Preliminary Study On The Effect Of Berberine Combined With Sorafenib Or Oxaliplatin On Hepatoma Cells

Objective:To investigate the effects of berberine combined with sorafenib or oxaliplatin on the proliferation and apoptosis of human hepatocellular carcinoma cells in vivo,providing basic theories for clinical application.Methods:(1)human hepatoma cells SMMC-7721,HepG2 was purchased from Shanghai Institute of Life Sciences,Chinese Academy of Sciences.(2)Experimental groups: control group,Berberine group of different concentrations(5,25,50,100,200)?mol/L,Oxaliplatin group of different concentrations(1.5625,3.125,6.25,12.5,25,50)?mol/L,Sorafenib group of different concentrations(1,2,4,8)?mol/L;100?mol/L Berberine+4?mol/L Sorafenib combination group,100?mol/L Berberine group,4?mol/L Sorafenib group,control group;50?mol/L Berberine+3.125?mol/L Oxaliplatin group,50?mol/L Berberine group,3.125?mol/L Oxaliplatin group,control group.(3)MTS assay was used to detect the viability of hepatocellular carcinomaSMMC-7721 and HepG2 cells after exposed to the intervention groups.Cell morphology,volume and density were examined by crystal violet staining.(4)The proliferation of SMMC-772 and HepG2 cells was detected by EdU cell proliferation assay.Cell colony formation assay was used to analyze the growth inhibition induced by berberine combined with sorafenib or oxaliplatin in SMMC-7721 and HepG2 cells.(5)The expression of cleaved-caspase 3,cleaved-PARP,Bcl-2 and VEGF in hepatoma cells SMMC-7721 and HepG2 was analyzed using Western blot.(6)Statistical analysis: Statistics were expressed using Graphpad Prism 6,mean± standard deviation data.One-way analysis of variance was used for comparison between groups,P<0.05 indicates statistically significant difference.Results:(1)The cell viability of SMMC-7721 and HepG2 cells decreased in a concentration-dependent manner after treated with berberine,sorafenib,oxaliplatin and 100?mol/L Berberine combined with4?mol/L Sorafenib and 100?mol/L Berberine combined with3.125?mol/L Oxaliplatin(P<0.05).The cell survival rate of 100?mol/L Berberine combined with 4?mol/L Sorafenib and 100?mol/L Berberine combined with 3.125?mol/L Oxaliplatin after 24 h,48h,72 h was lower than that of single drug group(P<0.05).Crystal violet staining also showed that: the cells in the control group were large in volume,diverse morphology,and the largest density,cells in the Berberine,Sorafenib andOxaliplatin single groups were smaller in volume,the spindle shape,smaller density,the combined Berberine+Sorafenib,Berberine+Oxaliplatin groups were smallest in volume,long shuttle shape,and the smallest density.(2)The result of EdU cell proliferation assay showed that compared with the control group,the proliferation of the Berberine alone group,the Sorafenib alone group,and the Oxaliplatin alone group cells decreased;while the proliferation of the Berberine+Sorafenib combined group and the Berberine+Oxaliplatin combined group cells was less than that of the single drug groups.The DNA replication of S phase in the combined group was less than that of the single drug group.(3)To verify the effect of Berberine combined with Sorafenib using colony formation assay,SMMC-7721 and HepG2 cells were treated with 100?mol/L Berberine combined with 4?mol/L Sorafenib for 3 days.The results showed that berberine can enhance the proliferation inhibition caused by sorafenib in SMMC-7721 and HepG2cells.(4)Western-blot showed that cleaved-PARP and cleaved-caspase 3in the combined group were up-regulated significantly.The anti-apoptotic protein Bcl-2 and VEGF in the Berberine+Sorafenib group were decreased compared with their single drug groups.Conclusion:(1)Berberine and Sorafenib alone or in combination can inhibit the growth and induce apoptosis of human hepatoma cells SMMC-7721 and HepG2.Both Berberine and Oxaliplatin alone or incombination can inhibit the proliferation and induce apoptosis of human hepatoma cells SMMC-7721.(2)Combination of Berberine and Sorafenib,Berberine and Oxaliplatin significantly inhibited the proliferation of human hepatoma cells compared with the single-drug groups.With the prolongation of drug treatment time,the growth of human hepatoma cells in Berberine+Sorafenib group and Berberine+Oxaliplatin group was more obvious than that in single-drug group.SMMC-7721 cells are both more sensitive than HepG2 and have lower survival rates,whether they are Chinese traditional medicine(Berberine)or chemotherapy(Sorafenib).(3)Berberine and Oxaliplatin,Sorafenib combination group and single drug group can induce apoptosis of hepatoma cells by the Caspase cascade.Berberine + Sorafenib group induced apoptosis pathway with the reduction of anti-apoptotic protein Bcl-2 and blood vessels generation factor VEGF production.