大黄素在体外诱导人肝癌细胞HepG2发生凋亡的初步研究

背景与目的:大黄素(3-甲基-1,6,8-三羟蒽醌)是大黄等多种中药的有效成分之一。研究表明大黄素对于乳腺癌细胞、肺癌细胞的生长具有抑制作用,但目前大黄素抗肿瘤的作用机理尚不明确。本研究拟讨论大黄素在体外对肝癌细胞HepG2的作用及其机理。方法:应用MTT、软琼脂克隆形成、DNAladder凝胶电泳及流式细胞术等方法,研究中药单体大黄素对肝癌细胞HepG2生长增殖的影响以及作用机理。结果:大黄素在较低浓度可抑制肿瘤细胞的生长,MTT实验测得的半数抑制浓度(IC50)为(36±2.6)μg/ml。在软琼脂实验中,大黄素可以浓度依赖的方式抑制细胞克隆的形成。经DNAladder及流式细胞仪检测发现,大黄素能诱导HepG2细胞凋亡,与阴性对照相比,随着药物浓度从10μg/ml增加到20μg/ml,AnnexinV染色细胞显著增多,由27.3%增至59.6%;当药物浓度增至40μg/ml时,培养液中几乎无活细胞,由碘化丙啶和AnnexinV双重标记的凋亡后期以继发性坏死细胞为主。结论:中药单体大黄素在体外能抑制肝癌细胞HepG2的生长增殖,并能诱导该细胞凋亡。     

MiR-122促进原发性肝细胞癌Huh-7细胞凋亡

目的 探讨miR-122对原发性肝细胞癌(hepatocellular carcinoma,HCC)细胞性状的影响。方法 HepG2细胞、Huh-7细胞分别转染miR-122、anti-miR-122,转染Mock组作为阴性对照,分别于转染后24 h、48 h用流式细胞法、TUNEL法检测细胞凋亡及细胞周期。结果 转染后24 h、48 h,转染miR-122、Mock的HepG2细胞两组间细胞凋亡率均无统计学意义(P＞0.05),而在转染后48 h,与Mock组Huh-7细胞相比,转染anti-miR-122的Huh-7细胞凋亡率明显降低(P＜0.05);转染后24 h、48 h,转染miR-122、Mock的HepG₂细胞,各细胞周期组份均无统计学意义(P＞0.05),但于转染后48 h,转染anti-miR-122的Huh-7细胞可见G₀-G₁期细胞明显多于Mock组,S期、G₂-M期细胞则显著少于Mock组(P＜0.05)。结论 miR-122表达水平的下降能够抑制某些HCC细胞如Huh7细胞凋亡,miR-122可能作为一种内源性凋亡调控因子,在肝脏组织中发挥抗肿瘤作用。     

乳腺癌不同肺转移潜能细胞株表达差异蛋白及其临床意义

背景与目的：使用蛋白质组技术，我们鉴定出不同肺转移潜能MDA—MB-435乳腺癌细胞之间11个表达存在差异的蛋白，本研究试图探讨其中3个蛋白，dB—crystatlin（CRAB）、热休克蛋白60（HSP60）和peroxiredoxin 6（PDX6），与乳腺癌转移的关系。方法：使用实时定量RT—PCR的方法验证3个蛋白在不同肺转移潜能细胞株之问mRNA水平的差异。进一步使用免疫组化的方法检测2003年1月～2004年1月，32例乳腺癌标本中3个蛋白的阳性率。结果：肺高转移潜能细胞株CRAB，HSP60和PDX6的mRNA水平分别为其亲代细胞的0．24倍，3．15倍和2．43倍。CRAB在淋巴结阴性乳腺癌患者和淋巴结阳性乳腺癌患者中的阳性率分别为52．9％和33．3％；HSP60在2组患者中的阳性率分别为35．3％和73．3％；PDX6在2组患者中的阳性率分别为29．4％和60．0％。结论：HSP60水平的升高可能与乳腺癌侵袭能力增加相关，具体机制尚待进一步研究。     

