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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.

Hydrogen sulfide (H2S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H2S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H2S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H2S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H2S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H2S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H2S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants dl-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H2S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H2S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.

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.

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. Addition of AP39 (30-300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30-100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

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

Angiogenesis is a major physiological response to ischemia that involves development of new capillaries and collateral circulation from preexisting vessels, and endothelial cell (EC) proliferation and migration are crucial events for this process. Epalrestat, an aldose reductase inhibitor, is used as a therapeutic agent for the treatment of diabetic peripheral neuropathy. We examined the effect of epalrestat on the proliferation and migration of a mouse microvascular endothelial cell line (bEnd3 cells) measured by scratch wound assay and cell proliferation assay. Epalrestat significantly enhanced the proliferation and migration of bEnd3 cells. The addition of epalrestat resulted in the phosphorylation of Akt and p70 S6 kinase (p70S6K), and this effect was attenuated by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR), inhibited p70S6K phosphorylation only. Both rapamycin and LY294002 inhibited epalrestat-induced proliferation and migration. These data suggest that epalrestat may activate PI3/Akt/mTOR signaling and that this pathway contributes to the proliferation and migration of EC.

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.

We have conducted a phenotypic screening in endothelial cells exposed to elevated extracellular glucose (an in vitro model of hyperglycemia) to identify compounds that prevent hyperglycemia-induced reactive oxygen species (ROS) formation without adversely affecting cell viability. From a focused library of >6,000 clinically used drug-like and pharmacologically active compounds, several classes of active compounds emerged, with a confirmed hit rate of

Various forms of circulatory shock (including septic shock) lead to an impairment of vascular function, which importantly contributes to the development of multiple organ failure and mortality. Such dysfunction of blood vessels consists of two principal components: vascular smooth muscle (VSM) dysfunction, and endothelial dysfunction. The VSM dysfunction (progressive, therapy-resistant loss of VSM responsiveness to vasoconstrictor catecholamines, such as noradrenaline) leads to a progressive deterioration of blood pressure in patients with circulatory shock. The endothelial dysfunction (loss of the ability of the endothelium to produce nitric oxide and other endothelium-derived factors) contributes to the impairment of microvascular blood flow, to the enhanced adhesion and activation of neutrophils and platelets, to coagulation problems, and perfusion/metabolism mismatch in the affected organs. Here we overview the vascular regulatory functions of the novel gasotransmitter hydrogen sulfide (H2S), with an emphasis on its potential role in the pathogenesis of vascular dysfunction in circulatory shock. We first review the roles of endogenously produced or exogenously administered H2S on vascular function. Next, we review the results of published studies using shock models induced by bacterial lipopolysaccharide, and by cecal ligation and puncture, a polymicrobial model of sepsis showing overproduction of H2S. Finally, we summarize the potential mechanisms by which H2S may contribute to vascular dysfunction in shock and show an example of how the vascular response to H2S is altered in a rat model of endotoxemia. In addition, we outline the potential means by which modulation of H2S (pharmacological inhibition of its biosynthesis or therapeutic donation) may affect the outcome in circulatory shock.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.

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 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.

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H 2 S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro “hyperglycemia”) induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H 2 S. Replacement of H 2 S or overexpression of the H 2 S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H 2 S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H 2 S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE −/− mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocin-induced diabetes in rats resulted in a decrease in the circulating level of H 2 S; replacement of H 2 S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H 2 S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H 2 S catabolism form a positive feed-forward cycle. H 2 S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

