HERG K^＋通道对VEGF诱导的肝癌细胞迁移和侵袭的影响

目的研究HERG K＋通道对VEGF诱导的肝癌细胞在侵袭和迁移方面的调节作用。方法运用膜片钳技术分别检测正常肝细胞系L-02和肝癌细胞系SMMC-7721中HERG K＋通道的表达情况;采用Bodyen-Chamber系统检测在HERG K＋通道特异性抑制剂E-4031作用后,对VEGF诱导的SMMC-7721细胞侵袭力和迁移潜能方面的影响;ELISA法检测E-4031处理SMMC-7721细胞后,培养基上清中的VEGF水平的变化。结果 HERG K＋通道在SMMC-7721细胞中表达,而在L-02细胞中不表达;且VEGP诱导的SMMC-7721细胞侵袭和迁移现象可被E-4031呈剂量依赖性地抑制;在阻断HERG K＋通道后上清中VEGF水平明显降低。结论肝癌细胞中存在HERG K＋通道,它可通过调控VEGF分泌水平来影响肝癌细胞的侵袭和迁移能力。由此,HERG K＋通道将有可能成为诊断肝癌和判断预后的新标志物及治疗的新靶位。     

干扰素-α对人肝癌细胞增殖及侵袭的抑制作用

目的:研究人干扰素-α对人肝癌细胞系BEL-7402细胞增殖及侵袭的抑制作用.方法:常规培养的BEL-7402细胞加入人干扰素-α(1×106U/L)共同孵育24 h后,以MTT法检测干扰素-α对肿瘤细胞增殖的影响;Boyden小室法检测肝癌细胞侵袭能力的变化;明胶酶谱法检测肝癌细胞基质金属蛋白酶的变化.免疫组化法检测肿瘤细胞增殖核抗原的表达;TUNEL法检测肿瘤细胞的凋亡.结果:人干扰素-α可以明显抑制体外BEL-7402细胞的增殖(抑制率为20.2%);干扰素-α作用后在Boyden小室穿膜的肿瘤细胞数明显减少(抑制率为23.9%);肝癌细胞基质金属蛋白酶的分泌没有明显变化.干扰素-α组肿瘤细胞增殖核抗原的表达明显下降,凋亡细胞数明显增加.结论:低剂量的干扰素-α可以直接抑制肝癌细胞株BEL-7402的增殖,对BEL-7402细胞的侵袭有明显的抑制作用;可以抑制肿瘤细胞增殖核抗原的表达并诱导细胞凋亡.     

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。     

肝癌干细胞逃避5-氟尿嘧啶单药杀伤可能是造成化疗失败的重要原因

目的通过观察5-氟尿嘧啶(5-FU)处理人肝癌细胞系BEL-7402和SMMC-7721,分析细胞生物学特性的变化。方法采用Cell Counting Kit-8法检测5-FU作用前后肝癌细胞系BEL-7402和SMMC-7721增殖能力的变化;采用流式细胞法检测细胞周期组成变化和细胞群体中肿瘤干细胞标志物EpCAM、ABCG2和ICAM-1阳性细胞的比例变化。结果 10μg.ml-15-FU作用48h后,BEL-7402和SMMC-7721细胞增殖均明显受抑制(P<0.05);两细胞系实验组静息期(G0/G1期)细胞比例较对照组均明显升高(P<0.05);两细胞系中肿瘤干细胞标志物阳性细胞比例均明显升高(P<0.05)。结论肝癌细胞系BEL-7402及SMMC-7721中的肿瘤干细胞能够逃避一定剂量5-FU的杀伤作用。肝癌干细胞逃避5-FU单药杀伤可能是造成临床化疗失败的重要原因。     

蟾毒灵对人肝癌细胞增殖与侵袭的影响

目的:研究蟾毒灵(bufalin,Bu)对肝癌BEL-7402细胞生长、迁移侵袭的作用.方法:体外培养人肝癌BEL-7402细胞,应用CCK-8比色法、流式细胞仪、Transwell小室迁移侵袭实验观测不同浓度和作用时间Bu对肝癌细胞增殖、迁移、侵袭的影响.结果:Bu明显抑制肝癌BEL-7402细胞生长和增殖,呈浓度-时间效应关系;0.085μg/mL Bu分别作用于肝癌BEL-7402细胞48、72 h,肝癌细胞周期阻滞于G2/S期[48 h(G2:t=-6.618,P<0.01;S:t=-5.339,P<0.01),72 h(G2:t=-14.273,P<0.01;S:t=-4.812,P<0.01)];0.085μg/mL Bu作用肝癌BEL-7402细胞72 h,显著抑制肝癌细胞迁移(t=11.717,P<0.01)和侵袭(t=5.437,P<0.01).结论:Bu能抑制肝癌细胞迁移、侵袭,并且通过阻滞肝癌细胞周期于G2/S期,抑制肝癌细胞增殖,其生长抑制作用呈时间和剂量依赖效应.     

