JNK信号通路介导ghrelin对乳腺癌MDA-MB-231细胞耐药蛋白表达的影响

目的探讨ghrelin调控c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)信号转导通路对乳腺癌细胞P-糖蛋白表达的影响。方法培养人乳腺癌MDA-MB-231细胞,分别加入生长激素释放肽(ghrelin,100 nmol/L,干预72小时)、多柔比星(0.8 mg/L,干预72小时)、ghrelin联合多柔比星(ghrelin 100 nmol/L,多柔比星0.8 mg/L,干预72小时)。采用MTT方法检测细胞增殖,Western blot法检测细胞JNK、P-糖蛋白(P-glycoprotein,P-gp)的表达及p-JNK水平。结果 MTT检测显示,人乳腺癌MDA-MB-231细胞在ghrelin的干预下增殖(P<0.01),存活率在多柔比星的干预下降低(P<0.01),ghrelin联合多柔比星能够抑制多柔比星对MDA-MB-231细胞的杀伤作用(P<0.01)。ghrelin组JNK表达及p-JNK水平上升(均P<0.01),且P-gp蛋白表达上升(P<0.05);多柔比星组JNK表达及p-JNK水平下降(均P<0.01);ghrelin联合多柔比星较多柔比星组JNK表达及p-JNK水平增加,且P-gp表达明显上升(均P<0.05)。结论 ghrelin激活JNK信号通路干预乳腺癌细胞P-gp表达增加从而抑制多柔比星诱导乳腺癌细胞凋亡。     

ghrelin调控ERK信号通路对乳腺癌细胞多药耐药的影响及机制

目的:探讨ghrelin调控ERK信号传导通路对乳腺癌细胞多药耐药的影响及机制.方法:人乳腺癌细胞MDA-MB-231细胞培养,设立对照组、多柔比星组、gbrelin组、ghrelin联合多柔比星组、ghrelin联合多柔比星加PD098059抑制剂组,采用流式细胞法检测细胞凋亡,Western-blot方法检测细胞ERK、p-ERK、P-gp蛋白的表达.结果:在ghrelin干预下人乳腺癌MDA-MB-231细胞凋亡率最低(P＜0.01),在多柔比星的干预下人乳腺癌MDA-MB-231细胞凋亡率最高(P＜0.01),ghrelin联合多柔比星能够抑制多柔比星对人乳腺癌MDA-MB-231细胞的促凋亡作用(P＜0.01),ghrelin联合多柔比星加ERK信号通路特异性阻滞剂PD98059能够减弱ghrelin联合多柔比星对人乳腺癌MDA-MB-231细胞凋亡的抑制作用(P＜0.01).多柔比星组与ghrelin组相比ERK表达及p-ERK水平明显下降(P＜0.05)、且ghrelin组与多柔比星组相比P-gp蛋白表达明显上升(P＜0.05),ghrelin联合多柔比星组与多柔比星组相比ERK表达及p-ERK水平明显增加(P＜0.05)、且P-gp蛋白表达明显增加(P＜0.05),ghrelin联合多柔比星加抑制剂组与ghrelin联合多柔比星组相比ERK表达及p-ERK水平明显下降(P＜0.01),P-gp蛋白表达无显著性差异(P=0.07).结论:一定浓度的ghrelin激活乳腺癌细胞ERK信号通路增加P-gp蛋白表达,从而抑制多柔比星诱导乳腺癌细胞凋亡而诱发耐药的产生,阻断ghrelin-ERK信号通路可能逆转乳腺癌细胞多药耐药的发生.     