瑞戈非尼联合哌立福辛诱导人肝癌HepG2细胞凋亡的分子机制研究

目的 探讨瑞戈非尼联合哌立福辛(AKT抑制剂)对人肝癌HepG2细胞凋亡的分子机制。 方法 不同浓度瑞戈非尼(1、5、10、15、20、25 μmol/L)处理肝癌细胞后,通过CCK-8检测其细胞存活率;再设置不同浓度瑞戈非尼(0、5、10、20 μmol/L)处理肝癌细胞后,流式细胞仪检测细胞凋亡;用免疫印迹法(Western blot)检测Bax、Bcl-2、p-AKT、AKT、p21、p27、Cleaved-caspase-8、Cleaved-caspase-9的表达情况;设置哌立福辛组(10 μmol/L)、瑞戈非尼组(20 μmol/L)及双药联合组(哌立福辛10 μmol/L联合瑞戈非尼20 μmol/L),检测细胞凋亡及相关蛋白表达变化。 结果 从5 μmol/L浓度起,瑞戈非尼可剂量依赖性的抑制肝癌细胞增殖(P＜0.05);Bcl-2及p-AKT表达下调,Bax、p21、p27、Cleaved-caspase-8、Cleaved-caspase-9表达上调(P＜0.05);瑞戈非尼和哌立福辛双药联合应用后,细胞凋亡率、Bax表达量明显增加(P＜0.05),Bcl-2、p-AKT表达量下降程度均较单独应用哌立福辛及瑞戈非尼明显(P＜0.05)。 结论 瑞戈非尼通过增加p21、p27、Bax、caspase-8、caspase-9的表达,减少Bcl-2、p-AKT的表达,来诱导肝癌细胞凋亡,并且瑞戈非尼与哌立福辛双药联合应用后,明显增加肝癌细胞凋亡率,为药物治疗肝癌提供新的理论基础和思路。     

芬太尼对人肝癌细胞bel-7404生长与凋亡的影响

目的探讨芬太尼对人肝癌细胞bel-7404生长及凋亡的影响。方法体外培养人肝癌bel-7404细胞,分F1、F2、F13、F4,4个实验组和1个对照组,实验组分别在RPMI-1640培养液中加入5ng／ml（F1）、50ng／ml（F2）、500ng／ml（F3）、5000ng／ml（F4）芬太尼,对照组不加芬太尼,所有样本孵育24h后,用倒置显微镜观察细胞形态学改变,应用MTT法和克隆形成实验检测细胞的增殖活性,应用流式细胞仪检测细胞凋亡率与细胞周期。结果各实验组人肝癌bel-7404细胞MTT和克隆形成率明显低于对照组（P〈0．01）,细胞凋亡百分比显著高于对照组（P〈0．05）。芬太尼浓度≥50ng／ml时,随着浓度的增加,凋亡率逐渐增高,细胞周期中G0／G1期比例逐渐增加,S期细胞比例逐渐减少,与对照组比较差异具有统计学意义（P〈0．05）。结论芬太尼可剂量依赖性抑制人肝癌细胞bel-7404的生长,干扰肝癌细胞增殖周期,诱导肝癌细胞凋亡。     

Acquisition of anoikis resistance reveals a synoikis-like survival style in BEL7402 hepatoma cells

Resistance to anoikis is a hallmark of human malignancies. Our results showed that hepatoma cells resisted anoikis by non-proliferation, non-apoptosis and cell cycle arrest which were termed synoikis-like. These synoikis-like cells are more resistant to extracellular stimuli and could spontaneously attach and proliferate again under suitable conditions, which indicate a reversible property of these cells. Microarray expression profile reveals the change of molecules involved in the synoikis-like hepatoma cells and our data indicated that ANGPTL4 contributed to anoikis resistance of hepatoma cells. These results demonstrated that hepatoma cells might resist anoikis through a synoikis-like survival style, which may facilitate tumor metastasis.     

