Intracellular Superoxide Radical Formation Decreases 6‐Mercaptopurine Uptake by MOLT‐4 Leukemia Cells

Abstract

BackgroundAcute lymphoblastic leukemia (ALL) is the most common cancer associated with childhood. 6‐Mercaptopurine (6‐MP) is a nucleobase analog drug used in the maintenance treatment phase of ALL. 6‐MP has numerous side‐effects associated with its use, which has both short‐term and long‐term implications which can impact patient compliance (especially in children). Our lab has established that SLC43A3, which encodes for the equilibrative nucleobase transporter 1 (ENBT1), is expressed in leukemia cells and transports 6‐MP. However, its modes of regulation are yet to be elucidated. A previous study in microvascular endothelial cells demonstrated that nucleobase uptake via ENBT1 was significantly decreased upon exposure to oxidative stress. Oxidative stress is very prevalent in cancer, including leukemia, suggesting that it could impact ENBT1 function and thus 6‐MP uptake and therapeutic activity.HypothesisENBT1 function will be reduced by oxidative stress and affect the cytotoxic efficacy of 6‐MP in leukemia cells.MethodsThe MOLT‐4 leukemia cell line was used for all experiments. Cell viability was assessed via the MTT assay. [14C]6‐MP was used to determine the functional kinetics of ENBT1 using an oil‐stop centrifugation assay. Compounds used for the studies on oxidative stress were tert‐butyl hydroperoxide (TBHP) and menadione (oxygen‐free radical generators). TBHP causes extracellular induction of oxidative stress, while menadione is primarily through an intracellular mechanism called redox cycling. Mn(III)tetrakis(1‐methyl‐4‐pyridyl)porphyrin (MnTMPyP) (superoxide dismutase mimetic) and N‐acetylcysteine (NAC) (antioxidant) were used to combat the effects caused by TBHP or menadione.ResultsIncubation of cells in TBHP and menadione for 30 min at 37℃ decreased cell viability only at the highest concentrations tested (100 ‐ 400 µM) (400 µM: 86 ± 4% and 72 ± 6%, respectively) Incubation with TBHP and menadione for 24 hours led to a greater loss of cell viability (Log EC50: ‐3.9 ± 0.1 and ‐4.3 ± 0.1, respectively). TBHP did not alter ENBT1‐mediated 6‐MP uptake. However, incubation with menadione at 100 µM for 30 min at 37℃ significantly decreased ENBT1‐mediated 6‐MP uptake (Vmax: Control – 77 ± 3 pmol/µL/sec; Menadione – 57 ± 3 pmol/µL/sec). The addition of the free radical scavenger MnTMPyP did not reverse the decrease caused by menadione. However, the addition of 1mM NAC caused a significant reversal of the reduction in 6‐MP uptake produced by menadione (Vmax: Menadione – 46 ± 2 pmol/µL/sec; Menadione + NAC – 55 ± 2 pmol/µL/sec).ConclusionsMenadione but not TBHP caused a significant decrease in ENBT1 functional activity, suggesting it was due to intracellular superoxide formation, thus supporting our hypothesis. The reduction caused by menadione could be reversed with NAC. This study confirms that ENBT1 function, and thus 6‐MP uptake by leukemia cells, is modified by oxidative stress, thus opening up potential therapeutic avenues in ALL treatment.