Study On Apoptosis Of Colon Cancer Cells Induced By Novel Isothiourea Modified Curcumin Pyrimidine Derivative 1g And Its Mechanism

Objective:A novel curcumin derivative 1g can inhibit the proliferation of colon cancer in vitro and in vivo.The purpose of this study was to investigate the role of 1g in inducing apoptosis of colon cancer cells,especially mitochondrial apoptosis and endoplasmic reticulum stress induced by reactive oxygen species.Methods:1.Detection of cell activity,apoptosis,cell cycle and membrane potential:Bioinformatics method was used to analyze differentially expressed mRNAs.CCK-8method was used to observe the inhibitory effect of 1g on four colon cancer cell lines HCT116,HT29,SW480 and LOVO.The apoptosis rate of HCT116 and HT29 treated with 1g was detected by Annexin V-FITC/PI double staining.Western Blotting was used to detect the expression of Cleaved-PARP,Cleave-Caspase 9 and Cleaved-Caspase 3apoptosis proteins in HCT116 cells after 1g treatment.The effects of 1g on cell cycle detection points of HCT116 and HT29 were detected by flow cytometry(FACS),and the expressions of p-Rb,Cyclin D1,p21 and p53 were detected by Western Blotting.The expression of Bcl-2,Bax and Cyto C was detected by Western Blotting,and then the effect of 1g on mitochondrial membrane potential of HCT116 and HT29 was detected by JC-1 staining.2.Detection and intervention of ROS: DCFH-DA staining was used to detect the production of reactive oxygen species after 1g treatment of HCT116 cells.The expression of Bcl-2,Bax,Cyto C,Cleaved-PARP,Cleaved-caspase 9,Cleaved-caspase 3,cyclin D1 protein in HCT116 and HT29 cells pretreated with N-acetyl-L-cysteine(NAC,reactive oxygen scavenger)and then treated with 1g was detected by Western Blotting.The apoptosis rate and activity of HCT116 and HT29 cells pretreated with NAC and then treated with 1g were detected by Annexin V-FITC/PI and CCK-8.3.Endoplasmic reticulum stress: The mRNA expression of GRP78 and CHOP after 1g treatment of HCT116 was detected by qRT-PCR.The expressions of PERK,p-PERK,pEIF2α,ATF4,CHOP,IRE1α and BIP in HCT116 cells were detected by Western Blotting.The expressions of PERK,p-PERK,p-EIF2α,ATF4,CHOP,IRE1α and BIP in HCT116 cells were detected by Western Blotting.Western Blotting was used to detect the expression of endoplasmic reticulum stress-related proteins in HCT116 and HT29 cells after NAC pretreatment and 1g treatment.The expression of PERK,p-PERK and CHOP protein in HCT116 cells pretreated with GSK2606414,an inhibitor of p-PERK,and then treated with 1g was detected by Western Blotting.4.Balb/c nude mice transplanted tumor: Balb/c nude mice were subcutaneously injected with HCT116 cells to establish nude mice model,which was divided into four groups: Control,1g(20 mg/kg),1g(40 mg/kg)and Cur(40 mg/kg).After drug treatment,the body weight and tumor volume of mice were observed.The expressions of apoptotic protein Cleaved-caspase 3 and endoplasmic reticulum stress protein CHOP were detected by Western Blotting,and the inhibitory effect of 1g on tumor in vivo was observed by hematoxylin-eosin staining.Results:1.According to KEGG database analysis,the apoptosis pathway of HCT116 cells induced by 1g is ranked first.Through CCK-8,it is concluded that 1g can effectively inhibit the proliferation of colon cancer HCT116,HT29,SW480 and LOVO cells in a time-and dose-dependent manner.According to apoptosis FACS,1g induces apoptosis of HCT116 and HT29 cells.For HCT116 cells,apoptosis is concentration-dependent and 1g with the same dose can promote apoptosis more than Cur.1g upregulates the expression of p21 and p53 protein in HCT116 cells,and inhibits the expression of Cyclin D1 and pRb,thus inducing cell cycle arrest in G0/G1 phase of colon cancer cells.1g induced changes in mitochondrial membrane potential of HCT116 and HT29,and increased expression of Bax/Bcl-2 and Cyto C protein.2.1g can stimulate HCT116 to produce reactive oxygen species in a time-and dosedependent manner.However,after pretreatment with NAC,the generation of reactive oxygen species was eliminated.According to Western Blotting,NAC reversed the protein expressions of Bcl-2,Bax,Cyto C,Cleaved-PARP,Cleaved-caspase 9,Cleaved-caspase 3and cyclind1 in HCT116 and HT29 cells.The apoptosis rate and cell activity were also reversed.3.The mRNA expression of GRP78 and CHOP increased in a time-and concentrationdependent manner after 1g treatment of HCT116.The expression of p-PERK,p-EIF2α,ATF4,CHOP and BIP reached the peak at the corresponding time after 1g treatment.The proteins of p-PERK,p-EIF2α,ATF4,CHOP and BIP increased with the increase of 1g concentration in corresponding time.After pretreatment with GSK2606414,an inhibitor of p-PERK,the protein expressions of p-PERK and CHOP were reversed,which again proved that 1g induced endoplasmic reticulum stress in colon cancer cells.4.The four groups of nude mice did not show significant weight difference and body shape change during administration.In 1g and Cur treatment groups,the growth of tumor was inhibited,but the inhibitory effect of 1g was significantly higher than that of Cur group,and the tumor volume of 1g(40 mg/kg)group was the smallest.Similarly,the expression of Cleave-caspase 3 and CHOP protein in 1g(40 mg/kg)group was significantly higher than that in Cur(40 mg/kg)group.A large number of pathological mitotic features were found in tumor tissues treated with 1g.Conclusion:This study not only found that 1g inherited the safety of curcumin,but also found that 1G stimulated colon cancer cells to produce excessive reactive oxygen species is one of the key factors to induce cell apoptosis.