紫杉醇诱导MCF-7乳腺癌细胞凋亡的蛋白质组研究

目的 研究紫杉醇诱导MCF-7乳腺癌细胞凋亡的分子机制。方法 以100nmol／L紫杉醇作用MCF-7细胞24h后提取细胞总蛋白,利用蛋白质组技术,对紫杉醇诱导的MCF-7乳腺癌细胞凋亡前后的细胞蛋白进行二维电泳图谱分析,通过质谱鉴定差异蛋白质。结果 通过对凋亡前后的蛋白质组二维电泳图谱分析,找到了17个差异较大的蛋白质,其中6个蛋白质通过质谱得到了鉴定,它们是烯醇化酶、细胞核氯离子通道蛋白、角蛋白8、核糖体蛋白S12、galectin-1和Hint。结论 通过蛋白质组技术,发现了在紫杉醇诱导MCF-7乳腺癌细胞凋亡前后发生变化的6种蛋白质;这些差异蛋白质在紫杉醇诱导细胞凋亡中发挥一定的作用。     

结直肠癌化疗敏感性相关蛋白的蛋白质组学初步研究

目的:本研究应用蛋白质组学技术建立化疗敏感性不同的结直肠癌组织总蛋白双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)图谱,并鉴定部分差异表达蛋白,以发现与结直肠癌化疗敏感性有关的蛋白.方法:收集临床诊断为晚期结直肠癌的病例,肠镜活检获取新鲜结直肠癌标本后液氮保存备用,根据肿瘤药物敏感实验分为化疗高敏感组和化疗低敏感组.提取组织总蛋白,采用双向凝胶电泳技术得到各组凝胶图谱;采用PD-quest 7.3软件进行图像的合成、对比和差异分析,识别化疗高敏感组和化疗低敏感组之间差异表达的蛋白斑点;选取差异蛋白质点,胶内酶解后行肽指纹图分析及网上数据库检索,鉴定差异蛋白质;应用Western印迹法检测部分差异蛋白的表达情况.结果: 建立了化疗高敏感组和化疗低敏感组的双向凝胶电泳图谱,多数蛋白质点集中于pH 4～8、相对分子质量为(20～100)×10~3.高敏感组和低敏感组的电泳图谱中平均蛋白质点数分别为(842±23)个和(793±19)个,2组平均匹配率为90.7%,2组间差异表达蛋白质点数为(79.00±13.56)个;选择30个差异蛋白质点进行质谱分析,经数据库查询鉴定出9个差异表达蛋白.结论:在化疗敏感性不同的结直肠癌中存在蛋白质表达的差异,这些差异表达蛋白可能与化疗敏感性有关,并可能用于化疗敏感性的预测.     

Butein induces G2/M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation

We investigated the molecular effects of 3,4,2′,4′-tetrahydroxychalcone (butein) treatment in two human hepatoma cancer cell lines–HepG2 and Hep3B. Butein treatment inhibited cancer cell growth by inducing G₂/M phase arrest and apoptosis. Butein-induced G₂/M phase arrest was associated with increased ATM, Chk1, and Chk2 phosphorylations and reduced cdc25C levels. Additionally, butein treatment enhanced inactivated phospho-Cdc2 levels, reduced Cdc2 kinase activity, and generated reactive oxygen species (ROS) that was accompanied by JNK activation. The extent of butein-induced G₂/M phase arrest significantly decreased following pretreatment with N-acetyl-l-cysteine or glutathione and following JNK phosphorylation reduction by SP600125. Both N-acetyl-l-cysteine and glutathione also decreased butein-mediated apoptosis. Taken together, these results imply a critical role of ROS and JNK in the anticancer effects of butein.     

