Anti-hepatoma cells function of luteolin through inducing apoptosis and cell cycle arrest

The aim of this study is to explore the apoptotic induction and cell cycle arrest function of luteolin on the liver cancer cells and the related mechanism. The liver cancer cell line SMMC-7721, BEL-7402, and normal liver cells HL-7702 were treated with different concentrations of luteolin. Cell proliferation ability was tested. Morphological changes of the apoptotic cells were observed under inverted fluorescence microscope after Hoechst33342 staining. We investigated the effect of luteolin on cell cycling and apoptosis with flow cytometry. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Caspases-3 and Bcl-2 family proteins expression were analyzed by real-time PCR. Cell proliferation of SMMC-7721 and BEL-7402 were inhibited by luteolin, and the inhibition was dose–time-dependent. Luteolin could arrest the cells at G1/S stage, reduce mitochondrial membrane potential, and induce higher apoptosis rate and the typical apoptotic morphological changes of the liver carcinoma cells. Q-RT-PCR results also showed that luteolin increased Bax and caspase-3 expression significantly and upregulated Bcl-2 expression in a dose-dependent manner in liver carcinoma cells. However, the normal liver cells HL-7702 was almost not affected by luteolin treatment. Luteolin can inhibit SMMC-7721 and BEL-7402 cell proliferation in a time- and dose-dependent manner. And the mechanism maybe through arresting cell cycle at phase G1/S, enhancing Bax level, reducing anti-apoptotic protein Bcl-2 level, resulting in activating caspase-3 enzyme and decrease of mitochondrial membrane potential, and finally leading to cell apoptosis.     

siRNA沉默RhoC表达诱导人肝癌BEL7402细胞凋亡

目的:研究siRNA沉默RhoC基因表达对人肝癌细胞BEL7402凋亡的影响及其机制,为肝癌的基因治疗提供实验依据。方法:构建RhoC-siRNA真核表达载体pU6mRFPRhoC-siRNA,转染BEL7402细胞,激光共聚焦显微镜检测转染效率,RT-PCR和Western blotting鉴定RhoC基因沉默效果;流式细胞术、琼脂糖凝胶电泳和瑞氏染色检测BEL7402细胞凋亡,RT-PCR检测细胞凋亡相关基因Bcl-2和Bax的表达。结果:成功构建pU6mRFPRhoC-siRNA重组载体,转染BEL7402细胞的效率为70%,RT-PCR和Western blotting检测RhoC基因沉默效率分别为85%和82%。pU6mRFPRhoC-siRNA转染组BEL7402细胞凋亡显著高于未转染BEL7402细胞和pU6mRFP Scramble-siRNA转染组BEL7402细胞[(21.00±2.23)%vs(6.47±1.64)%、(4.63±0.47)%,P<0.01)],pU6mRFPRhoC-siRNA转染组BEL7402细胞DNA呈典型的梯状条带,瑞氏染色见转染组BEL7402细胞中有大量凋亡...     

RhoC在肝癌细胞生长中的作用及分子机制

目的 研究RhoC在肝癌细胞生长中的作用及分子机制,为抑制肝癌细胞生长寻找分子靶点.方法 构建siRNA-RhoC载体,建立RhoC基因沉默的肝癌细胞株.采用四甲基偶氮唑蓝(MTT)法检测细胞生长,硝酸银染色检测细胞增殖,平板克隆实验检测细胞克隆形成能力,流式细胞术(FACS)检测细胞周期,逆转录聚合酶链反应(RT-PCR)检测细胞周期蛋白表达.PcDNA3-RhoC转染肝细胞,建立RhoC高表达细胞株,检测RhoC基因对正常肝细胞生长的影响.结果 siRNA-RhoC载体转染Bel7402细胞后,RhoC基因表达的抑制效率为82.3%.细胞培养第4天,RNAi组吸光度(A)值为0.41±0.10,显著低于Bel7402细胞组(0.73±0.11,P＜0.05)和阴性对照组(0.71±0.07,P＜0.05).此后,RNAi组的细胞增殖速度持续低于Bel7402细胞组和阴性对照组,直到第7天实验结束.RNAi组的细胞核平均银染颗粒数明显少于Bel7402细胞组和阴性对照组(分别为1.23±0.35、3.47±0.93和3.17±0.78,均P＜0.01);G0/G1期细胞显著升高[分别为(73.14±5.93)%、(57.05±5.97)%和(52.99±4.80)%,均P＜0.05];cyclin D1表达下降(分别为0.45±0.21、1.25±0.24和1.12±0.15,均P＜0.05);CDK4表达下降(分别为0.55±0.08、1.18±0.32和1.10±0.29,均P＜0.05);p16表达升高(分别为1.07±0.23、0.36±0.12和0.35±0.13,均P＜0.01);p21表达升高(分别为0.42±0.12、0.17±0.06和0.19±0.08,均P＜0.05).HL7702细胞转染PcDNA3-RhoC质粒后,RhoC 高表达,至转染的第3天,RhoC转染组A值为0.83±0.10,显著高于HL7702细胞组(0.54±0.11,P＜0.05)和空载体对照组(0.58±0.55,P＜0.05).其后,RhoC转染细胞生长速度持续高于HL7702细胞组和空载体对照组,直至第7天实验结束.结论 RhoC是调控肝癌细胞生长的关键分子,是抑制肝癌细胞生长的良好分子靶点.

Abstract：

Objective To clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.Methods siRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup.Cell growth was assessed by MTT assay.AgNORs staining was applied to determine cell proliferation.Plate cell clone test was conducted to examine the capacity of cell clone formation.FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins.In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.Results The inhibition rate of RhoC was 82.3%.From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ±0.10 vs.0.73 ±0.11and 0.71 ±0.07 respectively, P ＜0.05).AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ±0.35 vs.3.47 ±0.93 and 3.17 t0.78,P ＜0.01 ).Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42 ) % vs.(70.58 ± 10.10) %and (69.83 ± 14.77) %, respectively, P ＜ 0.01].Cell cycle analysis by FACS showed that G0/G1 cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ±5.93)% vs.(57.05 ± 5.97 ) % and (52.99 ± 4.80) %, P ＜ 0.05].Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1 (0.45 ±0.21 vs.1.25 ± 0.24 and 1.12 ± 0.15, respectively, P ＜ 0.05 ) and CDK4 (0.55 ± 0.08 vs.1.18 ± 0.32and 1.10 ± 0.29, respectively, P ＜ 0.05 ); the following genes were notably increased: p16 (1.07 ± 0.23vs.0.36±0.12 and 0.35 ±0.13, respectively, P ＜0.01)and p21 (0.42 ±0.12 vs.0.17 ±0.06 and 0.19±0.08, respectively, P ＜ 0.05).RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line.From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups(0.83 ±0.10 vs.0.54 ±0.11 and 0.58 ±0.55, respectively, P ＜ 0.05 ).Conclusions RhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.     

雷氏大疣蛛蛛毒对人肝癌BEL-7402 细胞增殖的抑制作用及其机制的研究

背景与目的蜘蛛毒素抗肿瘤的研究迄今还是未知领域。本研究探讨雷氏大疣蛛(Macrotheleraveni)蛛毒对BEL-7402细胞增殖的抑制作用,进一步探讨其作用机制。方法采用MTT法测定了雷氏大疣蛛蛛毒(10、20、40、80滋g/ml)对BEL-7402细胞增殖作用的影响;采用[3H]-TdR掺入法检测蛛毒加入前后BEL-7402细胞DNA的变化;利用流式细胞光度术(FCM)探讨雷氏大疣蛛蛛毒对BEL-7402细胞凋亡率和细胞周期的影响;利用Westernblot方法进一步检测雷氏大疣蛛蛛毒对细胞周期相关基因c-myc的变化。结果雷氏大疣蛛蛛毒对BEL-7402细胞增殖有较强的抑制作用(P<0.05),IC50为20滋g/ml,时效和量效关系良好。雷氏大疣蛛蛛毒可以抑制BEL-7402细胞DNA的合成。流式细胞仪检测发现经过雷氏大疣蛛蛛毒作用下的BEL-7402细胞凋亡率增加,细胞周期阻滞在G0/G1期。Westernblot方法进一步检测表明雷氏大疣蛛蛛毒作用于BEL-7402细胞72h后,c-myc基因表达减弱。结论雷氏大疣蛛蛛毒就可以抑制人肝癌BEL-7402细胞的增殖和DNA的合成。其药理作用机制可能是除了诱导凋亡外,主要是使细胞周期相关基因c-myc表达减弱,导致细胞周期的变化。     

