Exploration Of Effects And Mechanism On Hepatocellular Carcinoma With Sorafenib And Adriamycin In A Different Order

ObjectiveTo explorate effects and mechanism on hepatocellular carcinoma with Sorafenib and Adriamycin different order,providing a new theoretical basis for clinical treatment of liver cancer.MethodsHepatocellular carcinoma HepG2 were selected for drugs studing:1.Sorafenib and Adriamycin treated HepG2 cells with different concentration,such as(0,1,2,5,8,10 pmol/L)or(0,1,2,4,6,8?mol/L),detecting the effects of Sorafenib and Adriamycin on cell HepG2 activity by MTT assay.Drawing the drug-validity curve with the concentration gradient as the transverse coordinate and cell activity as the longitudinal coordinate.Calculating the IC50 of Sorafenib and Adriamycin to screen the following experimental drug concentration.Setting up Control group,Sorafenib group(2?mol/L),Adriamycin group(1?mol/L),Combination with different order group.Combination with different order group such as:Combination group,Sorafenib after one hour to join Adriamycin group(S1 A),Sorafenib after four hours to join Adriamycin group(S4A),setting S6A,S8A,A1 S,A4S,A6S,A8S with equivalent principle.Detectting the cell activity of each group by MTT assay.The most obvious effect group(S6A)was screened.2.Effects of Sorafenib and Adriamycin alone or in a different combination on cell proliferation.To observe the cell cloning ability of each group.Determining the inhibition of cell proliferation by single or combination.The levels of proliferative-related protein P-AKT,AKT,P-ERK and ERK were detected by Western Blot.3.Effects of Sorafenib and Ariamycin alone or in a different combination on apoptosis.The cell nucleus were observed by Hoechst 33342 staining,apoptosis rate was detected by the flow cytometry and expression of apoptosis protein Bax and Bcl-2 were detected by Western Blot.4.Effects of Sorafenib and Adriamycin alone or in a different combination on cell invasion and migration.The cell invasiveness and migration ability was examined by the Transwell invasiveness and migration experiment.The cell migration ability was examined by the cell scratch experiment in each group.Effects of Sorafenib and Adriamycin on cell invasion and migration were observed.5.The ROS level of cells with Sorafenib and Adriamycin alone or in a different combination by ROS kit.The mitochondrial membrane potential was examined by JC-1 kit in cells.Results1.Sorafenib and Adriamycin decreased activity of HepG2 cell in a concentration gradient dependence.Sorafenib IC5o was 5.003 umol/L.Adriamycin IC50 was 2.665 ?mol/L.Compared with thesingle drug group,the combination group inhibited the cell activity(P<0.05).Compared with the single drug group and the combined drug group,the combined order drug group(S6A)inhibited the cell activity obviously(P<0.01).2.The ability of cell cloning was obviously inhibited in the combined order drug group(S6A).Compared with the single drug group,the combination group,the combined order drug group(A6S),the difference is statistically significant(P<0.01).The results of Western Blot showed that the levels of P-AKT and P-ERK protein expression in the S6A group were significantly reduced,which had no effect on the expression of AKT and ERK protein.The levels of proliferative protein expression were obviously inhibited in the combined order drug group(S6A)group(P<0.01).3.The apoptotic nuclear morphology was obviously induced by the combined order drug group(S6A).Nuclear concentration and brightness of the combined order drug group(S6A)were observed by Hoechst 33342 staining.Compared with the single drug group,the combination group,the combined order drug group(A6S),nuclear changed are evident.Apoptosis was detected by the flow cytometry,Sorafenib group cell apoptosis rate was(12.1 ±0.9)%,Adriamycin group cell apoptosis rate was(13.7 ± 0.6)%,the Combination goup cell apoptosis rate was(25.2 ± 1.1)%,the Combined order drug group(A6S)cell apoptosis rate was(25.9±9)%.Compared with each group,the death rate(36.8 ± 0.5)%of the combined order drug group(S6A)increased significantly(P<0.05),the difference is statistically significant.4.Observating of the Transwell invasions,migration experiments and the cell scratch experiments.Compared with each drug group,the combined order drug group(S6A)obviously inhibited the cell invasion and migration ability(P<0.05).5.The level of ROS was significantly increased in the combined order drug group(S6A).Compared with each drug group,the ROS product was significantly increased(P<0.05).The JC-1 results showed that the ratio of Red/Green light decreased significantly,the membrane potential of mitochondria decreased significantly.Conclusions1.Combined Sorafenib with Adriamycin order administration(S6A)inhibited the proliferation,invasion and migration.Promoting apoptosis of HepG2 cells.Compared with each drug,effects were the most obvious.2.Combined Sorafenib with Adriamycin different order increased the ROS level and decreased the mitochondrial membrane potential.