| Primary hepatic carcinoma(PHC) is considered as one of the most common cancer in the world.And it has been ranked as the second cause of cancer mortality in China.The potentially curative therapies of late stage hepatocellular carcinoma (HCC) are low effective,so finding active and reasonable treatments is significant.Sorafenib is a multikinase inhibitor that has shown to block tumor cell proliferation and angiogenesis by inhibiting serine/threonine kinases,as well as the receptor tyrosine kinases vascular endothelial growth factor receptor 2(VEGFR2), vascular endothelial growth factor receptor 3(VEGFR3),platelet-derived growth factor receptor(PDGFR),FLT3 and C-KIT.In 2007 ASCO manual meeting,a phaseⅢrandomized placebo-controlled trial(SHARP trial) was reported.It confirmed that Sorafenib improved 44%of HCC patients' survival time than the placebo,MST was 10.7 months and TTP was 5.5 months in the Sorafenib team.At the end of 2007, FDA approved sorafenib for patients with inoperable liver cancer.Arsenic trioxide (As2O3) has shown remarkable curative effect in the treatment of acute promyelocytic Leukemia(APL).Studies have showed that As2O3 could inhibition of HCC in vitro and in vivo.As Sorafenib and As2O3 has different mechanism of inducing apoptosis and inhibiting proliferation on tumor cell,it is possible that the two drugs can treat HCC through different ways and targets.This study was designed to investigate that the nature of sorafenib combined with As2O3 on hepatocellular carcinoma cells.The first part:Inhibitory proliferation effect of Sorafenib combined with Arsenic trioxide on hepatocellular carcinoma cellsMethods:1.HepG2 human HCC tumor cells were cultured with RPMI 1640 containing 10%fetal calf serum.Cells in exponentially growing period were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group,As2O3-treated group and combination treatment group.MTT assay analyzed cells treated with Sorafenib(1.5,3,6μmol/L) alone,As2O3(2,4μmol/L) alone,Sorafenib combined As2O3 and cells of control group for 24, 48 and 72 hours respectively.The inhibitory rates of cells were calculated according OD value.2.Statistical analysis:All datas were analysised by SPSS 13.0 statistical software. Datas were expressed as(?)±s.The inhibitory rates were analyzed by One-way ANOVA followed by post hoc test.P＜0.05 was considered to be significant.Results:1.The MTT result indicated:Sorafenib,As2O3 alone or together could inhibit the proliferation of HepG2 cells(P＜0.05).Sorafenib combined with As2O3 enhanced inhibition of proliferation in HepG2 cells(P＜0.05).2.There was a synergistic effect in Sorafenib combined with As2O3 at 24h,48h,72h on HepG2 cells(P＜0.05).Conclusion:Sorafenib,As2O3 alone or together could inhibit the proliferation of HepG2 cells, the combination of Sorafenib and As2O3 showed stronger inhibition.The nature of the interaction of combination was synergistic.The second part:Induction apoptosis effect of Sorafenib combined with Arsenic trioxide on hepatocellular carcinoma cellsMethods:1.HepG2 human HCC tumor cells were cultured as the above mentioned.Cells of exponentially growing period were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group(Sorafenib 3μmol/L), As2O3-treated group(As2O3 4μmol/L) and combination treatment group(Sorafenib 3μmol/L+As2O3 4μmol/L).2.Group as the above mentioned,the apoptosis rate of HepG2 cells labeled by Annexin V-FITC/PI was observed by FCM.3.Group as the above mentioned,cells collected and that changes of mitochondrial membrane potential(Δψm) labeled by Rhodamine123 was examined by FCM.4.Group as the above mentioned,the activity of caspase-3 were examined by colorimetric.5.Statistical analysis:All datas were analysised by SPSS 13.0 statistical software. Datas were expressed as(?)±s.The data were analyzed by One-way ANOVA followed by post hoc test.P＜0.05 was considered to be significant.Results:1.Compared with untreated control,Sorafenib,As2O3 alone and together induced more HepG2 cells apoptosis(P＜0.05).Induction of cell apoptosis was enhanced by combinaton of Sorafenib and As2O3 compared with each agent alone.2.The mitochondrial membrane potential(Δψm) was decreased by As2O3,sorafenib alone or combination.The effect of the agents combined was stronger than that alone (P＜0.05).3.The activity of the caspase-3 increased in the three treated groups.At the same time,the combination group increased more significantly than the Sorafenib group, As2O3 group alone respectively.Conclusion:Sorafenib combined with As2O3 showed synergistic effect on induction apoptosis of HepG2 cells.The mechanisms were closely related to the promotion of apoptosis by influence several signaling pathways such as the mitochondria-dependent and caspase- dependent ways. |
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