PDGF-D对人肝癌细胞增殖及VEGF表达影响研究*

目的：研究血小板源性生长因子D（PDGF-D）对人肝癌细胞株BEL-7402增殖及其血管内皮生长因子（VEGF）表达的影响。方法：体外培养肝癌细胞株BEL-7402和肝癌旁非瘤性细胞株QSG-7701,采用RT-PCR 方法检测PDGF-D 与PDGFR βmRNA 在BEL-7402和QSG-7701的表达情况;将浓度分别为0（对照）、5、10、20、50、100、200 μ g/mL 的人重组PDGF-DD蛋白加入BEL-7402中,采用四甲基偶氮唑蓝比色法检测肝癌细胞的生长曲线;流式细胞仪检测细胞周期变化;半定量RT-PCR 检测VEGF及PDGFR β mRNA 表达情况,ELISA 检测PDGF-DD干预细胞后培养上清中VEGF蛋白的表达情况。结果：PDGF-D 及PDGFR βmRNA 在BEL-7402中高表达,与QSG-7701相比差异有统计学意义（P<0.05）。 PDGF-DD干预细胞后,可促进人BEL-7402增殖,浓度为100 μ g/mL 时达最高峰;细胞周期分布变化,G0/G1 期细胞数减少,S 期细胞数增加,与对照组相比差异有统计学意义;RT-PCR 结果显示,VEGF 及PDGFR β RI 值,实验组（除5 μ g/mL 组外）与对照组相比差异有统计学意义;ELISA 结果显示,加入PDGF-DD各浓度组VEGF蛋白浓度较对照组增高,差异有统计学意义,并呈量效依赖性关系。结论：PDGF-DD能促进BEL-7402的增殖,上调PDGFR β 及VEGF的表达。PDGF-D 及其信号传导系统在肝癌的发生、发展中可能发挥重要的作用,可作为肝癌预后预测指标和治疗靶点。     

家蝇抗菌肽Cecropin对人肝癌BEL-7402细胞周期的影响及其机制

目的探讨家蝇抗菌肽Cecropin对人肝癌BEL-7402细胞周期的影响及其机制。方法人肝癌细胞BEL-7402经家蝇抗菌肽Cecropin作用后,光学显微镜观察细胞生长情况,PI单染流式细胞术检测各组细胞周期分布情况,并采用间接免疫荧光标记技术通过流式细胞仪检测细胞周期检测点激酶1(CHK1) 和细胞周期依赖性蛋白激酶2(CDK2)。结果光学显微镜下,对照组细胞形态规则,呈梭形贴壁生长,抗菌肽组细胞部分变圆,贴壁不佳,数量减少;细胞增殖周期发生改变, G₀/G₁与G₂/M期细胞比例降低,S期细胞比例增高,与对照组相比差异有统计学意义(P<0.05) ,并且细胞周期调控关键蛋白分子CHK1和CDK2的表达增高。结论家蝇抗菌肽Cecropin可通过调节CHK1和CDK2的表达影响BEL-7402细胞增殖周期。     

Growth arrest and apoptosis of the human hepatocellular carcinoma cell line BEL-7402 induced by melittin

OBJECTIVE: The aim of this study was to investigate the cellular effects of melittin on the growth and apoptosis of human hepatocellular carcinoma (HCC) cells and to provide the molecular mechanism for potential application of a recombinant adenovirus carrying the melittin gene (Ad-rAFP-Mel) in the treatment of liver cancer. METHODS: Human HCC cells (BEL-7402) were infected with Ad-rAFPMel at different times. In vitro cell growth was determined by MTT assay. Cellular apoptosis was evaluated quantitatively and qualitatively by phase-contrast microscopy, transmission electron microscopy, DNA ladder electrophoresis, TUNEL staining and flow cytometry. RESULTS: Ad-rAFP-Mel infection had an inhibitory effect on the proliferation of BEL-7402 cells. The morphological changes of apoptosis were confirmed by microscopy and DNA electrophoresis. The ultrastructural characteristics of apoptotic cells, such as chromatin condensation and nuclear fragmentation, were also observed by electron microscopy in the Ad-rAFP-Mel-infected cells. Ad-rAFPMel infection markedly induced cellular apoptosis, and Fas expression on Bel-7402 cells infected by Ad-rAFPMel was up-regulated. CONCLUSION: The fact that melittin can induce apoptosis of the HCC cell line BEL-7402 leads us to consider adenovirus-mediated delivery of melittin as a promising approach for the treatment of HCC. However, the underlying mechanism needs to be further investigated.     

