Helicobacter pylori-activated fibroblasts as a silent partner in gastric cancer development.

The discovery of Helicobacter pylori (Hp) infection of gastric mucosa leading to active chronic gastritis, gastroduodenal ulcers, and MALT lymphoma laid the groundwork for understanding of the general relationship between chronic infection, inflammation, and cancer. Nevertheless, this sequence of events is still far from full understanding with new players and mediators being constantly identified. Originally, the Hp virulence factors affecting mainly gastric epithelium were proposed to contribute considerably to gastric inflammation, ulceration, and cancer. Furthermore, it has been shown that Hp possesses the ability to penetrate the mucus layer and directly interact with stroma components including fibroblasts and myofibroblasts. These cells, which are the source of biophysical and biochemical signals providing the proper balance between cell proliferation and differentiation within gastric epithelial stem cell compartment, when exposed to Hp, can convert into cancer-associated fibroblast (CAF) phenotype. The crosstalk between fibroblasts and myofibroblasts with gastric epithelial cells including stem/progenitor cell niche involves several pathways mediated by non-coding RNAs, Wnt, BMP, TGF-ß, and Notch signaling ligands. The current review concentrates on the consequences of Hp-induced increase in gastric fibroblast and myofibroblast number, and their activation towards CAFs with the emphasis to the altered communication between mesenchymal and epithelial cell compartment, which may lead to inflammation, epithelial stem cell overproliferation, disturbed differentiation, and gradual gastric cancer development. Thus, Hp-activated fibroblasts may constitute the target for anti-cancer treatment and, importantly, for the pharmacotherapies diminishing their activation particularly at the early stages of Hp infection.

Direct tool for quantitative analysis of cell/object dynamic behavior - metastasis and far beyond.

INTRODUCTION: The dynamics and depth of invasion as well as the ability of cancer cells to penetrate the walls of  lymphatic or blood vessels represent critical survival-influencing factors in cancer patients. Depending on the cell type and tissue environment, cancer cell invasion differ in terms of motility mechanism and migration modes. Thus, there is the need of effective models allowing not only for single cell invasion potential assessment but also for collective migration and expansive growth evaluation in 3D microenvironment e.g. basement membranes. To meet this task, the specimens should be compared and analyzed in terms of the dynamics of movement and the evolution of the shape. OBJECTIVES: Our main objective was development of the mathematical method that enables fast and credible calculation of parameters of shape and position, namely standard deviations (σX, σY), centroid position (µX, µY) and correlation coefficient ρ, based only on the contour of the aggregate. METHODS: In order to accomplish this goal we measured geometrical properties of aggregates of RGM1 cells seeded in 3D Geltrex basement membrane. Referential microscopic images were taken 24 and 48 h after seeding and cell group dynamics was registered over 8 h periods using time lapse microscopy. RESULTS: Based on gathered data, we managed to develop and fully test universal numerical tool allowing for estimation of statistical parameters of cell groups and aggregates which then allows for the precise evaluation of their behavior within microenvironment with time. CONCLUSION: We conclude, that our tool is suitable for any research on the metastatic potential and motility of cancer cells in a given microenvironment, regardless of the migration mechanism, which together with the advanced analysis like cell single-cell transcriptomic, proteomic, and chromatin accessibility data may allow to identify precise targets for anti-cancer therapies, to predict the degree of malignancy of neoplastic lesions as well as it can be useful during architecting therapeutic strategies. Moreover, the developed tool seems to be broadly applicable for assessment of behavioural dynamics of any population.

Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori-activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype.

