Versatile roles of cysteine persulfides in tumor biology.

Rewiring the transsulfuration pathway is recognized as a rapid adaptive metabolic response to environmental conditions in cancer cells to support their increased cysteine demand and to produce Reactive Sulfur Species (RSS) including hydrogen sulfide (H2S) and cysteine persulfide. This can directly (via RSS) or indirectly (by supplying Cys) trigger chemical or enzyme catalyzed persulfidation on critical protein cysteine residues to protect them from oxidative damage and to orchestrate protein functions, and thereby contribute to cancer cell plasticity. In this review key aspects of persulfide-mediated biological processes are highlighted and critically discussed in relation to cancer cell survival, bioenergetics, proliferation as well as in tumor angiogenesis, adaptation to hypoxia and oxidative stress, and regulation of epithelial to mesenchymal transition.

Cystathionine ß-synthase overexpression drives metastatic dissemination in pancreatic ductal adenocarcinoma via inducing epithelial-to-mesenchymal transformation of cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancer types with a constant rise in global incidence. Therefore, better understanding of PDAC biology, in order to design more efficient diagnostic and treatment modalities, is a priority. Here we found that the expression levels of cystathionine ß-synthase (CBS), a transsulfuration enzyme, is markedly elevated in metastatic PDAC cells compared to cell lines isolated from non-metastatic primary tumors. On human immunohistochemical samples from PDAC patients we also found higher CBS staining in cancerous ductal cells compared to in non-tumor tissue, which was further elevated in the lymph node metastasis of the same patients. In mice, orthotopically injected CBS-silenced T3M4 cells induced fewer liver metastases compared to control cells indicating important roles for CBS in PDAC cancer cell invasion and malignant transformation. Wound healing and colony formation assays in cell culture confirmed that CBS-deficient metastatic T3M4 and non-metastatic BxPC3 primary tumor cells migrate slower and have impaired anchorage-independent growth capacities compared to control T3M4 cells. CBS silencing in T3M4 cells lowered WNT5a and SNAI1 gene expression down to levels that were observed in BxPC3 cells as well as resulted in an increase in E-cadherin and a decrease in Vimentin signals in mouse tumors when injected orthotopically. These observations suggested a primary role for the epithelial to mesenchymal transformation of cancer cells in CBS-mediated tumor aggressiveness. Under normal conditions, STAT3, an upstream regulator of Wnt signaling pathways, was less phosphorylated and more oxidized in shCBS T3M4 and BxPC3 compared to control T3M4 cells, which is consistent with decreased transcriptional activity at lower CBS levels due to less protection against oxidation. Sulfur metabolome analyses suggested that this CBS-mediated protection against oxidative modifications is likely to be related to persulfide/sulfide producing activities of the enzyme rather than its canonical function to produce cystathionine for cysteine synthesis. Taken together, CBS overexpression through regulation of the EMT plays a significant role in PDAC cancer cell invasion and metastasis.

Reprogrammed transsulfuration promotes basal-like breast tumor progression via realigning cellular cysteine persulfidation.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast tumors with poor prognosis and limited molecular-targeted therapy options. We show that BLBC cells have a high Cys demand and reprogrammed Cys metabolism. Patient-derived BLBC tumors from four different cohorts exhibited elevated expression of the transsulfuration enzyme cystathione ß-synthetase (CBS). CBS silencing (shCBS) made BLBC cells less invasive, proliferate slower, more vulnerable to oxidative stress and cystine (CySSCy) deprivation, prone to ferroptosis, and less responsive to HIF1-α activation under hypoxia. shCBS xenograft tumors grew slower than controls and exhibited impaired angiogenesis and larger necrotic areas. Sulfur metabolite profiling suggested that realigned sulfide/persulfide-inducing functions of CBS are important in BLBC tumor progression. Supporting this, the exclusion of serine, a substrate of CBS for producing Cys but not for producing sulfide/persulfide, did not exacerbate CySSCy deprivation-induced ferroptosis in shCBS BLBC cells. Impaired Tyr phosphorylation was detected in shCBS cells and xenografts, likely due to persulfidation-inhibited phosphatase functions. Overexpression of cystathione γ-lyase (CSE), which can also contribute to cellular sulfide/persulfide production, compensated for the loss of CBS activities, and treatment of shCBS xenografts with a CSE inhibitor further blocked tumor growth. Glutathione and protein-Cys levels were not diminished in shCBS cells or xenografts, but levels of Cys persulfidation and the persulfide-catabolizing enzyme ETHE1 were suppressed. Finally, expression of enzymes of the oxidizing Cys catabolism pathway was diminished, but expression of the persulfide-producing CARS2 was elevated in human BLBC tumors. Hence, the persulfide-producing pathways are major targetable determinants of BLBC pathology that could be therapeutically exploited.

