The Research Of The Correlation Between The Effect Of MTOR Inhibitors On Non-small Cell Lung Cancer Cell Lines And LKB1 Gene

Objective:Lung cancer has become one of the world’s malignant tumor which the incidence rate and mortality rate of the fastest growing and most serious harm to human health.However,NSCLC accounted for a high proportion in lung cancer,and the incidence rate is rising year by year.Therefore,the research on NSCLC is particular important.Now it is widely believed that,the development of tumor is a multistage,biological process.One stage is the most important tumor suppressor gene inactivation in cancer.In recent years,a new cancer gene LKB1,also called STK11(serine/threonine protein kinase 11)in tumor caused great concern.Studies have been known,LKB1 can participate in a variety of biological activity in cells and the control and regulated of the cell energy,cell proliferation,cell cycle,apoptosis and cell polarity.Many study have shown that,somatic mutations in the LKB1 gene are rare in sporadic tumors,but in NSCLC mutation rate is as high as 35%.So the study of LKB1 signal pathway in NSCLC treatment w:ill bring hope.The LKB1 signal transduction pathway understanding is not much,the most clear pathway is LKB1-AMPK-mTOR.Many study have shown that the abnormal activation of mTOR pathway is related the pathogenesis of tumor.Therefore,mTOR inhibitor have become a ideal target drug in resistant to NSCLC.It has been found that there are 4 applications of mTOR inhibitors have been effective in clinical.The study will explore the role of LKB1 mTOR inhibitors in different cells.Through the detection of expression level of protein and affection the growth and proliferation of cells,and provide a new way for the treatment of NSCLC.Methods:1 Harvest the cell line of A549、H460、H1792、Calul,and detect p-S6K、p-4EBP1 expression by Western-blot analysis.2 Using 10nm/L Rapamycin function A549、H460、H1792、Calul,2 hours,each with a blank control,and detect p-4EBP1 expression by Western-blot analysis.3 Harvest the cell lysate of A549、H460、H1792、Calul,using different concentration Rapamycin、Rad001(0.0.1nm/L、1nm/L、10nm/L、100nm/L、1000nm/L)function the above cell line,72 hours.The MTS and SRB two kinds of experimental method for the detection of toxic effects of Rapamycin、Rad001 in lung cancer cell lines;Harvest the homologous cell lines,H1299-PLK0.1、H1299-LKB1shRNA,using Rapamycin,repeat the experiment.4 Using 10nm/L Rapamycin function A549、H460、H1792、Calul,2 hours,each with a blank control,and detect P-AKT expression by Western-blot analysis.5 Using lOum/L LY and 10nm/L Rapamycin function A549、H1792,2 hours,each with a blank control,and detect P-AKT expression by Western-blot analysis.Cytotoxicity experiments at the same time.Results:1 p-S6K、p-4EBP1 expression level in the LKB1 mutant cells lines(A549.H460)than in the wild type LKB1 cell lines(H1792、Calul)was significantly increased.2 In the A549、H460 H1792、Calul.after treatment,the expression level of p-4EBP1 decreased compare with blank control group.3 Using different concentration Rapamycin,RadOO1function the above cell line,72 hours.MTS shown that with the increase of drug concentration,the inhibition of the 2 drug on the cell grown rate was<40%;SRB shown that,the inhibition of the Rad001 on the cell grown rate was<30%.Using different concentration Rapamycin function H1299-PLK0.1、H1299-LKB1shRNA,72 hours.MTS shown that with the increase of drug concentration,the inhibition of the drug on the 2cell lines grown rate was<10%,and no significant difference was found between the inhibition rate.4 After the Rapamycin functioned,P-AKT expression level in the cells lines(A549、H460、H1792、Calul)was significantly increased.5 After the Rapamycin and LY functioned,P-AKT expression level in the cells lines(A549、H1792)was significantly decreased.In cell toxicity experiment,the inhibition rate in combination drug usage is high.Conclusion:1 LKB1-AMPK-mTOR signal transduction pathway in LKB1 mutant NSCLC cells in the activate state,and mTOR inhibitors can inhibit the signaling pathway.2 Weather the LKB1 gene expression or not,does not affect the sensitivity to mTOR inhibition,and mTOR inhibition could’ not inhibit the growth of cell lines,3 Toxic effect of Mtor inhibitor on NSCLC cell lines are very weak,and no correlation with LKB1gene.4 PI3K inhibitors and mTOR inhibitors used in combination,the former can partly offset the latter feedback on P-AKT activation,toxicity experiments show the synergy.