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CRMP2/DPYL2 is an effector protein in the semaphorin signaling pathway that controls cytoskeletal dynamics, linking extracellular signals to the formation of axonal networks. CRMP2 is regulated by post-translational modifications including a dithiol-disulfide redox switch. The mechanisms of reduction of this switch were established, the signal-induced oxidation, however, remained unclear. Here, we show that CRMP2 is oxidized through a redox relay involving the flavin-mooxygenase MICAL1 and the peroxidase Prx1 as specific signal transducers. Using molecular oxygen and electrons provided by NADPH, MICAL produces hydrogen peroxide and specifically oxidizes Prx1 through direct interactions between the proteins. Subsequently, Prx1 oxidizes CRMP2. The lack of any components of this redox relay dysregulates neurite outgrowth. Consequently, both oxidation and reduction of CRMP2 require reducing equivalents in the form of NADPH.

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.

Thioredoxins (Trxs) provide electrons to essential cellular processes such as DNA synthesis. Here, we characterize human and murine Trx1 as new iron-sulfur proteins. The [2Fe-2S] cluster is complexed using cysteinyl side chains 32 and 73 in a dimeric holocomplex. Formation of the holo-dimer depends on small structural changes of the loop connecting helices three and four and is stabilized by the formation of a direct electrostatic interaction between Lys72 and Asp60 of two monomers. The not strictly conserved Cys73 in vertebrates co-evolved with the regulation of cellular iron homeostasis through the iron-regulatory proteins (IRP). Active apo-Trx1 is required for the reduction of cysteinyl residues in IRP1 and its binding to the iron-responsive elements in the mRNA encoding hypoxiainducible factor (HIF) 2α. Depletion of Trx1 increased the mRNA levels of HIF2α, an important target of IRP1. Hence, translation of the HIF2α mRNA requires either sufficient iron-supply or the lack of reducing power of the Trx system under iron-limiting conditions. Only then, HIF2α protein may accumulate under hypoxic conditions to transcriptionally regulate processes like erythropoiesis.Significance StatementThioredoxins are, in general, cofactor-less key proteins in redox regulation and provide electrons to many essential cellular processes such as DNA synthesis. 55 years after its discovery, we show that mammalian thioredoxin 1 coordinates an iron-sulfur cluster using one of its active site cysteinyl residues and a non-conserved additional cysteinyl residue located outside the active site. This particular residue co-evolved with the vertebratespecific iron regulatory system. Our study demonstrates that this system is regulated by thioredoxin 1 at the level of the iron-regulatory protein 1, thus linking redox and iron homeostases.

Cold physical plasma is a partially ionized gas investigated as a new anticancer tool in selectively targeting cancer cells in monotherapy or in combination with therapeutic agents. Here, we investigated the intrinsic resistance mechanisms of tumor cells towards physical plasma treatment. When analyzing the dose-response relationship to cold plasma-derived oxidants in 11 human cancer cell lines, we identified four ‘resistant’ and seven ‘sensitive’ cell lines. We observed stable intracellular glutathione levels following plasma treatment only in the ‘resistant’ cell lines indicative of altered antioxidant mechanisms. Assessment of proteins involved in GSH metabolism revealed cystine-glutamate antiporter xCT (SLC7A11) to be significantly more abundant in the ‘resistant’ cell lines as compared to ‘sensitive’ cell lines. This decisive role of xCT was confirmed by pharmacological and genetic inhibition, followed by cold physical plasma treatment. Finally, microscopy analysis of ex vivo plasma-treated human melanoma punch biopsies suggested a correlation between apoptosis and basal xCT protein abundance. Taken together, our results demonstrate that xCT holds the potential as a biomarker predicting the sensitivity of tumor cells towards plasma treatment. Keywords: Cancer, Glutathione, kINPen, Melanoma, Plasma medicine

Malignant melanoma is an aggressive cancer that develops drug resistance leading to poor prognosis. Efficient delivery of chemotherapeutic drugs to the tumor tissue remains a major challenge in treatment regimens. Using murine (B16) and human (SK-MEL-28) melanoma cells, we investigated traditional cytotoxic agents in combination with cold physical plasma-derived oxidants. We report synergistic cytotoxicity of doxorubicin and epirubicin, and additive toxicity of oxaliplatin with plasma exposure in coefficient of drug interaction analysis. The combination treatment led to an increased DNA damage response (increased phosphorylation of ATM, γ-H2AX foci, and micronuclei formation). There was also an enhanced secretion of immunogenic cell death markers ATP and CXCL10 in cell culture supernatants following combination treatment. The observed synergistic effects in tumor cells was due to enhanced intracellular doxorubicin accumulation via upregulation of the organic cationic transporter SLC22A16 by plasma treatment. The doxorubicin uptake was reversed by pretreating cells with antioxidants or calcium influx inhibitor BTP2. Endoribonuclease-prepared siRNAs (esiRNA)-mediated knockdown of SLC22A16 inhibited the additive cytotoxic effect in tumor cells. SK-MEL 28 and THP-1 monocytes co-culture led to greater THP-1 cell migration and SK-MEL-28 cytotoxicity when compared with controls. Taken together, we propose pro-oxidant treatment modalities to sensitize chemoresistant melanoma cells towards subsequent chemotherapy, which may serve as therapeutic strategy in combination treatment in oncology.

