Metabolic Profiling of Multicell Tumor Spheroids by NADH Fluorescence and Spatially-Resolved Oximetry

BACKGROUND: Cancer cells utilize aerobic glycolysis rather than oxidative phosphorylation to generate most cellular ATP (Warburg phenomenon). In non-transformed differentiated cells, the adenine nucleotide translocator (ANT) catalyzes exchange of ATP for ADP across the mitochondrial inner membrane. Bongkrekic acid (BA) and carboxyatractyloside (CATR) specifically inhibit ANT. Here, our AIM was to assess whether mitochondrial ATP translocation in cancer cells depends on ANT. METHODS: Mitochondrial membrane potential (DJ) was assessed by confocal microscopy of tetramethylrhodamine methylester (TMRM) fluorescence. Respiration by HepG2 and A549 cells was determined with a Seahorse XF24 Analyzer. RESULTS: In rat hepatocytes, respiratory inhibition by myxothiazol (MYX) slightly decreased DJ, but subsequent oligomycin (OL), BA or CATR collapsed DJ, indicating that mitochondrial hydrolysis of glycolytic (cytosolic) ATP sustainsDJ. In HepG2 and A549 cells, MYX also slightly decreasedDJ, and subsequent OL collapsed DJ. By contrast to hepatocytes, BA and CATR added after MYX did not collapse DJ, whereas 2-deoxyglucose (2-DG), a glycolytic inhibitor, added after MYX, MYXþBA and MYXþCATR did collapse DJ. OL but not BA or CATR alone decreased respiration in both cell lines, whereas BA and CATR inhibited hepatocyte respiration. ANT2 is the predominant ANT isoform expressed in cancer cells, and 2-DG after MYX in ANT2 knockdown cells depolarized mitochondria. CONCLUSION: In cancer cells ANT is not the principal ATP transporter responsible for mitochondrial uptake of glycolytic ATP from the cytosol. Moreover, ANT2 deficiency does not alter uptake of glycolytic ATP into mitochondria. Warburg metabolism, therefore, appears to utilize an alternative pathway for entry of ATP into mitochondria.