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OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and exploreits potential mechanisms.METHODS ULK1 expression was analyzed by The Cancer Genome Atlas(TCGA)analysis and tissue microarray(TMA)analysis.ULK1agonist was designed by using in silico screening,as well as modified by chemical synthesis and screened by kinase and anti-proliferative activities.The amino acid residues that key to the activation site of LYN-1604 were determined by site-directed mutagenesis,as well as in vitro kinase assay and ADP-Glo kinase assay.The mechanisms of LYN-1604 induced cell death were investigated by fluorescence microscope,western blotting,flow cytometry analysis,immunocytochemistry,as well as si RNA and GFP-m RFP-LC3 plasmid transfections.Potential ULK1 interactors were discovered by performing comparative microarray analysis and the therapeutic effect of LYN-1604 was assessed by xenograft breast cancer mouse model.RESULTS We found that ULK1 was remarkably downregulated in breast cancer tissue samples,especial y in triple negative breast cancer(TNBC).32 candidate smal molecules were synthesized,and we discovered a small molecule named LYN-1604 as the best candidate ULK1agonist.Additionally,we identified that three amino acid residues(LYS50,LEU53 and TYR89)were key to the activation site of LYN-1604 and ULK1.Subsequently,we demonstrated that LYN-1604 could induce autophagy-associated cell death via ULK complex(ULK1-m ATG13-FIP200-ATG101)in MDA-MB-231 cells.We also found that LYN-1604 induced cell death involved in ATF3,RAD21 and caspase 3,accompanied with autophagy and apoptosis.Moreover,we demonstrated that LYN-1604 had a good therapeutic potential on TNBC by targeting ULK1-modulated cell death in vivo.CONCLUSION We discovered a small molecule(LYN-1604)has therapeutic potential by targeting ULK1-modulated cell death associated with autophagy and apoptosis of TNBC in vitro and in vivo,which could be utilized as a new anti-TNBC drug candidate.
OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and explore potential mechanisms. METHODS ULK1 expression was analyzed by The Cancer Genome Atlas (TCGA) analysis and tissue microarray (TMA) analysis. ULK1 antagonist was designed by using in silico screening, as well as modified by chemical synthesis and screened by kinase and anti-proliferative activities. amino acid residues that key to the activation site of LYN-1604 were determined by site-directed mutagenesis, as well as in vitro kinase assay and ADP-Glo kinase assay. These mechanisms of LYN-1604 induced cell death were investigated by fluorescence microscope, western blotting, flow cytometry analysis, immunocytochemistry, as well as si RNA and GFP-m RFP-LC3 plasmid transfections were discovered. by performing comparative microarray analysis and the therapeutic effect of LYN-1604 was evaluated by xenograft breast cancer mouse model .RESULTS We found that ULK1 wa s remarkably downregulated in breast cancer tissue samples, especial y in triple negative breast cancer (TNBC). 32 candidate smal molecules were synthesized, and we discovered a small molecule named LYN-1604 as the best candidate ULK1agonist. Additionally, we identified that three amino (ULK1-m ATG13-FIP200-ATG101) that could induce autophagy-associated cell death via LYN-1604 and ULK1.Subsequently, we demonstrated that LYN- in MDA-MB-231 cells. We also found that LYN-1604 induced cell death involved in ATF3, RAD21 and caspase 3, accompanied by autophagy and apoptosis. More than, we demonstrated that LYN-1604 had a good therapeutic potential on TNBC by targeting ULK1-modulated cell death in vivo. CONCLUSION We discovered a small molecule (LYN-1604) has therapeutic potential by targeting ULK1-modulated cell death associated with autophagy and apoptosis of TNBC in vitro and in vivo, which could be utilized as a new anti -TNBC drug candidate.