Cystathionine Beta-synthase Expression Research Articles

Abstract 2980: Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs)

Abstract Introduction: Our group has recently demonstrated that CBS, one of the H2S-producing enzymes, is abundantly overexpressed in human colorectal cancer (CRC) tissue specimens resulting in an increased H2S production. In order to extend our observations, we have now compared, using immunohistochemistry, CBS expression in carcinoma-associated fibroblasts (CAFs) compared to normal colonic fibroblasts (NCFs) in human colon adenocarcinoma samples. The overall goal of our studies is to characterize how the CBS/H2S axis contributes to the malignant phenotype of the CAFs in the tumor microenvironment. Methods: Human CAFs and NCFs were collected from CRC under an IRB approved protocol and cultured in vitro. Western blot analysis of both NCF and CAF was performed to determine CBS protein expression levels. SiRNA/shRNA-mediated silencing of CBS in CAFs was performed. A pharmacological inhibitor of CBS, aminooxyacetic acid (AOAA), in both CAF and NCF cultures was also utilized. Hydrogen sulfide (H2S) levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. We also performed proliferation assays using a coulter counter in CAFs, NCFs, and CAFs subjected to siRNA/shRNA-mediated CBS silencing or AOAA. Control cells were transduced with shNT vectors. Cells were also treated with the slow-releasing H2S donor GYY4137. We also conducted cell migration assays in transwell chambers (migration of CAFs toward conditioned media (CM) collected either from HCT116 cells or normal colon mucosal cells (NCM356). Finally, the XF24 Extracellular Flux Analyzer (Seahorse) was used to measure bioenergetic function in CAFs subjected to siRNA/shRNA-mediated silencing of CBS or treated with AOAA. Results: CBS protein expression and H2S production in CAFs was increased compared to NCFs. The H2S donor, GYY 4137, increased the proliferation rate of CAFs but not of NCFs. CBS silencing or AOAA treatment reduced CAF migration toward conditioned media from HCT116 cells. Genetic or pharmacological inhibition of CBS in CAF cells suppressed key bioenergetic parameters including basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity compared to NCF cells or CAF shNT cells. Conclusions: Thus, CBS/H2S axis serves as a mechanism to support CAFs’ function. In these cells, H2S contributes to maintaining their bioenergetic functions, which, in turn, promotes their proliferation and migration toward colon cancer cells. We hypothesize that these processes may contribute CRC progression. Citation Format: Katalin Modis, Manjit Maskey, Paul Johnson, Csaba Szabo, Mark R. Hellmich, Celia Chao. Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2017-2980

H2S and homocysteine control a novel feedback regulation of cystathionine beta synthase and cystathionine gamma lyase in cardiomyocytes

Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/−, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.

DNA hypomethylation of CBS promoter induced by folate deficiency is a potential noninvasive circulating biomarker for colorectal adenocarcinomas.

Aberrant DNA methylation patterns, which induced by folate deficiency, play important roles in tumorigenesis of colorectal cancer (CRC). Some DNA methylation alterations can also be detected in cell-free DNA (cfDNA) of patients’ plasma, making cfDNA an ideal noninvasive circulating biomarker. However, exact DNA methylation alterations induced by folate deficiency in tumorigenesis of CRC and exact potential circulating cfDNA methylation biomarker are still unclear. Therefore, DNA methylation patterns of the normal human colon mucosal epithelial cell line (NCM460), cultured with normal or low folate content, were screened and the DNA hypomethylation of cystathionine-beta-synthase (CBS) promoter was further validated in vitro and vivo. Then, the correlation analysis between folate level, DNA methylation alteration in promoter and expression of CBS was carried out in vitro and vivo. Further, the methylation patterns of CBS promoter in plasma cfDNA were detected and statistically correlated with pathological parameters and clinical outcome. Our study showed that DNA hypomethylation in CBS promoter, induced by folate deficiency, would lead to up-regulation of CBS both in vitro and vivo. Patients with cfDNA hypomethylation of CBS promoter in plasma were correlated with high tumor stage and poor clinical outcome. In addition, cfDNA hypomethylation of CBS promoter in plasma was shown to be an independent prognostic factor for recurrence and cancer-related death in CRC. Our results indicated that DNA hypomethylation of CBS promoter induced by folate deficiency could serve as a potential noninvasive circulating biomarker and may be helpful in developing more effective prognostic markers for CRC.

