Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.

Inhibitory effect of parvovirus H-1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multiplicity of infection (MOI) of H-1 given, survival of the QGY-7703 cells was found to be decreased. H-1 DNA amplification level at 30 h postinfection(p.i.) was detected to be 7.4 times higher than that at 2 h by dispersed cells assay, while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells (new-born human kidney cell line, NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay. The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2 h postinfection was observed to be prevented in 2 groups with given MOI 25 and 50. The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20 d latent period. It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.

Effect of 80.55 MeV//u^(12)C^(6+) Ions on Radiosensitivity and Cell Cycle of Human Hepatoma Cell Lines

In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u12C6＋ ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 （survival fraction at 2 gray） of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to 12C^6＋ ions.

ApoB-containing lipoproteins promote infectivity of chlamydial species in human hepatoma cell line

AIM:To evaluate the direct binding of two main chlamydial biovars(C.trachomatis and C.pneumoniae) to plasma lipoproteins and its effect on chlamydial infection rate in human hepatoma cell line(HepG2 cells). METHODS:Murine plasma lipoproteins were fractionated and isolated using fast-performance liquid chromatography(FPLC),spotted on nitrocellulose membrane and incubated with chlamydial suspensions. Direct binding of chlamydial particles to lipoprotein fractions has been studied using lipopolysaccharide-specific antibodies in immuno-dot blot binding assay and immunoprecipitation analysis.Immunostaining protocol as well as flow cytometry analysis have been employed to study the infectivity rate of chlamydial species in HepG2 cells. RESULTS:Elementary bodies of both C.trachomatis and C.pneumoniae bind ApoB-containing fractions of plasma lipoproteins.That binding becomes stronger when heat-denatured FPLC fractions are used, suggesting a primary role of apolipoproteins in interaction between chlamydial particle and lipoprotein. Both chlamydial biovars efficiently propagate in human hepatoma cell line-HepG2 cells even in serum free conditions forming late-stage inclusion bodies and releasing extracellular elementary bodies.Preincubation of C.trachomatis and C.pneumoniae with native ApoB-containing lipoproteins enhances the rate of chlamydial infection in HepG2 cells.CONCLUSION:A productive infection caused by C. trachomatis and C.pneumoniae may take place in human-derived hepatocytes revealing hepatic cells as possible target in chlamydial infection.Obtained results may suggest the participation of lipoprotein receptors in the mechanism of attachment and/or entry of chlamydial particles into target cells.

Effects of Terminalia arjuna bark extract on apoptosis of human hepatoma cell line HepG2

瞄准：调查尾器 arjuna 的效果(T。arjuna ) 人的肝细胞瘤房间线(HepG2 ) 和它在 apoptosis 的正式就职的可能的角色上的摘录。方法：人的肝细胞瘤房间与 T 的 ethanolic 摘录的不同集中被对待。arjuna 和它的细胞毒性效果被尝试平底锅蓝色排除方法和 lactate 脱氢酶漏试金测量。Apoptosis 被光和荧光分析显微镜的方法，和 DNA 破碎。apoptosis 的机制与 p53 和 caspase-3 蛋白质的表示被学习。谷胱甘肽(GSH ) 内容也在 T 以后在 HepG2 房间被测量。arjuna 处理。结果：T。arjuna 以一种集中依赖者方式禁止了 HepG2 房间的增长。Apoptotic 形态学在与 T 对待的 HepG2 房间被观察。在 60 和 100 mg/L 的集中的 arjuna。DNA 破碎， p53 的累积和 procaspase-3 蛋白质的劈开与 T 在处理以后在 HepG2 房间被观察。arjuna。GSH 的弄空在与 T 对待的 HepG2 房间被观察。arjuna。结论：T。arjuna 在 HepG2 房间在试管内导致了细胞毒性。HepG2 房间的 Apoptosis 可能由于 DNA 损坏和 apoptotic 蛋白质的表示。GSH 的弄空可以涉及 HepG2 房间的 apoptosis 的正式就职。

Influence of Toxoplasma gondii on in vitro proliferation and apoptosis of hepatoma carcinoma H7402 cell

