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Disulfiram/copper causes redox-related proteotoxicity and concomitant heat shock response in ovarian cancer cells that is augmented by auranofin-mediated thioredoxin inhibition.

Papaioannou M, Mylonas I, Kast RE, Brüning A - Oncoscience (2013)

Bottom Line: Within few hours of application, disulfiram caused irreversible cell damage associated with pronounced induction of the inducible heat shock proteins HSP70, HSP40, and HSP32.The small heat shock protein HSP27 was found to be covalently dimerized via oxidized disulfide bonds and precipitated in para-nuclear protein aggregates.Simultaneous inhibition of the cellular thioredoxin system by auranofin further enhanced the cytotoxic effect of disulfiram.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics/Gynecology, University Hospital Munich, Germany.

ABSTRACT
A valuable strategy to develop new therapeutic options for a variety of diseases has been the identification of new targets and applications for already approved drugs, the so-called drug repositioning. Recurrent ovarian cancer is a nearly incurable malignancy for which new and effective treatments are urgently needed. The alcohol-deterring drug disulfiram has been shown to cause preferential cell death in a variety of cancer cells. In this study, it is shown that disulfiram mediates effective cell death in ovarian cancer cells by promoting a pro-oxidative intracellular environment in a copper-dependent mechanism. Within few hours of application, disulfiram caused irreversible cell damage associated with pronounced induction of the inducible heat shock proteins HSP70, HSP40, and HSP32. The small heat shock protein HSP27 was found to be covalently dimerized via oxidized disulfide bonds and precipitated in para-nuclear protein aggregates. Simultaneous inhibition of the cellular thioredoxin system by auranofin further enhanced the cytotoxic effect of disulfiram. These data indeed indicate that the combination of two approved drugs, the anti-alcoholic disulfiram and the anti-rheumatic auranofin, may be of interest for the treatment of recurrent and genotoxic drug-resistant ovarian cancer by inducing a proteotoxic cell death mechanism.

No MeSH data available.


Related in: MedlinePlus

Subcellular distribution of heat shock proteins in response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 8 h with 1 μM disulfiram/copper, separated into a crude cytosolic and nuclear extract as described in the Methods section, and analyzed by Western blot analysis under reducing conditions. GAPDH was used as a cytosolic marker, the transcription factor MTA2 as a nuclear marker.
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Figure 4: Subcellular distribution of heat shock proteins in response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 8 h with 1 μM disulfiram/copper, separated into a crude cytosolic and nuclear extract as described in the Methods section, and analyzed by Western blot analysis under reducing conditions. GAPDH was used as a cytosolic marker, the transcription factor MTA2 as a nuclear marker.

Mentions: The specific activation of inducible heat shock proteins by disulfiram/copper indicates the accumulation of misfolded or damaged proteins and the likelihood of cytotoxic protein aggregates. Heat shock proteins are distributed in various intracellular organelles and their organelle-specific accumulation may also help in identifying the primary location of disulfiram/copper-induced cell damage. We therefore generated crude extracts of cytosolic and nuclear proteins which were analyzed by immunoblot analysis for heat shock protein expression. Cell fractionation analysis revealed HSP70 protein expression in both cytosolic and nuclear compartments and disulfiram/copper markedly increased HSP70 levels in both compartments. In contrast to HSP70, total expression level of HSP27 was not increased by disulfiram/copper, although its expression in the crude nuclear extract appeared to be slightly enhanced by disulfiram/copper treatment (Fig. 4). This was markedly more pronounced with the transcription factor HSF1, of which a large proportion shifted from a primarily cytosolic fraction to the nuclear fraction after disulfiram/copper treatment. In the presence and absence of disulfiram/copper, localization of the NFκB subunit p65 (Rel A) and its cytosolic inhibitor IκB was found to be primarily cytosolic, indicating no specific nuclear p65 translocation and NFκB activation by disulfiram/copper.