JNK/FOXO3信号通路介导黄连素促人肺腺癌PC-9细胞凋亡的研究

  目的  探讨黄连素（berberine,Ber）诱导肺腺癌PC-9细胞凋亡及c-Jun氨基末端激酶（c-Jun N-terminal kinase,JNK）/转录因子叉头蛋白3（forkhead box protein O3,FOXO3）信号通路的作用机制。  方法  实验采用完全随机化的分组方法,分为对照组、Ber组（30、60 μM）,分别检测各组PC-9细胞活力值、凋亡率、ROS含量、caspase 3活性、线粒体膜电位,以及JNK/FOXO3通路和凋亡相关蛋白含量的变化。SP600125特异性抑制JNK（磷酸化）激活后,Ber（0、60 μM）处理细胞24 h,重复上述检测。  结果  Ber有效抑制PC-9细胞活力,促进细胞凋亡（P < 0.05）,显著降低PC-9细胞线粒体膜电位,增加ROS含量和caspase 3活性（P < 0.05）,并呈浓度依赖效应;Western blot检测结果显示Ber上调p-JNK、FOXO3和Bax的含量（P < 0.05）,下调p-FOXO3和Bcl-2的含量（P < 0.05）;SP600125特异性抑制JNK激活后,拮抗Ber下调p-FOXO3含量及其促PC-9细胞凋亡作用（P < 0.05）。  结论  Ber可有效抑制肺腺癌PC-9细胞活力、促进细胞凋亡和氧化应激损伤,作用机制可能与上调p-JNK、FOXO3含量和抑制FOXO3磷酸化有关。      

热化疗联合作用抑制人小细胞肺癌细胞增殖的机制

目的 研究热化疗联合作用对肺部肿瘤细胞生长的抑制及其可能的机制。方法 参考临床常用剂量,采用43 ℃加热联合120 μg/L紫杉醇、43 ℃加热联合120 μg/L紫杉醇及20 μmol/L JNK特异抑制剂SP600125以及单纯使用120 μg/L紫杉醇处理H446细胞,以未处理的H446细胞作对照。应用噻唑蓝比色法检测各处理方式下细胞增殖率的变化,通过蛋白免疫印迹法检测JNK磷酸化水平和Caspase 3表达的变化,流式细胞术检测细胞凋亡率,运用SPSS 13.0对数据进行统计分析。结果 热化联合组细胞增殖率最低(P<0.05);JNK磷酸化水平在热化联合组中表达明显增高(P<0.05),SP600125可抑制其磷酸化(P<0.05);热化联合组的Caspase 3增高(P<0.05),SP600125使其表达减少(P<0.05);热化联合组细胞凋亡率增加(P<0.05),SP600125组细胞凋亡率减少(P<0.05)。结论 热化疗联合应用可以明显增加紫杉醇对H446细胞生长的抑制作用,且这种抑制作用可能是通过激活JNK信号转导通路,继而通过Caspase 3途径激活细胞凋亡来实现的。     

组蛋白去乙酰化酶抑制剂LBH589对急性髓系白血病细胞株HL60/ADM增殖、凋亡及耐药的影响

目的 探讨组蛋白去乙酰化酶抑制剂LBH589对急性髓系白血病细胞株HL660/ADM的增殖、凋亡和耐药的影响.方法 采用不同浓度LBH589处理耐药的HL60/ADM细胞,四甲基偶氮唑蓝（MTT）法检测细胞增殖和多柔比星作用24h IC50值,AnnexinV-FITC/PI荧光染色流式细胞术检测细胞凋亡、多柔比星摄取率和多药耐药蛋白1（MRP1）表达,评估LBH589逆转耐药效应.Westemblot检测p53、Akt、p-Akt、组蛋白-3、乙酰化组蛋白-3、β-actin蛋白表达.结果 10～80 nmol/L LBH589能够抑制HL60/ADM细胞增殖和诱导凋亡,70 nmol/LLBH589作用60 h抑制效果最佳.20 nmol/LLBH589显著下调HL60/ADM细胞表面MRP1的表达[（93.90±4.20）％比（76.19±6.53）％,P＜0.05]、提高HL60/ADM细胞多柔比星摄取率[（8.53±0.68）％比（25.67±1.34）％,P＜ 0.01]、降低多柔比星24 hIG0值[（6.833±0.319） μg/ml比（1.382±0.104）μg/ml,P＜ 0.01],其逆转耐药倍数为4.9倍.LBH589处理HL60/ADM细胞24、48 h,乙酰化组蛋白-3相对表达水平均高于LBH589处理前（P＜0.01）,处理后24h和48 h p-Akt相对表达水平分别为1.07±0.09和0.59±0.01,低于处理前表达水平（2.03±0.12）（P＜0.01）,p53蛋白相对表达水平分别为0.57±0.04和1.31±0.09,明显高于处理前的表达（0.21±0.02）（P＜ 0.01）.结论 LBH589通过阻断PI3K-Akt通路、下调其MRP1的表达及提高多柔比星摄取率有效地抑制HL60/ADM细胞的增殖和诱导其凋亡,并逆转其耐药.     

