AT7867,a Novel AKT And S6K1 Duel Inhibitor,Inhibits Human Colorectal Cancer Cells:a Mechanism Study

Objective:The potential effect and underlying mechanism of AT7867,a novel AKT and S6K1 duel inhibitor,in colorectal cancer cells was evaluated here.Methods:1.MTT assay,trypan blue staining assay and clonogenicity assay were applied to test the potential role of AT7867 on human colorectal cancer survival,death and proliferationrespectively.2.Caspase activity assay,Histone DNA ELISA assay,and Annexin V-PI FACS assay were applied to test colorectal cancer apoptosis with AT7867 treatment.3.Western blot assay was performed to test signaling changes of AKT in colorectal cancers with AT7867 treatment.4.Test changes of AT7867's cytotoxicity in colorectal cancers with constitutively-active AKT1 treatment.5.Test changes of AT7867's cytotoxicity in colorectal cancers after knock-down AKT1.6.Western blot assay was performed to test signaling changes of SphKl in colorectal cancers with AT7867 treatment.7.Test changes of ceramide level in colorectal cancers with AT7867 treatment and the cytotoxicity of ceramide.8.Test changes of ceramide level and signaling changes of SphKl in colorectal cancers with AT7867 treatment after knock-down AKT1.9.HT-29 xenograft nude mice model was applied,the in vivo anti-colorectal cancer activity by AT7867 was analyzed.Results:1.AT7867 inhibited survival and proliferation of established(HT-29,HCT116 and DLD-1 lines)and primary human CRC cells.2.AT7867 provoked dose-dependent apoptosis in the CRC cells.3.AT7867 blocked AKT-S6K1 activation in CRC cells.Restoring AKT-S6K1 activation,via expression of a constitutively-active AKT1("ca-AKT1"),only partially attenuated AT7867-induced HT-29 cell death.4.AT7867 dose-dependently inhibited sphingosine kinase 1(SphKl)activity to promote pro-apoptotic ceramide production in HT-29 cells.AT7867 promote ceramide production and promote cell apoptosis in HT-29 cells.5.SphKl activation and the cellular ceramide level were no change in HT-29 cells with AT7867 treatment after ca-AKT1.6.SphKl activation and the cellular ceramide level were no change in HT-29 cells with AT7867 treatment after knock-down AKT1.7.Intraperitoneal injection of AT7867 inhibited HT-29 xenograft tumor growth in nude mice.8.AKT-S6K1 activation was also inhibited in AT7867-treated HT-29 tumors.Conclusions:AT7867 inhibits CRC cells via AKT-dependent and AKT-independent mechanisms.