三氧化二砷对前列腺癌DU-145细胞周期阻滞及凋亡的影响

目的探讨三氧化二砷(As2O3)对前列腺癌非雄激素依赖细胞系DU-145细胞的增殖抑制作用及对细胞周期和凋亡的影响.方法应用体外细胞生长抑制试验(MTT比色法)研究As2O3对DU-145细胞生长的影响,流式细胞仪检测细胞周期分布情况及凋亡情况,琼脂糖凝胶电泳检测细胞DNA的变化.结果As2O3对DU-145细胞的增殖有明显的抑制作用,随着药物浓度增加和作用时间延长,凋亡细胞明显增加,出现G2/M期阻滞和DNA的断裂.结论As2O3可明显抑制前列腺癌DU-145细胞的增殖,诱导细胞凋亡,抑制细胞周期.     

PI3K/AKT/FOXO1信号通路参与复方中药CFF-1诱导的前列腺癌细胞凋亡和周期阻滞

目的:研究复方中药CFF-1诱导前列腺癌细胞的凋亡作用及其相关分子机制的探讨。方法:通过形态学观察、噻唑蓝(MTT)比色实验、CCK-8实验测定前列腺癌细胞的存活能力;采用DAPI染色法测定细胞核凝缩破裂;运用Annexin V-FITC/PI双染流式细胞术测定前列腺癌细胞的凋亡率。再通过PI单染流式细胞术测定前列腺癌细胞的周期变化;以及采用蛋白质免疫印迹实验检测PI3K/AKT/FOXO1信号通路以及其下游凋亡相关蛋白和周期相关蛋白的表达。结果:复方中药CFF-1使前列腺癌细胞周期阻滞在G1期,并呈浓度依赖性降低细胞的存活能力、增加细胞核凝缩破裂以及核小体的形成,显著提高前列腺癌细胞的凋亡率。在分子机制上,CFF-1呈现浓度依赖性下调PI3K/AKT活性,降低FOXO1磷酸化水平,进而上调FOXO1的转录活性,最终影响凋亡相关基因和周期相关基因的表达。结论:CFF-1对前列腺癌细胞的生长有显著的抑制作用,并且通过PI3K/AKT/FOXO1信号通路诱导前列腺癌细胞周期阻滞并发生凋亡,对治疗前列腺癌具有潜在的临床应用价值。     

增韧基团——姜黄素酯对前列腺癌细胞体外靶向影响

目的 以酯键链接增强基团,制备姜黄素前体,观察其对前列腺癌细胞和正常二倍体细胞生长活性影响的差异.方法 叔丁氧羰基(Boc)-苯丙氨酸酯姜黄素单酯作用于17组人前列腺癌DU-145细胞6～24h后,噻唑蓝(MTT)药物敏感实验检测细胞生长活性,流式细胞术检测细胞凋亡和比率,透射电镜观察细胞超微结构改变.1 ～7d后,采用计数法测定细胞生长曲线.同法作用于入主动脉平滑肌(HASMC)细胞,作为对照组.结果 10 ～40μmol/L的Boc-苯丙氨酸酯姜黄素单酯作用于人前列腺癌DU-145细胞6～24h后,DU-145细胞生长抑制率为7.37％～66.87％ (P＜0.05),呈浓度、时间依赖性;部分细胞出现凋亡形态学改变,FSC-SSC散点图可见24 h DU-145细胞凋亡比率分别为37.84％ ～47.12％ (P ＜0.05).对照组HASMC细胞凋亡比率为0.94％ ～4.23％(P＜0.05),较同浓度姜黄素降低.结论 姜黄素前体化合物能在体外有效诱导人前列腺癌DU-145细胞凋亡,对正常二倍体细胞的抑制作用较低.     

