| Objective:To investigate nimotuzumab’s effect on the sensitivity of EC cell to radiotherapy and discuss the associated possible mechanisms, aiming at providing evidence for clinical application.Methods:1 The expression of EGFR on esophageal carcinoma cell line: ECA-109、TTN、TE-1、TE-13、YES-2、EC9706 and KYES-450 was detected by Western blot.2 The ECA-109 cells were divided into 4 groups: control group, irradiation group(4Gy), medicine group(900n M nimotuzumab), and combined group(900n M nimotuzumab + 4Gy irradiation). The expression of EGFR and p-EGFR were detected by Western Blot.3 The sensitivity of ECA-109 cells to radiation was detected by clone formation assay.4 Cell cycle distribution and apoptosis rate of each group were detected with flow cytometry to assay.5 We used confocal microscopy to detect the differences of nucleus γH2AX foci formation among the ECA-109 cell groups.6 The expression of Akt、p-Akt、DNA-PKcs、p-DNA-PKcs andγH2AX protein of each group was detected by Western Blot.Result: 1 EGFR protein expression in various EC cell linesEGFR was expressed in ECA-109、TTN、TE-1、TE-13、YES-2、EC9706 and KYES-450 cell lines. The differences of EGFR expression level among these seven kinds of cell lines were of significance(F=10.00, P<0.05). Furthermore, the expression of EGFR in ECA-109 cells was higher than those in TTN、TE-1、TE-13、YES-2、EC9706 and KYES-450(P<0.05), while the differences between YES-2, EC9706 and KYES-450 were not significant(P>0.05). 2 Effects of nimotuzumab on the expression of EGFR and p-EGFR of ECA-109 cellAmong 4 ECA-109 groups, there were no significant difference of the EGFR expression level. Compared with the control group, the expression of p-EGFR in radiation group was significantly higher(P<0.05). In addition, compared with the radiation group and medicine group, the expression of p-EGFR in combined group was significantly lower(P<0.05). 3 Effects of nimotuzumab on the sensitivity of ECA-109 cells to radiationClone formation assay showed that: The value of Dq、D0 and SF2 in Nimotuzumab combined with radiation group was significantly lower than those in the radiation group(p<0.05) with SER equal to 1.35. 4 Effects of nimotuzumab on cell cycle and apoptosis of ECA-109 cellsFlow cytometry results showed that: Nimotuzumab increased G1/G0 phase arrest rates and combined group could obviously increase G2/M phase arrest.The value of control group、radiation group、medicine group and combined group are as follows:(2.76 ± 0.23)%,(19.75 ± 1.11)%,(9.54 ± 0.52)% and(41.31 ± 1.52)%. Compared with radiation groupapoptos is in the combination group increased obviously. 5 Study on mechanisms of nimotuzumab’s effect on sensitivity of ECA-109 to radiation5.1 Confocal microscopy results showed that: Confocal microscopy to detect changes each group of esophageal ECA-109 nucleus γH2AX foci formation, the red and purple highlights for γH2AX foci. Combined group than irradiation group and medicine group γH2AX foci significantly increased(P <0.05).5.2 Western blot results showed that: The expressions of Akt and DNA-PKcs protein were not of significant difference in various groups(P>0.05). Compared with those in the control group, p-Akt and p-DNA-PKcs in the radiation group were impressively higher. Compared with the radiation group and medicine group, p-Akt and p-DNA-PKcs protein expression in the consociation group were significantly decreased(P<0.05), γH2AX in the radiation group and the medicine group increased significantly compares with the control group(P<0.05). Compared with the radiation group and monotherapy group, γH2AX protein expression in the consociation group significantly was increased(P<0.05).Conclusions:Nimotuzumab could increase esophageal cancer ECA-109 cells sensitivity to radiotherapy and the possible mechanisms might be that nimotuzumab could inhibit the downstream signaling of EGFR and down-regualte the expression of p-Akt to block DNA damage repair. |
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