The Effect And Mechanism Of The Fusion Protein Anti-EGFR-iRGD On Radiation Sensitization In Gastric Cancer

Radiotherapy is an important treatment for patients with local advanced gastric cancer,and the radiation sensitizer can improve the therapeutic effect of radiotherapy.However,currently the radiotherapy sensitizers are mostly chemotherapeutic or targeted drugs.The former increases both the effectiveness of radiotherapy and the side effects and the latter will possibly induce drug resistance.High expression of epidermal growth factor receptor(EGFR)is observed in some patients with gastric cancer,suggesting poor prognosis and resistance to radiotherapy.We constructed a fusion protein(anti-EGFR-iRGD)that is a bispecific antibody with high penetration against tumor and investigated its antitumor effect on gastric cancer cells.This paper attempts to deal with(1)the sensitivity and safety evaluation of fusion protein anti-EGFR-iRGD in radiotherapy sensitization for gastric cancer;and(2)the mechanism underlying the effects of fusion protein anti-EGFR-iRGD on the radiotherapy sensitization of gastric cancer.Part ? In vivo and in vitro evaluation of the Fusion Protein anti-EGFR-iRGD for Gastric Cancer sensitizationObjective:To investigate the effect of fusion protein anti-EGFR-iRGD on the sensitization of gastric cancer in vivo and in vitro,and evaluate the safety in vivo of fusion protein anti-EGFR-iRGD combined with radiotherapy.Methods:Western blotting is used to confirm the expression of EGFR in three gastric adenocarcinoma cells(SNU-719,BGC-823 and HGC-27).The cytotoxicity and radiosensitization of the fusion protein anti-EGFR-iRGD is investigated by clonogenic assay and MTT test.The flow cytometry and Western blotting are used to explore the radiosensitization effect of fusion protein anti-EGFR-iRGD in gastric adenocarcinoma cells(SNU-719,BGC-823 and HGC-27).A subcutaneous tumor model of BGC-823 in nude mice is constructed to investigate the effects of anti-tumor,weight loss and the in vivo safety of fusion protein anti-EGFR-iRGD combined with radiotherapy.Results:1.The expression level of EGFR in gastric adenocarcinoma cells by Western blotting is ranked as HGC-27 < SNU-719 < BGC-823.2.MTT assay is used to detect the cytotoxic effects of different concentrations of fusion protein anti-EGFR-iRGD on three gastric adenocarcinoma cells.The fusion protein anti-EGFR-iRGD produces better inhibitory effect on the proliferation of SNU-719 and BGC-823 cells,and the cytotoxic effect depends on the concentration.The fusion protein anti-EGFR-iRGD showes no cytotoxic effect on HGC-27.3.Cloning test confirmes that the fusion protein anti-EGFR-iRGD shows radiosensitization effect in gastric adenocarcinoma cells with high expression of EGFR(SNU-719 and BGC-823),however,there is no radiosensitization effect in gastric cancer cell(HGC-27)without EGFR expression.4.Flow cytometry analysis in SNU-719 and BGC-823 cell lines indicates that the number of apoptotic cells in combined treatment group(radiotherapy pretreated with fusion protein anti-EGFR-iRGD)compared to the single group(fusion protein group or radiotherapy group)increases significantly(P < 0.0001).In the HGC-27 cell line,the apoptosis of the combined group doesn't increase significantly(P > 0.05).5.The fusion protein anti-EGFR-iRGD combined with radiotherapy significantly inhibited the growth of subcutaneous tumor in tumor bearing nude mice(BGC-823).There is no significant difference in the body weights of the nude mice between the fusion protein anti-EGFR-iRGD group and the control group while there is significant difference between the control group against the radiotherapy group and the combined treatment group.However,there shows no significant difference in the body weights of the nude mice between 3 treatment groups(P>0.05).Conclusion:The fusion protein anti-EGFR-iRGD better inhibits the proliferation of gastric cancer cells with expression of EGFR in a concentration-dependent manner and enhances the apoptosis of gastric adenocarcinoma cells with the expression