全反式维甲酸诱导前列腺癌DU145细胞凋亡中caspase-3及其细胞骨架蛋白F-actin的变化

目的探讨全反式维甲酸(ATRA)诱导雄激素非依赖型前列腺癌细胞系DU145凋亡过程中半胱氨酰天冬氨酸特异性蛋白酶(caspase)3及其细胞骨架蛋白F-actin的变化。方法应用吖碇橙(AO)染色,荧光显微镜观察细胞凋亡形态,流式细胞术(FACScan)测定ATRA对DU145细胞凋亡峰的形成,应用Westernblot测定ATRA在DU145细胞凋亡过程中caspase-3表达的变化,免疫荧光染色观察F-actin纤丝的形态。结果荧光显微镜观察细胞凋亡数目增多,48h时凋亡细胞比例为85.0%,流式细胞术检测可观察到凋亡峰,Westernblot检测显示裂解的caspase-3表达增多,免疫荧光染色可见F-actin的正常结构被破坏。结论ATRA诱导DU145细胞凋亡可能通过caspase-3介导,并导致细胞骨架蛋白破坏。     

前列消癥汤对前列腺癌DU-145 细胞凋亡及周期的影响

目的:观察前列消癥汤对前列腺癌DU-145细胞系的细胞凋亡及周期的影响,探讨前列消癥汤治疗前列腺癌的生物学机制。方法:采用流式细胞术测定不同浓度前列消癥汤对前列腺癌DU-145细胞凋亡及细胞周期分布的影响。结果:前列消癥汤在一定浓度范围内抑制DU-145细胞增殖,并呈现出一定的量效关系。与对照组比较,中、高剂量组均能够显著增加早期凋亡、晚期凋亡,具有统计学差异(中剂量组P<0.05,高剂量组P<0.01)。中剂量与高剂量组的S期细胞比例分别为(37.96±2.24)%和(43.77±2.48)%,均显著高于对照组的(31.69±1.29)%,差异有统计学意义(中剂量组P<0.05,高剂量组P<0.01)。结论:前列消癥汤能够诱导前列腺癌DU-145细胞的早期及晚期凋亡,并随着剂量的增加,促进凋亡的作用也随着增加。前列消癥汤主要抑制前列腺癌DU-145细胞从S期向G2期的转变。     

肿瘤坏死因子凋亡诱导配体诱导结肠癌细胞凋亡的机制

目的探讨肿瘤坏死因子凋亡诱导配体(TRAIL)诱导结肠癌细胞发生凋亡的机制。方法流式细胞仪技术、酶联免疫吸附技术、荧光显色技术检测500μg／L TRAIL和／或Caspase抑制剂Z-VAD．fmk处理后,结肠癌细胞SW1116在不同时点(0、2、4、6、24 h)凋亡情况、Caspase-3酶活性及4 h线粒体△ψm、cardiolipin、细胞ROS变化。结果 TRAIL可引起结肠癌细胞凋亡,并于 4 h达凋亡高峰,凋亡指数为32．98％;并出现线粒体△ψm下降、cardiolipin丢失增加及Caspase-3酶活性增加,4 h达到最大峰值为(37．56±2．572)μmol／L·hr-1·mg-1蛋白。但TRAIL所诱导的细胞凋亡作用可被Caspase抑制剂Z-VAD．fmk所抑制。同时证实TRAIL所诱导的结肠癌细胞凋亡与细胞氧自由ROS的生成无关。结论 TRAIL通过Caspase依赖方式引起线粒体△ψm下降、cardi- olipin丢失增加导致线粒体内膜损伤,从而诱导细胞发生凋亡。     

Lupeol对肝癌SMMC-7721细胞增殖及凋亡的影响

【摘要】〓目的〓探讨羽扁豆醇(lupeol)对人肝癌SMMC-7721细胞生长和凋亡的作用及机制。方法〓采用MTT法检测lupeol对肝癌细胞 SMMC-7721和正常肝细胞L-02的增殖抑制作用,流式细胞法检测细胞凋亡情况,Western blot检测TRAIL受体及抗凋亡蛋白表达变化,比色法分析caspase-8、-9、-3酶活性改变。结果〓lupeol对SMMC-7721细胞具有明显的增殖抑制作用(IC50=40 μmol/L),而对正常肝细胞L-02无细胞毒作用;lupeol在30、40、50 μmol/L浓度范围诱导SMMC-7721细胞48小时的凋亡率分别为5.5%、12.6%、28%;抗凋亡蛋白c-FLIPL表达下调,caspase-8及caspase-3活性增强均呈剂量依赖性。结论〓Lupeol通过下调c-FLIPL表达激活caspase通路诱导肝癌细胞凋亡,对肝癌细胞增殖发挥抑制作用。     

