Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation.

A special in vitro model maintained with ultrathin cardiac slices with a preserved architecture, multi-cellularity, and physiology of the heart tissue was used. In our experiments, we performed label-free quantitative SWATH-MS proteomic analysis of the adult myocardial slices in vitro after biomimetic electromechanical stimulation. Rat myocardial slices were stretched to sarcomere lengths (SL) within the physiological range of 1.8-2.2 µm. Electromechanically stimulated slices were compared with slices cultured without electromechanical stimulation (unloaded and nonstimulated-TW) on a liquid-air interface and with fresh myocardial slices (0 h-C). Quantitative (relative) proteomic analyses were performed using a label-free SWATH-MS technique on a high-resolution microLC-MS/MS TripleTOF 5600+ system (SCIEX). The acquired MS/MS spectra from the DDA LC-MS/MS analyses of the rat heart samples were searched against the UniProt Rattus norvegicus database (version of 15.05.2018) using the Paragon algorithm incorporated into ProteinPilot 4.5 (SCIEX) software. The highest number of differential proteins was observed in the TW group-121 when compared to the C group. In the 1.8 and 2.2 groups, 79 and 52 proteins present at a significantly different concentration from the control samples were found, respectively. A substantial fraction of these proteins were common for two or more comparisons, resulting in a list of 169 significant proteins for at least one of the comparisons. This study found the most prominent changes in the proteomic pattern related to mitochondrial respiration, energy metabolism, and muscle contraction in the slices that were stretched and fresh myocardial slices cultured without electromechanical stimulation.

A practical guide for investigating cardiac physiology using living myocardial slices.

Ex vivo multicellular preparations are essential tools to study tissue physiology. Among them, the recent methodological and technological developments in living myocardial slices (LMS) are attracting increasing interest by the cardiac research field. Despite this, this research model remains poorly perceived and utilized by most research laboratories. Here, we provide a practical guide on how to use LMS to interrogate multiple aspects of cardiac function, structure and biochemistry. We discuss issues that should be considered to conduct successful experiments, including experimental design, sample preparation, data collection and analysis. We describe how laboratory setups can be adapted to accommodate and interrogate this multicellular research model. These adaptations can often be achieved at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software, which is essential to broaden the utilization of this method. We will also highlight how current measurements can be improved to further enhance data quality and reliability to ensure inter-laboratory reproducibility. Finally, we summarize the most promising biomedical applications and envision how living myocardial slices can lead to further breakthroughs.

A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site.

As the hormone gastrin promotes gastrointestinal (GI) cancer progression by triggering survival pathways, regulation of gastrin expression at the translational level was explored. Sequence within the 5' untranslated region of a gastrin transcript expressed in GI cancer cells was investigated, then cloned into a bicistronic vector upstream of firefly luciferase and transfected into a series of GI cancer cell lines. Firefly luciferase activity was measured relative to that of a cap-dependent Renilla luciferase. A gastrin transcript that was different from that described in Ensembl was expressed in GI cancer cells. Its transcription appears to be initiated within the region designated as the gene's first intron. In GI cancer cells transfected with the bicistronic construct, firefly luciferase activity increased 8-15-fold compared with the control vector, and there was a further induction of the signal (up to 25-fold) following exposure of the cells to genotoxic stress or hypoxia, suggesting that the sequence acts as an internal ribosome entry site. These data suggest that the gastrin transcript within GI cancer cells contains an internal ribosome entry site that may allow continued expression of gastrin peptides when normal translational mechanisms are inactive, such as in hypoxia, thereby promoting cancer cell survival.

Pre-clinical evaluation of a new orally-active CCK-2R antagonist, Z-360, in gastrointestinal cancer models.

