Carbon monoxide being hydrogen sulfide and nitric oxide molecular sibling, as endogenous and exogenous modulator of oxidative stress and antioxidative mechanisms in the digestive system

Oxidative stress reflects an imbalance between oxidants and antioxidants in favor of the oxidants capable of evoking tissue damage. Like hydrogen sulfide (H2S) and nitric oxide (NO), carbon monoxide (CO) is an endogenous gaseous mediator recently implicated in the physiology of the gastrointestinal (GI) tract. CO is produced in mammalian tissues as a byproduct of heme degradation catalyzed by the heme oxygenase (HO) enzymes. Among the three enzymatic isoforms, heme oxygenase-1 (HO-1) is induced under conditions of oxidative stress or tissue injury and plays a beneficial role in the mechanism of protection against inflammation, ischemia/reperfusion (I/R), and many other injuries. According to recently published data, increased endogenous CO production by inducible HO-1, its delivery by novel pharmacological CO-releasing agents, or even the direct inhalation of CO has been considered a promising alternative in future experimental and clinical therapies against various GI disorders. However, the exact mechanisms underlying behind these CO-mediated beneficial actions are not fully explained and experimental as well as clinical studies on the mechanism of CO-induced protection are awaited. For instance, in a variety of experimental models related to gastric mucosal damage, HO-1/CO pathway and CO-releasing agents seem to prevent gastric damage mainly by reduction of lipid peroxidation and/or increased level of enzymatic antioxidants, such as superoxide dismutase (SOD) or glutathione peroxidase (GPx). Many studies have also revealed that HO-1/CO can serve as a potential defensive pathway against oxidative stress observed in the liver and pancreas. Moreover, increased CO levels after treatment with CO donors have been reported to protect the gut against formation of acute GI lesions mainly by the regulation of reactive oxygen species (ROS) production and the antioxidative activity. In this review, we focused on the role of H2S and NO molecular sibling, CO/HO pathway, and therapeutic potential of CO-releasing pharmacological tools in the regulation of oxidative stress-induced damage within the GI tract with a special emphasis on the esophagus, stomach, and intestines and also two solid and important metabolic abdominal organs, the liver and pancreas

Inhibitory effect of selenomethionine on carcinogenesis in the model of human colorectal cancer in vitro and its link to the Wnt/β-catenin pathway

Selenium compounds have been implicated as anticancer agents; however, the mechanism of their inhibitory action against cancer development has not been extensively investigated. A constitutive activation of the Wnt/β-catenin pathway is a central event in colorectal carcinogenesis. In this pathway, excessive cell proliferation is initiated by generation of β-catenin followed by overexpression of proto-oncogenes, such as c-Myc. It is believed that under physiological conditions the level of c-Myc is efficiently controlled by accessibility of the β-catenin protein through the process of phosphorylation by glycogen synthase kinase 3β (GSK-3β). Here, we determined whether selenomethionine (SeMet) can inhibit cell growth and affect the Wnt/β-catenin pathway in the HT-29 human colorectal cancer cells in vitro. The effective cytotoxic doses of SeMet have been selected after 48 h of incubation of this compound with colorectal cancer HT-29 cell line. MTT assay was used to assess cell viability and the protein and mRNA levels of β-catenin and c-Myc were determined by Western blotting and qPCR, respectively. SeMet potently inhibited growth of HT-29 cells, significantly decreased level of the β-catenin protein and mRNA concentration, down-regulated the c-Myc gene expression and up-regulated the pro-apoptotic Bax protein level. Moreover, SeMet increased the level of GSK-3β phosphorylated at serine 9 (S9) and significantly increased the level of β-catenin phosphorylated at S33 and S37. We conclude that SeMet suppresses growth of HT-29 colorectal cancer cells by a mechanism linked to the Wnt/β-catenin pathway, however, degradation of β-catenin may occur independently of GSK-3β catalytic activity and its phosphorylation status

Evidence for cytoprotective effect of carbon monoxide donor in the development of acute esophagitis leading to acute esophageal epithelium lesions

