Cyclosporine A Enhances Arsenic Trioxide-Induced Autophagic Cell Death In Malignant Glioma Cell Line U87-MG

Recent studies showed that arsenic trioxide (ATO) provided a novel approach to treat human acute promyelocytic leukemia (APL), multiple myeloma cells, androgen-independent prostate cancer, and hepatocellular carcinoma. Many investigations were done to explore the mechanism of ATO-induced cell death. ATO has been reported to induce apoptosis in APL cell lines, neuroblastoma cell lines, multiple myeloma cell and human gastric cancer cells through the activation of caspase 3. However, in malignant glioma cells, ATO induced autophagic cell death in stead of apoptosis, suggesting that ATO might be a novel pro-autophagic agent in treatment for malignant gliomas.Cyclosporine A (CSA), which is widely used in organ transplantation and in the treatment of autoimmune disorders, could induce growth arrest and/or apoptosis a wide spectrum of transformed cells, including rodent and human glioma cells. Many reports showed that CSA as a synergistic agent can enhance drug-induced apoptosis. Such as colchicine-induced apoptosis in rat cerebellar granule neurons, docetaxel-induced apoptosis in human gastric carcinoma cells, arsenic trioxide-induced apoptosis in NB4 cells, taxol-induced apoptosis of human urinary bladder cancer cells. However, it has not been reported that CSA can enhance autophagic cell death.In this study, we attempt to explore whether ATO-induced autophagy and growth inhibition could be enhanced by cyclosporine A (CSA) in malignant glioma cell line U87-MG. Our results showed that low dose of CSA (2μM) significantly enhanced ATO-induced growth inhibition and autophagic cell death. The IC50 value was 4.78μM in the ATO-treated group, which decreased to 1.28μM in the CSA/ATO-treated group. ATO treatment caused cell arrest at G0/G1 and S phases, which significantly increased by CSA pretreatment. Moreover, cell death increased by 7.5～9.1-folds in the CSA/ATO-treated group than that in the CSA-treated group or in the ATO-treated group. This type of cell death was similar with the type induced by the typical autophagy inducer agent rapamycin, which characterized with accumulation of acridine orange and upregulation of Beclin-1. The acridine orange staining was detected both through fluorescence microscope and flow cytometry. The results indicated that CSA (2μM) treatment increased 3-folds acridine orange accumulation. This phenomenon was confirmed by detection of the expression of Beclin-1. We therefore conclude that CSA can enhance ATO-induced autophagic cell death of U87-MG. Our results suggest that the combined treatment with ATO and CSA is an effective novel strategy to treat drug-resistant gliomas or complement programmed cell death-based therapies.