BACKGROUND: Gastrin has a role in gastrointestinal (GI) malignancy. This study provides pre-clinical evaluation of a novel, orally-active gastrin/cholecystokinin-2 receptor (CCK-2R) antagonist, Z-360. METHODS: (125)I gastrin-17 (G17) displacement and G17-stimulated calcium assays were used in classical CCK-2R-transfected cell lines. Akt phosphorylation was assessed by Western blotting. Z-360 efficacy in vivo was evaluated in three human xenograft models, and microvessel density and apoptosis in these models were investigated by immunohistochemistry. RESULTS: Z-360 inhibited (125)I G17 binding to cells expressing CCK-2R, and G17-stimulated signalling. Reduced Akt phosphorylation in an oesophageal cell-line treated with Z-360 was reversed by co-treatment with G17. Z-360 increased survival in a gastric ascites model (p=0.011) and decreased tumour growth in a hepatic metastasis model (81%, p=0.02). In an orthotopic pancreatic model, Z-360 combined with gemcitabine decreased final tumour weight compared to single agents (84%, p=0.002) and there was increased apoptosis and decreased microvessel density in ex vivo tumour tissue. CONCLUSIONS: These results show that the orally-active CCK-2R antagonist, Z-360 has high sub-nM affinity for classical CCK-2R, is well tolerated in vivo and exerts an anti-tumour effect.

De-regulation of the sonic hedgehog pathway in the InsGas mouse model of gastric carcinogenesis.

This study investigated sonic hedgehog (Shh) signalling in gastric metaplasia in the insulin-gastrin (InsGas) hypergastrinaemic mouse +/- Helicobacter felis (H. felis) infection. Sonic hedgehog gene and protein expression was reduced in pre-metaplastic lesions from non-infected mice (90% gene reduction, P<0.01) compared to normal mucosa. Sonic hedgehog was reactivated in gastric metaplasia of H. felis-infected mice (3.5-fold increase, P<0.01) compared to pre-metaplastic lesions. Additionally, the Shh target gene, glioma-associated oncogene (Gli)-1, was significantly reduced in the gastric glands of InsGas mice (75% reduction, P<0.05) and reactivated with H. felis infection (P<0.05, base of glands, P<0.01 stroma of metaplastic glands). The ability of H. felis to activate the Shh pathway was investigated by measuring the effect of target cytokine, interleukin-8 (IL-8), on Shh expression in AGS and MGLVA1 cells, which was shown to induce Shh expression at physiological concentrations. H. felis induced the expression of NF-kappaB in inflammatory infiltrates in vivo, and the expression of the IL-8 mouse homologue, protein KC, in inflammatory infiltrates and metaplastic lesions. Sonic hedgehog pathway reactivation was paralleled with an increase in proliferation of metaplastic lesions (15.75 vs 4.39% in infected vs non-infected mice, respectively, P<0.001). Furthermore, Shh overexpression increased the growth rate of the gastric cancer cell line, AGS. The antiapoptotic protein, bcl-2, was expressed in the stroma of infected mice, along with a second Shh target gene, patched-1 (P=0.0001, stroma of metaplastic gland). This study provides evidence suggesting reactivation of Shh signalling from pre-metaplastic to advanced metaplastic lesions of the stomach and outlines the importance of the Shh pathway as a potential chemoprophylactic target for gastric carcinogenesis.

PTHrP increases transcriptional activity of the integrin subunit alpha5.

Increasing evidence is emerging highlighting the role of parathyroid hormone-related protein (PTHrP) during metastasis by regulating cell adhesion. The current study demonstrated that modulation of PTHrP expression by PTHrP overexpression and small interfering RNA-induced silencing resulted in changes in cell adhesion and integrin expression. RNA interference of endogenous PTHrP caused a significant reduction in cell adhesion of a breast cancer cell line to collagen type I, fibronectin and laminin (P<0.05) and of a colon cancer cell to collagen type I and fibronectin (P<0.05). Overexpression of PTHrP induced a significant increase in cell adhesion of colon (P<0.0001) and breast (P<0.05) cancer cells to the same extracellular matrix proteins. These PTHrP-mediated effects were attributed to changes in integrin expression as the differences in adhesion profile correlated with the integrin expression profile. In an attempt to elucidate the mechanism whereby PTHrP regulates integrin expression, promoter activity of the integrin alpha5 subunit was analysed and significant increases in transcriptional activity were observed in PTHrP overexpressing cells (P<0.0001), which was dependent on nuclear localisation. These results indicate that modulation of cell adhesion is a normal physiological action of PTHrP, mediated by increasing integrin gene transcription.

