Ian L.P. Beales

Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells

Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1beta-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-kappaB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-kappaB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.

Statins Inhibit Proliferation and Induce Apoptosis in Barrett's Esophageal Adenocarcinoma Cells

OBJECTIVES:The incidence and mortality rates from esophageal adenocarcinoma (EAC) are rapidly increasing in the western world. Chemoprevention is being advocated to reduce the burden of disease. Statins are used clinically to treat hypercholesterolemia, and have an excellent safety profile. Statins reduce the intracellular availability of several biosynthetic intermediates important in intracellular signaling. We hypothesized that statins may effect EAC proliferation or apoptosis.METHODS:The OE33 and BIC-1 EAC cell lines and simvastatin, lovastatin, and pravastatin were studied. Proliferation was quantified by thiazoyl blue colormetric and bromodeoxyuridine incorporation assays. Apoptosis was determined using assays for intracellular nucleosomes and caspase-3 activity. Detection of phosphorylated kinases, affinity precipitation, immunoblotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to determine the effects on intracellular signaling.RESULTS:All three statins reduced viable cell number and inhibited proliferation in a similar dose-dependent manner. Statins induced apoptosis and enhanced the antiproliferative effect of NS-398, a selective cyclooxygenase (COX)-2 inhibitor. The effects were dependent on farnesylation, but not geranylgeranylation, of intracellular targets, and statins reduced serum-stimulated Ras activity . Simvastatin inhibited activation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) but not c-Jun NH2-terminal kinase or p38 mitogen-activated protein (MAP) kinase. Statin treatment increased messenger RNA (mRNA) and protein expression of the proapoptotic proteins Bax and Bad, but protein levels of the antiapoptotic proteins B-cell lymphoma (Bcl)-2 and Bcl-XL were unchanged.CONCLUSIONS:Statins inhibit proliferation and induce apoptosis in EAC cells via inhibition of Ras farnesylation and inhibition of the ERK and Akt signaling pathways. Statins may have some potential as chemopreventative and adjuvant chemotherapeutic agents in EAC.

The anti-apoptotic and growth stimulatory actions of leptin in human colon cancer cells involves activation of JNK mitogen activated protein kinase, JAK2 and PI3 kinase/Akt

Background and aimsObesity is a major risk factor for the development of colon cancer. Secretion of the hormone leptin from adipocytes is increased in obesity, and serum levels are proportional to body fat mass. Serum leptin levels are an independent risk factor for colon cancer. Leptin receptors are expressed in normal, premalignant and malignant colonic epithelia. We have investigated the effects of leptin on proliferation and apoptosis of colonic cancer cells and the early signalling events involved.MethodsProliferation of HT-29 colon cancer cells in response to leptin was assessed by 3-[4, 5-dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was quantified by enzyme-linked immunosorbent assay (ELISA) for intracellular nucleosomes. Signalling pathways involved were determined by using specific inhibitors, quantification of phosphorylated active intermediates and ELISA of active nuclear-translocated transcription factors.ResultsLeptin stimulated HT-29 cell proliferation and inhibited both serum-starvation and celecoxib-induced apoptosis. The proliferative and anti-apoptotic effects of leptin were abolished by inhibition of JAK2 with AG490, phosphatidylinositol 3′-kinase (PI3 kinase) with LY294002 and c-Jun NH2-terminal kinase (JNK) with SP600125. Stimulation of HT-29 cells with leptin increased phosphorylation of JAK2, Akt and JNK. Activation of JAK2 was upstream of PI3 kinase/Akt but not of JNK. Activation of JAK2 was followed by activation and nuclear translocation of STAT3 and JNK activation led to increased activator protein 1 (AP-1) transcriptional activity.ConclusionsLeptin stimulates proliferation and inhibits apoptosis in human colon cancer cells and may be an important factor in the increased incidence of colon cancer in obesity. This effect involves JAK2, PI3 kinase and JNK and activation of the oncogenic transcription factors signal transducer and activator of transcription (STAT)3 and AP-1.

Efficacy of Helicobacter pylori eradication therapies: a single centre observational study

BackgroundMany Helicobacter pylori eradication regimens have been described. There are little data reporting their efficacy or integration in routine clinical practice. The overall results of eradication therapy in a cohort of patients are described and an algorithm for management outlined.Methods469 patients receiving eradication therapy in routine clinical practice were evaluated. The successes of individual regimes as first, second and third line therapy were determined.ResultsOverall success after one, two and three courses of therapy were 73% (95% confidence intervals 69–77%), 94% (91–96%) and 98% (97–99%) respectively. 10 different regimens, including many non-recommended ones were used as primary therapy. Ranitidine bismuth citrate-amoxicillin-clarithromycin triple therapy (94.8%, 90–99%) was significantly more effective than any other combination as primary therapy, including all proton pump inhibitor based triple therapies. Quadruple therapy with bismuth chelate-proton pump inhibitor-tetracycline and a nitroimidazole (70%, 52–88%) and ranitidine bismuth citrate-based triple therapy (73%, 56–90%) where more effective second line combinations than proton pump inhibitor-triple therapies (37.5%, 12–58%). Third line therapy directed by the results of sensitivity testing improved eradication compared to further empirical antibiotics. The use of a proton pump inhibitor with clarithromycin and a nitroimidazole as initial therapy was associated with a significantly worse overall eradication rate than other combinations.ConclusionsHelicobacter pylori eradication rates can be maximised by using ranitidine bismuth citrate-clarithromycin-amoxicillin containing triple therapy, followed by bismuth and nitroimidazle containing second-line therapy, with third line combinations directed by sensitivity testing. Proton pump inhibitor-clarithromycin-metronidazole combinations should be avoided.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

Background & Aims Barretts esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barretts and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. Methods We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. Results We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09–1.18; P = 1.8 × 10−11) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86–0.93; P = 7.5 × 10−9). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87–0.93; P = 3.72 × 10−9). Conclusions We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Leptin synergistically enhances the anti-apoptotic and growth-promoting effects of acid in OE33 oesophageal adenocarcinoma cells in culture.

Obesity and gastro-oesophageal reflux are the main predisposing factors for oesophageal adenocarcinoma. We have examined the effects of transient acid exposure and leptin on OE33 oesophageal adenocarcinoma cells. Leptin and acid individually stimulated proliferation and inhibited apoptosis and the combination was synergistic. Leptin receptor protein levels were unchanged by acid exposure. The COX-2 inhibitor NS 398 blocked the effects of acid and leptin but while both acid and leptin individually significantly increased PGE2 production and COX-2 mRNA levels, the combination was not more effective than either stimulant alone. Leptin synergistically enhanced acid-stimulated EGFR and ERK phosphorylation but did not further increase JNK or p38 MAP kinase phosphorylation. Specific EGFR and ERK inhibitors reduced the effects of leptin and acid alone and in combination. The combination of increased circulating leptin levels in obesity and transient reflux of gastric acid may promote oesophageal carcinogenesis by increasing proliferation and inhibiting apoptosis.

Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

BackgroundHelicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study.MethodsAGS cells were cultured with IL-1β. DNA synthesis was assed by [3H]thymidine incorporation and total viable cell numbers by MTT assay.ResultsIL-1β dose dependently increased DNA synthesis and cell numbers. The enhanced proliferation was blocked by interleukin-1 receptor antagonist. Addition of neutralising antibody to GM-CSF reduced IL-1β-stimulated proliferation by 31 ± 4 %. GM-CSF alone significantly stimulated proliferation. Addition or neutralisation of IL-8 had no effect on basal or IL-1β-stimulated proliferation. The tyrosine kinase inhibitor genistein completely blocked IL-1β-stimulated proliferation and inhibition of the extracellular signal related kinase pathway with PD 98059 inhibited IL-1β stimulated proliferation by 58 ± 5 %.ConclusionsIL-1β stimulates proliferation in gastric epithelial cells. Autocrine stimulation by GM-CSF contributes to this proliferative response. Signalling via tyrosine kinase activity is essential to the mitogenic response to IL-1β. The extracellular signal related kinase pathway is involved in, but not essential to downstream signalling. IL-1β may contribute to the hyperproliferation seen in H. pylori- infected gastric mucosa, and be involved in the carcinogenic process.