敲除人乳腺癌MCF-7细胞中PTEN基因对JNK通路活性的影响

背景与目的:PTEN与多种肿瘤发生发展密切相关,大量研究表明PTEN基因通过直接或间接作用整合复杂的信号网络系统,并影响靶分子及其下游信号级联反应.本研究探讨敲除MCF-7细胞中PTEN基因对JNK通路活性的影响.方法:PTEN反义寡核苷酸转染后,激光共聚焦显微镜检测MCF-7细胞内PTEN蛋白表达:流式细胞术、四甲基偶氮唑蓝(MTT)及Western blot法分别检测SP600125诱导的细胞早期凋亡和细胞周期改变、细胞增殖抑制、细胞中JNK及其下游底物ATF-2、C-Jun的磷酸化水平.结果:FTEN反义寡核苷酸有效封闭MCF-7细胞中PTEN蛋白表达:SP600125(10 μmol/L)诱导已敲除PTEN的MCF-7细胞发生早期凋亡.凋亡率达(32.4±2.4)%,细胞发生G1期阻滞、细胞增殖明显受抑制,与SP600125组、反义组细胞相比差异有显著性(P<0.05);反义+SP600125组MCF-7细胞中磷酸化JNK及其下游底物ATF-2及C-Jun磷酸化水平下调.结论:MCF-7细胞中JNK通路激活与PTEN表达水平相关,PTEN缺失使MCF-7细胞中JNK通路活化,细胞对JNK相关抑制剂的敏感性增加.     

mTOR信号通路调控结直肠癌LoVo/ADR细胞多药耐药性的可能机制

目的:通过雷帕霉素(rapamycin,RAPA)抑制哺乳动物雷帕霉素靶分子(mammalian target of rapamycin,mTOR)通路,观察结直肠癌多药耐药LoVo/ADR细胞对多柔比星的敏感性、自噬、凋亡及多药耐药基因1(multidrug resistance gene 1,MDR1)表达的变化,探讨mTOR通路调控结直肠癌多药耐药的可能机制.方法:多柔比星联合RAPA作用后,MTT法检测多柔比星对LoVo/ADR细胞的半数抑制浓度(half inhibitory concentration,IC50)值;多柔比星和RAPA单独或联合作用后,在透射电子显微镜和荧光显微镜下观察LoVo/ADR细胞自噬体的形成,FCM分析细胞的自噬率和细胞凋亡率;RAPA作用后,RT-PCR和免疫细胞化学法分别检测LoVo/ADR细胞中MDR1 mRNA和P-糖蛋白的表达.结果:25和50 μmol/L RAPA作用下,多柔比星对LoVo/ADR细胞的IC50值均低于不加RAPA的对照组,差异有统计学意义(P＜0.05).对照组LoVo/ADR细胞中很少或几乎看不到自噬体或点状绿色荧光分布,细胞自噬率为(2.9±0.4)％;多柔比星组和RAPA组LoVo/ADR细胞中可见自噬体形成,散在点状绿色荧光分布于细胞质及细胞核周围,细胞自噬率分别为(35.5±5.4)％和(46.7±6.7)％,与对照组比较差异有统计学意义(P＜0.05);多柔比星联合RAPA组LoVo/ADR细胞中可见大量自噬体形成,细胞的自噬率为(73.1±7.4)％,显著高于多柔比星或RAPA单独作用组(P＜0.05).RAPA组和多柔比星组的细胞凋亡率分别为(2.06±0.43)％和(48.39±6.47)％,多柔比星组的细胞凋亡率高于对照组(2.23±0.50)％(P＜0.05);多柔比星联合RAPA组的细胞凋亡率为(79.43±8.28)％,高于多柔比星组(P＜0.05).RAPA作用后,LoVo/ADR细胞中MDR1 mRNA和P-糖蛋白表达下调.结论:抑制mTOR通路具有逆转结直肠癌细胞多药耐药性的作用,其机制可能与RAPA促使耐药细胞自噬、凋亡及下调MDR1 mRNA的表达有关.     