Exposure to acidic gastric content due to malfunction of lower esophageal sphincter leads to acute reflux esophagitis (RE) leading to disruption of esophageal epithelial cells. Carbon monoxide (CO) produced by heme oxygenase (HMOX) activity or released from its donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) was reported to protect gastric mucosa against acid-dependent non-steroidal anti-inflammatory drug-induced damage. Thus, we aimed to investigate if CO affects RE-induced esophageal epithelium lesions development. RE induced in Wistar rats by the ligation of a junction between pylorus and forestomach were pretreated i.g. with vehicle CORM-2; RuCl3; zinc protoporphyrin IX, or hemin. CORM-2 was combined with NG-nitro-L-arginine (L-NNA), indomethacin, capsazepine, or capsaicin-induced sensory nerve ablation. Esophageal lesion score (ELS), esophageal blood flow (EBF), and mucus production were determined by planimetry, laser flowmetry, histology. Esophageal Nrf-2, HMOXs, COXs, NOSs, TNF-α and its receptor, IL-1 family and IL-1 receptor antagonist (RA), NF-κB, HIF-1α, annexin-A1, suppressor of cytokine signaling (SOCS3), TRPV1, c-Jun, c-Fos mRNA/protein expressions, PGE2, 8-hydroxy-deoxyguanozine (8-OHdG) and serum COHb, TGF-β1, TGF-β2, IL-1β, and IL-6 content were assessed by PCR, immunoblotting, immunohistochemistry, gas chromatography, ELISA or Luminex platform. Hemin or CORM-2 alone or combined with L-NNA or indomethacin decreased ELS. Capsazepine or capsaicin-induced denervation reversed CORM-2 effects. COHb blood content, esophageal HMOX-1, Nrf-2, TRPV1 protein, annexin-A1, HIF-1α, IL-1 family, NF-κB, c-Jun, c-Fos, SOCS3 mRNA expressions, and 8-OHdG levels were elevated while PGE2 concentration was decreased after RE. CO donor-maintained elevated mucosal TRPV1 protein, HIF-1 α, annexin-A1, IL-1RA, SOCS3 mRNA expression, or TGF-β serum content, decreasing 8-OHdG level, and particular inflammatory markers expression/concentration. CORM-2 and Nrf-2/HMOX-1/CO pathway prevent esophageal mucosa against RE-induced lesions, DNA oxidation, and inflammatory response involving HIF-1α, annexin-A1, SOCS3, IL-1RA, TGF-β-modulated pathways. Esophagoprotective and hyperemic CO effects are in part mediated by afferent sensory neurons and TRPV1 receptors activity with questionable COX/PGE2 or NO/NOS systems involvement

Molecular Profile of Barrett's Esophagus and Gastroesophageal Reflux Disease in the Development of Translational Physiological and Pharmacological Studies

Barrett's esophagus (BE) is a premalignant condition caused by gastroesophageal reflux disease (GERD), where physiological squamous epithelium is replaced by columnar epithelium. Several in vivo and in vitro BE models were developed with questionable translational relevance when implemented separately. Therefore, we aimed to screen Gene Expression Omnibus 2R (GEO2R) databases to establish whether clinical BE molecular profile was comparable with animal and optimized human esophageal squamous cell lines-based in vitro models. The GEO2R tool and selected databases were used to establish human BE molecular profile. BE-specific mRNAs in human esophageal cell lines (Het-1A and EPC2) were determined after one, three and/or six-day treatment with acidified medium (pH 5.0) and/or 50 and 100 µM bile mixture (BM). Wistar rats underwent microsurgical procedures to generate esophagogastroduodenal anastomosis (EGDA) leading to BE. BE-specific genes (keratin (KRT)1, KRT4, KRT5, KRT6A, KRT13, KRT14, KRT15, KRT16, KRT23, KRT24, KRT7, KRT8, KRT18, KRT20, trefoil factor (TFF)1, TFF2, TFF3, villin (VIL)1, mucin (MUC)2, MUC3A/B, MUC5B, MUC6 and MUC13) mRNA expression was assessed by real-time PCR. Pro/anti-inflammatory factors (interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, tumor necrosis factor α, interferon γ, granulocyte-macrophage colony-stimulating factor) serum concentration was assessed by a Luminex assay. Expression profile in vivo reflected about 45% of clinical BE with accompanied inflammatory response. Six-day treatment with 100 µM BM (pH 5.0) altered gene expression in vitro reflecting in 73% human BE profile and making this the most reliable in vitro tool taking into account two tested cell lines. Our optimized and established combined in vitro and in vivo BE models can improve further physiological and pharmacological studies testing pathomechanisms and novel therapeutic targets of this disorder

The effect of oxidized low density lipoproteins (oxLDL) on cytokine (TNF, IL-10) production by human monocyte subpopulations stimulated with different Toll-like (TLR) receptor ligands.