Reciprocal regulation of eNOS, H2S and CO-synthesizing enzymes in human atheroma: Correlation with plaque stability and effects of simvastatin

The molecular and cellular mechanisms underlying plaque destabilization remain obscure. We sought to elucidate the correlation between NO, H2S and CO-generating enzymes, nitro-oxidative stress and plaque stability in carotid arteries. Carotid atherosclerotic plaques were collected from 62 patients who had undergone endarterectomy due to internal artery stenosis. Following histological evaluation the plaques were divided into stable and unstable ones. To investigate the impact of simvastatin we divided patients with stable plaques, into those receiving and to those not receiving simvastatin. Expression and/or levels of p-eNOS/eNOS, pAkt/t-Akt, iNOS, cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE), heme oxygenase-1(HO-1), soluble guanyl cyclase sGCα1, sGCβ1, NOX-4 and HIF-1α were evaluated. Oxidative stress biomarkers malondialdehyde (MDA) and nitrotyrosine (NT) were measured. NT levels were decreased in stable plaques with a concomitant increase of eNOS phosphorylation and expression and Akt activation compared to unstable lesions. An increase in HIF-1α, NOX-4, HO-1, iNOS, CBS and CSE expression was observed only in unstable plaques. 78% of patients under simvastatin were diagnosed with stable plaques whereas 23% of those not receiving simvastatin exhibited unstable plaques. Simvastatin decreased iNOS, HO-1, HIF-1α and CSE whilst it increased eNOS phosphorylation. In conclusion, enhanced eNOS and reduced iNOS and NOX-4 were observed in stable plaques; CBS and CSE positively correlated with plaque vulnerability. Simvastatin, besides its known effect on eNOS upregulation, reduced the HIF-1α and its downstream targets. The observed changes might be useful in developing biomarkers of plaque stability or could be targets for pharmacothepary against plaque vulnerability.

Augmented H2S production via cystathionine-beta-synthase upregulation plays a role in pregnancy-associated uterine vasodilation.

Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy.

Altered metabolic homeostasis between vitamin D and long chain polyunsaturated fatty acids in preeclampsia

Sub-optimal maternal nutrition may result in pregnancy complications like preeclampsia. Preeclampsia is known to be of placental origin and a major cause of maternal morbidity and mortality worldwide. Our earlier studies suggest that altered metabolism of folic acid, vitamin B12 and long chain polyunsaturated fatty acid (LCPUFAs) in the one carbon cycle increases homocysteine levels in preeclampsia. Recent reports indicate that vitamin D deficiency may also have a role in preeclampsia, although the mechanisms are unclear. A disturbed one carbon cycle can influence methylation patterns of various genes involved in placental development. Altered expression of cystathionine beta synthase (CBS) gene can result in hyperhomocystenemia. Higher homocysteine levels are known to increase reactive oxygen species (ROS) production which in turn leads to increased expression of phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2). Higher expression of PLA2 and COX-2 can influence the release of arachidonic acid (AA) from membrane phospholipid and result in increased conversion to thromboxane. Vitamin D [1,25(OH)2D3] is known to induce the CBS gene expression while it can suppress the oxidative stress-induced COX-2 up-regulation and thromboxane production. Based on this, we propose a novel hypothesis that a disturbed vitamin D and LCPUFA metabolism influence the regulation of the one carbon cycle which will trigger inflammation through oxidative stress in preeclampsia. This may lead to altered feto-placental growth and development in preeclampsia.

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Abstract Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p<0.001) and number (p<0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1857.

Mucosal acidification increases hydrogen sulfide release through up-regulating gene and protein expressions of cystathionine gamma-lyase in the rat gastric mucosa.