miR-16对人肝癌细胞BEL-7402增殖与凋亡的影响

背景与目的：miR-16和miR-15a基因复合体位于人13q14区域的DLEU2基因内含子内,是目前公认的抑癌基因之一。该基因区域的缺失与多种实体肿瘤有关,miR-16同时促进肿瘤细胞的凋亡。该研究旨在探讨miR-16对BEL-7402肝癌细胞增殖与凋亡的影响。方法：人肝癌细胞BEL-7402分为miR-16感染组(加入LV-hsamiR-16-1慢病毒)和阴性对照组(加入阴性对照病毒),采用倒置荧光显微镜观察细胞绿色荧光的强度;采用细胞计数试剂盒(cell counting kit-8,CCK-8)检测miR-16对BEL-7402肝癌细胞增殖的影响;采用流式细胞术分析miR-16对人肝癌细胞BEL-7402的细胞周期与凋亡的影响。结果：CCK-8检测结果显示,感染组细胞增殖能力明显降低(P<0.05);流式细胞术检测结果显示,阴性对照组BEL-7402细胞周期中G₁期细胞百分率数值明显下降,但S及G₂/M期细胞的百分率均明显上升(P<0.05)。miR-16促进BEL-7402肝癌细胞凋亡。结论：miR-16抑制BEL-7402肝癌细胞的增殖并促进其凋亡,miR-16有望成为临床肝癌靶向治疗的新靶点。     

雷帕霉素对人肝癌细胞BEL-7402体外抗肿瘤作用研究

目的:观察雷帕霉素体外对肝癌细胞BEL-7402生长、细胞凋亡以及细胞转移能力作用。方法:以5nmol/L,10nmol/L,20nmol/L,30nmol/L,40nmol/L和50nmol/L不同浓度的雷帕霉素作用于体外培养的BEL-7402细胞,MTT法检测细胞生长抑制率,应用流式细胞仪检测细胞凋亡,Hoechst33258荧光染色法观察细胞凋亡时的形态学变化,细胞粘附分析及体外侵袭试验观察肿瘤细胞的转移能力。结果:雷帕霉素可显著的抑制BEL-7402细胞的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系。雷帕霉素作用肝癌细胞BEL-740248h后,在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征。雷帕霉素能抑制其粘附性和侵袭性,具有剂量依赖性,随浓度增加而抑制增加。结论:雷帕霉素不仅能诱导BEL-7402肝癌细胞凋亡,抑制细胞的生长,而且同时抑制肿瘤细胞的粘附性和侵袭性,阻止肿瘤细胞的转移,雷帕霉素可能成为一种潜在的抗肝癌药物。     

siRNA表达载体介导的RNA干扰对肝癌细胞周期蛋白E1表达抑制研究

目的探讨经载体介导的RNA干扰对肝癌细胞周期蛋白E1表达抑制效率。方法针对周期蛋白E1基因设计并合成特定的RNA干扰模板片段，连接到pSilencer1．0-U6载体上构建siRNA真核表达载体；脂质体转染法将上述重组质粒转人BEL-7402肝癌细胞株。RT—PCR分析周期蛋白E1表达抑制率；流式细胞术测细胞周期S期和凋亡细胞比率；MTT比色法测定活细胞数并绘制细胞生长曲线。结果重组载体介导肝癌细胞BEL-7402周期蛋白E1抑制率达62％；转染重组质粒32h测得S期细胞为19％，空载体转染对照为27％；转染重组质粒96h流式细胞术测得凋亡细胞为13．1％，高于空载体对照的6．6％；细胞生长曲线作图表明，重组载体转染组细胞生长明显减缓。结论肝癌细胞周期蛋白E1的表达可被载体介导诱发的RNA干扰成功抑制，进而导致细胞生长受抑并诱导凋亡；本研究为探索肝癌的RNAi治疗提供了初步的实验依据。     

