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.     

As2O3联合L-OHP对HepG2肝癌细胞株的体外抑制作用

目的探讨As2O3联合L-OHP对HepG2肝癌细胞株的体外抑制作用。方法采用MTT法（四唑盐比色法）动态观察As2O3联合L-OHP对HepG2肝癌细胞的生长抑制作用,并采用两药相互作用系数（CDI）来评价两药相互作用的性质;应用流式细胞术（FCM）检测凋亡率和细胞周期分布。结果 As2O3与L-OHP联合应用,对HepG2肝癌细胞株的生长抑制、诱导凋亡作用均较相应的单药增强。流式细胞直方图上可见明显的＂凋亡峰＂,且As2O3使肝癌细胞周期阻滞于G2/M期,L-OHP使细胞周期阻滞于S期和G2/M期,两药合用使细胞周期阻滞于S期和G2/M期。结论中低浓度As2O3与L-OHP联合作用具有显著的协同效益,其主要机制可能是As2O3联合L-OHP诱导肝癌细胞凋亡作用及增强细胞周期阻滞作用。     

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。     

转录共激活子TAZ对肝癌细胞株MHCC97H增殖能力的影响

目的 观察转录共激活子TAZ对肝癌细胞株MHCC97H的增殖能力的影响,以期对阐明原发性肝细胞癌(HCC)的发生发展机制提供1个新的研究思路.方法 应用细胞转染、RNA干扰(RNAi)以及过表达技术,在肝癌细胞株MHCC97H中干预TAZ的表达.在转染后提取细胞RNA和总蛋白,应用qRT-PCR和Western blot等方法检测TAZ、Nek7的表达.选用细胞计数kit-8(CCK-8)法检测细胞活性和增殖能力.结果 与NC组相比,转染了TAZ特异性靶向干扰片段之后,TAZ的mRNA与蛋白表达水平均出现下调,差异具有统计学意义(P＜0.0001);Nek7表达也出现下降,差异具有统计学意义(P＜0.0001);功能实验(CKK8)发现MHCC97H细胞活性和增殖能力也降低,差异具有统计学意义(P＜0.001).另一方面,过表达了TAZ之后,Nek7表达水平也相应上调了,差异具有统计学意义(P＜0.0001);MHCC97H细胞活性和增殖能力也提高,差异具有统计学意义(P＜0.001).结论 转录共激活子TAZ能通过调控Nek7的表达对肝癌细胞MHCC97H的增殖能力产生影响,该发现对于阐明TAZ在肝癌发生发展过程中的作用提供一定的实验基础.     

Chloroquine and Valproic Acid Combined Treatment in Vitro has Enhanced Cytotoxicity in an Osteosarcoma Cell Line

Choroquine (CQ) and valproic acid (VPA) have been extensively studied for biological effects. Here, wefocused on efficacy of combined CQ and VPA on osteosarcoma cell lines. Viability of osteosarcoma cell lines (U20Sand HOS) was analyzed by MTT assay. Apoptotic assays and colony formation assays were also applied. ROSgeneration and Western Blotting were performed to determine the mechanism of CQ and VPA combination inthe process of apoptosis. The viability of different osteosarcoma cell lines significantly decreased after CQ andVPA combination treatment compared with either drug used alone, and apoptosis was increased significantly.ROS generation was triggered leading to expression of apoptosis related genes being increased and of antiapoptoticrelated genes being decreased. From our data shown here, CQ and VPA combination treatment invitro enhanced cytotoxicy to osteosarcoma cells.     

康莱特抑制肝癌细胞HepG2 增殖的实验研究

目的：探讨康莱特注射液（KLT）抑制肝癌细胞HepG2增殖的作用机制。方法：以低、中、高剂量（分别为5μl／ml、10μl／ml、15μl／ml）的KLT处理肝癌细胞HepG2，用免疫组化法检测周期素D1（cyclin D1）、周期素E（cvclinE）蛋白在肝癌细胞HepG2中的表达，用MTT比色法观察KLT时肝癌细胞HepG2生长的抑制作用．用流式细胞仪检测细胞周期分布；另设盐水对照组。结果：1）KLT对肝癌细胞HepG2有明显的抑制效应，且具有时间、剂量依赖关系。2）低、中、高剂量组KLT使G1期细胞分别上升至66．5％、70．1％、75．8％，明显高于对照组（P〈0、05）：中、高剂量组KLT使S期细胞分别下降至13．2％、20．0％，明显低于对照组（P〈0．05）。3）低、中、高剂量组KLT使肝癌细胞cvclinD1蛋白水平分别减少26.3％、35．1％、45．6％，与对照组比较P均〈0．05；使cyclinE蛋白水平分别减少18．8％、27．1％、33.3％．与对照组比较P均〈0．05。结论：KLT对肝癌细胞HepG2有明显的抑制效应，其抑制效应具有时间、剂量依赖关系．其机理是通过抑制下游cyclinD1、cyclinE表达阻止细胞进入S期。     