Despite of the improvement in gastric cancer (GC) therapies patients still suffer from cancer recurrence and metastasis. Recently, the high ratio of these events combined with increased chemoresistance has been related to the asymptomatic Helicobacter pylori (Hp) infections. The limited efficiency of GC treatment strategies is also increasingly attributed to the activity of tumor stroma with the key role of cancer-associated fibroblasts (CAFs). In order to investigate the influence of Hp infection within stromal gastric tissue on cancer initiation and progression, we have exposed normal gastric epithelial cells to long-term influence of Hp-activated gastric fibroblast secretome. We have referred obtained results to this secretome influence on cancer cell lines. The invasive properties of cells were checked by time-lapse video microscopy and basement membrane assays. The expression of invasion-related factors was checked by RT-PCR, Western Blot, immunofluorescence and Elisa. Hp-activated gastric fibroblast secretome induced EMT type 3-related shifts of RGM1 cell phenotype; in particular it augmented their motility, cytoskeletal plasticity and invasiveness. These effects were accompanied by Snail1/Twist activation, the up-regulation of cytokeratin19/FAP/TNC/Integrin-ß1 and MMPs, and by the induction of cMethigh/pEGFRhigh phenotype. Mechanistic studies suggest that this microevolution next to TGFß relies also on c-Met/EGFR signaling interplay and engages HGF-Integrin-Ras-dependent Twist activation leading to MMP and TNC upregulation with subsequent positive auto- and paracrine feedback loops intensifying this process. Similar shifts were detected in cancer cells exposed to this secretome. Collectively, we show that the secretome of Hp-infected fibroblasts induces reprogramming/microevolution of epithelial and cancer cells towards type 3 EMT-related invasive phenotype in a manner reciprocally reliant next to TGFß on cMet/Integrin-ß1/p-EGFR-dependent axis. Apparently, the phenotypical plasticity of Hp-activated fibroblast reprogrammed gastric epithelial cells determines their susceptibility to the pro-invasive signaling, which results in re-organization of gastric niches and provides the cues for GC promotion/progression.

Role of Obesity, Physical Exercise, Adipose Tissue-Skeletal Muscle Crosstalk and Molecular Advances in Barrett's Esophagus and Esophageal Adenocarcinoma.

Both obesity and esophageal adenocarcinoma (EAC) rates have increased sharply in the United States and Western Europe in recent years. EAC is a classic example of obesity-related cancer where the risk of EAC increases with increasing body mass index. Pathologically altered visceral fat in obesity appears to play a key role in this process. Visceral obesity may promote EAC by directly affecting gastroesophageal reflux disease and Barrett's esophagus (BE), as well as a less reflux-dependent effect, including the release of pro-inflammatory adipokines and insulin resistance. Deregulation of adipokine production, such as the shift to an increased amount of leptin relative to "protective" adiponectin, has been implicated in the pathogenesis of BE and EAC. This review discusses not only the epidemiology and pathophysiology of obesity in BE and EAC, but also molecular alterations at the level of mRNA and proteins associated with these esophageal pathologies and the potential role of adipokines and myokines in these disorders. Particular attention is given to discussing the possible crosstalk of adipokines and myokines during exercise. It is concluded that lifestyle interventions to increase regular physical activity could be helpful as a promising strategy for preventing the development of BE and EAC.

Bioactive compounds from Lactarius deterrimus interfere with the invasive potential of gastric cancer cells.

Stomach cancer is the 4th most common cancer diagnosed worldwide. Despite intensive research on its etiopathology, its treatment strategies have not changed in the last 50 years. Mushrooms have recently attracted much attention as the source of bioactive compounds that can potentially complement cancer therapies. Here, we extracted a phenolic fraction from Lactarius deterrimus and analyzed its composition and bioactivity against the gastric cancer (AGS) cells. The complexity of L. deterrimus compounds was revealed by an HPLC assay, and was accompanied by cytostatic, cytotoxic and anti-invasive effects of the L. deterrimus extract (LDE). These are illustrated by inhibition of the AGS cells' proliferation, metabolic activity and motility, and by induction of the cytoskeleton rearrangements. Apparently, these effects are exerted via activation of intracellular oxidative stress and decreased ATP production in AGS cells that could not be compensated by induction of autophagy. Less severe LDE effects were seen on physiology of normal gastric fibroblasts; however, inhibition of their motility indicates that LDE can interfere with gastric cancer development via an effect on stromal cells. Along with the observed synergy of LDE and cisplatin/5-fluorouracil effects on AGS cells, our data show the potential of LDE for supplementation of the gastric cancer therapy.

Intestinal Alkaline Phosphatase Combined with Voluntary Physical Activity Alleviates Experimental Colitis in Obese Mice. Involvement of Oxidative Stress, Myokines, Adipokines and Proinflammatory Biomarkers.

