Hypoxia-Induced Changes in L-Cysteine Metabolism and Antioxidative Processes in Melanoma Cells.

This study was performed on human primary (WM115) and metastatic (WM266-4) melanoma cell lines developed from the same individual. The expression of proteins involved in L-cysteine metabolism (sulfurtransferases, and cystathionine ß-synthase) and antioxidative processes (thioredoxin, thioredoxin reductase-1, glutathione peroxidase, superoxide dismutase 1) as well as the level of sufane sulfur, and cell proliferation under hypoxic conditions were investigated. Hypoxia in WM115 and WM266-4 cells was confirmed by induced expression of carbonic anhydrase IX and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 by the RT-PCR and Western blot methods. It was shown that, under hypoxic conditions the inhibition of WM115 and WM266-4 melanoma cell proliferation was associated with decreased expression of thioredoxin reductase-1 and cystathionine ß-synthase. These two enzymes may be important therapeutic targets in the treatment of melanoma. Interestingly, it was also found that in normoxia the expression and activity of 3-mercaptopyruvate sulfurtransferase in metastatic WM266-4 melanoma cells was significantly higher than in primary melanoma WM115 cells.

Sulfurtransferases and Cystathionine Beta-Synthase Expression in Different Human Leukemia Cell Lines.

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 the proliferation of various types of leukemic cells by modulating the activity of sulfurtransferases, cystathionine beta-synthase, and, consequently, the change of intracellular level of sulfane sulfur as well as H2S and reactive oxygen species production.

Sulfur Administration in Fe-S Cluster Homeostasis.

Mitochondria are the key organelles of Fe-S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe-S clusters. The newly formed cluster can be utilized by mitochondrial Fe-S protein synthesis or undergo further transformation. Mitochondrial Fe-S cluster biogenesis components are required in the cytosolic iron-sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe-S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe-S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe-S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe-S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme's attenuated activity.

Combination of ERK2 and STAT3 Inhibitors Promotes Anticancer Effects on Acute Lymphoblastic Leukemia Cells.

BACKGROUND/AIM: Deregulated activation of signaling through the RAS/RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAS/RAF/MEK/ERK) and signal transducer and activator of transcription (STAT) pathways is involved in numerous hematological malignancies, making it an attractive therapeutic target. This study aimed to assess the effect of the combination of ERK2 inhibitor VX-11e and STAT3 inhibitor STA-21 on acute lymphoblastic leukemia cell lines REH and MOLT-4. MATERIALS AND METHODS: REH and MOLT-4 cell lines were cultured with each drug alone and in combination. Cell viability, ERK activity, cell cycle distribution, apoptosis and oxidative stress induction were assessed by flow cytometry. Protein levels of STAT3, phospho-STAT3, protein tyrosine phosphatase 4A3 (PTP4A3), survivin, p53 and p21 were determined by western blotting. RESULTS: VX-11e in combination with STA-21 significantly inhibited cell viability, induced G0/G1 cell-cycle arrest, enhanced production of reactive oxygen species, and induced apoptosis. These effects were associated with an increased level of p21 protein in REH cells and with reduced levels of phopho-STAT3, survivin and PTP4A3 proteins in MOLT-4 cells. CONCLUSION: Our findings provide a rationale for combined inhibition of RAS/RAF/MEK/ERK and STAT3 pathways in order to enhance anticancer effects against acute lymphoblastic leukemia cells.

Targeting the MAPK/ERK and PI3K/AKT Signaling Pathways Affects NRF2, Trx and GSH Antioxidant Systems in Leukemia Cells.

The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.

Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells.

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Inhibition of Human Neuroblastoma Cell Proliferation by N-acetyl-L-cysteine as a Result of Increased Sulfane Sulfur Level.

BACKGROUND/AIM: In various cancer cells, the level of sulfane sulfur-containing compounds is decreased compared to normal cells. In the present study the effect of N-acetyl-L-cysteine (NAC), which acts as a precursor of H2S synthesis, on the human neuroblastoma SH-SY5Y cell proliferation, the activity of 3-mercaptopyruvate sulfurtransferase (MPST), and the level of sulfane sulfur were investigated. MATERIALS AND METHODS: SH-SY5Y cells were treated with NAC, while untreated cells were used as the control. The toxicity of NAC on the cells was studied by the LDH cytotoxicity assay; cell proliferation was examined by the MTT method. MPST activity and sulfane sulfur level were also analyzed in the NAC-treated cells. RESULTS: The addition of NAC to the medium, in non-cytotoxic concentrations, resulted in inhibition of the SH-SY5Y cell proliferation after 48 h of culture. The MPST activity and the level of sulfane sulfur-containing compounds were also elevated under the same culture conditions. CONCLUSION: The antiproliferative activity of NAC in the SH-SY5Y cells was associated with an increase in the MPST activity and consequently with an increase in the intracellular level of sulfane sulfur in these cells.

