Amiodarone-induced autophagy protects human epithelial lung cancer cells from apoptosis

Abstract

Autophagy is an essential process that mediates degradation of intracellular protein and organelles. Autophagy is long known to provide a survival advantage to cells undergoing nutrient deprivation or other stresses by clearing damaged proteins, organelles, pathogens or providing the cell with energy and nutrient, and has essential roles in survival, development and homeostasis. However, Autophagy has emerged as another major programmed mechanism to control life and death much like programmed cell death is for apoptosis in eukaryotes. Autophagy and apoptosis are not mutually exclusive pathway sharing many of the same molecules. Amiodarone, a bi-iodinated benzofuran derivative, is an effective class Ⅲ antiarrhythmic agent commonly used to treat supraventricular and ventricular arrhythmias. This drug tends to accumulate in several organs, including the lungs. It has been associated with a variety of adverse events and the most serious is amiodarone-induced pulmonary toxicity (AIPT). AIPT occurs in approximately 5% of treated patients and could lead to death. Amiodarone has been reported that it induced apoptosis and autophagy. In present study, we investigated the effect of amiodarone-induced autophagy on apoptosis in human epithelial lung cancer cells (H460 cells). We examined cell viability, apoptotic cell death and the expression of autophagic proteins and formation of autophagosomes in cells treated with amiodarone. Amiodarone had cytotoxicity and induced apoptosis. Autophagy was detected in amiodarone-treated cells as indicated by conversion of LC3 protein, and reduction of P62. The appearance of amiodarone-induced punctated staining of autophagosome-associated LC3-Ⅱ upon EGFP-LC3 transfection and ultrastructural TEM analysis provided further evidence for autophagy. Autophagy is induced through both mTOR-dependent and independent manner. mTOR, an evolutionarily-conserved protein kinase, is regulated by AMPK and Akt. Amiodarone activated AMPK and reduced Akt resulting inhibition of mTOR and its downstream. The autophagy inhibitor, 3-methyladenine, enhanced cytotoxicity and inhibited the formation of autophagosome but induced apoptosis as indicated by activation of apoptotic proteins, We further used siRNA against Atg5 and Atg7, essential for formation of autophagosome to confirm the protective effect of autophagy in amiodarone-induced apoptosis. The data was consistent with the result using 3-MA. Our results demonstrated that induction of autophagy suppressed amiodarone-induced apoptosis, and prolongs survival of H460 cells.