Effects of hydroxyapatite nanoparticles on proliferation and apoptosis of human hepatoma BEL-7402 cells

AIM: To study the effect of hydroxyapatite (HAP) nanoparticleson human hepatoma cell line BEL-7402 in vitro.METHODS: The human hepatoma cell line BEL-7402 wascultured and treated with HAP nanoparticles at variousconcentrations. Growth suppression was detected with MTTcolorimetric assay, cell apoptotic alterations were evaluatedby cytochemical staining (Hoechst 33258), transmissionelectron microscopy (TEM), and flow cytometry (FCM).RESULTS: HAP nanoparticles inhibited the growth ofhepatoma cells in a dose-dependent manner, with IC50 valuesof 29.30 mg/L. Treated with 50-200 mg/L HAP nanoparticlesfor 48 h, BEL-7402 cells apoptosis with nuclear chromatincondensation and fragmentation as well as cell shrinkageand the formation of apoptotic bodies were observed undercytochemical staining and transmission electron microscopy.FCM analysis showed hypodiploid peaks on histogram, theapoptotic rates at the concentrations of 50, 75, 100, 150and 200 mg/L of HAP nanoparticles were 20.35±2.23%,25.35±1.92%, 29.34±4.61%, 44.92±3.78 % and53.64±3.49%, respectively, which were all significantlyhigher than that of control group 2.23±0.14%. There wasa significant correlation between HAP nanoparticleconcentration and apoptotic rate (r=0.994, P＜0.01).CONCLUSION: HAP nanoparticles not only inhibitproliferation but also induce apoptosis of human hepatoma cell line BEL-7402 in vitro.     

HERG1 K+通道在肝癌细胞系BEL-7402中的表达及其意义

[目的]观察HERG1 K 通道在肝癌细胞系BEL-7402的表达,探索其在肝癌发生与发展中的作用。[方法]RT-PCR方法检测hepg1在肝癌细胞系BEL-7402细胞和正常肝细胞系L-02细胞中表达;四甲基偶氮唑盐比色法(MTT法)研究HERG1 K 通道对BEL-7402细胞和L-02细胞增殖作用;以流式细胞仪检测该通道对肝癌细胞系BEL-7402细胞周期的影响。[结果]HERG1 K 通道在肝癌细胞中表达,而在正常肝细胞中不表达,该通道特异性抑制剂E-4031可以显著抑制BEL-7402细胞的增殖,而对不表达herg1基因的L-02细胞增殖不起作用;E-4031抑制BEL-7402细胞HERG1 K 通道后可导致G1期细胞显著上升。[结论]HERG1 K 通道可能是一个潜在的癌基因,可促进肝癌细胞的增殖;参与调控BEL-7402细胞周期的G1期进程。通过抑制该通道的功能,或下调其表达抑制该肿瘤的发生与发展;HERG1 K 通道可能是肝癌治疗的一个潜在的药物靶点和诊断性生物标志。     

参桃软肝丸抑制人肝癌细胞增殖及增强LAK抑瘤活性的实验研究

目的：研究中药复方参桃软肝丸对人肝癌细胞BEL－7402、SMMC－7721增殖的影响,探讨其抗癌作用机制。方法：参桃软肝丸灌胃给药后,采集大鼠血清,处理人肝癌细胞BEL－7402、SMMC－7721,MTT法观察细胞增殖;并与淋巴因子激活的杀伤细胞（LAK）共同处理以上两种细胞,结果：参桃软肝丸具有显著抑制BEL－7402、SMMC－7721增殖的活性,结论：抑制人肝癌细胞增殖,提高LAK活性可能是参桃软肝丸抗肝癌的主要作用机制。     