Caspase-3在雷帕霉素诱导肝癌细胞BEL-7402凋亡中的作用

背景与目的:雷帕霉素是从吸水性链霉菌(Streptomyceshygroscopicus)发酵液中提取,最近研究发现雷帕霉素具有免疫抑制作用和广泛的抗肿瘤作用。本研究主要探讨Caspase-3在雷帕霉素诱导肝癌细胞BEL-7402凋亡中的作用。方法:以不同浓度(5、10、20、30、40、50nmol/L)的雷帕霉素作用于BEL-7402细胞,MTT法检测细胞生长抑制率,流式细胞术检测细胞凋亡率,Hoechst33258荧光染色法观察细胞凋亡形态学变化,采用Caspase-3试剂盒和Westernblot蛋白电泳测定Caspase-3活性。结果:雷帕霉素可显著抑制BEL-7402细胞的生长,诱导细胞发生凋亡,具有量-效与时-效关系;雷帕霉素作用BEL-7402细胞48h后,Hoechst33258荧光染色可见核浓缩及核碎裂等典型的细胞凋亡特征;凋亡过程中Caspase-3酶原蛋白的激活,出现20ku亚单位,Caspase-3特异性抑制剂z-DEVD-FMK能阻断凋亡的发生。结论:雷帕霉素能抑制BEL-7402细胞的生长,诱导细胞凋亡发生,Caspase-3酶的激活在雷帕霉素诱导细胞凋亡机制中起到重要作用。     

雷帕霉素诱导人肝癌细胞BEL-7402凋亡中Bcl-2作用研究

目的探讨雷帕霉素(rapamycin,RAPA)体外对肝癌BEL-7402细胞生长抑制及诱导细胞凋亡中的作用及Bcl-2变化在凋亡机制中的意义。方法以5、10、20、30、40和50nmol/L不同浓度的RAPA作用于体外培养的BEL-7402细胞,MTT法检测细胞生长抑制率,应用流式细胞仪检测细胞凋亡,Hoechst33258荧光染色法观察细胞凋亡时的形态学变化,Westernblot观察Bcl-2、Bcl-xl和bax等凋亡相关表达变化。结果RAPA可显著抑制BEL-7402细胞的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系。RAPA作用肝癌细胞BEL-740248h后,在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征,凋亡过程中伴有抗凋亡蛋白Bcl-2、Bcl-xl表达的降低和促凋亡蛋白bax上调。结论RAPA可能通过诱导抗凋亡蛋白Bcl-2、Bcl-xl表达的降低和促凋亡蛋白bax表达上调而诱导凋亡发生,抑制BEL-7402细胞的生长。     

抗高迁移率族蛋白1中和抗体对肝癌细胞BEL-7402/ADM药物敏感性的影响

目的:探讨抗高迁移率族蛋白1（high mobility group box-1,HMGB1）中和抗体对肝癌细胞药物敏感性的影响及可能机制。方法:采用阿霉素（adriamycin,ADM）小剂量持续诱导法建立肝癌细胞耐药细胞株BEL-7402/ADM。实验分为BEL-7402/ADM组、ADM组及ADM+抗HMGB1中和抗体组,MTT法检测细胞对ADM的敏感性及抗HMGB1中和抗体对细胞的低毒性,Annexin V-FITC流式细胞法检测细胞凋亡情况,酶标法检测Caspase-9、Caspase-3活性,Western-blot方法检测核因子-κB（nuclear factor-κB,NF-κB）亚单位p-p65、Bcl-2蛋白的表达。结果:ADM对亲本株的半数抑制浓度(IC50)为0.23 μg/mL,对肝癌细胞耐药株BEL-7402/ADM的IC50上升至4.07 μg/mL,耐药指数为17.7。联合抗HMGB1中和抗体能明显提升ADM对BEL-7402/ADM细胞增殖抑制率,IC50下降至0.91 μg/mL;加用逆转浓度（19 ng/mL）的抗HMGB1中和抗体组的BEL-7402/ADM细胞凋亡率较单用ADM组的凋亡率明显上升（P＜0.01）。与BEL-7402/ADM组相比,ADM组细胞的Caspase-9、Caspase-3活性有一定程度上升（P＜0.01）,联合抗HMGB1中和抗体作用后,Caspase-9、Caspase-3活性增强则更加明显（P＜0.01）。BEL-7402/ADM组及ADM组间p-p65、Bcl-2表达无显著差异（P＞0.05）,加用抗HMGB1中和抗体处理后,细胞p-p65、Bcl-2表达明显下降（P＜0.01)。结论:抗HMGB1中和抗体对BEL-7402/ADM细胞耐药性有逆转作用,其机制可能与下调NF-κB介导的Bcl-2表达,促进肝癌细胞凋亡有关。     

