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The thiosemicarbazone Me₂NNMe₂ induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

ABSTRACT: Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me₂NNMe₂ differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me₂NNMe₂- (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me₂NNMe₂ (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca²⁺ and ER thiol redox homeostasis. Our findings indicate that compounds like Me₂NNMe₂ are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.

SUBMITTER: Hager S

PROVIDER: S-EPMC6189190 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

Hager Sonja S Korbula Katharina K Bielec Björn B Grusch Michael M Pirker Christine C Schosserer Markus M Liendl Lisa L Lang Magdalena M Grillari Johannes J Nowikovsky Karin K Pape Veronika F S VFS Mohr Thomas T Szakács Gergely G Keppler Bernhard K BK Berger Walter W Kowol Christian R CR Heffeter Petra P

Cell death & disease 20181015 11

Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me2NNMe2 differs from Triapine not on ...[more]

PMID: 30323190

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Project description:This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide Isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5-10nM, can utilize a range of thiol substrates (including 5µM dithiothreitol, 10µM reduced RNase thiols, and 5mM glutathione; GSH), and can operate from pH 6-9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-?-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1µM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.

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The thiosemicarbazone Me₂NNMe₂ induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

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The thiosemicarbazone Me<sub>2</sub>NNMe<sub>2</sub> induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

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The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

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The thiosemicarbazone Me&lt;sub&gt;2&lt;/sub&gt;NNMe&lt;sub&gt;2&lt;/sub&gt; induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

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OmicsDI is part of the ELIXIR infrastructure

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The thiosemicarbazone Me₂NNMe₂ induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

The thiosemicarbazone Me<sub>2</sub>NNMe<sub>2</sub> induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.