The role of ROS in MAPK-dependent autophagy involved in phorbol myristate acetate-induced macrophage differentiation of HL-60 leukemia cells

Abstract

Background: Reactive oxygen species (ROS) have been implicated in autophagy induction and mitogen activated protein kinases (MAPK) activation which both participate in the differentiation of hematopoietic and leukemic cells. We assessed the role of ROS in MAPK activation and autophagy induction in phorbol myristate acetate-(PMA) induced macrophage differentiation of HL-60 leukemia cells. Material and methods: The macrophage markers CD11b, EGR1, CSF1R, and IL-8 were assessed by RT-qPCR and flow cytometry. The activation of MAPK was assessed by ERK and JNK immunoblotting, while autophagy was monitored by LC3-II and p62 immunoblotting. Pharmacological inhibition was used to determine the role of MAPK and autophagy in HL60 cell differentiation. Intracellular ROS production was determined by flow cytometric analysis of the green fluorescence emitted by non-selective redox-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate. Antioxidant N-acetylcysteine (NAC) was used to determine the role of ROS in MAPK activation, induction of autophagy and HL-60 macrophage differentiation. Results: PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by elevated expression of macrophage markers CD11b, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase of autophagic flux. Pharmacological inhibition of ERK or JNK suppressed PMA-triggered autophagy induction and differentiation of HL-60 cells into macrophage-like cells. PMA increased the intracellular ROS generation and the antioxidant NAC reduced the expression of macrophage markers EGR-1, CSF1R, IL-8 and CD11b in PMA-treated HL-60 cells. NAC also blocked PMA-induced LC3-II and ERK phosphorylation, but only slightly reduced the phosphorylation of JNK and did not affect the levels of p62. Conclusion: Our study revealed the partial involvement of ROS in MAPK-dependent autophagy in the differentiation of HL60 cells, indicating ROS/MAPK-mediated autophagy for further investigation in differentiation therapy of AML.