iNOS initiates and sustains metabolic arrest in hypoxic lung adenocarcinoma cells: mechanism of cell survival in solid tumor core

Abstract

Nitric oxide (NO) modulates cellular metabolism by competitively inhibiting the reduction of O2at respiratory complex IV. The aim of this study was to determine whether this effect could enhance cell survival in the hypoxic solid tumor core by inducing a state of metabolic arrest in cancer cells. Mitochondria from human alveolar type II-like adenocarcinoma (A549) cells showed a fourfold increase in NO-sensitive 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescence and sixfold increase in Ca2+-insensitive NO synthase (NOS) activity during equilibration from Po2s of 100→23 mmHg, which was abolished by Nω-nitro-l-arginine methyl ester-HCl (l-NAME) and the inducible NOS (iNOS) inhibitor, N6-(1-iminoethyl)-l-lysine dihydrochloride (l-NIL). Similarly, cytosolic and compartmented DAF-FM fluorescence increased in intact cells during a transition between ambient Po2and 23 mmHg and was abolished by transfection with iNOS antisense oligonucleotides (AS-ODN). In parallel, mitochondrial membrane potential (ΔΨm), measured using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), decreased to a lower steady state in hypoxia without change in glycolytic rate, adenylate energy charge, or cell viability. However, l-NAME or iNOS AS-ODN treatment maintained ΔΨmat normoxic levels irrespective of hypoxia and caused a marked activation of glycolysis, destabilization energy charge, and cell death. Comparison with other cancer-derived (H441) or native tissue-derived (human bronchial epithelial; alveolar type II) lung epithelial cells revealed that the hypoxic suppression of ΔΨmwas common to cells that expressed iNOS. The controlled dissipation of ΔΨm, absence of an overt glycolytic activation, and conservation of viability suggest that A549 cells enter a state of metabolic suppression in hypoxia, which inherently depends on the activation of iNOS as Po2falls.