ADAMTS expression in colorectal cancer.

ADAMTSs are a family of secreted proteinases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). By acting on a large panel of extracellular substrates, they control several cell functions such as fusion, adhesion, proliferation and migration. Through their thrombospondin motifs they also possess anti-angiogenic properties. We investigated whether ADAMTSs participate in colorectal cancer progression and invasion. Their expression was investigated at both mRNA and protein levels. Using RT-PCR, the expression of ADAMTS-1, -4, -5 and ADAMTS-20 was estimated in colorectal tumors of different cancer stage and anatomic site and 3 cell lines of different aggressiveness. An overexpression of ADAMTS-4 and -5 was observed, especially in tissue samples, whereas ADAMTS-1 and -20 were found to be down-regulated. Western blot analysis further supported the RT-PCR findings, revealing in addition the degradation of ADAMTS-1 and -20 in cancer. In situ expression and localization of ADAMTS-1, -4, -5 and -20 was also investigated by immunohistochemical analysis. Our data suggest a positive correlation between ADAMTS-4 and -5 expression and cancer progression, in contrast with the anti-angiogenic members of the family, ADAMTS-1 and -20, which were found to be down-regulated. Our findings support the notion that overexpression of ADAMTS-4 and ADAMTS-5 in colorectal cancer might be a possible invasive mechanism of cancer cells in order to degrade proteoglycans of ECM.

Chondroitin synthases I, II, III and chondroitin sulfate glucuronyltransferase expression in colorectal cancer.

Glycosaminoglycans undergo significant structural alterations in cancer, namely in terms of their sulfation pattern and hydrodynamic size. Numerous studies have focused on this issue, and have demonstrated that glycosaminoglycans play a crucial role in cancer growth and invasion. However, the majority of the enzymes involved in glycosaminoglycan alterations have yet to be examined in detail. The present study focused on the expression of chondroitin-synthesizing enzymes in colorectal cancer. Specimens from healthy controls and cancer patients were subjected to RT-PCR analysis after RNA isolation, and to Western blotting after sequential extraction. The results indicated that chondroitin polymerizing factor and glucuronyltransferase gradually increased with cancer stage, and were expressed at much higher levels in adenomas compared to adjacent normal tissue. The opposite profile was obtained for chondroitin synthase I. Chondroitin synthase III was present at low levels in all the samples examined; however, its expression was higher in the samples from the cancer patients than in those from the healthy controls. It can therefore be concluded that, among the various factors regulating the structure of glycosaminoglycans in cancer, the differential expression of chondroitin-synthesizing enzymes is of the most significance.

Involvement of hyaluronidases in colorectal cancer.

BACKGROUND: Hyaluronidases belong to a class of enzymes that degrade, predominantly, hyaluronan. These enzymes are known to be involved in physiological and pathological processes, such as tumor growth, infiltration and angiogenesis, but their exact role in tumor promotion or suppression is not clear yet. Advanced colorectal cancer is associated with elevated amounts of hyaluronan of varying size. The aim of the present study was therefore to illuminate the importance of hyaluronidases in colon carcinoma progression. METHODS: The patients' samples (macroscopically normal and cancerous) were subjected to sequential extraction with PBS, 4 M GdnHCl and 4 M GdnHCl --1% Triton X-100. The presence of the various hyaluronidases in the extracts was examined by zymography and western blotting. Their expression was also examined by RT-PCR. RESULTS: Among hyaluronidases examined, Hyal-1, -2, -3 and PH-20 were detected. Their activity was higher in cancerous samples. Hyal-1 and Hyal-2 were overexpressed in cancerous samples, especially in advanced stages of cancer. Both isoforms were mainly extracted with PBS. Hyal-3 was observed only in the third extract of advanced stages of cancer. PH-20 was abundant in all three extracts of all stages of cancer. The expression of only Hyal-1 and PH-20 was verified by RT-PCR. CONCLUSION: A high association of hyaluronidases in colorectal cancer was observed. Each hyaluronidase presented different tissue distribution, which indicated the implication of certain isoforms in certain cancer stages. The results provided new evidence on the mechanisms involved in the progression of colorectal cancer.

