Protection Against Arsenic Trioxide-Induced Autophagic Cell Death in U118 Human Glioma Cells by Use of Lipoic Acid

Abstract

P-698 Introduction: Exposure to toxic heavy metals has become an increasingly recognized source of illness worldwide. Arsenic is an environmental toxicant found naturally in ground water. Neurotoxic effects have been reported in clinical cases and animal experiments with chronic exposure to arsenic. Epidemiological studies also have suggested a correlation between arsenic exposure and potential neurotoxicity. Lipoic acid is a thiol-compound naturally occurring in plants and animals, which is thought to be a strong antioxidant and possess neuroprotective effects. The objective of this study was to determine if the AS2O3-induced neurotoxic effects could be prevented by lipoic acid. The U118 human glioma cell was selected as a research model. Methods: In this study, U118 cells were treated with As2O3 alone or in combination with lipoic acid, and the viability of the cells was determined with MTT assay. DNA fragmentation assay and flow cytometric analysis were performed to confirm the apoptotic cells with Annexin V-PI staining. Supravital cell staining with acridine orange was performed to detect the autophagic cell death. The regulatory proteins which were related to apoptosis was determined by immunoblotting analysis, using antibodies including HSP70, p53, p21/Cip1, Bax, and Bcl2. Results: The viability assays revealed that 50% mortality of the AS2O3-treated cells that died within 24 hrs was reversed by lipoic acid. The lipoic acid treatment promoted long-term survival in cells treated with AS2O3 and provided more than 80% protection during days 2–3. We also found that autophagic, but not apoptotic cell death was significantly induced by AS2O3 in U118 cells, and that AS2O3-mediated autophagic cell death was nearly completely attenuated by lipoic acid. The down-regulation of p53 and Bax proteins and the up-regulation of Bcl-2 and HSP-70 proteins were involved in AS2O3-mediated autophagic cell death. Discussion and Conclusions: The present study demonstrated for the first time that AS2O3-induced neurotoxicity could be completely attenuated by lipoic acid in U118 cells. Apoptotic neuronal cell death was suggested as a general mechanism underlying arsenic neurotoxicity. However, when U118 cells were exposed to AS2O3, autophagy but not apoptosis was induced. Such results implied that U118 cells response to AS2O3 must be regulated by different signaling mechanisms rather than the general apoptotic mechanism. Our results provide the molecular basis for the lipoic acid prevention of AS2O3-induced cell death in U118 cells; such observations may have significance in clinical application.