Cyclooxygenase/lipoxygenase shunting lowers the anti-cancer effect of cyclooxygenase-2 inhibition in colorectal cancer cells.

BACKGROUND: Arachidonic acid metabolite, generated by cyclooxygenase (COX), is implicated in the colorectal cancer (CRC) pathogenesis. Inhibiting COX may therefore have anti-carcinogenic effects. Results from use of non-steroidal anti-inflammatory drugs inhibiting only COX have been conflicting. It has been postulated that this might result from the shunting of arachidonic acid metabolism to the 5-lipoxygenase (5-LOX) pathway. Cancer cell viability is promoted by 5-LOX through several mechanisms that are similar to those of cyclooxygenase-2 (COX-2). Expression of 5-LOX is upregulated in colorectal adenoma and cancer. The aim of this study was to investigate the shunting of arachidonic acid metabolism to the 5-LOX pathway by cyclooxygenase inhibition and to determine if this process antagonizes the anti-cancer effect in colorectal cancer cells. METHODS: Three colorectal cancer cell lines (HCA7, HT-29 & LoVo) expressing 5-LOX and different levels of COX-2 expression were used. The effects of aspirin (a non-selective COX inhibitor) and rofecoxib (COX-2 selective) on prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) secretion were quantified by ELISA. Proliferation and viability were studied by quantifying double-stranded DNA (dsDNA) content and metabolic activity. Apoptosis was determined by annexin V and propidium iodide staining using confocal microscopy, and caspase-3/7 activity by fluorescent substrate assay. RESULTS: COX inhibitors suppressed PGE2 production but enhanced LTB4 secretion in COX-2 expressing cell lines (P <0.001). The level of COX-2 expression in colorectal cancer cells did not significantly influence the anti-proliferative and pro-apoptotic effects of COX inhibitors due to the shunting mechanism. CONCLUSIONS: This study provides evidence of shunting between COX and 5-LOX pathways in the presence of unilateral inhibition, and may explain the conflicting anti-carcinogenic effects reported with use of COX inhibitors.

The relationship between smoking and severe dysplastic disease in patients with Barrett's columnar-lined oesophagus.

The aim of this study was to examine the relationship between smoking and oesophageal high-grade dysplasia (HGD) or adenocarcinoma (AC) in a large cohort of patients with Barrett's columnar-lined oesophagus (CLO). A total of 1280 patients diagnosed with CLO and registered with the UK National Barrett's Oesophagus Registry were included. Data, including smoking habits, were collected from the patient's notes and development of HGD or AC noted. Analysis was performed with SPSS using logistic regression for calculation of odds ratios (ORs) for development of HGD/AC. Data on smoking habits were available in 956 (74.6%) patients. There was no significant difference between smokers and nonsmokers in mean age (P=0.877) or length of follow-up (P=0.359). There was a significant risk of HGD/AC in patients with any history of smoking compared with those who had never smoked (P<0.001, OR 2.81). Ex-smokers of 10 years or more remained at a significantly higher risk of HGD/AC compared with those who had never smoked (P=0.001, OR 3.37). Current smokers were not at a significantly higher risk of HGD/AC compared with ex-smokers (P=0.857) nor were those who smoked at least 20 a day compared with those who smoked fewer than 20 a day (P=0.632). In patients with CLO, smoking appears to be a significant risk factor for the development of severe dysplastic disease; however, we did not observe a dose-dependent effect of smoking on progression of disease.

Endothelin-1 stimulates colon cancer adjacent fibroblasts.

Endothelin-1 (ET-1) is produced by and stimulates colorectal cancer cells. Fibroblasts produce tumour stroma required for cancer development. We investigated whether ET-1 stimulated processes involved in tumour stroma production by colonic fibroblasts. Primary human fibroblasts, isolated from normal tissues adjacent to colon cancers, were cultured with or without ET-1 and its antagonists. Cellular proliferation, migration and contraction were measured. Expression of enzymes involved in tumour stroma development and alterations in gene transcription were determined by Western blotting and genome microarrays. ET-1 stimulated proliferation, contraction and migration (p < 0.01 v control) and the expression of matrix degrading enzymes TIMP-1 and MMP-2, but not MMP-3. ET-1 upregulated genes for profibrotic growth factors and receptors, signalling molecules, actin modulators and extracellular matrix components. ET-1 stimulated colonic fibroblast cellular processes in vitro that are involved in developing tumour stroma. Upregulated genes were consistent with these processes. By acting as a strong stimulus for tumour stroma creation, ET-1 is proposed as a target for adjuvant cancer therapy.

