Post-surgical chemotherapy versus surgery alone for resectable gastric cancer.

BACKGROUND: For gastric cancer surgery is the mainstay treatment. Chemotherapy seems to improve the survival results. But chemotherapy is not a complication-free therapy and its role has been questioned by some trials. OBJECTIVES: To determine whether post-surgical chemotherapy should be used routinely in resectable gastric cancer. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, and Science Citation Index Expanded (July 2013). SELECTION CRITERIA: Randomised controlled trials (RCT) comparing post-surgical chemotherapy versus surgery alone for resectable gastric cancer. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trials for inclusion and independently extracted the data. We analysed the data with both the fixed-effect and the random-effects models using the RevMan analysis software. We calculated the hazard ratio (HR) with 95% confidence interval (CI) based on intention-to-treat or available case analysis. MAIN RESULTS: The authors identified 34 studies (7824 patients) reporting overall survival (OS) and only 15 reporting disease free survival (DFS) as well. Post-surgical chemotherapy showed an improvement in OS (HR 0.85; 95% CI 0.80 to 0.90) and an improvement in DFS (HR 0.79; 95% CI 0.72 to 0.87), although all the trials had a high risk of bias.The planned analysis of quality of life, return to work, and number of hospital admissions was impossible to complete as the outcome data for the analysis were not available from any trials. AUTHORS' CONCLUSIONS: Post-surgical chemotherapy should be used routinely for resectable gastric cancer where possible. Further RCTs are needed to determine the role at each stage 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.

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.

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.

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.

In vivo factors influencing tumour M2-pyruvate kinase level in human pancreatic cancer cell lines.

In tumour cells, the tetramer/dimer ratio of the pyruvate kinase isoenzyme type M2 (M2-PK) determines whether glucose carbons are degraded to lactate with production of energy (tetrameric form) or are channelled into synthetic processes (dimeric form). The influence of different tumour microenvironment conditions on the tetramer/dimer ratio of M2-PK and cell doublings were investigated in a non-metastatic and metastatic pancreatic cancer cell line. The metastatic Colo357 cells contained about fourfold more M2-PK protein and about 3.5-fold more dimeric M2-PK than the non-metastatic Panc-1 cells. In Colo357 cells hypoxia, glucose starvation as well as acidification induced an increase of the dimeric form of M2-PK, whereas in Panc-1 cells no effect on M2-PK was observed. Under hypoxia in Colo357 cells, the dimerization and inactivation of M2-PK results in an inhibition of cell proliferation, whereas under glucose starvation and acidification the dimerization of M2-PK allowed further cell doublings. M2-PK expression and the quaternary structure of M2-PK are influenced by the tumour metastatic potential. The quaternary structure of M2-PK may be differently affected by hypoxia, glucose starvation and acidification with severe consequences on cell doublings.

Lowering the apoptotic threshold in colorectal cancer cells by targeting mitochondria.

BACKGROUND: Colorectal cancer is the third most-common cancer and the second most-common cause of cancer related death in UK. Although chemotherapy plays significant role in the treatment of colorectal cancer, morbidity and mortality due to drug resistance and cancer metastasis are yet to be eliminated. Recently, doxycycline has been reported to have cytotoxic and anti-proliferating properties in various cancer cells. In this study, whether doxycycline was apoptosis threshold lowering agent in colorectal cancer cells by targeting mitochondria was answered. RESULTS: This study showed dose-dependent cytotoxic effects of cisplatin, oxaliplatin and doxycycline in HT29 colorectal cancer cells. Doxycycline showed inhibition of cytochrome-c-oxidase activity in these cells over a time-period. The pre-treatment of doxycycline reported statistically significant increased cytotoxicity of cisplatin and oxaliplatin compared to cisplatin and oxaliplatin alone. The caspase studies revealed significantly less expression and activity of caspase 3 in HT29 cells pre-treated with doxycycline compared to the cells treated with cisplatin and oxaliplatin alone. CONCLUSIONS: It was concluded that doxycycline lowered the apoptotic threshold in HT 29 cells by targeting mitochondria. This also raised possible caspase-independent mechanisms of apoptosis in HT29 cells when pre-treated with doxycycline however this needs further research work.

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.

Routinely diagnosed low-grade dysplasia in Barrett's oesophagus: a population-based study of natural history.

