NADPH Oxidase Regulates Cytarabine-Induced Apoptotic Death in Acute Myeloid Leukemia Cells

Cellular metabolism and oxidative stress are important in the biology and pathophysiology of malignancies. Both increased reactive oxygen species [ROS] levels and induction of anti-oxidative pathways have been described in several malignancies, and may be modulate tumor biology and susceptibility to chemotherapy. Limited studies point to metabolic pathways, including ROS production, influencing pathogenesis and chemo-sensitivity of leukemia. NADPH oxidase is a critical enzyme in antimicrobial host defense and its activation results in ROS generation in myeloid leukemia cells. Our prior studies show that NADPH oxidase can activate Nrf2, a transcriptional factor that induces anti-oxidant and cytoprotective pathways. However the role of NADPH oxidase in chemotherapy-mediated apoptosis induction in leukemic cells is not well-known.

NADPH oxidase-derived ROS will increase sensitivity of AML cells to chemotherapy, whereas Nrf2 will be associated with chemotherapy resistance

We evaluated the role of NADPH oxidase and Nrf2 in regulating cytarabine-induced cell death in wild-type [WT] and engineered PLB-985 cells, a human acute myelomonocytic leukemia cell line derivative. NADPH oxidase-deficient PLB-985 cells were generated by recombination with mutant gp91^phox, a necessary component of NADPH oxidase. Nrf2-deficient cells were generated by shRNA (Nrf2shRNA) and depletion (>70%) of Nrf2 mRNA was confirmed by quantitative-PCR. WT and engineered PLB-985 cells were treated with cytarabine (12.5 to 750ng/ml for 24 – 48 hours) and cell death was determined by trypan blue exclusion and Annexin V/7-AAD staining.

NADPH oxidase-deficient PLB-985 cells were significantly more resistant to cytarabine compared to WT cells. Cytarabine (500 ng/ml for 48h) induced apoptotic cell death in 25% of NADPH oxidase-deficient vs. 53% of WT PLB-985 cells. Additional dose-response studies confirmed a significant effect of NADPH oxidase in potentiating cytarabine-induced cell death. Nrf2shRNA PLB-985 cells had either similar or modestly increased susceptibility to cytarabine-induced cell death compared to WT PLB-985 cells with empty vectors. NADPH-deficient/Nrf2shRNA PLB-985 cells had similar susceptibility to cytarabine as NADPH-deficient cells with empty vector.

Our results show that NADPH oxidase potentiates apoptotic cell death by cytarabine in a myelomonocytic leukemia cell line. However, we did not observe a consistent effect of Nrf2 depletion on apoptotic cell death by cytarabine. These studies suggest that modulation of redox-stress may be a potential therapeutic approach in AML that merits further study.

No relevant conflicts of interest to declare.