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 Urotensin II (U-II) is a cyclic peptide originally isolated from the teleost neurosecretory system and subsequently identified in other species, including man. U-II was identified as the natural ligand of an orphan G-protein coupled receptor (UT receptor). U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. U-II caused both constrictor and vasodilator effect, depending by vascular bed. The in vivo functional consequences of U-II on the cardiovascular hemodynamics are not clearly understood. Aim To investigate the presence of UT receptor and the effect of U-II in human corpus cavernosum (HCC) strips. To evaluate the effect of U-II in vivo in anesthetized rats. Methods UT receptor expression as protein and as mRNA were assessed by Western blot and reverse transcriptase polymerase chain reaction. Next, the UT receptor localization was evaluated by immunohystochemical analysis. By using HCC strips, with or without endothelium, the effect of U-II (0.1 nM–10 µM) was evaluated. In order to asses the nitric oxide (NO) involvement, the strips were incubated with N (G)-nitro-L-arginine methyl ester (NO synthase inhibitor, 100 µM). U-II (0.1, 0.3, 1.0 nmol/rat) effect in vivo was studied in anesthetized rats by monitoring the intracavernous and systemic blood pressure. Main Outcome Measures HCC expresses the UT receptor and its activation, by UII, causes an endothelium- and NO-dependent relaxation. Results UT receptor is expressed in human and rat corpus cavernosum. In HCC UT receptor is localized on endothelial cells. U-II significantly relaxed HCC strips in endothelium- and –NO-dependent fashion. The peptide caused a significant increase in intracavernous pressure in anesthetized rats. Conclusion This study demonstrates that UT receptor is expressed on the endothelium of HCC. U-II/UT receptor system is involved in HCC function and it involves endothelium and NO pathway. Thus, U-II/UT receptor pathway could be involved in erectile function. d'Emmanuele di Villa Bianca R, Cirino G, Mitidieri E, Coletta C, Grassia G, Roviezzo F, Grieco P, Novellino E, Imbimbo C, Mirone V, and Sorrentino R. Urotensin II: A novel target in human corpus cavernosum.

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.

We investigated the regulation of mitochondrial poly(ADP-ribose) polymerase 1 (PARP1) by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) system during oxidative stress in U937 monocytes. Oxidative stress induced an early (10 minutes) mitochondrial DNA damage, and concomitant activation of PARP1 in the mitochondria. These early events were followed by a progressive mitochondrial oxidant production and nuclear PARP1 activation (by 6 hours). These processes led to a functional impairment of mitochondria, culminating in cell death of mixed (necrotic/apoptotic) type. β-Adrenoceptor blockade with propranolol or inhibition of its downstream cAMP/PKA signaling attenuated, while β-adrenoceptor agonists and cAMP/PKA activators enhanced, the oxidant-mediated PARP1 activation. In the presence of cAMP, recombinant PKA directly phosphorylated recombinant PARP1 on serines 465 (in the automodification domain) and 782 and 785 (both in the catalytic domain). Inhibition of the β-adrenergic receptor/cAMP/PKA axis protected against the oxidant-mediated cell injury. Propranolol also suppressed PARP1 activation in peripheral blood leukocytes during bacterial lipopolysaccharide (LPS)-induced systemic inflammation in mice. We conclude that the activation of mitochondrial PARP1 is an early, active participant in oxidant-induced cell death, which is under the control of β-adrenoceptor/cAMP/PKA axis through the regulation of PARP1 activity by PARP1 phosphorylation.

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.

Emerging work demonstrates the dual regulation of mitochondrial function by hydrogen sulfide (H2 S), 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 H2 S on complex IV is enhanced, which may shift the balance of H2 S from protective to deleterious. Several pathophysiological conditions are associated with an overproduction of H2 S (e.g. sepsis), while in other disease states H2 S levels and H2 S 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 H2 S-producing enzyme cystathionine β-synthase (CBS), and utilize its product, H2 S, 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 H2 S-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.

Recent data show that lower concentrations of hydrogen sulfide (H2S), as well as endogenous, intramitochondrial production of H2S by the 3-mercaptopyruvate (3-MP)/3-mercaptopyruvate sulfurtransferase (3-MST) pathway serves as an electron donor and inorganic source of energy to support mitochondrial electron transport and ATP generation in mammalian cells by donating electrons to Complex II. The aim of our study was to investigate the role of oxidative stress on the activity of the 3-MP/3-MST/H2S pathway in vitro. Hydrogen peroxide (H2O2, 100-500 μM) caused a concentration-dependent decrease in the activity of recombinant mouse 3-MST enzyme. In mitochondria isolated from murine hepatoma cells, H2O2 (50-500 μM) caused a concentration-dependent decrease in production of H2S from 3-MP. In cultured murine hepatoma cells H2O2, (3-100 μM), did not result in overall cytotoxicity, but caused a partial decrease in basal oxygen consumption and respiratory reserve rapacity. The positive bioenergetic effect of 3-MP (100-300 nM) was completely abolished by pre-treatment of the cells with H2O2 (50 μM). The current findings demonstrate that oxidative stress inhibits 3-MST activity and interferes with the positive bioenergetic role of the 3-MP/3-MST/H2S pathway. These findings may have implications for the pathophysiology of various conditions associated with increased oxidative stress, such as various forms of critical illness, cardiovascular diseases, diabetes or physiological aging.