亚砷酸对肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响

目的探讨亚砷酸（AA）对人肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响。方法采用MTT比色法检测从作用后的BEL-7402细胞增殖抑制率,流式细胞术检测BEL-7402细胞周期及凋亡细胞,HE染色法观察凋亡细胞的形态,RT-PCR检测BEL．7402细胞的Bcl-2 mRNA,免疫组化法检测细胞的Bcl-2蛋白。结果1．0—8．0μmol／L的AA可使BEL-7402细胞增殖抑制率上升,能诱导BEL-7402细胞凋亡并阻滞细胞周期于S、G2／M期,呈剂量依赖性;8．0μmol／L的AA作用BEL-7402细胞48h后,细胞呈现明显的凋亡形态改变,其Bcl-2 mRNA及蛋白表达明显减弱。结论AA体外有抑制BEL-7402细胞增殖及诱导凋亡的作用,且呈时间、剂量依赖性,其作用机制可能与降低其Bcl-2表达有关。     

Alpha-fetoprotein-specific transfer factors downregulate alpha-fetoprotein expression and specifically induce apoptosis in Bel7402 alpha-fetoprotein-positive hepatocarcinoma cells

Aim: To investigate the mechanisms of AFP-specific transfer factors (AFP-TF) in induced Bel7402 cells apoptosis. Further, we investigate the interaction between AFP-TF and AFP in the apoptosis. Methods: Bel7402 and HepG2 AFP-positive hepatocarcinoma cell lines, SK-Hep-1 AFP-negative hepatocarcinoma cell line and Changliver normal liver cell line are used. Cell viability is evaluated by MTT assay and apoptosis is measured by Hoechst33342 staining and TUNEL assay. FACS is used to analyze the cell cycle. AFP expression is examined by RT-PCR, Western blotting and immunocytochemistry. The interaction between AFP-TF and AFP in the apoptosis is investigated by addition of AFP in cultures or AFP transfection in Bel7402 cells prior to AFP-TF treatment. Mitochondrial membrane potential (DeltaPsi(m)) and intracellular Ca2+ concentration are respectively measured by Rhodamine123 and Fluo-3 AM Ester. Western blotting detects the involvement of several apoptosis-related proteins. Finally, caspase-3 and Caspase-9 activity are respectively examined. Results: AFP-TF can induce apoptosis in Bel7402 and HepG2 AFP-positive hepatocarcinoma cells, but not SK-Hep-1 and Changliver cells. AFP-mRNA level changes little in apoptotic Bel7402 cells; while AFP expression is downregulated and uniformly dispersed throughout the whole cell. Addition of exogenous AFP or overexpression of intracellular AFP can reduce such apoptotic effect. Besides, apoptotic Bel7402 cells show a disruption of DeltaPsi(m), an immediate elevation of Ca2+ concentration, a prominently decreased ratio of bcl-2 to bax, a release of cytochrome c from mitochondria to cytosol, and ultimately an activation of caspase-9 and caspase-3. Conclusion: AFP-TF induced Bel7402 cells apoptosis is mitochondrial-dependent and is mediated by the interaction of AFP-TF with intracellular AFP.     

Effect of arsenic trioxide on human hepatocarcinoma in nude mice

AIM: To study the effect of arsenic trioxide (As(2)0(3)) on human hepatoma cell line BEL-7402 in vivo. METHODS: Human hepatoma cell line BEL-7402 cultured in vitro was inoculated into nude mice and arsenic trioxide, 5-Fu and saline were injected into abdominal cavity of the nude mice respectively. The volumes of tumor and general conditions of the nude mice and structural changes of the liver and kidney were observed. Morphologic changes were studied under electron microscope. Expression of AFP was investigated by immunohistochemical method. RESULTS: As(2)O(3) could inhibit the growth of tumor. The tumor growth inhibitory rate in mice treated with 2.5 mg/kg As(2)O(3) was 53.42% on the tenth day. The tumor growth inhibitory rate in mice treated with 5 mg/kg As(2)O(3) was 79.28% on the fifth day and 96.58% on the tenth day respectively. As(2)O(3) did not damage the liver and kidney of nude mice, or affect the blood system. Typical apoptotic morphological changes were found under electron microscope, and the change of mitochondria was obvious. The expression rate of AFP declined after treatment. CONCLUSION: Arsenic trioxide can induce apoptosis of human hepatoma cells, and inhibit proliferation of tumor with no obvious side effects on liver and kidney.     

