Hydrogen sulfide generation from l -cysteine in the human glioblastoma-astrocytoma U-87 MG and neuroblastoma SHSY5Y cell lines*

Hydrogen sulfide (H 2 S) is endogenously synthesized from l -cysteine in reactions catalyzed by cystathionine beta- synthase (CBS, EC 4.2.1.22) and gamma-cystathionase (CSE, EC 4.4.1.1). The role of 3-mercaptopyruvate sulfur- transferase (MPST, EC 2.8.1.2) in H 2 S generation is also considered; it could be important for tissues with low CTH activity, e.g. cells of the nervous system. The expression and activity of CBS, CTH, and MPST were detected in the human glioblastoma-astrocytoma (U-87 MG) and neuroblastoma (SHSY5Y) cell lines. In both cell lines, the expression and activity of MPST were the highest among the investigated enzymes, suggesting its possible role in the generation of H 2 S. The RP-HPLC method was used to determine the concentration of cystathionine and alpha-ketobutyrate, products of the CBS- and CTH-catalyzed reactions. The difference in cystathionine levels between cell homogenates treated with totally CTH-inhibiting concentrations of dl -propargylglycine and without the inhibitor was used to evaluate the activity of CBS. The higher expression and activity of CBS, CTH and MPST in the neuroblastoma cells were associated with more intensive generation of H 2 S in the presence of 2 mM cysteine. A threefold higher level of sulfane sulfur, a potential source of hydrogen sulfide, was detected in the astrocytoma cells in comparison to the neuroblastoma cells.

Enzymatic reactions involve l-cysteine hydrolysis by CBS to l-serine and H 2 S, l-cystine transformation by CTH into thiocysteine, pyruvate (PA) and ammonia and subsequent thiocysteine transformation into H 2 S and CysSR (S-thiolane). Another pathway includes the transformation of l-cysteine into 3-mercaptopyruvate (3MP) by cysteine aminotransferase (CAT) and, subsequently, 3MP desulfuration catalyzed by MPST resulting in H 2 S and pyruvate formation (Scheme 1A). H 2 S is formed in a redox reaction between thiosulfate or RSSH (persulfides) and biological thiols such as reduced glutathione (GSH) (Scheme 1B) (Libiad et al., 2014;Predmore et al., 2012). An additional pathway for the production of 3-MP and H 2 S from d-cysteine by d-amino acid oxidase provides protection of cerebellar neurons from oxidative stress (Shibuya et al., 2013).
Astrocytes secrete and store antioxidative compounds, such as glutathione or ascorbate (Bartosz, 2006;Bèlanger & Magistretti, 2009, Zabłocka & Janusz, 2007. The cells play an important role in supplying precursors necessary for GSH synthesis in the neurons. GSH captured by astrocytes from the extracellular space is degraded in a reaction catalyzed by γ-glutamyl-transpeptidase (γ-GT) to free cysteine (Zabłocka & Janusz, 2007) (Scheme 2). The main transport system for cysteine in the astrocytes and neurons is mediated by the Na + dependent X AG and ASC (alanine-serine-cysteine) systems (Shanker et 2016_1394 al., 2001aShanker et al., 2001b). The degradation product returns to neuron cells, where it is used as a substrate for glutathione synthesis. When compared to the neurons, astrocytes have the higher level of GSH, both in vivo and in cell cultures . Functional neuron-glial cell interrelations provide an important mechanism participating in brain functions control (Scheme 2). The anaerobic conversion of cysteine can lead to the formation of hydrogen sulfide (H 2 S). It is known that endogenously formed H 2 S acts as a neuromodulator and neuroprotector in the brain (Panthi et al., 2016;Paul & Snyder, 2015). By means of activating NMDA receptors and increasing the response of peripheral neurons, H 2 S may play a significant role in processes associated with memorization and learning (Ishigami et al., 2009;Shibuya et al., 2009). An increased synthesis of H 2 S has been observed in patients with Down's syndrome and septic shock, while its decreased generation has been noted in Alzheimer's disease. H 2 S activates TRPA1 channels in the astrocytes in a similar way, but not as efficiently as polysulfides (Kimura, 2013;Moore & Whiteman, 2005). H 2 S has antioxidative properties and it increases the production of glutathione in neural cells (Kimura et al., 2010;Kimura & Kimura, 2004).
