Hippo pathway - brief overview of its relevance in cancer.

The Hippo pathway is the major regulator of organ growth and proliferation. Described initially in Drosophila, it is now recognized as one of the most conserved molecular pathways in all metazoan. Recent studies have revealed the Hippo signalling pathway might contribute to tumorigenesis and cancer development. The core components of the Hippo pathway include the mammalian sterile 20-like kinases (MSTs), large tumour suppressor kinases (LATSs), the adaptor proteins Salvador homologue 1 (SAV1, also called WW45) and Mps One Binder kinase activator proteins. The major target of the Hippo core kinases is the mammalian transcriptional activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). In cancer, the Hippo signalling is inactivated and YAP and TAZ are activated and free to translocate into the nucleus to promote cell proliferation. Nuclear YAP/TAZ activate or suppress transcription factors that regulate target genes involved in cell proliferation, tissue growth, control of organ size and shape or metastasis. The Hippo signalling pathway that controls the most important cellular processes like growth and division appears to be a very promising research subject in the field of cell biology and tissue engineering. It consists of elements that in the cell play the roles of tumour suppressors as well as oncogenes. This 'Janus like' - an opposite activity hidden within one and the same signalling pathway represents a significant obstacle for studying it. This property of the Hippo pathway is worth remembering, as it will appear several times during the discussion of its properties. Here, we will review certain data regarding biology of the Hippo signalling and its interplay with other prominent signalling pathways in the cell, its relevance in cancer development and therapies that might target elements of the Hippo pathway in most human cancers.

Melatonin metabolite, N(1)-acetyl-N(1)-formyl-5-methoxykynuramine (AFMK), attenuates acute pancreatitis in the rat: in vivo and in vitro studies.

Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 µg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.

Recurrent mutations of BRCA1, BRCA2 and PALB2 in the population of breast and ovarian cancer patients in Southern Poland.

BACKGROUND: Mutations in the BRCA1, BRCA2 and PALB2 genes are well-established risk factors for the development of breast and/or ovarian cancer. The frequency and spectrum of mutations in these genes has not yet been examined in the population of Southern Poland. METHODS: We examined the entire coding sequences of the BRCA1 and BRCA2 genes and genotyped a recurrent mutation of the PALB2 gene (c.509_510delGA) in 121 women with familial and/or early-onset breast or ovarian cancer from Southern Poland. RESULTS: A BRCA1 mutation was identified in 11 of 121 patients (9.1 %) and a BRCA2 mutation was identified in 10 of 121 patients (8.3 %). Two founder mutations of BRCA1 accounted for 91 % of all BRCA1 mutation carriers (c.5266dupC was identified in six patients and c.181 T > G was identified in four patients). Three of the seven different BRCA2 mutations were detected in two patients each (c.9371A > T, c.9403delC and c.1310_1313delAAGA). Three mutations have not been previously reported in the Polish population (BRCA1 c.3531delT, BRCA2 c.1310_1313delAAGA and BRCA2 c.9027delT). The recurrent PALB2 mutation c.509_510delGA was identified in two patients (1.7 %). CONCLUSIONS: The standard panel of BRCA1 founder mutations is sufficiently sensitive for the identification of BRCA1 mutation carriers in Southern Poland. The BRCA2 mutations c.9371A > T and c.9403delC as well as the PALB2 mutation c.509_510delGA should be included in the testing panel for this population.

Kynuramines induce overexpression of heat shock proteins in pancreatic cancer cells via 5-hydroxytryptamine and MT1/MT2 receptors.

Kynuramines, metabolites of melatonin and L-tryptophan, are synthesized endogenously by oxygenases or in spontaneous reaction by an interaction with free radicals. We have reported previously that melatonin stimulates expression and phosphorylation of heat shock protein (HSP) 27, as well as production of HSP70 and HSP90αß in pancreatic carcinoma cells (PANC-1). Based on those results, we hypothesized that above processes could have been involved in the interruption of intrinsic proapoptotic pathway. Herein, we report that incubation of PANC-1 cells with N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) or with L-kynurenine (L-KYN) lead to the overexpression of heat shock protein synthesis and these effects are partially reversed by 5-HT3 or MT1/MT2 receptor antagonists. PANC-1 cells in culture were treated with AFMK or L-KYN, with non selective MT1/MT2 receptor antagonist (luzindole), with 5-HT2 and 5-HT3 receptor antagonists (ketanserin and MDL72222), or combination of these substances. Both AFMK and L-KYN significantly decreased cytoplasmic HSP27 and this effect was presumably due to increased of its phosphorylation and consequent nuclear translocation, confirmed by immunoprecipitation of phosphorylated form of HSP27. These changes were accompanied by marked augmentation of HSP70 and HSP90αß in the cytosolic fraction. Pretreatment of cell cultures with luzindole or MDL72222 followed by the addition of AFMK or L-KYN reversed the stimulatory effects of these substances on HSP expression in PANC-1 cells, whereas ketanserin failed to influence mentioned above phenomenon. We conclude that activation of HSPs in pancreatic carcinoma cells seems to be dependent on an interaction of AFMK or L-KYN with MT1/MT2 or/and 5-HT3 receptors.