This study was performed to investigate the effects of mucosal acidification on mRNA expression and protein synthesis of cystathionine gamma lyase (CSE), cystathionine beta synthase (CBS), and mucosal release of H2S in gastric mucosa in rats. Thirty-two rats were randomly assigned into 4 groups (8 in each), including: the control group, HCl (10 mM) treated group, HCl (100 mM) treated group, and one group to study the effect of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Anesthetized rats underwent tracheostomy and midline laparotomy. Ninety min after the instillation of neutral or acidic solutions, animals were sacrificed and the gastric mucosa was collected to measure the H2S concentration by ELISA method and to quantify mRNA expression of CSE and CBS by quantitative real-time PCR (qRT-PCR). Protein synthesis was also detected by Western blot method. Mucosal acidification with 10 and 100 HCl, significantly increased mucosal levels of H2S (P<0.01 and P<0.001) and mRNA (P<0.01 and P<0.001) and protein expressions of CSE (P<0.01 and P<0.001). L-NAME treatment reversed H2S release to control level. Our findings indicated that mucosal acidification with HCl increased mucosal release of H2S through upregulation of CSE gene and its protein expression. This effect is mainly mediated through the involvement of nitric oxide.

Gastric acid induces mucosal H2S release in rats by upregulating mRNA and protein expression of cystathionine gamma lyase.

It is well known that hydrogen sulfide (H2S) protects the gastric mucosa against gastric acid and other noxious stimulants by several mechanisms but until now the effect of gastric acid on H2S production has not been evaluated. This study was performed to determine the effect of basal and stimulated gastric acid secretion on mRNA and protein expression of cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS), and on mucosal release of H2S in rats. Seventy-two male rats were randomly assigned into 9 groups (8 in each)-control, distention, and pentagastrin-induced gastric acid secretion groups. The effects of 15% alcohol solution, propargylglycine (PAG), L-NAME, and pantoprazole were also investigated. Under anesthesia, animals underwent tracheostomy and midline laparotomy. A catheter was inserted into the stomach through the duodenum for gastric washout. At the end of the experiments, the animals were killed and the gastric mucosa was collected to measure H2S concentration and to quantify mRNA expression of CSE and CBS by quantitative real-time PCR, and expression of their proteins by western blot. Basal and stimulated gastric acid secretion increased mucosal levels of H2S, and mRNA and protein expression of CSE. Pantoprazole and L-NAME reversed H2S release and restored protein expression of CSE to the control level. Pantoprazole, but not propargylglycine, pretreatment inhibited the elevated level of protein expression of eNOS in response to distention-induced gastric acid secretion. Our findings indicated that NO mediated the stimulatory effect of gastric acid on H2S release and protein expression of CSE.

Commentary on: Expression of cystathionine beta-synthase and histopathological observations in placentas of patients with Down syndrome.

Commentary on: Expression of cystathionine beta-synthase and histopathological observations in placentas of patients with Down syndrome.

Neuroprotection of up-regulated carbon monoxide by electrical acupuncture on perinatal hypoxic-ischemic brain damage in rats.

This study investigated the neuroprotection and potential mechanism of carbon monoxide (CO) against perinatal hypoxic-ischemic brain damage in rats by electrical acupuncture (EA). Animal behavior, morphological changes, cystathionine beta-synthase (CBS), hypoxia-inducible factor-1α (HIF-1α), and heme oxygenase-1 (HO-1) expression levels, and CO content in rat cortex cells were determined. Results demonstrated that EA treatment decreased the slope behavior and increased the overhang behavior of perinatal rats. The treatment also decreased the number of positive cells. The activator and inhibitor of CBS aggravated and remitted the hypoxic damage in cortex cells, respectively. EA treatment decreased CBS expression level and increased HO-1 and HIF-1α expression levels in perinatal rat cortex cells. Compared with the control groups, the CO content of cortex cells in the EA treatment group significantly increased (**p < 0.01). We hypothesized that EA treatment increases cortical CO content to protect against hypoxic damage via the hydrogen sulfide/CBS-CO/HO-1-HIF-1α system. This study provided a significant reference for EA therapy and cued a novel protective mechanism for cerebral palsy.