The study was conducted to determine the activity and expression of the enzymes: CBS, CTH and MPST involved in the production of H 2 S in the human glioblastoma-astrocytoma (U-87 MG) and neuroblastoma (SHSY5Y) cell lines. The results of our previous studies (Jurkowska et al. 2011) showed the expression of CTH and MPST genes in the human neoplastic cell lines: astrocytoma U373 and neuroblastoma SH-SY5Y. The CTH and MPST enzymes, through an increase in sulfane sulfur levels, might increase H 2 S levels. The RP-HPLC method was used to detect and determine the amount of direct and indirect products of the CBS-and CTH-catalyzed reactions, such as cystathionine, cysteine, and glutathione. The difference in the cystathionine level between the cells incubated with totally CTH-inhibiting concentrations of dl-propargylglycine (PPG) and without the inhibitor was used to evaluate the activity of CBS. Differences in the expression and activity of CBS, CTH, and MPST point to a higher intensity of H 2 S generation in the neuroblastoma cells, which was confirmed by the higher level of H 2 S in SHSY5Y cells determined using the H 2 S trapping method of Kartha et al. (2012).
Cytotoxicity of l-cysteine. The cells were seeded in triplicates into 96-microwell plates at density of 20 × 10 3 cells/well and incubated for 24 h with or without 2 mM l-cysteine in DMEM medium supplemented with 10% FBS. Colorimetric assay was performed according to the manufacturer instructions (Cytotoxicity Detection Kit, Roche, Thermo Fisher Scientific). Absorbance of the colored product -formazane -was measured at 490 nm by microreader (EPOCH, BioTEK).
Expression of MPST, CTH, CBS in cell lines. RNA extraction. The total RNA was extracted using TRIZOL, according to the protocol provided by the manufacturer. The quality of RNA samples was determined by spectrophotometric analysis (A 260 /A 280 ) and electrophoresis in 2.5% agarose gel followed by staining with ethidium bromide.
cDNA Synthesis and RT-PCR analysis. Expression of MPST, CTH, CBS and β-actin was analyzed with RT-PCR as previously described by Jurkowska et al. (2011) with modifications. Amplification of cDNA samples was performed in a 12.5 µl reaction volume containing: 1 µl of synthesized cDNA, 0.2 µM of each of gene-specific primer pair, 0.04 U/µl of DNA polymerase in 10 mM Tris-HCl buffer pH 8.8, 0.2 mM of each dNTPs and nuclease-free water. The temperature profile of RT-PCR amplification for the MPST consisted of activation of Taq polymerase at 94°C for 5 min, denaturation of cDNA at 94°C for 30 s, primer annealing at 56°C for 30 s, elongation at 72°C for 2 min for the following 28 cycles and was finished by the extension step for 8 min. For the CTH gene, after the initial denaturation for 5 min at 94°C, amplification was performed under the following conditions: 94°C for 30 s, 51°C for 1 min and 72°C for 8 min for 28 cycles, with the final incubation at 72°C for 10 min. For the CBS gene, after the initial denaturation for 5 min at 94°C, amplification was performed under the following conditions: 94°C for 30 s, 60°C for 30 s and 72°C for 2 min for 38 cycles, with the final incubation at 72°C for 8 min. For β-actin gene, after the initial denaturation for 5 min at 94°C, amplification was performed under the following conditions: 94°C for 30 s, 54°C for 30 s and 72°C for 2 min for 28 cycles, with the final incubation at 72°C for 8 min. The following specific primers (Oligo Company) were used: MPST -F: 5'TCTTCGACATCGACCAGTGC' and R: 5'TGTGAAGGGGATGTTCACGG3' CTH -F: 5'GCAAGTGGCATCTGAATTTG3' and R: 5'CCCATTACAACATCACTGTGG3' CBS -F: 5'CGCTGCGTGGTCATTCTGCC3' and R: 5'TCCCAGGATTACCCCCGCCT3' β-actin -F: 5'CTGTCTGTCACCACCAT3' and R: 5'GCAACTAAGTCATAGTCCGC3' β-actin was used as the internal standard to normalize all samples for potential variations in mRNA content. PCR reaction products were separated electrophoretically in a 2.5% agarose gel, stained with ethidium bromide and directly visualized under UV light and photographed.