力达霉素联合硼替佐米的抗骨髓瘤作用及对MAPKs表达的影响

目的 研究力达霉素(lidamycin, LDM)联合硼替佐米(bortezomib, BZM)的抗骨髓瘤作用及对丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs)的影响,并探讨MAPKs在两药联合抗骨髓瘤中的作用。方法 选取适当的药物浓度和通路抑制剂浓度,MTS法检测细胞增殖情况;Westernblot 检测相关蛋白及蛋白磷酸化水平。结果 BZM能增强LDM对骨髓瘤细胞的增殖抑制作用,LDM激活c-Jun氨基末端激酶(c-Jun NH2-terminal kinase, JNK)、p38 MAPK的表达和细胞外信号调节激酶(Extracellular signal regulated rotein kinase, ERK),两药联合后可使JNK和p38 MAPK的激活显著增强,而ERK的激活显著下降。JNK抑制剂(SP600125)、p38抑制剂(SB203580)和MEK抑制剂(U0126) 3种抑制剂单独作用对细胞的增殖抑制作用均不明显,但SP600125或SB203580分别与LDM联合BZM合用后均降低了两药联合对细胞的增殖抑制作用,而U0126与LDM联合BZM合用后提高了两药联合对细胞的增殖抑制作用。结论 LDM通过进一步激活JNK、p38 MAPK和降低ERK的激活来增强BZM抗骨髓瘤敏感度。     

去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡

目的：研究去甲斑蝥素(norcantharidin，NCTD)抑制小鼠肺纤维瘤(L929)细胞的作用机制。方法：用MTT法测定细胞生长抑制率。采用Hoechst 33258染色、DNA凝胶电泳和乳酸脱氢酶(LDH)活力检测细胞凋亡。用免疫印迹法(Western blot)分析了细胞外信号调节激酶(extracellular signal-regulated kinase，ERK)和磷酸化ERK、c-Jun N-末端激酶(C-Jun N-terminal kinase，JNK)和磷酸化JNK蛋白表达的变化。结果：NCTD诱导L929细胞凋亡。JNK抑制剂(SP600125)与ERK抑制剂(PD98059)，可明显抑制NCTD对细胞的杀伤作用。同时磷酸化ERK和磷酸化JNK表达增加。结论：NCTD对L929细胞的细胞毒作用是通过诱导其凋亡而发生的，且呈时间剂量依赖性。这种作用可能与JNK，ERK通路激活有关。     

