Anti-leukemic activity of the arginase inhibitor nor−NOHA in ARG2-expressing cells.

(A) Schematic of cellular arginine metabolism. ARG2 converts arginine into ornithine and urea, a reaction which can be inhibited by the arginase inhibitor nor−NOHA. (B) Anti-leukemic activity of nor−NOHA. K562 cells were treated with vehicle (DMSO) or increasing concentrations of nor–NOHA (0.1, 0.5 or 1mM) under 21% or 1.5% O2 for 72 hours, and cell viability determined by Annexin V/ 7−AAD staining. (C) Effect of arginase inhibition on cellular amino acid composition. K562 cells were treated with 500 μM nor–NOHA under 21% or 1% O2 for 48 hours. The metabolites were extracted and subjected to quantification by LC−MS (average of 3 experiments). (D) Ornithine does not rescue cell death induced by nor–NOHA. Experiments were as in (B), and ornithine (1mM) was added at the beginning of incubation (average of 3 experiments). (E) Cell lines expressing and upregulating ARG2 under hypoxia are more sensitive to nor−NOHA-induced cell death than those without ARG2 upregulation. Leukemia and myeloma cell lines were grown under 21% or 1.5% O2 for 48 hours, and ARG2 expression determined by western blot on cell lysates. (F) The indicated cell lines were grown at 21% or 1.5% O2 for 72 hours, and the effect of nor−NOHA on cell viability was analysed by Annexin V/ 7−AAD staining (average of 4 experiments).