Objective:To discuss the influence of tachyzoite of Toxoplasma gondii（T.gondii） RH strain on proliferation and apoptosis of hepatoma carcinoma（HCC） H7402 cell.Methods:The HCC H7402 cell in logarithmic phase and tachyzoite of T.gondii RH strain in different concentrations（1×107/mL,2×107/mL.4×107/mL,8×107mL and 16×107/mL） were co-cultured.CCK-8was utilized to determine the inhibition rate of T.gondii tachyzoite on H7402 cell growth.Flow cytometry was used to detect the change of cell cycle.RT-PCR method was used to detect the expression of cyclinB1 and cdc2-two genes related to cell cycle.Western blot method was used to detect the expression of apoptosis-related proteins Caspase-3 and Bcl-2.Results:The tachyzoite of T.gondii RH strain can inhibit the proliferation of HCC H7402 cells.The inhibition rate of tumor cell growth increased with the increase of concentration of T.gondii tachyzoite.With the increase of concentration of T.gondii tachyzoite,the proportion of G0/G1 phase of H7402 cell increased,the proportion of S phase decreased,and PI value decreased accordingly.The expression of cyclinB1 and cdc2 genes decreased with the increase of the concentration of T.gondii tachyzoite.With the increase of the concentration of tachyzoite of T.gondii RH strain,the expression quantity of Caspase-3 in H7402 cell increased,but the expression quantity of Bcl-2protein decreased.Conclusions:T.gondii can inhibit the in vitro proliferation of HCC H7402 cell,and induce its apoptosis.This effect shows a trend of concentration-dependent increase.Moreover,it is related to the down-regulation of cyclinB1 and cdc2（cell cycle-related genes）,the increase of apoptosis-related protein Caspase-3.and the decreasc of Bcl-2 expression.

虫草素对人肝癌Bel-7402细胞抑制及作用机制的研究

虫草素是虫草的主要活性成分,具有抗肿瘤、抑菌、抑制病毒、抑制蛋白质激酶活性等作用[1-2]。研究证明虫草素能抑制mRNA的合成,诱导细胞凋亡,促进细胞分化,对多种肿瘤细胞的生长具有抑制作用[1-2]。诱导肿瘤细胞凋亡以及作用靶点已成研究的焦点。为探讨虫草素诱导肿瘤细胞凋亡的新靶点。

40种香豆素类化合物对人胃癌细胞株BGC和人肝癌细胞株BEL-7402细胞生长抑制活性的筛选

目的:寻找中药中抗肿瘤活性化合物和先导化合物,为开发新药奠定基础。方法:采用人胃癌细胞株BGC和人肝癌细胞株BEL-7402体外培养法,鉴定受试的40种香豆素类化合物对其生长的抑制作用。结果:伞形花内酯刘寄奴酸酯、木桔素、环氧酸橙皮油素、酸橙皮油素、前胡酮、芸香苦素、牛防风素和异香柑内酯对人胃癌细胞株BGC细胞的生长有一定程度的抑制作用,且呈浓度-效应关系;伞形花内酯刘寄奴酸酯、异虎耳草素、前胡素和二氢山芹醇当归酸酯对人肝癌细胞株BEL-7402细胞的生长有一定程度的抑制作用,且呈浓度-效应关系。结论:香豆素类化合物是潜在的抗肿瘤药物。

OA对人肝细胞HL-7702和肝癌细胞Bel-7402增殖的影响和对细胞凋亡的诱导作用研究

以人肝细胞HL-7702和肝癌细胞Bel-7402为研究对象,通过MTT法、细胞外部形态观察、Annexin V-FITC&PI双染法及TUNEL法等方法,研究了腹泻性贝毒的主要组分大田软海绵酸(Okadaic acid,OA)对其增殖的影响及对细胞凋亡的诱导作用。结果表明:OA对人两株细胞的增殖均有显著的抑制作用,24 h IC50分别为65ng/mL和60 ng/mL,且这种抑制作用呈剂量依赖性。OA导致两株细胞的形态均发生明显变化:细胞变圆,脱壁,细胞膜形成泡状突起,最终形成膜包裹的凋亡小体。Annexin V-FITC和PI双染法及TUNEL法检测均发现:OA能够诱导两株细胞发生不同程度的凋亡,且凋亡细胞的比例与OA的浓度相关。以上结果表明:OA能够通过抑制细胞增殖和诱导细胞凋亡而对人肝细胞和肝癌细胞造成影响,且这种影响的程度与OA的浓度和作用时间密切相关。