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.

Thioglycine and l-thiovaline are stable under acidic and basic conditions but in the presence of bicarbonate they liberate the gasotransmitter H(2)S. In cells both thioamino acids were proven to enhance cGMP formation and promote vasorelaxation in mouse aortic rings. Given that H(2)S is known to lower arterial hypertension, reduce oxidative stress and exhibit cardioprotective effects in preclinical models, H(2)S donors hold promise as novel treatments for cardiovascular diseases.

Although few experimental studies have been handled so far to exploit the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in the production of dry powders for antibiotic inhalation, there has been no comprehensive study on the role played by NP composition. In this work, we try to shed light on this aspect by designing and developing a pulmonary delivery system for antibiotics, such as tobramycin (Tb), based on PLGA NPs embedded in an inert microcarrier made of lactose, referred to as nano-embedded micro-particles (NEM). At nanosize level, helper hydrophilic polymers were used to impart the desired surface, bulk and release properties to PLGA NPs prepared by a modified emulsion-solvent diffusion technique. Results showed that poly(vinyl alcohol) (PVA) and chitosan (CS) are essential to optimise the size and modulate the surface properties of Tb-loaded PLGA NPs, whereas the use of alginate (Alg) allows efficient Tb entrapment within NPs and its release up to one month. Optimized formulations display good in vitro antimicrobial activity against P. aeruginosa planktonic cells. Furthermore, spray-drying of the NPs with lactose yielded NEM with peculiar but promising flow and aerosolization properties, while preserving the peculiar NP features. Nonetheless, in vivo biodistribution studies showed that PVA-modified Alg/PLGA NPs reached the deep lung, while CS-modified NPs were found in great amounts in the upper airways, lining lung epithelial surfaces. In conclusion, PLGA NP composition appears to play a crucial role in determining not only the technological features of NPs but, once processed in the form of NEM, also their in vitro/in vivo deposition pattern. © 2011 Elsevier B.V.

Over two-thirds of colorectal cancer patients will develop liver metastases. The current standard-of-care is treatment with the chemotherapeutic agents 5-flurouracil alone or in combination with oxaliplatin (OX) or irinotecan; however, these drugs are often ineffective. We have previously demonstrated that CBS is selectively upregulated in colorectal cancer cells and stimulates hydrogen sulfide (H 2 S)-mediated cell migration and invasion in vitro , processes that define the metastatic phenotype. The aim of this study was to evaluate whether CBS inhibition with aminooxyacetic acid (AOAA) alone or in combination with OX reduced liver metastases in vivo . Human HCT116 colon cancer cells, which express high levels of CBS, were transfected with a luciferase reporter gene. Cells were injected into the cecal submucosa of athymic nude mice. Mice were treated either with vehicle, AOAA (SQ QOD at 9 mg/kg), OX (IP Qwk at 10 mg/kg) or AOAA plus OX (N = 6 per group) for 3 weeks. In vivo metastasis was measured (photons/s) using the IVIS Spectrum Imaging System. Post-harvest, liver metastases were visually counted on serial H& E stained sections and quantified using a weighted scale based on the number of cells within each metastatic focus. In vivo imaging revealed a significant decrease in luciferase activity in the AOAA plus OX group when compared to either AOAA or OX alone ( p = 0.04 and 0.008, respectively). Histological quantification of liver metastasis confirmed the in vivo findings. In conclusion, our data indicate that CBS inhibition enhances the efficacy of OX in reducing metastatic tumor burden.