Cytoplasmic alpha-fetoprotein functions as a co-repressor in RA-RAR signaling to promote the growth of human hepatoma Bel 7402 cells

The role of AFP in the retinoic acid-RAR signaling pathway was investigated in human hepatoma Bel 7402 cells. The results showed that AFP and RAR-β were co-localized and interacted in cytoplasm. AFP may inhibit translocation of RAR-β into the nucleus via competitive binding to RAR-β with ATRA, which was reversed by AFP-siRNA transfection. Our data suggest that the ATRA resistance of Bel 7402 cells is at least in part attributable to their high level of cytoplasmic AFP. Thus, by counteracting the effect of AFP, it may be possible to increase the sensitivity of tumor cells to ATRA.     

Icariin,a natural flavonol glycoside,induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway

In this study, the anticancer effect of icariin, a natural flavonol glycoside, against human hepatoma SMMC-7721 cells and the underlying mechanisms were investigated. Icariin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, icariin induced a sustained activation of the phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 and ERK1/2, and SP600125 (an inhibitor of JNK) almost reversed icariin-induced apoptosis in SMMC-7721 cells. In addition, icariin provoked the generation of reactive oxygen species (ROS) in SMMC-7721 cells, while the antioxidant N-acetyl cysteine almost completely blocked icariin-induced JNK activation and apoptosis. Taken together, these findings suggest that icariin induces apoptosis through a ROS/JNK-dependent mitochondrial pathway.     

二甲双胍体外诱导人肝癌Huh-7细胞凋亡及其作用机制

目的 探讨二甲双胍对人肝癌Huh-7细胞增殖、凋亡的影响及其可能的作用机制.方法 二甲双胍干预Huh-7细胞后,四甲基偶氮唑蓝(MTT)法检测其对细胞活力及生长抑制的影响;Western blot检测凋亡相关蛋白及CD133蛋白的变化;流式细胞术检测细胞凋亡及肝癌干细胞相关标志物CD133的表达;无血清悬浮培养观察二甲双胍对肿瘤干细胞球形成的影响;逆转录聚合酶链反应(RT-PCR)检测其对肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达的影响.以加入与实验组相同的培养液但未给予药物处理的组为对照组.结果 MTT检测结果显示,与对照组比较,随着二甲双胍浓度的增高,肝癌Huh-7细胞的增殖率下降,差异均有统计学意义(均P＜0.05).10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的早期凋亡率为(22.29±0.80)％,对照组的早期凋亡率为(6.70±0.50)％,差异有统计学意义(P ＜0.05);10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的晚期凋亡率为(13.87±1.20)％,对照组的晚期凋亡率为(1.10±0.02)％,差异有统计学意义(P＜0.05).25 mmol/L二甲双胍干预48 h后细胞的早期凋亡率为(15.28±2.10)％,晚期凋亡率为(25.89±2.30)％,均高于对照组(均P＜0.05).Western blot检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h后,p-Akt、Bcl-2/Bax比值表达下调,PTEN表达上调.随着二甲双胍浓度的增加,CD133蛋白的表达下调.流式细胞仪检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h前后,Huh-7细胞中CD133+细胞的表达率分别为(40.7±2.1)％和(14.6±1.85)％ (P ＜0.05).在低黏附培养皿、无血清培养液的生长环境中,对照组细胞球体积较大,中心厚重,周围清亮,折光度强;10mmol/L二甲双胍组细胞球体积较小,球体折光度较弱,细胞球数量少于对照组.RT-PCR检测结果显示,肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达水平均下调.结论 二甲双胍能显著抑制人肝癌Huh-7细胞增殖,促进凋亡.其机制可能与抑制肝癌PI3 K/Akt信号通路和CD133+肿瘤干细胞的表达有关.     