ENO3在肝癌中的表达及其对奥沙利铂化疗敏感度的影响

目的 研究ENO3基因在肝癌中的表达及其是否可影响肝癌细胞对奥沙利铂的敏感度并探讨可能机制。方法 qRT-PCR、免疫组织化学法检测48例肝细胞肝癌组织和正常肝组织中ENO3的表达。构建ENO3过表达质粒,转染MHCC97H细胞和HepG2细胞;实验分为空载组（Vector组）、ENO3过表达组（ENO3组）、空载+OXA组（Vector+OXA组）和ENO3过表达+OXA组（ENO3+OXA组）。CCK-8法、克隆形成法检测细胞增殖能力,流式细胞术检测细胞凋亡率;Western blot法检测细胞中凋亡相关蛋白Bcl-2、Bax和Caspase-3的表达水平。结果 肝癌组织中ENO3的表达量明显低于正常肝组织。ENO3过表达组细胞ENO3的mRNA水平与蛋白水平较空载组均明显升高。过表达ENO3联合奥沙利铂后,与空载+OXA组相比,ENO3过表达+OXA组细胞活力降低、凋亡增加;Bcl-2蛋白表达减少,Bax、Caspase-3蛋白表达增加。结论 ENO3在肝癌组织中表达下降,过表达ENO3可通过促进凋亡增强肝癌细胞对奥沙利铂的敏感度。     

Comparison of Serum Survivin and Alpha Fetoprotein in Egyptian Patients with Hepatocellular Carcinoma Associated with Hepatitis C Viral Infection

Background: Survivin is specific antiapoptotic gene product expressed in a variety of human neoplasmswhose overexpression might assist in early diagnosis and as a prognostic marker. Objectives: The aim was to evaluate the plasma levels of survivin and alpha fetoprotein in patients with chronic hepatitis C viral infection (HCV) with and without hepatocellular carcinoma (HCC). Subjects: 70 subjects were divided into: a control group (Group I) ( 20 healthy volunteers ) and two patients groups: Group II, HCV group (20 patients); and Group III, HCC with HCV(30 patients ). Methods: Thorough physical examination, ultrasonography of the abdomen, laboratory investigations (liver profile, anti-HCV antibodies, hepatitis B surface antigen, Alpha fetoprotein (chemiluminometry) and Survivin (ELISA)) were performed. Results: There was a significant increase in survivin level in HCV patients (Group II) when compared to the control group (p=0.039), along with a significant increase in AFP in Groups II and III when compared to Group I (P<0.001 for both). AFP also distinguished between the two HCV groups. The best generated cut off value for AFP was 10.9 ng/ml and for survivin 13.7 pg/ml. Serum survivin diagnostic sensitivity was 53.3%, diagnostic specificity 62.5% and efficiency 58.6%, in contrast to 100%, 92.5% and 95.7%, respectively, for AFP. Conclusions: While survivin showed significant increase in the HCV group, its diagnostic performance was lower and it proved to be less reliable as a tumor marker for HCC than did AFP.     

Comparative Analysis of the Effects of 17-Beta Estradiol on Proliferation,and Apoptosis in Hepatocellular Carcinoma Hep G2 and LCL-PI 11 Cell Lines

Background: Phytoestrogens are a group of natural compounds with estrogen-like activity and similar structureto estradiol that structurally mimic the mammalian estrogen 17-β estradiol (E2). They have a biphasic effect and exertpleiotropic effects which induce or inhibit estrogen action by activation/inhibition of the estrogen receptors (ERs).These compounds can induce apoptosis at high concentrations. The previous finding indicated that E2 inhibited cellgrowth and induced apoptosis in hepatocellular carcinoma (HCC) PLC/PRF/5 cell line. The aim of the present studywas to investigate the apoptotic and proliferative effects of E2 on hepatocellular carcinoma HepG 2 and LCL-PI 11cells. Methods: The Hep G2 and LCL-PI 11 cells were cultured and treated with E2 for different time periods and thenMTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide] assay and flow cytometry assay weredone to determine cell viability and cell apoptosis respectively. Results: E2 had inhibitory and apoptotic effects on HepG2 cell line, whereas it indicated a biphasic effect on LCL-PI 11 cell line. The half-maximum inhibitory concentration(IC50) value was 3 μM. The inhibitory effect of E2 on Hep G2 cells was observed with all concentrations of E2 (P<0.087), whereas E2 showed a biphasic effect on LCL-PI 11. This compound induced significant apoptosis in Hep G2cell line at the all treatment times versus control groups, whereas, in the LCL-PI 11 cell, significant apoptotic cells wereobserved after 72 and 96h (P <0.001). Conclusion: E2 can inhibit cell growth and induce apoptosis in hepatocellularcarcinoma HepG 2 and LCL-PI 11 cell lines.     