herg1基因调控慢性髓细胞白血病细胞的增殖和细胞周期

目的:研究herg1基因在慢性髓细胞白血病(CML)细胞的表达及其对CML细胞增殖和细胞周期的调控作用.方法:应用RT-PCR方法测定33例初治慢性髓细胞白血病患者和10例健康志愿者的骨髓细胞herg1基因的表达,检测特异性抑制剂处理后CML细胞株K562增殖凋亡和细胞周期的变化.结果:①在初发CML病例中,herg1 mRNA的表达率是72.7% (24/33),高于正常对照(20%)(P<0.01).CML加速期和急变期的herg1 mRNA的表达率分别为75%和90.9%,高于正常对照(P<0.01),但是herg1的表达与CML的临床分期并无相关性(P>0.05).②E-4031(1 μmol/L)可呈时间依赖性抑制细胞增殖,不诱导明显凋亡.③E-4031阻滞K562细胞周期于G1期.结论:CML细胞herg1基因表达失调,IHERG抑制剂E-4031对K562细胞的增殖和细胞周期具有调控作用,herg1基因可能与CML发病机制相关.     

hergl基因调控慢性髓细胞白血病细胞的增殖和细胞周期

目的：研究hergl基因在慢性髓细胞白血病（CML）细胞的表达及其对CML细胞增殖和细胞周期的调控作用。方法：应用RT-PCR方法测定33例初治慢性髓细胞白血病患者和10例健康志愿者的骨髓细胞hergl基因的表达，检测特异性抑制剂处理后CML细胞株K562增殖凋亡和细胞周期的变化。结果：①在初发CML病例中，hergl mRNA的表达率是72．7％（24／33），高于正常对照（20％）（P〈0．01）。CML加速期和急变期的hergl mRNA的表达率分别为75％和90．9％，高于正常对照（P〈O．01），但是hergl的表达与CML的临床分期并无相关性（P〉0．05）。②E-4031（1μmol／L）可呈时间依赖性抑制细胞增殖，不诱导明显凋亡。③E-4031阻滞K562细胞周期于G1期。结论：CML细胞hergl基因表达失调IHERG抑制剂E-4031对K562细胞的增殖和细胞周期具有调控作用，hergl基因可能与CML发病机制相关。     

甘露糖受体在肝细胞癌中的表达及意义

目的:观察甘露糖受体(mannose receptor,M R)在肝细胞癌组织/肝癌细胞系中的表达,探讨其与肝细胞癌发生、发展及恶性程度的关系.方法:应用免疫组织化学检测50例肝细胞癌组织及癌旁组织、10例正常肝组织中MR的表达,免疫荧光法、Western blot法检测肝癌细胞系、肝细胞系中MR的表达.结果:免疫组织化学染色:肝细胞癌组织中M R的表达水平明显高于癌旁组织及正常肝组织(86%vs 76%,30%,P0.05).免疫荧光:MR在肝癌细胞系BEL-7402、Hep G2和人肝细胞系HL-7702中均有表达.在肝癌细胞系BEL-7402、Hep G2上有很强的MR表达,在肝细胞系HL-7702上MR的表达分布明显较少.Western blot法:MR在肝癌细胞系Hep G2、BEL-7402和人肝细胞系HL-7702上均有表达,肝癌细胞BEL-7402中MR的表达水平明显高于肝癌细胞Hep G2(P0.01)、肝细胞HL-7702(P0.01).结论:MR在肝细胞癌组织/肝癌细胞系中高表达提示可能与肝细胞癌的发生、发展及恶性程度密切相关.     