Despite striking advances in the treatment of metastasized melanoma, the disease is often still fatal. Attention is therefore paid towards combinational regimens. Oxidants endogenously produced in mitochondria are currently targeted in pre-clinical and clinical studies. Cytotoxic synergism of mitochondrial cytochrome c oxidase (CcO) inhibition in conjunction with addition of exogenous oxidants in 2D and 3D melanoma cell culture models were examined. Murine (B16) and human SK-MEL-28 melanoma cells exposed to low-dose CcO inhibitors (potassium cyanide or sodium azide) or exogenous oxidants alone were non-toxic. However, we identified a potent cytotoxic synergism upon CcO inhibition and plasma-derived oxidants that led to rapid onset of caspase-independent melanoma cell death. This was mediated by mitochondrial dysfunction induced by superoxide elevation and ATP depletion. This observation was validated by siRNA-mediated knockdown of COX4I1 in SK-MEL-28 cells with cytotoxicity in the presence of exogenous oxidants. Similar effects were obtained with ADDA 5, a recently identified specific inhibitor of CcO activity showing low toxicity in vivo. Human keratinocytes were not affected by this combinational treatment, suggesting selective effects on melanoma cells. Hence, targeting mitochondrial CcO activity in conjunction with exogenous pro oxidant therapies may constitute a new and effective melanoma treatment modality.

Due to its functional duality in toxicity as well as signaling, research on therapeutic manipulation of reactive oxygen species (ROS) in cancer has been futile. Targeting melanoma cell death with excess amounts ROS, however, is difficult as these cells are highly resistant towards oxidative stress, creating a need for alternative and combinational approaches. Tumor metabolism mainly relies on aerobic glycolysis. We investigated a potential synergism of mitochondrial complex IV inhibition and exogenous-derived oxidants with both single treatment modalities being essentially non-toxic in melanoma cells. In this study, we investigated murine (metastatic B16F10 and non-metastatic B16F0) as well as human (SK-Mel 28) melanoma cells, and compared them with murine (primary fibroblasts) and human (HaCaT keratinocytes) non-cancer cells. Mitochondrial complex IV inhibition was realized using sodium azide (NaN3) and potassium cyanide (KCN) and pharmacological inhibitor ADDA5. Exogenous oxidants were generated using cell culture medium that has been treated with the cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). This plasma-treated medium was subsequently applied to different cells. Metabolic activity was assessed by fluorescence quantification of resazurin-to-resorufin transformation. ATP levels were monitored by a luminescence assay. Total cell counts, intracellular ROS levels (DCF), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). For tumor cells, also 3D spheroid models were investigated for viability by sytox green staining. ADDA5, NaN3 and KCN treatment alone failed to induce a significant alteration in metabolic activity in cancer cells. We then exposed the cells pretreated with NaN3 (100-500 µM) or KCN (250-500 µM) to plasma-treated medium. There was a significant inhibition of metabolic activity and rapid induction of cell death 6 h following treatment in melanoma cells compared to cell receiving plasma-treated medium alone. Increase in cellular superoxide levels and decrease in mitochondrial membrane potential and ATP levels was observed preceding cell death. However, no caspase 3 cleavage was observed upon immunoblotting. siRNA mediated knockdown of COX4a led to increased sensitivity of tumor cells towards plasma-treated medium. Furthermore, we validated our findings using a pharmacological inhibitor of complex IV ADDA5 in a tumor spheroid model and demonstrated that complex IV inhibition supplemented with plasma-treated medium, triggered significant cell death via caspase independent mechanism in melanoma cells.

Introduction Chemotherapeutic agents remain as the essential regimen against malignant melanoma. Variety of combination therapies have been employed to sensitize tumor cells, circumvent drug resistance, and reduce systemic toxicity. However, there is still need for innovative combinational approaches to resolve these difficulties. In the current study, we investigated the potential synergism of cold physical plasma in conjunction with chemotherapeutic agents and identified mechanisms of synergistic immunogenic cell death (ICD) in tumor cells in vitro Methods In this study, we pretreated murine B16F10 and human SK-Mel 28 melanoma cells with increasing concentrations of Doxorubicin, Epirubicin, Oxaliplatin, Vorinostat and SAHA for 24h. This was followed by 30 secs exposure cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). After 6-hour incubation, IC50 was determined using alamar blue assay and compared with single treatment modalities. Cell culture supernatants were used to quantify markers of immunogenic cell death (CART, ATP, HMGB1, CXCL10) by ELISA, immunofluorescence and flow cytometry. Total cell counts, intracellular superoxide levels (DHE), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). Co culture and transwell migration assays was performed on THP1 monocytes and RAW 264.7 macrophages. For SK-Mel 28 tumor cells, 3D spheroid models were also investigated. Results Combination therapy of chemotherapeutic agents and cold physical plasma significantly decreased the IC50 values in melanoma cell lines in comparison to single treatment modalities in 2D and 3D culture conditions. The tumor cell killing was mediated by elevation of superoxide and loss of mitochondrial membrane potential. Co culturing SK-Mel 28 cells with THP1 monocytes or RAW macrophages with B16F10 following combination therapy led to pronounced tumor cell killing. ICD markers such as ATP, HMGB1, CART, and CXCL10 were induced in both single treatment conditions however were significantly elevated upon combination therapy demonstrating a synergistic effect. Transwell migration assay revealed migration of THP1 monocytes and RAW 264.7 macrophages following incubation with conditioned medium. In this study, we have demonstrated that cold physical plasma is a valuable tool to induce ICD in tumors pretreated with conventional chemotherapeutic agents.