Cells in the stromal microenvironment facilitate colorectal cancer progression and “co-evolve” with the malignant epithelial cells. It is well established that the tumor stroma contributes to the growth and metastatic spread of colorectal cancer. We have recently reported that colorectal cancer cells (CRC) express high levels of cystathionine-β-synthetase (CBS) and produce high amounts of hydrogen sulfide (H2S) when compared to the normal adjacent colonic epithelium. However, the effects of CRC-derived H2S on CRC-associated fibroblast (CAF) activity are unknown. The aim of this study was to determine whether H2S stimulates CAF proliferation and/or migration. Human CAFs from CRC were collected under an IRB-approved protocol and cultured. The xCELLigence system was used to assess cell proliferation for 144 h. For migration assays, 10[5] CAFs were suspended in serum-free media containing 0.1% BSA and added to 8 μm pore Transwell chambers. Cells were allowed to migrate toward the bottom chamber containing tumor cell conditioned media (CM) either with or without the slow-release H2S donor GYY4137. Migrated cells were quantified by visual counting. The results showed that GYY4137 (30 μM–3 mM) significantly increased CAF proliferation. Moreover, GYY4137 (1 mM) enhanced the migration of CAFs toward tumor-conditioned medium. Thus, the data demonstrate that H2S enhances CAF cell proliferation and migration in vitro. We hypothesize that cancer cell-derived H2S may play a role in promoting CAF proliferation and this effect may be part of a complex system of reciprocal interactions between fibroblasts and cancer cells important for tumor progression and spread.

Previous studies reported both beneficial and detrimental roles of hydrogen sulfide (H2S) in various models of critical illness. The goal of our study was to compare the outcome of septic shock in wild-type mice and in mice deficient in cystathionine-gamma-lyase (CSE). Mice were subjected to sepsis by cecal ligation and puncture and survival was monitored for 48 h. In a separate study, animals were sacrificed at 24 h post-CLP, vascular function was evaluated ex vivo and indices of organ function were measured. At 30 h, there was 20% mortality in CSE−/− mice, while no mortality in the wild-type controls. By 36 h, the mortality increased to 50% in CSE−/− mice, while it remained low (10%) in the wild-type controls. However, by 46 h, the two survival curves converged, and showed a similar, 60% mortality in both groups. Analysis of the plasma markers of organ injury revealed slight trends for a CLP-induced increase in the liver injury markers ALT and AST and the pancreatic injury marker amylase in wild-type mice. More pronounced increases were noted in the same parameters in the CSE−/− mice. Blood urea nitrogen levels were elevated 3-fold over baseline in wild-type mice subjected to CLP and 4-fold over baseline in CSE−/− mice. There was an impairment of endothelium-dependent relaxation responses in the thoracic aorta after CLP, which was more pronounced in the CSE−/− mice. These data indicate that CSE, and its product, H2S, exerts protective effects against the development of sepsis, especially in the early stages of the disease.

