Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Emodin

Previous experiments have shown that emodin is highly active in suppressing the proliferation of several tumor cell lines. However, it is not clear that emodin can induce growth inhibition of hepatoma cells. We have found that emodin induces apoptotic responses in the human hepatocellular carcinoma cell lines (HCC) Mahlavu, PLC/PRF/5 and HepG2. The addition of emodin to these three cell lines led to inhibition of growth in a time-and dose-dependent manner. Emodin generated reactive oxygen species (ROS) in these cells which brought about a reduction of the intracellular mitochondrial transmembrane potential (Δ Ψ _m), followed by the activation of caspase–9 and caspase–3, leading to DNA fragmentation and apoptosis. Our findings demonstrate that ROS and the resulting oxidative stress play a pivotal role in apoptosis. Preincubation of hepatoma cell lines with the hydrogen peroxide-scavenging enzyme, catalase (CAT) and cyclosporin A (CsA), partially inhibited apoptosis. These results demonstrate that enhancement of generation of ROS, Δ Ψ _mdisruption and caspase activation may be involved in the apoptotic pathway induced by emodin.     

Dryocrassin ABBA Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells Through a Caspase-Dependent Mitochondrial Pathway

Background: Biological and pharmacological activities of dryocrassin ABBA, a phloroglucinol derivative extracted from Dryopteris crassirhizoma, have attracted attention. In this study, the apoptotic effect of dryocrassin ABBA on human hepatocellular carcinoma HepG2 cells was investigated. Materials and Methods: We tested the effects of dryocrassin ABBA on HepG2 in vitro by MTT, flow cytometry, real-time PCR, and Western blotting. KM male mice were used to detect the effect of dryocrassin ABBA on H22 cells in vivo. Results: Dryocrassin ABBA inhibited the growth of HepG2 cells in a concentration-dependent manner. After treatment with 25, 50, and 75 g/mL dryocrassin ABBA, the cell viability was 68%, 60% and 49%, respectively. Dryocrassin ABBA was able to induce apoptosis, measured by propidium iodide (PI)/annexin V-FITC double staining. The results of real-time PCR and Western ting showed that dryocrassin ABBA up-regulated p53 and Bax expression and inhibited Bcl-2 expression which led to an activation of caspase-3 and caspase-7 in the cytosol, and then induction of cell apoptosis. In vivo experiments also showed that dryocrassin ABBA treatment significantly suppressed tumor growth, without major side effects. Conclusions: Overall, these findings provide evidence that dryocrassin ABBA may induce apoptosis in human hepatocellular carcinoma cells through a caspase-mediated mitochondrial pathway.     

康莱特注射液诱导人肝癌细胞凋亡的研究

目的：探讨康莱特注射液诱导人肝癌细胞凋亡的作用。为康莱特注射液治疗肝癌提供理论依据。方法：采用免疫组织化学及透射电镜技术观察康莱特注射液对肝癌细胞凋亡的影响。结果：肝癌细胞经康莱特注射液培养24h后,其凋亡的百分率明显高于对照组和空白组,有显著性差异（P＜0．05）,且组织细胞凋亡在50％以上者占44．12％（15／34）,而对照组及空白组无1例凋亡超过50％;电镜相可见较为典型的细胞凋亡的形态学变化,细胞核固缩,染色质凝集,呈新月型紧贴于核膜周边,核碎裂,凋亡小体形成等。结论：康莱特注射液能诱导肝癌细胞发生凋亡;诱导细胞凋亡可是康莱特注射液治疗作用的重要机制。     

