Investigate The Mechanism Of LCL161 Combined With Caspase Inhibitors In Promoting Apoptosis Of Breast Cancer Resistant Cells

Objective:1.Investigate the effect of SMAC analogue LCL161 combined with Caspase inhibitors(CI)on drug resistant cells of breast cancer.2.The proteomics technique of TMT combined with mass spectrometry was used to screen the response differential proteins of breast cancer drug-resistant cells under the action of LCL161 combined with CI,and through bioinformatics analysis,the mutual regulatory relationship between differential proteins and their involved signaling pathways were explored.3.Identify Differences target protein,using the parallel reaction monitoring(parallel reaction monitoring,the PRM)technology for validation of screening proteins.To study the changes of drug-resistant breast cancer cells treated with LCL161 combined with CI,explore the possible mechanism of LCL161 combined with CI to promote the apoptosis of drug-resistant breast cancer cells,and provide research clues for the subsequent treatment of breast cancer resistance and drug targeting markers.Methods:1.Cck-8 method was used to detect the effect of LCL161 combined with CI on the survival rate of Mc F-7 /DDP in drug-resistant human breast cancer cells.2.The isotopic labeling and absolute quantitative(Tandem Mass Tag,TMT)techniques were used to screen the significantly differentially expressed proteins in breast cancer drug-resistant cells before and after LCL161 combined with CI treatment(Fisher's exact test,p<0.05),and bioinformatics analysis was performed on all the differentially expressed proteins.3.Parallel reaction monitoring(PRM)was used to identify the target proteins of LCL161 combined with CI to promote the apoptosis of drug-resistant breast cancer cells.Results:1.When combined with CI,LCL161 increased the death of drug-resistant cells in breast cancer compared with LCL161 alone.2.TMT technology screened significantly different proteins :A total of 5949.0 proteins were identified in the study,among which 5220.0 contained quantitative information.Under the condition of 1.2,P<0.05,we found that 92 proteins were up-regulated and 114 proteins were down-regulated in the comparison group.Using gene ontology to analyze the expression of differentially expressed proteins from the perspective of biological activities of proteins mainly includes aspects of cellular processes,binding and integration,catalytic activity,and signaling activity..The results of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway showed that the differential proteins concentrated in the processes of cell proliferation,cell signaling and nutrient metabolism,etc2.The PRM validation.Based on the results of TMT and bioinformatics analysis,combined with the principle of protein selection,4 key proteins that are considered to be combined with LCL161 to promote the apoptosis of breast cancer resistant cells were selected and verified by PRM.The overall results of TMT and PRM were consistent,so the 4 proteins selected may be the key proteins for LCL161 combined with CI to promote the apoptosis of breast cancer resistant cells.Conclusions:1.LCL161 can promote apoptosis of MCF-7/DDP cells under Caspase inhibition.2.206 significantly differential proteins and 24 related pathways co-changed proteins were more or less involved in the molecular mechanism of LCL161 combined with CI to promote apoptosis in breast cancer drug-resistant cells.3.PRM verified the expression levels of 4 differentially expressed proteins consistent with the TMT results,and the verified proteins may be the key proteins of LCL161 combined with CI to promote the apoptosis of drug-resistant breast cancer cells.