Cystathionine Promotes the Proliferation of Human Astrocytoma U373 Cells.

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.

New aspects of antiproliferative activity of 4-hydroxybenzyl isothiocyanate, a natural H2S-donor.

The effect of 4-hydroxybenzyl isothiocyanate (HBITC), a natural H2S-donor from white mustard seeds (Sinapis alba), on the proliferation of human neuroblastoma (SH-SY5Y) and glioblastoma (U87MG) cells was studied and some aspects of the mechanism of its activity were suggested. The inhibition of both SH-SY5Y and U87MG cell proliferation was associated with an increase in the thiosulfate level, the number of cells with the inactive form of Bcl-2 protein, and with a decrease of mitochondrial membrane potential. Interestingly, HBITC results in downregulation of p53 protein and upregulation of p21 protein levels in SH-SY5Y cells. In the presence of elevated levels of H2S and thiosulfate, the sulfhydryl groups of p53 protein as well as Bcl-2 protein could be modified via HBITC-induced S-sulfuration or by oxidative stress. It seems that the induction of p21 protein level is mediated in SH-SY5Y cells by p53-independent mechanisms. In addition, HBITC-treatment caused downregulation of the level of mitochondrial rhodanese and 3-mercaptopyruvate sulfurtransferase, and consequently increased the level of the reactive oxygen species in SH-SY5Y cells.

Colonic hydrogen sulfide produces portal hypertension and systemic hypotension in rats.

Hydrogen sulfide, a toxic gas, at low concentrations is also a biological mediator in animals. In the colon, hydrogen sulfide is produced by intestinal tissues and gut sulfur bacteria. Gut-derived molecules undergo liver metabolism. Portal hypertension is one of the most common complications contributing to the high mortality in liver cirrhosis. We hypothesized that the colon-derived hydrogen sulfide may affect portal blood pressure. Sprague-Dawley rats were maintained either on tap water (controls) or on water solution of thioacetamide to produce liver cirrhosis (CRH-R). Hemodynamics were measured after administration of either saline or Na2S, a hydrogen sulfide donor, into (1) the colon, (2) the portal vein, or (3) the femoral vein. Expression of enzymes involved in hydrogen sulfide metabolism was measured by RT-PCR. CRH-R showed a significantly higher portal blood pressure but a lower arterial blood pressure than controls. Saline did not affect hemodynamic parameters. In controls, intracolonic hydrogen sulfide decreased arterial blood pressure and portal blood flow but increased portal blood pressure. Similarly, hydrogen sulfide administered into the portal vein decreased arterial blood pressure but increased portal blood pressure. In contrast, hydrogen sulfide administered into the systemic vein decreased both arterial and portal blood pressures. CRH-R showed significantly greater responses to hydrogen sulfide than controls. CRH-R had a significantly higher liver concentration of hydrogen sulfide but lower expression of rhodanese, an enzyme converting hydrogen sulfide to sulfate. In conclusion, colon-administered hydrogen sulfide increases portal blood pressure while decreasing the systemic arterial blood pressure. The response to hydrogen sulfide is more pronounced in cirrhotic rats which show reduced hydrogen sulfide liver metabolism. Therefore, colon-derived hydrogen sulfide may be involved in the regulation of portal blood pressure, and may contribute to portal hypertension. Impact statement Accumulating evidence suggests that gut-derived molecules affect the control of the circulatory system. Mechanisms controlling liver circulation have been profoundly studied; however, the effects of gut bacteria-derived molecules on portal blood pressure have not been established. In the colon, hydrogen sulfide is produced by intestinal tissues and gut sulfur bacteria. We found that colon-administered hydrogen sulfide increases portal blood pressure while decreasing the systemic arterial blood pressure. The hemodynamic response to hydrogen sulfide was more pronounced in cirrhotic rats which showed reduced hydrogen sulfide liver metabolism, i.e. lower expression of rhodanese, an enzyme converting hydrogen sulfide to sulfate. We propose that colon-derived hydrogen sulfide may affect the regulation of portal and arterial blood pressures and may be involved in portal hypertension.