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.     

siRNA干扰MTH1基因对肝癌HepG2细胞凋亡和迁移的影响

目的 应用siRNA干扰技术抑制人肝细胞癌系HepG2细胞MTH1基因表达,并研究MTH1基因干扰后对HepG2细胞凋亡和迁移能力的影响。方法 设计并化学合成3对特异性MTH1-siRNA,采用阳离子脂质体法瞬间转染肝癌HepG2细胞,离体培养肝癌HepG2细胞,并设正常对照组、阴性对照组及MTH1-siRNA1、MTH1-siRNA2、MTH1-siRNA3转染组。采用蛋白印迹（Western blot）法检测转染MTH1-siRNA后HepG2细胞MTH1蛋白水平表达的变化,并筛选干扰效果最佳序列。运用流式细胞技术和划痕实验检测转染siRNA-MTH1后HepG2细胞凋亡、体外迁移能力的变化。结果 MTH1-siRNA3转染组干扰效果与正常组和阴性对照组相比效果具有统计学意义（P=0.002, P=0.005）。MTH1-siRNA3转染组细胞凋亡率高于正常组及阴性对照组,差异有统计学意义（P=0.012, P=0.013）;MTH1-siRNA3转染组细胞的划痕24、36 h愈合率均低于正常组和阴性对照组,差异有统计学意义（P=0.001, P=0.000）。结论 MTH1在肝癌细胞的凋亡及迁移中起重要作用,干扰MTH1的表达能促进肝癌细胞凋亡并降低肝癌细胞的迁移能力。     

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.     

RASSF1A基因对肝癌细胞化疗药物敏感性的影响

目的探讨RASSF1A基因的表达对肝细胞肝癌化疗药物敏感性的影响。方法利用已构建成功的稳定表达野生型和突变型RASSF1A基因的肝癌细胞株QGY-7703,化疗药物Mitomycin、Adriamycin、Etoposide、5-Fluorouracilur、Cisplatin分别作用各细胞株后,比较生长抑制率、细胞周期及P53、P21、Bax、Caspase-3蛋白的表达水平。结果野生型RASSF1A的表达可提高Mitomycin诱导的肝癌细胞生长抑制率、凋亡发生率（P<0.05）和Caspase-3的活性,对P53、P21、Bax基因的蛋白表达水平没有影响。结论野生型RASSF1A基因可提高肝癌细胞对Mitomycin的敏感性。     

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。     

组蛋白去乙酰化酶2在宫颈鳞癌组织中的表达及临床意义

目的 探讨组蛋白去乙酰化酶2( HDAC2)在宫颈鳞癌组织中的表达及临床意义.方法 采用免疫组化和原位杂交法检测80例宫颈鳞癌组织及25例正常宫颈黏膜组织中HDAC2蛋白及mRNA的表达情况,并分析其表达与宫颈鳞癌临床病理学特征的关系.结果 HDAC2蛋白在宫颈鳞癌组织中的阳性表达率(65.0％)显著高于正常宫颈黏膜组...     

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。     

NS-398联合表阿霉素对肝癌HepG2细胞增殖及survivin表达的影响

[目的]探讨表阿霉素联合COX-2抑制剂NS-398对HepG2细胞增殖及survivin表达的影响.[方法]不同浓度表阿霉素单药或联合不同浓度NS-398分别作用于肝癌HepG2细胞株,MTT法测定表阿霉素联合NS-398对细胞生长的抑制率,RT-PCR和免疫荧光法分别检测HepG2细胞中survivin mRNA与survivin蛋白表达量的变化.[结果]单药表阿霉素对HepG2细胞有显著的抑制作用,且与NS-398联合应用后抑制作用更明显.正常无药对照组HepG2细胞中survivin蛋白和mRNA均呈强表达,表达水平分别为68.970±1.156和0.919±0.021;且在胞浆中survivin蛋白表达强于胞核.表阿霉素组HepG2细胞中survivin蛋白(47.630±0.863)和mRNA (0.749±0.014)水平均明显降低,联合组survivin蛋白(27.544±0.748)和mRNA(0.481±0.012)水平降低更显著(F=1065.233,P＜0.05;F=304.553,P＜0.05).[结论]NS-398联合表阿霉素可明显抑制HepG2细胞生长,NS-398可显著增强表阿霉素的抗癌作用;两者联合可明显下调HepG2细胞株中survivin蛋白和mRNA的表达.