3-methyladenine protects melanoma cells against energy stress-induced necrosis by autophagy-independent decrease in oxidative stress and partial involvement of JNK

Abstract

We investigated the effect of 3-methyladenine (3MA), a class III phosphatidylinositol 3- kinase (PI3K)-blocking autophagy inhibitor, on the melanoma cell death induced by simultaneous inhibition of glycolysis by 2-deoxyglucose (2DG) and mitochondrial respiration by rotenone. We have elsewhere shown that 2DG/rotenone caused oxidative stress, ATP depletion, swelling of mitochondria, ultimately leading to necrosis. Energy stress is known to induce autophagy, a tightly regulated self-degradation process, which by recycling damaged organelles and macromolecules provides building blocks and energy. However, 2DG/rotenone did not induce proautophagic beclin-1 expression and autophagic flux in melanoma cells despite activation of AMP-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin complex 1 (mTORC1). 3MA, but not autophagy inhibition with other PI3K and lysosomal inhibitors, attenuated 2DG/rotenone-induced mitochondrial damage, oxidative stress, ATP depletion, and cell death. 3MA increased both AMPK and mTORC1 activation in energy stressed cells, but neither AMPK nor mTORC1 inhibition reduced its cytoprotective effect. 3MA reduced superoxide generation and c-Jun N-terminal kinase (JNK) activation, and both antioxidant and JNK blockade mimicked its protective activity. Therefore, 3MA prevents energy stress-triggered melanoma cell death through autophagy-independent decrease of oxidative stress and JNK activation. Our results warrant caution in use of 3MA as an autophagy inhibitor.