首页|NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line
NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line
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AIM: To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53(N15)-antennapedia (Ant), and study its effect on HepG2 cells.METHODS: Plasmid pLenti6/V5-NT4 p53(N15)-Ant was constructed incorporating the following functional regions, including signal peptide sequence and pro-region of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma (HepG2) cells were used for transfection. 3-[4,5-dimethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, transmission electron microscopy (TEM) and flow cytometric analysis (FCM) were employed to investigate the effects of LV-NT4(Si)-p53(N15)-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4(Si)-p53(N15)-Ant on tumor growth in nude mice.RESULTS: LV-NT4(Si)-p54(N15)-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4(Si)-p53(N15)-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4(Si)-p53(N15)-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4(Si)-p53(N15)-Ant (P < 0.01). Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant, but no significant changes in HepG2 cells infected with LV-EGFP. Changes were observed in ultra-structure of HepG2 cells infected with LV-IMT4(Si)-p53(N15)-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4(Si)-p53(N15)-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4(Si)-p53(N15)-Ant treatment group (682 IU/L) than in control group (45 IU/L, P < 0.01). The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4(Si)-p5J(N15)-Ant could induce two different kinds of cell death: necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4(Si)-p53(N15)-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4(Si)-p53(N15)-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight.CONCLUSION: LV-NT4(Si)-p53(N15)-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.
gene therapylentivirus vectoranticancernecrosisLV-NT4(Si)-p53(N15)-Anthepatocellular carcinoma cell line
Li-Ping Song、Yue-Ping Li、Ning Wang、Wei-Wei Li、Juan Ren、Shu-Dong Qiu、Quan-Ying Wang、Guang-Xiao Yang
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Department of Radiotherapy Oncology, First Hospital, Medical School of Xi'an Jiao Tong University, Xi'an 710061, Shaanxi Province, China
Center for Reproductive Medicine, Affiliated Hospital of Hainan Medical College, Haikou 570102, Hainan Province, China
Department of Anatomy and Histology & Embryology, Medical school of Xi'an Jiao Tong University, Xi'an 710061, Shaanxi Province, China
Xi'an Huaguang Bioengineering Co, Xi'an 710061, Shaanxi Province, China
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