P91: Cystathionine-beta-synthase expression in medulla oblongata can be up-regulated by in utero cigarette exposure in neonatal rats

H2S has been found to act as a neuroprotectant. Cystathionine-beta-synthase (CBS) is one of three enzymes for production of H2S in mammals. The present study was conducted to investigate whether CBS expression can be affected by in utero cigarette smoke exposure (CS) in the neonatal rats. SD pregnant rats were randomly divided into control and CS groups. The neonatal offspring rats were used in experimental observation. The methylene blue assay method was used to examine H2S production rate by medulla oblongata. CBS mRNA and protein expression in medulla oblongata were analysed by RT-PCR and Western blot, respectively, and the levels of CBS expression in neurons and glia of medullary nuclei were examined with immunohistochemical technique. In medulla oblongata of CS group, compared to control group, H2S production rate was increased (P

P27 Single nucleotide polymorphisms of the maternal cystathionine-beta-synthase gene are associated with preeclampsia

Preeclampsia (PE), a severe pregnancy complication characterized by maternal hypertension and proteinuria, is associated with endothelial dysfunction. Homocysteine (converted by cystathionine-beta-synthase (CBS)) and H 2 S levels are altered during PE and placental CBS gene expression is decreased. As since single nucleotide polymorphisms (SNPs) can affect CBS expression and/or function, we studied CBS SNPs in patients with PE. Controls ( n = 81), severe ( n = 60) and mild PE ( n = 39) cases were genotyped for 6 tag SNPs in the CBS locus. Plasma homocysteine, cysteine and cystathionine during pregnancy were analyzed and urinary sulfate was measured after pregnancy in 28 of the cases and controls. Women with severe PE less often had the minor allele for rs11203172 (OR[95%CI] = 0.46[0.22–0.95], p p p In conclusion, CBS SNPs are associated with decreased and increased risk to develop PE. Decreased cysteine concentrations in healthy pregnant women having the minor allele for rs11203172, may be due to an increased conversion of cysteine to H 2 S by CBS. These women may have higher H 2 S levels, positively affecting placentation and vascular function during pregnancy and decreasing their risk to develop PE.

Hippocampal cystathionine beta synthase in young and aged mice

Cystathionine beta synthase (CBS) is the main contributor to the production of hydrogen sulfide (H2S) in the brain. Exogenously administered H2S has been reported to protect neurons against hypoxic injury, ischemia and LPS-induced neuro-inflammation and in the facilitating of long term potentiation (LTP). Dysregulation of CBS leads to different diseases, which all have mental retardation in common. Although multiple studies have implicated a link between the CBS/H2S pathway and neurodegeneration, no studies have been performed examining the pathway in healthy aging animals. We hypothesize that CBS/H2S pathway plays an important role in the protection of learning and memory functions in the brain at the level of the hippocampus. Thus, we studied a set of 8 young (4 months) and 14 aged (24 months (n=6) and 28 months (n=8)) C57Bl6 mice. The 24-month-old mice displayed a significant decrease of CBS immunoreactivity in the MoDG only, compared to 4-month-old mice. In 28-month-old mice, we observed a significant increase of CBS immunoreactivity in the MoDG, compared to 4-month-old mice. When comparing 28-month-old mice to 24-month-old mice, all areas showed a significant increase of CBS immunoreactivity. Thus, throughout aging, CBS expression is maintained in the hippocampus, and many other forebrain regions as well. Mice at the unusual age of 28 months even have a higher hippocampal CBS expression than young mice. Maintenance (and increase) of CBS levels may sustain memory and learning by precluding neuronal loss in areas of the hippocampus.