The role of glutamate oxaloacetate transaminases in sulfite biosynthesis and H2S metabolism.

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are two rare genetic disorders that are caused by impairment of the mitochondrial enzyme sulfite oxidase. Sulfite oxidase is catalyzing the terminal reaction of cellular cysteine catabolism, the oxidation of sulfite to sulfate. Absence of sulfite oxidase leads to the accumulation of sulfite, which has been identified as a cellular toxin. However, the molecular pathways leading to the production of sulfite are still not completely understood. In order to identify novel treatment options for both disorders, the understanding of cellular cysteine catabolism - and its alterations upon loss of sulfite oxidase - is of utmost importance. Here we applied a new detection method of sulfite in cellular extracts to dissect the contribution of cytosolic and mitochondrial glutamate oxaloacetate transaminase (GOT) in the transformation of cysteine sulfinic acid to sulfite and pyruvate. We found that the cytosolic isoform GOT1 is primarily responsible for the production of sulfite. Moreover, loss of sulfite oxidase activity results in the accumulation of sulfite, H2S and persulfidated cysteine and glutathione, which is consistent with an increase of SQR protein levels. Surprisingly, none of the known H2S-producing pathways were found to be upregulated under conditions of sulfite toxicity suggesting an alternative route of sulfite-induced shift from oxidative to H2S dependent cysteine catabolism.

Interplay of Liver-Heart Inflammatory Axis and Cannabinoid 2 Receptor Signaling in an Experimental Model of Hepatic Cardiomyopathy.

BACKGROUND AND AIMS: Hepatic cardiomyopathy, a special type of heart failure, develops in up to 50% of patients with cirrhosis and is a major determinant of survival. However, there is no reliable model of hepatic cardiomyopathy in mice. We aimed to characterize the detailed hemodynamics of mice with bile duct ligation (BDL)-induced liver fibrosis, by monitoring echocardiography and intracardiac pressure-volume relationships and myocardial structural alterations. Treatment of mice with a selective cannabinoid-2 receptor (CB2 -R) agonist, known to attenuate inflammation and fibrosis, was used to explore the impact of liver inflammation and fibrosis on cardiac function. APPROACH AND RESULTS: BDL induced massive inflammation (increased leukocyte infiltration, inflammatory cytokines, and chemokines), oxidative stress, microvascular dysfunction, and fibrosis in the liver. These pathological changes were accompanied by impaired diastolic, systolic, and macrovascular functions; cardiac inflammation (increased macrophage inflammatory protein 1, interleukin-1, P-selectin, cluster of differentiation 45-positive cells); and oxidative stress (increased malondialdehyde, 3-nitrotyrosine, and nicotinamide adenine dinucleotide phosphate oxidases). CB2 -R up-regulation was observed in both livers and hearts of mice exposed to BDL. CB2 -R activation markedly improved hepatic inflammation, impaired microcirculation, and fibrosis. CB2 -R activation also decreased serum tumor necrosis factor-alpha levels and improved cardiac dysfunction, myocardial inflammation, and oxidative stress, underlining the importance of inflammatory mediators in the pathology of hepatic cardiomyopathy. CONCLUSIONS: We propose BDL-induced cardiomyopathy in mice as a model for hepatic/cirrhotic cardiomyopathy. This cardiomyopathy, similar to cirrhotic cardiomyopathy in humans, is characterized by systemic hypotension and impaired macrovascular and microvascular function accompanied by both systolic and diastolic dysfunction. Our results indicate that the liver-heart inflammatory axis has a pivotal pathophysiological role in the development of hepatic cardiomyopathy. Thus, controlling liver and/or myocardial inflammation (e.g., with selective CB2 -R agonists) may delay or prevent the development of cardiomyopathy in severe liver disease.

Disruption of Renal Arginine Metabolism Promotes Kidney Injury in Hepatorenal Syndrome in Mice.