JNK抑制剂对D-氨基葡萄糖衍生物诱导Eca-109细胞Caspase-3活化的影响

目的:观察特异性c-Jun氨基末端激酶(JNK)抑制剂SP600125对D-氨基葡萄糖衍生物(COPADG)诱导的Eca-109细胞Caspase-3激活及细胞凋亡的影响,并探讨COPADG诱导Eca-109细胞凋亡的潜在分子机制。方法:体外培养Eca-109细胞,以COPADG及SP600125与细胞作用,细胞间接免疫荧光染色观察P-JNK蛋白表达的改变,流式细胞术检测细胞凋亡率及Caspase-3活性的变化。结果:经SP600125处理后,COPADG诱导的Eca-109细胞P-JNK蛋白表达明显减弱,凋亡率明显减低,Caspase-3活性显著下调,与COPADG单作用组之间有显著性差异。结论:SP600125能够显著抑制COPADG诱导Eca-109细胞Caspase-3激活以及COPADG诱导Eca-109细胞凋亡,并间接证明JNK信号转导通路在COPADG诱导Eca-109细胞凋亡过程中发挥着重要作用。     

双硫仑联合Cu对淋巴瘤Raji细胞增殖与凋亡影响及其机制的探讨

目的:研究双硫仑(Disulfiram,DS)联合Cu离子对Burkitt’s淋巴瘤细胞株(Raji细胞)增殖和凋亡的影响及其与JNK和NF-κB通路的关系。方法:不同浓度DS和DS/CuMTT法检测对Raji细胞的增殖抑制作用;AnnexinⅤ-FITC/PI流式细胞术检测不同浓度DS和DS/Cu作用Raji细胞后凋亡细胞比例;蛋白质印迹法检测Cu、DS及DS/Cu处理细胞后p65、p-JNK及c-jun蛋白表达的变化。结果:DS和DS/Cu对Raji细胞均具有抑制增殖作用,作用72h两组IC50值分别为(0.793±0.08)和(0.085±0.015)μmol/mL,DS/Cu对Raji细胞增殖抑制作用显著强于DS单药,P=0.000。DS单药及DS/Cu对Raji细胞均有诱导凋亡作用,但DS/Cu诱导Raji细胞凋亡比例显著高于DS单药,P=0.001。蛋白质印迹法显示DS及DS/Cu作用Raji细胞后p-JNK及c-jun蛋白表达水平均显著增加、p65蛋白表达水平显著下调。结论:DS/Cu及DS对Raji细胞均有抑制增殖及诱导凋亡作用,其中DS/Cu的作用显著强于DS单药,其机制与同时抑制NF-κB和活化JNK通路有关。     

A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G(2)-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G(2)-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G(2)-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G(2)-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G(2)-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.     

雷公腾内酯与多柔比星诱导耐药白血病细胞凋亡协同作用及其机制的探讨

目的：以耐多柔比星（adriamycin,ADM）的人急性髓系白血病（acute myeloid leukemia,AML）耐药细胞株HL60/ADM为研究对象,探讨IC20浓度雷公腾内酯（triptolide,TPL）能否提高ADM诱导耐药白血病细胞凋亡及其与Nrf2通路的关系。方法：流式细胞仪检测空白对照组、IC20浓度TPL单药组、ADM单药组和TPL联合ADM组处理HL-60/ADM后细胞凋亡率;实时荧光定量PCR检测各个处理组作用后,Nrf2及其下游基因醌氧化还原酶（quinone oxidoreductase,NQO1）、谷胱甘肽还原酶（glutathione reductase,GSR）及血红素加氧酶1（heme oxygenase 1,HO-1）的表达水平变化;蛋白质印迹法检测各处理组作用后Nrf2蛋白表达变化。结果：TPL单药组细胞凋亡率为（5.28±0.80）%,与空白对照组的（7.09±0.46）%比较差异无统计学意义,P=0.226;但该浓度TPL可使ADM组细胞凋亡率由（19.55±1.70）%提高到（72.62±4.83）%,是ADM单药组的3.71倍,P〈0.001。空白对照组、TPL单药组、ADM单药组及双药联合组Nrf2mRNA表达水平分别为1、0.742±0.052、0.619±0.042和0.241±0.010,NQO1分别为1、0.363±0.075、0.228±0.053和0.050±0.034;GSR分别为1、0.268±0.042、0.231±0.106和0.038±0.017;HO-1分别为1、0.495±0.023、0.282±0.099和0.048±0.036;各用药组Nrf2及其下游基因NQO1、GSR及HO-1的mRNA表达水平较对照组均出现显著下调,P〈0.001;其中双药联合组下调程度最大。蛋白质印迹法结果显示,用药组及联合组Nrf2表达水平较对照组均有不同程度下调,其中联合组下调最为明显。结论：IC20浓度雷公腾内酯可显著提高ADM诱导耐药白血病细胞凋亡,其分子机制与下调Nrf2通路有关。     

