Hydrogen sulfide generation from l-cysteine in the human glioblastoma-astrocytoma U-87 MG and neuroblastoma SHSY5Y cell lines (original) (raw)
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Acta Neurobiologiae Experimentalis, 2005
3-mercaptopyruvate sulfurtransferase (3-MST) has emerged as one of the significant sources of biologically active sulfur species in various mammalian cells. The current study was designed to investigate the functional role of 3-MST's catalytic activity in the murine colon cancer cell line CT26. The novel pharmacological 3-MST inhibitor HMPSNE was used to assess cancer cell proliferation, migration and bioenergetics in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). 3-MST expression was detected by Western blotting; H 2 S production was measured by the fluorescent dye AzMC. The results show that CT26 cells express 3-MST protein and mRNA, as well as several enzymes involved in H 2 S degradation (TST, ETHE1). Pharmacological inhibition of 3-MST concentration-dependently suppressed H 2 S production and, at 100 and 300 µM, attenuated CT26 proliferation and migration. HMPSNE exerted a bell-shaped effect on several cellular bioenergetic parameters related to oxidative phosphorylation, while other bioenergetic parameters were either unaffected or inhibited at the highest concentration of the inhibitor tested (300 µM). In contrast to 3-MST, the expression of CBS (another H 2 S producing enzyme which has been previously implicated in the regulation of various biological parameters in other tumor cells) was not detectable in CT26 cells and pharmacological inhibition of CBS exerted no significant effects on CT26 proliferation or bioenergetics. In summary, 3-MST catalytic activity significantly contributes to the regulation of cellular proliferation, migration and bioenergetics in CT26 murine colon cancer cells. The current studies identify 3-MST as the principal source of biologically active H 2 S in this cell line.
The Therapeutic Potential of Cystathionine β-Synthetase/Hydrogen Sulfide Inhibition in Cancer
Antioxidants & Redox Signaling, 2015
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 (H 2 S) from cystathionine b-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 H 2 S overproduction in tumor cells as a tumorgrowth 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 H 2 S 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 upregulation of cancer cells and the delineation of the interactions of H 2 S 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 H 2 S in cancer into human diagnostic and therapeutic approaches. Antioxid. Redox Signal. 22, 424-448. Biological Effects of H 2 S with Relevance for Cancer Biology H 2 S, as a vasodilator and pro-angiogenic mediator Vasorelaxation is one of the first recognized biological effects of H 2 S. Often compared with NO, H 2 S exerts a concentration-dependent vasodilatory effect in blood vessels. The mechanisms of H 2 S-mediated vasodilation include the activation of K ATP channels, a variety of other channels, inhibition of phosphodiesterases, and a synergy with NO (132).
International Journal of Molecular Sciences
The S-Allyl-L-cysteine (SAC) component of aged garlic extract (AGE) is proven to have anticancer, antihepatotoxic, neuroprotective and neurotrophic properties. γ-Cystathionase (CTH), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) are involved in H2S/sulfane sulfur endogenous formation from L-cysteine. The aim of the study was to determine the effect of SAC on MCF-7 cells survival and apoptosis, which is a widely known approach to reduce the number of cancer cells. An additional goal of this paper was to investigate the effect of SAC on the activity and expression of enzymes involved in H2S production. The experiments were carried out in the human breast adenocarcinoma cell line MCF-7. Changes in the cell viability were determined by MTT assay. Cell survival was determined by flow cytometry (FC). Changes in enzymes expression were analyzed using Western blot. After 24 h and 48 h incubation with 2245 µM SAC, induction of late apoptosis was observed. A d...