Intestinal alkaline phosphatase (IAP) is an essential mucosal defense factor involved in the process of maintenance of gut homeostasis. We determined the effect of moderate exercise (voluntary wheel running) with or without treatment with IAP on the course of experimental murine 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis by assessing disease activity index (DAI), colonic blood flow (CBF), plasma myokine irisin levels and the colonic and adipose tissue expression of proinflammatory cytokines, markers of oxidative stress (SOD2, GPx) and adipokines in mice fed a standard diet (SD) or high-fat diet (HFD). Macroscopic and microscopic colitis in sedentary SD mice was accompanied by a significant decrease in CBF, and a significant increase in the colonic expression of tumor necrosis factor-alpha (TNF-α), IL-6, IL-1ß and leptin mRNAs and decrease in the mRNA expression of adiponectin. These effects were aggravated in sedentary HFD mice but reduced in exercising animals, potentiated by concomitant treatment with IAP, especially in obese mice. Exercising HFD mice demonstrated a substantial increase in the mRNA for adiponectin and a decrease in mRNA leptin expression in intestinal mucosa and mesenteric fat as compared to sedentary animals. The expression of SOD2 and GPx mRNAs was significantly decreased in adipose tissue in HFD mice, but these effects were reversed in exercising mice with IAP administration. Our study shows for the first time that the combination of voluntary exercise and oral IAP treatment synergistically favored healing of intestinal inflammation, strengthened the antioxidant defense and ameliorated the course of experimental colitis; thus, IAP may represent a novel adjuvant therapy to alleviate inflammatory bowel disease (IBD) in humans.

Long-Term Helicobacter pylori Infection Switches Gastric Epithelium Reprogramming Towards Cancer Stem Cell-Related Differentiation Program in Hp-Activated Gastric Fibroblast-TGFß Dependent Manner.

Helicobacter pylori (Hp)-induced inflammatory reaction leads to a persistent disturbance of gastric mucosa and chronic gastritis evidenced by deregulation of tissue self-renewal and local fibrosis with the crucial role of epithelial-mesenchymal transition (EMT) in this process. As we reported before, Hp activated gastric fibroblasts into cells possessing cancer-associated fibroblast properties (CAFs), which secreted factors responsible for EMT process initiation in normal gastric epithelial RGM1 cells. Here, we showed that the long-term incubation of RGM1 cells in the presence of Hp-activated gastric fibroblast (Hp-AGF) secretome induced their shift towards plastic LGR5+/Oct4high/Sox-2high/c-Mychigh/Klf4low phenotype (l.t.EMT+RGM1 cells), while Hp-non-infected gastric fibroblast (GF) secretome prompted a permanent epithelial-myofibroblast transition (EMyoT) of RGM1 cells favoring LGR-/Oct4high/Sox2low/c-Myclow/Klf4high phenotype (l.t.EMT-RGM1 cells). TGFß1 rich secretome from Hp-reprogrammed fibroblasts prompted phenotypic plasticity and EMT of gastric epithelium, inducing pro-neoplastic expansion of post-EMT cells in the presence of low TGFßR1 and TGFßR2 activity. In turn, TGFßR1 activity along with GF-induced TGFßR2 activation in l.t.EMT-RGM1 cells prompted their stromal phenotype. Collectively, our data show that infected and non-infected gastric fibroblast secretome induces alternative differentiation programs in gastric epithelium at least partially dependent on TGFß signaling. Hp infection-activated fibroblasts can switch gastric epithelium microevolution towards cancer stem cell-related differentiation program that can potentially initiate gastric neoplasm.

Interaction of epidermal growth factor with COX-2 products and peroxisome proliferator-activated receptor-γ system in experimental rat Barrett's esophagus.

Mixed acidic-alkaline refluxate is a major pathogenic factor in chronic esophagitis progressing to Barrett's esophagus (BE). We hypothesized that epidermal growth factor (EGF) can interact with COX-2 and peroxisome proliferator-activated receptor-γ (PPARγ) in rats surgically prepared with esophagogastroduodenal anastomosis (EGDA) with healthy or removed salivary glands to deplete salivary EGF. EGDA rats were treated with 1) vehicle, 2) EGF or PPARγ agonist pioglitazone with or without EGFR kinase inhibitor tyrphostin A46, EGF or PPARγ antagonist GW9662 respectively, 3) ranitidine or pantoprazole, and 4) the selective COX-2 inhibitor celecoxib combined with pioglitazone. At 3 mo, the esophageal damage and the esophageal blood flow (EBF) were determined, the mucosal expression of EGF, EGFR, COX-2, TNFα, and PPARγ mRNA and phospho-EGFR/EGFR protein was analyzed. All EGDA rats developed chronic esophagitis, esophageal ulcerations, and intestinal metaplasia followed by a fall in the EBF, an increase in the plasma of IL-1ß, TNFα, and mucosal PGE2 content, the overexpression of COX-2-, and EGF-EGFR mRNAs, and proteins, and these effects were aggravated by EGF and attenuated by pioglitazone. The rise in EGF and COX-2 mRNA was inhibited by pioglitazone but reversed by pioglitazone cotreated with GW9662. We conclude that 1) EGF can interact with PG/COX-2 and the PPARγ system in the mechanism of chronic esophagitis; 2) the deleterious effect of EGF involves an impairment of EBF and the overexpression of COX-2 and EGFR, and 3) agonists of PPARγ and inhibitors of EGFR may be useful in the treatment of chronic esophagitis progressing to BE.NEW & NOTEWORTHY Rats with EGDA exhibited chronic esophagitis accompanied by a fall in EBF and an increase in mucosal expression of mRNAs for EGF, COX-2, and TNFα, and these effects were exacerbated by exogenous EGF and reduced by removal of a major source of endogenous EGF with salivectomy or concurrent treatment with tyrphostin A46 or pioglitazone combined with EGF. Beneficial effects of salivectomy in an experimental model of BE were counteracted by PPARγ antagonist, whereas selective COX-2 inhibitor celecoxib synergistically with pioglitazone reduced severity of esophageal damage and protected esophageal mucosa from reflux. We propose the cross talk among EGF/EGFR, PG/COX-2, and proinflammatory cytokines with PPARγ pathway in the mechanism of pathogenesis of chronic esophagitis progressing to BE and EAC.

