Cytotoxicity of Arginine Deprivation to AML Cells is Mediated by Autophagy

Abstract

In this study, we assess the activation of autophagy in AML cells following arginine deprivation, the mechanism of its activation, and its impact on cell cytotoxicity. Arginine deprivation was induced using a pegylated recombinant human Arginase I [HuArgI(Co)-PEG5000], in which the two Mn2+ ions of the active site have been replaced with Co2+ ions. AML cell lines were tested, and activation of autophagy was determined by staining for autophagosomes on flow cytometry. The impact of autophagy on cell cytotoxicity of arginine deprivation was assessed through inhibition of autophagy using the downstream autophagy inhibitor chloroquine (CQ). [HuArgI(Co)-PEG5000]-induced arginine deprivation led to significant and sustained activation of autophagy starting at 24 hours and lasting up to 120 hours following arginine deprivation in all cell lines. Inhibition of autophagy, using chloroquine, led to a significant decrease in cell cytotoxicity evident at 48 hours and up to 120 hours. This indicates that activation of autophagy in response to arginine deprivation, is leading to AML cell death (death by autophagy). This was further confirmed by the fact that cell death observed following arginine deprivation in AML cells was caspase-independent and nonapoptotic. Moreover, AML cells were shown to overexpress the major key player in the biosynthesis of L-arginine; Argininosuccinate Synthetase-1 (ASS-1) upon arginine deprivation without it conferring resistance to death by autophagy induced by HuArgI(Co)-PEG5000. Addition of the ROS scavenger N-acetyl-cysteine (NAC) repressed the autophagic response and reversed cytotoxicity of arginine deprivation to AML cells, indicating that the activation of autophagy is induced following ROS generation. Finally, our study demonstrates that in AML cells, arginine deprivation induces autophagy activation leading to cell death irrespective the overexpression of ASS-1. We have also shown that the activation of autophagy and the subsequent death by autophagy of AML cells is induced through generation of ROS.