塞来昔布对人肝癌细胞株增殖及凋亡的影响

[目的]探讨环氧合酶-2(COX-2)抑制剂塞来昔布对体外培养的肝癌细胞的抗增殖及诱导凋亡作用。[方法]培养肝癌细胞株hepG2,用不同浓度的塞来昔布进行干预,MTT法检测对细胞生长的抑制作用;流式细胞术检测细胞凋亡情况及对细胞周期的影响。[结果]与对照组相比,随着塞来昔布剂量和作用时间的增加,MTT显示细胞的吸光度值逐渐降低,塞来昔布作用24、36、48h,对hepG2细胞的中效抑制浓度(IC50)分别为94.2μmol/L、78.3μmol/L和55.7μmol/L,相关系数分别为0.97、0.94和0.99;随着塞来昔布浓度的增加和作用时间的延长,细胞的凋亡率升高;塞来昔布作用48h后,随着药物浓度的增加G0/G1期细胞比例增加,S期细胞比例下降。[结论]塞来昔布能抑制hepG2细胞增殖,促进细胞凋亡,并呈剂量和时间依赖性。     

三氧化二砷对人肝癌HepG2细胞内活性氧水平的影响

[目的]探讨三氧化二砷（As2O3）对人肝癌细胞生长抑制和诱导凋亡作用及其对细胞内活性氧（ROS）水平的影响。[方法]用MTT法、DNALadder检测及流式细胞术等观察As2O3对人肝癌细胞株HepG2的生长抑制和诱导凋亡作用及细胞内ROS的变化。[结果]As2O3能明显抑制HepG2细胞的增殖,并呈时间和剂量依赖性。DNALadder检测示,121μmol／L As2O3处理HepG2细胞48h开始出现DNALadder条带。流式细胞仪分析显示As2O3处理人肝癌细胞HepG2 12、24、36h后细胞内ROS水平较对照组明显升高（P〈0．01）,且呈时间依赖性。[结论]As20,可通过抑制人肝癌细胞HepG2增殖和提高细胞内ROS水平诱导细胞凋亡而发挥抗肿瘤作用。     

CA3诱导肝癌HepG2细胞凋亡及其机制CSCD

目的探讨CA3(CIL56)对肝癌HepG2细胞增殖及凋亡的影响及其机制。方法体外培养HepG2细胞,分别加入不同浓度CA3(0、0.25、0.5、1.0、2.0和4.0 mmol/L)作用24和48 h。增强型CCK-8试剂检测不同浓度CA3对细胞增殖的影响;流式细胞术检测细胞凋亡率;检测细胞内活性氧(ROS)的变化;Western blot法分析YAP1、Bcl-2、Bax、Caspase-3蛋白的表达。结果增强型CCK-8试剂检测结果显示,0.25、0.5、1.0、2.0、4.0 mmol/L的CA3作用HepG2细胞24和48 h后,细胞增殖率分别为(80.5±0.3)%、(79.4±0.2)%、(76.2±0.2)%、(76.4±0.1)%、(49.3±0.4)%和(75.3±0.2)%、(64.8±0.3)%、(48.4±0.2)%、(32.2±0.4)%、(31.9±0.2)%。流式细胞术结果显示,CA3浓度升高至2 mmol/L时,凋亡率增加到58.48%。CA3处理后,HepG2细胞内活性氧产量增加,YAP1、Bcl-2蛋白表达降低,Bax、Caspase-3表达增高。结论 CA3可抑制HepG2细胞增殖并诱导凋亡,其机制可能与细胞内活性氧产量增加,调节YAP1、Bcl-2、Bax及Caspase-3蛋白的表达有关。     

Induction of Apoptosis by IGFBP3 Overexpression in Hepatocellular Carcinoma Cells

Background: The insulin-like growth factor (IGF) system comprises a group of proteins that play key rolesin regulating cell growth, differentiation, and apoptosis in a variety of cellular systems. The aim of this studywas to investigate the role of insulin-like growth factor binding protein 3 (IGFBP3) in hepatocellular carcinoma.Materials and Methods: Expression of IGF2, IGFBP3, and PTEN was analyzed by qRT-PCR. Lentivirus vectorswere used to overexpress IGFBP3 in hepatocellular carcinoma cell (HCC) lines. The effect of IGFBP3 onproliferation was investigated by MTT and colony formation assays. Results: Expression of IGF2, IGFBP3, andPTEN in several HCC cell lines was lower than in normal cell lines. After 5-aza-2’-deoxycytidine/trichostatin Atreatment, significant demethylation of the promoter region of IGFBP3 was observed in HCC cells. Overexpressionof IGFBP3 induced apoptosis and reduced colony formation in HUH7 cells. Conclusions: Expression of IGF2,IGFBP3, and PTEN in several HCC cell lines was lower than in normal cell lines. After 5-aza-2’-deoxycytidine/trichostatin A treatment, significant demethylation of the promoter region of IGFBP3 was observed in HCCcells. Overexpression of IGFBP3 induced apoptosis and reduced colony formation in HUH7 cells.     