Helicobacter pylori-activated gastric fibroblasts induce epithelial-mesenchymal transition of gastric epithelial cells in vitro in a TGF-ß-dependent manner.

BACKGROUND: Colonization of the gastric mucosa with Helicobacter pylori (Hp) leads to the cascade of pathologic events including local inflammation, gastric ulceration, and adenocarcinoma formation. Paracrine loops between tissue cells and Hp contribute to the formation of gastric cancerous loci; however, the specific mechanisms underlying existence of these loops remain unknown. We determined the phenotypic properties of gastric fibroblasts exposed to Hp (cagA+vacA+) infection and their influence on normal epithelial RGM-1 cells. MATERIALS AND METHODS: RGM-1 cells were cultured in the media conditioned with Hp-activated gastric fibroblasts. Their morphology and phenotypical changes associated with epithelial-mesenchymal transition (EMT) were assessed by Nomarski and fluorescence microscopy and Western blot analysis. Motility pattern of RGM-1 cells was examined by time-lapse video microscopy and transwell migration assay. The content of TGF-ß in Hp-activated fibroblast-conditioned media was determined by ELISA. RESULTS: The supernatant from Hp-activated gastric fibroblasts caused the EMT-like phenotypic diversification of RGM-1 cells. The formation of fibroblastoid cell sub-populations, the disappearance of their collective migration, an increase in transmigration potential with downregulation of E-cadherin and upregulation of N-cadherin proteins, prominent stress fibers, and decreased proliferation were observed. The fibroblast (CAF)-like transition was manifested by increased secretome TGF-ß level, α-SMA protein expression, and its incorporation into stress fibers, and the TGF-ßR1 kinase inhibitor reduced the rise in Snail, Twist, and E-cadherin mRNA and increased E-cadherin expression induced by CAFs. CONCLUSION: Gastric fibroblasts which are one of the main targets for Hp infection contribute to the paracrine interactions between Hp, gastric fibroblasts, and epithelial cells. TGF-ß secreted by Hp-activated gastric fibroblasts prompting their differentiation toward CAF-like phenotype promotes the EMT-related phenotypic shifts in normal gastric epithelial cell populations. This mechanism may serve as the prerequisite for GC development.

Role of Helicobacter pylori infection in cancer-associated fibroblast-induced epithelial-mesenchymal transition in vitro.

BACKGROUND: Major human gastrointestinal pathogen Helicobacter pylori (H. pylori) colonizes the gastric mucosa causing inflammation and severe complications including cancer, but the involvement of fibroblasts in the pathogenesis of these disorders in H. pylori-infected stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts. Their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue; however, the interaction between H. pylori and fibroblasts remains unknown. We determined the effect of coincubation of normal rat gastric fibroblasts with alive H. pylori (cagA+vacA+) and H. pylori (cagA-vacA-) strains on the differentiation of these fibroblasts into cells possessing characteristics of cancer-associated fibroblasts (CAFs) able to induce epithelial-mesenchymal transition (EMT) of normal rat gastric epithelial cells (RGM-1). MATERIALS AND METHODS: The panel of CAFs markers mRNA was analyzed in H. pylori (cagA+vacA+)-infected fibroblasts by RT-PCR. After insert coculture of differentiated fibroblasts with RGM-1 cells from 24 up to 48, 72, and 96 hours, the mRNA expression for EMT-associated genes was analyzed by RT-PCR. RESULTS: The mRNA expression for CAFs markers was significantly increased after 72 hours of infection with H. pylori (cagA+vacA+) but not H. pylori (cagA-vacA-) strain. Following coculture with CAFs, RGM-1 cells showed significant decrease in E-cadherin mRNA, and the parallel increase in the expression of Twist and Snail transcription factors mRNA was observed along with the overexpression of mRNAs for TGFßR, HGFR, FGFR, N-cadherin, vimentin, α-SMA, VEGF, and integrin-ß1. CONCLUSION: Helicobacter pylori (cagA+vacA+) strain induces differentiation of normal fibroblasts into CAFs, likely to initiate the EMT process in RGM-1 epithelial cell line.