P23 CBS gene variants are associated with conditions characterized by ischemia

The CBS gene regulates the expression of cystathionine beta-synthase (CBS), a key enzyme in the production of hydrogen sulfide (H 2 S) by conversion of homocysteine (Hcy). H 2 S is a gasotransmitter that protects cells against ischemic damage by exerting vasodilatory, anti-inflammatory and antioxidant effects. Both renal transplantation and preeclampsia (PE) are conditions marked by ischemia. Renal ischemia/reperfusion impairs CBS activity and consequently H 2 S production. Partial restoration of CBS activity causes an increase in H 2 S levels and a reduction of ischemia/reperfusion damage. Also, H 2 S is involved in placental vascular tone regulation. Furthermore, preeclamptic women have hyperhomocysteinemia and a decreased placental expression of the CBS gene. We therefore hypothesize that variations in the CBS gene caused by single nucleotide polymorphisms (SNPs) affect both susceptibility to ischemic damage in renal transplantation and the onset of PE. The genotype of 1271 renal transplantation donor and recipient pairs in the University Medical Centre Groningen were determined for seven tag SNPs in the CBS locus. These SNPs were analyzed for association with primary non-function (PNF), delayed graft function, first years biopsy proven acute rejection, death-censored graft survival and patient survival. In the Oslo University Hospital, 99 controls and 60 severe and 39 mild cases of PE were genotyped for six of these seven tag SNPs. Severe and mild cases of PE were subdivided into early- (<34 weeks) and late-onset (>34 weeks of pregnancy). The SNPs were then analyzed for association with the onset of PE. Univariate analysis showed improved graft survival in kidney transplant recipients that homozygously carry the minor allele of rs11203172 (HR[95%CI] = 0.124[0.017–0.882], p = 0.013 (homozygous carriers of the minor allele versus others)). Furthermore, no PNF was seen in these patients. Multivariate analyses showed no significant associations, most probably due to the relatively low number of patients in this group and the number of parameters studied. Univariate analysis also showed patients carrying the minor allele of rs11203172 to have a reduced risk to develop severe PE (OR[95%CI] = 0.54[0.21–0.94], p = 0.023). Conversely, the minor allele of rs234706, which is known to be associated with low Hcy, increased the risk to develop mild, late-onset PE (2.10[1.15–3.85], p = 0.016). SNPs in the CBS gene are associated with both kidney graft survival and the risk to develop PE. Changes of the function or expression of CBS caused by SNPs may affect ischemic conditions through decreased H 2 S production or Hcy accumulation. Since CBS is markedly reduced under ischemic conditions we assume that these findings are of considerable importance. Further characterization of the effects of these CBS gene variants and their alleged role in ischemia is warranted.

GW24-e1000 Danhong injection combined with Adipose tissue-derived stem cells transplantation promotes angiogenesis after hind limb ischaemia in diabetic nude mice through a endogenous hydrogen sulfide dependent mechanism

ObjectivesTo investigate the efficacy of Danhong injection (DH) combined with Adipose tissue-derived stem cells (ADSCs) transplantation on angiogenesis in diabetic hind limb ischaemia model and the related molecular mechanism.MethodsMale nude...

Assessment of immune function in Down syndrome patients

In Down syndrome (DS), trisomy 21 leads to overexpression of gene coding for specific enzymes. This overexpression translates directly into biochemical aberrations that affect multiple interacting metabolic pathways which culminates in cellular dysfunction and contributes to the unique pathogenesis of DS. The aim of this study is to evaluate parameters of immune response in terms of cytokines [tumor necrosis factor-α (TNF-α) and interlukin-2 (IL-2)] together with the quantitative expression of cystathionine beta synthase (CBS), whose transsulfuration pathway generates cysteine and hydrogen sulfide (H2S). H2S is known to boost host defense at physiological concentrations and to display cytotoxic activity at higher concentrations. Calcineurin activity (CAN) was also measured as its dysregulation has been shown to cause immune suppression. Subjects were 60 DS patients vs. 30 age and socioeconomic matching healthy controls. In their blood, the cytokines: TNF-α and IL-2, together with CBS and its by product H2S as well as CAN activity, were measured. Results showed that CBSmRNA relative expression (0.56±.06 vs. 0.32±.02), plasma H2S (72±8.5 vs. 50.8±4.1) and TNF-α (8.11±.01 vs. 3.6±0.9) were significantly higher among DS patients compared to controls, while CAN (0.27±0.1 vs. 0.45±0.2 units), was significantly decreased in blood of DS patients compared to controls. IL-2 (36.4±2.6 vs. 37.4±0.9) showed no significant difference between DS patients and controls. Accordingly it can be concluded that excessive synthesis of multiple gene products derived from overexpression of the genes present on chromosome 21 may be the cause for decreased immunity in DS patients compared to controls.