苯丁酸钠体外对人肝癌细胞HepG2增殖和凋亡的影响

[目的]探讨组蛋白去乙酰化酶抑制剂苯丁酸钠（PB）体外对人肝癌细胞HepG2增殖和凋亡的影响。[方法]不同浓度苯丁酸钠处理HepG2,应用MTT比色法观察苯丁酸钠对HepG2的生长抑制作用,落射荧光显微镜、DNA电泳和流式细胞仪观察HepG2的凋亡,West-ernblot检测苯丁酸钠处理前后HepG2细胞中Bcl-2、Bax蛋白表达水平的变化。[结果]苯丁酸钠2mmol/L、4mmol/L、8mmol/L作用48h对细胞的抑制率分别为19.41%、39.03%、42.19%,作用72h对细胞的抑制率分别为27.42%、57.11%、70.31%,并诱导细胞凋亡;4mmol/L苯丁酸钠作用HepG248h细胞凋亡率明显高于对照组。落射荧光显微镜和DNA电泳均观察到苯丁酸钠作用后HepG2细胞出现凋亡细胞的特征性变化。苯丁酸钠处理HepG2后Bcl-2蛋白表达减少,Bax蛋白表达增加。[结论]苯丁酸钠体外抑制HepG2增殖并促进其凋亡,其作用机制可能是通过降低细胞内的Bcl-2蛋白,增加细胞内Bax蛋白而实现的。     

Survivin反义寡核苷酸对肝癌细胞增殖和凋亡的影响

目的:观察Survivin反义寡核苷酸对SMMC-7721细胞增殖、凋亡的影响。方法:设计合成特异性Survivin反义寡核苷酸,转染肝癌SMMC-7721细胞,MTT法测定Survivin ASODN对细胞增殖抑制情况的影响,FCM法检测对细胞周期、凋亡及Survivin蛋白表达的影响。结果:Survivin ASODN可抑制SMMC-7721细胞的生长增殖,并呈浓度和时间依赖性。ASODN转染组可诱导SMMC-7721细胞凋亡(P<0.01),细胞周期阻滞于G2/M期(P<0.05)。ASODN转染组Survivin蛋白表达水平显著降低(P<0.01)。结论:Survivin ASODN能下调SMMC-7721细胞Survivin表达,并可抑制其增殖并诱导凋亡。     

羧基末端结合蛋白1对肝癌细胞HepG2增殖的影响

目的 研究羧基末端结合蛋白1（C-terminal binding protein1,CtBP1）对人肝癌细胞HepG2增殖的影响及机制。 方法 采用CtBP1 siRNA转染HepG2细胞,实验分为4个组：空白对照组、转染试剂对照组、阴性序列对照组与干扰组。于转染后不同的时间收集细胞,用MTT法检测细胞增殖、流式细胞术检测细胞周期及凋亡情况。结果 CtBP1 siRNA转染有效抑制了HepG2细胞中CtBP1蛋白表达,与空白对照组比较,转染后72 h后,CtBP1蛋白下调了57.80%。 CtBP1沉默后细胞生长受到抑制（P＜0.05）,转染后24、48、72、96 h生长抑制率分别为22.34%、52.15%、53.97%和54.77%;而对细胞周期分布无明显影响（P＞0.05）; 但CtBP1表达下调有促凋亡作用,与对照组相比,干扰组的细胞凋亡率显著升高（P＜0.05）。结论 CtBP1沉默能抑制HepG2细胞增殖,其机制是通过促凋亡来实现的。CtBP1能促进肿瘤生长和抑制凋亡,是潜在的肿瘤治疗靶点。     

