To explore the effects and molecular mechanisms of tenacissoside G (TG) on the proliferation of colorectal cancer,we determined the proliferation inhibitory effects of TG on colorectal cancer (CRC) cell lines RKO and LoVo by CCK-8 assay and colony formation.After 48 h treatment of TG,flow cytometry was used to analyze the cell cycle distribution of CRC cell lines;the comet assay and immunofluorescence were adopted to detect the DNA damage degree and the expression level of γ-H2AX;and the expression level of cell cycle and apoptosis related proteins was detected by Western blot.The results showed that TG significantly inhibited the proliferation and colony formation of RKO and LoVo in a dose and time dependent manner.After 48 h treatment of TG,the IC₅₀ values of TG on RKO and LoVo were 91.71 and 88.34 μM respectively;the proportion of cells of G0/G1 phase significantly increased (P< 0.01) and marked DNA damage was detected with an obvious increase in the expression of γ-H2AX.The expression of cell cycle related proteins including CDK2,CDK4,CDK6,Cylin D1,and Cyclin E was remarkably downregulated (P < 0.01),however,the expression of cleaved Caspase3,cleaved PARP,p-ATM,p-CHK1,and p-p53 was remarkably upregulated (P < 0.01).Meanwhile,p-ATM/ATM,p-CHK2/CHK2 and p-p53/p53 were significantly increased (P< 0.01).In conclusion,TG exerted proliferation inhibition through DNA damage,which further activated cell cycle arrest and apoptosis mediated by ATM-CHK2-p53 signaling pathway.