A possible mechanism of inhibition of U87MG and SH-SY5Y cancer cell proliferation by diallyl trisulfide and other aspects of its activity.

The study was conducted to elucidate the mechanism of antiproliferative and antioxidative action of diallyl trisulfide (DATS), a garlic-derived organosulfur compound. Changes in the L-cysteine desulfuration, and the levels of cystathionine and non-protein thiols in DATS-treated human glioblastoma (U87MG) and neuroblastoma (SH-SY5Y) cells were investigated. The inhibition of proliferation of the investigated cells by DATS was correlated with an increase in the inactivated form of Bcl-2. In U87MG cells, an increased level of sulfane sulfur and an increased activity of 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese, the enzymes involved in sulfane sulfur generation and transfer, suggest that DATS can function as a donor of sulfane sulfur atom, transferred by sulfurtransferases, to sulfhydryl groups of cysteine residues of Bcl-2 and in this way lower the level of active form of Bcl-2 by S-sulfuration. Diallyl trisulfide antioxidative effects result from an increased level of cystathionine, a precursor of cysteine, and an increased glutathione level. MPST and rhodanese, the level of which is increased in the presence of DATS, can serve as antioxidant proteins.

Identification of Taurine-Responsive Genes in Murine Liver Using the Cdo1-Null Mouse Model.

The cysteine dioxygenase (Cdo1)-null mouse is unable to synthesize hypotaurine and taurine by the cysteine/cysteine sulfinate pathway and has very low taurine levels in all tissues. The lack of taurine is associated with a lack of taurine conjugation of bile acids, a dramatic increase in the total and unconjugated hepatic bile acid pools, and an increase in betaine and other molecules that serve as organic osmolytes. We used the Cdo1-mouse model to determine the effects of taurine deficiency on expression of proteins involved in sulfur amino acid and bile acid metabolism. We identified cysteine sulfinic acid decarboxylase (Csad), betaine:homocysteine methytransferase (Bhmt), cholesterol 7α-hydroxylase (Cyp7a1), and cytochrome P450 3A11 (Cyp3a11) as genes whose hepatic expression is strongly regulated in response to taurine depletion in the Cdo1-null mouse. Dietary taurine supplementation of Cdo1-null mice restored hepatic levels of these four proteins and their respective mRNAs to wild-type levels, whereas dietary taurine supplementation had no effect on abundance of these proteins or mRNAs in wild-type mice.

Na2S, a fast-releasing H2S donor, given as suppository lowers blood pressure in rats.

BACKGROUND: Hydrogen sulfide (H2S) is involved in blood pressure control. The available slow-releasing H2S-donors are poorly soluble in water and their ability to release H2S in biologically relevant amounts under physiological conditions is questionable. Therefore, new slow-releasing donors or new experimental approaches to fast-releasing H2S donors are needed. METHODS: Hemodynamics and ECG were recorded in male, anesthetized Wistar Kyoto rats (WKY) and in Spontaneously hypertensive rats (SHR) at baseline and after: 1) intravenous (iv) infusion of vehicle or Na2S; 2) administration of vehicle suppositories or Na2S suppositories. RESULTS: Intravenously administered vehicle and vehicle suppositories did not affect mean arterial blood pressure (MABP) and heart rate (HR). Na2S administered iv caused a significant, but transient (2-5min) decrease in MABP. Na2S suppositories produced a dose-dependent hypotensive response that lasted ∼45min in WKY and ∼75-80min in SHR. It was accompanied by a decrease in HR in WKY, and an increase in HR in SHR. Na2S suppositories did not produce a significant change in corrected QT, an indicator of cardiotoxicity. Na2S suppositories increased blood level of thiosulfates, products of H2S oxidation. CONCLUSIONS: Na2S administered in suppositories exerts a prolonged hypotensive effect in rats, with no apparent cardiotoxic effect. SHR and WKY differ in hemodynamic response to the H2S donor. Suppository formulation of fast-releasing H2S donors may be useful in research, if a reference slow-releasing H2S donor is not available.

Similar effect of sodium nitroprusside and acetylsalicylic acid on antioxidant system improvement in mouse liver but not in the brain.