Detection of H 2 S. The H 2 S produced during the incubation of the cell culture with H 2 S-releasing compounds was trapped as zinc sulfide in the zinc agarose layer according to Kartha et al. (2012). The standard curve was linear at the concentration range of 0-250 µM with correlation coefficient of 0.994.
Enzymes assay. Cell homogenization. U-87 MG and SHSY5Y cells (3.5-5 × 10 6 cells) were suspended in 0.1 M phosphate buffer pH 7.5, in the proportion of 1 mln cells/0.07 ml of the buffer, sonicated 3 × 5 s at 4°C (BandelinSonoplus GM 70). After centrifugation at 1600 × g for 10 min, the supernatant was used for the determination of protein concentration, sulfane sulfur levels and the activity of MPST and CTH. For RP-HPLC analyses cells were suspended in 0.1 ml 0.9% NaC/l 70% PCA/1 mM BPDS. The sediment was separated by centrifugation at 1400 × g for 10 min, and supernatant was stored at -80°C until analysis.
MPST activity. MPST activity was assayed according to the method of Valentine and Frankelfeld, (1974) following a procedure described in our earlier paper (Wróbel et al. 2004). The incubation mixture contained: 250 µl of 0.12 M sodium phosphate buffer, pH 8.0, 50 µl of 0.5M sodium sulfite, 50 µl of 0.15 M dithiothreitol, 50 µl of homogenates, 50 µl of H 2 O and 50 µl of 0.1 M 3-mercaptopyruvate acid sodium salt in a final volume of 500 µl. Mixture was incubated for 15 min. To stop the reaction 250 µl of 1.2 M PCA was added. Samples were centrifuged at 1600 × g for 5 min, and 100 µl of supernatant was transferred to 1350 µl of mixture that contained: 1200 µl of 0.12 M sodium phosphate buffer, pH 8.0, 100 µl of 0.1 M N-ethylmaleimide, 50 µl of NADH 5 mg/ml. After equilibration at 37°C, 2.5 µl of lactate dehydrogenase (7 IU) was added, and the decrease in absorbance was measured at 340 nm. The enzyme activity was expressed as nmoles of pyruvate produced during 1 min incubation at 37°C per 1 mg of protein.
CTH activity. Cystathionase activity was determined using Matsuo and Greenberg's method (1958) with modifications described by Czubak et al. (2002). The incubation mixture contained: 25 µl of 1.3 mM PLP, 25 µl of 0.02 mM EDTA, 250 µl of 45 mM cystathionine solution in 0.1 M phosphate buffer, pH 7.5 (2.5 mg of cystathionine per sample) and 75 µl of homogenate and 0.1 M phosphate buffer, pH 7.5 containing 0.05 mM 2-mercaptoethanolin in the final volume of 650 µl. The reaction was stopped after 15 min of incubation at 37°C by placing 125 µl of the incubation mixture in 25 µl of 10% PCA. Samples were centrifuged at 1600 × g for 10 min, and 25 µl of supernatant was transferred to 625 µl of 0.194 mM NADH solution and kept at 37°C. Control samples, without 45 mM cystathionine, were prepared in the same way as the examined samples. After 10 s of the measurement (absorbance at 340 nm), 25 µl (9.06 IU) of lactate dehydrogenase (LDH) was added and measurement was continued up to 180 s. The difference between the initial value of absorbance (before adding LDH) and the lowest value (after adding LDH) corresponded to the amount of alpha-ketobutyrate formed in the course of the cystathionase reaction. Cystathionase activity was expressed as nmoles of α-ketobutyrate formed during 1min incubation at 37°C per 1 mg of protein.