Atherosclerosis is considered to be a chronic inflammatory disease closely linked to increased concentration of oxidized low density lipoproteins (oxLDL) and to various infectious agents present both in peripheral blood and in vascular subendothelium. Monocytes and monocyte-derived macrophages are the key cells of immunological system involved in the progression of the disease. Monocytes are heterogenic population both in relation to the morphology, expression of membrane molecules as well as to the generation of cytokines. They posses unique ability to recognize infectious factors and to respond to the presence of oxLDL via integrating the signals coming simultaneously from these both types of molecules. Stimulation of membrane Toll-like receptors (TLR) by LPS, activating the network of intracellular signals, results in raised generation of pro- (TNF) an anti-inflammatory (IL-10) cytokines. In addition, it is commonly accepted that the presence oxLDL is able to modulate the generation of cytokines. It seems that distemper of the profile of cytokines released by monocytes may be the whys and wherefores of pathophysiological abnormalities determining atherosclerosis risk factors. In this dissertation we undertake investigations to define functional differences between subpopulations of human monocytes on the ground of expression of their CD14 and CD16 receptors. This was achieved by measuring and analyzing the differences between concentrations of pro-inflammatory TNF and anti-inflammatory IL-10 among subpopulations of monocytes stimulated by different ligands of TLR receptors. In parallel, the effect of oxLDL on the capacity of the monocyte subpopulations to generate TNF and IL-10 was studied.It has been found that human monocyte subpopulations stimulated by ligands of TLR2 and TLR4 receptors (stLPS, upLPS and PgLPS) differ individually one to the other in their capacity to generate TNF and IL-10. Under stimulation by all studied ligands monocytes characterized by expression of CD16 receptors generate much higher concentration of TNF than CD16-. Moreover, comparison of subpopulation CD14high and CD14dim among CD16+ monocytes allows us to state that CD14dim when stimulated with stLPS or PgLPS generates more TNF than CD14high. Nevertheless independently on the type of stimulation, among all of monocyte subpopulations, subpopulation of CD14high CD16+ exhibits the highest capacity to generate IL-10.The presence of oxLDL markedly inhibits the generation of IL-10 both in stimulated monocytes and in its all subpopulations. Moreover, for unclear reasons in monocytes CD16+ level of inhibition of the generation of IL-10 is slightly lower. OxLDL also inhibit the generation of TNF when stimulated by stLPS or upLPS. In this case however the extent of inhibition is rather low and it looks comparable with every individual subpopulation investigated. Interestingly, no effect of oxLDL, or even the effect of increased generation of TNF, was observed in monocytes as well as in its subpopulation CD16- under stimulation with PgLPS. Contrary to the above in monocytes that expressed CD16 receptors the presence of oxLDL slightly inhibits the generation of TNF.Our results tend towards better understanding of immunological processes involved in etiopathogensis of atherosclerosis.Miażdżyca uważana jest aktualnie za przewlekły stan zapalny, u którego podstaw leży między innymi narastające w przestrzeni podśródbłonkowej stężenie utlenionych lipoprotein o niskiej gęstości (oxLDL) oraz obecność różnorodnych czynników infekcyjnych. Za kluczowe komórki układu odpornościowego, zaangażowane w patomechanizm miażdżycy, uznano monocyty oraz powstające z nich makrofagi. Monocyty są populacją wykazującą znaczną heterogenność pod względem morfologii, profilu wydzielania cytokin oraz ekspresji antygenów powierzchniowych. Komórki te za pośrednictwem znajdujących się na ich powierzchni receptorów TLR posiadają zdolność rozpoznawania czynników infekcyjnych oraz reagowania na obecność oxLDL, a więc integrowania sygnałów płynących od tych dwóch typów cząsteczek. Stymulacja receptorów TLR czynnikami infekcyjnymi inicjuje aktywację sieci sygnalizacji wewnątrzkomórkowej, prowadząc do produkcji cytokin pro- (TNF) i przeciwzapalnych (IL-10). Równocześnie zaobserwowano, że obecności oxLDL skutkuje zaburzeniem profilu tej produkcji, co może przyczyniać się do rozwoju wielu patofizjologicznych nieprawidłowości, stanowiąc ostatecznie czynnik ryzyka rozwoju miażdżycy.W niniejszej pracy podjęto próbę określenia wybranych funkcjonalnych różnic pomiędzy subpopulacjami monocytów, definiowanych na podstawie ekspresji cząsteczek CD14 oraz CD16 i zrealizowano to poprzez zbadanie różnic w stymulowanej ligandami TLR produkcji cytokin: prozapalnej (TNF) i przeciwzapalnej (IL-10). W tych samych subpopulacjach analizowano także wpływ oxLDL na produkcję TNF i IL-10. Wykazano, że poszczególne subpopulacje monocytów ludzkich stymulowane ligandami receptorów TLR4 oraz TLR2 (stLPS, upLPS oraz PgLPS) różnią się między sobą zdolnością do produkcji TNF i IL-10. W przypadku wszystkich badanych ligandów receptorów TLR monocyty wykazujące ekspresję CD16 produkują znacznie większe ilości TNF niż monocyty CD16-. Wśród monocytów CD16+ porównanie populacji CD14high i CD14dim pozwoliło stwierdzić, że ta ostania produkuje wyraźnie więcej TNF indukowanego stLPS oraz PgLPS. Natomiast niezależnie od rodzaju stymulacji głównym producentem IL-10 okazała się subpopulacja CD14highCD16+.Zaobserwowano, że obecność oxLDL podczas stymulacji monocytów oraz ich poszczególnych subpopulacji, skutkuje wyraźnym obniżeniem produkcji IL-10. Ponadto stwierdzono, że w populacji monocytów CD16+ hamowanie to jest nieco mniejsze. OxLDL wykazują również hamujący, aczkolwiek nie tak silny, wpływ na produkcję TNF stymulowaną stLPS i upLPS. W tym przypadku stopień zahamowania produkcji cytokiny jest podobny w każdej z badanych subpopulacji. Co istotne, obecność oxLDL nie ma wpływu, a nawet nasila produkcję TNF indukowaną PgLPS w monocytach oraz ich subpopulacji CD16-. W przeciwieństwie do tego, w komórkach wykazujących ekspresje CD16, produkcja ta jest nieznacznie ograniczona.Uzyskane wyniki stanowią wkład w wiedzę zbliżającą nas do poznania istoty procesów immunologicznych odpowiedzialnych za etiopatogenezę miażdżycy