布洛芬抑制人肝癌细胞BEL-7402生长及机制的初步研究

背景与目的：近来发现,临床上常用的非甾体类抗炎药(non-steroidal anti-inflammatory drugs,NSAIDs)可以降低多种癌症的发病率。布洛芬(ibuprofen)作为一种常用的NSAIDs,对肝癌是否具有抑制作用,国内外鲜有报道。本研究初步探讨布洛芬抑制肝癌细胞BEL-7402的作用,并研究其相关机制。方法：肝癌细胞BEL-7402分为对照组和不同浓度布洛芬处理组,药物处理0、24、48和72 h后用MTT法检测各组细胞增殖抑制率;流式细胞术检测各组细胞的周期分布;细胞分析仪检测各组细胞活力及细胞凋亡;蛋白[质]印迹(Western blot)检测各组细胞增殖性核抗原(PCNA)、细胞周期蛋白(Cyclin D1)、B细胞淋巴瘤/白血病-2 (Bcl-2)和环氧合酶-2(COX-2)蛋白的表达;ELISA法检测细胞培养上清液中前列腺素E₂(PGE₂)蛋白表达水平。结果：布洛芬组中BEL-7402细胞增殖受到抑制,且抑制作用呈时间和剂量依赖性(P<0.05)。2.0 mmol/L布洛芬组48 h细胞活力明显低于对照组［(47.87±5.23)% vs (88.93±5.49)%］,G₀/G₁期细胞比例明显高于对照组［(80.04±3.61)%vs (62.36±8.33)%］,细胞早期凋亡率明显高于对照组［(36.65±10.07)% vs (9.81±6.80)%］,差异有统计学意义(P<0.05)。布洛芬作用于细胞48 h后,PCNA、Cyclin D1、Bcl-2以及COX-2蛋白表达与对照组相比显著减少(P<0.05);细胞培养上清液中PGE₂蛋白表达量与对照组［(23.98±4.89) ng/L vs (68.70±9.43) ng/L］相比,显著降低(P<0.01)。结论：布洛芬能够抑制肝癌细胞BEL-7402增殖与活力,阻滞细胞周期,促进细胞凋亡,其作用机制可能与抑制COX-2及PGE₂表达有关。     

Waltonitone induces human hepatocellular carcinoma cells apoptosis in vitro and in vivo

Waltonitone, a new ursane-type pentacyclic triterpene isolated from Gentian waltonii Burkill significantly inhibited human hepatocellular carcinoma BEL-7402 cells growth. Apoptosis induced by waltonitone was characterized by AO/EB staining and flow cytometric analysis. Apoptosis microarray assay results showed BCL-2 family genes might especially play an important role in waltonitone-induced apoptosis. RT-PCR and Western blotting analysis showed that waltonitone could induce tumor cell apoptosis via both death receptor and mitochondria pathways. Meanwhile, the inhibitory effect of waltonitone was examined in vivo using BEL-7402 tumor cells xenografted into athymic mice model. In summary, these studies demonstrated that waltonitone might inhibit hepatocellular carcinoma cells growth and induce apoptosis in vitro and in vivo.     

免疫杀手细胞的体外扩增方法及其对肝癌细胞BEL-7402的杀伤作用

目的:探讨免疫杀手细胞(immune-killer cells,IKCs)的体外扩增方法及其对人肝癌细胞BEL-7402的杀伤作用。方法:改良自然杀伤细胞培养扩增法扩增IKCs细胞,流式细胞仪分析IKCs表型,台盼蓝染色计数法观察细胞增殖状况,AlamarBlue法观察IKCs细胞对BEL-7402细胞的杀伤作用,用BALB/c裸鼠模型检测IKCs细胞对肝癌动物模型的治疗作用。结果:IKCs细胞培养14 d的细胞总数扩增300倍以上,主要由CD3~+CD56~+、CD3~-CD56~+和CD8~+细胞亚群组成,CD3~+CD56~+和CD3~-CD56~+比例达85%以上,CD8~+比例达到77%以上。IKCs细胞对肝癌BEL-7402细胞的杀伤实验显示,当效靶比为25:1时和100:1时,其杀伤活性分别为66.5%和97.8%(P<0.01)。裸鼠肝瘤模型治疗实验显示,IKCs细胞对早期和晚期人肝癌BEL-7402细胞裸鼠模型均有显著的治疗作用,与对照组比较,其抑瘤率达69%以上(P<0.01)。结论:本实验建立的肿瘤免疫杀手细胞IKCs的培养扩增方法是可行的、有效的,有望成为一种新的肿瘤免疫细胞治疗方法。     