Bcl-2 overexpression attenuates SP600125-induced apoptosis in human leukemia U937 cells

SP600125 is a specific inhibitor of c-Jun N-terminal kinase (JNK) that is known to strongly induce apoptosis and block cell cycle progression in G₂/M phase. In this study, we demonstrated that treatment of U937 cells with SP600125 resulted in significant G₂/M cell cycle arrest that was due to decreased cyclin B1 and cdc25c protein levels. Moreover, SP600125 promoted LDH release and DNA fragmentation that was associated with caspase-3 activation and degradation of its substrates. In contrast, overexpression of the antiapoptotic protein Bcl-2 rendered leukemia cells resistant to SP600125-induced apoptosis, but more sensitive to G₂/M phase arrest and endoreduplication (>4N DNA). Overexpression of Bcl-2 significantly inhibited SP600125-induced caspase-3 activation and degradation of its substrates, and sustained expression levels of the IAP-2 proteins following SP600125 treatment. The inhibitory effect of Bcl-2 on apoptosis was attenuated by treatment with the small molecule Bcl-2 inhibitor, HA14-1. These data provide important mechanistic insights related to Bcl-2-mediated resistance to SP600125-induced apoptosis, and induction of G₂/M phase arrest and endoreduplication.     

Knockdown of CD44 enhances chemosensitivity of acute myeloid leukemia cells to ADM and Ara-C

It is known that chemoresistance is a major cause of treatment failure in acute myeloid leukemia (AML). Substantial data indicate that the CD44 adhesion molecule is strongly expressed on AML blasts and that it can also inhibit apoptosis. Our study shows that drug resistance of the AML cell line HL60/ADM is due to overexpression of CD44. In an in vitro study, we knocked down CD44 in the HL60/ADM cell line using small interfering RNA (siRNA). Cell proliferation and the 50 % inhibitory concentrations (IC₅₀) were determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis and intracellular ADM accumulation were detected by flow cytometry. Expression of CD44, Bcl-2, c-Myc were assayed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The results indicate that the expression of CD44 in HL60/ADM cell line was much higher than in HL60 cell, and siRNA targeted CD44 (siRNA/CD44) could silence its expression in both mRNA and protein levels effectively. siRNA/CD44 substantially induces cell apoptosis, inhibits cell proliferation, enhances susceptibility to ADM and Ara-C, and at the same time increases intracellular ADM accumulation even reverses chemoresistance to ADM and Ara-C. Furthermore, by qRT-PCR and Western blot, we found that siRNA/CD44 decreases Bcl-2 and c-Myc synthesis. Our study provides a novel clue that CD44 plays a significant role in the chemoresistance of AML cells to Ara-C and ADM. Moreover, this provides a new direction to the approaches that combination therapy including targeting CD44 may overcome drug resistance and improve treatment effects.     