Introduction Although the pathophysiological roles of hydrogen sulfide (H2S) have been demonstrated or proposed in many diseases, the role of endogenously produced H2S in the pathogenesis of cancer has not yet been explored. Using surgical specimens from human colorectal cancer patients, and human colorectal cancer cell lines and corresponding control tissues and cells, we have investigated the potential changes in the expression of H2S-producing enzymes in colorectal cancer cells, and the potential role of endogenous H2S in the proliferation and invasion of colorectal cancer cells in vitro. Methods Freshly resected human colorectal tumor tissue, along with a specimen of patient-matched normal mucosa (wide margin), was collected under an IRB approved protocol and snap frozen in liquid N2. The colon cancer cells line HT-29 and the non-tumorigenic colon epithelial cell line derived from the normal margin of a rectal cancer specimen, NCM356 (NCM) were cultured in DMEM supplemented with 10% fetal bovine serum. HCT116 and LoVo cells were cultured in McCoy’s 5A and F-12, respectively. For detection of H 2S production in lysates of NCM or HCT116 cells, the amperometric H2S-specific sensor or the methylene blue assay were used. In addition, Western blotting for CSE, CBS and 3-MST was conducted in cell and tissue homogenates. 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. To overexpress CBS in the normal colon mucosa cell line NCM, gene transfer was accomplished using an adenovirus at a MOI of 10, with a green fluorescent protein – expressing adenovirus used as a control. For cellular migration and invasion assays, 105cells were suspended in serum-free media containing 0.1% BSA and then added to 6.5 mm Transwell chambers (8 mm pore) uncoated (migration assay) or coated with a thin layer (45 μl) of growth factor reduced matrigel (cell invasion assay). Cells were allowed to migrate toward the bottom chamber containing fibroblast (NIH-3T3 cells) conditioned media at 37 °C for 6 or 24 h for cell migration and invasion, respectively. Migrated and invaded cells were then fixed in Carson’s fixative, stained in 0.33% toluidine blue and quantified by visual counting. For HCT116-EAhy926 co-culture experiments, the cancer cell line was grown to confluence on the bottom wells of the transwell chambers containing 600 μl of DMEM media. Endothelial cells were grown until 60–70% confluent and then trypsinized, suspended at a density of 105 cells/ml in DMEM and added to the upper chamber (100 μl of the cell suspension). Cell migration was then assessed. For assessment of cell proliferation, the xCELLigence system (Roche) was used. Results Comparison of human colon cancer specimens with patient-matched normal mucosa tissue revealed the selective upregulation of the H2S-producing enzyme cystathionine-β-synthase (CBS) in the cancers. In contrast, the expression of the other two H2S-producing enzymes, cystathionine-γ-lyase (CSE) and 3-MST remained unchanged. Similar to colon tumors, colon adenocarcinoma-derived cell lines (HCT116, HT-29, LoVo) exhibited the selective upregulation of CBS, when compared to NCM cells. Homogenates of the patient-derived colon tumor specimens, as well as homogenates of the colon cancer-derived cell lines exhibited increased rates of H2S production, which was blocked by the CBS inhibitor aminooxyacetic acid (AOAA), but not by the CSE inhibitor propargylglycine PAG. Gene specific shRNA sequences in lentiviral vectors were used to suppress the expression of either CBS or CSE in HCT116 cells. Densitometric analyses of Western blots revealed an approximately 50% decrease in CBS expression with comparable reductions in both cell proliferation and H2S production. In contrast to CBS, silencing of CSE or the pharmacological CSE inhibitor propargylglycine (PAG, 3 mM) did not significantly affect either HCT116 cell proliferation or H2S production. To further define the function of CBS in colon cancer cells, we treated both NCM (which express CSE, but only low levels of CBS), and HCT116 cells (which overexpress CBS) with the pharmacological CBS inhibitor, amino-oxyacetic acid (AOAA). Consistent with the CBS knockdown experiments, AOAA treatment inhibited the growth of HCT116 colon cancer cells, but did not affect the proliferation of the slower-growing non-malignant NCM356 cell line. Conversely, forced overexpression of CBS in NCM356 cells significantly increased their basal rate of proliferation. AOAA treatment also suppressed the migration and invasion of HCT116 cells, while the presence of the H2S donor NaHS (30 μM) in the lower chamber slightly enhanced HCT116 cell migration and invasion in serum-free media. Inhibition of CBS with AOAA reduced endothelial cell migration in colon cancer/endothelial cell co-cultures. Conclusions The H2S-producing enzyme CBS is selectively overexpressed in human colorectal cancer cells, and significantly contributes to their proliferation, migration and invasion. Together with additional in vitroand in vivo data presented at the current meeting, these data support the view that CBS-derived H2S serves as an endogenous cancer cell survival factor, and identify CBS as a potential future antitumor drug target.