The dynamics of heat shock system activation in Monomac-6 cells upon Helicobacter pylori infection.

Immune system cells, particularly phagocytes, are exposed to direct contact with pathogens. Because of its nature - elimination of pathogenes - their cytoprotective systems supposed to be quick and forceful. Physiological consequence of phagocytosis for the phagocyte is the apoptotic death to prevent the eventual survival of bacteria as intracellular parasites. However, in some cases, defense systems used by the bacteria force the immune cells to prolong the contact with the pathogen for its effective elimination. Experiments were performed on Monomac-6 cells exposed to live CagA, VacA expressing Helicobacter pylori (H. pylori) over different period of time. Total cellular RNA, cytoplasmic and nuclear proteins were isolated for polymerase chain reaction, Western-blot and electrophoretic mobility shift assay, respectively. We found that Monomac-6 cells infection with H. pylori resulted in the translocation of the entire cellular content of the heat shock protein 70 (HSP70) into the cytoplasm, where its presence could protect cell against toxic products of engulfed bacteria and premature apoptosis. At the same time the nuclear translocation of heat shock factor 1 (HSF-1) and activation of HSP70 gene transcription was noticed. Action of HSP70 might to postpone monocyte apoptosis through protecting cytoplasmic and nuclear proteins from damaging effect of bacterial products, what could be the defending mechanism against the toxic stress caused by engulfed bacteria and provide the immune cell with the sufficient amount of time required for neutralization of the bacteria from phagosomes, even at the expense of temporary lack of the protection of nuclear proteins.

Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.

Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.

Effect of 5-week moderate intensity endurance training on the oxidative stress, muscle specific uncoupling protein (UCP3) and superoxide dismutase (SOD2) contents in vastus lateralis of young, healthy men.

In the present study fifteen male subjects (age: 22.7 ± 0.5 years; BMI: 23.5 ± 0.6 kg x m⁻²; VO2(max) 46.0 ± 1.0 mL x kg⁻¹ x min⁻¹) performed 5 week moderate intensity endurance training. The training resulted in a significant increase in maximal oxygen uptake (VO2(max)) (P=0.048) and power output reached at VO2(max) (P=0.0001). No effect of training on the uncoupling protein 3 (UCP3) content in the vastus lateralis was found (P>0.05). The improvement of physical capacity was accompanied by no changes in cytochrome-c and cytochrome-c oxidase contents in the vastus lateralis (P>0.05). However, the training resulted in an increase (P=0.02) in mitochondrial manganese superoxide dismutase (SOD2) content in this muscle. Moreover, a significant decrease (P=0.028) in plasma basal isoprostanes concentration [F2isoprostanes](pl) accompanied by a clear tendency to lower (P=0.08) gluthatione disulfide concentration [GSSG](pl) and tendency to higher (P=0.08) total antioxidant capacity (TAC) was observed after the training. We have concluded that as little as 5 weeks of moderate intensity endurance training is potent to improve physical capacity and antioxidant protection in humans. Surprisingly, these effects occur before any measurable changes in UCP3 protein content. We postulate that the training-induced improvement in the antioxidant protection at the muscle level is due to an increase in SOD2 content and that therefore, the role of UCP3 in the enhancement of physical capacity and antioxidant protection, at least in the early stage of training, is rather questionable.

Only live Helicobacter pylori is capable of caspase-3 dependent apoptosis induction in gastric mucosa epithelial cells.

Physiological process of cell death, apoptosis, plays a beneficial role in organism survival, but in some pathologies, like gastric Helicobacter pylori (Hp) infection, this process may turn against the host organism causing tissue damage. Knowledge of the mechanisms controlling apoptosis may have potential significance in treatment of these pathologic states. Therefore, we sought to determine whether apoptosis induced in the gastric epithelial cells exposed to live Hp involves the alteration in heat shock protein 70 (HSP70) expression and activation of caspase-3 in peroxisome proliferator-activated receptors (PPARgamma dependent manner). Experiments were performed with KATO III, gastric epithelial cells, exposed to CagA and Vac A positive live Hp, water Hp extracts or Hp culture supernatant over different time periods. Total cellular RNA and proteins were isolated for PCR, western-blot and EMSA studies. Genomic DNA was isolated to analyze apoptosis status. We propose new model of Hp induced HSP70 dependent, caspase-3 executed apoptosis in human gastric epithelium. KATO III cells exposed to Hp, showed an increase in caspase-3 activity accompanied and preceeded by activation of nuclear translocation of PPARg peaking at 48 h of culture. Moreover, heat shock factor 1 (HSF-1) bound up with phosphorylated STAT-3 was unable to activate HSP70 protein synthesis in KATO III exposed to Hp. Lack of protective effect of HSP70, activation of caspase-3--dependent apoptosis pathway caused by Hp and alteration of the bax/bcl-2 cellular equilibrium led to gastric epithelial cell death. The observed phenomenon might be helpful in understanding of the mechanism of Hp related gastrointestinal tract diseasess, especially gastric cancer.