Moderate exercise training attenuates the severity of experimental rodent colitis: the importance of crosstalk between adipose tissue and skeletal muscles.

Although progress has been recently made in understanding of inflammatory bowel diseases (IBD), their etiology is unknown apart from several factors from adipose tissue and skeletal muscles such as cytokines, adipokines, and myokines were implicated in the pathogenesis of ulcerative colitis. We studied the effect high-fat diet (HFD; cholesterol up to 70%), low-fat diet (LFD; cholesterol up to 10%), and the normal diet (total fat up to 5%) in rats with TNBS colitis forced to treadmill running exercise (5 days/week) for 6 weeks. In nonexercising HFD rats, the area of colonic damage, colonic tissue weight, the plasma IL-1ß, TNF-α, TWEAK, and leptin levels, and the expression of IL-1ß-, TNF-α-, and Hif1α mRNAs were significantly increased and a significant fall in plasma adiponectin and irisin levels was observed as compared to LFD rats. In HFD animals, the exercise significantly accelerated the healing of colitis, raised the plasma levels of IL-6 and irisin, downregulated the expression of IL-1ß, TNF-α, and Hif1α, and significantly decreased the plasma IL-1ß, TNF α, TWEAK, and leptin levels. We conclude that HFD delays the healing of colitis in trained rats via decrease in CBF and plasma IL-1ß, TNF-α, TWEAK, and leptin levels and the release of protective irisin.

Exogenous asymmetric dimethylarginine (ADMA) in pathogenesis of ischemia-reperfusion-induced gastric lesions: interaction with protective nitric oxide (NO) and calcitonin gene-related peptide (CGRP).

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide (NO) synthesis inhibitor and pro-inflammatory factor. We investigated the role of ADMA in rat gastric mucosa compromised through 30 min of gastric ischemia (I) and 3 h of reperfusion (R). These I/R animals were pretreated with ADMA with or without the combination of L-arginine, calcitonin gene-related peptide (CGRP) or a small dose of capsaicin, all of which are known to afford protection against gastric lesions, or with a farnesoid X receptor (FXR) agonist, GW 4064, to increase the metabolism of ADMA. In the second series, ADMA was administered to capsaicin-denervated rats. The area of gastric damage was measured with planimetry, gastric blood flow (GBF) was determined by H2-gas clearance, and plasma ADMA and CGRP levels were determined using ELISA and RIA. ADMA significantly increased I/R-induced gastric injury while significantly decreasing GBF, the luminal NO content, and the plasma level of CGRP. This effect of ADMA was significantly attenuated by pretreatment with CGRP, L-arginine, capsaicin, or a PGE2 analogue. In GW4064 pretreated animals, the I/R injury was significantly reduced and this effect was abolished by co-treatment with ADMA. I/R damage potentiated by ADMA was exacerbated in capsaicin-denervated animals with a further reduction of CGRP. Plasma levels of IL-10 were significantly decreased while malonylodialdehyde (MDA) and plasma TNF-α contents were significantly increased by ADMA. In conclusion, ADMA aggravates I/R-induced gastric lesions due to a decrease of GBF, which is mediated by a fall in NO and CGRP release, and the enhancement of lipid peroxidation and its pro-inflammatory properties.

Role of angiotensin-(1-7) in gastroprotection against stress-induced ulcerogenesis. The involvement of mas receptor, nitric oxide, prostaglandins, and sensory neuropeptides.

