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Transforming growth factor-beta1 (TGF-beta1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF-beta1 levels in homogenates of colorectal neoplasia. In contrast to total TGF-b levels, showing a stepwise increase in the mucosa-adenoma-carcinoma sequence, active TGF-beta1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF-beta1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF-beta1 levels correlated significantly with plasminogen activator inhibitor (PAI)-1, alpha-smooth muscle actin (SMA) and several matrix-remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF-beta1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF-beta1 and SMA expression in tumors indicates that tumor-promoting myofibroblasts might arise as a result of increased TGF-beta1 activation. These data underline the significance of the interaction between malignant cells and (myo)-fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer.

Purpose: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy., Experimental Design: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay., Results: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001)., Conclusion: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.

Aim: To study the (functional) relevance of single nucleotide polymorphisms (SNPs) in genes encoding matrix metalloproteinases (MMP)-1, -2, -3, -9, tissue inhibitors of metalloproteinases (TIMP)-1, -2 and tumor necrosis factor (TNF)-alpha in the etiopathogenesis of inflammatory bowel diseases (IBD), that may enhance susceptibility and/or disease severity., Methods: Genomic DNA from 134 Crohn' s disease (CD), 111 ulcerative colitis (UC) patients and 248 control subjects was isolated from resected intestinal tissue or blood. Allelic composition at SNP loci was determined by PCR-RFLP or tetra primer ARMS PCR., Results: The TIMP-1 genotype TT in women and T in men at SNP +372 T/C was found to increase CD susceptibility (39% vs 23.8%, P = 0.018 and 67.9% vs 51.6%, P = 0.055, respectively), while women with this genotype were less prone to development of fistulae during follow-up (41.4% vs 68.3%, P = 0.025). Male IBD or CD patients carrying the TIMP-1 +372 T-allele expressed lower levels of TIMP-1 in surgically resected macroscopically inflamed tissue (0.065 < P < 0.01). The 5T5T genotype at MMP-3 SNP -1613 5T/6T increased the chance of stenotic complications in CD during follow-up (91.2% vs 71.8%, P = 0.022) but seemed to protect against colonic involvement of this disease at first endoscopic/radiologic examination (35.3% vs 59.5%, P = 0.017)., Conclusion: Allelic composition at the examined SNPs in genes coding for TIMP-1 and MMP-3 affect CD susceptibility and/or phenotype, i.e., fistulizing disease, stricture pathogenesis and first disease localisation. These findings reinforce the important role of these proteins in IBD.

Background: Previous studies have shown an upregulation of matrix metalloproteinases (MMPs) in intestinal tissue of patients with inflammatory bowel disease (IBD) and significant clinical improvement after administration of the anti-TNF-a antibody infliximab. The aims of our study were to determine expression and secretion of MMP-1, -2, -3, -9, and their inhibitors TIMP-1, -2 by IBD versus control intestinal mucosa ex vivo and to assess the regulatory capacity by infliximab of the proteolytic phenotype., Methods: Intestinal mucosal explants from 20 IBD and 15 control patients were cultured with or without infliximab and/or the T-cell activator pokeweed mitogen (PWM). Explants and culture supernatants were analyzed for MMPs, TIMPs, and TNF-alpha protein, activity and/or mRNA levels. All patients were genotyped for functional TNF-alpha, MMP, and TIMP single nucleotide polymorphism (SNP) loci., Results: Expression of MMP and TIMP protein/activity in basal medium was higher in IBD versus control explants. Dependent on genotype at SNP loci, infliximab downregulated MMP-1, -3, and -9 relative to TIMP-1 and -2 and also decreased MMP-1 and -3 activities, while PWM enhanced these levels, partly counteracted again by infliximab. The expression of MMP-2 relative to TIMP did not change by treatment with infliximab and/or PWM., Conclusions: The high expression of MMPs in patients with IBD suggests a role for these proteinases in the pathogenesis of this disease. Infliximab seems to induce a genotype-associated matrix protective phenotype, which may contribute to the observed therapeutic efficacy of this drug in IBD, particularly at the mucosal surface.

Purpose: Metallothionein (MT) is a small protein with a high affinity for divalent heavy metal ions. This metalloproteinis involved in many (patho)physiological processes, like metal homeostasis and detoxification, cell proliferation, apoptosis, therapy resistance, and protection against oxidative damage. Alterations in the immunohistochemical expression of MT have been reported for various human tumors, and a high expression has been found to be associated with a poor clinical outcome. We showed previously that gastrointestinal cancer is accompanied by a decrease in MT expression, but the most malignant phenotypes had the highest MT levels. The purpose of the present study was to assess the clinical relevance of MT in gastrointestinal cancer., Experimental Design: In this study, we determined the MT levels, by radioimmunoassay, in intestinal tissue of 251 patients with colorectal cancer and 81 patients with gastric cancer and assessed the relation with the overall survival of these patients., Results: More than 74% of the carcinomas were found to have a lower MT level than their corresponding normal mucosa. In colorectal cancer patients, but not in gastric cancer patients, a high MT level in both the carcinomas and normal mucosa was, however, significantly associated with a poor overall survival, independently from clinicopathological features., Conclusions: Overexpression of MT in intestinal tissue of colorectal cancer patients is a prognostic marker for a poor overall survival. In gastric cancer, however, MT expression in the gastric mucosa is not of prognostic significance. This observation emphasizes the clinical relevance of this multifunctional metalloprotein in colorectal carcinogenesis and therapy.