Molecular mechanism of protection against chemically and gamma-radiation induced apoptosis in human colon cancer cells.

The involvement of peroxisome proliferator-activated receptors (PPARs) in the cancer cell elimination through apoptosis is a generally accepted fact. However, some reports indicate that the activation of PPARgamma is directly responsible for carcinogenesis. Caco-2 cells, a human adenocarcinoma cells, were used as a model of colon cancer. Cell cultures (5 x 10(6) cell per dish) were pretreated for 24 h with PPAR gamma agonists ciglitazone (CI, 1 x 10(-6)M) and retinoic acid (RA, 1 x 10(-6)M) and part of the cultures were subsequently subjected to gamma-radiation (photons) with therapeutic dose of 2,5 Gy. Total cellular RNA and proteins (cytoplasmic and nuclear) were isolated 24h after cultures irradiation or 48 h after stimulation in the non irradiated part of experiment to preserve the equal growth time for all samples. gamma-Irradiation of the cells abolished nuclear translocation of PPARgamma under its agonists treatment and preserved PPARgamma in the cytoplasmic pool. But it did not affect the HSP 70 expression in response to ciglitazone and retinoic acid. Moreover, combined gamma-irradiation and CI/RA treatment of the cells changed the equilibrium between Bax and Bcl-2 mRNA to anti apoptotic state with increased expression of Bcl-2 and almost abolished expression of Bax. In conclusion, this paper provides an evidence for the anti-apoptotic action of PPARgamma agonists used along with the gamma-radiation. Moreover, it shows that the up-regulated HSP70, in response to PPARgamma agonists in gamma-irradiated cultures promotes cell survival.

The relationship between the presence of Helicobacter pylori in the oral cavity and gastric in the stomach.

There are numerous studies suggesting that inflammation of the oral cavity caused by bacteria or fungi is accompanied by gastric inflammation. This is particularly relevant in patients using complete dentures. Since the presence of H. pylori in the oral cavity can be easily discovered by bacteria culture and that in the stomach by (13)C urea breath test (UBT) and histology of gastric endoscopic biopsy samples it is reasonably to state that the majority of the patients show the presence of bacterium in oral cavity and active gastric H. pylori infection. When comparing, however, the bacteria culture originating from the oral mucosa to those from the gastric mucosa, employing molecular biology examination, such as polymerase chain reaction (PCR), we found that the oral bacteria and those originating from stomach are completely different, suggesting that H. pylori may be present only transiently in oral cavity and does not play major role in gastric H. pylori infection. Thus, oral cavity does not serve as bacterial reservoir to infect gastric mucosa. Most important finding of our study is that patients with recognized inflammation in the oral cavity in the form of stomatitis prothetica hyperplasica both fibrosa as well as papillaris showed in nearly 100% gastric H. pylori infection, usually without the presence of the same bacterium in the oral cavity, suggesting that gastric H. pylori infection affects oral mucosa at distance by some, as yet, unknown mechanism.

Helicobacter pylori inhibits expression of heat shock protein 70 (HSP70) in human epithelial cell line. Importance of Cag A protein.

Heat shock proteins (HSP) are crucial for the maintenance of cell integrity under normal cell growth and at pathophysiological conditions such as colonization of gastric mucosa by Helicobacter pylori (Hp). The effect of Hp on mRNA expression for HSP70 in the gastric epithelial cells in vitro has been little studied and remains inconclusive. In this study we attempted to determine the alterations in gene expression for HSP70 induced by two live strains of Hp in the epithelial MKN7 cells. The following Hp strains were employed; 1) Hp strain expressing cagA and vacA, and 2) cagA and vacA negative Hp strain without or with addiction of exogenous recombinant protein CagA. MKN7 cells were incubated in a standard medium RPMI 1640 supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO2 and humidified atmosphere under basal condition or in a presence of Hp (1 x 10(9) CFU per dish) without or with the recombinant CagA (10 microg/ml of RPMI 1640 medium). After 3 h, 24 h and 48 h of incubation with Hp and in some experiments with the prolonged incubation time up to 72 h, the cells were harvested, the total cellular RNA was isolated and the expression of mRNA for HSP70 was determined by RT-PCR. The incubation of the MKN cells with CagA protein alone failed to affect significantly the expression of HSP70. In contrast, the strain Hp (cagA+, vacA+) inhibited in time-dependent manner the expression of mRNA for HSP70. When the MKN7 cells were coincubated with Hp (cagA+, vacA+) and exogenous CagA, the significant inhibition of the signal intensity for HSP70 mRNA was observed at 3 h and 24 h of incubation and these effects were followed by complete disappearance of the signal for HSP70 mRNA at 48 h. The incubation of MKN7 with Hp (cagA-, vacA-) also significantly attenuated the expression of HSP70 mRNA with the most pronounced inhibitory effect observed at 72 h of incubation with this Hp strain. Addition of the recombinant CagA to Hp (cagA-, vacA-) completely suppressed the expression of HSP70 at 48 h and 72 h after the end of incubation periods. We conclude that 1) both, Hp (cagA+, vacA+) and Hp (cagA-, vacA-) inhibit expression of HSP70 in MKN7 human gastric epithelial cells independently of the presence or absence of cagA gene, and that 2) recombinant CagA protein may exert biological activity in vitro via acceleration of inhibitory effect of Hp negative for Cag A and VacA on HSP70 expression in epithelial cells infected with this bacteria.

