1. Introduction
Mitochondria control the biological processes occurring in almost all cells of the human body, such as the synthesis of iron-sulfur clusters, apoptosis, cell signaling, maintenance of calcium homeostasis, and maintaining reactive oxygen species levels.[1, 2]Above all, mitochondria are renowned for synthesizing high energy phosphate bonds in the form of ATP during oxidative phosphorylation (OXPHOS). During the series of oxidation and reduction reactions within the mitochondrial complexes, electrons follow one of the two major electron transport pathways to finally reduce oxygen to water. One pathway is through complexes I, III, and IV using electrons from NADH, and the other is through complexes II, III, and IV using FADH2 from succinic acid.[3] The continuous pumping of protons increases the hydrogen ion concentration and reduces pH, eventually generating a membrane potential across the inner mitochondrial membrane. This membrane potential is utilized by complex V (ATP synthase) to generate ATP.[4-6]
The significance of OXPHOS function can again be emphasized by the fact that mitochondrial diseases arise from dysfunctional OXPHOS systems.[7] Defective complex I was found to cause inflammation and cell death.[8] Despite the significant influence of OXPHOS in many diseases, its function in cancer has been overlooked because it was previously thought that cancer cells mostly utilize aerobic glycolysis as their main energy-synthesizing process.[9] However, growing evidence has shown that OXPHOS function is upregulated in lung cancer,[10, 11] estrogen receptor (ER)-positive breast cancer,[12] pancreatic cancers,[13] Hodgkin’s lymphoma,[14] and other cancers.[15] In addition, blocking mitochondrial respiration resulted in suppressed tumor growth in xenograft models,[16] further proving that mitochondrial function is essential for tumor cell proliferation. Mitochondrial ATP synthesis function through the OXPHOS system can no longer be overlooked, even in cancer, for it can be representative of the mitochondrial robustness.
Despite the apparent significance of the mitochondrial OXPHOS system and ATP, there is not yet a single assay to measure the mitochondrial function related to ATP production. This study was thus designed to propose a new method for an accurate, direct, and real-time detection of mitochondrial function through sensitivity to activators and inhibitors in isolated mitochondria, cell lines, and peripheral blood mononuclear cells (PBMCs).