Disulfiram/copper causes redox-related proteotoxicity and concomitant heat shock response in ovarian cancer cells that is augmented by auranofin-mediated thioredoxin inhibition.

Papaioannou M, Mylonas I, Kast RE, Brüning A - Oncoscience (2013)

Subcellular distribution of heat shock proteins in response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 8 h with 1 μM disulfiram/copper, separated into a crude cytosolic and nuclear extract as described in the Methods section, and analyzed by Western blot analysis under reducing conditions. GAPDH was used as a cytosolic marker, the transcription factor MTA2 as a nuclear marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295765&req=5

Figure 4: Subcellular distribution of heat shock proteins in response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 8 h with 1 μM disulfiram/copper, separated into a crude cytosolic and nuclear extract as described in the Methods section, and analyzed by Western blot analysis under reducing conditions. GAPDH was used as a cytosolic marker, the transcription factor MTA2 as a nuclear marker.
Mentions: The specific activation of inducible heat shock proteins by disulfiram/copper indicates the accumulation of misfolded or damaged proteins and the likelihood of cytotoxic protein aggregates. Heat shock proteins are distributed in various intracellular organelles and their organelle-specific accumulation may also help in identifying the primary location of disulfiram/copper-induced cell damage. We therefore generated crude extracts of cytosolic and nuclear proteins which were analyzed by immunoblot analysis for heat shock protein expression. Cell fractionation analysis revealed HSP70 protein expression in both cytosolic and nuclear compartments and disulfiram/copper markedly increased HSP70 levels in both compartments. In contrast to HSP70, total expression level of HSP27 was not increased by disulfiram/copper, although its expression in the crude nuclear extract appeared to be slightly enhanced by disulfiram/copper treatment (Fig. 4). This was markedly more pronounced with the transcription factor HSF1, of which a large proportion shifted from a primarily cytosolic fraction to the nuclear fraction after disulfiram/copper treatment. In the presence and absence of disulfiram/copper, localization of the NFκB subunit p65 (Rel A) and its cytosolic inhibitor IκB was found to be primarily cytosolic, indicating no specific nuclear p65 translocation and NFκB activation by disulfiram/copper.

Bottom Line: Within few hours of application, disulfiram caused irreversible cell damage associated with pronounced induction of the inducible heat shock proteins HSP70, HSP40, and HSP32.The small heat shock protein HSP27 was found to be covalently dimerized via oxidized disulfide bonds and precipitated in para-nuclear protein aggregates.Simultaneous inhibition of the cellular thioredoxin system by auranofin further enhanced the cytotoxic effect of disulfiram.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics/Gynecology, University Hospital Munich, Germany.

ABSTRACT
A valuable strategy to develop new therapeutic options for a variety of diseases has been the identification of new targets and applications for already approved drugs, the so-called drug repositioning. Recurrent ovarian cancer is a nearly incurable malignancy for which new and effective treatments are urgently needed. The alcohol-deterring drug disulfiram has been shown to cause preferential cell death in a variety of cancer cells. In this study, it is shown that disulfiram mediates effective cell death in ovarian cancer cells by promoting a pro-oxidative intracellular environment in a copper-dependent mechanism. Within few hours of application, disulfiram caused irreversible cell damage associated with pronounced induction of the inducible heat shock proteins HSP70, HSP40, and HSP32. The small heat shock protein HSP27 was found to be covalently dimerized via oxidized disulfide bonds and precipitated in para-nuclear protein aggregates. Simultaneous inhibition of the cellular thioredoxin system by auranofin further enhanced the cytotoxic effect of disulfiram. These data indeed indicate that the combination of two approved drugs, the anti-alcoholic disulfiram and the anti-rheumatic auranofin, may be of interest for the treatment of recurrent and genotoxic drug-resistant ovarian cancer by inducing a proteotoxic cell death mechanism.

No MeSH data available.


Related in: MedlinePlus