土槿皮乙酸对BEL-7402细胞凋亡、线粒体膜电位及COX-2蛋白表达的影响

目的:探讨土槿皮乙酸(PAB)对人肝癌细胞株BEL-7402增殖和凋亡的影响及其影响机制.方法:体外培养肝癌BEL-7402级胞,用不同浓度PAB作用不同时间后,MTT比色法检测PAB对细胞增殖的影响;流式细胞仪分析PAB对细胞周期和凋亡的影响:吖啶橙染色荧光显微镜下观察PAB引起的肝癌细胞株BEL-7402形态学变化;JC-1检测线粒体膜电位的变化:Western blot方法检测PAB对COX-2表达的影响.结果:PAB作用细胞24,48,72h,细胞增殖受到抑制,且表现为剂量依赖性和时间依赖性;PAB使肝癌细胞细胞周期阻滞于G2/M期;与对照组相比,2.O,4.0 μmol/L PAB作用24h后,细胞凋亡率明显增加,差异显著(19.06%±3.87%,31.19%±1.46% vs 0.10%±0.08%,P<0.05);吖啶橙染色发现1.0μmol/L PAB作用24h,细胞内即有凋亡小体出现:与对照组相比,0.5μmol/L PAB作用24h即可降低BEL-7402线粒体膜电位(P<0.05);不同浓度PAB作用24h后,随PAB浓度增加COX-2蛋白表达递减.结论:PAB通过降低BEL-7402线粒体膜电位、减少COX-2的表达抑制细胞生长,诱导细胞凋亡.     

siRNA沉默Cyclin E基因对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响

目的:观察siRNA沉默Cyclin E基因表达对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响.方法:构建2个靶向Cyclin E基因siRNA载体,转染人肝癌HepG2、SMMC-7721和BEL-7402细胞.RT-PCR、Western blot检测转染后HepG2、SMMC-7721和BEL-7402细胞Cyclin E基因mRNA和蛋白表达水平.CCK-8试验、软琼脂克隆形成实验检测HepG2、SMMC-7721和BEL-7402细胞增殖、克隆形成能力.流式细胞术、transwell试验分别检测HepG2、SMMC-7721和BEL-7402细胞周期和侵袭能力.结果:构建的2个Cyclin E基因siRNA载体插入序列与所设计序列均一致;转染HepG2、SMMC-7721和BEL-7402细胞后,干扰1组、干扰2组与空白对照组和阴性对照组比较,C y c l i n E m R N A和蛋白表达量均显著降低(P<0.05),细胞生长速度延缓,软琼脂细胞集落形成数、穿透细胞数均显著降低(P<0.05),S和G2/M期细胞比例减少,G0/G1期细胞比例增加.结论:沉默肝癌细胞Cyclin E表达水平,可有效抑制细胞生长、增殖和侵袭能力.     

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.     

人肝癌细胞系Slit/Robo启动子甲基化状态及基因表达的研究

目的 研究Slit/Robo信号通路相关基因Slit1、Slit2、Slit3和Robo1、Robo3在多种人肝癌细胞系中的表达及甲基化状态,探讨与肝癌发生和发展的关系. 方法提取9种人肝细胞癌细胞株(Hep3B、HepG2、PLC/PRF/5/PRF/5、SMMC-7721、BEL-7402、MHCC97-H,MHCC97-L、LM3、LM6)及对照细胞株L02的基因组DNA和总RNA,采用半定量逆转录聚合酶链反应技术和甲基化特异性聚合酶链反应技术检测Slit1、Slit2,Slit3和Robo1,Robo3基因的基因表达水平与启动子甲基化状态.实验数据应用Paired t检验. 结果 Slit1、Slit2、Slit3基因除个别细胞株外,在不同转移潜能细胞株中均发生了DNA甲基化,同时Slit1和Slit3在mRNA水平几乎均不表达,Slit2基因表达程度在不同转移潜能的细胞株之间存在差异,随着转移潜能的增加表达大致呈下降趋势.作为Slit2受体的Robo1基因在10株肝癌细胞株中均发生甲基化修饰,但除在SMMC-7721、BEL-7402、L02不表达外,其余7种细胞株均有表达.Robo3基因相关CpG岛在9种肝癌细胞株中均未发生甲基化,同时其在mRNA水平均无表达. 结论 Slit/Robo可能在肝癌发生和发展中发挥作用.而Robo3则在肝癌中不发生表达而且其表达沉默可能不受甲基化方式调控.     