CBS activity. The activity of CBS was examined in cells homogenates in the presence of DL-homoserine as substrate after 15 minutes incubation at 37°C according to the description in Bronowicka-Adamska et al. (2011). PPG, in the concentration of 0.7 mM, was used to completely inhibit the activity of CTH in both cell lines. The level of cystathionine was determined using the HPLC method described by Bronowicka-Adamska et al. (2015). The CBS activity was expressed as pmoles of cystathionine formed during 1min incubation at 37°C per 1 mg of protein.
Sulfane sulfur. Sulfane sulfur was determined by the method of Wood, (1987), based on cold cyanolysis and colorimetric detection of ferric thiocyanate complex ion. Incubation mixtures in a final volume 880 µl contained: 20 µl of 1 M ammonia solution, 20 µl of homogenate, 740 µl of H 2 O and 100 µl of 0.5 M sodium cyanide. Incubation was performed for 45 min at room temperature. After incubation, thiocyanate was estimated calorimetrically at 460 nm after the addition of 20 µl of 38% formaldehyde and 40 µl of ferric nitrate reagent. Sulfane sulfur level was expressed as nmoles of SCNproduced per 1 mg of protein.
Proteins. Protein concentration was determined with the method of Lowry et al. (1951) using crystalline bovine serum albumin as a standard. Protein concentration measurement with Bradford assay was used for the determination of protein in Western blotting analysis (Bradford, 1976).

Statistical analysis.
All results were expressed as means ± S.D. The significance of the differences between controls and investigated samples were calculated using Student's Test (P<0.05). Each experiment was repeated minimum three times.

RESULTS AND DISCUSSION
The studies showed U-87 MG and SHSY5Y cells capacity of hydrogen sulfide formation from l-cysteine and an increased level of hydrogen sulfide in the neuroblastoma -SHSY5Y cells (Fig. 1) by about 20%, as compared to the control cells without l-cysteine, and only by about 5% in the glioblastoma-astrocytoma (U-87 MG cells) after 24 h of incubation with 2 mM l-cysteine (Fig. 1). The cytotoxic effect on the SHSY5Y and U-87 MG cell lines after 24 hours of incubation with 2 mM lcysteine was lower than 10% for both cell lines. Table 1 shows the activity of CTH, MPST, CBS and the level of sulfane sulfur in the SHSY5Y and U-87 MG cells. One can summarize that all the enzymatic pathways (Scheme 1) generating H 2 S can occur in the investigated cell lines. CBS is regarded as the principal enzyme responsible for H 2 S synthesis in the brain, while CTH plays a similar role in the circulatory system (Abe & Kimura, 1996). More than tenfold higher CBS activity and three fold higher MPST activity were estimated in the neuroblastoma cells, as compared to the astrocytoma cells. Therefore, the neuroblastoma cells have potentially higher capacity of H 2 S generation from cysteine than astrocytoma. In the neuroblastoma cells, specific activity of MPST being the highest among the investigated enzymes and the highest expression of MPST ( Fig. 2 and The experiments were carried out for control homogenates of U-87 MG, SHSY5Y cells with 2 mM l-cysteine as the main endogenous substrate for the hydrogen sulfide producing enzymes. The data represent the mean value from three independent experiments. Statistical analysis was performed using the Student's t-test (*P<0.05).

Figure 2. RT-PCR analysis of MPST, CBS and CTH expression in SHSY5Y and U-87 MG cell lines.