Background Hydrogen sulfide (H2S) and nitric oxide (NO) are recognized as essential endogenous gaseous signaling molecules involved in a variety of homeostatic and disease processes. Recent studies suggest that multiple interactions exist between H2S and NO, and indicate that these interactions are crucial in regulating biological responses of vascular cells. However, the precise nature and mechanisms of the interactions between these two gasotransmitters remain incompletely understood. The goal of the current study was to investigate whether angiogenesis and vasodilation require the simultaneous production of H2S and NO. Methods Cultured murine bEnd.3 endothelial cells were used for the studies. CSE was silenced using siRNA. eNOS and PKG were inhibited by pharmacological inhibitors. H2S production was measured by the methylene blue method. Burn-induced wound healing was measured in rats by planimetry. Results Exposure of endothelial cells to a H2S donors (NaHS) increased intracellular cGMP in a NO-dependent manner and activated protein kinase G (PKG) as evidenced by phosphorylation of its downstream substrate, VASP. Inhibition of eNOS or PKG abolished the H2S-stimulated angiogenic response. Inhibition of eNOS also attenuated H2S-stimulated vasorelaxation. These findings demonstrate the requirement of NO in vascular H2S signaling, and the convergence of the vascular actions of H2S and NO to the cGMP/PKG pathway. Conversely, silencing of the H2S-producing enzyme cystathionine-gamma-lyase (CSE) abolished NO-stimulated cGMP accumulation and angiogenesis and attenuated the NO-induced vasorelaxation, indicating the requirement of H2S in the vascular actions of NO. Moreover, acetylcholine-induced, endothelium-dependent relaxations were attenuated after silencing of CSE. VEGF, a pro-angiogenic and vasorelaxant hormone is associated with the production of both NO and H2S: VEGF-induced angiogenic and vasorelaxant responses are attenuated either by the inhibition of NO or by the silencing of CSE. A key site of the cooperative interaction between NO and H2S involves the cGMP/PKG pathway: H2S exerted a potent inhibitory effect on phosphodiesterase 5 (PDE5) activity in vitro leading to cGMP elevation and PKG activation. Another site of the interaction between H2S and NO is Akt: H2S increased the phosphorylation (activation) of Akt, as well as the phosphorylation of eNOS at Ser1177. The in vivo relevance of the current findings is highlighted by the fact that H2S-induced wound healing in rats and angiogenesis in the matrigel plug model in mice, were suppressed by pharmacological inhibition (L-NAME) or genetic ablation of eNOS (eNOS−/− mice). Conclusions In conclusion, NO and H2S are mutually required for the control of angiogenesis and vasorelaxation.

Previous studies reported both stimulatory and inhibitory effects of hydrogen sulfide (H2S) donors on the proliferation and viability of in various human cancer cell lines. The aim of this study was to test whether the contradictory data in the literature may be explained by the bell-shaped dose–response character of H2S. Using HCT116 colon cancer cells, proliferation rates were assessed in the presence of different concentrations (0.03–3 mM) of either NaHS (fast-release) or GYY4137 (slow-release) H2S donors, with or without pretreatment with the cystathionine-β-synthase (CBS) inhibitor aminooxyacetic acid (AOAA). In the absence of AOAA, low concentrations (0.03–0.3 mM) of NaHS stimulated proliferation whereas higher concentrations (1–3 mM) inhibited growth. CBS inhibition with AOAA shifted the dose–response to the left, decreasing basal proliferation but also limiting toxicity only to the cells exposed to 3 mM NaHS. In the absence of AOAA, all concentrations of GYY4137 (0.03–3 mM) stimulated HCT116 cell proliferation over the basal growth rate. However, only the highest concentration of GY4137 (3 mM) stimulated HCT116 cells when CBS was inhibited. These data demonstrate that CRC cell proliferation exhibits a biphasic (bell-shaped) dose–response to H2S. The precise nature of the cellular response (increased or decreased growth) is determined by rate H2S production (fast- vs. slow-release H2S donors) as well as the concentration of donor relative to the endogenous (basal) level of CBS-dependent H2S production. Our model provides a useful framework to reconcile some of the controversies in the literature regarding the role of H2S in the regulation of cancer cell proliferation.

We have previously shown that H2S, as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important source of hydrogen sulfide (H2S) in endothelial cells. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have adverse effect on cellular homeostasis. Given the importance of H2S as a vasorelaxant, angiogenesis stimulator and positive bioenergetic factor, here we tested whether a dysfunction of the 3-MST/H2S system develops in hyperglycemia/diabetes and studied its contribution to the development of diabetes-associated vascular complications. 3-mercaptopyruvate (3-MP, the substrate of 3-MST) displayed many characteristics of a H2S donor. Intraperitoneal 3-MP (1 mg/kg) raised plasma H2S levels in rats. 3-MP induced angiogenesis in vitro (10–1000 μM; bEnd3 cells) and in vivo (0.3–1 mg/kg; matrigel assay in mice). 3-MP also facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased the bioenergetic function of mitochondria. In vitro hyperglycemia or in vivo streptozotocin diabetes resulted in endothelial dysfunction mitochondrial dysfunction, impaired angiogenesis and reduced wound healing and was associated with a suppression of the vasodilatory, pro-angiogenic, wound-healing stimulating and bioenergetic effects of 3-MP. Systemic treatment with the lipoic acid restored the effect of 3-MP in diabetes, improving endothelial function, cellular bioenergetics and wound healing. We conclude that diabetes is associated with an impairment of the 3-MST/H2S pathway, and speculate that this may contribute to the pathogenesis of diabetic complications.