AIMS: To examine the natural history of columnar-lined oesophagus with routinely diagnosed low-grade dysplasia and ascertain the risk of oesophageal adenocarcinoma development. METHODS AND RESULTS: A multicentre retrospective cohort study of 283 patients with low-grade dysplasia. Follow-up data were obtained from examination of hospital records. One hundred and forty-four patients had biopsies prior to low-grade dysplasia diagnosis and 217 had follow-up biopsies after index low-grade dysplasia diagnosis. In these patients the incidence of high-grade dysplasia and adenocarcinoma combined was 4.6% per annum and of adenocarcinoma alone was 2.7% per annum. At most recent follow-up, 43 (19.8%) had persistent low-grade dysplasia, 37 (17.1%) had changes indefinite for dysplasia and 108 (49.8%) had non-dysplastic columnar-lined oesophagus. When prevalent cases were excluded (those occurring within 1 year of index low-grade dysplasia diagnosis), the annual incidence of high-grade dysplasia and adenocarcinoma combined was 2.2% and of adenocarcinoma alone was 1.4%. The relative risk for adenocarcinoma development in low-grade dysplasia compared with non-dysplastic columnar-lined oesophagus was 2.871 (P = 0.002). CONCLUSIONS: Low-grade dysplasia has a threefold increased risk of progression to cancer compared with non-dysplastic epithelium, but in the majority of patients dysplasia is not subsequently detected.

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.

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.

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.

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.

[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.

Inducing apoptosis of human colon cancer cells by an IGF-I D domain analogue peptide.

BACKGROUND: The resistance of tumour cells to apoptosis is a major contributor to the limited effectiveness of chemotherapies. Insulin-like growth factor I (IGF-I) has potential to protect cancer cells from variety of apoptotic challenges. This study was carried out to investigate the effect of a novel IGF-I receptor antagonist on apoptosis in colon cancer cells. RESULTS: We have designed and synthesised a novel antagonist of IGF-I receptor. The effect of this antagonist on human colon cancer cell proliferation was examined by a non-radioactive assay; the apoptosis was revealed by determining the activities of cellular caspases3/7, 8 and 9. The apoptosis pathways were investigated by examining the levels of pro-apoptosis proteins with Western blotting. Following 40 hours treatment with the novel antagonist peptide, colon cancer cell Caspase 3/7 activities increased 2-7 times; Caspase 8 activities increased 2-5 times and Caspase 9 increased 1.2-1.6 times. The proliferation of cancer cell was inhibited by 14-15%. The data showed that the antagonist induced colon cancer cell apoptosis and inhibited cancer cell proliferation. The different changes of Caspase 3/7, 8 and 9 activities suggested that the extrinsic pathways may play a major role in the antagonist peptide-induced apoptosis. CONCLUSION: This is the first report on this novel antagonist to induce human colon cancer cell apoptosis and inhibit cancer cell proliferation. These results suggest that IGF-I receptor antagonists may have the potential to be developed as a novel therapy for colon cancers in the future.

Improved screening for anal neoplasia by immunocytochemical detection of minichromosome maintenance proteins.

PURPOSE: Early detection of anal intraepithelial neoplasia (AIN) and anal squamous cell carcinoma (SCC) by screening will improve clinical outcome. Assessment of anal cytology samples using routine Papanicolaou testing suffers from shortcomings in sensitivity and/or specificity, suggesting that screening tests based on biomarkers may be of value. We tested the suitability in this context of minichromosome maintenance (MCM) proteins, accurate markers of the deregulated cell cycle entry that characterizes malignancy and premalignancy. EXPERIMENTAL DESIGN: We undertook an initial immunohistochemical study of 54 anal tissue samples and validated our findings using an independent prospective cohort study of 235 anal cytology samples from 144 subjects. RESULTS: In the progression from normal anal epithelium through AIN to SCC, there was increasing expression of MCM2 and MCM5, including in the superficial epithelial third, the source of the majority of cells collected by anal swab. The median labeling indices (LI) for MCM2 and MCM5 in the superficial third of AIN2/3 and SCCs combined were 90.2% and 84.0%, respectively. MCM LIs in the superficial layers were significantly greater than LIs for Ki67, an alternative marker of cell cycle entry (P<0.0001). By immunocytochemistry using a mixture of anti-MCM2 and anti-MCM5 antibodies, immunopositive cells were readily identified in anal cytology samples, even at low magnification. MCM testing showed sensitivity for AIN2/3 of 84% (95% confidence interval, 75,93) and for AIN1/viral changes of 76% (68, 84), with overall specificity (for any lesion) of 77% (64, 90). CONCLUSIONS: MCMs are promising biomarkers for improving detection of AIN and SCC in anal cytology samples.