G17DT: an antigastrin immunogen for the treatment of gastrointestinal malignancy.

G17DT (Gastrimmune) is an antigastrin-17 immunogen, raising antibodies that blockade gastrin-stimulated tumor growth. It has completed Phase III trials in patients with pancreatic cancer, and Phase III trials in gastric cancer are planned. Preclinical studies have confirmed that the G17DT-induced antibodies both reduce gastrin-17-stimulated gastric acid secretion and inhibit gastrin from interacting with the cholecystokinin-2 receptor. The efficacy of both passive and active immunization with G17DT has been established in a number of tumor systems, with additive effects demonstrated in combination chemotherapy in pancreatic, colon and gastric tumor models. Phase I/II studies in advanced gastrointestinal malignancies have shown no systemic or autoimmune reactions to active immunization with G17DT. The use of an optimized dose and dosing schedule has yielded a high proportion of antibody responders (70%), with minimal side effects and antibody titers measurable within 2 - 4 weeks. Phase II trials of G17DT in combination with chemotherapy have also been conducted in gastric and colorectal cancer. A Phase III, multicenter, double-blind, randomized, controlled trial of G17DT versus placebo in patients with advanced pancreatic cancer confirmed improved survival of patients in the G17DT group through an intention-to-treat analysis. The results of a randomized, double-blind, multinational, multicenter study of G17DT in combination with gemcitabine versus placebo and gemcitabine in patients with advanced pancreatic cancer failed to show improved overall survival except on subset analysis of patients with high antibody titers. Therefore, G17DT represents an emerging new modality for gastrointestinal malignancy.

Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells.

Gastrin isoforms, acting through a variety of receptors, have proliferative and anti-apoptotic effects on gastrointestinal (GI) cancers. A small interfering RNA (siRNA) targeting the gastrin gene was used to investigate the role of endogenous gastrin in GI cancer cell survival. Downregulation of the gastrin gene in siRNA-transfected cells was measured using real-time reverse transcriptase-PCR. The most effective siRNA was tested in a panel of GI cancer cell lines at various concentrations and time points, and the effect on cell survival and apoptosis was measured using methyl thiazoyl tetrazolium (MTT) and caspase 3 activation assays. Gastrin siRNA reduced gene expression by more than 90% in a range of GI cancer cell lines. Downregulation of the gastrin gene was dose-dependent and effective over approximately 1 week in vitro. However, downregulation at the protein level was delayed by 3-4 days. Gastrin siRNA-transfected cells showed up to a 60% reduction in growth and up to a 50% increase in apoptosis compared with control siRNA-transfected cells. The effects were most marked in the cell line with the highest constitutive level of gastrin gene expression (human metastatic colon, C170HM2) and in epidermal growth factor (EGF)-treated cells as the gastrin promoter contains an EGF-response element, gERE. The ability of the siRNAs to reduce survival of these GI cell lines is further evidence of the importance of autocrine and/or intracrine gastrin loops in GI cancer, where expression of the gastrin gene and autonomous gastrin appears widespread.

Androgen receptors may act in a paracrine manner to regulate oesophageal adenocarcinoma growth.

BACKGROUND: The role of androgen receptors (ARs) in tumorigenesis, including transcription of fibroblast growth factors (FGFs), is established in prostate cancer. This study examined the role of ARs and FGFs in oesophageal adenocarcinoma (EAC), where tumour incidence in males is higher. METHODS: AR gene expression was analysed using quantitative RT-PCR; AR, fibroblast growth factor receptor-1 (FGFR-1) and fibroblast growth factor-8 isoform b (FGF-8b) protein by immunohistochemistry; and serum steroid levels (testosterone, progesterone, luteinising hormone and follicle stimulating hormone (FSH)) by immunoassay. A human oesophageal adenocarcinoma cell line was grown subcutaneously in nude mice. RESULTS: AR gene expression was of significantly higher levels than oesophageal adenocarcinomas (n=21, p=0.002) and in the squamous carcinoma line (OE21) compared with the adenocarcinoma lines (OE33 and OE19). Median serum testosterone levels in oesophageal carcinoma patients were higher than in age-matched controls (p=0.01) and reduced postoperatively, in patients undergoing curative resection (p=0.006). No significant differences were observed in hormones except FSH, where preoperative levels were significantly higher in the EAC group. AR protein was expressed in normal oesophageal squamous epithelial cells and also in the stroma of 18/23 EAC samples. FGFR-1 protein was expressed in malignant epithelium of 23/23 tumour samples. OE19 xenografts grew faster in male versus female mice (tumour weight at day 21, 1.14 g and 0.28 g, respectively, p=0.005) and had elevated FGF receptor expression. CONCLUSIONS: AR expressed in the stroma of oesophageal adenocarcinomas may induce paracrine effects following stimulation by androgens (including tumour-derived), possibly via FGFs, including FGF-8b.