Cystathionine Promotes the Proliferation of Human Astrocytoma U373 Cells
Anticancer Research, 2018
Background/Aim: In certain cancers, accumulation of cystathionine has been observed. The present study investigated the effect of cystathionine on astrocytoma (U373) cell proliferation, the activity of γ-cystathionase (CTH) and changes in thiols levels. Materials and Methods: The methods used in the study included cytotoxicity assay, crystal violet staining method, CTH activity assay and reverse phase-high performance liquid chromatography (RP-HPLC). Results: The addition of cystathionine to the culture medium resulted in an increase of cystathionine level in U373 cells after 24 h of culture. Reduction of intracellular cystathionine level after 48 and 72 h of culture was associated with increased L-cysteine and L-cystine levels and stimulation of cell proliferation. Interestingly, a decrease in intracellular L-cysteine and L-cystine levels during the first hours of culture was observed. Conclusion: Elevated levels of cystathionine resulted in increased U373 cell proliferation by increasing the L-cysteine levels and GSH/GSSG ratio (especially after 72 h of the culture), but not with a simultaneous increase in the levels of total glutathione. Cystathionine is known as an intermediary metabolite in L-cysteine synthesis from methionine through the transsulfuration pathway. Endogenous synthesis of cystathionine, a sulfur-containing metabolite, is catalyzed by cystathionine βsynthase (CBS) via β-replacement reaction in which serine condenses with homocysteine in a pyridoxal-5'phosphatedependent manner. Cystathionine is subsequently converted to L-cysteine, α-ketobutyrate and ammonia by the action of cystathionine γ-lyase (CTH) (Figure 1). L-cysteine is catabolized by desulfuration pathways (generating sulfane sulfur-containing compounds and H 2 S) and oxidative pathways (production of taurine and sulfate) and is used for glutathione and proteins 3501
The Quantitative Significance of the Transsulfuration Enzymes for H 2 S Production in Murine Tissues
Antioxidants & Redox Signaling, 2011
The enzymes of the transsulfuration pathway, cystathionine b-synthase (CBS) and cystathionine c-lyase (CSE), are important for the endogenous production of hydrogen sulfide (H 2 S), a gaseous signaling molecule. The relative contributions of CBS and CSE to H 2 S generation in different tissues are not known. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H 2 S generation in these tissues and in brain at saturating substrate concentrations. We show that CBS protein levels are significantly lower than those of CSE; 60-fold and 20-fold in liver and kidney, respectively. Each enzyme is more abundant in liver compared with kidney, twofold and sixfold for CBS and CSE, respectively. At high substrate concentrations (20 mM each cysteine and homocysteine), the capacity for liver H 2 S production is approximately equal for CBS and CSE, whereas in kidney and brain, CBS constitutes the major source of H 2 S, accounting for *80% and *95%, respectively, of the total output. At physiologically relevant concentrations of substrate, and adjusting for the differences in CBS versus CSE levels, we estimate that CBS accounts for only 3% of H 2 S production by the transsulfuration pathway enzymes in liver. Antioxid. Redox Signal. 15, 363-372.
Sulfurtransferases and Cystathionine Beta-Synthase Expression in Different Human Leukemia Cell Lines
Biomolecules
The studies concerned the expression of sulfurtransferases and cystathionine beta-synthase in six human leukemia cell lines: B cell acute lymphoblastic leukemia-B-ALL (REH cells), T cell acute lymphoblastic leukemia-T-ALL (DND-41 and MOLT-4 cells), acute myeloid leukemia—AML (MV4-11 and MOLM-14 cells), and chronic myeloid leukemia—CML (K562 cells). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed to determine the expression of thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, gamma-cystathionase, and cystathionine beta-synthase on the mRNA and protein level. Interestingly, we found significant differences in the mRNA and protein levels of sulfurtransferases and cystathionine beta-synthase in the studied leukemia cells. The obtained results may contribute to elucidating the significance of the differences between the studied cells in the field of sulfur compound metabolism and finding new promising ways to inhibit th...
Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase
Antioxidants & Redox Signaling
Aims: The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and cystathionine gammalyase are thought to be the major source of hydrogen sulfide (H 2 S). In this study, we assessed the role of CBS in H 2 S biogenesis. Results: We show that despite discouraging enzyme kinetics of alternative H 2 S-producing reactions utilizing cysteine compared with the canonical condensation of serine and homocysteine, our simulations of substrate competitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine on CBS, thus leading to generation of appreciable amounts of H 2 S. The leading H 2 S-producing reaction is condensation of cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine is more abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the main determinant of CBS H 2 S productivity. Abundance of cysteine over serine, for example, in plasma, allowed for up to 43% of CBS activity being responsible for H 2 S production, while excess of serine typical for intracellular levels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine and cysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H 2 S pool. Innovation: Our study characterizes the H 2 S-producing potential of CBS under biologically relevant conditions and highlights the serine-to-cysteine ratio as the main determinant of H 2 S production by CBS in vivo. Conclusion: Our data clarify the function of CBS in H 2 S biogenesis and the role of thioethers as surrogate H 2 S markers. Antioxid. Redox Signal. 28, 311-323.
Biomolecules, 2021
This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese (TST, EC: 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC: 2.8.1.2) and cystathionine γ-lyase (CTH, EC: 4.4.1.1) in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line (NMuNG, ATCC: CRL-1636), mouse mammary gland tumor (4T1, ATCC: CRL-2539), mouse fibroblast (MEF, ATCC: SCRC-1008), mouse melanoma (B16-F1, ATCC: CRL-6323), human colorectal adenocarcinoma (Caco-2, ATCC: HTB-37), human embryonic kidney (HEK-293, ATC...
Proceedings of the National Academy of Sciences of the United States of America, 2013
The physiological functions of hydrogen sulfide (H2S) 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 H2S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H2S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H2S 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 capa...