The Effect And Molecular Mechanism Of Multikinase Inhibitor PKC412 In Colorectal Cancer Cells

Objective:To investigate the effects of multikinase inhibitor PKC412 on human colorectal cancer in vitro and in vivo,and to further clarify the molecular mechanism underlying the anti-tumor effects of PKC412 against colorectal cancerMethods:MTT assay,trypan blue staining assay and clonogenicity assay were applied to test the potential role of PKC412 on colorectal cancer survival,death and proliferation,respectively.Annexin V-PI FACS assay,Histone DNA ELISA assay,Caspase activity assay and western blot assay were applied to test colorectal cancer apoptosis,caspase activity and caspase expression with PKC412 treatment.The effect of caspase inhibitor on the anti-colorectal cancer cells of PKC412 was also investigated.PI-FACS assay was applied to examine cell cycle progression in PKC412-treated colorectal cancer cells.Role of Bc1-2 in PKC412-mediated anti-colorectal cancer cell acti'vity was tested by knocking down Bc1-2 and forced Bc1-2 overexpression.Western blot assay was performed to test AKT signaling changes in colorectal cancers with PKC412 treatment.HT-29 xenograft nude mice model was applied to analyze the in vivo anti-colorectal cancer activity by PKC412 and the AKT activation in PKC412-treated tumor tissues was also tested by IHC staining assay.Results:PKC412 was cytotoxic and anti-proliferative against colorectal cancer cell line in tine-and dose-dependent manners.PKC412 dose-dependentfy induced apoptosis activation in colorectal cancer cells and increased activity of caspase-3 and caspase-9 in HT-29 cells.Pre-treatment with the caspase inhibitors largely attenuated PKC412-nduced HT-29 cell viability reduction and cell death.PKC412 induced G2-M arrest in colorectal cancer cell's cycle progression.ABT-737,a Bc1-2 inhibitor or Bc1-2 siRNA knockdown,dramatically potentiated PKC412's lethality against colorectal cancer cells.Forced Bcl-2 over-expression,on the other hand,attenuated PKC412's cytotoxicity.AKT activation was inhibited by PKC412 in colorectal cancer cells.Reversely,expression of constitutively-active AKT1(CA-AKT1)decreased the PKC412's cytotoxicity against HT-29 cells.PKC412 oral administration(100 mg/kg body weight,daily)suppressed AKT activation and inhibited HT?29 tumor growth in nude mice.Conclusions:PKC412 inhibits the survival and proliferation of colorectal cancer cells,but also induces apoptosis and G2-M cycle arrest.lts anti-colorectal cancer cell activity in vitro and in vzvo could be due to inhibition of AKT signaling.Be1-2 could be a primary resistance factor of PKC412 in colorectal cancer cells.These results indicate that PKC412 may have potential value for colorectal cancer treatment.