蛋白酶体抑制剂对肝癌HepG2细胞BAG3表达影响及其机制探讨

目的：探讨4种蛋白酶体抑制剂硼替佐米（bortezome,BZ）、环氧甲酮四肽（epoxomycin,Epox）、乳孢素（1actacystin,Lacta）和MG132单独作用或者联合JNK特异性抑制剂SP600125,对肝癌HepG2细胞内BAG3蛋白表达的影响及分子机制。方法：空白对照、100nmol／LBZ、100nmol／LEpox、500nmol／LLacta和2umol／LMG132单独或联合50μmol／LSP600125处理HepG2细胞;实时定量RT-PCR检测蛋白酶体抑制剂对肝癌HepG2细胞内BAG3基因mR—NA表达水平影响,蛋白质印迹法检测蛋白酶体抑制剂单独或者联合JNK激酶特异性抑制剂对BAG3蛋白表达水平的影响;利用MTT试剂盒检测蛋白酶体抑制剂细胞存活率,Hoechest33258染色检测细胞凋亡率。结果：蛋白酶体抑制剂BZ、Epox、Lacta和MG132均不同程度上调BAG3基因的mRNA和蛋白表达水平,而SP600125显著抑制蛋白酶体抑制剂引起的BAG3表达上调。MTT结果显示,SP600125显著抑制HepG2细胞对蛋白酶体抑制剂的敏感性;进一步Hoechst33258染色结果显示,4种蛋白酶体抑制剂联合SP600125作用引起HepG2细胞的凋亡率分别为（49．2±3．2）％、（58．7±3．5）％、（56．8±3．4）％和（53．4±3．3）％,明显高于蛋白酶体抑制剂单独作用组的（7．2±2．8）％、（16．7±3．2）％、（12．3±2．9）％和（11．4±3．0）％,P〈0．05。结论：蛋白酶体抑制剂通过JNK信号通路上调BAG3的表达,抑制JNK活性增加了HepG2细胞对蛋白酶体抑制剂的敏感性。     

Radix Tetrastigma Hemsleyani Flavone Induces Apoptosis in Human Lung Carcinoma A549 Cells by Modulating the MAPK Pathway

Radix Tetrastigma Hemsleyani Flavone (RTHF) is widely used as a traditional herb for its detoxificationand anti-inflammation activity. Recently, several studies have shown that RTHF can inhibit growth and induceapoptosis in human cancer cell lines. However, the mechanisms are not completely understood yet. In this studywe investigated the potential effects of RTHF on growth and apoptosis in human lung adenocarcinoma A549 cellsas well as its mechanisms. A549 cells were treated with RTHF at various concentrations for different times. Invitro the MTT assay showed that RTHF had obvious anti-proliferation effects on A549 cells in a dose- and timedependentmanner. Cell morphological changes observed by inverted microscope and Hoechst33258 methodswere compared with apoptotic changes observed by fluorescence microscope. Cell apoptosis inspected by flowcytometry showed significant increase in the treatment group over the control group (P<0.01). Expression ofapoptosis related Bax/Bcl-2, caspases and MAPK pathway proteins were detected by Western blotting. Theresults showed that RTHF up-regulated the Bax/Bcl-2 ratio and cle-caspase3/9, cle-PARP expression in a dosedependentmanner. Expression of p-p38 increased, p-ERK decreased significantly and that of p-JNK was littlechanged in the RTHF group when compared with the control group. These results suggest that RTHF mightexert anti-growth and apoptosis activity against lung cancer A549 cells through activation of caspases and Bcl-2family proteins and the MAPK pathway, therefore presenting as a promising therapeutic agent for the treatmentof lung cancer.     

Bcl-2反义肽核酸诱导HL60细胞凋亡

目的　探讨不同结构的反义药物对HL6 0细胞系生物学活性的影响。方法　应用细胞计数、细胞形态观察和流式细胞术观察并比较反义肽核酸和反义寡核苷酸对白血病细胞HL6 0生物学活性的影响。结果　 10 μmol/L靶向bcl mRNA蛋白编码区的反义肽核酸能有效地抑制HL6 0细胞的生长、下调bcl 2蛋白的水平及诱导细胞凋亡。 10μmol/L同样靶点的反义肽核酸和反义寡核苷酸作用HL6 0细胞 72小时 ,细胞凋亡的百分率分别为 17.8± 1.5 3 ,13.17± 1.12 ,统计学上有显著性差异。结论　反义Bcl 肽核酸能诱导HL6 0细胞的调亡 ,比反义寡核苷酸有更好的反义作用。