Pretreatment with insulin-like growth factor I protects skeletal muscle cells against oxidative damage via PI3K/Akt and ERK1/2 MAPK pathways.

Oxidative stress has an important role in the pathogenesis of many muscle diseases. The major contributors to oxidative stress in muscle tissue are reactive oxygen species such as oxygen ions, free radicals, and peroxides. Insulin-like growth factor I (IGF-I) has been shown to increase muscle mass and promote muscle cell proliferation, differentiation, and survival. We, therefore, hypothesized that IGF-I might also be cytoprotective for muscle cells during oxidative stress. Exogenous hydrogen peroxide (H(2)O(2)) was used to induce oxidative stress/damage in two types of skeletal muscle cells. Apoptotic pathways were assessed after the oxidative damage and the effects of IGF-I on oxidative stress in muscle cells were examined. Different IGF-I sub-pathways were analyzed with measurement of the expression of pro-and anti-apoptotic proteins. It was found that H(2)O(2) diminishes muscle cell viability and induces a caspase-independent apoptotic cell death. Pretreatment with IGF-I protects muscle cells from H(2)O(2)-induced cell death and enhances muscle cells survival. This effect appears to result from the promotion of the anti-apoptotic protein, Bcl2. Further investigation shows that protection is via an IGF-I sub-pathway: PI3K/Akt and ERK1/2 MAPK pathways. Protecting muscle cells from oxidative damage presents a potential application in the treatment of the muscle wasting, which appears in many muscle pathologies including Duchenne muscle dystrophy and sarcopenia.

Ischemic preconditioning of small bowel mitigates the late phase of reperfusion injury: heme oxygenase mediates cytoprotection.

BACKGROUND: Ischemia and reperfusion (IR) injury of the intestine is a major cause of morbidity and mortality following small bowel transplantation. The current study evaluates the effect of ischemic preconditioning (IPC) on the intestinal microcirculation in the late phase of IR injury of the intestine. METHODS: Sixty rats were randomly allocated to 5 study groups (n = 12 per group): (1) sham, (2) IR (3) IPC, (4) pyrrolidine dithiocarbamate (PDTC) (HO-1 inducer), and (5) zinc protoporhyrin (ZnPP) (HO-1 inhibitor). Mucosal perfusion and leukocyte-endothelial interactions were measured with the aid of an intravital microscope. At the end of the experiments, blood samples for lactate dehydrogenase (LDH) levels and biopsies of ileum for histologic evaluation were obtained. RESULTS: IPC significantly improved the mucosal perfusion and decreased the leukocyte-endothelial interactions. Histologic examination showed that ileal mucosa was significantly less injured in the IPC and PDTC groups as compared with the IR group. CONCLUSIONS: IPC protects the intestine from late reperfusion injury. HO-1 is involved in this protection. These findings may be of significant importance in clinical small bowel transplantation.

[The role of insulin-like growth factors (IGF) in cell division processes and in malignancy]. / Az inzulinszeru növekedési faktor (IGF) szerepe a sejtosztódási folyamatokban és a daganatokban.

Extensive research is being carried out to identify the role of insulin-like growth factor (IGF) in cellular development and tumorigenesis. There is substantial experimental and clinical evidence now that IGF and the related signalling pathways have important roles in regulating cellular proliferation, promoting cellular differentiation and anti-apoptotic effect. Significant amount of IGF is produced locally by neoplastic tissue, which gets into the circulation and adds to the naturally liver-generated and circulating amount. The IGF binding proteins (IGFBP) modulate the bioavailability of IGFs. Upon ligand binding to the receptor, the intrinsic tyrosine kinase activity initiates the phosphatidylinositol-3-kinase (PI3-K) and p38 mitogen activated protein kinase (MAPK) pathway; these have a summon effect on cell cycle. The ligand and the receptor biosynthesis are reviewed, as well as the signal transduction system and the IGF' role in neoplasm. Finally, the therapeutic modalities are surveyed with the preclinical drug's main features.

