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Colon cancer cells contain high levels of cystathlonlne-β-synthase (CBS). Its product, hydrogen sulfide (H2S), promotes the growth and proliferation of colorectal tumor cells. To improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle or AOAA (0.1, 0.5 or 1 mg/kg/d) for 3 wks. Tumor growth was significantly reduced by 9 mg/kg/d AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/d. Thus, the in vivo efficacy of YD0171 is nine times higher than that of AOAA. YD0171 (1 mg/kg/d) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/d) also reduced tumor growth in patient-derived tumor xenograft bearing athymic mice. YD0171 (3 mg/kg/d) induced the regression of established HCT116 tumors in vivo. A 5-d safety study in mice demonstrated that YD0171 at 20 mg/kg/d (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

Significance: Cancer represents a major socioeconomic problem; there is a significant need for novel therapeutic approaches targeting tumor-specific pathways. Recent Advances: In colorectal and ovarian cancers, an increase in the intratumor production of hydrogen sulfide (H2S) from cystathionine β-synthase (CBS) plays an important role in promoting the cellular bioenergetics, proliferation, and migration of cancer cells. It also stimulates peritumor angiogenesis inhibition or genetic silencing of CBS exerts antitumor effects both in vitro and in vivo, and potentiates the antitumor efficacy of anticancer therapeutics. Critical Issues: Recently published studies are reviewed, implicating CBS overexpression and H2S overproduction in tumor cells as a tumor-growth promoting “bioenergetic fuel” and “survival factor,” followed by an overview of the experimental evidence demonstrating the anticancer effect of CBS inhibition. Next, the current state of the art of pharmacological CBS inhibitors is reviewed, with special reference to the complex pharmacological actions of aminooxyacetic acid. Finally, new experimental evidence is presented to reconcile a controversy in the literature regarding the effects of H2S donor on cancer cell proliferation and survival. Future Directions: From a basic science standpoint, future directions in the field include the delineation of the molecular mechanism of CBS up-regulation of cancer cells and the delineation of the interactions of H2S with other intracellular pathways of cancer cell metabolism and proliferation. From the translational science standpoint, future directions include the translation of the recently emerging roles of H2S in cancer into human diagnostic and therapeutic approaches. Antioxid. Redox Signal. 22, 424–448.

Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1 mM, while 3 mM was inhibitory. Longer-term (72 h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects.

Until recently, hydrogen sulfide (H2S) was exclusively viewed a toxic gas and an environmental hazard, with its toxicity primarily attributed to the inhibition of mitochondrial Complex IV, resulting in a shutdown of mitochondrial electron transport and cellular ATP generation. Work over the last decade established multiple biological regulatory roles of H2S, as an endogenous gaseous transmitter. H2S is produced by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). In striking contrast to its inhibitory effect on Complex IV, recent studies showed that at lower concentrations, H2S serves as a stimulator of electron transport in mammalian cells, by acting as a mitochondrial electron donor. Endogenous H2S, produced by mitochondrially localized 3-MST, supports basal, physiological cellular bioenergetic functions; the activity of this metabolic support declines with physiological aging. In specialized conditions (calcium overload in vascular smooth muscle, colon cancer cells), CSE and CBS can also associate with the mitochondria; H2S produced by these enzymes, serves as an endogenous stimulator of cellular bioenergetics. The current article overviews the biochemical mechanisms underlying the stimulatory and inhibitory effects of H2S on mitochondrial function and cellular bioenergetics and discusses the implication of these processes for normal cellular physiology. The relevance of H2S biology is also discussed in the context of colonic epithelial cell physiology: colonocytes are exposed to high levels of sulfide produced by enteric bacteria, and serve as a metabolic barrier to limit their entry into the mammalian host, while, at the same time, utilizing it as a metabolic ‘fuel’. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8

Emerging work demonstrates the dual regulation of mitochondrial function by hydrogen sulfide (H2S), including, at lower concentrations, a stimulatory effect as an electron donor, and, at higher concentrations, an inhibitory effect on cytochrome C oxidase. In the current article, we overview the pathophysiological and therapeutic aspects of these processes. During cellular hypoxia/acidosis, the inhibitory effect of H2S on complex IV is enhanced, which may shift the balance of H2S from protective to deleterious. Several pathophysiological conditions are associated with an overproduction of H2S (e.g. sepsis), while in other disease states H2S levels and H2S bioavailability are reduced and its therapeutic replacement is warranted (e.g. diabetic vascular complications). Moreover, recent studies demonstrate that colorectal cancer cells up-regulate the H2S-producing enzyme cystathionine β-synthase (CBS), and utilize its product, H2S, as a metabolic fuel and tumour-cell survival factor; pharmacological CBS inhibition or genetic CBS silencing suppresses cancer cell bioenergetics and suppresses cell proliferation and cell chemotaxis. In the last chapter of the current article, we overview the field of H2S-induced therapeutic ‘suspended animation’, a concept in which a temporary pharmacological reduction in cell metabolism is achieved, producing a decreased oxygen demand for the experimental therapy of critical illness and/or organ transplantation. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8