扶正抑瘤中药复方对人前列腺癌DU-145细胞凋亡影响及机制研究

目的探讨具有扶正抑瘤功效的中药复方对人前列腺癌DU-145细胞凋亡的影响作用及机制。方法采用体外细胞培养的研究方法,用25μl/mL、50μl/mL、100μl/mL、200μl/mL的中药复方干预DU-145细胞生长48h。以流式细胞仪检测细胞凋亡情况并计算凋亡率;以免疫细胞化学法和MIAS图像分析系统检测Bax、Capase-3和TNF-α3种凋亡相关蛋白的表达情况。结果中药复方作用DU-145细胞48h后,DU-145细胞生长呈抑制状态,细胞凋亡百分率增高(P0.05或P0.01),并呈一定的量效关系;细胞3种凋亡相关蛋白Bax,Caspase-3和TNF-α表达均增高(P0.05或P0.01)。结论中药复方可抑制DU-145细胞增殖,并诱导DU-145细胞凋亡,其机制与"线粒体通路"中Bax、Caspase-3表达增高以及"死亡受体通路"中TNF-α表达增高有关。     

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。     

肿节风对人前列腺癌DU-145细胞PI3K/Akt/mTOR信号传导通路的影响

目的:探讨中药肿节风溶液对人前列腺癌DU-145细胞PI3K/Akt/mTOR信号传导通路的影响.方法 采用细胞培养的方法,用不同浓度的肿节风溶液作用于DU-145细胞48h,用MTT法检测肿节风溶液对DU-145细胞增殖的影响,用RT-PCR检测用药后细胞PI3K、Akt、mTOR和P70(S6K)的基因表达情况.结果:肿节风溶液作用48h后,DU-145细胞增殖受到抑制,并表现为量效关系;DU-145细胞的PI3K、Akt、mTOR和P70(S6K)的表达均呈不同程度的抑制,但不表现为量效关系.结论:肿节风溶液能抑制人前列腺癌DU-145细胞增殖,其抑制细胞增殖的机制与其抑制了细胞的PI3K/Akt/mTOR信号传导通路的作用有关.     

改良消减杂交方法克隆前列腺癌细胞凋亡相关基因

目的：建立前列腺癌细胞系DU－145凋亡细胞模型,克隆与凋亡相关的基因,了解凋亡产生的分子机制。方法：以全反式维甲酸（all-trans retinoic acid)诱导前列腺癌细胞系DU－145细胞凋亡,观察细胞形态及超微结构的改变,采用基于PCR技术的改良消减杂交方法克隆前列腺癌细胞凋亡相关基因。结果：在维甲酸诱导的前列腺癌细胞DU－145凋亡过程中,有TNF及c-erb B-2基因的参与,并成功克隆出一条可能与凋亡密切相关的未知基因pcar,已被Genebank收录,登录号为AF174394。结论：维甲酸诱导前列腺癌细胞DU－145凋亡过程有多基因参与,其中一些基因仍然是未知的。     

siRNA靶向沉默B7-H4基因对人前列腺癌DU145细胞增值和凋亡的影响

目的研究siRNA沉默B7-H4基因对人前列腺癌DUl45细胞增殖和凋亡的影响。方法以脂质体Lipofeclami-ne“2000（Lipo）为载体转染siRNA-B7-H4至DUl45细胞,应用RT-PCR和WesternBlot检测B7-H4表达水平,CCK-8法检测细胞增殖的变化,Annexinv／PI双染流式细胞术检测细胞凋亡情况。结果与空白组和阴性对照组（NC）相比,转染B7-H4-siRNA的细胞B7-H4mRNA和蛋白表达明显减少（P〈0．05）,DUl45转染B7-H4siRNA后,增殖能力减弱,凋亡显著。结论通过B7-H4-siRNA抑制DUl45细胞B7-H4的表达,可以抑制细胞增殖,促进其凋亡,表明B7-4在前列腺癌的发生发展中发挥重要作用。     

