In Vitro And In Vivo Study On The Radiosensitizing Effect Of 17AAG-cypate Polymeric Micelle In Human Lung Cancer A549 Cells

PurposeTo explore the mechanism of 17AAG-cypate polymeric micelles containing 17AAG and cypate enhance the radiosensitivity in human lung cancer A549 cells.Methods and Materials(1)The preparation and characterization of 17AAG-cypate polymeric micelles:17AAG-cypate polymeric micelles were constructed by solvent dialysis method and organic solvent evaporation method.The characteristics of 17AAG-cypate polymeric micelles were investigated by transmission electron microscopy as well as the nano-particle and zeta potential analyzer.(2)To evaluate the in vivo anti-tumor efficacy of the 17AAG-cypate polymeric micelles under the laser and X-ray irradiation:A549 lung adenocarcinoma tumor-burdened nude mice were prepared as first.Then the therapeutic effect was evaluated by the volume change of tumors and HE staining during the treatment.Besides,The toxicity effect of 17AAG-cypate polymeric micelles was measured by the weight change as well as blood routine and blood biochemical indexes.Immunohistochemistry was used to detect the expression of Ki67 and CD31 in tumor tissues to analyze its effects on tumor tissue proliferation ability and microvessel density.The effect on apoptosis of transplanted tumor tissue was analyzed by using TUNEL and Western blot.(3)To evaluate the in vitro anti-tumor activity mechanism of the 17AAG-cypate polymeric micelles under the laser and X-ray irradiation:Cell cycle and cell apoptosis were detected by flow cytometry,Cellular senescence was analyzed by ?-galactose glucoside enzyme dyeing experiment.The repair of DNA double-strand break were detected by immunofluorescence experiment and Western blot was used to investigate the related protein level.Results1.17AAG-cypate polymer micelles containing 17AAG and cypate were successfully constructed,the appearance of the micelles has rounded surrounding and spherical by the transmission electron microscope,the average particle size is(73.7 ±0.07 nm),the zeta potential is(-7.70±1.22)mV,and the poly dispersion index(PDI)is 0.234.The size distribution of micelles is relatively uniform.2.In vivo:(1)The nude mice model experiment showed that 17AAG-cypate polymer micelles combined with X-ray and laser irradiation significantly inhibited the growth of tumor efficiently,and enhanced the radiosensitivity of human lung cancer xenograft tumor.(2)The blood routine and blood biochemical results showed that no toxic effect was found in hematopoietic system,liver and kidney of nude mice after the treatment of 17AAG-cypate polymer micelles,and there was no difference in the weight of mice during the treatment.(3)Immunohistochemistry results showed that 17AAG-cypate micelles combined with X-ray and laser irradiation could reduce the expression of Ki67 and CD31 in tumor tissues.(4)The results of TUNEL and immunohistochemistry showed that 17AAG-cypate micelles combined with X-ray and laser irradiation could dramatically increase the number of TUNEL positive cells in tumor tissues and increase the expression level of cleaved-caspase3.Western blot showed that the expression of total PARP and BCL2 protein decreased,while the expression of cleaved-PARP and cleaved-caspase3 protein increased.3.In virto:(1)Flow cytometry results showed that 17AAG-cypate micelles combined with X-ray and laser irradiation could cause significant G2/M phase arrest and cell apoptosis.(2)The results of ?-galactosidase staining assay showed that 17AAG-cypate micelles combined with X-ray and laser irradiation could significantly promote the proportion of the cellular senescence.(3)Immunofluorescence experiments showed that 17AAG-cypate micelles combined with X-ray and laser irradiation could significantly increase the DNA-damage sites and delay the repair of DNA damage.ConclusionsIn vivo experiments,17AAG-cypate micelles combined with X-ray and laser irradiation could enhance the radiosensitivity of human lung cancer xenograft tumor by affecting the proliferation,apoptosis,and angiogenesis;in vitro experiments,17AAG-cypate micelles combined with X-ray and laser irradiation could increase the radiosensitivity of A549 cells by various ways,including inducing apoptosis,triggering cell senescence,prolonging G2/M phase arrest,increasing the number of DNA double-strand breaks,and reducing the repair of DNA damage.