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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)-p53(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 signif icant changes in HepG2 cells infected with LV-EGFP.Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4(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 signif icantly 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)-p53(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.
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 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) -p53 (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 (Si) -p53 (N15) -Ant and were 33.9% and 95.8% respectively after 72 h infection with LV-NT4 (Si) -p53 (N15) -Ant TEM showed morphological changes in HepG2 cells infected with LV-NT4 (Si) -p53 (N15) -Ant, but no signif icant changes in HepG2 cells infected with LV-EGFP.Changes were observed in ultra-structure of HepG2 cells infected with LV -NT4 (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 ) -Ant and LV-EGFP, which showed that LDH release was signif icantly higher in LV-NT4 (Si) -p53 (N15) -Antreatment 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) -p53 (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 specifically inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight.