N-acetylcysteine serves as substrate of 3-mercaptopyruvate sulfurtransferase and stimulates sulfide metabolism in colon cancer cells

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Modulation of hydrogen sulfide metabolism: new pharmacological targets in cancer and amyotrophic lateral sclerosis therapy

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule with a key role in human (patho)physiology. In human cells, H2S is synthetized by cystathionine β-synthase (CBS), cystathionine γ-lysase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST), and is oxidatively catabolized in the mitochondrion by sulfide:quinone oxidoreductase (SQR). H2S homeostasis relies on a fine balance between its catabolism and biosynthesis. Dysregulation of H2S metabolism has been associated to neurodegeneration and cancer. Reprogramming of H2S metabolism was shown to have pro-survival effects in cancer cells and, recently, N-acetylcysteine (NAC) was suggested to exert its antioxidant effects by increasing intracellular levels of persulfides and polysulfides (collectively known as “sulfane sulfur species”). In paper 1, the effect of NAC on the H2S metabolism of SW480 colon cancer cells was investigated. After exposing cells to 10 mM NAC for 24 hours, both MST and SQR displayed enhanced expression and activity. Moreover, NAC has proven to persist inside colon cancer cells over the 24-hour treatment and to act as a substrate for human MST, as shown working on the isolated recombinant enzyme. Altogether, these findings demonstrate that NAC stimulates H2S metabolism in colon cancer cells and serves as substrate for human MST. Hypoxia is currently recognized as a hallmark of the microenvironment of solid tumors, typically associated to malignant phenotypes. An increasing body of evidence suggests that H2S can increase cell resistance to hypoxia, while supporting angiogenesis, energy metabolism and drug resistance in cancer. Available information on the role played by H2S in the tumor microenvironment was reviewed in paper 3. In paper 2, working on SW480 colon cancer cells, the effect of hypoxia on the ability of cells to metabolize H2S was evaluated by high resolution respirometry. Hypoxia was found to decrease the mitochondrial content and the overall H2S-consuming activity of cells, while inducing a mitochondrial enrichment in SQR. These findings suggest that under hypoxic conditions cancer cells undergo adaptive changes to ensure higher intracellular H2S levels with pro-survival effects and, concomitantly, protection of cell respiration from H2S poisoning. In the central nervous system, H2S plays a central role in the regulation of numerous physiological processes, including neurotransmission and cytoprotection. Dysregulation of sulfide metabolism has been associated to cognitive disturbances like in Down’s syndrome and neurodegeneration like in Parkinson’s and Alzheimer’s disease. Recently, it was suggested an involvement of H2S in the etiology of amyotrophic lateral sclerosis (ALS). In the provisional paper 5, working on in vivo (Drosophila melanogaster) and in vitro ALS models, the effect of H2S on ALS-associated neurotoxicity was investigated. Of interest, knock down or pharmacological inhibition of the two H2S-synthetizing enzymes CBS and CSE was found to result in ameliorative phenotypes. These results triggered additional, still on-going investigations aimed at elucidating the impact of H2S in ALS and the underlined molecular mechanisms. Given the role of H2S in human (patho)physiology, there is an urgent need for inhibitors of the human H2S-synthetizing enzymes with pharmacological potential. In paper 4, a small library of newly synthesized pyridine derivatives was screened for the ability of these compounds to inhibit the human CBS, CSE and MST, as recombinantly produced in Escherichia coli and purified. By combining a wide range of biophysical and biochemical techniques, two compounds with similar molecular scaffold were found to weakly inhibit both CBS and CSE. In this study, a robust methodological platform for compound screening was set-up, and two hit compounds were identified which will be used as a starting point for future screening campaigns. The new knowledge acquired here, while deepening our understanding of the role of H2S in human (patho)physiology and, more specifically, in tumorigenesis and neurodegeneration, will hopefully set the basis for innovative diagnostic and therapeutic approaches

N-Acetylcysteine Serves as Substrate of 3-Mercaptopyruvate Sulfurtransferase and Stimulates Sulfide Metabolism in Colon Cancer Cells

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST

Endogenously produced hydrogen cyanide serves as a novel mammalian gasotransmitter

<p>The deposit contains RNA-seq data analysed with gene set enrichment analysis (GSEA) of HepG2 cells treated with Glycine (10 mM) vs Control (vehicle). Gene sets and their annotation are examined using the Molecular Signatures Database (MSigDB) hallmark gene sets.</p&gt