Growth factors enhance endothelial progenitor cell proliferation under high-glucose conditions.

BACKGROUND: The purpose of this study was to investigate the impact of growth regulators, including growth hormone (GH), insulin-like growth factor 1 (IGF-1), and mechano growth factor (MGF), on endothelial progenitor cell (EPC) proliferation at different glucose concentrations. MATERIAL/METHODS: EPCs were isolated and cultured from peripheral blood samples of healthy volunteers and immunocytochemically characterized after 7 days. The effects of glucose and growth regulators on EPC proliferation were determined with the Alamar Blue and Trypan Blue assays. The effect of glucose supplementation at 2.5, 11.1, and 25.0 mM was examined using cells seeded at densities of 15000, 30000, and 45000 cells/ml. RESULTS: For the GH-treated cells, enhancement of EPC proliferation was detected in the samples supplemented with 11.1 and 25.0 mM glucose. A slight elevation in EPC proliferation was only observed in the IGF-1-treated cells supplemented with 25.0 mM glucose. Significant enhancement of EPC proliferation was observed in MGF-treated cells supplemented with 11.1 and 25.0 mM glucose. All three growth factors demonstrated enhancement of cellular proliferation when the cells were supplemented with 25.0 mM glucose. No enhancement of EPC proliferation by the growth factors was detected in any of the cells supplemented with 2.5 mM glucose. CONCLUSIONS: GH, IGF-1, and MGF enhance EPC proliferation under 25.0 mM glucose conditions. The presence of these growth regulators in EPC culture may contribute to protecting EPCs from high-glucose conditions. This action may be of therapeutic relevance contributing to beneficial cardiovascular effects for diabetic patient.

In vitro small intestinal epithelial cell growth on a nanocomposite polycaprolactone scaffold.

Tissue engineering of the small intestine remains experimental despite worldwide attempts to develop a functional substitute for short bowel syndrome. Most published studies have reported predominant use of PLLA (poly-L-lactide acid)/PGA (polyglycolic acid) copolymer as the scaffold material, and studies have been limited by in vivo experiments. This lack of progress has inspired a fresh perspective and provoked further investigation and development in this field of tissue engineering. In the present paper, we exploit a relatively new nanocomposite of POSS (polyhedral oligomeric silsesquioxane) and PCL [poly(caprolactone-urea)urethane] as a material to develop porous scaffolds using a solvent casting/particulate leaching technique to fabricate porous scaffolds in different pore sizes and porosities. Scaffolds were characterized for pore morphology and porosity using scanning electron microscopy and micro-computed tomography. Rat intestinal epithelial cells were then seeded on to the polymer scaffolds for an in vitro study of cell compatibility and proliferation, which was assessed by Alamar Blue and lactate dehydrogenase assays performed for 21 days post-seeding. The results obtained demonstrate that POSS-PCL nanocomposite was produced as a macroporous scaffold with porosity over the range of 40-80% and pore size over the range of 150-250 microm. This scaffold was shown to support epithelial cell proliferation and growth. In conclusion, as a further step in investigating small intestinal tissue engineering, the nanocomposite employed in this study may prove to be a useful alternative to poly(lactic-co-glycolic acid) in the future.

Aspirin is not chemoprotective for Barrett's adenocarcinoma of the oesophagus in multicentre cohort.