miRNA-195 sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w

The role of microRNA-195 in developing acquired drug resistance in hepatocellular carcinoma cells was investigated. Expression pro?ling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miRNA-195 in BEL-7402/5-FU cells. Overexpression of miRNA-195 sensitized BEL-7402/5-FU cells to anticancer drugs. Consistent with these findings, miR-195 antisense oligonucleotide induced drug resistance in BEL-7402/5-FU cells. Also, the basal levels of the anti-apoptotic protein Bcl-w were high in BEL-7402/5-FU cells and miR-195 overexpression repressed Bcl-w protein level and inhibited the luciferase activity of a Bcl-w 3' untranslated region-based reporter construct in both BEL-7402/5-FU and BEL-7402 cells. These results indicate that miR-195 could improve the drug sensitivity at least in part by targeting Bcl-w to increase cell apoptosis in hepatocellular carcinoma cells.     

复方苦参注射液对SGC-7901，HepG2和BEL-7402肿瘤细胞作用的实验研究

 目的 通过观察复方苦参注射液（岩舒注射液）对SGC-7901，HepG2和BEL-7402肿瘤细胞的作用，探讨其在恶性肿瘤治疗中的作用机制。方法 本实验选用SGC-7901、HepG2和BEL-7402细胞，采用MTT法检测岩舒注射液对于各肿瘤细胞的体外杀伤率，流式细胞仪检测岩舒注射液作用后的肿瘤细胞的细胞周期、凋亡率及bcl-2、CD44V6和nm23基因表达的变化。结果 岩舒注射液浓度达到150 μl／ml时HepG2，SGC-7901和BEL-7402细胞存活率最低。岩舒注射液终浓度100 μl／ml作用后的HepG2细胞G0／G1期、G2／M期明显增高，S期的细胞数量明显减少，凋亡率（2.8 %）明显高于对照组（0.2 %）；SGC7901细胞G2／M期细胞明显增多，S期细胞明显减少，凋亡率（4.6 %）明显高于对照组（0.8 %）；100 μl／ml岩舒注射液浓度作用后的nm23为97.85 %，明显高于对照组（41.01 %），CD44V6为10.29 %明显低于对照组（12.26 %）。结论 岩舒注射液对于SGC-7901，HepG2和BEL-7402细胞具有明显的体外杀伤作用，并影响SGC-7901、HepG2的细胞周期，促进其凋亡；同时能够明显的抑制BEL7402细胞体内CD44V6的表达，促进抑转移因子nm23的表达，具有明显的抑制肿瘤转移的作用。     

RNA干扰抑制高迁移率族蛋白-1表达对人视网膜母细胞瘤细胞增殖和凋亡的影响

目的:研究siRNA抑制高迁移率族蛋白-1(high mobility group protein box-1, HMGB1)表达对人视网膜母细胞瘤细胞增殖和凋亡的影响。方法:RT-PCR和Western blot检测HMGB1在人视网膜母细胞瘤中的表达。体外化学合成靶向HMGB1 siRNA,RT-PCR和Western blot检测其抑制效率。MTT法检测HMGB1沉默后Y79细胞的增殖情况。Caspase-3活性检测试剂盒检测HMGB1沉默后Y79细胞的凋亡情况。采用流式细胞仪来分析细胞周期的分布以及细胞凋亡率的变化。结果:HMGB1 mRNA和蛋白在人视网膜母细胞瘤细胞中高表达,siRNA抑制HMGB1表达后,Y79细胞增殖抑制,促进凋亡。结论:抑制HMGB1的表达可以降低人视网膜母细胞瘤细胞的增殖,并促进其凋亡,为人视网膜母细胞瘤的生物学治疗提供新的思路。     

Characterization and Resistance Mechanisms of A 5-fluorouracilresistant Hepatocellular Carcinoma Cell Line

Purpose: The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especiallyintrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC.In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU)-resistant human HCC cell line. Methods: BEL-7402/5-FU cells were established through continuous culturingparental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivityto cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates werecalculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessedunder optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity weremeasured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FUcells were detected by immunocytochemistry. Results: Compared to its parental cells, BEL-7402/5-FU cells hada prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drugefflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructuresoccurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitantdecrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increaseddrug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase(TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated byE-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Conclusion:Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanismof BEL-7402/5-FU.