Mcl-1 Participates In The Experimental Study Of EGFR And ALK Inhibitors Sensitivity In NSCLC Cells

Mcl-1(myeloid cell leukemia-1, Mcl-1) is a member of the Bcl-2(B cell lymphoma-2, Bcl-2) anti apoptotic protein family, which is involved in the regulation of cell apoptosis and survival. Mcl-1 is widely expressed in many normal tissues, which is of great significance for maintaining the maturation and differentiation of many kinds of cells. The up regulation of Mcl-1 expression suggested that the occurrence of tumor, Mcl-1 overexpression in many human cancers, and the overexpr ession of Mcl-1 could induce the tolerance of cells to some therapeutic drugs. Thus, Mcl-1 can be used as a new target of cancer therapy, and play an important role in inducing apoptosis of tumor cells. In this study, we observe the effect and mechanism of Mcl-1 on EGFR and ALK inhibitor sensitivity in NSCLC cells, the PC-9 cells of EGFR mutation and H3122 cells of EML4-ALK fusion as the research object.Section 1: Detection of EGFR and ALK inhibitor induced apoptosis channel in NSCLC cells and molecular proteins in the process of apoptosis by Western blot method. The results showed that EGFR and ALK inhibitors by regulating the expression of Bcl-2 family protein, so that Caspase-3 pathway activation, inducing apoptosis of NSCLC cells, and Mcl-1 is a key molecule in the process of apoptosis.Section 2: Observe the effect of Mcl-1 on EGFR and ALK inhibitor sensitivity in NSCLC cells. The expression of Mcl-1 was detected by quantitative real-time PCR?Western blot and co-immunoprecipitation,the results showed that EGFR and ALK inhibitors were not down regulated Mcl-1 at the transcriptional level, but down regulated Mcl-1 at the level of protein degradation. By the MTT experiment detected there are some EGFR wild type patients sensitive to Gefitinib in clinical. Then the expression level of Mcl-1 and apoptosis of sensitive cell and resistant cell to Gefitinib after being treated with Gefitinib were examined by Western blot method. The results showed that with the increase of the concentration of the drug, the sensitive cell line increased apoptotic fragments and the expression of Mcl-1 decreased, the resistant cell have no apoptotic fragments and the expression of Mcl-1 was not decreased. Flow cytometry showed that cell apoptosis was significantly increased when Mcl-1 was specific knockouted of resistant cell.Section 3: To investigate the mechanism of Mcl-1 on EGFR inhibitor sensitivity in EGFR mutant cells. Through Western blot method to detect the expression level of Mcl-1 of PC-9 cell after being treated with Gefitinib?Akt inhibitor and Erk inhibitor. It was found that the EGFR inhibitor inhibited the expression of p GSK3? by down regulating pAkt, which increased the phosphorylation level of Mcl-1, and then was degraded by ubiquitin. By co- immunoprecipitation and Western blot proved that FBW7 is the ubiquitin ligase that mediates Mcl-1 ubiquitin modification, down regulation of FBW7 expression can inhibit the degradation of Mcl-1 protein.