ABSTRACT
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 0.5 μmol/L,1.0 μmol/L,5.0 μmol/L and 10.0 μmol/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 (10 pmol/L),15 Gy irradiation and 15 Gy irradiation+Met (10 μmol/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,15 Gy 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 10.0 μmol/L was significantly stronger than that of 5.0 μmol/L (P＜0.001).The colone formation rate 34.0％,24.0％,22.3％ and 14.0％ in the control,Met,15 Gy 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 15 Gy irradiation and 15Gy irradiation+Met groups (both P＜0.01).Compared with the 15 Gy 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 15 Gy+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 15 Gy 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 15 Gy+ 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 15 Gy 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.
ABSTRACT
Objective@#To investigate the percentage of myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) in peripheral blood of nasopharyngeal cancer (NPC) patients undergoing concurrent chemoradiotherapy or radiotherapy alone.@*Methods@#Sixty NPC patients who received radiotherapy or concurrent chemoradiotherapy from September 2012 to November 2015 and 20 healthy individuals were included in this study. For the patients, the blood samples were collected at four time points: pre-radiation (Pre-RT), reaching a dose of 40 Gy (RT-40 Gy), finishing radiation (RT-finish) and three months after finishing radiation (3m-post-RT). Flow cytometry was used to evaluate the percentage of Treg (CD4+ CD25+ CD127low/-) and MDSC (HLA-DR-CD11b+ CD33+ ) cells in peripheral blood.@*Results@#Treg and MDSC cells were present in peripheral blood lymphocytes of healthy individuals as a percentage of (7.50±1.62)% and (1.08±0.48)%, respectively. The proportions of peripheral Treg cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (8.42± 1.52)%, (9.10±1.57)%, (8.87±1.56)% and (7.31±1.43)%, respectively, showing a statistically significant difference between Pre-RT and the other groups (P<0.05). At Pre-RT point, the percentage of Treg cells in Stage Ⅲ-Ⅳ patients [(8.63±1.39)%] was higher than that in Stage Ⅰ-Ⅱ [(7.65±1.94)%, P=0.042]. Moreover, the proportions of peripheral MDSC cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (2.14±1.21)%, (4.08±1.90)%, (3.76±1.31)% and (1.52±0.88)%, respectively. The percentages of MDSC cells at RT-40 Gy and RT-finish points were significantly higher than those at Pre-RT, while the percentage of MDSC cells at 3m-post-RT was significantly lower than those at Pre-RT (P<0.05). At Pre-RT point, the percentage of MDSC cells in Stage Ⅲ-Ⅳ patients [(2.25±1.26)%] was higher than that in Stage Ⅰ-Ⅱ [(1.35±0.66)%, P=0.007]. At RT-finish point, the proportions of MDSC and Treg cells in patients with Ⅲ-Ⅳ grade of radiation induced oral mucositis [(4.41±1.27)% and (9.91±1.23)%] were significantly higher than those in Ⅰ-Ⅱ grade patients [(3.15±1.04)% and (8.41±1.52)%, both of P<0.05].@*Conclusions@#The proportions of MDSC and Treg cells in initial treated NPC patients are higher than healthy individuals, and they are also associated with the tumor stages. During the concurrent chemoradiotherapy and radiation, the percentage of MDSC and Treg cells is elevated, suggesting a decreased immune activity. The increase of MDSC and Treg cells is related to radiation induced oral mucositis.