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Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

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.

SUBMITTER: Smith MR

PROVIDER: S-EPMC4732023 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

Smith M Ryan MR Vayalil Praveen K PK Zhou Fen F Benavides Gloria A GA Beggs Reena R RR Golzarian Hafez H Nijampatnam Bhavitavya B Oliver Patsy G PG Smith Robin A J RA Murphy Michael P MP Velu Sadanandan E SE Landar Aimee A

Redox biology 20160108

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 ...[more]

PMID: 26774751

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Project description:Hydrogen sulphide (H(2)S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H(2)S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H(2)S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 ?M NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F(v)/F(m)) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H(2)S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome oxidase (CCO) were down-regulated after exposure to the optimal concentration of H(2)S. These findings suggest that increases in RuBISCO activity and the function of thiol redox modification may underlie the amelioration of photosynthesis and that H(2)S plays an important role in plant photosynthesis regulation by modulating the expression of genes involved in photosynthesis and thiol redox modification.

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Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

Publications

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

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