Angiotensin-(1-7) [Ang-(1-7)] is a major vasoactive metabolite of angiotensin I (Ang I), both being important components of the renin-angiotensin system (RAS). Ang-(1-7) acting via Mas receptor was documented in kidneys, heart, brain, and gastrointestinal (GI)-tract. We studied the gastroprotective activity of exogenous Ang-(1-7) in rats exposed to water immersion and restraint stress (WRS) without or with A-779 [d-Ala7-Ang-(1-7), an antagonist of Ang-(1-7) Mas receptors], AVE 0991 (5-formyl-4-methoxy-2-phenyl-1[[4-[2-(ethylaminocarbonylsulfonamido)-5-isobutyl-3-thienyl]-phenyl]-methyl]-imidazole), the agonist of Ang-(1-7) receptor, as well as the inhibition of nitric-oxide (NO) synthase, the suppression of cyclo-oxygenase (COX)-1 (indomethacin, SC-560 [5-(4-chloro-phenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl-pyrazole]), the activity COX-2 (rofecoxib), and denervation with capsaicin. The mRNA expression of constitutively expressed nitric-oxide synthase (cNOS), inducible nitric-oxide synthase (iNOS), interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α was analyzed by reverse transcription polymerase chain reaction. The WRS lesions were dose-dependently reduced by pretreatment with Ang-(1-7), which also caused an increase in gastric blood flow (GBF) and luminal content of NO. COX-1 and COX-2 inhibitors or L-NNA (N5-[imino(nitroamino)methyl]-L-ornithine) reversed the reduction in lesion number and the rise in GBF evoked by Ang-(1-7). Ang II augmented the WRS lesions, decreased GBF and increased the plasma IL-1ß and TNF-α levels. Capsaicin denervation attenuated the reduction of Ang-(1-7)-induced gastric lesions and the rise in GBF; these effects were restored by supplementation with calcitonin gene-related peptide (CGRP). The cNOS mRNA was upregulated while iNOS, IL-1ß and TNF-α mRNAs were downregulated in Ang-(1-7)-pretreated rats. We conclude that Ang-(1-7), in contrast to Ang II, which worsened WRS ulcerogenesis, affords potent gastroprotection against WRS ulcerogenesis via an increase in GBF mediated by NO, endogenous prostaglandins, sensory neuropeptides, and anti-inflammatory action involving the inhibition of proinflammatory markers iNOS, IL-1ß, and TNF-α.

New satiety hormone nesfatin-1 protects gastric mucosa against stress-induced injury: mechanistic roles of prostaglandins, nitric oxide, sensory nerves and vanilloid receptors.

Nesfatin-1 belongs to a family of anorexigenic peptides, which are responsible for satiety and are identified in the neurons and endocrine cells within the gut. These peptides have been implicated in the control of food intake; however, very little is known concerning its contribution to gastric secretion and gastric mucosal integrity. In this study the effects of nesfatin-1 on gastric secretion and gastric lesions induced in rats by 3.5h of water immersion and restraint stress (WRS) were determined. Exogenous nesfatin-1 (5-40µg/kg i.p.) significantly decreased gastric acid secretion and attenuated gastric lesions induced by WRS, and this was accompanied by a significant rise in plasma NUCB2/nefatin-1 levels, the gastric mucosal blood flow (GBF), luminal NO concentration, generation of PGE2 in the gastric mucosa, an overexpression of mRNA for NUBC2 and cNOS, as well as a suppression of iNOS and proinflammatory cytokine IL-1ß and TNF-α mRNAs. Nesfatin-1-induced protection was attenuated by suppression of COX-1 and COX-2 activity, the inhibition of NOS with L-NNA, the deactivation of afferent nerves with neurotoxic doses of capsaicin, and the pretreatment with capsazepine to inhibit vanilloid VR1 receptors. This study shows for the first time that nesfatin-1 exerts a potent protective action in the stomach of rats exposed to WRS and these effects depend upon decrease in gastric secretion, hyperemia mediated by COX-PG and NOS-NO systems, the activation of vagal and sensory nerves and vanilloid receptors.

The impact of asymmetric dimethylarginine (ADAMA), the endogenous nitric oxide (NO) synthase inhibitor, to the pathogenesis of gastric mucosal damage.