Gastric MALT-lymphoma, gastrin and cyclooxygenases.

Malt-lymphoma, gastrin and COX-2 interaction. Low grade, mucosal associated lymphoid tissue (MALT)-lymphoma is an unique among gastric malignancies where causal involvement of Helicobacter pylori (H. pylori) infection has been proposed based on complete regression of the tumor following the eradication therapy. In this report ten primary, low-grade MALT-lymphomas have been examined before and 6 months after one week of successful eradication therapy (clarithromycin + amoxicillin + omeprazole). Gastric biopsy samples from tumor and intact antrum and corpus mucosa were obtained during endoscopy before and after eradication for assessment of expression of gastrin and gastrin receptor (CCKB-R) as well as cyclooxygenase (COX)-1 and COX-2 using RT-PCR. The gastric lumen and serum gastrin and mucosal and tumor tissue PGE2 biosynthesis were determined by RIA before and after H. pylori eradication. Eradication of H. pylori resulted in complete endoscopic and histological remission of MALT-lymphoma in 9 out of 10 patients as assessed 6 months after this eradication. Before eradication, the mRNA expression for gastrin and CCKB-R as well as mRNA expression for COX-1 and COX-2 were observed in tumor tissue and infected mucosa, while corpus mucosa expressed only CCKB-R and antrum mucosa only gastrin. Six months upon the eradication when MALT-lymphoma completely regressed both endoscopically and histologically in 9 of 10 tested subjects, the expression of gastrin and COX-2 disappeared from the former area of MALT-lymphoma tumor. Gastrin mRNA remained detectable only in antrum mucosa, CCKB-R mRNA in corpus mucosa and COX-1 mRNA both in antrum and corpus mucosa. Gastric luminal and serum gastrin levels and gastric mucosa and tumor PGE2, which were greatly elevated before eradication, became normalized after this procedure. This study demonstrates that low-grade MALT-lymphoma is linked to H. pylori infection which may promote the expression and excessive release of gastrin and COX-2 expression that could be involved in the pathogenesis of MALT-lymphoma.

Molecular basis of colorectal cancer - role of gastrin and cyclooxygenase-2.

BACKGROUND: Tumors arising in the colorectal area have worldwide distribution and concern mostly older population being attributed to genetic, dietary and hormonal factors but most recently also to infection with Helicobacter pylori (HP). Both, HP discovery and molecular biology of colorectal cancer have been recently considered as two of ten greatest advances of gastroenterology at the dawn of 3rd millenium but little information is available regarding the relationship between the HP and colorectal cancer. Since HP infection is usually accompanied by an increase in plasma level of gastrin, which is also recognized as a trophic hormone for the colonic epithelium and a potent mitogen capable to induce cyclooxygenase-2 (COX-2), we decided 1) to compare the seroprevalence of HP, its cytotoxic protein, CagA, and cytokines (TNFalpha, IL-1beta and IL-8) in colorectal cancer patients, before and after removal of cancer, with those in age- and gender-matched controls; 2) to determine the gene expression of gastrin and gastrin receptors (CCK(B)-R) in colorectal cancer tissue, 3) to assess the plasma levels and tumor tissue contents of gastrin, 4) to examine the mRNA expression of cyclooxygenase COX-1 and COX-2 cancer tissue and intact colonic mucosa. MATERIAL AND METHODS: The trial material included 80 patients with colorectal cancers and 160 age- and gender-matched controls. Anti-HP IgG, anti-CagA IgG seroprevalence and cytokine levels were estimated by ELISA tests. Gene expressions of gastrin, CCK(B)-R, COX-1, COX-2 and Bax and Bcl2 was examined using RT-PCR, while gastrin was measured by RIA. RESULTS: The HP IgG seroprevalence, especially that expressing CagA, was significantly higher in colorectal cancer patients than in controls and did not change one week after tumor resection while plasma cytokines were significantly reduced after this operation. Gastrin and CCK(B)-R mRNA were detected in the cancer tissue and the resection margin and similarly COX-2 mRNA was expressed in most of cancers and their resection margin but not in intact colonic mucosa where only COX-1 was detected. The colorectal cancer tissue contained several folds more immunoreactive gastrin than cancer resection margin and many folds more than the intact colonic mucosa. CONCLUSIONS: 1) Colorectal carcinoma and its resection margin overexpress gastrin and receptors for gastrin (CCK(B)-R), and COX-2; 2) here, we propose that an increased plasma level of gastrin should be considered as suitable biomarker of colorectal cancer, 3) HP infection may contribute to colonic cancerogenesis by enhancing expression of gastrin and COX-2, they may account for stimulation of the tumor growth, angiogenesis and reduction in apoptosis as evidenced an increased ratio of mRNA expression for anti-apoptotic Bcl2 over proapoptotic Bax proteins and 4) HP positive patients who develop colorectal cancer should be subjected to the HP eradication; this is expected to reduce hypergastrinemia and to attenuate COX-2 expression. Our final conclusion would be: treatment of patients with colorectal cancer with COX-2 selective inhibitors now gained a strong support as a preventive measure.