Cystathionine-β-synthase: molecular regulation and pharmacological inhibition

Cystathionine-β-synthase (CBS), the first (and rate-limiting) enzyme in the transsulfuration pathway, is an important mammalian enzyme in health and disease. Its biochemical functions under physiological conditions include the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor) and the generation of hydrogen sulfide (H2S), a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system. CBS is up-regulated in several diseases, including Down syndrome and many forms of cancer; in these conditions, the preclinical data indicate that inhibition or inactivation of CBS exerts beneficial effects. This article overviews the current information on the expression, tissue distribution, physiological roles, and biochemistry of CBS, followed by a comprehensive overview of direct and indirect approaches to inhibit the enzyme. Among the small-molecule CBS inhibitors, the review highlights the specificity and selectivity problems related to many of the commonly used “CBS inhibitors” (e.g., aminooxyacetic acid) and provides a comprehensive review of their pharmacological actions under physiological conditions and in various disease models

The Role of Organosulfur Compounds as Nrf2 Activators and Their Antioxidant Effects

Abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling has become a key pathway for cellular regulation against oxidative stress and inflammation, and therefore an attractive therapeutic target. Several organosulfur compounds are reportedly activators of the Nrf2 pathway. Organosulfur compounds constitute an important class of therapeutic agents in medicinal chemistry due to their ability to participate in biosynthesis, metabolism, cellular functions, and protection of cells from ox- idative damage. Sulfur has distinctive chemical properties such as a large number of oxidation states and versatility of reactions that promote fundamental biological reactions and redox biochemistry. The presence of sulfur is responsible for the peculiar features of organosulfur compounds which have been utilized against oxidative stress-mediated diseases. Nrf2 activation being a key therapeutic strategy for oxidative stress is closely tied to sulfur-based chemistry since the ability of compounds to react with sulfhydryl (-SH) groups is a common property of Nrf2 inducers. Although some indi- vidual organosulfur compounds have been reported as Nrf2 activators, there are no papers with a collective analysis of these Nrf2-activating organosulfur compounds which may help to broaden the knowledge of their therapeutic potentials and motivate further research. In line with this fact, for the first time, this review article provides collective and comprehensive information on Nrf2-activating organosulfur compounds and their therapeutic effects against oxidative stress, thereby enriching the chemical and pharmacological diversity of Nrf2 activators

The multifaceted roles of sulfane sulfur species in cancer-associated processes

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H2S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H2S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H2S binding to heme iron centers or H2S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H2S but also as key mediators of some of the biological effects commonly attributed to H2S, the multifaceted role of these species in cancer biology is reviewed here with reference to H2S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties

Sequential Accumulation of ‘Driver’ Pathway Mutations Induces the Upregulation of Hydrogen-Sulfide-Producing Enzymes in Human Colonic Epithelial Cell Organoids

Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential ‘driver’ mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H2S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H2S producing enzymes. Western blotting was used to detect the expression of the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H2S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H2S levels were detected by live-cell imaging using a fluorescent H2S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression—in particular in its truncated (45 kDa) form—as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H2S-producing enzymes coincided with the downregulation of the H2S-degrading enzyme SQR, increased H2S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H2S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes

H 2 S metabolism in colon cancer cells: Effect of hypoxia

Hydrogen sulfide (H2S) plays key roles in human (patho)physiology. Synthesized endogenously by known enzymatic systems, H2S is mainly metabolized through a mitochondrial sulfide-oxidizing pathway that comprises sulfide:quinone oxidoreductase (SQR) and a few other enzymes. H2S degradation through this pathway is coupled to electron injection into the respiratory chain and, thus, to stimulation of ATP synthesis. In cancer cells, H2S was reported to be synthesized at high levels and to stimulate energy metabolism and cell proliferation. Under hypoxic conditions, commonly found in the microenvironment of solid tumours, H2S is known to be more stable and to be overproduced with pro-survival effects. Here, H2S catabolism was investigated in colon cancer model cells grown under either normoxic (20% O2) or hypoxic (1% O2) conditions, comparing their maximal ability to dispose H2S at the level of the mitochondrion by high resolution respirometry. Intriguingly, cell exposure to hypoxic conditions was found to have effects on H2S metabolism, both in functional terms and at the level of protein expression. The potential implications of these findings will be discussed