Knockdown Of TIGAR Enhances The Antitumor Activity Of β-Escin On The Colorectal Carcinoma Cells And Its Underlying Mechanisms

Aim: To observe the effects of β-escin on TIAGR expression and its role in the antitumor activity of β-escin on the colorectal carcinoma cells. To explore the underlying mechanisms of the effect of TIGAR expression on the antitumor activity of β-escin on colorectal carcinoma cells.Methods: The expression of TIGAR protein after β-escin treatment was detected by Western Blot. Transient transfection of TIGAR siRNA was used to knockdown TIGAR expression and CCK8 test or colony formation analysis were used to detect cancer cell survival after TIGAR was knocked down with or without treatment of β-escin. Annexin V-FITC/PI double dyes were used to stain the cells and flow cytometry assay(FCM) was performed to observe cell apoptosis. Apoptosis-related proteins were detected by Western Blot. Intracellular ROS levels were analyzed by DCFH2-DA and DHE. Comet assay was used to explore DNA damage. Protein levels related to DNA damage repair such as p-ATM and Cdk5 were detected by Western Blot.Results: To detect the effect of β-escin on TIGAR expression in HCT116 cells, HCT116 cells were treated with different concentration of β-escin for different length of time and the protein of TIGAR expression was detected by Western Blot. Results showed that TIGAR was increased after the treatment with β-escin after HCT116 cells treated with β-escin at a low concentration for short length of time. The expression of TIGAR decreased if the concentration of β-escin was high or if HCT 116 cells were treated with β-escin for a long time. To further detect the role of TIGAR expression on the antitumor activity of β-escin on the colorectal carcinoma cells, TIGAR siRNA was used to knockdown TIGAR expression. Results showed that knockdown of TIGAR obviously inhibited cell viability and increased cell apoptosis detected by CCK8 assay, colony formation assay and FCM assay.Many mechanisms are involved in the treatment of cancer cells. ROS adaptive response and cell DNA damage response were important in the treatment of cancer cells. TIGAR was reported to inhibit cell glycolysis, induce pentose phosphate pathway(PPP) to generate NADPH and to lower ROS levels. Thus to reduce ROS associated cell apoptosis, ROS induced cell autophagy and ROS induced DNA damage. So we further detected the underlying mechanisms of TIGAR on the antitumor activity of β-escin to the colorectal carcinoma cells from two aspects including cell apoptosis and cell DDR.Firstly, knockdown of TIGAR induced cell apoptosis after HCT116 cells treated with β-escin. The increased cell apoptosis was caused by the increased ROS levels and activated Caspase-3 activity. It was indicated that knockdown of TIGAR enhanced the antitumor activity of β-escin which was associated with knockdown of TIGAR increasing cell apoptosis.Secondly, knockdown of TIGAR enhanced the DNA damage of HCT116 cells treated with β-escin.The effects were partly reversed by the supplementation of PPP products NADPH. Further studies revealed that TIGAR regulated the phosphorylation of ATM, a key protein in DDR, through Cdk5. TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway. To sum up, TIGAR regulates DDR through PPP and Cdk5-ATM pathway in HCT116 cells after treatment of β-escin and the regulation of TIGAR on DDR was related with the antitumor activity of β-escin on the colorectal carcinoma cells.Conclusion: β-escin induced TIGAR expression in HCT116 cells. Knockdown of TIGAR increased the antitumor activity of β-escin on the colorectal carcinoma cells and the mechanisms of apoptosis and DDR were involved in. The protective effect of TIGAR on colorectal carcinoma cells survival suggests that TIGAR would be a therapeutic target for colorectal carcinoma which provides theoretical foundation for cancer therapy especially the antitumor activity of β-escin on the colorectal carcinoma.