Mechanisms Of Mcl-1 In Radioresistance Of Radiation-resistant Lung Adenocarcinoma Cells

Background : Lung cancer is the highest mortality malignancy worldwide,a serious threat to human life.Radiation therapy(Radiotherapy,RT)has become an important means of limited lung cancer and palliative care,the ultimate goal of radiotherapy is long-term eradication of cancer cells,radiation failure mainly manifested in two aspects,one is outside the radiation recurrence,one is within the radiation recurrence,the former is due to the distant metastasis before radiation,and the latter may be attributed to the radiation resistance.A large number of strong evidence shows that deoxyribonucleic acid(DNA)is the main target of the biological effects caused by ionizing radiation,and cluster DNA damage is the symbol of ionizing radiation,and the lethal biological effects caused by radiation are mainly DSB and base damage.With a higher frequency of Mcl-1 su Bclone acquisition in lung tumors after TP53 mutation in Chinese lung adenocarcinoma patients,targeting Mcl-1not only acts as an apoptotic switch,but also is a key determinant of DNA damage response activation and DNA damage repair.Objectives : Build a radiation resistant lung adenocarcinoma cell model;verify the radioresistance of radiation tolerant cell model,clarify whether the anti-apoptotic protein Mcl-1 in apoptostant cell model mediabnormal apoptotic pathway to enhance cell radiation resistance;explore the related mechanism of Mcl-1 regulating apoptosis and DNA damage repair to enhance radiation resistance of radiation resistant lung adenocarcinoma cells.Methods: Simulation of clinical treatment gives a certain dose of ionizing radiation to cells to build a model of radiation-resistant lung adenocarcinoma cells.CCK8 experiments and clone formation experiments verified the radioresistance of radioresistant cells.The effect of X-ray on apoptosis and cell cycle changes were determined by flow cytometry.Single cell gel electrophoresis and reactive oxygen species tested the effect of X ray on DNA damage levels in radiorestant cells.The expression levels of apoptosis-related proteins such as Mcl-1 as well as DNA damage repair proteins in radiorestant cells were determined by Western Blot.The binding effects of proteins such as Mcl-1 and DNA damage repair were determined by coimmunoprecipitation.Intracellular cololocation expression of Mcl-1 and APE1 was determined by immunofluorescence assay.Results: The radiation A549-RR cell model was constructed by giving 45 Gy X-ray irradiation of A549 cells(5Gy 9 times),And 2Gy of weekly low-dose X-ray irradiation can maintain the radioresitivity of the cells;The proliferation of radiorestant cells does not slow down,cell cycle changes,reduced apoptosis;The DNA damage of radiorestant cells is weakened and enhanced after irradiation;The anti-apoptotic protein Mcl-1 is overexpressed in radioresistant A549-RR cells and interacts with the pro-apoptotic protein Bax;The phosphorylation level of the antiapoptotic protein Mcl-1 was slightly downregulated;The DNA damage repair proteins APE1 and Rad51 were upregulated in radiation-resistant lung adenocarcinoma cells,Interference with Mcl-1expression by si RNA downregulates the expression levels of the DNA repair proteins APE1 and Rad51;Inhibition of Mcl-1 with small molecule compounds enhances radiation sensitivity in radiorestant cells.Conclusions:In this study,a radiation-tolerant cell model was successfully constructed and found that the anti-apoptotic protein Mcl-1was upregulated in the radiation-tolerant lung adenocarcinoma cell model and inhibited cell apoptosis by interacting with the proapoptotic protein Bax,by a mechanism possibly related to the wnt /-catenin signaling pathway.In this model,DNA damage after irradiation is reduced,DNA damage repair protein expression is enhanced,and targeted inhibition of Mcl-1 can downregulate the expression of homologous recombinant protein Rad51 and base excision repair protein APE1,restore the radiotherapy sensitivity of radiation-resistant lung adenocarcinoma cells,and enhance the comprehensive treatment effect of lung adenocarcinoma.