44种生物碱类化合物对人胃癌细胞株BGC和人肝癌细胞株BEL-7402细胞增殖抑制活性的筛选

目的:从中药中筛选有抗肿瘤活性的化合物。方法:应用人胃癌细胞株BGC和人肝癌细胞株BEL-7402筛选44种生物碱类化合物对其增殖的抑制作用。结果:伊波加因、哈尔明碱、黄连碱、小檗碱、延胡索碱、小檗胺、吐根碱、马兜铃酸I、番茄碱苷、澳洲茄次碱、茄次碱、金鸡宁、罂粟碱和酒石酸麦角胺对人胃癌细胞株BGC细胞的增殖有一定程度的抑制作用,且呈浓度-效应关系;最强者为黄连碱、延胡索碱、小檗胺、吐根碱、番茄碱苷、澳洲茄次碱和金鸡宁,其次为伊波加因、哈尔明碱、小檗碱、马兜铃酸I、茄次碱、罂粟碱和酒石酸麦角胺。蛇根精、γ-崖椒碱、黄连碱、小檗碱、延胡索碱、吐根碱、马兜铃酸I、茄次碱和喜树碱对人肝癌细胞株BEL-7402细胞的增殖有一定程度的抑制作用,且呈浓度-效应关系;最强者为吐根碱、小檗碱、延胡索碱和喜树碱,其次为黄连碱、马兜铃酸I和茄次碱。结论:上述生物碱类化合物可能是含有该生物碱、有抗肿瘤活性中药的有效成分,亦可能成为开发抗肿瘤新药的候选药物。

青蒿酯钠抗人肝癌(BEL-7402)与诱导凋亡

应用体内外抑瘤实验、细胞形态学观察、流式细胞仪、Westernblot实验进行检测和观察，探讨青蒿酯钠抗肿瘤作用及机制。结果表明，青蒿酯钠对人肝癌有体内、外抗肿瘤作用；诱导人肝癌细胞凋亡，可能是其抗肿瘤作用机制之一；诱导体外人肝癌细胞凋亡通过P53非依赖性途径。

抗癌抗生素力达霉素诱导人肝癌BEL-7402细胞死亡的特征

目的 研究抗癌抗生素力达霉素 (LDM )诱导人肝癌BEL 740 2细胞死亡的特征。方法 用荧光染料Hoechst 33342和PI联染 ;琼脂糖凝胶电泳检测 ;流式细胞术检测等。结果 LDM 1μmol·L-1处理该细胞后 6h ,可观察到一种有别于典型凋亡的染色质凝集方式 :核膜一直保持完整 ,细胞仍贴壁 ,无凋亡小体形成 ;琼脂糖凝胶电泳检测到DNA梯带。流式细胞术检测到的G1亚峰 ,仅在LDM处理BEL 740 2细胞后 2 4h出现。LDM处理BEL 740 2细胞后 6h ,caspase 3,6的活性分别增高 5 ,4倍。染色质开始凝集的时间比caspase 6活性达到高峰的时间早。结论 力达霉素诱导人肝癌BEL 740 2细胞死亡的特征有别于典型的凋亡 。

