Olorunseun O. Ogunwobi

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.

Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways

Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from chemical transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined and functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics. 1MEA cells express and secrete more HGF than BNL cells. There was significantly decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1, vimentin, snail and slug in 1MEA cells. There was also increased expression of cyclooxygenase-2 (COX-2), Akt and phosphorylated Akt (pAkt) in 1MEA cells. Moreover, 1MEA cells had increased migratory capacity inhibited by inhibition of COX-2 and Akt but not extracellular signal regulated kinase (ERK). Molecular mesenchymal characteristics of 1MEA cells were reversed by inhibition of COX-2, Akt and ERK. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, snail, slug, COX-2, Akt, pAkt and increased migration, invasiveness and clonogenicity. We conclude that development of HCC is associated with upregulation of HGF which promotes EMT and carcinogenesis via upregulation of COX-2 and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC.

Epigenetic Upregulation of HGF and c-Met Drives Metastasis in Hepatocellular Carcinoma

Hepatocyte growth factor (HGF) and its receptor, c-Met, are important regulators of growth and differentiation of healthy hepatocytes. However, upregulation of HGF and c-Met have been associated with tumor progression and metastasis in hepatocellular carcinoma (HCC). Hematogenous dissemination is the most common route for cancer metastasis, but the role of HGF and c-Met in circulating tumor cells (CTCs) is unknown. We have isolated and established a circulating tumor cell line from the peripheral blood of a mouse HCC model. Our studies show that these CTCs have increased expression of HGF and c-Met in comparison to the primary tumor cells. The CTCs display phenotypic evidence of epithelial-mesenchymal transition (EMT) and the EMT appears to be inducible by HGF. Epigenetic analysis of the c-Met promoter identified significant loss of DNA methylation in CTCs which correlated with overexpression of c-Met and increased expression of HGF. Six specific CpG sites of c-Met promoter demethylation were identified. CTCs show significantly increased tumorigenicity and metastatic potential in a novel orthotopic syngeneic model of metastatic HCC. We conclude that during hematogenous dissemination in HCC, CTCs undergo EMT under the influence of increased HGF. This process also involves up regulation of c-Met via promoter demethylation at 6 CpG sites. Consequently, targeting HGF and c-Met expression by CTCs may be a novel non-invasive approach with potential clinical applications in HCC management.

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.

Cyclooxygenase-2 and Akt mediate multiple growth-factor-induced epithelial-mesenchymal transition in human hepatocellular carcinoma

Background and Aim:  Cancer invasion and metastasis are characterized by epithelial‐mesenchymal transition (EMT). Hepatocellular carcinoma (HCC) causes metastasis and significant mortality. Elucidating factors promoting EMT in HCC are necessary to develop effective therapeutic strategies.

Intrahepatic Cholangiocarcinoma Progression: Prognostic Factors and Basic Mechanisms

In this review, we will examine various molecular biomarkers for their potential to serve as independent prognostic factors for predicting survival outcome in postoperative patients with progressive intrahepatic cholangiocarcinoma. Specific rodent models of intrahepatic cholangiocarcinoma that mimic relevant cellular, molecular, and clinical features of the human disease are also described, not only in terms of their usefulness in identifying molecular pathways and mechanisms linked to cholangiocarcinoma development and progression, but also for their potential value as preclinical platforms for suggesting and testing novel molecular strategies for cholangiocarcinoma therapy. Last, recent studies aimed at addressing the role of desmoplastic stroma in promoting intrahepatic cholangiocarcinoma progression are highlighted in an effort to underline the potential value of targeting tumor stromal components together with that of cholangiocarcinoma cells as a novel therapeutic option for this devastating cancer.

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

Therapeutic and prognostic importance of epithelial–mesenchymal transition in liver cancers: Insights from experimental models

Hepatocellular carcinoma (HCC) and hepatic metastases of colon cancers are malignant conditions of the liver that cause significant morbidity and mortality worldwide. Experimental models suggest that both conditions are characterized by epithelial-mesenchymal transition (EMT). Whilst ongoing research efforts aim to definitively clarify its role in human cancer patients, data from experimental models have unraveled a number of potential therapeutic targets as well as markers of prognostic importance. This area of research is generating intense interest amongst both basic scientists and clinicians. Some questions have been answered, but many important issues remain unresolved. We expect that in the near future, studies of human tissues can definitively clarify the role of EMT in the development and progression of human malignant diseases of the liver and that further studies can be carried out to determine how best to target aspects of the process for the treatment of patients with hepatocellular carcinoma and hepatic metastases of colon cancers.