The effect of jaundice on the generation of anti-gastrin antibodies in G17DT immunized patients with advanced pancreatic cancer.

AIM: The aim of this study was to determine the ability of G17DT to generate anti-gastrin antibodies in jaundiced patients with biliary obstruction due to advanced pancreatic cancer. METHODS: G17DT was administered to 41 patients with advanced pancreatic adenocarcinoma by intramuscular (i.m.) injection at a dose of 250mcg at weeks 0, 1 and 3 of the study. RESULTS: Thirty-five of 41 patients participating in the study were categorized as responders in terms of their gastrin-17 antibody response. There was no correlation between the maximum G17 antibody response and the bilirubin level at either week 0 or week 12. The median survival of patients from the time of the first injection of G17DT was 204 days with 25% of patients surviving for or=305 days. CONCLUSION: This study shows that G17DT administered to jaundiced patients with advanced pancreatic cancer is immunogenic and well tolerated.

Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion.

AIMS: Matrix metalloproteinase (MMP) activity is increased after radiation. The aims of this study were to assess the time course of this increase and its effects on malignant cell invasion. METHODS: Colorectal cancer (HCT 116, LoVo, C 170 HM 2, CaCO-2), fibroblast (46-BR.IGI, CCD-18 Co) and fibrosarcoma (HT1080) cell lines were irradiated at 4 gray (4 Gy) and matrix metalloproteinase gene and protein expression examined over a 96 h period by real time polymerase chain reaction and gelatin zymography. Invasion was assessed on Matrigel. Human rectal tumour MMP expression was compared before and after long course radiotherapy. RESULTS: Radiation increased MMP gene expression of tumour cell lines, and resulted in increased MMP protein activity in the HT1080 line. HT1080 and HCT 116 in monoculture and LoVo in co-culture were more invasive after radiation at 48 h in vitro, but long course radiotherapy did not result in a consistent increase in MMP expression from human rectal tumour biopsies. CONCLUSIONS: Radiation results in increased MMP expression for a limited time period. This results in an early increase in cell line invasion. Further clinical research is required to clarify if MMP inhibition given perioperatively following radiotherapy decreases local recurrence rates.

Inhibition of matrix metalloproteinase activity and growth of gastric adenocarcinoma cells by an angiotensin converting enzyme inhibitor in in vitro and murine models.

INTRODUCTION: Angiotensin converting enzyme (ACE) shares structural homology with the matrix metalloproteinase family of proteolytic enzymes (MMPs) responsible for degradation of the extracellular matrix (ECM). ACE inhibitors have been reported to protect against cancer in patients. The aim of this study was to determine whether the ACE inhibitor, captopril, could impair the activity of MMPs and impact on tumour invasion and growth in a cell line and murine model. METHODS: For proof of principle, the protein activity of human MMP-2 and MMP-9 produced by the HT1080 fibrosarcoma cell line was detected using gelatin zymography. Gene expression was determined by real time reverse transcriptase PCR and tumour cell invasion using Matrigel invasion chambers. The effect of captopril on the in vivo growth of MGLVA-1 human gastric adenocarcinoma xenografts was evaluated in a nude mouse model. RESULTS: Captopril inhibited activity of secreted MMP-9 and MMP-2, however, gene expression in HT1080 remained unaltered. Invasion of HT1080 cells was inhibited by 48% (p<0.001). Tumour size was reduced by 40-50% with 0.4 mg/ml captopril (p<0.01) and when combined with cisplatin the inhibition increased to 71% (p<0.05). DISCUSSION: ACE inhibitors inhibit the activity of secreted MMP-2 and -9 by a mechanism similar to synthetic MMP inhibitors. ACE inhibitors have previously been shown to inhibit tumour growth, however; this is the first study to demonstrate inhibition of a human gastric xenograft, both alone and in combination with cisplatin. These results support further investigation into the anticancer effects of ACE inhibitors.