Synergetic anticancer effect of combined quercetin and recombinant adenoviral vector expressing human wild-type p53, GM-CSF and B7-1 genes on hepatocellular carcinoma cells in vitro

AIM: This study investigated the anti-cancer effect of combined quercetin and a recombinant adenovirus vector expressing the human p53, GM-CSF and B7-1 genes (designated BB-102) on human hepatocellular carcinoma (HCC) cell lines in vitro. METHODS: The sensitivity of HCC cells to anticancer agents was evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The viability of cells infected with BB-102 was determined by trypan blue exclusion. The expression levels of human wild-type p53, GM-CSF and B7-1 genes were determined by Western blot, enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis, respectively. The apoptosis of BB-102-infected or quercetin-treated HCC cells was detected by terminal deoxynucleotidyl transferase (TdT) assay or DNA ladder electrophoresis. RESULTS: Quercetin was found to suppress proliferation of human HCC cell lines BEL-7402, HuH-7 and HLE, with peak suppression at 50 micromol/L quercetin. BB-102 infection was also found to significantly suppress proliferation of HCC cell lines. The apoptosis of BB-102-infected HCC cells was greater in HLE and HuH-7 cells than in BEL-7402 cells. Quercetin did not affect the expression of the three exogenous genes in BB-102-infected HCC cells (P>0.05), but it was found to further decrease proliferation and promote apoptosis of BB-102-infected HCC cells. CONCLUSION: BB-102 and quercetin synergetically suppress HCC cell proliferation and induce HCC cell apoptosis, suggesting a possible use as a combined anti-cancer agent.     

Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling

AIM: To assess the impact of eukaryotic elongation factor 1 alpha 2(e EF1A2) on hepatocellular carcinoma(HCC) cell proliferation, apoptosis, migration and invasion, and determine the underlying mechanisms.METHODS: e EF1A2 levels were detected in 62 HCC tissue samples and paired pericarcinomatous specimens, and the human HCC cell lines SK-HEP-1, Hep G2 and BEF-7402, by real-time PCR and immunohistochemistry. Experimental groups included e EF1A2 silencing in BEL-7402 cells with lentivirus e EF1A2-sh RNA(KD group) and e EF1A2 overexpression in SK-HEP-1 cells with e EF1A2 plasmid(OE group). Non-transfected cells(control group) and lentivirusbased empty vector transfected cells(NC group) were considered control groups. Cell proliferation(MTT and colony formation assays), apoptosis(Annexin V-APC assay), cell cycle(DNA ploidy assay), and migration and invasion(Transwell assays) were assessed. Protein levels of PI3K/Akt/NF-κB signaling effectors were evaluated by Western blot.RESULTS: e EF1A2 m RNA and protein levels were significantly higher in HCC cancer tissue samples than in paired pericarcinomatous and normal specimens. SK-HEP-1 cells showed lower e EF1A2 m RNA levels; Hep G2 and BEL-7402 cells showed higher e EF1A2 m RNA levels, with BEL-7402 cells displaying the highest amount. Efficient e EF1A2 silencing resulted in reduced cell proliferation, migration and invasion, increased apoptosis, and induced cell cycle arrest. The PI3K/Akt/NF-κB signaling pathway was notably inhibited. Inversely, e EF1A2 overexpression resulted in promoted cell proliferation, migration and invasion.CONCLUSION: e EF1A2, highly expressed in HCC, is a potential oncogene. Its silencing significantly decreases HCC tumorigenesis, likely by inhibiting PI3K/Akt/NF-κB signaling.     