Heterogeneity in plasminogen activator (PA) levels in human prostate cancer cell lines: increased PA activity correlates with biologically aggressive behavior

Plasminogen activators (PA), particularly the lower Mr urokinase (u-PA) type, have been associated with tumor cell invasion and metastasis. We have examined the expression of PA by two human prostate cancer cell lines (PC-3 and DU-145) using functional and immunologic techniques. The culture media and cell extracts of the more aggressive PC-3 cell line contained more than two-fold greater PA activity than the relatively indolent DU-145 cell line. Zymographic studies identified the PA expressed as u-PA. PC-3 cells expressed an additional lower molecular weight form of u-PA not noted in DU-145 cells. Heterogeneity in u-PA expression was shown by the fibrin lysis assay, immunohistochemistry, and dual parameter flow cytometry indicating the presence of phenotypically divergent cell populations. Increased u-PA expression may identify those tumor cells that possess aggressive biological potential.     

核小体结合蛋白调控Survivin基因对前列腺癌DU145凋亡的作用

目的探讨抑制人核小体结合蛋白1（NSBP1）基因后促进非激素依赖性前列腺癌细胞系DU145凋亡的作用机制。方法通过慢病毒lentivirus-NSBP1转染非激素依赖性前列腺癌细胞系DU145后,MTT法观察细胞活性变化,流式细胞仪检测细胞的凋亡情况,应用Western-blot技术检测NSBP1、Survivin蛋白的变化情况。结果抑制NSBP1表达水平后非激素依赖性前列腺癌细胞系DU145细胞活性降低,凋亡增加,同时Survivin蛋白的表达也随之降低。结论通过慢病毒转染抑制NSBP1的表达可以促进非激素依赖性前列腺癌细胞系DU145凋亡,NSBP1可能是通过调控Survivin基因的变化影响细胞的增值凋亡。     

Mechanism of Brefeldin A-Induced Growth Inhibition and Cell Death in Human Prostatic Carcinoma Cells

The mechanism of growth inhibition and triggering of cell death by the antibiotic brefeldin A (BFA) was investigated in human prostatic cancer DU-145 cells. After cells were cultured with various concentrations of BFA, cell number and viability were determined at specified times. Compared with untreated cells, a drastic growth reduction (>80%) with approximately 50% cell death was observed in the cells cultured with BFA (30 ng/mL) for 72 h. Cell-cycle analysis using flow cytometry revealed that such growth inhibition was associated with approximately 85% reduction in the S-phase population, indicating the inhibition of the G(1)-S phase progression. Western blots further showed that cell-cycle-dependent kinases (cdk2 and cdk4), cyclin D(1), and p53 were all downregulated, whereas WAF1 (p21) was upregulated with BFA treatment. Possible induction of apoptosis by BFA was also assessed by TUNEL assay and by DNA analysis using agarose gel electrophoresis. The TUNEL assay demonstrated the positive staining of BFA-treated cells, and gel electrophoresis confirmed nucleosomal DNA ladder formation. Thus, these results suggest that growth inhibition of DU-145 cells by BFA is attributable mainly to a G1 cell-cycle arrest through the modulation of specific cell-cycle regulators. The accompanying cell death may follow a p53-independent apoptotic pathway.     

Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway,impairing Ras membrane localization in prostate cancer cells

PURPOSE: Metastasis to bone is an important cause of morbidity in advanced prostate cancer. Despite the typically sclerotic nature of prostatic bone metastases osteolysis has a significant role in the pathogenesis of this disease. The nitrogen containing bisphosphonates (N-BPs), such as pamidronate and zoledronic acid, have greatly enhanced potency for inhibiting bone resorption and inducing apoptosis in osteoclasts. We investigated the effects of N-BPs on prostate cancer cells. MATERIALS AND METHODS: Cell viability was determined with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeyhoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) dye reduction assay. Cell cycle analysis, DNA fragmentation and caspase 3 activity were assessed using flow cytometry. Ras, Bcl-2 and Bax were quantified by Western blotting. RESULTS: Pamidronate and zoledronic acid decreased cell viability in the 3 human cell lines DU145, PC3 and LNCaP. These effects were associated with changes in cell cycle distribution, induction of DNA fragmentation and a decrease in the Bcl-2-to-Bax ratio, which are features of apoptotic cell death. Pre-incubation with caspase inhibitors attenuated the effects of zoledronic acid and caspase 3 activity was demonstrated in treated DU145 cells. Zoledronic acid induced loss of cell viability in DU145 cells was prevented by co-treatment with farnesol, suggesting that N-BPs cause inhibition of the mevalonate pathway and Ras prenylation. A decrease in active, membrane bound Ras in zoledronic acid treated DU145 cells was shown by Western blot analysis. CONCLUSIONS: N-BPs induce apoptosis in prostate cancer via a caspase dependent mechanism. They have effects on protein prenylation via inhibition of the mevalonate pathway and impair membrane localization of Ras in prostate cancer cells.     

