Transfection of p27^kip1 enhances radiosensitivity induced by ^60Co γ-irradiation in hepatocellular carcinoma HepG2 cell line

AIM: To study the cell cycle alterations of human hepatoma cell line HepG2 in vitro after ^60Co γ-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to γ-irradiation in HepG2 transiently transfected with wild type p27^kip1. METHODS: The proliferation of HepG2 cells was evaluated with MTT assay, and the cell cycle profile and apoptosis were assessed by cell morphology, DNA fragmentation analysis and flow cytometry. HepG2 cells were transfectedwith p27^kip1 wild type by using Lipofectamine (LF2000), and the expression and subcellular localization of p27^kip1 in HepG2 were detected by immunocytochemistry.RESULTS: ^60Co γ-irradiation inhibited the growth of HepG2 cells in a dose-dependent manner. Apoptosis of HepG2 cells was induced 48 h after ~, ray exposure. Furthermore research was carried out to induce exogenous expression of p27^kip1 in HepG2. The expression of p27^kip1 induced G0/G1 phase arrest in HepG2 cells. The overexpression of p27^kip1 enhanced ^60Co γ-irradiation-induced radiosensitivity in HepG2 cells. CONCLUSION: Overexpression of p27^kip1 is a rational approach to improve conventional radiotherapy outcomes, which may be a possible strategy for human hepatoma therapy.     

胰岛素对人肝细胞L02水通道蛋白9表达的影响及其调控通路

Objective To explore the effects of insulin on the expression and the regulatory pathway of AQP9 in normal human liver cells. Methods Normal human liver cells L02 were cultured and treated with PI3K inhibitor LY294002, AKT inhibitor A-443654, MAPK inhibitors SB2030580 and insulin at different concentrations respectively. The AQP9 mRNA and protein expressions were detected with semi-quantitative RT-PCR andWestern blot respectively. Results The insulin (100nmol/L～500nmol/L) treatment decreased the expression of AQP9 in normal human liver cells (P < 0.05) concentration dependency, and the expression of AQP9 began to reduce from 3 hours of insulin stimulation (P < 0.05), especially at insulin treatment for 12 hours (P < 0.05); Incubated with the selective inhibitor of PI3K(LY294002) and AKT(A-443654), the inhibitory effects of insulin on AQP9 expression decreased (P < 0.05); but it did not change significantly by blocking the MAPK signaling pathway. Conclusion The insulin treatment inhibited the expression of AQP9 and the PI3K/akt signal transduction pathwsy was involved in the mechanism.     

胰岛素对人肝细胞L02水通道蛋白9表达的影响及其调控通路

Objective To explore the effects of insulin on the expression and the regulatory pathway of AQP9 in normal human liver cells. Methods Normal human liver cells L02 were cultured and treated with PI3K inhibitor LY294002, AKT inhibitor A-443654, MAPK inhibitors SB2030580 and insulin at different concentrations respectively. The AQP9 mRNA and protein expressions were detected with semi-quantitative RT-PCR andWestern blot respectively. Results The insulin (100nmol/L～500nmol/L) treatment decreased the expression of AQP9 in normal human liver cells (P < 0.05) concentration dependency, and the expression of AQP9 began to reduce from 3 hours of insulin stimulation (P < 0.05), especially at insulin treatment for 12 hours (P < 0.05); Incubated with the selective inhibitor of PI3K(LY294002) and AKT(A-443654), the inhibitory effects of insulin on AQP9 expression decreased (P < 0.05); but it did not change significantly by blocking the MAPK signaling pathway. Conclusion The insulin treatment inhibited the expression of AQP9 and the PI3K/akt signal transduction pathwsy was involved in the mechanism.     

Involvement of fatty acid-CoA ligase 4 in hepatocellular carcinoma growth： Roles of cyclic AMP and p38 mitogen-activated protein kinase