This review was designed to provide an update on the role of asymmetric arginine (ADMA), the endogenous inhibitor of nitric oxide (NO) synthase in the pathophysiology of the upper gastrointestinal (GI) tract. Numerous studies in the past confirmed that NO is a multifunctional endogenous gas molecule involved in most of the body organs' functional and metabolic processes including the regulation of gastrointestinal (GI) secretory functions, motility, maintenance of GI integrity, gastroprotection and ulcer healing. NO is metabolized from L-arginine by enzymatic reaction in the presence of constitutive NO synthase. In upper GI tract, NO acts as a potent vasodilator known to increase gastric mucosa blood flow, regulates the secretion of mucus and bicarbonate, inhibits the gastric secretion and protects the gastric mucosa against the damage induced by a variety of damaging agents and corrosive substances. In contrast, ADMA first time described by Vallance and coworkers in 1992, is synthesized by the hydrolysis of proteins containing methylated arginine amino acids located predominantly within the nucleus of cells. This molecule has been shown to competitively inhibit NO synthase suggesting its regulatory role in the functions of vascular endothelial cells and systemic circulation in humans and experimental animals. Nowadays, ADMA is a potentially important risk factor for coronary artery diseases and a marker of cardiovascular risk. Increased plasma levels of ADMA have been documented in several conditions that are characterized by endothelial dysfunction, including hypertension, hypercholesterolemia, hyperglycemia, renal failure and tobacco exposure. The role of ADMA in other systems including GI-tract has been so far less documented. Nevertheless, ADMA was shown to directly induce oxidative stress and cell apoptosis in gastric mucosal cells in vitro and to contribute to the inflammatory reaction associated with major human pathogen to gastric mucosa, Helicobacter pylori (H.pylori). Infection of gastric mucosa with this germ or H. pylori water extract led to marked increase in the plasma concentration of ADMA and significantly inhibited bicarbonate secretion, considered as one of the important components of upper GI-tract defense system. When administered to rodents, ADMA aggravated gastric mucosal lesions injury induced by cold stress, ethanol and indomethacin and this worsening effect on gastric lesions was accompanied by the significant increase in the plasma level of ADMA. This exaggeration of gastric lesions by ADMA was coincided with the inhibition of NO, the suppression of gastric blood flow and excessive release of proinflammatory cytokine TNF-α. This metabolic analog of L-arginine applied to rats was exposed to water immersion and restraint stress and ischemia-reperfusion, causing an elevation of plasma levels of ADMA and gastric MDA content, which is the marker of lipid peroxidation. These effects, including the rise in the plasma levels of ADMA in rats with stress and ischemia-reperfusion-induced gastric lesions, were attenuated by concomitant treatment with L-arginine, the substrate for NO-synthase, and superoxide dismutase (SOD), a reactive oxygen metabolite scavenger added to ADMA. We conclude that ADMA could be considered as an important factor contributing to the pathogenesis of gastric mucosal damage and inflammatory reaction in H. pylori-infected stomach due to inhibition of NO, suppression of GI microcirculation, and the proinflammatory and proapoptotic actions of this arginine analog.

Involvement of cytoskeleton in orientation of cell division in contact guided cells.

Single human skin fibroblasts and the skin keratinocyte cell line HaCaT show contact guidance and elongate along narrow (1-2 microm) scratches in glass substratum. During cell division these cells orientate their mitotic spindles along the long axis of the cell. Immunofluorescence staining of actin, tubulin, chromatin, and the nuclear NuMA protein complex demonstrated that cell elongation along scratches is accompanied by a corresponding rearrangement in the cytoskeleton. The results and literature suggest the following steps in the interplay between outside-in and inside-out signalling in the regulation of cell division orientation by extracellular factors. The interaction of cell surface with an anisotropy in the local environment causes changes in F-actin organization, cell elongation and alignment of stress fibres along the cell axis. This is accompanied by a corresponding reorientation of microtubules. Microtubules mediate between cell shape changes dependent upon cell interaction with substratum or other cells, the cortical actin and the position of centrosomes. Centrosomes determine the position and orientation of the mitotic spindle. The astral and central microtubules of the mitotic spindle control the localization of contraction-relaxation in the cell cortex and the position of the constriction ring and cell division plane.

Surface anisotropy orients cell divisions in contact guided cells.

The orientation of cell elongation and the plane of cell division were determined in cells growing under isotropic conditions on a plain glass surface or under anisotropic conditions on a scratched glass surface. Four cell lines were analysed, each showing various degrees of contact guidance. Human skin fibroblasts and the skin keratinocyte cell line HaCaT oriented randomly on the smooth isotropic surface of glass, grew and divided randomly. By contrast, on an anisotropic scratched surface these cells showed contact guidance, elongated along scratches, and their planes of division were perpendicular to the long axis of the cell. In these two cell types there was a high degree of correlation between the cell alignment and the plane of cell division, which shows that extracellular factors can influence or even determine the latter. In cell lines in which contact guidance under anisotropic conditions was less evident, viz. the lung endothelial cell line HLMEC and the skin endothelial cell line HSkMEC, the alignment of cell division planes was less ordered. This report concentrates on quantitative phenomenological descriptions of the orientation of cell division as determined by contact guidance.