Cell Death Induced by Baicalein in Human Hepatocellular Carcinoma Cell Lines

We examined the action of baicalein, a flavonoid contained in the herbal medicine sho-saiko-to (TJ-9), on three cell lines of human hepatocellular carcinoma (HCC). Treatment with baicalein strongly inhibited the activity of topoisomerase II and suppressed the proliferation of all three HCC cell lines. But the mode of cell death induced by baicalein differed according to the cell line. Baicalein induced apoptosis in a concentration-dependent mannner in only one cell line, and an increased concentration of baicalein produced cell death via necrosis in the other two lines. These results suggest that the inhibition of topoisomerase II is not by itself sufficient for induction of apoptosis, and that there is a more important mechanism which can account for the difference in susceptibility of cells to apoptosis induced by baicalein.     

肿瘤抗原致敏树突状细胞协同CD3AK细胞的抗肿瘤作用

目的:观察雷公藤内酯醇是否能抑制肿瘤细胞增殖,并探讨其是否通过诱导肿瘤细胞凋亡发挥抗肿瘤作用.方法:选择人宫颈癌细胞株HeLa为研究对象,以MTr实验检测肿瘤细胞的增殖,以AnnexinV/PI染色检测细胞的凋亡,R123染色检测线粒体膜电势的变化.Hoechst 33258荧光染色法观察细胞形态学改变,琼脂糖凝胶电泳检测染色体DNA断裂.结果:雷公藤内酯醇能明显抑制HeLa细胞的增殖;AnnexinV/PI染色结果证明雷公藤内酯醇能诱导HeLa细胞凋亡,R123染色结果说明雷公藤内酯醇诱导的细胞凋亡与线粒体膜电势的变化有关.雷公藤内酯醇处理的HeLa细胞可见凋亡特有的形态学及生物化学改变,DNA电泳呈梯状条带.结论:雷公藤内酯醇可以通过诱导肿瘤细胞凋亡而发挥抗肿瘤作用.     

雷公藤内酯醇对人宫颈癌细胞的凋亡诱导效应

目的:探讨腺病毒介导AFP基因修饰的DC(AFP-DC)瘤苗经不同途径免疫后机体抗肿瘤免疫应答反应.方法:采用皮下注射、静脉注射和瘤体注射三种途径回输AFP-DC瘤苗,比较观察AFP-DC瘤苗对荷瘤小鼠免疫治疗作用,应用4h51cr释放杀伤实验、T细胞与NK体内剔除实验等方法,观察AFP-DC瘤苗对荷瘤小鼠免疫治疗作用及保护性免疫反应.结果:皮下注射AFP-DC瘤苗治疗效果在抑制肿瘤生长、延长小鼠存活期方面都明显优于瘤体内注射或尾静脉注射(P＜0.05),AFP-DC瘤苗体内能更有效地诱导特异CTL细胞毒活性,能使免疫动物产生一定的免疫保护作用,抵抗肿瘤细胞的再攻击.在AFP-DC瘤苗诱导抗肿瘤免疫排斥反应过程中,必需有CD4 T和CD8 T细胞的参与;而在其效应阶段,则依赖于CD8 T细胞的参与,CD4 T细胞为非必需;在免疫诱导及效应阶段剔除NK细胞对抗肿瘤免疫应答无明显影响.结论:皮下注射AFP-DC瘤苗能有效诱导机体产生抗肿瘤免疫反应,为DC介导的肝癌免疫治疗开辟了新的途径.     

