Effect Of Activating Transcription Factor-3(ATF3) On The Biological Characteristics Of Colon Cancer Cell HCT116and ATF3is Up-regulated Upon Proteasome Inhibitor Carfilzomib

Objective: colorectal cancer is the forth leading cause of cancer death in the worldwide. Although the diagnosis and treatment of colon cancer is improved, the relative5-years survival rate is only53.8-65.2%. Activating transcription factor3(ATF3) is a member of the ATF/cyclic AMP response element-binding (ATF/CREB) family of transcription factors. and has a dichotomous role both oncogene and tumor suppressor in cancer. This study, lentivirus was used to mediate ATF3overexpression in colorectal cancer cells line HCT116. We want to explore the biology change such as proliferation, cancer-initiation feature, tumor migration, invasion and EMT and the possible molecular mechanism after ATF3overexpression. In addition we found a proteasome inhibitor Carfilzomib can regulate ATF3expression and inhibit cell growth in HCT116.Methods: HCT116cells were infected by lentivirus LV5and LV5-ATF3,then selected by puromycin to pick out stably expressing HCT116/Control and HCT116/ATF3pool. The detection of cell proliferation used MTS cell activity assay,FACS cell cycle assay,FACS AnexinV-7-AAD assay and colony formation assay; The detection of cancer-initiating feature used spheroid formation assay and FACS;The detection of cell migration and invasion ability use scratch assay,transwell migration assay and transwell invasion assay; The detection of EMT used light microscopy observation.The associated gene mRNA and protein expression was measured by real-time qPCR and Western Blot. Results: HCT116/ATF3colon cancer cells significantly decrease MTS OD(490nm)and the number of clone formation (p<0.05) and decrease the expression level of survivin,PCNA, cyclin D1and increase the expression of PARP compared to the HCT116/Control; HCT116/ATF3colon cancer cells significantly decrease the percentage of CD133+CD24+cells, the average diameter and the number of floating spheroid (p<0.05) and decrease the expression level of CD133, CD24, Ep-CAM, CD166, EZH2,beta-catenin and ID1compared to the HCT116/Control.HCT116/ATF3colon cancer cells significantly decrease wound healing ability and the number of transmembranes cells in transwell migration assay and transwell invasion assay (p<0.05)and downregulate the expression level of MMP2MMP9and upregulate the expression level of TIMP-1TIMP-2compared to the HCT116/Control. HCT116/ATF3colon cancer cells change cell morphology from mesenchymal cell to epithelial cell and upregulate the expression of E-cadherin and downregulate the expression of twist, snail and slug compared to the HCT116/Control. Carfilzomib inhibit HCT116poliferation and promote apoptosis was measured by MTS OD(490nm) and Anexinâ…¤-7-AAD with increasing dosage and arrest in G0/G1phase was measured by FACS cell cycle analysis.Carfilzomib can be a dose and time dependent manner induced the expression of ATF3.Conclution:in concution,we successfully built stable overexpression cell line HCT116/Control and HCT116/ATF3mediated by lentivirals.ATF3inhibit HCT116proliferation through dowregulation of Survivin,CyclinD1and PCNA and upregulatedion of PARP;ATF3inhibit HCT116cancer-initiation feature through dowregulation of CD133, CD24, Ep-CAM, CD166, EZH2,beta-catenin and ID1.ATF3inhibit HCT116metastasis through downregulation MMP9and MMP2and upregulation of TIMP-1and TIMP-2.ATF3reverse EMT feature through upregulation of E-cadherin and downregulation of sanil,twist and slug.Proteasome inhibitor Carfilzomib can induce ATF3expression and inhibit proliferation in HCT116.