Alterations of glycosaminoglycan disaccharide content and composition in colorectal cancer: structural and expressional studies.

The glycosaminoglycans are implicated in many processes important in the growth and progression of malignant tumors. In the present study glycosaminoglycans were purified from healthy, macroscopically normal and cancerous specimens of different anatomic sites and different stages of cancer and analyzed by FACE after chondroitinases and sulfatases digestion. The cancerous samples contained increased levels of 6-sulfated unsaturated disaccharides compared to macroscopically normal and healthy samples, the increase being stage-related. The differences in sulfation were found to be related to the anatomic site and the stage of cancer. RT-PCR analysis of 4-sulfotransferase mRNA revealed its presence in decreasing amounts as the stage of the cancer increased. Furthermore, the percent content of hyaluronan disaccharides was elevated in macroscopically normal samples compared to the cancerous, and in addition, it was much more elevated than that of healthy samples. Haluronan levels increase with stage in cancerous tissues. Therefore, it could be concluded that the glycosaminoglycans in colorectal cancer are biosynthetically directed to contribute in different ways depending on the cancer stage and anatomical site.

The greatly increased amounts of accumulated versican and decorin with specific post-translational modifications may be closely associated with the malignant phenotype of pancreatic cancer.

Pancreatic carcinoma (PC) is a cancer type with highly malignant growth and dissemination pattern of which the mechanisms are poorly understood. However, the malignant phenotype is closely linked to extracellular matrix (ECM) of which proteoglycans (PGs) and hyaluronan (HA) play a crucial role in the control of tumor progression and metastasis. In this study, we demonstrated that versican and decorin, two different PGs with contradictory roles and functions in the pathobiology of cancer, were the main matrix PGs in PC presenting a great increase 27- and 7-fold, respectively, in comparison to normal pancreas (NP). PC was characterized by the disproportional increase of versican compared to decorin, about 4 to 1, with a concurrent increase of HA, which may be closely associated with the growth and aggressiveness of this carcinoma. Significant specific post-translational modifications were also observed in both versican and decorin regarding the type, hydrodynamic size, sulfation pattern and extent of uronate epimerization of their glycosaminoglycan chains (GAGs). In particular, chondroitin sulphate (CS) was the predominant GAG type in both PC-associated versican and decorin. The CS of PC-decorin was increased 11-fold, compared to NP in which dermatan sulfate (DS) was the predominant GAG type in both PGs. The sulfation pattern of GAG chains was significantly altered in PC, since 6-sulfated disaccharides predominated in both versican and decorin with a marked presence of non-sulfated disaccharides accompanied by lower hydrodynamic sizes of both CS and DS chains compared to NP. In conclusion, all these findings agree with the highly malignant phenotype of this cancer and, thus, more studies need to be addressed on the roles of the post-translational modifications of versican and decorin in the biology of cancer.

Changes of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the serum of patients with autoimmune diseases: association with age and disease activity.

Matrix metalloproteinases participate in the degradation of the extracellular matrix proteins and are regulated mainly by their respective tissue inhibitors. In a variety of inflammatory connective tissue diseases, variations in the tissue content of both metalloproteinases and tissue inhibitors have been reported. The purpose of this study was to determine the serum levels of metalloproteinases and tissue inhibitors in patients with autoimmune diseases and compare with those of healthy individuals of similar age. The metalloproteinase content was analyzed by zymography and it was found that the serum levels of metalloproteinase-2 and metalloproteinase-9 of all autoimmune disease samples were decreased, in all diseases examined and independently of clinical activity, while those of active metalloproteinase-9 were significantly elevated. Both tissue inhibitors were quantitated by direct enzyme-linked immunosorbent assay and were also found decreased in autoimmune disease samples, confirming the balance that should exist in the secretion of metalloproteinases and tissue inhibitors. These results suggested that the increased active form of metalloproteinase-9, together with the decreased concentration of tissue inhibitor-2, could be used for diagnostic purposes and for the follow-up of patients with autoimmune diseases.