Effect of combined therapy of radiosurgery and temozolomide on U87-epidermal growth factor receptor and variant III cells and their xenograft

Abstract

Objective To explore the effect of radiosurgery in combination of temozolomide therapy in vitro and in vivo on invasion and endocrine function of human glioma cell line U87-epidermal growth factor receptor and variant III (EGFRvIII) and their xenograft. Methods (1) Human glioma U87-EGFRvIII cells were routinely cultured; CCK-8 was used to detect the effect of temozolomide at different concentrations on proliferation of U87-EGFRvIII cells, and the semi-inhibitory concentration (IC50) of temozolomide was calculated; the U87-EGFRvIII cells were divided into control group, 6 Gy radiotherapy group and 12 Gy radiotherapy group, and the expressions of invasion-related proteins and gene repair proteins in the three groups were detected by Western blotting after 0, 6 and 12 Gy edge-dose irradiation, respectively; the U87-EGFRvIII cells were divided into control group, 6 Gy radiotherapy group and 6 Gy radiation combined with temozolomide treatment group (5 mmol/L temozolomide was added after 6 Gy irradiation), and 24 h after each treatment, Western blotting was used to detect the expressions of invasion related proteins and gene repair related proteins in the three groups. (2) Nude mouse transplantation intracranial glioma models were established with cell suspension of the control group, 6 Gy radiotherapy group and 6 Gy radiation combined with temozolomide treatment group; 7, 14, 21 d after transplantation, bioluminescence imaging (BLI) was employed to detect luciferase expression in nude mice in vivo and measurement of transplanted tumors was performed; survival curve and body mass curve were drew in nude mice; immunohistochemical staining was used to detect the expressions of EGFRvIII, epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF) and Ki67 in intracranial transplanted gliomas. Results (1) The IC50 of temozolomide on U87-EGFRvIII cells was 5 mmol/L; as compared with those in the control group, the expressions of EGFRvIII, EGFR, MMP-2, MMP-9, VEGF, MGMT, AKT-1, β-catenin and Ku70 significantly increased in the cells of the 6 Gy radiotherapy group, while the expressions of EGFRvIII, EGFR, MMP-2, MMP-9, VEGF, MGMT, AKT-1, β-catenin and Ku70 significantly decreased in the cells of the 12 Gy radiotherapy group as compared with those in the 6 Gy radiotherapy group (P<0.05); as compared with those in the 6 Gy radiotherapy group, the expressions of EGFRvIII, EGFR, MMP-2, MMP-9, VEGF, MGMT, AKT-1, β-catenin and Ku70 in cells of the 6 Gy radiotherapy combined with temozolomide group were statistically decreased (P<0.05). (2) As compared with mice in the control group and 6 Gy radiotherapy group, mice in the 6 Gy radiotherapy combined with temozolomide group had significantly lower cell tumorigenicity, significantly higher survival rate and body mass 21 d after transplantation, and significantly smaller volume of xenograft tumors (P<0.05); the expressions of EGFRvIII, EGFR, MMP-9, VEGF and Ki67 in tumor tissues of nude mice in the 6 Gy radiotherapy group were significantly decreased as compared with those in the control group; as compared with those in the 6 Gy radiotherapy group, the expressions of EGFRvIII, EGFR, MMP-9, VEGF and Ki67 in tumor tissues of mice in the 6 Gy radiotherapy group were significantly decreased (P<0.05). Conclusion The combined treatment of radiosurgery and temozolomide can more obviously inhibit the invasiveness of U87- EGFRvIII cells and it exerts a stronger inhibitory effect on tumorigenicity of U87-EGFRvIII cells in brain of nude rats. Key words: Epidermal growth factor receptor and variant III; Mutation; Radiotherapy; Temozolomide; Invasion; Proliferation