Dissociation of E-4031 from the HERG channel caused by mutations of an amino acid results in greater block at high stimulation frequency

OBJECTIVE: We have reported identification of the amino acid whose mutation reduces effects of quinidine on the HERG channel. Although the residue (isoleucine at 647) is not in the recently reported methanesulfonanilide binding site, a single concentration of E-4031 (10 microM) was less effective to I647 mutant channels than wild type HERG channel. We designed the present experiment to further investigate influence of mutations at 647 on the effects of methanesulfonanilides. METHODS AND RESULTS: HERG channels were expressed in Xenopus oocytes and their currents were measured by a two-microelectrode voltage clamp method. Of the two mutations initially studied (I647A and I647F), the I647F had a greater influence and differentially affected the effects of dofetilide and E-4031. The IC(50) for dofetilide of the two mutant channels (I647A and I647F) was increased only 2-fold, but the IC(50) for E-4031 was increased 6-fold (I647A) and 14-fold (I647F). Aromatic residues other than phenylalanine were then substituted for I647, and found to reduce the effects of E-4031. Whereas E-4031 dissociated from the mutant channels during rested state, dofetilide little dissociated. The mutant channels that showed recovery from E-4031 block were inhibited greater at 1 Hz than at 0.1 Hz. CONCLUSIONS: The present results indicate that dissociation of a drug from the HERG channel results in greater block at high frequency. Although the mechanism by which the mutations cause the dissociation of E-4031 is uncertain, it is noteworthy that one methanesulfonanilide dissociates from the channel more easily than another.     

Heat shock protein 70 chaperoned alpha-fetoprotein in human hepatocellular carcinoma cell line BEL-7402

AIM: To investigate the interaction between heat shock protein 70 (HSP70) and a-fetoprotein (AFP) in human hepatocellular carcinoma (HCC) cell line BEL-7402. METHODS: The expression and localization of HSP70 and AFP in human HCC cell line BEL-7402 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. The interaction between HSP70 and AFP in HCC cells was analyzed by immunoprecipitation and Western blot. RESULTS: Immunocytochemical staining detection showed that HCC cell BEL-7402 expressed a high level of HSP70 and AFP synchronously. Both were stained in cell plasma. AFP existed in the immunoprecipitate of anti-HSP70 mAb, while there was HSP70 in the immunoprecipitate of anti-AFP mAb. CONCLUSION: HSP70 chaperones AFP in human HCC cell BEL-7402. The interaction between HSP70 and AFP in human HCC cell can be a new route to study the pathogenesis and immunotherapy of HCC.     

Role of hERG1 K(+) channels in leukemia cells as a positive regulator in SDF-1a-induced proliferation

Previous work from our laboratory has confirmed that human ether-à-go-go-related gene 1 (hERG1) K(+) channels are constitutively expressed in leukemia cells and enhanced cell proliferation. More importantly, it has shown that stromal cell-derived factor-1a (SDF-1a) significantly increases hERG1 K(+) tail current and a specific hERG1 K(+) channels inhibitor significantly blocks SDF-1a-induced migration of leukemic cells. In this study, we investigated a possible regulatory effect of hERG1 K(+) channels upon SDF-1a-mediated cell proliferation as a mean to uncover new molecular events involved in bone marrow microenvironment and leukemogenesis. RT-PCR showed that SDF-1a enhanced hERG1 expression in a dose-dependent manner. Cell proliferation assay illustrated that SDF-1a promoted cell proliferation in a dose-dependent manner, whereas this effect was impaired by E-4031. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G(0)/G(1) arrest. Interestingly, E-4031 promoted SDF-1a-induced apoptosis in HL-60 and leukemic blasts, which markedly impaired the protection effect of SDF-1a in AML. Moreover, SDF-1a increased the expression of Wnt/beta-catenin target genes, including beta-catenin, cyclin-D1, and c-myc; however, this manner was abolished by blockage with the hERG1 K(+) channels. Taken together, our results provide evidence of a novel mechanism involved in the proliferative effects of SDF-1a and highlight hERG1 K(+) channels as a therapeutic target for leukemia treatment and prevention.     

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。     

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.     

Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma

AIM： To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene （LV-shMAT2B） on hepatocelluar carcinoma （HCC） cells. METHODS： We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine （SAMe） in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS： We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 （P = 0.054） and HepG2 （P = 0.031）. Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA （P = 0.047）, but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION： LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.