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.

TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

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.

Establishment of a human hepatoma multidrug resistant cell line in vitro

AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malignant hepatoma was incubated with a high concentration of cisplatin(CDDP) to establish a CDDP-resistant cell subline(SK-Hep-1/CDDP).The 50% inhibitory dose(IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cells were all evaluated using cell counting kit-8 assays.The distribution of the cell cycles were detected by flow cytometry.Expression of acquired multidrug resistance P-glycoprotein(MDR1,ABCB1) and multidrug resistance-associated protein 1(MRP1,ABCC1) was compared with that in parent cells by Western blotting and immunofluorescence combined with laser scanning confocal microscopy.RESULTS:The SK-Hep-1/CDDP cells(IC50 = 70.61 ± 1.06 μg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells(IC50 = 5.13 ± 0.09 μg/mL),and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin,5-fluorouracil and vincristine.Similar morphologies were determined in both SK-Hep-1 and SK-Hep-1/CDDP groups.The cell cycle distribution of the SK-Hep-1/CDDP cell line exhibited a significantly increased percentage of cells in S(42.2% ± 2.65% vs 27.91% ± 2.16%,P < 0.01) and G2/M(20.67% ± 5.69% vs 12.14% ± 3.36%,P < 0.01) phases in comparison with SK-Hep-1 cells,while the percentage of cells in the G0/G1 phase decreased(37.5% ± 5.05% vs 59.83% ± 3.28%,P < 0.01).The levels of MDR1 and MRP1 were overexpressed in the SK-Hep-1/CDDP cells exhibiting the MDR phenotype.CONCLUSION:Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of MDR1 and MRP1.

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.

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

AIM：To investigate the effects of Terminalia arjuna （T. arjuna） extract on human hepatoma cell line （HepG2） and its possible role in induction of apoptosis.METHODS： Human hepatoma cells were treated with different concentrations of ethanolic extract of T. arjuna and its cytotoxicity effect was measured by trypan blue exclusion method and lactate dehydrogenase leakage assay. Apoptosis was analyzed by light and fluorescence microscopic methods, and DNA fragmentation. The mechanism of apoptosis was studied with expression of p53 and caspase-3 proteins. Glutathione （GSH） content was also measured in HepG2 cells after T. arjuna treatment.RESULTS： T. arjuna inhibited the proliferation of HepG2 cells in a concentration-dependent manner. Apoptotic morphology was observed in HepG2 cells treated with T. arjuna at the concentrations of 60 and 100 mg/L. DNA fragmentation, accumulation of p53 and cleavage of procaspase-3 protein were observed in HepG2 cells after the treatment with T. arjuna. The depletion of GSH was observed in HepG2 cells treated with T. arjuna.CONCLUSION： T. arjuna induced cytotoxicity in HepG2 cells in vitro. Apoptosis of HepG2 cells may be due to the DNA damage and expression of apoptotic proteins. Depletion of GSH may be involved in the induction of apoptosis of HepG2 cells.

ESTABLISHMENT OF A MURINE ASCITES HEPATOMA CELL LINE H_(22)-F_(25)/L AND ITS BIOLOGICAL CHARACTERISTICS

Having been passed for 160 generations, a cell linedesignated as H22-F25/L was established from a murine tumorlymphatic metastatlc model H22-F25 which had been set up in our college. The cell line was in suspension culture with a rapid proliferation and stable growth. The peak tune of cell division and proliferation was 48 and 96 hours after culture. In a week, the cell number was Increased by 25 tunes. H22-F25/L still keeps the features of a poorly differentiated cancer. Its tumor inducing rate (in vivo)was 100% in 615 mice. Lymph node metastasis rate was 50% and pulmonary metastasis rate 10%. H22- F25/ L Is a population of heterogenetlc tumor cells Including 2 stem cell lines (the model number of chromosomes being 43 in 40% tumor cells and 86 in 32%) and some side lines. The common marker chromosomes M1, M2, M3 and M4 were present in all stem and side lines.

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone ⅡA could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6. 28μg/ml. After treatment with 1-10μg/ml tanshinone ⅡA for 72 h, BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at μg/ml concentration for 12 h> 24 h, 36 h, 48 h and 72 h were (2. 32±0. 16)%, (3. 01±0. 35) %, (3. 87±0. 43)%, (6. 73±0. 58)% and (20. 85 ± 1. 74) % respectively, which were all significantly higher than those in the control group (1. 07±0. 13) %. It is concluded that Tanshinone ⅡA could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.

