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.

Intracellular Ca2 + Imbalance Critically Contributes to Paraptosis

“…Me 2 NNMe 2 , which is a new derivative of α-N-heterocyclic thiosemicarbazones, induces major hallmarks of paraptotic cell death, including ER-derived vacuolation, mitochondrial swelling, activation of MEK/ERK signaling pathway, and caspase-independent cell death ( Hager et al, 2018 ). In this process, the copper complex of Me 2 NNMe 2 accumulates in the ER to inhibit the reductive potential of protein disulfide isomerase (PDI).…”

“…The resultant disruption of thiol redox homeostasis in the ER, in turn, activates protein kinase R (PKR)-like endoplasmic reticulum kinase signaling and the release of Ca 2+ ions from the ER. This prolonged Ca 2+ imbalance triggers the swelling of the ER and the mitochondria as well as mitochondrial membrane depolarization, leading to cell death ( Hager et al, 2018 ).…”

“…On the other hand, massive Ca 2+ release from the ER causes the prolonged mitochondrial Ca 2+ overload and excessive mitochondrial swelling ( Rovere et al, 2016 ; Marchi et al, 2018 ). Mitochondrial Ca 2+ overload has been commonly reported in paraptosis-associated cell death by curcumin ( Yoon et al, 2012 ), celastrol ( Yoon et al, 2014 ), hesperidin ( Yumnam et al, 2016 ), morusin ( Xue et al, 2018 ), δ-TT ( Fontana et al, 2020a ), IPH4 ( Yokoi et al, 2020 ), Me 2 NNMe 2 ( Hager et al, 2018 ), CGP-37157/PI ( Yoon et al, 2012 ), Ler/PI ( Lee et al, 2019 ), Lop/PI ( Kim et al, 2019 ), and Nutlin-3/PI ( Lee et al, 2017 ). Among them, the functional significance of MCU-mediated mitochondrial Ca 2+ overload as an early signal for paraptosis was confirmed by the reports that pharmacological and/or genetic inhibition of MCU attenuates the mitochondrial dilation and subsequent paraptotic cascades (i.e., ER stress, ER dilation, and subsequent cell death) induced by curcumin ( Yoon et al, 2012 ), celastrol ( Yoon et al, 2014 ), hesperidin ( Yumnam et al, 2016 ), CGP-37157/PI ( Yoon et al, 2012 ), and Ler/PI ( Lee et al, 2019 ).…”

“…Paraptosis is successfully inhibited by the translation inhibitor, cycloheximide (CHX) ( Sperandio et al, 2000 ), suggesting that inhibition of protein synthesis may prevent this cell death by reducing the potential burden of misfolded proteins and proteotoxicity ( Lee et al, 2016 ). The activation of mitogen-activated protein (MAP) kinases, such as c-Jun N-terminal protein kinases (JNKs), MAP kinase kinase 2 (MEK-2), and p38, has been found to be positively involved in the paraptosis induced by many natural products or chemicals ( Zhang et al, 2009 , 2010 ; Yoon et al, 2010 , 2012 , 2014 ; Wang et al, 2012 ; Yumnam et al, 2014 ; Hager et al, 2018 ; Han et al, 2018 ; Fontana et al, 2019 ; Yokoi et al, 2020 ), whereas AIP-1/Alix appears to be negatively involved in some cases of paraptosis ( Sperandio et al, 2004 ; Valamanesh et al, 2007 ; Yoon et al, 2010 , 2014 ; Han et al, 2018 ; Xue et al, 2018 ). The term “paraptosis-like cell death” has been used to describe the types of cell death accompanied by the dilation of either the mitochondria or the ER alone or those that do not share multiple biochemical features of paraptosis, including the activation of MAP kinases, the downregulation of AIP-1/Alix, and the inhibition of vacuolation and cell death by cycloheximide, etc.…”

Endoplasmic Reticulum Stress during Mammalian Follicular Atresia

“…ROS production can trigger paraptosis through PINK and mitophagy activation [108,109]. Covalent modifications of free sulfhydryl groups on proteins cause protein misfolding and the accumulation of misfolded proteins, leading to ER stress, CHOP activation, and paraptosis [110,111]. In malignant hepatoma cells with Bcl-xL-mediated apoptotic defects, the disruption of thiol homeostasis and treatment with doxorubicin and pyrrolidine dithiocarbamate induced paraptotic cell death [112].…”