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 Several lines of studies demonstrate the biphasic bioenergetic effect of hydrogen sulfide (H 2 S): at lower concentrations, endogenously produced and exogenously administered H 2 S stimulates mitochondrial electron transport and supports oxidative phosphorylation, whereas at higher concentration, H 2 S inhibits mitochondrial respiration by inhibiting mitochondrial Complex IV (cytochrome C oxidase).In addition, it has recently been demonstrated that in the mitochondrial matrix there is a distinct cAMP pool, which is not related to the cytosolic cAMP pool.Furthermore, a soluble adenylyl cyclase enzyme (sAC) has been localized to the mitochondrial matrix, which is involved in the maintenance of the intra-mitochondrial cAMP levels.Given the recently emerging evidence that (a) H 2 S is an endogenous inhibitor of cAMP and cGMP phosphodiesterases and that (b) intramitochondrial cAMP and PKA act an endogenous stimulators of mitochondrial electron transport and oxidative phosphorylation, the aim of the current study was to explore the potential role of mitochondrial cAMP and its downstream effector, protein kinase A (PKA) in the stimulatory bioenergetic effect of H 2 S in vitro . Methods The presence of PDE2A (a combined cAMP- and cGMP-degrading phosphodiesterase) and of PKA was investigated by western blotting of native isolated rat liver mitochondria, as well as its partially digested forms to yield mitoplasts and post-mitoplasts.In addition, freshly isolated rat liver mitochondria were subjected to H 2 S donor NaHS and analyzed by Extracellular Flux Analysis (Seahorse) in the absence or presence of various modulators of mitochondrial cAMP/PKA, followed by the measurement of oxygen consumption rate in the absence or presence of ADP, as well as a sequential administration of specific modulators (the ATP synthase inhibitor oligomycin, the mitochondrial uncoupler FCCP and the Complex III inhibitor antimycin) of mitochondrial electron transport chain complexes.The effect of the H 2 S donor NaHS on the activity of recombinant PDEA2 (an enzyme involved in the degradation of cAMP) was measured in a cell-free assay. Results Western blot analysis confirmed the presence of PDE2A (a combined cAMP- and cGMP-degrading phosphodiesterase) and of PKA in the mitochondrial preparations.Addition of the membrane-permeable cAMP analog 8-bromo-cAMP or the H 2 S donor NaHS to isolated rat liver mitochondria both increased oxygen consumption rate (OCR) and ATP turnover, as well as the maximal mitochondrial respiration elicited by the uncoupling agent FCCP. The positive bioenergetic effects of 8-bromo-cAMP and of H 2 S were both attenuated by the selective PKA inhibitor Rp-cAMP. H 2 S exerted a concentration-dependent inhibitory effect on the activity of recombinant PDE2A by H 2 S in vitro . Conclusions The current data indicate that H 2 S, in addition to acting as a direct electron donor to the mitochondria, stimulates mitochondrial electron transport and cellular bioenergetic effects by activating a secondary pathway that involves inhibition of phosphodiesterase activity, enhancement of cAMP levels and stimulation of mitochondrial PKA. Based on recent publications, the likely mode of the enhancement of mitochondrial electron transport by PKA may involve the direct phosphorylation of specific electron transport chain components.