Cancerogenesis in Helicobacter pylori infected stomach--role of growth factors, apoptosis and cyclooxygenases.

BACKGROUND: Epidemiological and animal studies demonstrated a link between gastric cancer (GC) or mucosal associated lymphoid tissue (MALT) lymphoma and chronic infection with Helicobacter pylori (H. pylori). The exact mechanism responsible for the development of GC and MALT-lymphoma in H. pylori-infected patients still remains obscure. This report is designed to overview the molecular biology, especially the gene expression and histochemical manifestation of gastrin and other growth factors such as transforming growth factor alpha (TGF alpha) and hepatocyte growth factor (HGF) in the GC before and after eradication of H. pylori. Furthermore, gene expression of cyclooxygenase-1 (COX-1) and COX-2 and apoptosis-related proteins such as Bax and Bcl-2 are discussed. MATERIAL AND METHODS: The findings originate from two series of patients; Series I involving 337 GC patients and 400 age- and gender-matched controls and series 2 including 20 MALT-lymphoma patients and 40 matched controls. RESULTS: An overall H.pylori-seropositivity reached about 80% in GC and about 90% in MALT-lymphoma, significantly higher than in non-cancer controls (60%). The prevalence of CagA-positive strains was about twice as high (about 70%) in GC and MALT-lymphomas as in sex- and age-matched controls. Expression of gastrin was detected in antrum of all tested patients but also in majority (90%) of GCs and MALT-lymphomas tumor tissue. HGF and TGF alpha were expressed more frequently in GC tissue than in normal fundic mucosa. COX-1 was similarly expressed in GC and MALT as in intact mucosa, while COX-2 mRNA was detected only in tumor tissue, being attenuated by H.pylori eradication in GC and abolished by this therapy in MALT-lymphoma. The plasma levels of alpha-amidated gastrin in GC and MALT were several folds higher than in controls. Gene expression of bcl-2 was detected in all, while bax--only in about 50% of GC samples. CONCLUSIONS: Infection with H. pylori, especially that expressing CagA-positivity, is primum movens in developing GC and MALT-lymphoma and the upregulation of growth factors, particularly of gastrin, and COX-2 and dysregulation of the Bax/Bcl-2 system seem to contribute to gastric cancerogenesis.

Helicobacter pylori infection, gastrin, cyclooxygenase-2, and apoptosis in colorectal cancer.

Helicobacter pylori (HP) infection is usually accompanied by an increased plasma level of gastrin, a potent mitogen able to induce cyclooxygenase (COX)-2. This study examined (a) the seroprevalence of HP, its cytotoxic protein, CagA, and cytokines (tumor necrosis factor alpha, interleukins 1beta and 8) in 80 patients with colorectal cancers, before and after the removal of tumor, compared with 160 age- and gender-matched controls; (b) the gene expression of gastrin and its receptors (CCKB-R) in the cancer tissue, (c) the plasma levels and tumor tissue contents of gastrin, and (d) the mRNA expression of COX-1, COX-2, and apoptotic proteins (Bax and Bcl2) in cancer tissue and intact colonic mucosa. Anti-HP IgG, anti-CagA IgG seroprevalence, and cytokine levels were analyzed by enzyme-linked immunosorbent assay tests; gene expressions of gastrin, CCKB-R, COX-1, COX-2, Bax, and Bcl2 by reverse transcriptase polymerase chain reaction; and gastrin by radioimmunoassay. The seroprevalence of HP, especially that expressing CagA, was significantly higher in cancer patients than in controls and did not change 1 week after tumor resection while plasma cytokines were significantly reduced after this operation. Both gastrin and CCKB-R mRNA were detected in the cancer tissue and the resection margin; similarly, COX-2 mRNA was expressed in most of cancers and their resection margin but not in intact colonic mucosa, where only COX-1 was detected. The colorectal cancer tissue contained several folds more immunoreactive gastrin than cancer resection margin and many folds more than the intact colonic mucosa. We conclude that colon adenocarcinoma and its resection margin overexpress gastrin, its receptors, CCKB-R, and COX-2, and that HP infection may contribute to colonic cancerogenesis via overexpression of gastrin and COX-2, which may account for the stimulation of the tumor growth and the reduction in apoptosis as documented by enhanced mRNA expression of anti-apoptotic Bcl2 over proapoptotic Bax proteins.