Barrett's columnar-lined oesophagus is the precursor lesion for oesophageal adenocarcinoma. The overall rate of progression to adenocarcinoma is 0.59% per annum. A large prospective multicentre trial is recruiting to assess the role of aspirin as a chemoprotective agent in prevention of development of cancer as well as cardiovascular protection in patients with Barrett's oesophagus. This retrospective analysis of the large UK National Barrett's Oesophagus Registry database seeks to analyse this question from within its large natural history study cohort. Multicentre UK retrospective cohort compared patients known to have been taking aspirin with those who did not take aspirin during the course of surveillance for columnar-lined oesophagus. End point was development of dysplasia or oesophageal adenocarcinoma. Analysis was undertaken using Cox's proportional hazard ratio. Total follow-up was 3683 patient-years. Eighty-six patients were taking aspirin, 650 were not taking aspirin (reference group). Numbers of patients developing all grades of dysplasia and adenocarcinoma were: 13 aspirin (15.1%) and 97 no aspirin (14.9%) (hazard ratio 0.723, 95% confidence interval 0.410-1.310, P = 0.294), high-grade dysplasia and adenocarcinoma: five aspirin (5.8%) and 25 no aspirin (3.8%) (hazard ratio 0.898, 95% confidence interval 0.340-2.368, P = 0.827) and adenocarcinoma: four aspirin (4.7%) and 16 no aspirin (2.5%) (hazard ratio 1.092, 95% confidence interval 0.358-3.335, P = 0.877). No significant difference was observed in hazard of developing dysplasia or adenocarcinoma between patients taking aspirin and those not taking aspirin during the course of follow-up of surveillance for columnar-lined oesophagus. In conclusion, no difference in risk of development of dysplasia or adenocarcinoma was observed between patients taking aspirin and those not taking aspirin in this large cohort.

The effect of consecutively larger doses of L-arginine on hepatic microcirculation and tissue oxygenation in hepatic steatosis.

BACKGROUND: Impairment of hepatic microcirculation in fatty liver is thought to render it more susceptible to the effects of ischaemia-reperfusion injury as compared to non-fatty liver grafts. The present study aimed to investigate the effect of consecutively larger doses of L-arginine on the hepatic microcirculation and tissue oxygenation of fatty liver. METHODS: Sprague-Dawley rats (200-250 g) were fed a liquid ethanol diet to induce hepatic steatosis or a normal diet for 6 weeks. Hepatic blood flow, microcirculation, tissue oxyhaemoglobin (HBO(2)) in response to consecutive intravenous bolus administrations of l-arginine (50 mg/kg, 100 mg/kg, 300 mg/kg and 500 mg/kg) or normal saline, were assessed. RESULTS: Baseline hepatic arterial flows and hepatic microcirculation values were significantly lower in steatotic livers vs. control livers. L-arginine significantly improved hepatic arterial, portal venous blood flow, hepatic microcirculation and tissue oxygenation in both fatty and control livers. CONCLUSIONS: The administration of NO in cumulatively larger doses is effective at improving hepatic blood flow, microcirculation and hepatic tissue oxygenation in steatotic liver and these results could form the basis of further work into using NO as a therapeutic tool to reclaim moderately steatotic grafts for use in liver transplantation.

Functional blocking of specific integrins inhibit colonic cancer migration.

For more effective oncological management of disseminated colorectal cancer, therapies must be devised that target the different individual stages of metastasis development. Recent work showed that integrin subunits alpha2, alpha6 and beta4 are involved in the colorectal cancer cell extravasation process. By means of Immunocytochemistry and Western blotting, it was shown that all three integrins are expressed not only in human colorectal cancer cells (HT29) but also in rat colonic cancer cells (DHDK12). Using in vivo models and intravital video microscopy techniques, it was shown that functional blocking of these integrin subunits by specific antibodies produced a significant reduction in cancer cell extravasation and migration. In conclusion, integrin subunits alpha2, alpha6 and beta4 are expressed in unrelated colorectal cancer cell strains and appear to play a key role in cancer cell migration.

IGF-I activates caspases 3/7, 8 and 9 but does not induce cell death in colorectal cancer cells.

BACKGROUND: Colorectal cancer is the third most common cancer in the western world. Chemotherapy is often ineffective to treat the advanced colorectal cancers due to the chemo-resistance. A major contributor to chemo-resistance is tumour-derived inhibition or avoidance of apoptosis. Insulin-like growth factor I (IGF-I) has been known to play a prominent role in colorectal cancer development and progression. The role of IGF-I in cancer cell apoptosis is not completely understood. METHODS: Using three colorectal cancer cell lines and one muscle cell line, associations between IGF-I and activities of caspase 3/7, 8 and 9 have been examined; the role of insulin-like growth factor I receptor (IGF-IR) in the caspase activation has been investigated. RESULTS: The results show that exogenous IGF-I significantly increases activity of caspases 3/7, 8 and 9 in all cell lines used; blocking IGF-I receptor reduce IGF-I-induced caspase activation. Further studies demonstrate that IGF-I induced caspase activation does not result in cell death. This is the first report to show that while IGF-I activates caspases 3/7, 8 and 9 it does not cause colorectal cancer cell death. CONCLUSION: The study suggests that caspase activation is not synonymous with apoptosis and that activation of caspases may not necessarily induce cell death.