In vivo models for early development of colorectal liver metastasis.

In Europe, colorectal cancer is the second most prevalent form of cancer diagnosed. Globally each year, almost one million cases of colorectal cancer are registered and almost half a million deaths are attributed to this disease. This high mortality is associated with the development of liver metastases. For oncological advances to occur, accurate in vivo models are required to study colorectal cancer metastasis development. These models, by increasing our understanding of the early stages of colorectal liver establishment, will facilitate the development of novel therapeutic interventions and allow the clinical effects of these interventions to be studied. By analysis of current in vivo models for early development of colorectal liver metastasis, this review examines available methods of the tumour cell preparation, introduction and monitoring in vivo. An insight into the technical problems which can occur will be discussed. The implications of these different techniques on the resulting metastasis picture will be analysed. Existing in vivo models are assessed regarding the accuracy of the metastatic picture they portray.

An in vivo rat model for early development of colorectal cancer metastasis to liver.

At diagnosis of colorectal cancer, approximately 25% of the patients have established colorectal liver metastasis. Optimal management of disseminated disease requires therapies targeting multiple stages in hepatic colorectal cancer metastasis development. To facilitate this, biologically accurate in vivo models are required. Early colonic cancer liver metastases development was studied using BDIX and Sprague-Dawley rat strains with human HT29 and rat DHDK12 colonic cancer cell lines. Different cancer cell-host combinations were used. Rat DHDK12 was previously chemically induced in the BDIX rat. Real-time intra-vital microscopy was employed to analyse the early development of liver metastases in four groups (n = 6 per group) (HT29-BDIX, DHDK12-BDIX, HT29-SD and DHDK12-SD). Data were compared using one-way anova with Bonferroni's multiple comparison test. The total number of tumour cells visualized, adherent cells within the hepatic sinusoids, extravasated tumour cells and migration rates were significantly higher in the DHDK12-BDIX combination. Maximum number of visualized cells and maximum migration rate were also significantly higher in this group. No significant differences were observed in these experimental parameters among the other three groups or in the haemodynamic parameters among all groups. In conclusion, cancer cell line-host selection has a significant effect on early colonic cancer liver metastasis development.

Quantitating therapeutic disruption of tumor blood flow with intravital video microscopy.

Vascular-disrupting agents (VDA) kill tumor cells by selectively disrupting blood circulation in tumors. In vivo analysis of this intensely studied class of anticancer agents is invaluable for preclinical assessment of pharmacodynamic end points and effective therapeutic windows. In this review, we consider the role of intravital video microscopy in measuring tumor vascular response to VDAs, the potential of which lies in the opportunity to quantitate specific variables and to obtain real-time information on how VDAs affect tumor microcirculation.

Insulin-like growth factor binding protein-4 gene therapy increases apoptosis by altering Bcl-2 and Bax proteins and decreases angiogenesis in colorectal cancer.

Insulin-like growth factors are known to inhibit apoptosis and promote tumour angiogenesis. Previously we have shown that insulin-like growth factor binding protein-4 (IGFBP-4) gene therapy increased apoptosis and decreased mitosis in colon cancer. In this experiment we used HT-29 colon cancer cells to induce subcutaneous cancers in nude mice and administered either the mammalian expression vector with IGFBP-4 insert or vector only around the tumour site. Three weeks after gene transfer, tumours were harvested and expressions of Bax, Bcl-2 and IGF-I receptor in tumours were determined by Western blotting and immunofluorescence. Micro-vessel counting was performed by immunostaining with CD34 and von Willebrand antibodies. Results showed that tumours treated with IGFBP-4 gene had higher expression of Bax, lower expression of Bcl-2 and IGF-I receptor. Bcl-2 was localised to tumour cell cytoplasm while Bax was expressed both in the interstitial area and cytoplasm. IGFBP-4 treatment also decreased micro-vessel count in tumour tissues. Micro-vessels were mainly located in the periphery and interstitial area. This experiment shows that IGFBP-4 gene therapy increases tumour apoptosis via altering the expressions of Bcl-2 and Bax and decreasing the angiogenesis in colorectal cancer.