The inflammatory process plays a crucial role in the onset and progression of several lung pathologies, including cystic fibrosis (CF),andthe involvement of NF-κBis widely recognized.Thespecific inhibition of NF-κB by decoy oligonucleotides delivered within the lung may be beneficial, although rationally designed systems are needed to optimize their pharmacological response. Prompted by this need, we have developed and tested in vivo an inhalable dry powder for the prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPPs) based on poly(lactic-co-glycolic) acid. First, LPPs containing dec- ODN (dec-ODN LPPs) were engineered to meet the aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release, and to preserve its structural integrity in lung lining fluids. We then investigated the effects of dec- ODN LPPs in a rat model of lung inflammation induced by the intratracheal aerosolization of LPS from Pseudomonas aeruginosa. Theresults show that a single intratracheal insufflation of dec-ODN LPPs reduced the bronchoalveolar neutrophil infiltration induced by LPS for up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated with reduced NF-κB/DNA binding activity, as well as reduced IL-6, IL-8, and mucin-2 mRNA expression in lung homogenates. We consider it noteworthy that the developed LPPs, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for the local treatment of inflammation associated with lung disease. Copyright © 2013 by the American Thoracic Society.

The physiological functions of hydrogen sulfide (H 2 S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H 2 S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H 2 S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H 2 S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H 2 S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H 2 S produced from CBS serves to ( i ) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and ( ii ) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H 2 S as a tumor growth factor and anticancer drug target.

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300-1,000 µM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 µM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6‑morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 µM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of inosine for protection against various forms of warm and cold liver ischemia is relevant.

BACKGROUND AND PURPOSE Hydrogen sulphide (H2S), considered as a novel gas transmitter, is produced endogenously in mammalian tissue from L-cysteine by two enzymes, cystathionine β-synthase and cystathionine γ-lyase. Recently, it has been reported that H2S contributes to the local and systemic inflammation in several experimental animal models. We conducted this study to investigate on the signalling involved in H2S-induced inflammation. EXPERIMENTAL APPROACH L-cysteine or sodium hydrogen sulphide (NaHS) was injected into the mouse hind paw and oedema formation was evaluated for 60 min. In order to investigate H2S-induced oedema formation, we used 5-HT and histamine receptor antagonists, and inhibitors of KATP channels or arachidonic acid cascade. Prostaglandin levels were determined in hind paw exudates by radioimmunoassay. Paws injected with L-cysteine or NaHS were examined by histological methods. KEY RESULTS Both NaHS and L-cysteine caused oedema characterized by a fast onset which peaked at 30 min. This oedematogenic action was not associated with histamine or 5-HT release or KATP channel activation. However, oedema formation was significantly inhibited by the inhibition of cyclooxygenases and selective inhibition of phospholipase A2. Prostaglandin levels were significantly increased in exudates of hind paw injected with NaHS or L-cysteine. The histological examination clearly showed an inflammatory state with a loss of tissue organization following NaHS or L-cysteine injection. CONCLUSIONS AND IMPLICATIONS Phospholipase A2 and prostaglandin production are involved in pro-inflammatory effects of H2S in mouse hind paws. The present study contributes to the understanding of the role of L-cysteine/H2S pathway in inflammatory disease.

Introduction: Bidirectional paracrine signaling between cancer cells and fibroblasts in the stroma facilitates tumor progression. We have shown that CRC cells upregulate cystathionine β synthase (CBS), resulting in H2S production, when compared to normal colonic epithelium, and that CRC-derived H2S increases CRC growth, migration, and tumor angiogenesis. The aims of this study are to determine whether H2S can promote CAF proliferation and migration, and stimulate NCF to develop into a CAF-like phenotype. Methods: Human CAF from CRC and NCF were collected under an IRB approved protocol and established in culture. CAF were grown for up to 120h in the presence of vehicle or a slow-release H2S donor, GYY4137. CAFs were allowed to migrate through transwell chambers toward conditioned media (CM) from HCT116 or normal colon mucosal cells (NCM356). Western blots for NCF and CAF were performed to determine CBS expression levels. Results: H2S donor GYY4137 (30-300μM) significantly increased proliferation of CAF, bu...