三氧化二砷诱导人肝癌细胞株HepG2凋亡的机制研究

目的：探讨三氧化二砷（As2O3）诱导人肝癌细胞株HepG2凋亡的作用机制。方法：采用MTT法观察不同浓度的As2O3对人类肝癌细胞株HepG2生长的抑制作用;以流式细胞术观察细胞的凋亡率;以Westernblot法检测JNK、p-JNK、Caspase-3及PARP蛋白在As2O3作用下及SP600125阻断JNK信号转导通路情况下的表达。结果：As2O3对体外生长的肝癌细胞HepG2具有明显抑制作用,并可诱导细胞凋亡。Westernblot结果显示,As2O3诱导肝癌细胞HepG2凋亡伴随着Caspase-3和PARP的活化;AS2O3作用于HepG2细胞10min后p-JNK蛋白表达开始增加,20min达到高峰,30min开始减少,总JNK蛋白的含量无明显改变,JNK的激活早于细胞凋亡;用SP600125预处理HepG2细胞株后,可以明显减少Caspase-3和PARP的活化。结论：As2O3通过诱导细胞凋亡抑制肝癌细胞株HepG2的增殖,细胞凋亡通过Caspase-3途径实现。JNK信号转导通路参与了As2O3诱导的HepG2凋亡反应,并位于Cas-pase-3的上游。     

法尼基转移酶抑制剂ManumyCin诱导HepG2细胞凋亡的研究

目的:研究法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞的抗肿瘤作用,并探讨其诱导凋亡的分子机制。方法:采用MTT(Methythiazolyltetrazolium)法观察法尼基转移酶抑制剂Manumycin对肝癌细胞株HepG2细胞增殖的抑制作用,荧光显微镜、DNA凝胶电泳、流式细胞术等技术检测细胞凋亡,应用Westernblot方法检测bcl-2、p53、bax的蛋白水平变化。结果:法尼基转移酶抑制剂Manumycin能明显抑制HepG2细胞的生长且呈浓度依赖性,其IC50为(17.65±0.58)μmol/L。荧光显微镜检查显示Manumycin处理的HepG2细胞DAPI染色后,细胞核内可见浓染致密的颗粒荧光,典型细胞可见新月型改变,固缩或片段化的核。DNA凝胶电泳可见典型的DNA梯形带。流式细胞DNA直方图上出现典型的亚二倍体“凋亡峰”,细胞凋亡与Manumycin作用的时间和浓度相关。Manumycin能时间依赖性地诱导HepG2细胞发生G2/M期阻滞。Manumycin处理HepG2细胞后,Westernblot检测结果显示p53蛋白表达明显增加,而bcl-2蛋白和bax蛋白表达无明显变化。结论:法尼基转移酶抑制剂Manumycin对人肝癌细胞株HepG2有强烈的细胞毒作用,其分子机制可能是诱导HepG2细胞凋亡。Manumycin诱导HepG2细胞凋亡与bcl-2蛋白和bax蛋白表达水平无关,而p53蛋白表达水平的上调可能在此过程中起了一定     

Effects of Genistein and Synergistic Action in Combination with Tamoxifen on the HepG2 Human Hepatocellular Carcinoma Cell Line

Introduction: The flavonoids comprise a diverse group of polyphenolic compounds with antioxidant activity that is present in edible plants like soybeans and soy products. In vivo studies have concentrated on the effects of flavonoids on cancer and genistein (GE), a soy-derived isoflavone, has been reported to reduce prostate, colon, hepatic and breast adenocarcinoma risk. Tamoxifen (TAM) is an important drug for cancer treatment worldwide, which can induce apoptosis in various cancers, including examples in the liver, breast and ovaries. The aim of the present study was to evaluate the effects of GE and TAM, alone and in combination, on proliferation and apoptosis in the human hepatocellular carcinoma (HCC) HepG2 cell line. Materials and Methods: HepG 2 cells were treated with GE, TAM and GE/TAM and then MTT and flow cytometry assays were conducted to determine effects on viability and apoptosis, respectively. Results: GE and TAM inhibited cell proliferation and induced apoptosis in the HepG 2 cell lines. Discussion: Our findings clearly indicated that GE and TAM may exert inhibitory and apoptotic effects in liver cancer cells. Conclusion: GE and TAM can significantly inhibit growth of HCC cells and play a significant role in apoptosis.     