Barrett's metaplasia progression towards esophageal adenocarcinoma : an attempt to select a panel of molecular sensors and to reflect clinical alterations by experimental models

The molecular processes that predispose the development of Barrett’s esophagus (BE) towards esophageal adenocarcinoma (EAC) induced by gastrointestinal reflux disease (GERD) are still under investigation. In this study, based on a scientific literature screening and an analysis of clinical datasets, we selected a panel of 20 genes covering BE- and EAC-specific molecular markers (FZD5, IFNGR1, IL1A, IL1B, IL1R1, IL1RN, KRT4, KRT8, KRT15, KRT18, NFKBIL1, PTGS1, PTGS2, SOCS3, SOX4, SOX9, SOX15, TIMP1, TMEM2, TNFRSF10B). Furthermore, we aimed to reflect these alterations within an experimental and translational in vitro model of BE to EAC progression. We performed a comparison between expression profiles in GSE clinical databases with an in vitro model of GERD involving a BE cell line (BAR-T) and EAC cell lines (OE33 and OE19). Molecular responses of cells treated with acidified bile mixture (BM) at concentration of 100 and 250 μM for 30 min per day were evaluated. We also determined a basal mRNA expression within untreated, wild type cell lines on subsequent stages of BE and EAC development. We observed that an appropriately optimized in vitro model based on the combination of BAR-T, OE33 and OE19 cell lines reflects in 65% and more the clinical molecular alterations observed during BE and EAC development. We also confirmed previous observations that exposure to BM (GERD in vitro) activated carcinogenesis in non-dysplastic cells, inducing molecular alternations in the advanced stages of BE. We conclude that it is possible to induce, to a high extent, the molecular profile observed clinically within appropriately and carefully optimized experimental models, triggering EAC development. This experimental scheme and molecular marker panel might be implemented in further research, e.g., aiming to develop and evaluate novel compounds and prodrugs targeting GERD as well as BE and EAC prevention and treatment