汉滩病毒诱导乳鼠组织热休克蛋白表达及其与病毒抗原蛋白的相关性

目的 探讨汉滩病毒（HV）感染诱导乳鼠组织热休克蛋白（HSPs)表达及热休克蛋白与病毒蛋白的相互关系。方法 出生后2-3d的昆明乳鼠，每只脑内接种100个半数致死量的HV0.03ml,8d后乳鼠发病死亡，取其脑组织一部分固定，石蜡包埋切片，用免疫组织化学法结合共聚焦显微镜检测组织中病毒抗原及HSP70的表达，一部分组织缺匀浆液，用ELISA，免疫共沉淀方法分析病毒抗原和HSP70的表达及关系。结果 HV感染诱导了乳鼠脑组织神经细胞高表达热休克蛋白（HSP）70。细胞内病毒抗原与HSP70有共定位关系；免疫共沉淀结果显示用HSP70mAb可以共沉淀感染乳鼠脑组织匀浆中的汉滩病毒核心抗原（HV-NP），用HV-NPmAb1A8可沉淀感染乳鼠脑组织匀浆中的HSP70，对照实验阴性，所用两种蛋白抗体间无交叉反应，因而HSP70与HV-NP结合形成复合物。结果是特异的，结论 HV感染诱导HSP70的表达，后者与HV-NP结合形成HSP70-病毒抗原肽复合物。该复合物可能在病毒感染和复制过程中发挥作用。     

Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells

AIM:To study the effects of Pinus massoniana bark extract (PMBE) on cell proliferation and apoptosis of human hepatoma BEL-7402 cells and to elucidate its molecular mechanism. METHODS:BEL-7402 cells were incubated with various concentrations (20-200 ug/mL) of PMBE for different periods of time. After 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by morphological observation, agarose gel electrophoresis, and flow cytometry analysis. Possible molecular mechanisms were primarily explored through immunohistochemical staining. RESULTS:PMBE (20-200 ug/mL) significantly suppressed BEL-7402 cell proliferation in a time-and dose-dependent manner. After treatment of BEL-7402 cells with 160 ug/mL PMBE for 24, 48, or 72 h, a typical apoptotic "DNA ladder"was observed using agarose gel electrophoresis. Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. Sub-G_1 curves were displayed by flow cytometry analysis. PMBE decreased the expression levels of Bcl-2 protein in a time-dependent manner after treatment of cells with 160 ug/mL PMBE. CONCLUSION:PMBE suppresses proliferation of BEL-7402 cells in a time-and dose-dependent manner and induces cell apoptosis by possibly downregulating the expression of the bcl-2 gene.     

Co-expression of heat shock protein 70 and glucose-regulated protein 94 in human gastric carcinoma cell line BGC-823

AIM:To investigate the co-expression and significance of heat shock protein 70 (HSP70) and glucose-regulated protein 94 (grp94) in human gastric carcinoma cell line BGC-823. METHODS: The expression and localization of HSP70 and grp94 in human gastric carcinoma cell line BGC-823 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. Flow cytometry was used to analyze the correlation between expression of HSP70, grp94 and cell cycle in BGC-823 cell line. RESULTS: Gastric cancer cell line BGC-823 expressed high level of HSP70 and grp94. The positive rate of HSP70 and grp94 was 84.9±4.94% and 79.6±5.16%, respectively. Both of them were stained in cell plasma. There was a significant difference compared with control group (1.9±0.94%, P<0.01). During the cell cycle, HSP70 and grp94 were continuously expressed in BGC-823. CONCLUSION: HSP70 and grp94 are highly expressed in human gastric carcinoma BGC-823 cells through the whole cell cycle. There is no relationship between expression of HSP70, grp94 and cell cycle.     

肝癌细胞株中肿瘤特异性抗原MAGE、GAGE、BAGE基因表达的研究

目的 研究MAGE、GAGE、BAGE基因在肝癌细胞株中的表达情况,评价这些肿瘤特异性抗原作为肿瘤分子标记以及肿瘤免疫治疗特异性靶位的可能性。 方法 用RT-PCR检测国内建株的肝癌细胞株SMMC-7721、QQY-7701、BEL-7402中MAGE-1、MAGE-3、GAGE1-8、GAGE1-2和BAGE基因mRNA的表达,以GAPDH基因作为检测内对照,并与非肿瘤肝穿组织比较。 结果 肝癌细胞株SMMC-7721表达MAGE-1和BAGE基因;QQY-7701表达MAGE-3和BAGE基因;BEL-7402表达MAGE-1和GAGE1-2基因;3株肝癌细胞至少表达其中一个基因。肝硬化病人肝穿刺组织中MAGE、GAGE、BAGE基因表达均为阴性。 结论 MAGE、GAGE、B A G E肿瘤特异性抗原可以作为肝癌早期诊断的分子标记,并具有作为肝癌免疫治疗特异性靶位的潜在价值。     

Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

AIM: To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402 xenografted tumors. METHODS: Immunohistochemistry analysis with an anti-endostatin antibody was preformed to detect endostatin protein expression in HCC BEL-7402 cells infected with Ad/hEndo. MTT assay was used to investigate the effects of Ad/hEndo on proliferation of human umbilical vein endothelial cells (HUVEC). Intra-tumoral injections of 1X10(9) pfu Ad/hEndo was given to treat BEL-7402 xenografted tumors in nude mice once weekly for 6 wk. Mice received injections of Ad/LacZ and DMEM were regarded as control groups. After intra-tumoral administration with Ad/hEndo, the endostatin mRNA expression in tumor tissue was analyzed by Northern blotting, and plasma endostatin levels were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: High level expression of endostatin gene was detected in the infected HCC BEL-7402 cells. Ad/hEndo significantly inhibited HUVEC cell proliferation by 57.2% at a multiplicity of infection (MOI) of 20. After 6-week treatment with Ad/hEndo, the growth of treated tumors was inhibited by 46.50% compared to the Ad/LacZ control group (t=2.729, P<0.05) and by 48.56% compared to the DMEM control group (t=2.485, P<0.05). The ratio of mean tumor volume in treated animals to mean tumor volume in the control animals (T:C ratio) was less than 50% after 24 d of treatment. Endostatin mRNA in tumor tissue was clearly demonstrated as a band of approximately 1.2 kb, which was the expected size of intact and functional endostatin. Plasma endostatin levels peaked at 87.52+/-8.34 ng/mL at d 3 after Ad/hEndo injection, which was significantly higher than the basal level (12.23+/-2.54 ng/mL). By d 7, plasma levels dropped to nearly half the peak level (40.34+/-4.80 ng/mL). CONCLUSION: Adenovirus-mediated human endostatin gene can successfully express endogenous endostatin in vitro and in vivo, and significantly inhibit the growth of BEL-7402 xenografted liver tumors in nude mice.     

腺病毒介导的人野生型p53、GM-CSF和B7-1基因转移诱导肝癌细胞的生长抑制和凋亡

目的 观察携带人野生型p53、GM－CSF和B7－1基因的重组腺病毒载体（BB－102）转染BEL－7402、HLE及HuH-7肝癌细胞后p53基因的表达,以及诱导肝癌细胞凋亡,影响肝癌细胞的增殖。方法 BB－102以MOI为50pfu/细胞感染肝癌细胞系BEL－7402（p53基因为野生型）、HLE及HuH-7(p53基因为突变型）。免疫组织化学法检测BB－102携带的p53基因的表达,TdT法原位检测肝癌细胞的凋亡。结果 BB－102携带的p53基因能在转染了BB－102的肝癌细胞中高效表达。转染BB－102后肝癌细胞生长明显受到抑制;染毒后第4－10d期间,BEL－7402、HLE及HuH-7三株肝癌细胞的平均受抑率分别为58．5％、81．5％及71．1％,其中对p53基因突变的肝癌细胞的抑制程度要大于对p53基因为野生型肝癌细胞的抑制程度。转染BB－102还能诱导肝癌细胞的凋亡。结论 BB－102通过其介导p53基因的表达抑制肝癌细胞的增殖,这为BB－102应用于肝癌的基因治疗提供实验依据。     

Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas／FasL and tumor necrosis factor related apoptosis-inducing ligand and its receptor of lymphocytes and liver cancer cells