Central leptin and cholecystokinin in gastroprotection against ethanol-induced damage.

BACKGROUND: Leptin, a product of the ob gene controlling food intake, has recently been detected in the stomach and shown to be released by cholecystokinin (CCK) and to induce gastroprotection against various noxious agents, but it is not known whether centrally applied leptin influences gastric secretion and mucosal integrity. AIMS: In this study we compared the effects of leptin and CCK-8 applied intracerebroventricularly (i.c.v.) on gastric secretion and gastric mucosal lesions induced by topical application of 75% ethanol. METHODS: Several major series of Wistar rats were used in this study. The effects of leptin or CCK applied i.c.v. on gastric secretion were examined using conscious rats with gastric fistulas. For the studies on gastroprotection the following series of rats were used to determine the effects of: (A) leptin and CCK applied centrally on this protection and the blockade of CCK(A) with loxiglumide (30 mg/kg i.p.) and CCK(B) receptors with RPR 102681 (30 mg/kg i.p.); (B) cutting of vagal nerves; (C) inactivation of sensory nerves by capsaicin (125 mg/kg s.c.); (D) inhibition of calcitonin gene-related peptide (CGRP) receptors with CGRP(8-37) (100 microg/kg i.p.), and (E) suppression of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v. ) on ethanol-induced gastric lesions in rats with or without the i.c.v. pretreatment with leptin or CCK-8. Rats were anesthetized 1 h after ethanol administration to measure the gastric blood flow (GBF) and then to determine the area of gastric lesions by planimetry. Blood was withdrawn for the measurement of plasma leptin and gastrin levels by radioimmunoassay and gastric biopsy samples were collected for the determination of cNOS and iNOS mRNA by RT-PCR. RESULTS: Leptin and CCK-8 (0.01-5 microg/kg i.c.v.) dose dependently attenuated gastric lesions induced by 75% ethanol; the doses reducing these lesions by 50% (ED(50)) were 0.8 and 1.2 microg/kg, respectively. The protective effects of leptin and CCK-8 applied i.c. v. were accompanied by a significant rise in plasma leptin level and an increase in GBF. Blockade of CCK(A) receptors with loxiglumide abolished the protective and hyperemic effects of CCK but not those of leptin, while RPR 10268, a specific antagonist of CCK(B) receptors, counteracted leptin-induced protection and the rise in the GBF but failed to influence those afforded by CCK-8. For comparison, pretreatment with peripheral CCK-8 or leptin (10 microg/kg i.p.) causing a similar rise in the plasma leptin level also significantly reduced gastric lesions induced by 75% ethanol. The protective and hyperemic effects of centrally administered leptin were abolished by vagotomy, producing a fall in plasma leptin levels, and significantly attenuated by sensory denervation with capsaicin, by pretreatment with the CGRP antagonist, CGRP(8-37), or with L-NAME. A strong signal for iNOS mRNA was recorded in the gastric mucosa of leptin- and CCK-8-treated animals, whereas cNOS mRNA was unaffected. CONCLUSIONS: (1) Central leptin exerts a potent gastroprotective action at a dose that has no influence on gastric secretion; (2) this protection depends upon CCK(B) receptors, vagal activity and sensory nerves, and involves hyperemia probably mediated by NO, and (3) leptin mimics the gastroprotective effect of CCK and may be implicated in the protective and hyperemic actions of this peptide on the rat stomach.

Helicobacter pylori-gastrin link in MALT lymphoma.

