Inhibitory Mechanism Of 8-O-Acetylharpagide On HCT116 Cells

Objective:To explore the mechanism of 8-O-Acetylharpagide inhibiting the proliferation of human colon cancer HCT116 cells.Methods:HCT116 cells were treated with different concentrations of 8-O-Acetylharpagide for 24,48 and 72h.and the toxicity of 8-O-Acetylharpagide on HCT116 cells was tested by CCK8.The morphological changes of HCT116 cells treated with 8-O-Acetylharpagide were observed with DiO green fluorescent probe.Cell migration assay was used to detect the effect of 8-O-Acetylharpagide on HCT116 cell invasion ability.The effect of 8-O-Acetylharpagide on HCT116 cell cloning ability was detected by plate clone formation assay.The effect of 8-O-Acetylharpagide on HCT116 cell cycle and apoptosis was detected by flow cytometry.High-throughput transcriptome sequencing was used to screen the differentially expressed genes of HCT116 cells treated with 8-O-Acetylharpagide.The mRNA expression levels of β-catenin,GSK-3β,APC,CyclinD1,Survivin,c-myc,Axin-1,Bax and Bcl-2 in HCT116 cells treated with 8-O-Acetylharpagide were detected by RT-qPCR.The protein expressions of β-catenin,GSK-3β,APC,CyclinD1,Survivin,c-Myc,Axin-1,Bax,Bcl-2,Pro-caspase3 and Cleaved-caspase3 in HCT116 cells treated with 8-O-Acetylharpagide were tested by Western Blot.Results:8-O-Acetylharpagide could significantly inhibit the growth of HCT116 cells after 24,48 and 72h.8-O-Acetylharpagide showed a concentration and time dependence.DiO green fluorescence staining showed that the number of HCT116 cells decreased significantly after 8-O-Acetylharpagide treatment.Transwell and plate cloning experiments showed that 8-O-Acetylharpagide could reduce the invasion and cloning ability of HCT116 cells.The flow cytometry showed that G1 phase arrest and increased apoptosis rate of HCT116 cells.Transcriptome sequencing showed that 8-O-Acetylharpagide induced a total of 1921 up-regulated mRNAs and 2208 down-regulated mRNAs in HCT116 cells.KEGG differential gene enrichment analysis showed that 8-O-Acetylharpagide mainly inhibited the growth of HCT116 cells through Wnt/β-catenin signaling pathway.RT-qPCR showed that compared with the control group,the mRNA expressions of β-catenin,CyclinDl,Survivin,Bcl-2 and c-Myc in HCT116 cells were significantly decreased after 8-O-Acetylharpagide treatment for 48h.And the mRNA of Bax was significantly up-regulated(P<0.05).While the mRNA expression of APC,GSK-3β and Axin-1 gene was up-regulated but the difference was not statistically significant(P>0.05).Western Blot results showed that β-catenin,c-Myc,Survivin,CyclinDl,Bcl-2,Pro-caspase3 proteins were significantly down-regulated.Bax,cleaved casepase3 proteins were significantly up-regulated(P<0.05).And APC,GSK-3β,Axin-1 proteins were up-regulated,but the difference was not statistically significant(P>0.05).Conclusion:8-O-Acetylharpagide can significantly reduce the survival rate of HCT116 cells.8-O-Acetylharpagide can also weaken the invasion and cloning ability of HCT116 cells,which induce the cell cycle arrest in the G1 phase and induce the cell apoptosis reaction in vitro.The inhibition of 8-O-Acetylharpagide on HCT116 cells is mainly achieved by inhibiting the Wnt/β-catenin signaling pathway.