Estradiol causes a dose-dependent stimulation of prostate growth in castrated beagle dogs

BACKGROUND: We analyzed the cytotoxic properties of the new heterodinucleoside phosphate dimer 5-FdU-NOAC, which is composed of the cytotoxic drugs 5-FdU and N(4)-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) against human prostate tumor cells. METHODS: 5-FdU-NOAC effects on cell proliferation, cell cycle distribution, thymidylate synthase activity, and apoptosis were investigated in vitro in the two human prostate carcinoma cell lines DU-145 and PC-3 and compared to cells treated with the corresponding single drugs 5-FdU and NOAC. RESULTS: Treatment of the cells with 5-FdU-NOAC resulted in IC(50) values of 3.9-5 microM and in a complete inhibition of cell proliferation at 200 microM after 96 hr compared to 5-FdU, where 10% of the cells remained resistant. Flow cytometric analysis revealed cell cycle perturbations in S-phase only in the DU-145 cells. 5-FdU-NOAC caused 50% inhibition of thymidylate synthase after 90 min at 0.6 microM in both cell lines. Apoptotic cell fractions in DU-145 (66%) and in PC-3 (34%) cells were found after treatment with 5-FdU-NOAC for 96 hr. DNA fragmentation further confirmed the induction of apoptosis. CONCLUSIONS: 5-FdU-NOAC inhibits thymidylate synthase and cell cycle progression causing proliferation arrest and apoptosis in DU-145 and PC-3 cells, suggesting a potential role of 5-FdU-NOAC for the treatment of prostate cancer.     

槲皮素及其7-OH衍生物对膀胱癌细胞增殖抑制作用及细胞周期的影响

【摘要】 目的 通过细胞试验及裸鼠移植瘤实验,研究槲皮素及其7-OH衍生物对人膀胱癌BIU-87细胞的增殖抑制作用及细胞周期的影响。方法〓体外实验:培养BIU-87细胞,分别加入槲皮素(QUE)及槲皮素单硫酸酯钠盐(SQMS),MTT实验测定并计算生长抑制率及半数抑制浓度IC50;流式细胞仪检测细胞周期。体内实验:观察不同剂量SQNS及槲皮素对BIU-87细胞裸鼠移植瘤生长的抑制作用;瘤体组织切片行HE染色和免疫组化染色检测Ki-67,CyclingB1,CyclingD1蛋白表达情况。结果〓槲皮素及SQMS体外均能抑制BIU-87细胞的增殖,相同浓度两药物间对比差异无统计学意义(P>0.05)。槲皮素使BIU-87细胞周期明显阻滞于G2/M期,而SQMS则主要阻滞于G0/G1期。在体内实验中,中等剂量的SQMS组抑瘤率与槲皮素组相近(P>0.05),CyclingD1表达比槲皮素组下调更明显(P<0.01),而槲皮素组CyclingB1表达比中剂量SQMS组下调更明显(P<0.05)。两组Ki-67表达无显著性差异(P>0.05)。结论〓槲皮素及其衍生物SQMS对膀胱癌细胞BIU-87有抑制增殖作用;中剂量的SQMS对成瘤生长的抑制作用与槲皮素作用相近;槲皮素对人膀胱癌细胞增殖抑制作用可能是通过下调CyclingB1的表达,阻滞细胞周期于G2/M期,而SQMS则可能是通过下调CyclingD1的表达,阻滞细胞周期于G0/G1期。