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.