of EGFR in tumor radiotherapy.The combined radiotherapy shows significant inhibitory effects on subcutaneous transplantated tumor in nude mice,but with good safety and no adverse reaction.The fusion protein anti-EGFR-iRGD is a highly selective radiosensitizer for the highly expressed EGFR in gastric adenocarcinoma cells and xenografts.Part ? The Mechanism Underlying the Effect of FusionProtein anti-EGFR-iRGD on Radiation Sensitization inGastric CancerObjective:To investigate the mechanism of the effects of fusion protein anti-EGFR-iRGD on the radiotherapy sensitization in gastric cancer.Methods:Immunohistochemical method is used to confirm the effect of radiotherapy on the expression of EGFR in gastric adenocarcinoma cell line BGC-823.The effect of fusion protein anti-EGFR-iRGD combined with radiotherapy on the expression of EGFR in gastric adenocarcinoma BGC-823 cells is examined by Western blotting.Immunofluorescence is used to confirm that radiotherapy enhances the penetration of fusion protein anti-EGFR-iRGD into solid tumor tissue.The effects of fusion protein anti-EGFR-iRGD combined with radiotherapy on the expression,proliferation and necrosis of BGC-823 cell xenografts in gastric adenocarcinoma are investigated by the immunohistochemical method.Results:1.After ionizing radiation,the expression of EGFR in gastric adenocarcinoma BGC-823 cells or tumor tissues up-regulates to certain degrees.However,it down-regulates after ionizing radiation that lagged 24 hours of the pretreatment on fusion protein anti-EGFR-iRGD(concentration of 100?g/ml).2.After pretreatment with fusion protein anti-EGFR-iRGD for 24 hours,the cells proportion in phase G1 increases by 16.54% in SNU-719 cells and 22.53% in BGC-823 cells respectively,while the cells proportion in phase S decreases by 8.52%and 10.37%.After pretreatment for 48 hours,the cells proportion in phase G1 increases by 13.48% in SNU-719 cells and 20.7% in BGC-823 cells respectively,but decreases by 8.52% and 9.29% for cells in phase S.However,only minor changes occurred in HGC-27 cells.3.After 24-hour radiation on the subcutaneous tumor in nude mice with gastric adenocarcinoma(BGC-823)and the injection of fusion protein anti-EGFR-iRGD through the tail vein,the fusion protein anti-EGFR-iRGD in irradiated tumor tissue is more extensively observed than un-irradiated tumor tissue.4.The expression of EGFR is the lowest in the combined treatment group,lower in both the radiotherapy group and the fusion protein anti-EGFR-iRGD group,but most in the control group.The number of Ki67 proliferating cells is the least in the combined treatment group and most in the control group while stands between in both anti-EGFR-iRGD group and radiotherapy group.TUNEL staining results showed that the number of apoptotic cells in the combined treatment group was slightly higher than that in the radiotherapy group and the fusion protein group.However,there was no statistical difference.5.There is almost no tumor necrosis in the control group,however there is a wider necrosis areas in fusion protein anti-EGFR-iRGD group and the radiotherapy group,while the widest necrosis area appears in combined treatment group.Conclusion:The expression level of EGFR in tumor cells increases after irradiation.After the treatment of radiotherapy combined with fusion protein anti-EGFR-iRGD,the expression level of EGFR in tumor cells decreases in both tumor cells and solid tumor tissues.The fusion protein anti-EGFR-iRGD regulates the cell cycle of the tumor,i.e.,increases the proportion of cells in phase G1 and reduces the proportion of cells in the phase S.Radiotherapy promote more fusion protein anti-EGFR-iRGD into the solid tumor tissue,which expands its distribution and enhances its anti-tumor effect.The fusion protein anti-EGFR-iRGD combined with radiotherapy enhances the effects of the radiotherapy to inhibit tumor cell proliferation and promote the tumor necrosis.The fusion protein anti-EGFR-iRGD is possibly an potential effective treatment for EGFR targeting therapy combined with radiotherapy.