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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.

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.

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.

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.

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.

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.