[Molecular action of insulin-sensitizing agents]. / Molekularne podstawy antynowotworowego dzialania uwrazliwiaczy na insuline.

Atypical endometrial hyperplasia has been associated with progression to endometrial cancer, the most common genital malignancy. There are multiple risk factors for endometrial cancer, such as early menarche, exogenous estrogen exposure, obesity and diabetes. Diabetics have a 3-4 fold relative risk of endometrial cancer. Also, several studies have demonstrated an association between insulin resistance and endometrial cancer. There is known the first description of atypical endometrial hyperplasia resistant to progestogen therapy, which was subsequently treated with an insulin-sensitizng agent, metformin. Metformin is a biguanide antihyperglycemic agent used in the treatment of adult-onset diabetes. Unlike the sulfonylureas, metformin does not act primarily by increasing insulin secretion. In contrast, metformin lowers the rate of gluconeogenesis in the presence of insulin. Therefore, it is considered an insulin-sensitizer. Increased insulin sensitivity may improve the metabolic effect of insulin and decrease its mitogenic effect by tissue-specific mechanisms. One explanation for tissue specific differences in insulin binding and action may be through the relative expression of the insulin receptor (IR) isoforms. The IR isoforms IR-A and IR-D differ by 12 amino acid residues, owing to the alternative splicing of exon. The IR-A is predominantly expressed in malignant tissues and may lead to mitogenic effects within the cell. The relative expressions of IR-A and IR-B in normal and malignant endometrial tissue is not known. Besides direct effects on the IR, several additional mechanisms have been proposed for the mitogenic effect of insulin in endometrial cancer. In addition to the possible direct mitogenic effects of insulin through the IR-A, insulin resistance may be associated with alterations in expression of insulin-like growth factors (IGFs) and the IGF binding proteins (IGFBPs) or may inhibit the protective effect of progestagens. Binding sites for IGF-1 and IGF-2 have been confirmed in both normal and malignant endometrium. Binding of IGF-1 is significantly higher in endometrial cancer compared to normal endometrium. In the Ishikawa human endometrial cancer cell line IGF-1 was a more potent mitogen than insulin or IGF-2. Insulin may increase mitogenicity by regulating the expression of IGFBPs. The IGFBPs are a family of proteins that have both proliferative and anti-proliferative effects. While all six high-affinity IGFBPs are expressed in the endometrium, IGFBP-1 is the best characterized. Hyperinsulinemia can decrease IGFBP-1 even in the presence of progesterone, perhaps inhibiting progesterone's protective effects. Interestingly, IGFBP-1 was undetectable or minimally expressed in endometrial cancers. Nestler discussed results of a 6-month treatment of 100 nonebese women with PCOS, which showed a somewhat greater effect of metformin than rosiglitazone and no benefit of administering both agents in combination. Long-term treatment with oral contraceptives decreases endometrial cancer, with a reduction in serum androgens and a decreases in hirsutism and acne, but may worsen insulin resistance and lead to deteriration in glucose tolerance. Insulin sensitizers, on the other hand, should decrease endometrial hyperplasia by inducing regular menses, but may not be as beneficial in improving androgen - related symptoms. Note that the Nurses Health Study (NHS) showed increased risk of diabetes in oral contraceptive users. These considerations may be related to the finding that women who used oral contraceptives have increased risk of myocardial infarction. Thus, in view of the particular increase in CVD risk among women with PCOS, one might be less likely to recommend oral contraceptives, while insulin sensitizers may be of particular benefit, decreasing androgens, improving ovulation and fertility, and reducing the risk of diabetes and CVD. Theoretically, metformin, a treatment which is now widely used to treat infertile women with PCOS, may have a role in preventing endometrial hyperstimulation by lowering insulin concentrations and restoring ovulation. However, the long-term effects of this drug in women with PCOS are not known and more studies are required before suggesting its use for preventing endometrial cancer.

[Skin cells as steroid target; the steroid action in some skin diseases and wound healing]. / Wplyw steroidów na komórki skóry; ich rola w wybranych schorzeniach skóry i procesie gojenia ran.

The paper is concerned with contemporary concepts on the role of androgens, estrogens, progesterone, mineralcorticosteroids, glucocorticoids and vitamin D3 and their signal transduction pathways in skin physiology, pathology and wound healing. The new therapeutic strategies based on mechanisms of steroid action have been presented. The classic, genomic pathway based on nuclear receptor activation is a well documented fact but less is known about indirect, nongenomic steroid effects. Steroids induced effects depend on their concentration, type of target cells and the receptors as well as accompanying side effects.