| [Objective]The present study was aimed to explore the toxic and radiosensitizing effects of gambogric acid(GA)on TE13 cells.We also discussded the underlying mechanisms,providing the theoretical foundation for the application of GA as radiosensitizer in clinic.[Materials and methods](1)Cell counting kit-8(CCK-8)assay was used to evaluate the proliferation inhibition function of GA in TE13 cells and to determine the best working concentrations of GA;(2)The clone forming experiment tested the radiotherapy sensitization effect of GA.The colonies which contained more than 50 cells were counted under the microscope;(3)Western blot(WB)was carried out to detect the expression levels of microtubule-associated protein 1 light chain 3(LC3),apoptosis related proteins caspase-3,caspase-8,caspase-9,phosphorylation-serine/threonine kinase(p-Akt)and phosphorylation-mammalian target kinase of rapamyci(p-mTOR)proteins;(4)The effects of different treatments on apoptotic rate of esophagus cancer cells were detected by flow cytometry;(5)Immunofluorescence was performed to monitor the distribution of LC3;(6)The cells were infected with red fluorescent protein(RFP)-green fluorescent protein(GFP)-LC3 adenovirus according to mRFP-GFP-LC3 instruction manual.The rationale of the method is based on the difference of pH between the neutral autolysosomes and acidic autophagosomes.RFP was used for marking and tracking LC3 while the degradation of GFP indicated the formation of autolysosomes.After the images were emerged,the yellow dots represent autophagosomes while the red ones represent autolysosomes.Images were captured by laser scanning confocal microscope(LSCM).By this way,the progress of autophagy in TE13 cells was monitored;(7)The mean intensities of DCFH-DA signal which represented the state of reactive oxygen species(ROS)were obtained by LSCM. [Results](1)The proliferative inhibition of TE13 cells caused by different concentrations of GA was in positive correlation to dose and time.The IC50 values were 1.47 and 1.14 ?mol/L for 24 and 48h,respectively;(2)The selection of working cincentrations of GA,time of exposure and the irradiation dose:we chose the 0.25 and 1 ?mol/L as the working concentrations,as the survival rates of TE13 cells didn't change significantly after treated with GA(0.25 or 1 ?mol/L)for 24h.After the cells were treated with different concentrations of GA combined with different irradiation doses,the results of WB showed that the expression of LC3-II/LC3-I reached the maximum ratio when the irradiation dose was 6Gy.(3)GA increase the radiosensitivity of human esophageal TE13 cells:after treated with GA(0.25 ?mol/L or 1 ?mol/L)for 24h,the clone formation rate of the combined therapy group was lower than irradiation(IR)alone group significantly.The SERD0 were 1.217 and 1.436;(4)GA can promote the apoptosis of TE13 cells;(5)GA induced the production of autophagy and impaired the autophagy flow:GA treatment alone group caused increased expression of LC3-II.Compared to the IR alone group,the combined treatment group increased the ratio of LC3-II/LC3-I.The experiment of adenovirus infection showed that in GA treatment alone group the number of autophagosomes increased significantly than in the blank group and the combined treatment induced more autophagosomes than irradiation alone.However.the number of autolysosomes didn't change significantly;(6)GA caused the increased load of ROS in cells and may play a part in radiosensitivity by affecting the Akt/mTOR pathway.LSCM was used to measure the intensities of intracellular ROS.The results showed that GA could lead to the accumulation of ROS and the inhibition of Akt/mTOR pathway.After adding ROS inhibitors,the expression of LC3-II,apoptosis related proteins and the rate of apoptosis were significantly decreased while the expression levels of p-Akt,p-mTOR were raised.[Conclusion]GA induced radiosensitivity involves apoptosis and autophagy which were regulated by ROS hypergeneration and Akt/mTOR pathway inhibition. |
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