The role of gastrin in colorectal carcinogenesis.

Colorectal cancer is one of the leading causes of cancer-associated death in the United States and United Kingdom. In England and Wales, it is the second most common cancer in women and the third most common in men. Currently, treatment options for this debilitating disease are limited and surgical resection is the only curative treatment available. Despite rapid advances in surgery, as well as in adjuvant therapies such as radiotherapy and chemotherapy, there has been only a relatively modest improvement in mortality. The majority of colorectal cancers are epithelial-derived adenocarcinomas and arise from benign adenomas through the gain of mutations in key genes. Gastrin, an important polypeptide hormone, responsible for gastric acid secretion has been found to be involved in tumourigenesis in the gastrointestinal tract. When aberrantly expressed, the gastrin and gastrin/CCK-2 receptor genes can mediate powerful down stream events; the gastrin gene can impart anti-apoptotic properties while the gastrin/CCK-2 receptor can activate the transcription of a number of factors including ligands of the epidermal growth factor (EGF) receptor, the REG protein and matrix metalloproteinases (MMPs). In colonic tumourigenesis, gene expression of both gastrin and the gastrin/CCK-2 receptor is activated within epithelial cells at an early stage of the adenoma-carcinoma sequence. This review details the role played by gastrin in the adenoma-carcinoma sequence of colorectal carcinogenesis.

A phase II study of G17DT in gastric carcinoma.

PURPOSE: G17DT is a gastrin immunogen, raising antibodies that blockade gastrin-stimulated growth. The aim of the study was to characterise antibody response and assess safety and tolerability of G17DT given to patients with gastric cancer. EXPERIMENTAL DESIGN: G17DT was administered to 52 patients with gastric adenocarcinoma at weeks 0, 2 and 6 by intramuscular injection at doses of 10, 100 and 250 microg. Antibody levels were measured by an ELISA assay. A radioligand displacement assay determined the ability of G17DT-immunised patients' sera to inhibit binding of 125IG17 to cholecystokinin (CCK)-2 receptors. RESULTS: By week 12 of the study, 6/12 evaluable stage I-III patients achieved an antibody response in the 10 microg group, 7/11 in the 100 microg group, and 11/12 in the 250 microg group. Stage IV patients dosed at 250 microg achieved a similar response rate to stage I-III patients dosed at 10 or 100 microg. G17DT was well tolerated in 47/52 patients. Two patients suffered significant adverse reactions including injection site pain and abscess. G17DT antibodies displaced iodinated gastrin from CCK-2 receptors, with the level of displacement correlating with antibody titre. CONCLUSIONS: G17DT immunisation is a well-tolerated method of raising functional antibodies to 17 amino acid gastrin forms in patients with gastric carcinomas.

Helicobacter pylori upregulates matrilysin (MMP-7) in epithelial cells in vivo and in vitro in a Cag dependent manner.

