(LNCaP：RBBP4 vs Ctrl ＝133．8±14．1 vs 48．6±11．9;DU145：RBBP4 vs Ctrl ＝118．2±10．5 vs 62．3±13．0,P ＜0．001)和侵袭(LNCaP：RBBP4 vs Ctrl ＝252．0±16．3 vs 82．5±12．6;DU145：RBBP4 vs Ctrl ＝232．8±9．2 vs 61．0±8．3,P ＜0．001)能力;RBBP4高表达可以刺激 DU145前列腺癌细胞的增殖并显著加快 DU145细胞移植瘤的生长速度(P ＜0．01).结论 RBBP4能刺激前列腺癌细胞的侵袭、迁移,促进前列腺癌的形成及生长.     

原癌基因N-MYC下游调节基因-2对前列腺癌细胞株PC3的体外增殖抑制作用

目的 观察原癌基因N-MYC下游调节基因-2(NDRG2)在不同前列腺癌细胞株中的表达水平和对PC3细胞的体外增殖抑制作用.方法 采用逆转录-聚合酶链反应(RT-PCR)及Western blot检测不同前列腺癌细胞株(PC3、LNCaP、DU145)的NDRG2表达水平,使用pAD_CMV腺病毒感染的方法 观察NDRG2对PC3细胞的作用,乳糖操纵子z(Lacz)为阳性对照,并设空白对照,噻唑蓝比色法(MTT法)绘制细胞生长曲线、流式细胞仪(FCM)检测感染48、72 h的细胞周期及凋亡.结果 3个前列腺癌细胞株的NDRG2表达水平相对较低,其中PC3细胞表达水平最低,细胞生长曲线显示NDRG2基因自感染36h开始能够抑制PC3细胞增殖(F≥12.43,P＜0.01),FCM检测发现,感染72 h时,包装有NDRG2基因腺病毒感染组同空白及阳性对照组比较,细胞G1期阻滞(3组分别为68.06%、50.33%和50.33%),细胞凋亡增加(3组分别为12.33%、3.21%和3.87%).结论 NDRG2基因可能参与了前列腺癌的发病,腺病毒介导的人NDRG2基因可明显抑制PC3细胞的增殖.     

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.     

PC-SPES: a unique inhibitor of proliferation of prostate cancer cells in vitro and in vivo

BACKGROUND: Management of prostate cancer that has spread beyond the capsule is a difficult problem. Innovative and nontoxic approaches to the disease are urgently required. Recently, a commercially available herbal mixture called PC-SPES showed potent antitumor activities on a variety of malignant cells in vitro. METHODS: PC-SPES was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of three human prostate cancer cell lines (LNCaP, PC-3, and DU 145). Western blot analysis examined the effect of PC-SPES on levels of p21(waf1), p27(kip1), Bcl-2, and E-cadherin in the three cell lines; and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay. Furthermore, the effect of oral PC-SPES (250 mg/kg/day) on growth of PC-3 and DU 145 tumors present in male BNX nu/nu triple immunodeficient mice was studied. LNCaP cells were not analyzed in mice because they grow only with difficulty in these immunodeficient mice. RESULTS: PC-SPES markedly inhibited clonal growth of LNCaP, PC-3, and DU 145 prostate cancer cells, with a 50% inhibition (ED50) at approximately 2 microl/ml. Pulse-exposure studies showed that a 5-day pulse-exposure to PC-SPES (2 microl/ml) in liquid culture achieved a 50% inhibition of PC-3 clonal growth in soft agar, suggesting that the growth inhibition mediated by the extracts remained after removal of PC-SPES. Cell cycle analysis using the prostate cancer cell lines found that PC-SPES induced a significant increase in the number of cells in G0-G1 and G2/M, with a concomitant decrease in the number of cells in S phase. PC-SPES (2 microl/ml, 4 days) increased slightly the levels of p21(waf1) in the three cell lines, decreased by 40% the levels of Bcl-2 in PC-3, and the levels of p27(kip1) and E-cadherin and telomerase were unchanged in each of the lines. In vivo treatment with oral PC-SPES of male BNX mice having DU 145 tumors produced significant inhibition of their growth (P < 0.001), with no objective side effects including blood chemistries, weights, or autopsy analysis. The PC-SPES showed no statistical effect on the in vivo growth of PC-3 cells. CONCLUSIONS: PC-SPES inhibits clonal proliferation of human prostate cancer cells both in vitro and in vivo, using a murine model.     

Expression of cyclin DI in human prostate cancer cell lines

Deregulation of cyclin expression has been found in many tumors. In this report, we studied expression of cyclin DI in three human prostate cancer cell lines: the androgen-dependent LNCaP and the androgen-independent PC3 and DU 145 cell lines. Northern blot analysis showed that DU145 and PC3 cells expressed more abundant cyclin DI than LNCaP cells. Southern blot analysis showed no evident gene amplification or rearrangement of cyclin DI in any of these cell lines. Serum starvation and replenishment were used in the cell culture to study the regulation of expression of cyclin DI. Cyclin DI mRNA expression was detected by Northern blot analysis when LNCaP cells grew in medium with serum but was not detected after serum withdrawal; however, cyclin DI mRNA was induced after serum was added. Cyclin DI mRNA expression by PC3 and DU 145 cells was detected both when they grew in medium with serum and after serum withdrawal, although expression decreased greatly after 24 hours in the PC3 cell line. Immunoprecipitation and immunohistochemical staining also showed that cyclin D I protein was always expressed in PC3 and DU 145 cells under different growth factor environment, whereas it decreased significantly in LNCaP cells deprived of serum and the level resumed again when serum was re-added. This suggests that expression of cyclin DI is regulated by exogenous growth factors in LNCaP cell line and becomes constitutive in PC3 and DU 145 cell lines.     

Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production

BACKGROUND: Anandamide (ANA) is an endogenous lipid which acts as a cannabinoid receptor ligand and with potent anticarcinogenic activity in several cancer cell types. METHODS: The inhibitory effect of ANA on the epidermal growth factor receptor (EGFR) levels expressed on the EGF-stimulated prostatic cancer cells LNCaP, DU145, and PC3 was estimated by ELISA tests. The anti-proliferative and cytotoxic effects of ANA were also evaluated on these human prostatic cancer cell lines by growth tests, flow cytometric analyses, trypan blue dye exclusion assays combined with the Papanicolaou cytological staining method. RESULTS: ANA induced a decrease of EGFR levels on LNCaP, DU145, and PC3 prostatic cancer cells by acting through cannabinoid CB(1) receptor subtype and this leaded to an inhibition of the EGF-stimulated growth of these cells. Moreover, the G(1) arrest of metastatic DU145 and PC3 growth was accompanied by a massive cell death by apoptosis and/or necrosis while LNCaP cells were less sensitive to cytotoxic effects of ANA. The apoptotic/necrotic responses induced by ANA on these prostatic cancer cells were also potentiated by the acidic ceramidase inhibitor, N-oleoylethanolamine and partially inhibited by the specific ceramide synthetase inhibitor, fumonisin B1 indicating that these cytotoxic actions of ANA might be induced via the cellular ceramide production. CONCLUSIONS: The potent anti-proliferative and cytotoxic effects of ANA on metastatic prostatic cancer cells might provide basis for the design of new therapeutic agents for effective treatment of recurrent and invasive prostatic cancers.     

DNA损伤对前列腺癌细胞株BRCA1蛋白表达的影响

目的:探讨肿瘤抑制基因BRCA1对DNA损伤的反应中的重要作用。明确BRCA1蛋白除了受转录控制、磷酸化和蛋白相互间作用等机制调节外,其功能也受BRCA1的亚细胞分布的功能调节。方法:以乳腺癌细胞株作为阳性对照组,对前列腺癌细胞株LNCaP,DU145和PC-3的BRCA1亚细胞分布免疫荧光染色,并对BRCA1的表达量进行Western印迹检测。结果:BRCA1蛋白存在于LNCaP,DU145和PC-3的前列腺癌细胞株中,而且DNA损伤后能够引起其出核现象,即BRCA1蛋白在细胞质的细胞分布比例由14%增加到40%(P<0.01),相反在细胞核其分布比例由46%减少到21%(P<0.01)。BRCA1这种DNA损伤后的胞质亚细胞分布比例增高仅在p53野生型前列腺癌细胞株出现,在p53突变型前列腺癌细胞株没有此现象。形态学和Western印迹法证实,LNCaP细胞DNA损伤后BRCA1蛋白在胞质、胞核间互动后其凋亡比例高达40%,细胞凋亡的标记蛋白(裂解后caspase-3活性片段)也明显增高。结论:BRCA1的胞质胞核重新分布可能是DNA损伤后的蛋白功能调节机制之一;BRCA1这种亚细胞分布改变需要p53的功能调节,与p53的状态及其功能相关,同时也引起细胞凋亡比例的提高。在前列腺癌细胞株BRCA1引起更高比例的细胞凋亡现象预示,BRCA1可能成为前列腺癌临床治疗的生物靶点。     

Antitumor activity and downregulation of pro-angiogenic molecules in human prostate cancer cells by a novel thiazolidione compound

BACKGROUND: Current treatments for prostate cancer are effective in many patients with locally advanced disease, but many of these patients eventually have recurrence. It is therefore important to develop alternative therapeutic agents with improved efficacy and tolerability. We recently identified a synthetic thiazolidin compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidione (DBPT), that induces apoptosis in human colon cancer cells, independent of p53 and P-glycoprotein status. Here, we investigated the antitumor properties and mechanisms of action of this compound in human prostate cancer cell lines. METHODS: The effect of DBPT on cell-cycle progression and apoptosis in LNCaP and DU145 cells was examined by flow cytometry and Western blotting. The effect of DBPT on pro-angiogenic molecules was analyzed by Western blotting and by an enzyme-linked immunosorbent assay. RESULTS: DBPT inhibited the growth of LNCaP and DU145 cells with 50% inhibitory concentrations ranging from 1.6 to 5.9 microM. Treating LNCaP and DU145 cells with DBPT led to a time-dependent cell-cycle arrest in the G(2)/M phase and increased levels of G(2)/M checkpoint proteins, such as cyclin B1, cdc25C, phosphorylated histone H(3), and MPM-2. DBPT induced the phosphorylation of Bcl-xL and Bim, and induced apoptosis, as evidenced by cleavage of caspase and poly (ADP-ribose) polymerase. DBPT also effectively induced apoptosis in Bcl-2-overexpressing DU145 cells. Furthermore, DBPT decreased hypoxia-inducible factor 1 alpha and vascular endothelial growth factor expression in LNCaP cells under both normoxia and hypoxia. CONCLUSIONS: DBPT can suppress proliferation, induce apoptosis, and down regulate pro-angiogenic molecules in prostate cancer cells, and might be useful in treating prostate cancer.