二甲双胍增强放射对CT26WT细胞及小鼠移植瘤效应机制研究

目的 探讨二甲双胍(Met)联合放射对结肠癌CT26WT细胞及移植瘤的抑制作用及其机制研究。方法 利用CellTiter-Glo化学发光细胞活性试验检测0.5、1.0、5.0、10.0μmol/L的Met对CT26WT细胞活力影响,克隆形成试验检测对照组、10.0μmol/L的Met、15Gy照射、15Gy+10.0μmol/L的Met组对CT26WT细胞的增殖抑制作用。构建Bablc小鼠皮下移植瘤模型,肿瘤体积>150mm³随机分对照组、单纯15Gy照射、Met组、15Gy+Met组,照射前24h给予小鼠750 mg/kg的Met,定期测量肿瘤体积及小鼠体重绘制肿瘤生长曲线及生存时间曲线。蛋白质印迹法检测上述处理条件下CT26WT细胞及移植瘤组织中P-H2AX、Sting蛋白表达;并利用免疫组化方法检测移植瘤组织中CD8a (+) T细胞的浸润情况。结果 0、0.5、1.0、5.0、10.0μmol/L的Met的相对细胞存活率分别为100%、87.9%、87.8%、87.3%、76.5%(P<0.05),其中10.0μmol/L较5.0μmol/L抑制作用更强(P<0.001)。克隆形成实验结果显示对照组、Met组、15Gy组、15Gy+Met组细胞克隆形成率分别为34.0%、24.0%、22.3%、14.0%(P<0.001)。与对照组比较,Met组、15Gy组、15Gy+Met组细胞内Sting蛋白表达分别增加2.99、1.37、4.41倍(P<0.001、<0.01、<0.001)。15Gy+Met组P-H2AX蛋白表达较15Gy组增加1.43倍(P<0.001)。移植瘤体积15Gy+Met组较对照组生长缓慢,最终结果为(1007.0±388.5)、(2639.0±242.9) mm³,(P<0.05),15Gy+Met组小鼠总生存期较对照组增加(48d︰32d,P<0.001)。移植瘤组织中P-H2AX、Sting蛋白表达量在15Gy+Met组较对照组分别增加8.8、1.6倍(P均<0.001)。15Gy+Met组CD8a (+) T细胞浸润在较对照组明显增高(P<0.01)。结论 Met与放射联合能协同抑制结肠癌细胞增殖、克隆形成,可能作用机制是通过加重DNA损伤、激活Sting信号通路导致肿瘤组织中CD8a (+) T细胞增加加强对肿瘤细胞的杀伤作用。

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) mm³ vs. (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.