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 upregulation 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 patientderived 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.

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. N itric oxide (NO) and hydrogen sulfide (H 2 S) are two endogenous gasotransmitters whose regulatory roles in the cardiovascular system include vasorelaxation and stimulation of angiogenesis (1, 2). In endothelial cells, NO is synthesized by the endothelial isoform of NO synthase (eNOS). The principal pathway of NO signaling involves binding to the heme moiety of the soluble guanylyl cyclase (sGC) and production of the second messenger cyclic guanosine 5′-monophosphate (cGMP), followed by the activation of protein kinase G (PKG) (3, 4). However, vascular H 2 S is generated from L-cysteine by two pyridoxal 5′-phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST); activation of the ATP-dependent potassium channel (K ATP channel), modulation of cell metabolism, and posttranslational protein modifications via sulfhydration have been identified as some of its key signaling pathways (5-7).It is generally assumed that the signaling pathways of NO and H 2 S are independent. In the ...

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'.

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.

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. Streptozotocininduced 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.