| Objective:Radiotherapy resistance in patients with non-small cell lung cancer(NSCLC)reduces patient survival and remains a significant challenge for the treatment of NSCLC.Studies have shown that autocrine affects the radiosensitivity of tumors,but in non-small cell lung cancer,the main role of autocrine is not yet clear.In this study,we investigated the effects of autocrine secretions on radiosensitivity of NSCLC cell lines(H460,H1299,and A549)and explore the main mechanism of autocrine secretions to enhance cell radiation resistance from the overall perspective.Methods:Human lung cancer cell lines(H460,H1299,and A549)were used as research objects,and the effects of autocrine secretions on the radiosensitivity of non-small cell lung cancer cells(H460,H1299,A549)were compared using a colony formation assay and Transwell co-culture.Secretion of 293T cells effects on the radiosensitivity of H460 cells.Clone formation experiments were performed to compare the effects of autocrine conditioned medium on the radiosensitivity of H460 cells;MTT assay was used to observe the effect of conditioned medium on the proliferation of H460 cells after irradiation;Flow cytometry was used to detect the effect of conditioned medium on the expression of reactive oxygen species and cell cycle in H460 cells after irradiation;The level of SOD1 mRNA in cells was detected by qRT-PCR,and the level of SOD1 protein in cells was detected by Western blotting;The changes of yH2AX in irradiated H460 cells cultured in conditioned media at different times were observed by immunofluorescence;Changes of ?H2AX and DNA repair related proteins in H460 cells were detected by Western blotting;Differential centrifugation to collect exosomes secreted by H460 cells;Western blotting and transmission electron microscopy experiments were performed to verify the exosomes;Clone formation assays were used to examine the effects of exosomes on radiosensitivity of H460 cells.Results:In this study,the NSCLC cell lines,H460 and H1299 cells,the autocrine factors enhance their radiation sensitivity;the autocrine effect of A549 cell line has no obvious effect on their radiation sensitivity;Paracrine effect of 293T cell line had no significant effect on radiosensitivity of H460 cells;Autocrine promotes the proliferation of H460 cells after irradiation,increases the expression of intracellular ROS and DNA repair capacity.H460 cells showed obvious G2/M cell cycle arrest after irradiation,and the ability of homologous recombination repair was obviously enhanced,while exosomes secreted by H460 cells had no influence on the radiation resistance.Conclusions:Non-small cell lung cancer cells can enhance DNA repair ability to increase their own radiation resistance through autocrine.Objective:By observing the effect of radiation-resistant H460R cells secretion on the radiation sensitivity of parental H460 cells and to explore the role of paracrine in the radiation tolerance delivery of non-small cell lung cancer and its possible mechanism.Methods:The experiment was divided into normal culture group,combined with H460R conditioned medium culture group,simple irradiation group and irradiation combined with H460R conditioned medium culture group.The colony formation assay was used to compare the radiosensitivity of H460 cells and H460R cells.The effect of H460R on the radiosensitivity of H460 cells was observed by using colony formation experiments combined with Tanswell and co-culture experiments.The effect of H460R conditioned medium on the formation of yH2AX foci in irradiated H460 cells was detected by immunofluorescence assay.Flow cytometry was used to detect the change of cell cycle of H460 cells after exposure to H460R conditioned medium.Western blot experiments were performed to observe the expression of DNA damage repair-related proteins.Results:Clone formation experiments showed that the radiosensitivity of H460 cells was higher than H460R cells.The clones of H460 cells treated with H460R-drived conditioned media had a significantly increased compared with the control group after irradiation;the number of yH2AX positive cells decreased;the G2/M phase arrest increased,DNA repair capacity increased.Conclusion:In the tumor microenvironment,the radiation-resistant H460R cells are able to enhance the radioresistance of the radiation-sensitive H460 cells by paracrine,which may be related to the promotion of DNA repair ability after irradiation. |
PDF Full Text Request