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.

EFFECT OF HEPATIC STIMULATOR SUBSTANCE (HSS) EXTRACTED FROM FETAL LIVER ON THE PROLIFERATION OF HUMAN ALEXENDER HEPATOMA CELLS

It's reported that hepatic stimulator substance (HSS) was extracte from the fetal liver of 4 - 6 months of fetus, and that the effect of HSS on the proliferation of human Alexender hepatoma cells was studied in this paper. The results showed that proliferation of Alexender cells varied with the amount of HSS in the culture medium, and the former was positively correlated with the latter significantly (P<0. 01). The study indicated that HSS from the fetal liver can stimulate the proliferation of human Alexender hepatoma cells.

骨肉瘤组织中GFRA1、FBN1表达水平及意义

目的探讨胶质细胞系源性神经营养因子受体1(GFRA1)、原纤维蛋白-1(FBN1)在骨肉瘤组织中表达水平及意义。方法收集2017年9月至2019年9月住院手术的66例骨肉瘤患者治疗精细切除骨肉瘤组织标本及癌旁组织标本,同时收集整理其临床分期、肿瘤直径、肿瘤分化程度等临床资料。采用免疫组织化学法检测GFRA1、FBN1蛋白表达;骨肉瘤组织GFRA1、FBN1表达与患者预后的关系采用Kaplan-Meier法分析;多因素Logistic回归分析骨肉瘤患者预后的影响因素。结果与癌旁组织相比,骨肉瘤组织中GFRA1、FBN1阳性表达率明显较高(P<0.05)。GFRA1、FBN1的表达与骨肉瘤患者的临床分期、分化程度、是否发生肺转移、软组织是否浸润有关(P<0.05),与患者性别、年龄、肿瘤直径、肿瘤位置无关(P>0.05);骨肉瘤组织GFRA1、FBN1阳性表达患者3年生存率低于FBN1阴性表达患者(P<0.05)。GFRA1、FBN1阳性表达、肿瘤转移、软组织浸润是骨肉瘤患者预后的独立危险因素(P<0.05)。结论GFRA1、FBN1的表达与骨肉瘤患者的临床病理特征及预后有关,可以作为骨肉瘤患者预后评估的指标。

生长抑素对人肝癌HepG2细胞增殖及cyclinD1、cyclinE蛋白表达的影响

目的探讨生长抑素治疗肝癌的作用机制。方法取对数生长期HepG2细胞以不含血清的培养液使其周期同步化，以含血清培养液继续培养24h后随机分为观察Ⅰ、Ⅱ、Ⅲ组及对照组，前三组分别加入质量浓度为100、200、400μg／（kg·d）的生长抑素，对照组加入等体积RPMll640培养液24—72h。MTT比色法观察各组细胞增殖抑制率，流式细胞仪检测细胞周期分布，免疫组化法检测周期素D1（cyclinD1）、周期素E（cyclinE）蛋白表达。结果观察Ⅰ、Ⅱ、Ⅲ组不同时间点细胞增殖抑制率均显著高于对照组，且同一时间点观察Ⅲ组〉观察Ⅱ组〉观察I组，同组中72h〉48h〉24h（P〈0．05、0．01）；观察Ⅰ、Ⅱ、Ⅲ组G，期细胞比例均显著高于对照组，观察Ⅱ、Ⅲ组S期细胞比例均显著低于对照组（P〈0．05、0．01）；观察Ⅰ、Ⅱ、Ⅲ组cyclinD1、cyclinE蛋白水平均显著低于对照组，且观察Ⅲ组〈观察Ⅱ组〈观察Ⅰ组（P〈0．05、0．01）。结论生长抑素可通过下调cyclinD1、cyclinE表达抑制HepG2细胞增殖，此可能为其治疗肝癌的作用机制之一。