BACKGROUND: There is accumulating evidence for the role of Helicobacter pylori in the development of gastric cancer as well as of lymphomas that arise in mucosa-associated lymphoid tissue (MALT). We reported recently that gastric cancer patients show high prevalence of cagA-positive H. pylori and express gastrin and gastrin receptors enabling them to stimulate tumour growth in autocrine fashion. AIMS: Since the H. pylori infection is considered to be more strongly associated with MALT lymphoma than with gastric cancer, we decided to determine the gastrin and its receptors' mRNA expression and gastrin content in this tumour as well as the release of this hormone both into plasma and gastric lumen. Twenty MALT lymphoma patients were compared with 100 age- and gender-matched controls with similar dyspeptic symptoms. RESULTS: The overall H. pylori seropositivity in MALT lymphoma was about 90% and CagA positivity was 70%, compared to 56% and 33%, respectively, in controls. The serum gastrin in MALT lymphoma was about sixfold higher than in controls while gastric luminal gastrin in these patients was over 70 times higher than in controls. Gastrin content in tumour was about 10-fold higher than in antral mucosa. Gastrin and gastrin-receptor (CCKB-receptor) mRNA were detected by reverse transcriptase-polymerase chain reaction in cancer tissue whilst in the fundic and antral mucosa, only enhanced expression of CCKB-receptor mRNA and gastrin mRNA was detected, respectively. Histamine stimulation in MALT lymphoma induced acid secretion that was only about 30% of control value due to atrophic gastritis. This study confirms an important role of CagA-positive H. pylori in the pathogenesis of MALT lymphoma and shows that this lymphoma is capable of synthesizing and releasing potent growth promoting gastrin, possibly due to the action on G-cells of H. pylori-originated Nalpha-methyl histamine and cytokines (tumour necrosis factor alpha and interleukin-8). CONCLUSIONS: Gastric MALT lymphoma is closely linked to CagA-positive H. pylori infection. Gastrin and its receptors may be implicated in the pathogenesis of gastric lymphoma.

Helicobacter pylori, gastrin and cyclooxygenases in gastric cancer.

BACKGROUND: Tumors arising in the stomach have worldwide distribution and the infection with Helicobacter pylori (HP) has been implicated in causation of this disease. The HP discovery, which is considered as the greatest advance of gastroenterology at the dawn of 3rd millennium, is accompanied by hypergastrinemia, which seems to play a key role in gastric cancerogenesis but no study was undertaken to assess the relationship between the HP infection and coexpression of gastrin and cyclooxygenases (COX), the rate limiting enzymes in the eicosanoids production. AIMS: Since gastrin is recognized as a effective gastric mitogen, it could be capable to induce COX-2, a potent tumor growth promoting and angiogenic factor, we decided 1) to compare the seroprevalence of HP and its cytotoxic protein, CagA, in gastric cancer patients with those in age- and gender-matched controls; 2) to determine the gene expression of gastrin and its receptors (CCK(B)-R) in gastric cancer, 3) to assess the plasma levels, gastric lumen and tumor tissue contents of gastrin and 4) to examine the mRNA and enzyme protein expression of COX-1 and COX-2 in cancer tissue and intact gastric mucosa before and after HP eradication. MATERIAL AND METHODS: The trial material included 20 patients with gastric cancers and 100 age- and gender-matched controls. Anti-HP and anti-CagA IgG seroprevalence was estimated by specific antisera using ELISA tests. Gene expressions of gastrin, CCK(B)-R, COX-1 and COX-2 was examined using RT-PCR with GAPDH as a reference and employing Western blot for COX-2 expression, while gastrin was measured by RIA. RESULTS: The seroprevalence of HP, especially that expressing CagA, was significantly higher in gastric cancers than in controls. Both gastrin and CCK(B)-R mRNA were detected by RT-PCR in the cancer tissue and similarly COX-2 mRNA and protein were found in most of cancers and in the HP infected antral mucosa but not in HP eradicated patients in whom only cancer tissue but not gastric mucosa expressed COX-2. The gastric cancer tissue contained 20 times more of immunoreactive gastrin than the HP infected antral gastric mucosa and following HP eradication the gastrin content in the tumor and antrum showed a marked and significant reduction. No significant change in CCK(B)-R expression was noticed before and after HP eradication in the tumor and the corpus mucosa. CONCLUSIONS: 1). Gastric carcinoma coexpresses gastrin, its receptors (CCK(B)-R), and COX-2; 2) HP infection may contribute to gastric cancerogenesis via gastrin andCOX-2 that may account for the stimulation of tumor growth, angiogenesis, and reduction in apoptosis 3) HP positive patients developing gastric cancer should be considered for HP eradication to reduce the HP provoked hypergastrinemia and COX-2 overexpression in the tumor tissue.

Leptin in gastroprotection induced by cholecystokinin or by a meal. Role of vagal and sensory nerves and nitric oxide.