胡桃醌不同给药途径对人肝癌细胞BEL-7402裸鼠皮下移植瘤生长的影响

目的采用裸鼠皮下移植瘤模型,通过不同给药途径对胡桃醌抗肿瘤活性和毒性进行评价。方法建立人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径观察胡桃醌抑制肿瘤生长的效果。结果①以600、300和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,发现该剂量胡桃醌对肿瘤生长没有明显的影响;NK细胞活性检测发现,600、300μg/kg胡桃醌对裸鼠免疫功能有影响(P均<0.01),150μg/kg胡桃醌则没有影响(P>0.05);与阳性对照组(5-Fu)相比,600μg/kg胡桃醌组NK细胞活性差异无显著性(P>0.05),300和150μg/kg胡桃醌组NK细胞活性差异有显著性(P<0.05,P<0.01),结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。②以4.5、3和1.5 mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,抑瘤率分别为为78.24%、66.57%、48.94%;4.5、3 mg/kg胡桃醌的抑瘤作用可与阳性对照组比拟(P均>0.05)。但4.5 mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。③以pH 7.4和pH 4.0的600、300和150μg/kg胡桃醌人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同pH(pH 7.4或4.0)600、300μg/kg的胡桃醌局部注射抑瘤作用与阳性对照组(5-Fu)组差异无显著性(P>0.05),而不同pH的150μg/kg胡桃醌抑瘤作用不明显。同一浓度不同pH药物的抑瘤作用差异无显著性(P均>0.05),但pH 4.0的胡桃醌组肿瘤细胞肝转移较少。结论胡桃醌不同给药途径均可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长,但有一定的毒副作用,药物安全范围较小。

半夏蛋白对人肝癌细胞Bel-7402的诱导凋亡作用

目的观察并比较半夏蛋白对体外培养的人肝癌细胞Bel-7402的生长抑制作用。方法应用流式细胞技术,检测半夏蛋白对人肝癌细胞Bel-7402的诱导凋亡作用,并观察了其诱导凋亡作用的量-效关系。结果60%硫酸铵沉淀蛋白没有明显诱导Bel-7402细胞凋亡的作用,而30%硫酸铵沉淀蛋白有诱导Bel-7402细胞凋亡的作用。30%沉淀进一步分离结果显示,只有0.1 mol/L NaCl洗脱峰有一定诱导Bel-7402细胞凋亡的作用。结论半夏蛋白有一定的诱导Bel-7402细胞凋亡作用,其主要成分可能存在于总有机酸部位。

MTT法测定稀土离子对BEL-7402及K562细胞的毒性及增殖毒性

用MTT法测定稀土离子在不同浓度、不同培养液中,与BEL 7402和K562细胞作用不同时间,对细胞的毒性和增殖毒性。结果表明,在含10%小牛血清培养液中,仅个别稀土离子在较高浓度时对BEL 7402细胞增殖有较弱的抑制作用;对于K562细胞,稀土离子在低浓度时对细胞增殖即表现出较强的抑制作用(P<0.05)。当培养液不含小牛血清时,较低浓度的稀土离子即可抑制BEL 7402细胞的增殖(P<0 05)。

雄黄抗人肝癌BEL-7402细胞作用

目的:观察雄黄体外抗人肝癌BEL-7402细胞作用和作用机制。方法:采用MTS法和细胞克隆实验法观察雄黄对BEL-7402细胞增殖的影响,应用PI/Hoechest33258双染法和流式细胞仪检测细胞凋亡,荧光分光光度计检测细胞内钙和线粒体膜电位。结果:与阴性对照组比,雄黄能剂量依赖性地抑制BEL-7402细胞的生长和克隆形成。雄黄作用后,BEL-7402细胞出现了凋亡形态学改变如染色质浓缩等;流式细胞仪检测显示,雄黄能剂量依赖性地诱导细胞凋亡,其中,225μg/ml雄黄引起BEL-7402细胞凋亡的百分率为(28.6±4.6)%。与阴性对照组比,雄黄作用后,BEL-7402细胞的细胞内钙升高和线粒体膜电位降低。结论:雄黄具有一定的抗肿瘤作用,其作用机制与升高细胞内钙、降低细胞线粒体膜电位并诱导细胞凋亡有关。

