Experimental Study Of A New MTOR Kinase Inhibitor CZ415 Against Human Osteosarcoma Cells

Objective:Using of molecular biology and cell biology to observe the new mTOR kinase inhibitor CZ415 anti OS effect in vitro system and to identify the molecular mechanism of CZ415 anti OS activity.Furthermore,the nude mice model was established to observe the effect of oral CZ4 15 anti OS cell activity in vivo.The aim of this research is to clarify the anti OS effect and molecular mechanism of CZ415.Methods:Cell experiments in vitro:1)MTT assay,clonogenicity assay and BrdU ELISA were preformed to test the potential activity of CZ415 on human osteosarcoma cells survival and proliferation.2)TUNEL staining assay,caspase-9 activity assay,and Histone DNA apoptosis ELISA assay were performed to test the potential role of CZ415 on human osteosarcoma cell apoptosis.3)Propidium iodide(PI)assay was utilized to test cell cycle in CZ415-treated OS cells.4)Western blot assay was showed to test signalings(mTOR,ERK)in CZ415-treated OS cells.5)CZ415's activity against OS cells was tested when ERK was inhibited(by MEK162/U0126)or silenced(by targeted ERK 1/2 shRNAs).Animal experimental in vivo:6)Mice U20S xenograft model was utilized to test the anti-tumor activity of CZ415,alone or in combination with MEK162.Results:Cell experiments in vitro:1)CZ415 potently inhibited survival and proliferation of established OS cell lines(U20S,MG-63 and SaOs2),and primary human OS cells.2)CZ415 provoked apoptosis and disrupted cell cycle progression in OS cells.3)CZ415 treatment in OS cells concurrently blocked mTORC1 and mTORC2 activation.4)ERK inhibition(by MEK162/U0126)or knockdown(by targeted ERK1/2 shRNAs)dramatically sensitized CZ415-induced OS cell death and apoptosis.5)CZ415 plus MEK162 co-administration led to in-activation of mTORC1/2(p-S6KI/p-AKT Ser473)and ERK(p-ERK1/2)in U20S tumor tissues.Animal experimental in vivo:6)CZ415 oral administration efficiently inhibited U20S tumor growth in mice.7)CZ415 activity's in vivo was further potentiated with co-administration of ERK-MAPK inhib tor MEK162.Conclusion:ERK inhibition sensitizes CZ415-induced anti-OS activity in vitro and in vivo.