| Objective: To study the effects of different concentrations of Koumine(Kou),Koumine combined with cisplatin on the proliferation activity of human hepatoma cell lines(Hep G2,7404,7402),as well as the effects of ROS,JNK1,Akt,Bcl-2,Bax changes in the expression levels of related genes,to explore whether there is a synergistic effect of koumine combined with cisplatin,and the effect of different concentrations of koumine on the regulation of proliferation in liver cancer cell lines and its possible molecular mechanism.The feasibility of applying to liver cancer treatment provides experimental basis.Methods: 1.Three kinds of hepatocellular carcinoma cell lines(Hep G2,7404,7402)were cultured in vitro;2.The effect of different concentrations of koumine group,cisplatin group,koumine combined with cisplatin group on hepatoma cells(Hep G2,7404,7402)was detected by MTT method,and calculate the IC50 value of each drug;use fluorescent DCFH-DA probe method to determine the level of intracellular ROS;plate colony formation assay to analyze the effect of drugs on the growth of liver cancer cell colonies;real-time quantitative PCR to detect ROS transcription levels of related genes(Akt,JNK1,Bcl-2 and Bax m RNA);Western blotting to evaluate the protein expression levels of Akt,JNK1/2,Bcl-2 and Bax;3.SPSS 21.0 software was applied for statistical analysis in the experiment.The measurement data was expressed by the mean ± standard deviation(`x ± s)and analyzed by the t test method.The counting data was analyzed by the chi-square test.Statistical difference is meaningful when P value below 0.05,and significantly meaningful when P value below 0.01.Results:(1)Tumor cell proliferation in three kinds of hepatoma cell lines(Hep G2,7404,7402)was dose-dependently inhibited after koumine treatment whose IC50 values were 90.32,107.96,89.59μg/m L,respectively.Koumine played an inhibitory role in a low concentration(10μg/m L)as least,and koumine had a stronger activation effect at a concentration of 20μg/m L,compared to that of the low concentration(≤10μg/m L);koumine combined with cisplatin group has a strong activation effect on the three kinds of liver cancer cells have obvious inhibitory effect,and its inhibitory effect is stronger than that of koumine group and cisplatin group,which is dose-dependent.(2)Reactive oxygen species content the detection experiment showed that the level of ROS in liver cancer cells in the koumine concentration of 20μg/m L was lower than that in the control group.With the increase of koumine concentration,increased the level of intracellular ROS.The level of ROS in liver cancer cells in the koumine combined with cisplatin group was higher than that in the koumine group and the cisplatin group;(3)Compared with the control group,the colony formation ability of the cells increased in the concentration of koumine 20μg/m L.With the increase of koumine concentration,the cell colony formation ability was significantly inhibited,and the koumine combined with cisplatin group inhibited the colony formation ability of liver cancer cells more obviously;(4)RT-PCR results showed that three liver cancer cells(7402,7404 and Hep G2 cells)were treated with different concentrations of koumine(respectively 20.0,120,and200 μg/m L)for 48 hours,and the results were contrast with the control group.Compared with other groups,JNK1 and Bax in the koumine concentration group of 20μg/m L were decreased(only 0.8-0.9 times compare with control group),and JNK1 and Bax were increased in the koumine concentration group of 120μg/m L group and the koumine concentration of 200μg/m L group;Compared with the control group,Bcl-2 in the koumine concentration of20μg/m L was increased by 1.32-1.94 times,and Akt was increased by1.37-1.76 times,while Bcl-2 in the koumine concentration of 120μg/m L and200μg/m L group decreased in a dose-dependent manner(0.47～0.55 folds and0.09～0.13 folds respectively)(P<0.01).The changes of Akt in the three groups of hepatoma cells were similar to that of Bcl-2.(5)The results of western blotting showed that the Akt level in the combination group of koumine and cisplatin was lower than that in the koumine group or the cisplatin group,while the level of JNK was significantly increased,and the expression of the anti-apoptotic protein Bcl-2 was down-regulated at the same time,up-regulated the expression of the pro-apoptotic protein Bax and increased Bax/Bcl-2.Conclusion: 1.Koumine combined with cisplatin can effectively inhibit the proliferation of human hepatoma cell lines(Hep G2,7404,7402),and has synergistic effect;2.Koumine has a strong activation effect at the concentration of 20μg/m L,considering the existence of Hormesis effect;3.The inhibitory effect of Koumine combined with cisplatin on hepatoma cell lines may be related to its effect on JNK/Bcl-2/Bax signaling pathway. |