Regular statin and aspirin use in patients with Barrett's oesophagus is associated with a reduced incidence of oesophageal adenocarcinoma.

Background Oesophageal adenocarcinoma (OAC) has a poor prognosis, and chemoprevention is an attractive option to reduce the burden of the disease. Hydroxyl-methyl-CoA reductase inhibitors (statins) have been shown to exert potentially useful anticancer effects against OAC cell lines, but there are only limited clinical data examining the effects of statins on the incidence of OAC. Aim To examine the association between statin use and the incidence of OAC. Methods We have carried out a case–control study comparing statin use between patients with an incident diagnosis of OAC and controls with nonprogressive Barrett’s oesophagus. Eighty-five cancer cases were compared with 170 age-matched and sex-matched controls. Risk factors and drug exposure were established using standardized interviews. Logistic regression was used to compare statin exposure and correct for confounding factors. Results Regular statin use was associated with a significantly lower incidence of OAC [uncorrected odds ratio (OR) 0.45, 95% confidence intervals 0.24–0.84)]. After correction for confounding variables including aspirin and NSAID use, statin use was still associated with a reduced incidence of OAC (OR 0.57, 0.28–0.94). Longer duration of statin use and higher doses were both associated with a significantly greater reduction in OAC. The combination of regular statin and aspirin use was associated with a significantly further reduced incidence of OAC (OR 0.31, 0.04–0.69). Conclusion In this observational case–control study, the regular use of statins was associated with a reduced incidence of OAC. The chemopreventative actions of statins, especially in conjunction with aspirin, deserve further study.

Activation of Akt is increased in the dysplasia-carcinoma sequence in Barrett's oesophagus and contributes to increased proliferation and inhibition of apoptosis: a histopathological and functional study

BackgroundThe incidence of oesophageal adenocarcinoma is increasing rapidly in the developed world. The serine-threonine protein kinase and proto-oncogene Akt has been reported to regulate proliferation and apoptosis in several tissues but there are no data on the involvement of Akt in oesophageal carcinogenesis. Therefore we have examined the activation of Akt in Barretts oesophagus and oesophageal adenocarcinoma and the functional effects of Akt activation in vitro.MethodsExpression of total and active (phosphorylated) Akt were determined in endoscopic biopsies and surgical resection specimens using immunohistochemistry. The functional effects of Akt were examined using Barretts adenocarcinoma cells in culture.ResultsIn normal squamous oesophagus, erosive oesophagitis and non-dysplastic Barretts oesophagus, phospho-Akt was limited to the basal 1/3 of the mucosa. Image analysis confirmed that Akt activation was significantly increased in non-dysplastic Barretts oesophagus compared to squamous epithelium and further significantly increased in high-grade dysplasia and adenocarcinoma. In all cases of high grade dysplasia and adenocarcinoma Akt was activated in the luminal 1/3 of the epithelium. Transient acid exposure and the obesity hormone leptin activated Akt, stimulated proliferation and inhibited apoptosis: the combination of acid and leptin was synergistic. Inhibition of Akt phosphorylation with LY294002 increased apoptosis and blocked the effects of acid and leptin both alone and in combination. Activation of Akt was associated with downstream phosphorylation and deactivation of the pro-apoptotic protein Bad and phosphorylation of the Forkhead family transcription factor FOXO1.ConclusionAkt is abnormally activated in Barretts oesophagus, high grade dysplasia and adenocarcinoma. Akt activation promotes proliferation and inhibits apoptosis in Barretts adenocarcinoma cells and both transient acid exposure and leptin stimulate Akt phosphorylation. Downstream targets of Akt include Bad and Forkhead transcription factors. Activation of Akt in obesity and by reflux of gastric acid may be important in the pathogenesis of Barretts adenocarcinoma

Clinical trial: oral prednisolone metasulfobenzoate (Predocol) vs. oral prednisolone for active ulcerative colitis

Background  Systemic corticosteroids are effective in ulcerative colitis but commonly cause side effects.