Uptake Rate of Cationic Mitochondrial Inhibitor MKT-077 Determines Cellular Oxygen Consumption Change in Carcinoma Cells

Abstract

ObjectiveSince tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mammary carcinoma cells would take up MKT-077, causing a decrease in oxygen metabolism related to drug uptake.MethodsR3230Ac rat breast adenocarcinoma cells were exposed to MKT-077. Cellular MKT-077 concentration was quantified using spectroscopy, and oxygen consumption was measured using polarographic electrodes. MKT-077 uptake kinetics were modeled by accounting for uptake due to both the concentration and potential gradients across the plasma and mitochondrial membranes. These kinetic parameters were used to model the relationship between MKT-077 uptake and metabolic inhibition. MKT-077-induced changes in oxygen consumption were also characterized in MDA-MB231 human breast carcinoma cells.ResultsCells took up MKT-077 with a time constant of ∼1 hr, and modeling showed that over 90% of intracellular MKT-077 was bound or sequestered, likely by the mitochondria. The uptake resulted in a rapid decrease in oxygen consumption, with a time constant of ∼30 minutes. Surprisingly the change in oxygen consumption was proportional to uptake rate, not cellular concentration. MKT-077 proved a potent metabolic inhibitor, with dose-dependent decreases of 45–73% (p = 0.003).ConclusionsMKT-077 caused an uptake rate-dependent decrease in cellular metabolism, suggesting potential efficacy for increasing tumor oxygen levels and radiosensitivity in vivo.

Selective Mitochondrial Uptake of MKT-077 Can Suppress Medullary Thyroid Carcinoma Cell SurvivalIn VitroandIn Vivo

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Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

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Dispersible oxygen microsensors map oxygen gradients in three-dimensional cell cultures

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“…Even ratiometric imaging with an oxygen-insensitive reference dye present is challenging, as photo-bleaching properties and excitation/emission properties will be different between the oxygen-sensitive and oxygen-independent dye. In contrast, measurement methods that depend on the indicator’s luminescent lifetime, such as phase fluorimetric measurements 34,38,48 , can usually eliminate inconsistencies that arise from intensity differences, caused by dye-loading variability, photobleaching, and optical path variability inherent to 3D tissues 48,49 . Using small, dispersible polydimethylsiloxane (PDMS)-based oxygen sensing microbeads with phase fluorimetry 18,19 we quantified significant differences in oxygen microenvironments between three different but commonly-used 3D culture formats.…”