Introduction In vitro data presented at the current Meeting demonstrate that the upregulation of the H 2 S-producing enzyme cystathionine-®-synthase (CBS) in colorectal cancer cells supports bioenergetics, proliferation and migration. Several prior studies have shown that H 2 S also functions as a pro-angiogenic factor. By using a combination of genetic and pharmacological approaches, we have investigated whether CBS activity in cancer cells plays a role in the tumor growth and angiogenesis in vivo . Methods To determine the effects of CBS and CSE gene silencing on HCT116 xenograft growth in vivo , cells were transduced with a lentiviral vector containing either an enzyme-specific shRNA sequence or a non-targeting control shRNA sequence. The resulting HCT116 cell lines, designated shCBS, shCSE and shNT, were then injected subcutaneously in the right and left dorsum or either Nu/nu Balb/C or athymic nude mice (8–10 week old females). Changes in tumor volume (i.e., growth) over time were assessed by transcutaneous measurement using a caliper and calculated with the formula V = ( π /6) hd 2 . After 3–4 weeks, tumor tissue and blood plasma were harvested and tumor wet weights, H 2 S production/mg tumor protein, H 2 S levels in plasma, and tumor micro-blood vessel density were quantified. Micro-blood vessels were visualized by immunostaining tumor tissue sections for the mouse endothelial cell marker CD31 (Promin1) and quantified using a Chakley reticle. In a second series of experiments, mice bearing HCT116 xenografts or low passage (P2 to P3) patient-derived tumor xenografts (PDTXs) were randomized and treated (via subcutaneous injection) either with vehicle [phosphate buffered saline (PBS)], the CBS inhibitor aminooxyacetic acid (AOAA, 9 mg/kg/day) or the CSE inhibitor propargylglycine (PAG, 50 mg/kg/day) 6 days/week for up to 2 weeks. In a final set of experiments, peritumoral microvascular blood flow was measured using a PeriFlux 5000 laser-Doppler flowmeter. Changes in the subcutaneous microcirculatory perfusion were detected following intratumoral injections of either L-cysteine (1 mg/kg) or AOAA (9 mg/kg). Each in vivo experiment was repeated at least twice and statistical analyses were performed using GraphPad Prism 5 software. Results Consistent with the in vitro findings, shRNA-mediated knockdown of CBS expression (shCBS) significantly reduced the growth rate and size (i.e. volume) of HCT116 tumor xenografts. In contrast, silencing of CSE (shCSE) did not affect tumor growth. As expected, silencing of CBS or CSE in the tumor cells did not alter circulating H 2 S levels in tumor-bearing mice. Similar to the lack of inhibition of tumor growth by CSE silencing, the CSE inhibitor PAG failed to affect tumor growth in vivo . CBS suppression caused a significant reduction in the density of CD31-positive blood vessels within the tumor tissue as well as the prevalence of larger blood vessels and the extent of vessel branching, consistent with the hypothesis that CBS-derived H 2 S acts locally in a paracrine manner to stimulate tumor angiogenesis. Since small-molecule inhibitors are potentially more amenable to therapeutic translation than shRNA-based strategies, we next tested the effects of AOAA on tumor growth in vivo . Similar to CBS knockdown, AOAA treatment significantly slowed the rate of HCT116 xenograft growth, resulting in tumors with reduced size and weight at the time of harvest. A reduction in the plasma concentration of H 2 S confirmed the inhibitory effect of AOAA on systemic H 2 S production. Also, in accordance with the role of H 2 S as a local vasodilator, direct injection of AOAA into the tumor parenchyma significantly reduced, while intra-tumor administration of the CBS substrate L-cysteine increased peritumor blood flow. Finally, we conducted a series of translational studies with PDTXs, an emerging preclinical model for anti-cancer drug development, which closely recapitulates both the genetic and cellular heterogeneity of the patient’s original tumor tissue. We evaluated the effects of AOAA treatment on mice bearing PDTXs derived from the primary adenocarcinoma of a patient with pathological stage III disease with an activating Kras mutation and with a marked upregulation of endogenous CBS. Following randomization, mice were treated daily either with AOAA or vehicle. AOAA-treated animals showed a reduced rate of PDTX growth. Importantly, the growth inhibitory effects of AOAA on PDTX growth were also observed using tumor tissue from two other patients with stage II disease that also possessed a mutant Kras allele. Summary data using the PDTXs from three different patients showed that AOAA significantly reduced the rate of PDTX growth over the time course of the treatment. Conclusions The current study supports the conclusion that colon cancer cells overexpress CBS, which produces H 2 S to drive cellular bioenergetics and increase cancer cell proliferation. In addition to these cell-autonomous effects, the enhanced H 2 S production by cancer cells exerts paracrine effects on the tumor microenvironment to promote angiogenesis and peritumoral vasorelaxation. These studies identify CBS-derived H 2 S as a tumor promoting factor and a potential future anticancer drug target.