Leptin, detected recently in the stomach, is a product of the ob gene released by cholecystokinin (CCK) and plays an important role in the control of food intake but its influence on gastroprotection against the damage caused by noxious agents has not been studied. This study was designed to compare the effects of leptin and cholecystokinin-8 (CCK-8) on gastric mucosal lesions induced by topical application of 75% ethanol or acidified aspirin. Four series of Wistar rats (A, B, C and D) were used to determine the effects of: (A) suppression of prostaglandin biosynthesis by indomethacin (5 mg/kg i.p.); (B) inhibition of nitric oxide (NO)-synthase by nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.v.); (C) blockade of sensory nerves by capsaicin (125 mg/kg s.c.) and (D) bilateral vagotomy, on the gastric lesions induced by intragastric (i.g.) application of ethanol with or without pretreatment with CCK-8, a known gastroprotective substance or leptin. CCK-8 (1-100 microg/kg i.p.) and leptin (0.1-50 microg/kg i.p.) dose dependently attenuated gastric lesions induced by 75% ethanol; the dose reducing these lesions by 50% being about 10 microg/kg and 8 microg/kg, respectively. The protective effects of CCK-8 and leptin were accompanied by a significant rise in gastric blood flow (GBF) and luminal NO concentration. Leptin was also effective to attenuate aspirin-induced damage and the accompanying fall in the GBF, whereas CCK-8 dose dependently worsened aspirin damage and failed to influence GBF. CCK (1-100 microg/kg i.p.), given in graded doses, produced a dose-dependent increase in the plasma leptin level and a rise of the expression of ob messenger RNA (mRNA) in gastric mucosa, the maximum being reached at a dose of 100 microg/kg. Pretreatment with CCK-8 (10 microg/kg i.p.) or with 8% peptone, that is known to stimulate CCK release, also produced a significant rise in plasma leptin levels and up-regulation of ob mRNA while reducing significantly the gastric lesions induced by 75% ethanol to the same extent as that induced by exogenous leptin (10 microg/kg i.p.). Indomethacin, which suppressed prostaglandin generation by approximately 90%, failed to influence leptin- or CCK-8-induced protection against ethanol, whereas L-NAME attenuated significantly CCK-8- and leptin-induced protection and hyperemia but addition to L-NAME of L-arginine, but not D-arginine, restored the protective and hyperemic effects of both hormones. The ob mRNA was detected as a weak signal in the intact gastric mucosa and in that exposed to ethanol alone but this was further enhanced after treatment with graded doses of CCK-8 or peptone meal applied prior to ethanol. We conclude that: (1) exogenous leptin or that released endogenously by CCK or meal exerts a potent gastroprotective action depending upon vagal activity, and involving hyperemia probably mediated by NO and sensory nerves but unrelated to endogenous prostaglandins; (2) leptin mimics the gastroprotective effect of CCK and probably mediates the protective and hyperemic actions of CCK in the rat stomach.

Helicobacter pylori induces apoptosis in gastric mucosa through an upregulation of Bax expression in humans.

BACKGROUND: Maintenance of gastric mucosal integrity depends on the balance between cell loss due to apoptosis and cell proliferation. Helicobacter pylori induces apoptosis in gastric epithelial cells, but the regulation of this process has been little studied. The Bcl-2 proteins are the best-studied family of proteins involved in the mechanism of apoptotic death. Some members of this family, such as Bcl-2, inhibit apoptosis, whereas others, such as Bax, induce it. The present study was performed to determine the apoptosis rate and mRNA and protein expression for Bax and Bcl-2 in the gastric mucosa of duodenal ulcer (DU) patients with H. pylori infection before and after H. pylori eradication. We recruited 8 H. pylori-negative control subjects and 20 DU patients (H. pylori-positive) given a 1-week triple therapy to eradicate H. pylori. The apoptosis was analyzed by means of terminal deoxyribonucleotide transferase-mediated digoxigenin-11-deoxyuridine triphosphate biotin nick-end labeling (TUNEL) staining, and the expression of mRNA for Bax and Bcl-2 by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot. In all patients gastritis was assessed histologically on the basis of the Sydney classification, the presence of H. pylori, and analysis of cagA status. RESULTS: All 20 DU patients were H. pylori-positive, and 18 (90%) were CagA-positive. The apoptotic cells were infrequently identified in gastric surface epithelium by TUNEL histochemistry in H. pylori-negative controls. In DU patients infected with H. pylori, apoptotic cells were more numerous and seen deep in the gastric glands. The infection was associated with significantly upregulated expression of mRNA and protein for Bax and suppressed mRNA and protein expression for Bcl-2, as determined using RT-PCR and Western blot analysis. The Bax overexpression was significantly stronger in the antrum than in the corpus of H. pylori-infected patients. Four weeks after the eradication a marked decrease of neutrophil infiltration, an improvement of the grade of gastritis (mononuclear infiltration), and significant reduction in apoptosis rate were observed. After eradication the Bax mRNA expression was still at an increased level, whereas the Bcl-2 mRNA expression remained suppressed. CONCLUSIONS: 1) H. pylori induces apoptosis in the gastric epithelium, at least in part, due to an upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2, and 2) Bax mRNA and protein expression was higher in the antrum than in the corpus, and this was probably due to greater inflammatory changes observed in the antrum than in the corpus.