Cell Surface GRP78 Mediated Up-regulation Of CRAF Promotes Sorafenib Resistance

ObjectiveHepatocellular carcinoma cells resistant key proteins cell surface GRP78 and CRAF,to explore the interaction between them,provide new clues for systemic clinical treatment of hepatocellular carcinoma.MethodsThis experiment used human hepatoma carcinoma cell line Hep G2 as he models,and the establishment of sorafenib resistant cell lines Hep G2-SR,the following studies: 1.Invasion by morphology and in vitro experiments,colony formation assay,MTT method and flow cytometry sorafenib in Hep G2 and Hep G2-SR efficacy,sensitivity between the two groups of cells to sorafenib.2.Western blot to detect and compare the expression before and after treatment with sorafenib two cells CRAF and p-ERK downstream of;after the drug-resistant down with sh RNA CRAF,MTT method and colony formation assay in vitro detection sorafenib Hep G2-SR efficacy,reduced Hep G2-SR and compare the sensitivity of the change before and after sorafenib.3.GST-pulldown,immunoprecipitation and immunofluorescence colocalization between GRP78 and detect the presence of CRAF interaction.Result 1.Hepatocellular carcinoma of liver cancer cells than the drug-resistant cell morphology changes polygonal asymmetrical,its invasiveness has been significantly improved.Liver cancer and liver cancer cells resistant cells differ in sensitivity to sorafenib,and within the effective range of concentrations,Hep G2-SR than Hep G2 sorafenib resistance stronger.2.Sorafenib CRAF and downstream of p-ERK expression in hepatoma cells treated drug significantly more than the untreated liver cancer cells;in the effective concentration range,Hep G2 cells,CRAF and downstream of the p-ERK expression showed a decreasing relationship,and Hep G2-SR in CRAF and downstream of the p-ERK expression did not change the trend;sh RNA down after CRAF,Hep G2-SR sensitivity to a difference of sorafenib,down after Hep G2-SR sorafenib resistance decreased significantly.Analysis conclusionLiver cancer and liver cancer cells compared to resistant cells,sorafenib more resistant.MEK signaling pathway in liver cancer cells vulnerable to the impact of medication sorafenib with increasing drug concentration,p-ERK1 / 2 expression decreased.In Sorafenib-related genes,CRAF expression in hepatoma cells are more resistant,so that sorafenib by CRAF inhibition of MEK signaling pathway is limited,so Sorafenib HCC-resistant cells can be maintained without active but stable MEK signaling pathway activity,resistance to sorafenib inhibits proliferation and promote apoptosis.Sorafenib HCC CRAF resistant cells can be combined with CRAF hepatoma cell surface,and jointly promote Sorafenib HCC cell resistance.Increase Sorafenib HCC cell resistance.