Chloroquine Enhances The Radiosensitivity Of Bladder Cancer Cells By Inhibiting Autophagy And Activating Apoptosis

?Purpose?Chloroquine was formerly used as an anti-malarial agent drug but has now been proven to be useful for various diseases.This study aimed to investigate the radiosensitizing effect of chloroquine in bladder cancer and its further mechanism.?Materials and methods?Effect of CQ on cell viability of EJ and T24 cells was determined by a Cell Counting Kit 8 assay.Then IC10 and IC50 of CQ were estimated by software,meanwhile a low cytotoxic concentrations of IC10 was chosen for the following experiments.The rates of cell survival were evaluated by clonogenic assays.The the radiobiology parameters were determined from the curve fitted by the single-hit multi target model[SF=1-(1-e^-D/D0)ⁿ].Besides,we used flow cytometry to detect cell apoptosis.Radiation-induced DNA double strand break was measured by the staining of?-H2AX.The level of autophagy was detected by western blot,immunofluorescence staining,and electron microscopy.Moreover,the radiosensitization effect of CQ in vivo was estimated by a xenograft model.?Result?The treatment with chloroquine alone inhibited the proliferation of bladder cancer cells in a dose-and time-dependent manner.Therefore,cells were treated at 48 h with low concentration of chloroquine?10?M?to investigate the radiosensitivity of these cell lines.Low cytotoxic concentrations of chloroquine enhanced the radiation sensitivity of bladder cancer cells with a sensitization enhancement ratio of 1.53 and 1.40.A combination of chloroquine and radiation enhanced the apoptosis rate of EJ and T24 cells and down-regulated the expression of Bcl-2 while up-regulating the expression of caspase-3.Chloroquine also obviously weakened the repair of radiation-induced DNA damage.The activity of autophagy was evaluated by western blotting analysis,immunofluorescence staining and electron microscopy.The accumulation of LC3II and p62 protein levels were both increased by the treatment of chloroquine.The result indicated that chloroquine blocked autophagic flux.The expression of LC3II increased,and the p62 protein level decreased in both EJ and T24 cells,which suggested that irradiation activated autophagy.Moreover,the expression levels of LC3II and p62 in the combined group were higher than irradiation alone,indicating that chloroquine inhibited autophagy induced by irradiation in bladder cancer.In vivo studies confirmed that the combinatorial use of CQ with irradiation significantly diminishes tumor burden compared to either treatment alone.?Conclusion?The present findings suggested a crucial role of autophagy in the modulation of radiosensitization in bladder cancer,and chloroquine increased the radiosensitivity in EJ and T24 cells mainly via inhibiting autophagy and activating apoptosis,highlighting a novel profile of chloroquine as a potential radiosensitizer for the treatment of bladder cancer.