3-Mercaptopyruvate sulfurtransferase supports endothelial cell angiogenesis and bioenergetics.

BACKGROUND AND PURPOSE: During angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis. EXPERIMENTAL APPROACH: To assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds. KEY RESULTS: 3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome. CONCLUSION AND IMPLICATIONS: 3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Comparison of the effects of e-cigarette vapor with cigarette smoke on lung function and inflammation in mice.

Electronic cigarettes (e-cigs) are advertised as a less harmful nicotine delivery system or as a new smoking cessation tool. We aimed to assess the in vivo effects of e-cig vapor in the lung and to compare them to those of cigarette smoke (CS). We exposed C57BL/6 mice for either 3 days or 4 wk to ambient air, CS, or e-cig vapor containing 1) propylene glycol/vegetable glycerol (PG:VG-Sol; 1:1), 2) PG:VG with nicotine (G:VG-N), or 3) PG:VG with nicotine and flavor (PG:VG-N+F) and determined oxidative stress, inflammation, and pulmonary mechanics. E-cig vapors, especially PG:VG-N+F, increased bronchoalveolar lavage fluid (BALF) cellularity, Muc5ac production, as well as BALF and lung oxidative stress markers at least comparably and in many cases more than CS. BALF protein content at both time points studied was only elevated in the PG:VG-N+F group. After 3 days, PG:VG-Sol altered tissue elasticity, static compliance, and airway resistance, whereas after 4 wk CS was the only treatment adversely affecting these parameters. Airway hyperresponsiveness in response to methacholine was increased similarly in the CS and PG:VG-N+F groups. Our findings suggest that exposure to e-cig vapor can trigger inflammatory responses and adversely affect respiratory system mechanics. In many cases, the added flavor in e-cigs exacerbated the detrimental effects of e-cig vapor. We conclude that both e-cig vaping and conventional cigarette smoking negatively impact lung biology.

Drug resistance induces the upregulation of H2S-producing enzymes in HCT116 colon cancer cells.

Hydrogen sulfide (H2S) production in colon cancer cells supports cellular bioenergetics and proliferation. The aim of the present study was to investigate the alterations in H2S homeostasis during the development of resistance to 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent. A 5-FU-resistant HCT116 human colon cancer cell line was established by serial passage in the presence of increasing 5-FU concentrations. The 5-FU-resistant cells also demonstrated a partial resistance to an unrelated chemotherapeutic agent, oxaliplatin. Compared to parental cells, the 5-FU-resistant cells rely more on oxidative phosphorylation than glycolysis for bioenergetic function. There was a significant increase in the expression of the drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP2A6 in 5-FU-resistant cells. The CYP450 inhibitor phenylpyrrole enhanced 5-FU-induced cytotoxicity in 5-FU-resistant cells. Two major H2S-generating enzymes, cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) were upregulated in the 5-FU-resistant cells. 5-FU-resistant cells exhibited decreased sensitivity to the CBS inhibitor aminooxyacetate (AOAA) in terms of suppression of cell viability, inhibition of cell proliferation and inhibition of oxidative phosphorylation. However, 5FU-resistant cells remained sensitive to the antiproliferative effect of benserazide (a recently identified, potentially repurposable CBS inhibitor). Taken together, the current data suggest that 5-FU resistance in HCT116 cells is associated with the upregulation of drug-metabolizing enzymes and an enhancement of endogenous H2S production. The anticancer effect of prototypical H2S biosynthesis inhibitor AOAA is impaired in 5-FU-resistant cells, but benserazide remains efficacious. Pharmacological approaches aimed at restoring the sensitivity of 5-FU-resistant cells to chemotherapeutic agents may be useful in the formulation of novel therapeutic strategies against colorectal cancer.

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine ß-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer.

Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60µM), tannic acid (IC50: ∼40µM) and benserazide (IC50: ∼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3µM) and NSC67078 (IC50: ∼1µM), while aurintricarboxylic acid (IC50: ∼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6µM), tannic acid (IC50: ∼20µM), benserazide (IC50: ∼20µM), and NSC67078 (IC50: ∼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

Molecular alterations of PI3K/Akt/mTOR pathway: a therapeutic target in endometrial cancer.

It is well established that the PI3K/Akt/mTOR pathway plays a central role in cell growth and proliferation. It has also been suggested that its deregulation is associated with cancer. Genetic alterations, involving components of this pathway, are often encountered in endometrial cancers. Understanding and identifying the rate-limiting steps of this pathway would be crucial for the development of novel therapies against endometrial cancer. This paper reviews alterations in the PI3K/Akt pathway, which could possibly contribute to the development of endometrial cancer. In addition, potential therapeutic targets of this pathway with emphasis on the mTOR inhibitors are also presented.