Glutamine-dependent effects of nitric oxide on cancer cells subjected to hypoxia-reoxygenation

Limited O-2 availability can decrease essential processes in energy metabolism. However, cancers have developed distinct metabolic adaptations to these conditions. For example, glutaminolysis can maintain energy metabolism and hypoxia signaling. Additionally, it has been observed that nitric oxide (NO) possesses concentration -dependent, biphasic effects in cancer. NO has potent anti-tumor effects through modulating events such as angiogenesis and metastasis at low physiological concentrations and inducing cell death at higher concentrations. In this study, Ewing Sarcoma cells (A-673), MIA PaCa, and SKBR3 cells were treated with DetaNONOate (DetaNO) in a model of hypoxia (1% O-2) and reoxygenation (21% O-2). All 3 cell types showed NO-dependent inhibition of cellular O2 consumption which was enhanced as O-2-tension decreased. L-Gln depletion sup-pressed the mitochondrial response to decreasing O-2 tension in all 3 cell types and resulted in inhibition of Complex I activity. In A-673 cells the O(2 )tension dependent change in mitochondrial O2 consumption and in-crease in glycolysis was dependent on the presence of L-Gln. The response to hypoxia and Complex I activity were restored by alpha-ketoglutarate. NO exposure resulted in the A-673 cells showing greater sensitivity to decreasing O2 tension. Under conditions of L-Gln depletion, NO restored HIF-1 alpha levels and the mitochondrial response to O-2 tension possibly through the increase of 2-hydroxyglutarate. NO also resulted in suppression of cellular bioenergetics and further inhibition of Complex I which was not rescued by alpha-ketoglutarate. Taken together these data suggest that NO modulates the mitochondrial response to O-2 differentially in the absence and presence of L-Gln. These data suggest a combination of metabolic strategies targeting glutaminolysis and Complex I in cancer cells.