Study of the mechanism of anti-tumor effect of Metformin-enhanced radiotherapy in CT26WT cell lines or mouse models with transplanted tumors

Objective To investigate the inhibitory effect and mechanism of Metformin (Met) combined with irradiation in CT26WT cell lines or mouse models with transplanted tumors. Methods CT26WT cell line was treated with 0.5μmol/L, 1.0μmol/L, 5.0μmol/L and 10.0μmol/L Met, and CellTiter Glo kit was used to detect the inhibitory effect of Met at different concentrations on the viability of CT26WT cells. CT26WT cell line was treated with the control, Met (10μmol/L), 15Gy irradiation and 15Gy irradiation+ Met (10μmol/L). Clone formation assay was employed to detect the cell proliferation activity. Bablc mouse models of transplanted tumors (tumor size>150 mm3) were established and randomly divided into the control, 15Gy irradiation, Met and 15Gy irradiation+ Met groups. Mice were given with 750 mg/kg Met at 24 h before irradiation. Transplanted tumor volume was measured regularly to delineate the growth curve of transplanted tumors and survival curve. The expression levels of P-H2AX and Sting proteins in CT26WT cells and transplanted tumors were detected by Western blot. The infiltration of CD8a (+ ) T cells in transplanted tumor tissues was detected by immunohistochemistry. Results The relative cell survival rate was 100%, 87.9%, 87.8%, 87.3% and 76.5% in the 0, 0.5, 1.0, 5.0 and 10.0μmol/L Met groups, respectively (all P<0.05). The inhibitory effect of 10.0μmol/L was significantly stronger than that of 5.0μmol/L (P<0.001). The colone formation rate 34.0%, 24.0%, 22.3% and 14.0% in the control, Met, 15Gy irradiation, Met+ 15Gy irradiation groups, respectively (all P<0.001). Western blot showed that compared with the control group, the expression of Sting protein was increased by 2.99-fold after Met treatment (P<0.001), and increased by 1.37-fold and 4.41-fold in the 15Gy irradiation and 15Gy irradiation+ Met groups (both P<0.01). Compared with the 15Gy irradiation group, the expression of P-H2AX protein was significantly increased by 1.43 times after treatment with 15Gy+ Met (P<0.001). The transplanted tumor growth curve showed that the transplanted tumor growth in the 15Gy+ Met group was slower than that in the control group[(1007.0±388.5) mm3vs. (2639.0±242.9) mm3, P<0.05)]. The overall survival time in the 15Gy irradiation+ Met group was 48 d, significantly longer than 32 d in the control group (P<0.001). Compared with the control group, the expression of P-H2AX and Sting proteins in the 15Gy+ Met group was increased by 8.8-fold and 1.6-fold (both P<0.001). Immunohistochemical staining showed that the infiltration of CD8a (+ ) T cells in the 15Gy irradiation+ Met group was significantly higher than that in the control group (P<0.01). Conclusions Met combined with radiotherapy can inhibit the proliferation and clone formation of colon cancer cells, probably by aggravating DNA damage and activating the Sting signaling pathway, eventually leading to the increase of CD8a (+ ) T cells in tumor tissues and enhancing the killing effect upon transplanted tumor cells. Key words: Metformin; Sting gene; H2AX gene; CT26WT cell line; Transplanted tumor/Bablc mouse