| ObjectiveTo investigate the effects of 3-bromopyruvate(3-Br PA)combined with sorafenib(SOR)on the proliferation and apoptosis of human hepatocellular carcinoma cells in vitro and in vivo,and its related molecular mechanism.Methods1.MTT assay was used to detect the viability of hepatocellular carcinoma Hep G2 and SMMC7721 cells after exposed to different concentrations of 3-Br PA(0,20,40,80,160?mol/L),SOR(0,2,4,8,16?mol/L),and 100 ? mol/L 3-Br PA combined with SOR(0,2,4,8,16?mol/L).Then the experiment was divided into four groups:control group,3-Br PA group,SOR group,3-Br PA+SOR group.2.The growth inhibition induced by 3-Br PA combined with SOR in Hep G2 and SMMC7721 cell was analyzed using Cell colony formation assay.3.DAPI staining was performed to demonstrate the cell nucleus changes.4.The apoptosis rate of cells was assessed using flow cytometry with PI staining.5.Changes of mitochondrial membrane potential in hepatocellular carcinoma Hep G2 and SMMC7721 cells treated with 3-Br PA combined with SOR for 24 h were analyzed using JC-1 kit.6.The intracellular ROS levels in hepatocellular carcinoma Hep G2 and SMMC7721 cells treated with 3-Br PA combined with SOR for 6h was measured by ROS kit.7.The intracellular ATP levels in hepatocellular carcinoma Hep G2 and SMMC7721 cells treated with 3-Br PA combined with SOR for 5h was assessed by the ATP Assay Kit.8.The expression of Mcl-1,PARP,Bcl-2 and Bax in hepatocellular carcinoma Hep G2 and SMMC7721 cells was analyzed using Western blot.9.The anti-tumor effects of 3-Br PA combined with SOR in vivo was explored in nude mice xenograft models.Results1.3-Br PA enhanced proliferation inhibition of sorafenib in hepatocellular carcinoma SMMC7721 and Hep G2 cells 1.1 The cell viability of Hep G2 and SMMC7721 cells decreased in a concentration dependent manner after treated with 3-Br PA,SOR,and 100? mol/L 3-Br PA combined with SOR(P<0.05).The cell survival rate of 100? mol/L 3-Br PA combined with SOR applied in Hep G2 and SMMC7721 cells after 24 h was lower than that of SOR alone(P<0.05).1.2 hepatocellular carcinoma Hep G2 and SMMC7721 cells were treated with 100?mol/L 3-Br PA combined with 16?mol/L SOR for 24 h.Compared with 3-Br PA group and SOR group,the intercellular spaces increased,and the number of suspension cells increased in 3-Br PA+SOR group under the microscope.1.3 To verify the effect of 3-Br PA combined with SOR in Hep G2 and SMMC7721 cells using Cell colony formation.Hep G2 and SMMC7721 cells were treated with 100?mol/L 3-Br PA combined with 16?mol/L SOR for 6-8 days.The results shows that 3-Br PA can enhance the proliferation inhibition caused by SOR in Hep G2 and SMMC7721 cells.2 3-Br PA enhance sorafenib-induced apoptosis in hepatocellular carcinoma SMMC7721 and Hep G2 cells2.1 Hepatocellular carcinoma Hep G2 and SMMC7721 cells were treated with 100?mol/L 3-Br PA combined with 16?mol/L SOR for 24 h,and the mitochondrial membrane potential was analyzed using JC-1 kit.The results showed that red fluorescence transited to green fluorescence in 3-Br PA+SOR group.The results illustrated that 3-Br PA enhance sorafenib-induced early-apoptosis in Hep G2 and SMMC7721 cells.2.2 100? mol/L 3-Br PA combined with 16?mol/L SOR was applied in Hep G2 and SMMC7721 cells after 24 h,DAPI staining showed that Hep G2 and SMMC7721 cell nucleus became significantly condensed and fragmented in 3-Br PA+SOR group.2.3 The apoptotic rate was(47.4±5.02)% in SMMC7721 cells and(46.2±4.14)% in Hep G2 cells after the combined treatment for 24 h,which were also markedly higher than 3-Br PA or SOR group(P<0.05).3 Effects of 3-Br PA combined with SOR on the cellular ATP levelsHepatocellular carcinoma Hep G2 and SMMC7721 cells were treated with 100?mol/L 3-Br PA combined with 16?mol/L SOR for 5h,and the intracellular ATP levels were measured by ATP kit.The intracellular ATP levels were(18.5±2.63)% in SMMC7721 cells and(22.3±4.14)% in Hep G2 cells in 3-Br PA+SOR group,which were markedly lower than 3-Br PA or SOR group(P<0.05).carcinoma Hep G2 and SMMC7721 cells.To inhibit cellular ROS production can significantly decrease apoptosis induced by 3-Br PA combined with SOR.4.3-Br PA combined with SOR can increase intracellular ROS production in hepatocellularHepatocellular carcinoma Hep G2 and SMMC7721 cells were treated with 100?mol/L 3-Br PA combined with 16?mol/L SOR for 6h,and the intracellular ROS production were measured by ROS kit.The intracellular ROS production was ignificantly increased in 3-Br PA+SOR group.To inhibit cellular ROS production by NAC(5 mmol/L)can significantly decrease apoptosis induced by 3-Br PA combined with SOR.5.Effects of 3-Br PA combined with SOR on apoptosis-related protein in Hep G2 and SMMC7721 cells.The expression of Mcl-1,PARP and Bcl-2 in hepatocellular carcinoma Hep G2 and SMMC7721 cells were down-regulated clearly in 3-Br PA+SOR group(P<0.05).6 Anti-tumor effect of 3-Br PA combined with SOR in nude mice.Anti-tumor effect of 3-Br PA combined with SOR was assessed in nude mice.The results showed that the average volumes in 3-Br PA+SOR group were 28.71±7.48 mm3,while the 3-Br PA group were 113.07±43.22 mm3,the SOR group were 157.42±47.04 mm3.compared with 3-Br PA group or SOR group,3-Br PA+SOR group had a good anti-tumor effect(P<0.05).HE staining indicated chaotic distribution of blood vessel and large necrotic areas in 3-Br PA+SOR group.Thus,3-Br PA enhanced the anti-tumor efficacy of SOR in vivo.Conclusion1.3-Br PA enhanced proliferation inhibition and apoptosis induced by SOR in hepatocellular carcinoma SMMC7721 and Hep G2 cells.2.3-Br PA combined with SOR can down-regulate the expression of Bcl-2,Mcl-1 and PARP in hepatocellular carcinoma Hep G2 and SMMC7721 cells.3.3-Br PA combined with SOR increase intracellular ROS production in hepatocellular carcinoma Hep G2 and SMMC7721 cells.To inhibit cellular ROS production cansignificantly decrease apoptosis induced by 3-Br PA combined with SOR.4.3-Br PA enhanced anti-tumor efficacy of SOR in nude mice. |
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