Tubular dysfunction is an important feature of renal injury in hepatorenal syndrome (HRS) in patients with end-stage liver disease. The pathogenesis of kidney injury in HRS is elusive, and there are no clinically relevant rodent models of HRS. We investigated the renal consequences of bile duct ligation (BDL)-induced hepatic and renal injury in mice in vivo by using biochemical assays, real-time polymerase chain reaction (PCR), Western blot, mass spectrometry, histology, and electron microscopy. BDL resulted in time-dependent hepatic injury and hyperammonemia which were paralleled by tubular dilation and tubulointerstitial nephritis with marked upregulation of lipocalin-2, kidney injury molecule 1 (KIM-1) and osteopontin. Renal injury was associated with dramatically impaired microvascular flow and decreased endothelial nitric oxide synthase (eNOS) activity. Gene expression analyses signified proximal tubular epithelial injury, tissue hypoxia, inflammation, and activation of the fibrotic gene program. Marked changes in renal arginine metabolism (upregulation of arginase-2 and downregulation of argininosuccinate synthase 1), resulted in decreased circulating arginine levels. Arginase-2 knockout mice were partially protected from BDL-induced renal injury and had less impairment in microvascular function. In human-cultured proximal tubular epithelial cells hyperammonemia per se induced upregulation of arginase-2 and markers of tubular cell injury. CONCLUSION: We propose that hyperammonemia may contribute to impaired renal arginine metabolism, leading to decreased eNOS activity, impaired microcirculation, tubular cell death, tubulointerstitial nephritis and fibrosis. Genetic deletion of arginase-2 partially restores microcirculation and thereby alleviates tubular injury. We also demonstrate that BDL in mice is an excellent, clinically relevant model to study the renal consequences of HRS. (Hepatology 2018; 00:000-000).

Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis.

Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvß6 signaling, as judged by its ability to inhibit these pathways in cnr1-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.

Cannabidiol Limits T Cell-Mediated Chronic Autoimmune Myocarditis: Implications to Autoimmune Disorders and Organ Transplantation.

Myocarditis is a major cause of heart failure and sudden cardiac death in young adults and adolescents. Many cases of myocarditis are associated with autoimmune processes in which cardiac myosin is a major autoantigen. Conventional immunosuppressive therapies often provide unsatisfactory results and are associated with adverse toxicities during the treatment of autoimmune myocarditis. Cannabidiol (CBD) is a nonpsychoactive constituent of marijuana that exerts antiinflammatory effects independent of classical cannabinoid receptors. Recently, 80 clinical trials have investigated the effects of CBD in various diseases from inflammatory bowel disease to graft versus host disease. CBD-based formulations are used for the management of multiple sclerosis in numerous countries, and CBD also received U.S. Food and Drug Administration approval for the treatment of refractory childhood epilepsy and glioblastoma multiforme. Herein, using a well-established mouse model of experimental autoimmune myocarditis (EAM) induced by immunization with cardiac myosin emmulsified in adjuvant resulting in T cell-mediated inflammation, cardiomyocyte cell death, fibrosis and myocardial dysfunction, we studied the potential beneficial effects of CBD. EAM was characterized by marked myocardial T-cell infiltration, profound inflammatory response and fibrosis (measured by quantitative real-time polymerase chain reaction, histology and immunohistochemistry analyses) accompanied by marked attenuation of both systolic and diastolic cardiac functions measured with a pressure-volume conductance catheter technique. Chronic treatment with CBD largely attenuated the CD3+ and CD4+ T cell-mediated inflammatory response and injury, myocardial fibrosis and cardiac dysfunction in mice. In conclusion, CBD may represent a promising novel treatment for managing autoimmune myocarditis and possibly other autoimmune disorders and organ transplantation.

Protection from Radiation-Induced Pulmonary Fibrosis by Peripheral Targeting of Cannabinoid Receptor-1.

Radiation-induced pulmonary fibrosis (RIF) is a severe complication of thoracic radiotherapy that limits its dose, intensity, and duration. The contribution of the endocannabinoid signaling system in pulmonary fibrogenesis is not known. Using a well-established mouse model of RIF, we assessed the involvement of cannabinoid receptor-1 (CB1) in the onset and progression of pulmonary fibrosis. Female C57BL/6 mice and CB1 knockout mice generated on C57BL/6 background received 20 Gy (2 Gy/min) single-dose thoracic irradiation that resulted in pulmonary fibrosis and animal death within 15 to 18 weeks. Some C57BL/6 animals received the CB1 peripherally restricted antagonist AM6545 at 1 mg/kg intraperitoneally three times per week. Animal survival and parameters of pulmonary inflammation and fibrosis were evaluated. Thoracic irradiation (20 Gy) was associated with marked pulmonary inflammation and fibrosis in mice and high mortality within 15 to 18 weeks after exposure. Genetic deletion or pharmacological inhibition of CB1 receptors with a peripheral CB1 antagonist AM6545 markedly attenuated or delayed the lung inflammation and fibrosis and increased animal survival. Our results show that CB1 signaling plays a key pathological role in the development of radiation-induced pulmonary inflammation and fibrosis, and peripherally restricted CB1 antagonists may represent a novel therapeutic approach against this devastating complication of radiotherapy/irradiation.

Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing.

Extracellular ATP binds to and signals through P2X7 receptors (P2X7Rs) to modulate immune function in both inflammasome-dependent and -independent manners. In this study, P2X7(-/-) mice, the pharmacological agonists ATP-magnesium salt (Mg-ATP; 100 mg/kg, EC50 ≈ 1.32 mM) and benzoylbenzoyl-ATP (Bz-ATP; 10 mg/kg, EC50 ≈ 285 µM), and antagonist oxidized ATP (oxi-ATP; 40 mg/kg, IC50 ≈ 100 µM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture-induced polymicrobial sepsis in mice. Our results with P2X7(-/-) bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloid-specific P2X7(-/-) mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 ≈ 0.25 µM) and pannexin channel inhibitor probenecid (10 mg/kg, IC50 ≈ 11.7 µM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial burden. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the treatment of sepsis.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

CD39 improves survival in microbial sepsis by attenuating systemic inflammation.

Sepsis remains the leading cause of morbidity and mortality in critically ill patients. Excessive inflammation is a major cause of organ failure and mortality in sepsis. Ectonucleoside triphosphate diphosphohydrolase 1, ENTPDase1 (CD39) is a cell surface nucleotide-metabolizing enzyme, which degrades the extracellular purines ATP and ADP, thereby regulating purinergic receptor signaling. Although the role of purinergic receptor signaling in regulating inflammation and sepsis has been addressed previously, the role of CD39 in regulating the host's response to sepsis is unknown. We found that the CD39 mimic apyrase (250 U/kg) decreased and knockout or pharmacologic blockade with sodium polyoxotungstate (5 mg/kg; IC50 ≈ 10 µM) of CD39 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture. CD39 decreased inflammation, organ damage, immune cell apoptosis, and bacterial load. Use of bone marrow chimeric mice revealed that CD39 expression on myeloid cells decreases inflammation in septic mice. CD39 expression is upregulated during sepsis in mice, as well as in both murine and human macrophages stimulated with Escherichia coli. Moreover, E. coli increases CD39 promoter activity in macrophages. Altogether, these data indicate CD39 as an evolutionarily conserved inducible protective pathway during sepsis. We propose CD39 as a novel therapeutic target in the management of sepsis.

Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis.

UNLABELLED: Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. CONCLUSION: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.

Role of poly(ADP-ribosyl)ation in a 'two-hit' model of hypoxia and oxidative stress in human A549 epithelial cells in vitro.

A preceding hypoxic insult can sensitize the cells or the organism to a subsequent, second insult. The aim of the present study was to investigate the molecular mechanism of this phenomenon (often termed 'two-hit' injury paradigm), in an in vitro model of hypoxia/oxidative stress injury in A549 epithelial cells, with special emphasis on the role of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) in the process. Pre-exposure of the cells to 24 h hypoxia significantly reduced intracellular glutathione (GSH) levels, reduced mitochondrial activity and adenosine triphosphate (ATP) levels. However pre-exposure to hypoxia failed to induce any change in PARP-1 expression and activation, DNA single­strand breaks or plasma membrane integrity. Pre-exposure to hypoxia markedly increased the sensitivity of the cells to subsequent oxidative stress-induced DNA damage. Hydrogen peroxide (H2O2) induced a concentration-dependent increase in DNA breakage, PARP activation, depletion of intracellular ATP, inhibition of mitochondrial activity and two distinct parameters that quantify the breakdown of plasma membrane integrity (propidium iodide uptake or lactate dehydrogenase release). PARP-1 activation played a significant role in the H2O2-induced cell death response because PARP activation, depletion of intracellular ATP, inhibition of mitochondrial activity, and the breakdown of plasma membrane integrity were attenuated in cells with permanently silenced PARP-1. Based on measurement of the endogenous antioxidant GSH, we hypothesized that the mechanism of hypoxia-mediated enhancement of H2O2 involves depletion of the GSH during the hypoxic period, which renders the cells more sensitive to a subsequent DNA single­strand break elicited by H2O2. DNA strand breakage then activates PARP-1, leading to the inhibition of mitochondrial function, depletion of ATP and cell necrosis. PARP-1 deficiency protects against the cytotoxicity, to a lesser degree, by protecting against GSH depletion during the hypoxic period, and, to a larger degree, by maintaining mitochondrial function and preserving intracellular ATP levels during the subsequent oxidative stress period.