The in-vivo effect of pyrrolidine dithiocarbamate on hepatic parenchymal microcirculation and oxygenation of the rat liver.

OBJECTIVE: Pyrrolidine dithiocarbamate has been shown to be a potent inducer of haemeoxygenase-1. This study investigated its in-vivo effects on systemic and hepatic microcirculatory perfusion. METHODS: Male Sprague-Dawley rats (n=12) were administered intravenously with pyrrolidine dithiocarbamate (10, 20 and 50 mg/kg body weight) or vehicle (0.2 ml physiological saline) served as control. Systemic and hepatic haemodynamics including arterial oxygen saturation, heart rate, mean arterial blood pressure and portal blood flow were monitored. Microcirculation in skeletal muscle and liver was measured by laser Doppler flowmetry and intravital fluorescence microscopy, whereas hepatic tissue oxyhaemoglobin and cytochrome oxidase CuA redox state, which is an indicative of extracellular and intracellular oxygenation were measured by near infrared spectroscopy. RESULTS: Pyrrolidine dithiocarbamate induced a dose-dependent increase in mean arterial blood pressure and skeletal muscle microcirculation. The hepatic parenchymal microcirculation was significantly improved and an increase in sinusoidal diameter and reduction in RBC velocity were observed. Pyrrolidine dithiocarbamate also showed beneficial effect on hepatic tissue oxygenation showed by an increase in oxyhaemoglobin and cytochrome oxidase CuA redox state as well. CONCLUSION: Pyrrolidine dithiocarbamate improves hepatic parenchymal microcirculation and tissue oxygenation, suggesting that it may be used as a potential agent in pharmacological preconditioning in the liver.

Apoptosis and colorectal cancer: implications for therapy.

Colorectal cancer (CRC) is characterized by the partial suppression of apoptosis, which in turn gives tumours a selective advantage for survival and can cause current chemotherapy approaches to be ineffective. Recent progress in understanding the mechanisms of apoptosis in colorectal carcinogenesis has provided potential new targets for therapy. Here, we review recent studies of the regulation of apoptosis and its role in CRC initiation and progression, and we discuss the relationship between chemoresistance and the suppression of apoptosis. Recent progress in targeting apoptotic pathways and their regulators provide strategies for the exploration of novel therapies for CRC.

Are newly diagnosed columnar-lined oesophagus patients getting younger?

OBJECTIVES: The prevalence of columnar-lined oesophagus seems to have increased steadily in the past three decades in Europe and North America. Although the vast majority of columnar-lined oesophagus will not progress to malignancy, it is nevertheless important to identify the risk factors associated with this condition. This study investigates whether there has been a change, at diagnosis, in age of columnar-lined oesophagus patients between 1990 and 2005, or an increase in the number of patients aged less than 50 years. METHODS: Data on age of diagnosis were abstracted from medical records of 7220 patients from 19 centres registered with UK National Barrett's Oesophagus Registry, between the years 1990 and 2005. Linear regression analysis was carried out to assess any trends in the mean age of diagnosis. RESULTS: Overall there was a mean decrease in age at diagnosis for each 1-year increase in time. This equated to a mean decrease of 3 years over the study period, 1990-2005 with the greatest difference being seen in female patients. About 18% of patients in the study were aged less than 50 years at the time of diagnosis. With this group also, the trend was similar, with an increase in the number of patients aged less than 50 years, at the time of diagnosis, with increasing years. CONCLUSION: The mean age of diagnosis of columnar-lined oesophagus has decreased between the years 1990 and 2005 in both men and women, more so in women. This is also reflected in an increase in newly diagnosed columnar-lined oesophagus patients below the age of 50 years.

