Combination of chemotherapy and physical plasma elicits melanoma cell death via upregulation of SLC22A16

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

Cytochrome C oxidase Inhibition and Cold Plasma-derived Oxidants Synergize in Melanoma Cell Death Induction

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

xCT (SLC7A11) expression confers intrinsic resistance to physical plasma treatment in tumor cells

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

Incidence, Risk Factors, and Outcomes of Intra-Abdominal Hypertension in Critically Ill Patients-A Prospective Multicenter Study (IROI Study)

To identify the prevalence, risk factors, and outcomes of intra-abdominal hypertension in a mixed multicenter ICU population. Prospective observational study. Fifteen ICUs worldwide. Consecutive adult ICU patients with a bladder catheter. None. Four hundred ninety-one patients were included. Intra-abdominal pressure was measured a minimum of every 8 hours. Subjects with a mean intra-abdominal pressure equal to or greater than 12 mm Hg were defined as having intra-abdominal hypertension. Intra-abdominal hypertension was present in 34.0% of the patients on the day of ICU admission (159/467) and in 48.9% of the patients (240/491) during the observation period. The severity of intra-abdominal hypertension was as follows: grade I, 47.5%; grade II, 36.6%; grade III, 11.7%; and grade IV, 4.2%. The severity of intra-abdominal hypertension during the first 2 weeks of the ICU stay was identified as an independent predictor of 28-and 90-day mortality, whereas the presence of intra-abdominal hypertension on the day of ICU admission did not predict mortality. Body mass index, Acute Physiology and Chronic Health Evaluation II score greater than or equal to 18, presence of abdominal distension, absence of bowel sounds, and positive end-expiratory pressure greater than or equal to 7 cm H2O were independently associated with the development of intra-abdominal hypertension at any time during the observation period. In subjects without intra-abdominal hypertension on day 1, body mass index combined with daily positive fluid balance and positive end-expiratory pressure greater than or equal to 7 cm H2O (as documented on the day before intra-abdominal hypertension occurred) were-associated with the development of intraabdominal hypertension during the first week in the ICU. In our mixed ICU patient cohort, intra-abdominal hypertension occurred in almost half of all subjects and was twice as prevalent in mechanically ventilated patients as in spontaneously breathing patients. Presence and severity of intra-abdominal hypertension during the observation period significantly and independently increased 28-and 90-day mortality. Five admission day variables were independently associated with the presence or development of intra-abdominal hypertension. Positive fluid balance was associated with the development of intra-abdominal hypertension after day 1474535542NIGMS NIH HHSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [U54 GM104940

Cysteinyl and methionyl redox switches: Structural prerequisites and consequences

Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications

Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity-Leads to common and unique interaction partners and functions.

The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPH-dependent Trx reductases

Combination of chemotherapy and physical plasma elicits melanoma cell death via upregulation of SLC22A16

Abstract 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

Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases

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

Cytochrome C oxidase Inhibition and Cold Plasma-derived Oxidants Synergize in Melanoma Cell Death Induction

Abstract 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