AIM: Fatty acid-CoA ligase 4 (FACL4) is an arachidonate-preferring enzyme which has been shown to be up-regulated in human colon cancer tissues and implicated in the colon tumorigenesis. The purpose of this study was to investigate the role of FACL4 in the human hepatocellular carcinoma (HCC) tumorigenesis and the specific signal pathways involved in this process. METHODS: We investigated the expression and regulation of FACL4 in HCC, adjacent non-tumorous liver tissues, and cell lines. RESULTS: In HCC patients, we demonstrated that FACL4 gene expression was markedly elevated in the cancerous tissues than in the adjacent non-cancerous liver tissues. In addition, several human hepatoma cell lines, including Hep3B and HepG2, expressed high levels of FACL4. Stable overex-pression of FACL4 knockdown plasmids (small interfering RNA, siRNA) to Hep3B cells significantly decreased FACL4 expression and subsequently limited the cell proliferation. Treatment of Hep3B cells with 8-bromo-cAMP and SB203508 (p38 MAPK inhibitor) significantly suppressed the FACL4 expression. CONCLUSION: FACL4 is involved in the HCC tumorigenesis and both cAMP and p38 MAPK pathways are associated with the regulation of FACL4 in HCC.     

Transfection of apoptosis related gene Fas ligand in human hepatocellular carcinoma cells and its significance in apoptosis

AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immuohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope. RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining. CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.     

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway

AIM： To investigate the signaling pathways implicated in phosphatidylethanolamine （PE）-induced apoptosis of human hepatoma HepG2 cells. METHODS： Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Cell cycle, apoptosis and mitochondrial transmembrane potential （△ψm） were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS： PE inhibited the growth of HepG2 cells in a dose- and time- dependent manner. It did not affect the cell cycle, but induced apoptosis. PE significantly decreased △ψm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a dose- and time-dependent manner. CONCLUSION： Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.     

PI3K/Akt/mTOR信号通路对大鼠肾小球系膜细胞增殖的调控作用探讨

目的:探讨磷脂酰肌醇-3(PI3K)/蛋白激酶(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对大鼠肾小球系膜细胞(MC)增殖的调控作用。方法:体外培养大鼠MC,随机分为正常对照组、表皮生长因子(EGF)(10ng/m L)组、PI3K抑制剂LY294002组(2μg/m L)、EGF联合LY294002组。采用四甲基偶氮唑蓝(MTT)法检测4组MC的增殖,流式细胞术检测MC周期,免疫荧光法检测系膜细胞m TOR的表达。结果:与正常对照组相比,EGF组能显著促进大鼠MC增殖,G0/G1期细胞减少,S期及G2/M期细胞增加,且m TOR表达增加;LY294002组大鼠MC增殖受抑制,G0/G1期细胞增加,S期及G2/M期细胞减少,且m TOR表达降低;与EGF组比较,EGF联合LY294002干预组MC增殖受抑制,G0/G1期细胞增多,S期及G2/M期细胞减少,且m TOR表达降低。结论:m TOR信号通路参与了大鼠肾小球MC增殖的调控,抑制该信号通路的活化可显著抑制MC的增殖和MC周期的进程。     

巨噬细胞游走抑制因子通过PI3K／Akt信号通路调控人结肠癌细胞株HT-29增殖和血管生成因子表达

背景：巨噬细胞游走抑制因子（MIF）是-种多功能细胞因子,其在结直肠癌发生、发展中的作用机制尚不明确。目的：探讨MIF是否通过P13K／Akt信号通路调控人结肠癌细胞增殖和血管生成因子表达。方法：人结肠癌细胞株HT-29分为对照组（RPMI-1640）、重组人MIF（rhMIF）组、PI3K抑制剂LY294002组和LY294002预处理联合rhMIF组,并予相应干预。以甲基噻唑基四唑（MTT）法检测HT-29细胞增殖状态,分别以逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检测血管内皮生长因子（VEGF）、白细胞介素-8（IL-8）mRNA表达和蛋白分泌,蛋白质印迹法检测Akt和磷酸化Akt（p—Akt）蛋白表达。结果：rhMIF对HT-29细胞具有促增殖作用,能增加细胞VEGF、IL-8 mRNA表达和蛋白分泌,并促进Akt蛋白磷酸化（P＜0．05）;而先予LY294002预处理可显著抑制rhMIF的上述作用．HT-29细胞增殖显著受抑（P＜0．05）,VEGF、IL-8mRNA表达、蛋白分泌以及p-Akt蛋白水平与对照组相比无明显差异。各组总Akt蛋白水平均无明显差异。结论：MIF诱导人结肠癌细胞增殖和血管生成因子表达可能是通过活化PI3K／Akt信号通路实现的。     

The role of XPB in cell apoptosis and viability and its relationship with p53, p21 and c-myc in hepatoma cells

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.