Hydrogen sulfide (H 2 S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H 2 S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H 2 S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H 2 S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H 2 S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H 2 S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H 2 S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections. IMPORTANCE RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.

Aims H2S is known to confer cardioprotection; however, the pathways mediating its effects in vivo remain incompletely understood. The purpose of the present study is to evaluate the contribution of cGMP-regulated pathways in the infarct-limiting effect of H2S in vivo . Methods and results Anaesthetized rabbits were subjected to myocardial ischaemia (I)/reperfusion (R), and infarct size was determined in control or H2S-exposed groups. The H2S donor sodium hydrosulfide (NaHS, an agent that generates H2S) increased cardiac cGMP and reduced the infarct size. The cGMP-dependent protein kinase (PKG)-I inhibitor DT2 abrogated the protective effect of NaHS, whereas the control peptide TAT or l-nitroarginine methyl ester (l-NAME) did not alter the effect of NaHS. Moreover, the KATP channel inhibitor, glibenclamide, partially reversed the effects of NaHS, whereas inhibition of mitochondrial KATP did not modify the NaHS response. NaHS enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner. To further investigate the role of PLN in H2S-mediated cardioprotection, wild-type and PLN KO mice underwent I/R. NaHS did not exert cardioprotection in PLN KO mice. Unlike what was observed in rabbits, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. Conclusions Our findings demonstrate (i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and (ii) contribution of nitric oxide to the H2S response is species-specific.

Introduction The goal of the current study was to investigate the effect of aging on the development of endothelial dysfunction in a murine model of sepsis, and to compare it with the effect of genetic deficiency of the endothelial isoform of nitric oxide synthase (eNOS). Methods Cecal ligation and puncture (CLP) was used to induce sepsis in mice. Survival rates were monitored and plasma indices of organ function were measured. Ex vivo studies included the measurement of vascular function in thoracic aortic rings, assessment of oxidative stress/cellular injury in various organs and the measurement of mitochondrial function in isolated liver mitochondria. Results eNOS deficiency and aging both exacerbated the mortality of sepsis. Both eNOS-deficient and aged mice exhibited a higher degree of sepsis-associated multiple organ dysfunction syndrome (MODS), infiltration of tissues with mononuclear cells and oxidative stress. A high degree of sepsis-induced vascular oxidative damage and endothelial dysfunction (evidenced by functional assays and multiple plasma markers of endothelial dysfunction) was detected in aortae isolated from both eNOS−/− and aged mice. There was a significant worsening of sepsis-induced mitochondrial dysfunction, both in eNOS-deficient mice and in aged mice. Comparison of the surviving and non-surviving groups of animals indicated that the severity of endothelial dysfunction may be a predictor of mortality of mice subjected to CLP-induced sepsis. Conclusions Based on the studies in eNOS mice, we conclude that the lack of endothelial nitric oxide production, on its own, may be sufficient to markedly exacerbate the severity of septic shock. Aging markedly worsens the degree of endothelial dysfunction in sepsis, yielding a significant worsening of the overall outcome. Thus, endothelial dysfunction may constitute an early predictor and independent contributor to sepsis-associated MODS and mortality in aged mice.