薯蓣皂苷对蛋鸡脂肪肝综合征肝损伤和氧化应激的缓解作用及其机制研究

旨在探究薯蓣皂苷(dioscin, Dio)对高能低蛋白饲喂诱导蛋鸡脂肪肝综合征脂代谢紊乱和氧化损伤的缓解作用及其机制的影响。以105只150日龄的蛋鸡作为试验对象,分为5组:对照组,HELP(高能低蛋白饲料)组,低剂量组(添加100 mg/kg Dio),中剂量组(添加500 mg/kg Dio),高剂量组(添加1 000 mg/kg Dio),饲喂3个月后剖检观察,测定肝组织AMP-依赖性蛋白激酶(AMPK)和核因子红细胞系2相关因子2(Nrf2)转录水平。体外以棕榈酸钠(PA)诱导鸡肝癌(LMH)细胞代谢紊乱,CCK-8和吖啶橙/碘化丙啶检测细胞死亡,尼罗红染色观察脂滴,荧光探针法检测活性氧(ROS)产生水平,Western blot检测脂代谢、抗氧化相关蛋白表达水平,免疫荧光检测Nrf2入核。结果:与对照组相比,HELP组蛋鸡肝脏发生明显的脂肪变性和出血,中剂量组明显缓解肝脏脂肪变性和出血,提高AMPKα和Nrf2转录水平,Dio缓解PA诱导的LMH细胞死亡,减少LMH细胞中脂滴沉积,并显著上调AMPK通路(P<0.05),同时减少ROS产生并增加Nrf2的表达及入核。综上,Dio能够缓解高能低蛋白饲喂诱导蛋鸡脂肪肝综合征脂代谢紊乱和氧化损伤,激活LMH细胞AMPK和Nrf2信号通路缓解PA诱导的LMH细胞脂代谢紊乱及氧化应激的发生,为其作为一种生理调节剂用于家禽脂肪肝及其相关的营养代谢症的防控提供理论依据。

小分子干扰RNA沉默肝癌细胞CDK2基因对RB、CyclinE、E2F1基因表达的影响

【目的】观察小分子干扰RNA(siRNA)沉默细胞周期素依赖性蛋白激酶(CDK2)基因后,细胞周期相关基因RB、CyclinE、E2F1在肝癌细胞SMMC7721中mRNA的表达。【方法】将前期研究中已构建成功并筛选出的最有效干扰抑制CDK2基因的siRNA序列片段,采用Lipofectamine TM2000脂质体转染法转染肝癌细胞株SMMC7721后分6组:重组质粒组190、重组质粒组191、SMMC7721肝癌组、转染试剂组、阴性对照组、空质粒组。采用实时荧光定量PCR法检测RB、CyclinE、E2F1 mRNA水平。【结果】CDK2的siRNA转染SMMC7721细胞后,RB基因mRNA表达上调,CyclinE、E2F1基因mRNA表达下调。【结论】抑制CDK2基因的表达能使肝癌细胞SMMC7721中RB基因mRNA表达上调,CyclinE、E2F1基因mRNA表达下调,提示肝癌细胞中RB、CyclinE、E2F1基因的表达与CDK2基因的表达具有明显的相关性。

和枢消积方对人肝癌细胞系HepG2增殖凋亡的影响

目的探讨和枢消积方对人肝癌细胞系HepG2增殖凋亡的影响,揭示可能的分子机制。方法使用和枢消积方作用于人肝癌细胞系HepG2,采用Cell Counting Kit-8(CCK-8)、平板克隆实验检测细胞增殖能力。采用蛋白质印迹法(Western blot)检测B淋巴细胞瘤-2基因(BCL-2)、BCL-2相关X蛋白(BAX)、丙型肝炎病毒(HCV)非结构5A蛋白(NS5A)反式激活基因13(NS5ATP13)及蛋白激酶B(AKT)/糖原合酶激酶(GSK)/雷帕霉素靶蛋白(MTOR)相关通路蛋白的表达水平。结果和对照组相比,和枢消积方各组可使HepG2细胞活性、增殖率显著降低(P<0.05);同时可上调BAX的蛋白表达,下调BCL-2及磷酸(p)-AKT、p-GSK、p-MTOR及NS5ATP13的表达(P<0.05),呈浓度依赖性。结论和枢消积方可以抑制HepG2细胞的增殖并诱导凋亡,这可能与和枢消积方抑制NS5ATP13的表达,继而抑制AKT/GSK/MTOR信号转导通路的活化有关。

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.