BACKGROUND AND AIMS: Matrix metalloproteinase-7 (MMP-7) is important in normal and pathological remodelling of epithelial-matrix interactions, and is upregulated in gastric cancer. Helicobacter pylori infection is the first stage in gastric carcinogenesis, and therefore our aim was to determine if H pylori upregulated gastric MMP-7 expression and if this was affected by strain virulence. METHODS: We took gastric biopsy specimens at endoscopy from H pylori infected (n = 17) and uninfected (n = 18) patients and assessed MMP-7 expression by ELISA, real time polymerase chain reaction (PCR), and immunohistochemistry (concentrating on epithelial cells in the proliferative zone). We PCR typed H pylori for cagE and vacA. We performed H pylori/cell line coculture studies with wild-type pathogenic and non-pathogenic H pylori strains and with CagE(-) and VacA(-) isogenic mutants. RESULTS: Gastric biopsy specimens from H pylori+ patients expressed higher levels of MMP-7 at the protein and mRNA levels in the antrum and corpus (for example, by ELISA: H pylori+ 0.182 OD units vH pylori- 0.059; p = 0.009 antrum). Epithelial cells from H pylori+ patients stained more intensely for MMP-7 than those from uninfected patients, including in the proliferative zone containing pluripotent cells (p<0.03 antrum, p<0.04 body). Upregulation of MMP-7 in epithelial cells was confirmed at the protein and mRNA levels by H pylori/cell line coculture. These experiments also showed that MMP-7 upregulation was dependent on an intact H pyloricag pathogenicity island but not on the vacuolating cytotoxin. CONCLUSION: We speculate that increased expression of MMP-7 in H pylori gastritis may contribute to gastric carcinogenesis.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Potential role of endocrine gastrin in the colonic adenoma carcinoma sequence.

The role of hyper-gastrinaemia in the incidence of colonic cancer remains to be clarified. The aim of this study was to determine whether cholecystokinin-2 (CCK-2) receptor expression predicts the sensitivity of human colonic adenomas to the proliferative effects of serum hyper-gastrinaemia. Gene expression of the classical (74 kDa) CCK-2 receptor in human colonic adenoma specimens and cell lines, was quantified by real-time PCR. Western blotting, using a CCK-2 receptor antiserum, confirmed protein expression. A transformed human colonic adenoma was grown in SCID mice, with hyper-gastrinaemia induced by proton pump inhibitors. CCK-2 receptor blockade was achieved by using neutralising antiserum. Both human colonic adenoma cell lines and biopsies expressed CCK-2 receptor mRNA at levels comparable with CCK-2 receptor transfected fibroblasts and oxyntic mucosa. Western blotting confirmed immunoreactive CCK-2 receptor bands localised to 45, 74 and 82.5 kDa. Omeprazole and lansoprazole-induced hyper-gastrinaemia (resulting in serum gastrin levels of 34.0 and 153.0 pM, respectively) significantly increased the weight of the human adenoma grafts (43% (P=0.016) and 70% (P=0.014), respectively). The effect of hypergastrinaemia on tumour growth was reversed by use of antiserum directed against the CCK-2 receptor. Hyper-gastrinaemia may promote proliferation of human colonic adenomas that express CCK-2 receptor isoforms.

Effect of preoperative radiotherapy on matrilysin gene expression in rectal cancer.

Matrilysin, a member of matrix metalloproteinase family, is believed to play a significant role in the growth and proliferation of colon cancer cells. Overexpression of the matrilysin gene has been shown to correlate with Dukes' stage and increased metastatic potential in colorectal cancer. The aim of this study was to evaluate the effect of preoperative high-dose radiotherapy (25 Gy in five fractions over 5 days) on matrilysin (MMP-7) gene expression, in patients with resectable rectal cancer, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Biopsy samples of tumour (n=30) and distant normal mucosa (n=12) from 15 patients were obtained pre- and post-radiotherapy. Messenger (m)RNA was extracted from all of the tissue samples and reverse transcribed to double-stranded cDNA. Quantitative RT-PCR was performed to study the effect of preoperative radiotherapy on matrilysin gene expression in both the tumour and normal mucosal specimens. Matrilysin mRNA values were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample. In 14 out of 15 cases, matrilysin mRNA was detected in the cancerous tissue. Although all six normal mucosal specimens expressed matrilysin mRNA, the levels were approximately 10-fold lower compared with those seen in the paired tumour samples. Preoperative radiotherapy led to a significant 6- to 7-fold increase (P=0.001) in the expression of matrilysin mRNA in rectal cancer tissue. In contrast, there was no significant change in the matrilysin mRNA expression of normal mucosal specimens post-radiotherapy. Preoperative high-dose radiotherapy upregulates matrilysin gene expression in rectal cancer. Matrilysin inhibition may be a useful preventive or therapeutic adjunct to radiotherapy in rectal cancer.