Although hydrogen sulfide (H₂S) is generally known as a mitochondrial poison, recent studies show that lower concentrations of H₂S play a physiological role in the stimulation of mitochondrial electron transport and cellular bioenergetics. This effect involves electron donation at Complex II. Other lines of recent studies demonstrated that one of the biological actions of H₂S involves inhibition of cAMP and cGMP phosphodiesterases (PDEs). Given the emerging functional role of the mitochondrial isoform of cAMP PDE (PDE2A) in the regulation of mitochondrial function the current study investigated whether cAMP-dependent mechanisms participate in the stimulatory effect of NaHS on mitochondrial function. In isolated rat liver mitochondria, partial digestion studies localized PDE2A into the mitochondrial matrix. NaHS exerted a concentration-dependent inhibitory effect on recombinant PDE2A enzyme in vitro. Moreover, NaHS induced an elevation of cAMP levels when added to isolated mitochondria and stimulated the mitochondrial electron transport. The latter effect was inhibited by Rp-cAMP, an inhibitor of the cAMP-dependent protein kinase (PKA). The current findings suggest that the direct electron donating effect of NaHS is amplified by an intramitochondrial cAMP system, which may involve the inhibition of PDE2A and subsequent, cAMP-mediated stimulation of PKA.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H???S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H???S are cystathionine ?? synthase (CBS) and cystathionine ?? lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H???S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: ??-Cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC?????? 14 ± 0.2 ??M vs. 40 ± 8 ??M respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC?????? 1.1 ± 0.1?????M); the IC?????? for AOAA for inhibiting CBS was 8.5 ± 0.7 ??M. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H???S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC?????? for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Purpose Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule with significant pathophysiological importance, but its role in retinal neovascular diseases is unknown. Hydrogen sulfide is generated from L-cysteine by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and/or 3-mercaptopyruvate sulfurtransferase (3-MST). The aim of this study was to investigate the role of H2S in retinal neovascularization (NV) in ischemia-induced retinopathy. Methods Studies were performed in a murine model of oxygen-induced retinopathy (OIR). Hydrogen sulfide was detected with a fluorescent assay. Western blots and immunohistochemistry were used to assess the changes of H2S-producing enzymes. Gene deletion and pharmacologic inhibition were used to investigate the role of H2S in retinal NV. Results Hydrogen sulfide production was markedly increased in retinas from OIR mice compared with those from room air (RA) controls. Cystathionine-β-synthase and CSE were significantly increased in OIR retinas, whereas 3-MST was not changed. Cystathionine-β-synthase was expressed throughout the neuronal retina and upregulated in neurons and glia during OIR. Cystathionine-γ-lyase was also localized to multiple retinal layers. Its immunoreactivity was prominently increased in neovascular tufts in OIR. Pharmacologic inhibition of CBS/CSE or genetic deletion of CSE significantly reduced retinal NV in OIR. Conclusions Our data indicate that the H2S-generating enzymes/H2S contributes to retinal NV in ischemia-induced retinopathy and suggest that blocking this pathway may provide novel therapeutic approaches for the treatment of proliferative retinopathy.

Hydrogen sulfide (H(2)S), a novel gaseous transmitter, is considered a physiological regulator of vascular homeostasis. Recent evidence suggests H(2)S as an endothelium-hyperpolarizing factor (EDHF) candidate. To address this issue, we evaluated the vascular effect of sodium hydrogen sulfide (NaHS), an H(2)S donor, on the rat mesenteric arterial bed. NaHS concentration-response curve was performed on preconstricted mesenteric arterial bed. To assess the contribution of EDHF, we performed a pharmacologic dissection using indomethacin, N(G)-nitro-l-arginine methyl ester (l-NAME), or apamin and charybdotoxin as cyclooxygenase, nitric-oxide synthase, and calcium-dependent potassium channel inhibitors, respectively. In another set of experiments, we used 4-(4-octadecylphenyl)-4-oxobutenoic acid, baicalein, or proadifen as phospholipase A(2) (PLA(2)), lipoxygenase, and cytochrome P450 inhibitors, respectively. Finally, an immunofluorescence study was performed to support the involvement of PLA(2) in mesenteric artery challenged by NaHS. NaHS promoted a dual vascular effect (i.e., vasoconstriction and vasodilation). l-NAME or baicalein administration affected neither NaHS-mediated vasodilation nor vasoconstriction, whereas apamin and charybdotoxin significantly inhibited NaHS-induced relaxation. Pretreatment with PLA(2) inhibitor abolished both the contracting and the relaxant effect, whereas P450 cytochrome blocker significantly reduced NaHS-mediated relaxation. The immunofluorescence study showed that NaHS caused a migration of cytosolic PLA(2) close to the nucleus, which implicates activation of this enzyme. Our data indicate that H(2)S could activate PLA(2), which in turn releases arachidonic acid leading, initially, to vasoconstriction followed by vasodilation mediated by cytochrome P450-derived metabolites. Because EDHF has been presumed to be a cytochrome P450 derivative of the arachidonic acid, our results suggest that H(2)S acts through EHDF release.