BACKGROUND: The aim of the present study was to analyze the relative antioxidant effects of acetylsalicylic acid (ASA) and sodium nitroprusside (SNP) in mouse liver and brain. METHODS: The activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase (CSE), functioning as antioxidant proteins and capable of producing H2S, was investigated in mouse liver and brain after intraperitoneal once a day administration of sodium nitroprusside (5 mg/kg body weight) or acetylsalicylic acid (500 mg/kg body weight) continued for 5 days. The tissues were homogenized and then the obtained supernatants were used for further determinations. At the same time, the levels of sulfane sulfur, reduced and oxidized glutathione, cysteine, cystine, and cystathionine were also studied in these tissues. RESULTS: Both ASA and SNP show a statistically significant increase of sulfurtransferases activities in liver. The mechanism of action of sodium nitroprusside appears to consist in liberation of nitric oxide (NO), an important signaling molecule in the mammalian body. SNP also releases cyanide ions, which are converted in the liver to thiocyanate by the enzyme rhodanese and/or MPST and/or γ-cystathionase - the activities of all the enzymes were elevated in reaction to SNP. The action of γ-cystathionase is dependent upon converting cystathionine to cysteine, a precursor of the major cellular antioxidant, glutathione. Under oxidizing conditions, an increase in cystathionine ß-synthase activity might indirectly result in an increase in the antioxidant glutathione level; this was reflected by the increased GSH/GSSG ratio in the liver, but not in the brain, where a trace activity of γ-cystathionase is normally detected. CONCLUSION: The results of the present investigations show that ASA and SNP may stimulate the GSH-dependent antioxidant system and protect liver cells from oxidative stress. An increased activity of the H2S-producing enzymes and the increased GSH/GSSG ratio may lead to an elevated level of H2S, a molecule with antioxidant properties. A similar effect was not observed in the brain. In case of both sodium nitroprusside and aspirin administration, homeostasis of sulfane sulfur level was noted in both the liver and brain.

Intracolonic hydrogen sulfide lowers blood pressure in rats.

Research suggests that hydrogen sulfide (H2S) is an important biological mediator involved in various physiological processes including the regulation of arterial blood pressure (BP). Although H2S is abundant in the colon, the effects of gut-derived H2S on the circulatory system have not yet been investigated. We studied the effects of intracolonic administration of Na2S, a H2S donor, on systemic hemodynamics. Hemodynamics were recorded in anesthetized, normotensive Wistar Kyoto and spontaneously hypertensive rats at baseline and after intracolonic injection of either saline (controls) or Na2S·9H2O saline solution at a dose range of 10-300 mg/kg of BW. The H2S donor produced a significant, dose-dependent decrease in mean arterial blood pressure (MABP), which lasted several times longer than previously reported after parenteral infusions (>90 min). The effect was more pronounced in hypertensive than in normotensive rats. The Na2S-induced decrease in MABP was reduced by pretreatment with glibenclamide, an inhibitor of ATP-sensitive potassium-channels. Na2S did not affect mesenteric vein blood flow. Rats treated with Na2S showed increased portal blood levels of thiosulfate and sulfane sulfur, products of H2S oxidation. In contrast, rats treated with neomycin, an antibiotic, showed significantly decreased levels of thiosulfate and sulfane sulfur, and a tendency for greater hypotensive response to Na2S. The H2S donor decreased heart rate but did not affect ECG morphology and QTc interval. In conclusion the gut-derived H2S may contribute to the control of BP and may be one of the links between gut microbiota and hypertension. Furthermore, gut-derived H2S may be a therapeutic target in hypertension.

Downregulation of hepatic betaine:homocysteine methyltransferase (BHMT) expression in taurine-deficient mice is reversed by taurine supplementation in vivo.