Intramitochondrial hydrogen sulfide production by 3-mercaptopyruvate sulfurtransferase maintains mitochondrial electron flow and supports cellular bioenergetics.

It is well established that exposure of mammalian cells to hydrogen sulfide (H(2)S) suppresses mitochondrial function by inhibiting cytochrome-c oxidase (CcOX; complex IV). However, recent experimental data show that administration of H(2)S to mammalian cells can serve as an electron donor and inorganic source of energy. The aim of our study was to investigate the role of endogenously produced H(2)S in the regulation of mitochondrial electron transport and oxidative phosphorylation in isolated liver mitochondria and in the cultured murine hepatoma cell line Hepa1c1c7. Low concentrations of H(2)S (0.1-1 µM) elicited a significant increase in mitochondrial function, while higher concentrations of H(2)S (3-30 µM) were inhibitory. The positive bioenergetic effect of H(2)S required a basal activity of the Krebs cycle and was most pronounced at intermediate concentrations of succinate. 3-mercaptopyruvate (3-MP), the substrate of the mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) stimulated mitochondrial H(2)S production and enhanced mitochondrial electron transport and cellular bioenergetics at low concentrations (10-100 nM), while at higher concentrations, it inhibited cellular bioenergetics. SiRNA silencing of 3-MST reduced basal bioenergetic parameters and prevented the stimulating effect of 3-MP on mitochondrial bioenergetics. Silencing of sulfide quinone oxidoreductase (SQR) also reduced basal and 3-MP-stimulated bioenergetic parameters. We conclude that an endogenous intramitochondrial H(2)S-producing pathway, governed by 3-MST, complements and balances the bioenergetic role of Krebs cycle-derived electron donors. This pathway may serve a physiological role in the maintenance of mitochondrial electron transport and cellular bioenergetics.

Hydrogen peroxide-induced poly(ADP-ribosyl)ation regulates osteogenic differentiation-associated cell death.

We set out to investigate the role of poly(ADP-ribosylation), the attachment of NAD(+)-derived (ADP-ribose)(n) polymers to proteins, in the regulation of osteogenic differentiation of SAOS-2 cells and mesenchymal stem cells. In osteogenic differentiation medium, SAOS-2 cells showed mineralization and expressed alkaline phosphatase and osteoblastic marker genes such as Runx2, osterix, BMP2, and osteopontin. The cells also released hydrogen peroxide, displayed poly(ADP-ribose) polymerase (PARP) activation, and showed commitment to cell death (apoptosis and necrosis). Scavenging reactive oxygen species by glutathione or decomposing hydrogen peroxide by the addition of catalase reduced differentiation, PARP activation, and cell death. We silenced the expression of the main PAR-synthesizing enzyme PARP-1 and the PAR-degrading enzyme poly(ADP-ribose) glycohydrolase (PARG) in SAOS-2 osteosarcoma cells (shPARP-1 and shPARG, respectively). Both shPARP-1- and shPARG-silenced cells exhibited altered differentiation, with the most notable change being increased osteopontin expression but decreased alkaline phosphatase activity. PARP-1 silencing suppressed both apoptotic and necrotic cell death, but the PARP inhibitor PJ34 sensitized cells to cell death, indicating that the effects of PARP-1 silencing are not related to the activity of the enzyme. PARG silencing resulted in more apoptosis and, in the last days of differentiation, a shift from apoptosis toward necrosis. In conclusion our data prove that hydrogen peroxide-induced poly(ADP-ribose) signaling regulates cell death and osteodifferentiation.

Poly(ADP-ribosyl)ation is a survival mechanism in cigarette smoke-induced and hydrogen peroxide-mediated cell death.

