Interaction Between Cdc20 And GRP78 Activating ERK Induces Sorafenib Resistance In HCC

ObjectiveTo investigate the interaction between cell-division cycle protein 20(Cdc20)and glucose-regulating protein 78(GRP78),determining the role and mechanism of Cdc20 and GRP78 in the development of sorafenib resistance in hepatocellular carcinoma(HCC),exploring the possibility of Cdc20 as a potential target for clinical treatment of sorafenib resistance in HCC.Methods1.Hepatocellular carcinoma Hep G2 cells were screened by increasing concentration of sorafenib to obtain sorafenib resistant Hep G2-SR cells,observing cells morphology by inverted microscope.Treated with sorafenib at gradient concentration,cell proliferation was detected by MTT and colony formation,as the expression of Cdc20,GRP78,DUSP6 and p-ERK was detected by Western blotting in Hep G2 and Hep G2-SR cells.2.Sorafenib-resistant cells Hep G2-SR were respectively inoculated on the dorsal side of the upper limb of BABL/C mice with 100,1000 and 10000 cells,and Hep G2 cells were inoculated on the contralateral side as the control group,observing the tumor-forming ability of nude mice.3.The proteins interacting with Cdc20 were screened by coimmunoprecipitation technique.The interaction between Cdc20 and GRP78 was detected by co-immunoprecipitation,and the protein localization of Cdc20 and GRP78 was detected by immunofluorescence assay.Results1.The cell morphology was observed by inverted microscope,Hep G2 cells grew well and Hep G2-SR cells were slender fusiform.MTT results showed that the survival rate of Hep G2-SR cells was significantly higher than that of Hep G2 cells,and the results of colony formation assay showed that the number of clones of Hep G2-SR cells was significantly higher than that of Hep G2 cells.Western blotting showed that compared with Hep G2 cells,the expression of Cdc20,GRP78 and p-ERK in Hep G2-SR cells was significantly increased,while the expression of DUSP6 was lower than that in the control group.2.The tumorigenesis ability of nude mice was observed.Compared with the nude mice inoculated with Hep G2 cells,the tumorigenesis ability of Hep G2-SR cells was significantly increased,and the tumorigenesis volume of Hep G2-SR cells increased with the increase of the number of Hep G2-SR cells.3.The results of co-immunoprecipitation showed that GRP78 may be a protein interacting with Cdc20.The results of co-immunoprecipitation showed that Cdc20 interacted with GRP78,and immunofluorescence showed that Cdc20 and GRP78 were co-located.ConclusionsCdc20 is overexpressed in sorafenib-resistant HCC cells and activates ERK by interacting with GRP78 to induce sorafenib resistance in HCC cells.Therefore,Cdc20 may be a potential target for the treatment of sorafenib-resistant HCC.