Radiotherapy is a widespread treatment in human solid tumors. However, therapy resistance and poor prognosis are still problems. Gambogic acid (GA), extracted from the dried yellow resin of gamboges, has an anticancer effect against various types of cancer cells. To explore the radiosensitivity of GA on esophageal cancer cell line TE13, cell viability was tested by Cell Counting Kit-8 (CCK-8) assay, colony formation assay was used to assess the effects of GA on the radiosensitivity of TE13, and flow cytometry was performed to meter the percentage of apoptosis. The protein levels of microtubule-associated protein 1 light chain 3 (LC3), caspase3, caspase8, casepase9, pAkt, and p-mammalian target of rapamycin (p-mTOR) were tested using Western blot. The distribution of LC3 was detected by immunofluorescence. Additionally, we also examined reactive oxygen species (ROS) expression by laser scanning confocal microscope (LSCM). The cells were transfected with adenovial vector to monitor the autophagy through the expression of green fluorescent protein (GFP-red fluroscent protein (RFP)-LC3. The rates of apoptotic cells in combined-treated TE13 increased significantly compared with the control groups in accordance with the results of Western blot. The clonogenic survival assay showed that GA enhances radiosensitivity with a sensitizing enhancement ratio (SER) of 1.217 and 1.436 at different concentrations. The LC3-II protein level increased in the combined group indicating the increase of autophagy incidence, and the results of GFP-RFP-LC3 experiment showed that GA may block the process of autophagic flux in TE13 cells. Moreover, we successfully demonstrated that ROS is involved in the induction of autophagy. ROS-mediated autophagy depends on the inhibition of the Akt/mTOR pathway. Taken together, GA induced radiosensitivity involves autophagy and apoptosis which are regulated by ROS hypergeneration and Akt/mTOR inhibition.
Keywords: Autophagy; Esophageal cancer; GFP–RFP; Gambogic acid; LC3; Radiosensitivity.