硒-甲基硒代半胱氨酸诱导肝癌HepG2细胞凋亡作用

[目的]探讨硒-甲基硒代半胱氨酸(MSC)对肝癌HepG2细胞的增殖抑制及诱导凋亡的作用.[方法]用不同浓度的MSC处理培养的HepG2细胞,MTT法和流式细胞仪分别检测其对细胞生长和细胞凋亡的影响,并检测半胱氨酸蛋白酶3,8,9(caspase-3,8,9)的活性.[结果]25μmol/L的MSC处理HepG2细胞24 h后,细胞生长受到明显抑制,出现细胞凋亡,呈现浓度和时间依赖关系.caspase-3,8,9的活性检测显示,25μmol/L MSC处理HepG2细胞24h,48 h后,caspase-3的活性分别升高38.50%和119.72%,caspase-8活性分别升高28.86%和89.42%,caspase-9活性分别升高31.94%和74.28%.50μmol/L的MSC处理24h,48h后,caspase-3活性分别升高82.56%和155.79%,caspase-8活性分别升高48.95%和167.84%,caspase-9活性分别升高55.94%和126.58%.[结论]MSC抑制HepG2细胞增殖,诱导其凋亡,其凋亡与caspase-3,8,9的活性增强有关.     

Effect of Paclitaxel-loaded Nanoparticles on the Viability of Human Hepatocellular Carcinoma HepG2 Cells

Objective: To explore effects of paclitaxel-loaded poly lactic-co-glycolic acid (PLGA) particles on the viabilityof human hepatocellular carcinoma (HCC) HepG2 cells. Materials and Methods: The viability of HepG2 cellswas assessed using MTT under different concentrations of prepared paclitaxel-loaded particles and paclitaxel(6.25, 12.5, 25, 50, and 100 mg/L), and apoptosis was analyzed using Hochest33342/Annexin V-FITC/PI combinedwith an IN Cell Analyzer 2000. Results: Paxlitaxel-loaded nanoparticles were characterized by narrow particlesize distribution (158.6 nm average particle size). The survival rate of HepG2 cells exposed to paclitaxel-loadedPLGA particles decreased with the increase of concentration and time period (P<0.01 or P<0.05), the dose- andtime-dependence indicating sustained release (P<0.05). Moreover, apoptosis of HepG2 cells was induced, againwith an obvious dose- and time-effect relationship (P<0.05). Conclusions: Paclitaxel-loaded PLGA particles caninhibit the proliferation and induce the apoptosis of HCC HepG2 cells. This new-type of paclitaxel carrier bodyis easily made and has low cost, good nanoparticle characterization and sustained release. Hence, paclitaxelloadedPLGA particles deserve to be widely popularized in the clinic.     

罗格列酮对肝癌HepG2细胞周期和凋亡的影响

目的研究罗格列酮对人肝癌HepG2细胞周期和凋亡的影响。方法设不同浓度罗格列酮组(10、25、50.0、100.0μg/ml)、2‰二甲基亚砜的RPMI1640培养基加细胞为阴性对照组。应用MTT法检测罗格列酮对肝癌HepG2细胞的增殖抑制和生长活性,流式细胞仪检测细胞周期和凋亡。结果罗格列酮对肝癌HepG2细胞具有较强的体外毒性,48 h IC50为41.6μg/ml。肝癌HepG2细胞活性随着用药剂量和作用时间的增加而不断下降,出现剂量-效应和时间-效应的关系。罗格列酮作用48 h后,肝癌HepG2细胞G_0/G_1期比例显著升高,S期细胞比例有所降低,到G_2/M期细胞的比例明显下降,并呈明显的剂量依赖性。罗格列酮对HepG2细胞作用48 h后可以诱导肿瘤细胞产生凋亡,并且呈剂量依赖性。结论罗格列酮能抑制肝癌HepG2细胞生长并促进其凋亡。