Preclinical Study Of Cinobufagin As A Promising Anti-Colon Cancer Agent,a Mechanism Study

Objective: We studied the anti-Colon cancer cell activity along with underlying signaling mechanisms by cinobufagin(CBG),a Traditional Chinese Medicine.Methods: MTT assay,Brd U ELISA assay,and clonogenicity assay were applied to test the potential effect of CBG on human Colon cancer survival and proliferation.TUNEL staining assay,Histone DNA ELISA assay,and Annexin V-PI FACS assay were applied to test Colon cancer apoptosis with CBG treatment.PI-FACS assay was performed to test Colon cancer cell cycle progression with/without CBG treatment.Western blot assay was performed to test signaling changes in Colon cancer with CBG treatment.HCT-116 xenograft nude mice model was applied,the in vivo anti-Colon cancer activity by CBG was analyzed.Results: CBG inhibited survival and proliferation of established and primary human Colon cancer cells.CBG activated apoptosis,but disrupted cell-cycle progression in the Colon cancer cells.Restoring AKT-S6K1 activation,via expression of a constitutively-active AKT1(“ca-AKT1”),only partially attenuated CBG-induced HT-29 cell death.CBG treatment in Colon cancer cells provoked endoplasmic reticulum stress(ER stress)activation.Contrarily,the ER stress inhibitor(salubrinal),the caspase-12 inhibitor(z ATADfmk)and CHOP silence(by targeted sh RNAs)remarkably attenuated CBG-induced Colon cancer cell death and apoptosis.Further studies demonstrated that CBG in-activated mammalian target of rapamycin complex 1(m TORC1).Introduction of a constitutively-active S6K1(“ca-S6K1”)restored proliferation of CBG-treated Colon cancer cells.CBG intraperitoneal injection suppressed HCT-116 xenograft tumor growth in the nude mice.CHOP upregulation and m TORC1 in-activation were also noticed in HCT-116 tumors with CBG administration.Conclusions: CBG inhibits Colon cancer cell growth in vitro and in vivo.ER stress activation and m TORC1 in-activation could be the primary mechanisms responsible for its actions in Colon cancer cells.