Proteomic analysis of liver cancer cells treated with suberonylanilide hydroxamic acid

Purpose Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumor activity in a variety of tumor cells. To evaluate if SAHA has an activity against liver cancer, and with an aim to identify the altered cellular factors upon SAHA treatment, human HepG2 cancer cell line was used as a model, and proteomic approach was utilized to elucidate the molecular mechanisms underlying SAHA’s antitumor activity. Methods Cell growth inhibition was measured by MTT method, and apoptosis was detected by means of flow cytometry analysis and TUNEL assay. Protein expression profiles were analyzed by 2-DE coupled with MALDI-Q-TOF MS/MS analysis. Results A total of 55 differentially expressed proteins were visualized by 2-DE and Coomassie Brilliant Blue (CBB) staining. Of these, 34 proteins were identified via MS/MS analysis. Among the identified proteins, six proteins also displayed significant expression changes at earlier time points upon SAHA treatment, and such alterations were further confirmed by semi-quantitative RT-PCR. Together, at both the mRNA and protein levels, SAHA suppressed the expression of reticulocalbin 1 precursor (RCN1), annexin A3 (ANXA3) and heat shock 27 kDa protein 1 (HSP27), while increasing the expression of aldose reductase (AR), triosephosphate isomerase 1 (TPI) and manganese superoxide dismutase (SOD2). Conclusion SAHA remarkably inhibited proliferation of HepG2 cancer cells, and induced apoptosis in vitro. Using proteomics approaches, a variety of differentially expressed proteins were identified in HepG2 cancer cells before and after treatment with SAHA. This study will enable a better understanding of the molecular mechanisms underlying SAHA-mediated antitumor effects at the protein level.     

Synthetic chenodeoxycholic acid derivative, HS-1200, induces apoptosis of human hepatoma cells via a mitochondrial pathway

We investigated whether HS-1200 has anti-proliferation effects on human hepatoma cells in vitro. Here, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) were observed after treatment of HS-1200, indicating the occurrence of apoptotic cell death, which was associated with up-regulation of Bax, cleaved-caspase-3 and cleaved-caspase-9. Inhibition of caspase-9 rescued HS-1200-induced apoptosis. Furthermore, cells treated with HS-1200 showed a reduction in mitochondrial membrane potential (Δψ_m) and caused cytochrome c release into the cytosol. The results indicated that synthetic chenodeoxycholic acid HS-1200 could induce cell apoptosis in BEL7402 human hepatoma cell line, via a Bax/cytochrome c/caspase-9 independent pathway. This study suggested that HS-1200 is potentially useful as an apoptosis inducer for the treatment of hepatocellular carcinoma.     

Vitamin K2 suppresses malignancy of HuH7 hepatoma cells via inhibition of connexin 43

The anti-cancer potential of vitamin K(2) (VK(2)) in hepatoma has gained considerable attention but the underlying mechanisms are unclear. Treatment of HuH7 hepatoma cells with VK(2) produced a normal liver phenotype. Following treatment of cells with VK(2), there was an increase in gap junctional intercellular communication activity, accompanied by up-regulation of connexin 32 (Cx32), dominantly expressed in normal hepatocyte. In contrast, Cx43 expression was inhibited. Moreover, the effect of VK(2) on Cx32 was abolished by over-expression of Cx43. Taken together, we propose that the anti-tumor effect of VK(2) is at least partly due to a decrease in Cx43 promoter activity.     

Curcumin induces apoptosis involving bax/bcl-2 in human hepatoma SMMC-7721 cells

Curcumin is a major active component of Curcuma aromatica salisb, which has been shown to inhibit proliferation of a wide variety of tumor cells. In this study, the molecular mechanisms of curcumin inducing apoptosis in human hepatoma SMMC-7721 cells were examined. We find that curcumin inhibits the growth of SMMC-7721 cells significantly in a concentration-depenent manner, with typical apoptotic morphological changes of cellular nuclei. Annexin-V/PI double staining detected by flow cytometry and expression of the relative apoptotic proteins (Bax, Bcl-2 and caspase-3) revealed a strong apoptosis-inducing competent of curcumin in SMMC-7721 cells. Curcumin increased the expression of bax protein while decreasing that of bc1-2 protein significantly. The results suggest that curcumin induction of apoptosis involves modulation of bax/bcl-2 in SMMC-7721 cells and provide a molecular basis for the development of naturally compounds as novel anticancer agents for human hepatomas.     