One set of representative results is shown. β-actin was used as the internal control. The length of the products: MPST, 227 bp; CBS, 300 bp; CTH, 300 bp; β-actin, 280 bp. Specific primers and temperature profiles for particular proteins are given in Material and Methods. 3) can both suggest that this enzyme plays a role in the generation of H 2 S. On the other hand, the sulfane sulfur level was more than threefold higher in the astrocytoma cells, which can suggest that a nonenzymatic release of H 2 S from sulfane sulfur-containing compounds (Scheme 2) is possible. It seems probable that in the neuroblastoma cells, H 2 S, functioning as a neurotransmitter, is synthesized in response to the signal-to-date. The astrocytoma cells, in turn, can release H 2 S from sulfane sulfur reserves to transmit a signal within the astrocyte network (Perea & Araque, 2003;Zabłocka & Janusz, 2007). The conditions under which physiological signals mobilize H 2 S from sulfane sulfur stores have not been elucidated so far (Paul & Snyder, 2015).
The U-87 MG astrocytoma cells had the higher level of GSH in comparison to the neuroblastoma cells, which confirms the differences in glutathione content between the astrocytes and neurons   (Fig. 4).The high levels of glutathione in the astrocytes seem to be essential for neurons protection e.g.: against the toxicity of reactive oxygen species. In the control cultures and cultures with PPG (inhibitor of CTH) added in the concentration of 0.7 mM, both the level of α-ketobutyrate and the level of cysteine were undetectable. The inhibition of CTH with PPG in the astrocytoma cells resulted in a diminished level of GSH after 15 min of incubation, but no such effect was seen in the neuroblastoma cells (Fig. 4). This may suggest that the pathway of cysteine generation through CTH-catalyzed reaction is important in the U-87 MG cells, while the SHSY5Y cells depend to a great extent on an exogenous source of cysteine. The importance of the transsulfuration pathway in astrocytes and glioblastoma/astrocytoma cells as a reserve pathway when the demand for glutathione is high was recently discussed by Mc-Bean (2012). Changes in cystathionine levels were not observed in either of the cell lines in response to PPG after 15 min of incubation (Fig. 4).

CONCLUSIONS
In the glioblastoma-astrocytoma (U-87 MG) and neuroblastoma (SHSY5Y) cells, the pathway catalyzed by enzymatic tandem CAT/MPST can play a role in the generation of hydrogen sulfide from cysteine. In the neuroblastoma cells, the pathway from methionine to cysteine through the CBS and CTH reactions seems to play a more significant role as compared to the astrocytoma cells. The higher activity and expression of enzymes involved in H 2 S generation from cysteine, in the neuroblastoma cells, provide an opportunity for more rapid response in H 2 S production than in the astrocytoma cells. However, in the astrocytoma cells, the elevation of H 2 S seems to be possible by releasing it from the pool of sulfane sulfur. Panthi et al. (2016) reviewed possible physiological roles of H 2 S in neurons protection from oxidative stress or in the upregulation of the GABA β-receptors at pre-and postsynaptic sites along with astrocytes roles in the regulation of neurotransmitter levels or neuronal excitability. (A) Western blotting analysis. Western blot analysis of MPST, CBS and CTH was performed for the cell lines using a mixture of rabbit-(MPST) and mouse-derived (CBS, CTH, β-actin and alpha-tubuline) primary antibodies with the appropriate mixture of alkaline phosphatase-conjugated secondary antibodies. One set of representative results is shown. β-Actin and alpha-tubuline were used as the internal control of protein loading; 20 µg of protein was added to each lane. For details see Material and Methods. (B) The relative intensity normalized to β-actin and alpha-tubulin. Analysis of protein bands imaged with the ChemiDoc TM MP. The relative intensity value is the average from three independent experiments. The relative intensity was normalized using β-actin and alphatubulin signals, the average of which was taken as one. The experiments were carried out for control homogenates of U-87 MG, SHSY5Y cells with or without PPG as the inhibitor of CTH in concentration of 0.7 mM for both cells lines. The homogenates were incubated with PPG for 15 minutes. The data represent the mean value from three independent experiments. A B