A Novel Mechanism of Formaldehyde Neurotoxicity: Inhibition of Hydrogen Sulfide Generation by Promoting Overproduction of Nitric Oxide

BackgroundFormaldehyde (FA) induces neurotoxicity by overproduction of intracellular reactive oxygen species (ROS). Increasing studies have shown that hydrogen sulfide (H2S), an endogenous gastransmitter, protects nerve cells against oxidative stress by its antioxidant effect. It has been shown that overproduction of nitric oxide (NO) inhibits the activity of cystathionine-beta-synthase (CBS), the predominant H2S-generating enzyme in the central nervous system.ObjectiveWe hypothesize that FA-caused neurotoxicity involves the deficiency of this endogenous protective antioxidant gas, which results from excessive generation of NO. The aim of this study is to evaluate whether FA disturbs H2S synthesis in PC12 cells, and whether this disturbance is associated with overproduction of NO.Principal FindingsWe showed that exposure of PC12 cells to FA causes reduction of viability, inhibition of CBS expression, decrease of endogenous H2S production, and NO production. CBS silencing deteriorates FA-induced decreases in endogenous H2S generation, neurotoxicity, and intracellular ROS accumulation in PC12 cells; while ADMA, a specific inhibitor of NOS significantly attenuates FA-induced decreases in endogenous H2S generation, neurotoxicity, and intracellular ROS accumulation in PC12 cells.Conclusion/SignificanceOur data indicate that FA induces neurotoxicity by inhibiting the generation of H2S through excess of NO and suggest that strategies to manipulate endogenous H2S could open a suitable novel therapeutic avenue for FA-induced neurotoxicity.

Frequent Epigenetic Silencing of the Folate-Metabolising Gene Cystathionine-Beta-Synthase in Gastrointestinal Cancer

BackgroundBoth gastric and colorectal cancers (CRC) are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG) silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS) functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.Methodology/Principal FindingsTo identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96) and 55% of gastric tumors (11 of 20). In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ2-test). However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.ConclusionA novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.

P39 Expression and localisation of H2S metabolising enzymes in the murine myocardium and liver

Background New interventions are required to prevent the loss of cardiomyocytes associated with myocardial ischemia and with the reperfusion injury that follows revascularisation. Recent studies have shown that compounds releasing hydrogen sulfide (H2S) can have cardioprotective effects [1] . However, these compounds are generally effective only at high (μM) concentrations and release is not well controlled, increasing the potential for cytotoxicity. H2S is produced endogenously and targeting the pathways that regulate availability in heart may be a more promising therapeutic approach. The present study aimed to characterise the distribution of the H2S synthesising enzymes, cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and mercaptopyruvate sulfurtransferase (MPST), and of H2S degrading enzyme, thiosulfate sulfurtransferase (TST) in the murine heart and in liver, an important site of H2S metabolism. Methods RNA and protein were prepared from heart and liver collected from 10 week old male C57BL/6J mice for quantitative RT-PCR and Western blotting. Further tissue was formalin fixed, processed and wax embedded before sectioning for immunohistochemistry. Results qRT-PCR showed high relative gene expression for all H2S metabolising enzymes in the liver. Cardiac tissue displayed high relative gene expression of TST & CSE, moderate expression of CBS and low expression of MPST. Western blots confirmed the presence of protein for TST & all H2S synthesising enzymes within heart & liver homogenates. Immunohistochemical staining of fixed tissue sections located CSE to vascular smooth muscle in the heart, while CBS & MPST were found in the vascular endothelium. Cardiomyocytes were immunoreactive for all enzymes. In hepatocytes, immunoreactive TST, CBS & MPST are found in hepatocytes throughout the liver, while CSE immunoreactivity is notably intensified around the central vein, responsible for hepatic venous return. Conclusion The murine myocardium and liver have the capacity to regulate local H2S availability via three separate synthetic pathways at least one degradative pathway which have distinctive cellular localisations. Future studies will investigate whether metabolic & synthetic enzyme expression and H2S availability is differentially regulated following ischaemia and reperfusion.