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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

Activation of apoptosis and the heat shock response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 24 h with either 1 μM disulfiram (DSF), 1 μM copper chloride (Cu), or a combination of both (DSF/Cu; 1 μM each) and analyzed by Western blot. Cell lysates for Western blot analysis were prepared with RIPA buffer and subsequently solubilized in mercaptoethanol-containing sample buffer (Roti-Load 1; Carl Roth, Karlsruhe, Germany) or non-reducing sample buffer (Roti-Load 3; Carl Roth, Karlsruhe, Germany) as indicated. cl.PARP = cleaved PARP.
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Figure 2: Activation of apoptosis and the heat shock response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 24 h with either 1 μM disulfiram (DSF), 1 μM copper chloride (Cu), or a combination of both (DSF/Cu; 1 μM each) and analyzed by Western blot. Cell lysates for Western blot analysis were prepared with RIPA buffer and subsequently solubilized in mercaptoethanol-containing sample buffer (Roti-Load 1; Carl Roth, Karlsruhe, Germany) or non-reducing sample buffer (Roti-Load 3; Carl Roth, Karlsruhe, Germany) as indicated. cl.PARP = cleaved PARP.

Mentions: To investigate the molecular mechanisms of disulfiram/copper-induced cytotoxicity, immunoblots on ovarian cancer cells, treated with disulfiram with and without copper chloride, were performed (Fig. 2). A strong cleavage of PARP (poly(ADP-ribose)-polymerase 1), a specific marker of apoptosis, was observed in cells that were treated with the combination of disulfiram and copper. The disulfiram/copper combination revealed further a pro-apoptotic c-jun N-terminal kinase (JNK) activation and mcl-1 downregulation, an anti-apoptotic protein targeted for degradation by oxidative stress-induced JNK phosphorylation [11]. A pronounced upregulation of HSP70 could also be observed in disulfiram/copper-treated ovarian cancer cells, which was associated with an increased molecular weight (activation by hyperphosphorylation [12]) of its transcription factor HSF1 (heat shock factor 1). Total expression of the small heat shock protein HSP27 appeared only slightly changed in disulfiram/copper - treated ovarian cancer cells, but phosphorylation analysis indicated posttranslational activation of HSP27. Posttranslational activation of HSP27 is further known to be associated with changes in the oligomerization status of HSP27 [13,14]. Immunoblot analysis of HSP27 expression under non-reducing conditions in the absence of mercaptoethanol revealed a marked shift of monomeric HSP27 to dimeric isoforms (Fig. 2).


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)

Activation of apoptosis and the heat shock response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 24 h with either 1 μM disulfiram (DSF), 1 μM copper chloride (Cu), or a combination of both (DSF/Cu; 1 μM each) and analyzed by Western blot. Cell lysates for Western blot analysis were prepared with RIPA buffer and subsequently solubilized in mercaptoethanol-containing sample buffer (Roti-Load 1; Carl Roth, Karlsruhe, Germany) or non-reducing sample buffer (Roti-Load 3; Carl Roth, Karlsruhe, Germany) as indicated. cl.PARP = cleaved PARP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Activation of apoptosis and the heat shock response by disulfiram/copperOVCAR3 and SKOV3 cells were treated for 24 h with either 1 μM disulfiram (DSF), 1 μM copper chloride (Cu), or a combination of both (DSF/Cu; 1 μM each) and analyzed by Western blot. Cell lysates for Western blot analysis were prepared with RIPA buffer and subsequently solubilized in mercaptoethanol-containing sample buffer (Roti-Load 1; Carl Roth, Karlsruhe, Germany) or non-reducing sample buffer (Roti-Load 3; Carl Roth, Karlsruhe, Germany) as indicated. cl.PARP = cleaved PARP.
Mentions: To investigate the molecular mechanisms of disulfiram/copper-induced cytotoxicity, immunoblots on ovarian cancer cells, treated with disulfiram with and without copper chloride, were performed (Fig. 2). A strong cleavage of PARP (poly(ADP-ribose)-polymerase 1), a specific marker of apoptosis, was observed in cells that were treated with the combination of disulfiram and copper. The disulfiram/copper combination revealed further a pro-apoptotic c-jun N-terminal kinase (JNK) activation and mcl-1 downregulation, an anti-apoptotic protein targeted for degradation by oxidative stress-induced JNK phosphorylation [11]. A pronounced upregulation of HSP70 could also be observed in disulfiram/copper-treated ovarian cancer cells, which was associated with an increased molecular weight (activation by hyperphosphorylation [12]) of its transcription factor HSF1 (heat shock factor 1). Total expression of the small heat shock protein HSP27 appeared only slightly changed in disulfiram/copper - treated ovarian cancer cells, but phosphorylation analysis indicated posttranslational activation of HSP27. Posttranslational activation of HSP27 is further known to be associated with changes in the oligomerization status of HSP27 [13,14]. Immunoblot analysis of HSP27 expression under non-reducing conditions in the absence of mercaptoethanol revealed a marked shift of monomeric HSP27 to dimeric isoforms (Fig. 2).

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