番茄红素对人肝癌细胞增殖和凋亡的影响及其机制

目的 研究番茄红素对人肝癌HepG2细胞增殖和凋亡的影响及其机制.方法 取对数生长期人肝癌HepG2细胞,分别给予不同浓度番茄红素(5、10、20 μg/ml)和顺铂(40 μg/ml)进行干预,48 h后通过四甲基偶氮唑蓝(MTT)比色法测定细胞增殖抑制率,通过流式细胞术检测细胞周期和细胞凋亡状况,采用Western blotting技术检测Bax、Bcl-2、激活型Caspase-3蛋白表达.结果 干预48 h后,空白对照组人肝癌HepG2细胞增殖抑制率为0,番茄红素(5、10、20 μg/ml)组和顺铂组分别为(21.3±4.2)％、(40.5±7.6)％、(63.8±9.1)％和(37.8±5.9)％,差异有统计学意义(F=37.905,P=0.000);它们分别与空白对照组比较,差异均具有统计学意义(t=208.124,P=0.000;t=394.637,P=0.000;t=628.592,P=0.000;t =257.168,P=0.000).番茄红素(10、20μg/ml)组细胞周期G0-G1期比例分别为(54.0±2.9)％、(67.3±3.6)％,与空白对照组(37.9±1.5)％比较,差异有统计学意义(t =4.508,P=0.024;t=10.673,P=0.006);番茄红素(10、20 μg/ml)组G2-M期比例分别为(8.5±0.6)％、(4.7±0.5)％,与空白对照组比较(18.4±0.8)％,差异有统计学意义(t=9.975,P=0.008;t=13.864,P=0.003).番茄红素组(5、10、20 μg/ml)和顺铂组细胞凋亡指数分别为(19.5±4.8)％、(43.0±9.2)％、(67.6±10.1)％和(36.9±6.8)％,与空白对照组[(3.6±1.7)％]比较均显著升高,差异有统计学意义(t=18.617,P=0.001;t=34.295,P=0.000;t=51.437,P=0.000;t =29.804,P=0.000).番茄红素(10、20 μg/ml)组和顺铂组Bel-2、Bax表达及Bax/Bcl-2比值分别为0.42±0.09、0.43 ±0.14、1.02±0.39,0.27±0.08、0.76 ±0.19、2.81 ±0.85和0.34 ±0.11、0.31 ±0.09、0.91 ±0.40,与空白对照组(0.59 ±0.17、0.18 ±0.06、0.31 ±0.12)比较,Bcl-2表达显著下调,差异具有统计学意义(t=4.327,P=0.023;t=11.064,P=0.006;t =5.182,P=0.018),Bax表达显著上调,差异具有统计学意义(t=9.837,P =0.008;t=17.349,P=0.001;t=10.165,P=0.007),Bax/Bcl-2比值显著升高,差异均具有统计学意义(t=11.521,P=0.006;t=18.194,P=0.001;t=9.537,P=0.008).番茄红素(5、10、20 μg/ml)组和顺铂组激活型Caspase-3蛋白表达分别为0.25 ±0.07、0.34 ±0.11、0.46 ±0.18和0.17 ±0.05,与空白对照组(0.08±0.03)比较,差异具有统计学意义(t=8.307,P=0.009;t=13.067,P=0.006;t=16.218,P=0.003;t=5.202,P=0.019).结论 番茄红素能够通过抑制细胞增殖并促进其凋亡而对人肝癌HepG2细胞生长起到一定的抑制作用,其机制可能与番茄红素能够影响细胞周期进程并调节凋亡相关基因蛋白表达有关.