The cysteine dioxygenase (Cdo1)-null and the cysteine sulfinic acid decarboxylase (Csad)-null mouse are not able to synthesize hypotaurine/taurine by the cysteine/cysteine sulfinate pathway and have very low tissue taurine levels. These mice provide excellent models for studying the effects of taurine on biological processes. Using these mouse models, we identified betaine:homocysteine methyltransferase (BHMT) as a protein whose in vivo expression is robustly regulated by taurine. BHMT levels are low in liver of both Cdo1-null and Csad-null mice, but are restored to wild-type levels by dietary taurine supplementation. A lack of BHMT activity was indicated by an increase in the hepatic betaine level. In contrast to observations in liver of Cdo1-null and Csad-null mice, BHMT was not affected by taurine supplementation of primary hepatocytes from these mice. Likewise, CSAD abundance was not affected by taurine supplementation of primary hepatocytes, although it was robustly upregulated in liver of Cdo1-null and Csad-null mice and lowered to wild-type levels by dietary taurine supplementation. The mechanism by which taurine status affects hepatic CSAD and BHMT expression appears to be complex and to require factors outside of hepatocytes. Within the liver, mRNA abundance for both CSAD and BHMT was upregulated in parallel with protein levels, indicating regulation of BHMT and CSAD mRNA synthesis or degradation.

Propargylglycine inhibits hypotaurine/taurine synthesis and elevates cystathionine and homocysteine concentrations in primary mouse hepatocytes.

Our investigation showed that hepatocytes isolated from cysteine dioxygenase knockout mice (Cdo1(-/-)) had lower levels of hypotaurine and taurine than Cdo1 (+/+) hepatocytes. Interestingly, hypotaurine accumulates in cultured wild-type hepatocytes. DL-propargylglycine (PPG, inhibitor of cystathionine γ-lyase and H2S production) dramatically decreased both taurine and hypotaurine levels in wild-type hepatocytes compared to untreated cells. Addition of 2 mM PPG resulted in the decrease of the intracellular taurine levels: from 10.25 ± 5.00 observed in control, to 2.53 ± 0.68 nmol/mg protein (24 h of culture) and from 17.06 ± 9.40 to 2.43 ± 0.26 nmol/mg protein (control vs. PPG; 48 h). Addition of PPG reduced also intracellular hypotaurine levels: from 7.46 ± 3.55 to 0.31 ± 0.12 nmol/mg protein (control vs. PPG; 24 h) and from 4.54 ± 3.20 to 0.42 ± 0.11 nmol/mg protein (control vs. PPG; 48 h). The similar effects of PPG on hypotaurine and taurine levels were observed in culture medium. PPG blocked hypotaurine/taurine synthesis in wild-type hepatocytes, suggesting that it strongly inhibits cysteinesulfinate decarboxylase (pyridoxal 5'-phosphate-dependent enzyme) as well as cystathionine γ-lyase. In the presence of PPG, intracellular and medium cystathionine levels for both wild-type and Cdo1 (-/-) cells were increased. Addition of homocysteine or methionine resulted in higher intracellular concentrations of homocysteine, which is a cosubstrate for cystathionine ß-synthase (CBS). It seems that PPG increases CBS-mediated desulfhydration by enhancing homocysteine levels in hepatocytes. There were no overall effects of PPG or genotype on intracellular or medium glutathione levels.

Is development of high-grade gliomas sulfur-dependent?

We characterized γ-cystathionase, rhodanese and 3-mercaptopyruvate sulfurtransferase activities in various regions of human brain (the cortex, thalamus, hypothalamus, hippocampus, cerebellum and subcortical nuclei) and human gliomas with II to IV grade of malignancy (according to the WHO classification). The human brain regions, as compared to human liver, showed low γ-cystathionase activity. The activity of rhodanese was also much lower and it did not vary significantly between the investigated brain regions. The activity of 3-mercaptopyruvate sulfurtransferase was the highest in the thalamus, hypothalamus and subcortical nuclei and essentially the same level of sulfane sulfur was found in all the investigated brain regions. The investigations demonstrated that the level of sulfane sulfur in gliomas with the highest grades was high in comparison to various human brain regions, and was correlated with a decreased activity of γ-cystathionase, 3-mercaptopyruvate sulfurtransferase and rhodanese. This can suggest sulfane sulfur accumulation and points to its importance for malignant cell proliferation and tumor growth. In gliomas with the highest grades of malignancy, despite decreased levels of total free cysteine and total free glutathione, a high ratio of GSH/GSSG was maintained, which is important for the process of malignant cells proliferation. A high level of sulfane sulfur and high GSH/GSSG ratio could result in the elevated hydrogen sulfide levels. Because of the disappearance of γ-cystathionase activity in high-grade gliomas, it seems to be possible that 3-mercaptopyruvate sulfurtransferase could participate in hydrogen sulfide production. The results confirm sulfur dependence of malignant brain tumors.

Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate.

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine ß-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.