Oridonin Down-regulates The Expression Of Mcl-1 Inducing The Apoptosis In Hepatocellular Carcinoma Cells

Objective1.Investigate the effects of Oridonin on the proliferation and apoptosis of human hepatocellular carcinoma cells.2.Research the mechanism of Oridonin inducing human hepatocellular carcinoma cells apoptosis.Methods1.HCCLM3 and HepG2 cells were treated with different concentrations of Oridonin,then CCK-8 assay was used to detect the proliferation.Cloning assay was used for the evaluation of clonal formation of HCCLM3 and HepG2 cells at different concentrations(0?5?10?20?M)of Oridonin.Apoptosis of liver cancer cells was assessed by Annexin-FITC/ PI and flow cytometry.2.Different concentrations(0?5?10?20?M)of Oridonin were dealed with HCCLM3 and HepG2 cells,then the apoptosis-associated protein was detected with Western Blot.After knocking out Mcl-1 involved in HCCLM3 and HepG2 cells by using siRNA technology,the changes of the apoptosis rate(control group,Oridonin 10?M group,siMcl-1 group,Oridonin 10?M + siMcl-1 group)were determined with flow cytometry.Results1.CCK-8 assay results showed that low concentration of Oridonin had a weak inhibition on HCCLM3 and HepG2 cells,while high concentration of Oridonin had a strong inhibition of the two cell lines,and the inhibition rate was dose-dependent.The result of cloning assay showed that the ability of inhibiting colony-formation was also dose-dependent.Flow cytometry results showed that Oridonin could induce apoptosis in HCCLM3 cells at concentration of 5?M?10?M and 20?M after 24 h applied,and the rates of apoptosis were 20.1% ? 41.5% ? 74.3%,which were significantly higher than that in control group(6.7%,P<0.05).To HepG2 cells,the rates of apoptosis were 20.3%?42.6%?69.2%,which were also significantly higher than that in control group(4.5%,P<0.05).2.Western blot protein detection showed that Oridonin could activated the expression of caspase-3 protein and PARP in HCCLM3 and HepG2 cells at concentration of 10?M after 24 h.The results also showed that Oridonin could down-regulate the expression of Mcl-1 protein significantly in HCCLM3 and HepG2 cells at concentration of 10?M and 20 ?M after 24 h,while had no significant effect on the expression of Bcl-2?Bcl-xL.After knocking out Mcl-1 involved in HCCLM3 and HepG2 cells by using siRNA technology,flow cytometry results showed that the rates of apoptosis(Oridonin 10?M group,siMcl-1 group,Oridonin 10?M + siMcl-1 group)were 39.2%?41.6%?43.7% in HCCLM3 cells,which were significantly higher than that in control group(5.3%,P<0.05),however the rates of apoptosis(Oridonin 10?M group,siMcl-1 group,Oridonin 10?M + siMcl-1 group)had no statistical difference.And to HepG2 cells,flow cytometry results showed that the rates of apoptosis(Oridonin 10?M group,siMcl-1 group,Oridonin 10?M + siMcl-1 group)were 40.3%?42.4%?44.8%,which were significantly higher than that in control group(4.6%,P<0.05),however the rates of apoptosis(Oridonin 10?M group,siMcl-1 group,Oridonin 10?M + siMcl-1 group)also had no statistical difference.At the same time we found that Oridonin could not increase significantly the rates of apoptosis in Oridonin 10?M + siMcl-1 HCCLM3 group and Oridonin 10?M +siMcl-1 HepG2 group,when compared to siMcl-1 HCCLM3 group and siMcl-1 HepG2 group.Conclusions1.Oridonin has the effects of inhibition to human hepatocellular carcinoma cells proliferation and induction of cells apoptosis.2.Oridonin may induce apoptosis of human hepatocellular carcinoma cells by down-regulating the expression of Mcl-1.