The Redox State of Cytochrome C Modulates Resistance to Methotrexate in Human MCF7 Breast Cancer Cells

Abstract

BackgroundMethotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells.ResultsCell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexate-induced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells.ConclusionsWe conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Altered ProteinS-Glutathionylation Identifies a Potential Mechanism of Resistance to Acetaminophen-Induced Hepatotoxicity

“…NAPQI induces mitochondrial permeability transition, resulting in depolarization of the mitochondrial membrane, uncoupling of oxidative phosphorylation, and activation of proapoptotic proteins, including the release of cytochrome c (Masubuchi et al, 2005;Lee et al, 2015). The ability of cytochrome c to activate the caspase pathway is dependent on its redox state, where oxidized cytochrome c stimulates apoptotic activation, whereas reduction of cytochrome c prevents activation (Borutaite and Brown, 2007;Barros et al, 2013). As the majority of S-glutathionylation reactions are believed to result in enzyme inactivation, it can be hypothesized that S-glutathionylation prevents transition to cellular necrosis by inhibiting this enzyme activity.…”

Effect of Paullinia cupana on MCF-7 breast cancer cell response to chemotherapeutic drugs

“…The selection of the antitumor agents was based on previous studies demonstrating their cytotoxic effect on MCF-7 breast cancer cells: Cyclophosphamide, a nitrogen mustard alkylating agent that forms irreversible DNA crosslinks leading to cell death (18); doxorubicin, an anthracycline antibiotic with a DNA intercalating effect; 5-fluorouracil, a pyrimidine analog that belongs to the family of antimetabolite drugs, causing irreversible inhibition of thymidylate synthase and, consequently, cell cycle arrest and apoptosis (19); paclitaxel, a mitotic inhibitor targeting tubulin, causing defects in mitotic spindle assembly and chromosome segregation (20); vinorelbine, a semi-synthetic vinca alkaloid with an antimitotic effect (21); gemcitabine, a nucleoside analog that adds a 'faulty' nucleoside during DNA synthesis, leading to cell apoptosis (22); and methotrexate, a folic acid analogue that prevents purine and pyrimidine synthesis, leading to the inhibition of DNA, RNA and protein synthesis (23). Based on previous studies investigating the effects of these chemotherapeutics on MCF-7 cells, their concentrations were as follows: 10 µM gemcitabine (22), vinorelbine and methotrexate (24); 2 µM 5-fluorouracil (25); 50 µM paclitaxel (26); 200 nM doxorubicin (19); and 5 mM cyclophosphamide (27).…”

Correlation between the potency of flavonoids for cytochrome c reduction and inhibition of cardiolipin‐induced peroxidase activity

“…Accordingly, several efficient reductants of Cyt c were found to prevent caspase activation and apoptosis in different cellular models and in heart ischemia . In other study, Cyt c ‐reducing agents, tetramethylphenylenediamine or reduced glutathione added to the cell medium in micromolar concentrations, and ascorbate at sub‐milimolar concentration, protected breast cancer cells and other cell lines against methotrexate‐induced death . Taking into account the key role of Cyt c in apoptosis, the possible modulation of Cyt c redox state by flavonoids required further investigation, as it may contribute to the anti‐apoptotic and protective capacities of these compounds.…”