| Aim : Multidrug resistance is a critical issue for the failure of liver cancer chemotherapy.To date,three generations of multidrug resistance reversal agents have been developed.But due to their poor efficacy and toxic side effects,the chinical trials have failed.The developing of new drug,which overcome drug resistance induced by liver cancer,has become important and urgent in oncology.According to our previous study,it was found that the 23 hydroxy betulinic acid derivative,B5G1,was able to kill a variety of tumor cells in vitro enviroment.In this thesis,the growth inhibitory effect of B5G1 on multidrug-resistant hepatocellular carcinoma cells will be further investigated.The corresponding mechanism can be revealed which possibly provides us a new notion to develop compounds for the treatment of multidrug resistance.Methods:(1)MTT assay and colony formation assay were used to detect the in vitro cytotoxic activity and proliferation inhibitory activity of B5G1 on hepatocellular carcinoma drug-resistant cell line Hep G2/ADM.(2)The effect of B5G1 on the apoptosis of Hep G2/ADM cells was detected by Annexin V-FITC/PI double staining assay.(3)GFP-LC3 transfection,LC3 immunofluorescence assay,and MDC staining assay were used to observe the accumulation of autophagosomes induced by B5G1 in Hep G2/ADM cells.(4)The ultrastructure of autophagosomes and mitochondria in B5G1-treated Hep G2/ADM cells were observed by transmission electron microscope.(5)Mitotracker staining assay was utilized to detect the effect of B5G1 on the numbers or the morphology of mitochondria in Hep G2/ADM cells.(6)Mitotracker staining assay,LC3,p62 and LAMP1 immunofluorescence as well as mt-Keima fluorescence assays were used to detect the colocalization between mitochondria and autophagosomes or lysosomes in Hep G2/ADM cells.(7)Western blot was used to evaluate the effect of B5G1 on the expression of the apoptosis-associated proteins Caspase-9,Caspase-8,Caspase-3,PARP,cytochrome c,the autophagy-related proteins LC3 B,p62,Atg5,Beclin-1,the mitochondrion-related proteins COX IV?TOM20,Mfn2,Parkin and PINK1.(8)si RNA Atg5,si RNA Beclin-1 and si RNA Parkin were used to detect the role of Atg5,Beclin 1 and Parkin in mitophagy induced by B5G1.(9)JC-1 staining was used to detect the effect of B5G1 on mitochondrial membrane potential of Hep G2/ADM cells.(10)The effect of B5G1 on mitochondrial ROS was detected by Mito SOX red staining assay.Results:(1)B5G1 significantly inhibited the proliferation of Hep G2/ADM cells in a dose-and time-dependent manner;the combination of B5G1 and verapamil did not alter the cytotoxicity of B5G1,indicating that B5G1 is not a substrate of P-gp.(2)B5G1 induced cytochrome c releasing from mitochondria,activating of Caspase-9,Caspase-3,and PARP.(3)B5G1 induced accumulation of autophagosomes in Hep G2/ADM cells.(4)B5G1 induced colocalization of mitochondria with autophagosomes and lysosomes,result in degradation of mitochondria.(5)B5G1 had no effect on the expression of Atg5 and Beclin-1.Knocking down of Atg5 and Beclin-1 did not affect B5G1-induced mitophagy.(6)B5G1 induced upregulation of PINK1 and mitochondrial anchoring of parkin to initiate mitophagy.The parkin si RNA inhibited B5G1-induced mitophagy.(7)B5G1 impaired mitochondria by inducing mitochondrial ROS release to active mitophagy.The addition of antioxidant NAC,reversed the decrease of mitochondrial membrane potential which inhibits B5G1-induced mitophagy and apoptosis.(8)Mdivi-1 or Parkin si RNA promoted the apoptosis of Hep G2/ADM cells,indicating that B5G1-induced mitophagy was a protective mechanism.Conclusion: B5G1 shows a significant inhibitory effect on Hep G2/ADM cells in vitro.B5G1 led to a massive release of mitochondrial ROS and mitochondrial damage,which initiating the mitochondrial apoptosis of Hep G2/ADM cells.It also induces PINK1/Parkin-dependent mitophagy which serves as a pro-survival role,inhibiting mitophagy sensitize Hep G2/ADM cells to B5G1. |
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