Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.
To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.
Advanced Technology Fusion, Graduate School of Science and Engineering, Saga University, Saga, Japan; and.
Eiji
E
Takahashi
Michihiko
M
Sato
D000693:Anaerobiosis; D000818:Animals; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D019069:Cell Respiration; D002522:Chlorocebus aethiops; D004579:Electron Transport; D042963:Electron Transport Complex II / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D064828:Prolyl Hydroxylases / Q000378:metabolism*; D064800:Prolyl-Hydroxylase Inhibitors / Q000494:pharmacology; D014162:Transfection; D059016:Tumor Microenvironment
D008164:Luminescent Proteins; D064800:Prolyl-Hydroxylase Inhibitors; D064828:Prolyl Hydroxylases; D042963:Electron Transport Complex II; D010100:Oxygen
anaerobic respiration; cancer cell line; hypoxia; hypoxia-inducible factor; prolyl hydroxylase domain-containing protein