Hydrogen sulfide (H2S) has emerged as a gaseous signaling molecule with important roles in neuromodulation, blood pressure regulation, angiogenesis, cardioprotection, cellular energetics and apoptosis. H2S is produced endogenously by three enzymes in the sulfur metabolic network, namely cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Although several compounds that inhibit CSE are available, no selective inhibitors for CBS or 3MST currently exist. In the present study we screened the LOPAC library for inhibitors of H2S-producing enzymes. The methylene blue assay was used to measure H2S production from recombinant enzymes. HCT116 and HT29 cells were used for MTT-based proliferation assays. Screening of the LOPAC library yielded several hit compounds that blocked H2S-synthesizing enzymes to varying extents. Among those, doxycyclin and demeclocycline were found to inhibit 3MST by approximately 50% at 100 uM, with doxycyclin being a more potent 3MST inhibitor than demeclocycline in the in vitro activity assay. Tetracyclin, meclocyclin, oxycyclin, methacyclin, tigecyclin and minocyclin did not affect 3MST activity when used up to 300 uM. From the above-mentioned tetracyclines, tigecyclin at 100 uM caused only a slight inhibition of both CBS and CSE; tigecyclin additionally inhibited CSE. In a cell-based assay, demeclocyclin, but not doxycyclin, inhibited the growth of HCT116 and HT29, two colon cancer cell lines in which CBS is highly expressed and its product, H2S supports cell proliferation. We conclude that based on its selectivity towards 3MST, demeclocyclin could serve as a lead compound that after undergoing optimization could yield future selective 3MST inhibitors.

Introduction Recent data indicate that hydrogen sulfide (H2S) has dual effects on cellular bioenergetics: promotion of electron transport at low (physiological) concentrations, and inhibition of mitochondrial respiration at higher (cytotoxic) concentrations. Based on recent data from our group, demonstrating the upregulation of the H2S-producing enzyme cystathionine-β-synthase (CBS) in surgical specimens from human colorectal cancer patients, and in human colon cancer cell lines, we have investigated the subcellular localization of CBS in HCT116 colon cancer cell line and explored the potential role of CBS-derived H2S in the regulation of their bioenergetic function in vitro. Methods HCT116 cells were cultured in McCoy’s 5A medium. For shRNA-mediated silencing of CBS and CSE, HCT116 cells were transduced with either a lentiviral vector containing shRNA sequences targeting CBS or CSE. A non-targeting control shRNA sequence (shNT) was used to control of off-target effects. In studies investigating the cellular localization of CBS, HCT116 cells were subjected subcellular fractionation by centrifugation. Mitochondrial and cytosolic fractions were analyzed by western blotting; membranes were probed overnight with anti-CBS (60 kDa), anti-β-actin (43 kDa), anti-LDH (35 kDa), anti-Tom20 (20 kDa) and anti-Complex IV (17 kDa) antibodies. In a separate set of experiments, the mitochondrial outer membrane was subjected to limited trypsin digestion, followed by Western blotting. The XF24 Extracellular Flux Analyzer (Seahorse) was used to measure bioenergetic function. Oxygen consumption rate (OCR) after oligomycin (1.5 μg/ml) was used to assess ATP production rate and OCR after FCCP (0.5 μM) to assess maximal mitochondrial respiratory capacity. 2-deoxyglucose (100 mM) was used to estimate cellular glycolytic dependency and antimycin A (2 μg/ml) and rotenone (2 μM) were used to inhibit the flux of electrons through complex III and I, to detect residual non-mitochondrial activity. For the measurement of glycolytic parameters, the changes in Proton Production Rate (PPR) were monitored in response to the sequential administration of D-glucose (10 mM), oligomycin (1.5 μM) and 2-deoxyglucose (100 mM), to assess glycolysis, maximal glycolytic capacity, glycolytic reserve capacity, and non-glycolytic acidification rate, respectively. GAPDH activity was determined by a kinetic assay. Bioenergetic measurements in mitochondria isolated from shNT and shCBS HCT116 cells were conducted in the absence or presence of L-cysteine (30 nM). Basal respiration (State 2) in the presence of succinate and rotenone was measured, followed by State 3 (phosphorylating respiration), in the presence of ADP. State 4o was determined after the addition of oligomycin, and maximal uncoupler-stimulated respiration (State 3u) was assessed in the presence of FCCP. Results Cell fractionation studies showed that a significant portion of the total amount of cellular CBS was associated with the mitochondria in HCT116 cells. A trypsin digestion assay on isolated mitochondria showed that CBS was primary associated with the outer mitochondrial membrane. In agreement with the physiological role of endogenous H2S in promoting cellular bioenergetics in various cell types including colonocytes, low concentrations of H2S caused a stimulation of mitochondrial function in mitochondria isolated from HCT116 cells. Moreover, shRNA-mediated silencing of CBS (but not of CSE) expression, or CBS inhibition with AOAA reduced basal cellular respiration, suppressed the calculated ATP synthesis, and attenuated the spare respiratory capacity. CBS silencing or CBS inhibition also reduced glycolytic functions, while CSE silencing was without significant effect. This latter effect may be attributed, at least in part, to inhibition of GAPDH activity: GAPDH activity was quantified in HCT116 cells, and it was reduced by CBS silencing. (GAPDH activity in wild-type and CBS silenced cells amounted to 6.2 ± 0.1 U/ml and 4.0 ± 0.1 U/ml, respectively, p Conclusions Prior studies have demonstrated that H2S donors can stimulate mitochondrial electron transport and ATP generation in various cell lines in vitro. Furthermore, H2S, via sulfhydration, has been shown to increase the catalytic activity of the glycolytic enzyme GAPDH. Consistent with these findings, our results show that both oxidative and glycolytic tumor cell metabolism is suppressed by shRNA-mediated silencing of CBS, or by treating cells with AOAA. Tumor bioenergetics relies both on oxidative phosphorylation, as well as glycolysis. Because proliferation, migration and invasion are energetically demanding processes, we hypothesize that CBS inhibition/silencing reduces the intracellular levels of H2S, which, in turn, contributes to the energy starvation of the tumor, impairing its growth. In addition to bioenergetic effects, we hypothesize that endogenous H2S may also contribute to tumor growth by a variety of mechanisms including activation of phosphoinositide-3-kinase and Akt kinase, inhibition of phosphatases and the regulation of cell cycle genes. Together with additional in vitro and in vivo data presented at the current meeting, these data support the view that CBS-derived H2S serves as an endogenous cancer cell bioenergetic factor, and identify CBS as a potential future antitumor drug target.

