Abstract
Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model.
Highlights
One of the hallmarks of cancer cell transformation is the dysregulation of energetic pathways [1]
butyl triphenylphosphonium (BTPP) decreased basal, non-mitochondrial respiration, ATP-dependent respiration, maximal respiration, and proton leak excluding reserve capacity, but at higher concentrations than those seen with iodobutyl triphenylphosphonium (IBTP), (1–10 mM); BTPP exhibited unique changes proton leak when compared to IBTP (0.1 mM)
We examined the functional effects of mitochondrial thiol modification through the use of a mitochondriatargeted electrophile, IBTP, metabolism, and enzyme activity and protein levels using both a breast cancer cell line (MB231) and an immortalized nontumorigenic epithelial cell line (MCF-10A)
Summary
One of the hallmarks of cancer cell transformation is the dysregulation of energetic pathways [1] This process, known as “metabolic reprogramming,” involves an increased reliance on glycolysis independent of oxygen levels to provide ATP, and to provide intermediates for new lipids, amino and nucleic acids needed for rapid cell proliferation. Upregulation of glutaminolysis within the mitochondrion allows for generation of ATP with high efficiency, while generating substrates required in protein synthesis serving as a complement to glycolysis for biomass synthesis and energy production [7]. MB231 cells represent a prototype of metabolically reprogrammed glutamine-dependent cancer cells These “triple-negative” cells are characterized by lacking estrogen, progesterone, and Her2/neu receptors. We show that mitochondrial thiol modification blocks energy production by decreasing protein levels and activity, depleting Krebs cycle intermediates and inhibiting oxidative phosphorylation, decreasing ATP levels. Our data suggest that likely mechanisms of IBTP inhibition of metabolism include decreasing enzyme activity and/or decreasing protein levels
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