AIM: To investigate the mechanism ofα-fetoprotein (AFP) in escaping from the host immune surveillance of hepatoc-ellular carcinoma. METHODS: AFP purified from human umbilical blood was administrated into the cultured human lymphoma Jurkat T cell line or hepatoma cell line, Bel7402 in vitro. The expression of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and its receptor (TRAILR) mRNA were analyzed by Northern blot and Western blot was used to detect the expression of Fas and Fas ligand (FasL) protein. RESULTS: AFP (20 mg/L) could promote the expression of FasL and TRAIL, and inhibit the expression of Fas and TRAILR of Bel7402 cells. For Jurkat cell line, AFP could suppress the expression of FasL and TRAIL, and stimulate the expression of Fas and TRAILR. AFP also could synergize with Bel7402 cells to inhibit the expression of FasL protein and TRAIL mRNA in Jurkat cells. The monoclonal antibody against AFP (anti-AFP) could abolish these functions of AFP. CONCLUSION: AFP is able to promote the expression of FasL and TRAIL in hepatoma cells and enhance the expression of Fas and TRAILR in lymphocytes. These could elicit the escape of hepatocellular carcinoma cells from the host's lymphocytes immune surveillance.     

Mitotic cell death in BEL-7402 cells induced by enediyne antibiotic lidamycin is associated with centrosome overduplication

AIM:Mitotic cell death has been focused on in tumor therapy.However,the precise mechanisms underlying it remainunclear.We have reported previously that enediyneantibiotic lidamycin induces mitotic cell death at lowconcentrations in human epithelial tumor cells.The aim ofthis study was to investigate the possible link betweencentrosome dynamics and lidamycin-induced mitotic celldeath in human hepatorna BEL-7402 cells.METHODS:Growth curve was established by MTT assay.Cell multinucleation was detected by staining with Hoechst33342.Flow cytometry was used to analyze cell cycle.Aberrant centrosomes were detected by indirectimmunofluorescence.Western blot and senescence-associated β-galactosidase (SA-β-gal) staining were usedto analyze protein expression and senescence-like phenotype,respectively.RESULTS:Exposure of BEL-7402 cells to a low concentrationof lidamycin resulted in an increase in cells containing multiplecentrosomes in association with the appearance ofmitotic cell death and activation of SA-β-gal in somecells,accompanied by the changes of protein expressionfor the regulation of proliferation and apoptosis.Themitochondrial signaling pathway,one of the major apoptoticpathways,was not activated during mitotic cell death.Theaberrant centrosomes contributed to the multipolar mitoticspindles formation,which might lead to an unbalanceddivision of chromosomes and mitotic cell death characterizedby the manifestation of multi- or micronucleated giant cells.Cell cycle analysis revealed that the lidamycin treatmentprovoked the retardation at G2/M phase,which might beinvolved in the centrosome overduplication.CONCLUSION:Mitotic cell death and senescence canbe induced by treatment of BEL-7402 cells with a lowconcentration of lidamycin.Centrosome dysregulation mayplay a critical role in mitotic failure and ultimate cell deathfollowing exposure to intermediate dose of lidamycin.     

Mitotic cell death in BEL-7402 cells induced by enediyne antibiotic lidamycin is associated with centrosome overduplication

AIM: Mitotic cell death has been focused on in tumor therapy.However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells.METHODS: Growth curve was established by Ml-l assay.Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle.Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescenceassociated β-galactosidase (SA-β-gal) staining were used to analyze protein expression and senescence-like phenotype,respectively.RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-β-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells.Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication.CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.     