Surveillance of Barrett's columnar-lined oesophagus in the UK: endoscopic intervals and frequency of detection of dysplasia.

OBJECTIVES: Endoscopic surveillance of patients with columnar-lined oesophagus (CLO) may identify those with early adenocarcinoma (AC). The benefits of surveillance are unproven and there is little evidence to support recommendations for precise endoscopic intervals. We sought to examine surveillance practice for CLO in the UK and the impact of endoscopic intervals on detection of dysplastic disease. METHODS: Eight hundred and seventeen patients with CLO, registered with the UK National Barrett's Oesophagus registry and undergoing surveillance were studied. Endoscopic intervals were calculated and frequency of detection of dysplastic disease analysed using chi2 test of association. Factors affecting surveillance intervals were analysed using multiple linear regression. RESULTS: 94.7% of patients with low-grade dysplasia (LGD), 95.0% with high-grade dysplasia (HGD) and 71.4% with AC were diagnosed on surveillance endoscopies. Mean endoscopic surveillance intervals varied between the centres from 1.07 to 1.63 years for nondysplastic CLO; 0.69-1.19 years for LGD, and 0.35-1.17 years for HGD; with overall mean surveillance intervals of 1.29, 1.01 and 0.44 years, respectively. When LGD was surveyed, significantly higher proportions of HGD/AC were detected at intervals of 3 months or less (P=0.013). Shorter endoscopic intervals were significantly associated with the presence of oesophageal strictures (P=0.002), ulcers (P=0.046), increasing patient age (P<0.001) and higher grade of dysplasia surveyed (P<0.001). CONCLUSION: A variation in surveillance practice for CLO was observed throughout the UK. A large proportion of dysplastic disease is detected on specific surveillance endoscopies. Shorter endoscopic intervals for surveillance of LGD are associated with an increased detection of HGD/AC.

Treatment modality and risk of development of dysplasia and adenocarcinoma in columnar-lined esophagus.

Columnar metaplasia is the precursor lesion for esophageal adenocarcinoma, resulting from prolonged gastroesophageal reflux. The influence of the efficacy of reflux control on the development of neoplastic change in columnar-lined esophagus is not established. This study compares the rate of development of dysplasia and adenocarcinoma in patients with columnar metaplasia of the esophagus between patients treated pharmacologically and those treated with antireflux surgery. This study is a retrospective review of a cohort of patients enrolled in a multicenter national registry involving 738 patients from seven UK centers. Forty-one were treated with antireflux surgery, 42 with H2 receptor antagonist, 532 with proton pump inhibitor, and 114 with a combination of these medications. Nine had none of these medications or surgery. Total follow-up was 3697 years. Mean age and follow-up for patients treated medically were 61.6 and 4.96 years and surgically were 50.5 and 6.19 years, respectively. No patient in the surgical group developed high-grade dysplasia (HGD) or adenocarcinoma. Twenty patients treated medically developed adenocarcinoma and 10 developed HGD. Hazards ratio comparing pharmacological to surgical therapy for development of all grades of dysplasia and adenocarcinoma 1.77 (P = 0.272). Log rank test comparing antireflux surgery to pharmacological therapy for development of HGD or adenocarcinoma P = 0.1287 and for adenocarcinoma P = 0.2125. Although there was a trend towards greater efficacy of antireflux surgery over pharmacological therapy in reducing the development of dysplasia and adenocarcinoma, this did not reach statistical significance.

Principles and applications of gene therapy in colon cancer.

Colorectal cancer is one of the leading causes of mortality and morbidity. Mutations and aberration of some of the genes may lead to colon cancer. With advancing knowledge, more and more defective genes can be identified. Theoretically, correction of these defective genes, selective overexpression of certain genes may lead to not only prevention of cancer development but also regression of existing cancer. Carcinogenesis is a multistep process and more than one gene may be altered. At present, the success of gene therapy as a stand alone therapy is limited by poor expression and long-term non-expression of transferred genes, immunological effect on the viral vectors, viraemia, leukaemia and occasional deaths. Further experiments and trials are underway to modify the vectors and genes to make sure gene therapy is safe, effective and long-lasting before it can be used in day-to-day practice.