Cigarette smoking can contribute to the development of many human diseases such as cardiovascular disease, lung cancer, asthma, and chronic obstructive pulmonary disease. Thousands of compounds are present in cigarette smoke, including a large number of reactive oxygen species that can cause DNA damage, leading to the activation of poly(ADP-ribose) polymerase (PARP) enzymes. The PAR polymer is degraded by poly(ADP-ribose) glycohydrolase (PARG). Here we have investigated the effects of cigarette smoke extract (CSE) on A549 human lung epithelial cells. CSE induced DNA damage (comet assay), PAR accumulation (immunofluorescence and immunoblotting), impaired proliferation (clonogenic survival assay and electric cell-substrate impedance sensing measurement), and cell death (MTT reduction, propidium iodide uptake, lactate dehydrogenase release). CSE-induced cell death was also characterized by mitochondrial depolarization but massive translocation of apoptosis-inducing factor could not be observed. To investigate the role of PARylation in CSE-induced oxidative stress, PARP-1- and PARG-silenced A549 cells were used. Silencing of both PARP-1 and PARG sensitized cells to CSE-induced toxicity: PARP-1- and PARG-silenced cell lines exhibited reduced clonogenic survival, displayed a delayed repair of DNA breaks, and showed higher levels of cytotoxicity. CSE triggered the production of mitochondrial superoxide and hydrogen peroxide. Addition of superoxide dismutase increased, whereas catalase abolished, CSE-induced PAR formation. In summary, our data show that the superoxide-hydrogen peroxide-DNA breakage pathway activates the PAR cycle by PARP-1 and PARG, which serves as a survival mechanism in CSE-exposed cells. Our data also raise the possibility that the PARP-1/PARG status of smokers may be an important determinant of the efficiency of DNA repair in their lungs and of their susceptibility to CS-induced carcinogenesis.

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation.

Hydrogen sulfide (H(2)S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H(2)S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H(2)S and NO exert their effects via separate pathways, the results of the current study show that H(2)S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H(2)S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H(2)S-stimulated angiogenic response, and attenuated H(2)S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H(2)S signaling. Conversely, silencing of the H(2)S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H(2)S in the vascular activity of NO. The actions of H(2)S and NO converge at cGMP; though H(2)S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H(2)S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H(2)S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H(2)S are mutually required for the physiological control of vascular function.

Short-term adaptation of rat intestine to ileostomy: implication for pediatric practice.

BACKGROUND: Surgical neonates with complex intestinal conditions, such as enterocolitis, midgut volvulus with bowel loss and multiple atresias, often require temporary stomas. Little is known on the postsurgical response of the altered gut segments, although adaptation is an important consideration in neonatal postoperative care, particularly after stoma closure. MATERIALS AND METHODS: Rats underwent bowel resection at a point 15 cm proximal to the ileocecal valve, and a split ileostomy was performed. On the 6th postoperative day the mucosal thickness was calculated with Soft Imaging System Analysis Pro, the rate of proliferation was measured following Ki67 immunohistochemistry and the apoptotic index was determined on sections stained with ApopTag Plus. The intestinal motor activity was recorded on isolated gut segments. Neuronal nitric oxide synthase (nNOS) expression and distribution was examined with NADPH-diaphorase histochemistry and Western blot analysis. RESULTS: An increased wet weight of the mucosa and a pronounced mucosal thickening were observed in the proximal functional bowel segment. Enterocyte proliferation rate was increased significantly, while the apoptotic index remained unchanged in the epithelial layer. The dilation of the gut lumen resulted in a morphological change in the nitrergic myenteric network with an overexpression of nNOS. As a consequence of the surgical procedure, the functional proximal gut segment showed strong and frequent contraction waves, with an enhanced responsiveness to cholinergic stimuli. CONCLUSIONS: The dilated functional bowel segment was characterized by hyperplasic changes in the mucosa and stronger mechanical activity with overproduction of nNOS. Although early restoration of intestinal continuity is recommended, our observations on adaptive changes may partly explain intestinal motility disorders after early stoma closure, suggesting the need for a careful approach to a redo-laparotomy.

Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells.

Activation of poly(ADP-ribose) polymerase-1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure. shPARG cells accumulated large amounts of poly(ADP-ribosyl)ated proteins and exhibited reduced PARP activation. Hydrogen peroxide-induced cell death is regulated by PARG in a dual fashion. Whereas the shPARG cell line (similarly to shPARP1 cells) was resistant to the necrotic effect of high concentrations of hydrogen peroxide, these cells exhibited stronger apoptotic response. Both shPARP1 and especially shPARG cells displayed a delayed repair of DNA breaks and exhibited reduced clonogenic survival following hydrogen peroxide treatment. Translocation of apoptosis-inducing factor could not be observed, but cells could be saved by methyl pyruvate and alpha-ketoglutarate, indicating that energy failure may mediate cytotoxicity in our model. These data indicate that PARG is a survival factor at mild oxidative damage but contributes to the apoptosis-necrosis switch in severely damaged cells.