抑制HSP70—2基因表达诱导肝癌细胞G2／M期阻滞的机制研究

目的探讨HSP70—2在肝癌组织中的表达，以及抑制HSP70—2表达对肝癌细胞生长和周期影响的详细作用机制。方法免疫组织化学检测HSP70—2在45例肝癌组织和癌旁组织中的表达，Westernblot检测HSP70—2在5种肝癌细胞株中的表达。设计合成针对HSP70—2基因的特异性shRNA，构建真核表达载体HSP70-2shRNA1和HSP70—2shRNA2。转染肝癌细胞后，MTT法检测细胞增殖，流式细胞仪检测细胞周期，Westernblot检测HSP70—2表达及细胞周期相关蛋白p-Cdc2（Tyr15）、Cdc25C、Cdc2和cyclinB1的表达变化。结果免疫组织化学检测显示，45例肝癌组织中有33例（73．3％）HSP70—2蛋白表达阳性，45例癌旁组织中仅4例（8．9％）表达阳性，表达差异有显著性（P〈0．05）。HSP70—2蛋白在HepG2、Bel-402、Huh-7、Hep3B、SMMC-7721肝癌细胞中的表达明显高于其在L02细胞中的表达。HSP70—2shRNA1和shRNA2均能有效抑制肝癌细胞HepG2和Bel-7402中HSP70-2的表达。与controlshRNA组相比，转染HSP70-2shRNA1和shRNA2组HepG2和Bel-7402细胞生长增殖速度均明显减慢，细胞周期表现为处于G1／M期的细胞比例显著增加。Westernblot检测发现，转染HSP70-2shRNA1和shRNA2后肝癌细胞中磷酸化的p-Cdc2（Tyr15）表达增加，Cdc25C、Cdc2和cyclinB1表达显著减少。结论HSPT0-2在肝癌中过表达，抑制HSP70．2表达可以显著抑制肝癌细胞的增殖，诱导细胞周期阻滞，其诱导G，／M期阻滞的机制是通过影响Cdc25C-Cdc2／cyclinB1通路来实现的。     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

Effect of arsenic trioxide on human hepatoma cell line BEL-7402 cultured in vitro

AIM:To study the effect of a varying concentrations of arsenic trioxide on human hepatoma cell line BEL-7402 cultured in vitro and its mechanism of action.METHODS:The BEL-7402 cells were treated with arsenic trioxide (at the concentrations of 0.5 1 2&mgr;mol/L, respectively) for 4 successive days. The cell growth and proliferation were observed by cell counting and cell-growth curve. Morphologic changes were studied with electronmicroscopy. Flow cytometry was used to assay cell-DNA distribution and the protein expression of Bcl-2 and Bax detected by immunocytochemical method.RESULTS:The cell growth was significantly inhibited by varying concentrations of arsenic trioxide as revealed by cell counting and cell-growth curve, which was dose- and time-dependent. Arsenic trioxide treatment at 0.5, 1 and 2&mgr;mol/L resulted in a sub-G1 cell peak, the apoptosis rate of the control group was 9.31% and that of 0.5&mgr;mol/L arsenic trioxide 15.53%, no significant difference was seen between the two.The apoptosis rates of 1,2&mgr;mol/L arsenic trioxide were 19.10% and 21.87% respectively, which were much higher (both P < 0.05). Decrease of G(0)/G(1) phase cells and increase of S phase cells were observed by flow cytometry, suggesting the inhibition effect of 0.5, 1, 2&mgr;mol/L arsenic trioxide on BEL-7402 cell lay in the G(0)/G(1) phase. Morphologic changes such as intact cell membrane, nucleic condensation, apoptotic body formation were seen under transmission electronmicrescopy, whereas the 0.5mol/L arsenic trioxide-treated BEL-7402 cells showed decrease of nucleocytoplasmic ratio, round nucleus, well-differentiated organelles in the cytoplasm. The processes and microvilli on the cell surface of the experimental groups under scanning electron microscopy were significantly decreased. High expressions of Bcl-2 and Bax were detected in 1 and 2&mgr;mol/L arsenic trioxide-treated cells, these were 46%, 87.33% and 83.08%, 95.83% respectively, among which that of Bax was more significant. Arsenic trioxide treatment at 0.5&mgr;mol/L resulted in a higher expression level of Bcl-2 and lower expression level of Bax,which were 8.81% and 3.83% respectively, as compared with that of the control group (15.33%) (P(1)<0.01, P(2)<0.01).CONCLUSION:Arsenic trioxide not only inhibited proliferation but also induced apoptosis of human hepatoma cell line BEL-7402. The induced-apoptosis effect of 1,2&mgr;mol/L arsenic trioxide was related to the expression level of Bcl-2 and Bax.