Effect of Calmodulin and Voltage-dependent Ca^(2+) Channel on the Proliferation of Heptoma Cells Induced by Epidermal Growth Factor

The effect of thyrosine kinase, calmodulin and voltage-dependent Ca 2+ channel on the proliferation of hepatoma cells induced by EGF was studied. Hepatoma cell line SMMC7721 was cultured in RPMI1640 serum-free medium. DNA synthesis rate of hepatoma cells was measured by 3H-TdR incorporation. 10 -9 mol/L EGF could significantly stimulate the proliferation of hepatoma cells (P<0.05), and this effect might be significantly inhibited by tyrosine kinase inhibitor (P<0.001). Calmodulin inhibitor W-7 had no effect on the basic phase of cultured hepatoma cells (P> 0.05), but it had very significantly inhibitory effect on the proliferation of hepatoma cells induced by EGF (P<0.001). Voltage-dependent Ca 2+ channel inhibitor Varapamil had no inhibition on the proliferation of hepatoma cells induced by EGF (P>0.05). It had no effect on the basic phase of cultured hepatoma cells (P>0.05). It is suggested that tyrosine kinase and Ca 2+-calmodulin-dependent pathway may play a critical role on the proliferation of heptoma cells induced by EGF, and voltage-dependent Ca 2+ channel is independent of the effect of EGF.

In vitro study on the morphology of human blood dendritic cells and LPAK cells inducing apoptosis of the hepatoma cell line

Objective To observe in vitro effects and morphological changes of human peripheral blood dendritic cells (DCs) on the ability of lymphokine and phytohaemagglutininum (PHA) activated killer (LPAK) cells to induce apoptosis of the human hepatoma cell line (BEL-7402， B).Methods Experimental groups were divided into LD group (DCs+L+B), L group (L+B)， D group (DCs+B) and B group. The methods of neutral red uptake, ordinary light microscopy, electron microscopy, TDT mediated X-dUTP nick end labeling (TUNEL) were used. Results The difference between the D group and the B group was not distinct (P>0.05). The difference between the LD group and the L group was distinct， with DCs+LPAK >LPAK (P<0.01) in cytotoxity. Apoptotic cells were TUNEL positive in light microscopy, and apoptotic nuclei were stained yellow brown and dark brown, with size and shape varying from cell to cell. Ultrastructural change in apoptotic tumor cells comprised of compaction and condensation of nuclear chromatin， and condensation of cytoplasm and apoptotic bodies. At the same time, LPAK cells manifested the characteristics of autophagic apoptosis, and there were some autophagic bodies in it. Conclusions The combination of human blood DCs and LPAK cells could induce apoptosis of BEL-7402 cells effectively, with some LPAK cells manifesting the characteristics of autophagic apoptosis.

阿霉素对热处理的人肝癌细胞-7721/Adm耐药株细胞毒性的影响

目的： 探讨比较阿霉素（ ADM）与 43℃加热单独或合并处理人肝癌细胞 7721敏感株（简称 7721细胞）和人肝癌细胞 7721/Adm耐药株（简称 7721/Adm细胞）的细胞毒作用。方法：以体外培养的人肝癌细胞 7721敏感株和已经建立的人肝癌细胞 7721/Adm耐药株为研究对象，采用水浴加温法 ,体外细胞毒试验（ MTT法） ,观察 ADM与加热处理对细胞生长抑制的影响；采用流式细胞技术检测热处理对 7721细胞和 7721/Adm细胞胞内阿霉素浓度的影响。结果： (1)热处理可以明显提高两种细胞对阿霉素的敏感性： 7721细胞、 7721/Adm细胞经阿霉素及 43.5℃热处理，其细胞存活率分别下降 35.2％（ 30 min）、 24.8％（ 60 min）和 29.4％（ 30 min）、 22.8％（ 60 min）； (2)流式细胞仪检测显示，热处理可明显提高这两种细胞尤其是 7721/Adm细胞内的阿霉素浓度： 7721细胞组提高 30.8％， HCC 7721/Adm组提高 51％。结论：热处理可以显著提高人肝癌细胞 7721敏感株和人肝癌细胞 7721/Adm耐药株对阿霉素的敏感性。