黄酮类化合物Davidigenin的合成及对人肝癌Bel-7402细胞的抗癌活性研究

目的合成黄酮类化合物Davidigenin(二氢异甘草素,DH-ILG),并对体外抗肝癌活性进行研究。方法对2,4-二羟基苯乙酮和对羟基苯甲醛进行羟基的保护,并通过羟醛缩合反应后行脱保护而合成异甘草素(ILG);通过2种催化氢化反应系统对ILG进行还原而制备目标化合物Davidigenin,对于氢化还原试剂进行筛选,用光谱方法对结构进行鉴定;应用人肝癌Bel-7402细胞株为体外模型,用MTT法观察Davidigenin对人肝癌细胞增殖的抑制活性。结果经2种催化氢化还原试剂得到的目标化合物产率分别为20%及91.45%;Davidigenin对人肝癌Bel-7402细胞的增殖有抑制活性,在浓度为25～200μg/mL范围内,其抑制率为2.76%～65.27%,且活性强度低于异甘草素。结论利用二氧化铂对查尔酮类化合物分子侧链中的α-β不饱和双键进行选择性还原反应的效果明显高于钯碳催化还原;人肝癌Bel-7402细胞对Davidigenin的抗癌活性具有一定的敏感性;查尔酮类化合物分子侧链中的α-β不饱和双键是其抗癌活性的必要条件。本研究对甘草查尔酮类化合物的抗癌构效关系研究及抗肝癌候选药物筛选奠定一定基础。

呫吨酮衍生物XP-15对BEL-7402细胞线粒体膜电位的影响

目的 :探讨呫吨酮并吡啶衍生物9-(2-吗啡啉乙酰胺基)-7H-吡啶并[4,3-c]呫吨-7-酮(XP-15)对BEL-7402细胞线粒体膜电位的影响。方法:通过MTT法、细胞形态学和克隆实验观察XP-15对BEL-7402细胞增殖的影响;用荧光分光光度计检测线粒体膜电位的影响。结果:高剂量的XP-15能明显抑制BEL-7402细胞增殖(P<0.05);对BEL-7402细胞克隆的抑制作用,呈剂量和时间依赖性。XP-15作用后,BEL-7402细胞的线粒体膜电位降低(P<0.05)。结论:XP-15诱导BEL-7402细胞凋亡的作用,可能与其降低线粒体膜电位有关。

Artemis对人肝癌细胞系BEL-7402/5FU DNA损伤的影响

目的探讨转染sh Artemis干扰质粒对人肝癌细胞BEL-7402/5FU DNA损伤的影响。方法将人肝癌细胞分为正常对照组、脂质体对照组、空质粒对照组和转染sh Artemis干扰质粒实验组(sh Artemis实验组);建立DNA损伤模型,分别用0.625、1.25、2.5、5.0和10.0μg/m L的丝裂霉素C作用BEL-7402/5FU细胞24和48 h,MTT法检测细胞活性,Western blot观察磷酸化组蛋白H_2AX(γ-H_2AX)的表达;转染Artemis干扰质粒,检测Artemis、γ-H_2AX表达;彗星实验检测DNA的损伤。结果 0.625、1.25、2.5、5.0和10.0μg/m L的丝裂霉素C作用48h后肝癌细胞活力明显下降(P<0.05);丝裂霉素C刺激细胞48 h后,γ-H_2AX的表达量随着药物浓度的增加而增加;转染sh Artemis干扰质粒后,γ-H_2AX的表达量较正常对照组增加(P<0.05);sh Artemis实验组较正常对照组拖尾DNA量增加(P<0.05)。结论丝裂霉素C能够诱导肝癌细胞损伤;转染sh Artemis干扰质粒促进丝裂霉素C诱导的人肝癌细胞BEL-7402/5FU DNA损伤。

复方斑蝥含药血清抑制人肝癌Bel-7402细胞增殖的机制研究

目的探讨复方斑蝥抑制人肝癌Bel-7402细胞增殖的作用机制。方法血清药理学方法制备复方斑蝥血清并处理人肝癌Bel-7402细胞,MTT方法观察其对人肝癌Bel-7402细胞增殖的抑制作用,RT-PCR技术检测其对c-Myc、p53基因表达的影响。结果复方斑蝥含药血清能够显著抑制Bel-7402细胞的增殖,并下调c-Myc和上调p53基因的表达。结论复方斑蝥可能通过影响细胞增殖相关基因的表达抑制Bel-7402细胞的增殖,可作为一种抗肝癌中药复方的研究原型。