Background In the field of environmental toxicology, H 2 S is viewed as a mitochondrial toxin: an inhibitor of mitochondrial electron transport by inhibiting cytochrome C oxidase (Complex IV).In bacteria and multicellular organisms living in oxygen-deprived environments, however, H 2 S can function as an electron donor and a source of energy. Recently, several studies suggested that this role of H 2 S as an electron donor and an inorganic source of energy may also exist in mammalian cells. The goal of our study was to define the relative role of these two processes in hepatic mitochondria and in hepatocytes. Methods Using Extracellular Flux Analysis technique (Seahorse XF24, a technique which measures oxygen consumption in a micro-chamber), in isolated rat liver mitochondria and cultured murine hepatocytes, we investigated the relative role of H 2 S and the Krebs-cycle-derived metabolite succinate in the regulation of mitochondrial electron transport and oxidative phosphorylation. In addition, we investigated the effect of the H 2 S precursor l -cysteine on mitochondrial H 2 S production, and on mitochondrial and cellular bioenergetics. H 2 S production from isolated mitochondria was measured by the methylene blue method. Results Succinate, at concentrations between 5 and 20 mM, induced a dose-dependent increase in mitochondrial electron transport. H 2 S, on its own (in the presence of ADP, but in the absence of succinate) was unable to stimulate mitochondrial electron transport. On the other hand, in the presence of 5 or 10 mM succinate (which partially stimulates mitochondrial electron transport), low concentrations of H 2 S (0.1–1 μM) elicited a significant increase in mitochondrial activity. Higher concentrations of H 2 S (3–30 μM) suppressed mitochondrial activity. In rat liver mitochondria, as well as in cultured hepatocytes, the H 2 S precursor l -cysteine also induced a similar biphasic effect on energetics: at lower concentrations (10–30 μM) it increased mitochondrial reserve capacity and oxygen consumption rate, while at higher concentrations (100–1000 μM) it became inhibitory. The l -cysteine-induced production of H 2 S in isolated mitochondria was confirmed by the methylene blue method. Conclusion H 2 S and l -cysteine appear to have a physiological role as a positive modulator of mitochondrial electron transport and its actions are dependent on a basal activity of the Krebs cycle. We hypothesize that both Krebs-cycle-derived electron donors and exogenously administered or endogenously produced H 2 S, as an electron donor, converge at Coenzyme Q. At higher concentrations of H 2 S or l -cysteine the inhibitory effect on Complex IV becomes predominant and mitochondrial function becomes suppressed; this finding is consistent with the inhibitory effect of H 2 S on Complex IV. The fact that the bioenergetic effects of H 2 S are most pronounced at intermediate concentrations of succinate may suggest that the bioenergetic effects of H 2 S may be most important a secondary/back-up/emergency ’fuel’ in cells where bioenergetic function is slightly impaired. The current result may help explain some of the biphasic or opposing biological effects of hydrogen sulfide on cell viability in physiology and pathophysiology.