Mechanism Of Apoptosis Induced By Binimetinib Through Death Receptor DR5 In Colon Cancer Cells

BackgroundColorectal cancer（CRC）is the most common fatal cancer in women and men worldwide.In 2020,CRC ranked second in mortality and third in morbidity.Binimetinib is a mitogenactivated protein kinase 1/2（MEK1/2）inhibitor currently in phase iii clinical trials.Apoptosis is a normal physiological process of highly regulated cell death that occurs in most multicellular organisms.There are two main apoptosis pathways: exogenous death receptor pathway and endogenous mitochondrial apoptosis pathway.Early studies have shown that Binimetinib induces apoptosis of colon cancer cells through endoplasmic reticulum stress signaling pathway,but the specific mechanism of Binimetinib inducing apoptosis remains to be further studied.ObjectivesTo explore the specific mechanism of Binimetinib inducing apoptosis of colon cancer cells,and to verify the effect of Binimetinib combined with chemotherapy drugs 5-Fu and TRAIL on colon cancer cells.It provides a new therapeutic idea for clinical treatment of colon cancer.Methods1.Colon cancer HCT-116 and Ca Co2 were treated with different concentrations of MEK1/2 inhibitor Binimetinib,and the changes of mitochondrial membrane potential were detected by flow cytometry2.Colon cancer cells HCT-116 and Ca Co2 were treated with different concentrations of MEK1/2inhibitor Binimetinib,and the expression changes of related proteins in endogenous apoptosis pathway were detected by WB.3.Colon cancer cells HCT-116 and Ca Co2 were treated with different concentrations of MEK1/2inhibitor Binimetinib,and the expression levels of apoptosis protein Cleaved caspase-8 were detected by WB4.CCK8 was used to detect the effects of MEK1/2inhibitor Binimetinib on the growth of HCT-116 and CaCo2 in shcaspase-8 colon cancer cells.5.Cleaved caspase-8 and Cleaved caspase-3 expressions of apoptosis-related proteins induced by MEK1/2inhibitor Binimetinib in shcaspase-8 colon cancer cells were detected by WB.6.Colon cancer cells HCT-116 and Ca Co2 were treated with different concentrations of MEK1/2inhibitor Binimetinib,and the expression levels of death receptor-related RNA were detected by QPCR.7.Colon cancer cells HCT-116 and Ca Co2 were treated with different concentrations of MEK1/2inhibitor Binimetinib,and the expression of induced DR5 protein was detected by WB.8.Interference with DR5,CCK8 was used to detect the inhibition of MEK1/2 inhibitor Binimetinib on the growth of HCT-116 and Ca Co2 in colon cancer cells.9.Interference with DR5,WB was used to detect DR5,Cleaved caspase-8,Cleaved caspase-3 protein expression levels in HCT-116 and Ca Co2 induced by MEK1/2 inhibitor Binimetinib.10.MEK1/2inhibitor Binimetinib combined with TRAIL and 5-FU,CCK8 was used to detect the growth of HCT-116 and Ca Co2 in colon cancer cells.11.MEK1/2inhibitor Binimetinib combined with TRAIL and 5-FU was used to detect the apoptosis rate of HCT-116 and Ca Co2 cells by flow cytometry.12.Cleaved caspase-8 and Cleaved caspase-3 were detected by WB in combination with MEK1/2 inhibitor Binimetinib,TRAIL and 5-FU.13.Construct subcutaneous tumor of colon cancer in nude mice,and MEK1/2 inhibitor Binimetinib combined with TRAIL and 5-FU to treat subcutaneous tumor in nude mice.Results1.Different concentrations of MEK1/2 inhibitor Binimetinib could not cause changes in mitochondrial membrane potential of HCT-116 and Ca Co2 in colon cancer2.Different concentrations of MEK1/2 inhibitor Binimetinib could not induce changes in the expression of related proteins in the endogenous apoptosis pathway of colon cancer cells.3.MEK1/2 inhibitor Binimetinib induced increased expression of apoptosis protein Cleaved caspase-8 in colon cancer cells4.MEK1/2 inhibitor Binimetinib inhibited the growth of shcaspase-8 colon cancer cells.5.Cleaved caspase-8 and Cleaved caspase-3 were reduced by MEK1/2inhibitor Binimetinib in shcaspase-8 colon cancer cells.6.MEK1/2 inhibitor Binimetinib induced increased expression of DR5 RNA in death receptor signaling.7.MEK1/2 inhibitor Binimetinib induces increased DR5 protein expression.8.Interference with DR5,MEK1/2 inhibitor Binimetinib inhibited the growth of colon cancer cells.9.By interfering with DR5,Cleaved caspase-8 and Cleaved caspase-3 proteins were reduced by MEK1/2 inhibitor Binimetinib in colon cancer cells.10.MEK1/2 inhibitor Binimetinib combined with TRAIL and 5-FU inhibited the survival of HCT-116 and Ca Co2 in colon cancer cells.11.MEK1/2 inhibitor Binimetinib combined with TRAIL and 5-FU induced increased apoptosis rate of HCT-116 and Ca Co2 in colon cancer cells.12.Cleaved caspase-8 and Cleaved caspase-3 expression were induced by MEK1/2 inhibitor Binimetinib combined with TRAIL and 5-FU.13.MEK1/2 inhibitor Binimetinib combined with TRAIL and 5-FU inhibited the growth of subcutaneous tumors in nude mice.ConclusionsMEK1/2 inhibitor Binimetinib did not induce apoptosis of HCT-116 and Ca Co2 through the mitochondrial pathway.MEK1/2 inhibitor Binimetinib induces apoptosis of HCT-116 and Ca Co2 through death receptor DR5.MEK1/2 inhibitor Binimetinib combined with chemotherapy drugs 5-FU and TRAIL could inhibit the growth of colon cancer cells and induce apoptosis of colon cancer cells.In vitro functional tests confirmed that MEK1/2 inhibitor Binimetinib combined with chemotherapy drugs 5-FU and TRAIL inhibited subcutaneous tumor growth in nude mice.It provides a new therapeutic idea for clinical treatment of colon cancer.