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Peccerella T, Lukan N, Hofheinz R, Schadendorf D, Kostrezewa M, Neumaier M, Findeisen P. Endoprotease Profiling with Double-Tagged Peptide Substrates: A New Diagnostic Approach in Oncology. Clin Chem 2010; 56:272-80. [DOI: 10.1373/clinchem.2009.133462] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Abstract
Background: The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens.
Methods: The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase “cancer procoagulant.”
Results: Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001).
Conclusions: The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.
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Affiliation(s)
- Teresa Peccerella
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Nadine Lukan
- III. Medical Clinic, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Ralf Hofheinz
- III. Medical Clinic, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Dirk Schadendorf
- Skin Cancer Unit, German Cancer Research Center Heidelberg, and Department of Dermatology, University Hospital of Mannheim, Mannheim, Germany
| | | | - Michael Neumaier
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Peter Findeisen
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
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Weiswald LB, Richon S, Validire P, Briffod M, Lai-Kuen R, Cordelières FP, Bertrand F, Dargere D, Massonnet G, Marangoni E, Gayet B, Pocard M, Bieche I, Poupon MF, Bellet D, Dangles-Marie V. Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness. Br J Cancer 2009; 101:473-82. [PMID: 19603013 PMCID: PMC2720229 DOI: 10.1038/sj.bjc.6605173] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Background: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. Methods: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. Results: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. Conclusion: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.
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Affiliation(s)
- L-B Weiswald
- IFR71 Sciences du Médicament, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 avenue de l'Observatoire, F-75006 Paris, France
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Kim YS, Hwang SY, Kang HY, Sohn H, Oh S, Kim JY, Yoo JS, Kim YH, Kim CH, Jeon JH, Lee JM, Kang HA, Miyoshi E, Taniguchi N, Yoo HS, Ko JH. Functional proteomics study reveals that N-Acetylglucosaminyltransferase V reinforces the invasive/metastatic potential of colon cancer through aberrant glycosylation on tissue inhibitor of metalloproteinase-1. Mol Cell Proteomics 2008; 7:1-14. [PMID: 17878270 DOI: 10.1074/mcp.m700084-mcp200] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
N-Acetylglucosaminyltransferase-V (GnT-V) has been reported to be up-regulated in invasive/metastatic cancer cells, but a comprehensive understanding of how the transferase correlates with the invasive/metastatic potential is not currently available. Through a glycomics approach, we identified 30 proteins, including tissue inhibitor of metalloproteinase-1 (TIMP-1), as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 was aberrantly glycosylated as characterized by the addition of beta1,6-N-acetylglucosamine, polylactosaminylation, and sialylation in GnT-V-overexpressing WiDr cells. Compared with normal TIMP-1, the aberrantly glycosylated TIMP-1 showed the weaker inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro. Integrated data, both of TIMP-1 expression level and aberrant glycosylation, could provide important information to aid to improve the clinical outcome of colon cancer patients.
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Affiliation(s)
- Yong-Sam Kim
- Daejeon-KRIBB-Fred Hutchinson Cancer Research Center Research Cooperation Center, Daejeon, Korea
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Cohen PA, Gower AC, Stucchi AF, Leeman SE, Becker JM, Reed KL. A neurokinin-1 receptor antagonist that reduces intraabdominal adhesion formation increases peritoneal matrix metalloproteinase activity. Wound Repair Regen 2007; 15:800-8. [DOI: 10.1111/j.1524-475x.2007.00291.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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Shen D, Deng C, Zhang M. Peroxisome proliferator-activated receptor gamma agonists inhibit the proliferation and invasion of human colon cancer cells. Postgrad Med J 2007; 83:414-9. [PMID: 17551074 PMCID: PMC2600042 DOI: 10.1136/pmj.2006.052761] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2006] [Accepted: 01/16/2007] [Indexed: 11/04/2022]
Abstract
BACKGROUND AND AIMS Data regarding the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) ligands on the invasive ability of colon cancer cells are currently limited. This study was designed to examine the effects of PPAR-gamma agonists on the proliferation and invasion of two colon cancer cells to identify the role of PPAR-gamma in colon cancer growth and metastasis. METHODS SW480 and LS174T cells were treated with PPAR-gamma ligands, pioglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), as well as their combinations with the PPAR-gamma antagonist GW9662. MTT assay was used to determine the antiproliferative effects. Cell cycle analysis was conducted by flow cytometry. The mRNA and protein expression were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot, respectively. The invasive ability of cells was determined by the BD BioCoat Matrige invasion chamber. RESULTS Pioglitazone and 15d-PGJ2 inhibited the proliferation of both colon cancer cell lines in a dose-dependent manner. This growth inhibitory effect was reversed by GW9662. Results from flow cytometry demonstrated G1 arrest following treatment with pioglitazone and 15d-PGJ2. The expression of matrix metalloproteinase-7 (MMP-7) was only detected in LS174T cells, while its tissue inhibitor-1 (TIMP-1) was expressed in both colon cancer cells. 15d-PGJ2 and pioglitazone downregulated MMP-7 expression and upregulated TIMP-1 expression. PPAR-gamma agonists can only inhibit invasive activity of LS174T cells. CONCLUSIONS PPAR-gamma agonists have inhibitory effects on the proliferation of colon cancer cell lines associated with G1 cell cycle arrest and invasive activity. The latter effect is demonstrated in certain cell lines through the down-regulation of MMP-7 synthesis.
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Affiliation(s)
- Dan Shen
- Department 1 of Internal Medicine & Geriatrics, Zhongnan Hospital, Wuhan University, Wuhan, PR China
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Rath T, Roderfeld M, Graf J, Wagner S, Vehr AK, Dietrich C, Geier A, Roeb E. Enhanced expression of MMP-7 and MMP-13 in inflammatory bowel disease: a precancerous potential? Inflamm Bowel Dis 2006; 12:1025-1035. [PMID: 17075343 DOI: 10.1097/01.mib.0000234133.97594.04] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Matrix metalloproteinases (MMPs) are responsible for the turnover and degradation of extracellular matrix. They play a crucial role in the growth and migration of colorectal carcinoma cells. Colorectal carcinomas are characterized by enhanced expression of MMP-2, MMP-9, MMP-7, and MMP-13. The aim of this study was to determine the expression levels of MMP-2, MMP-9, MMP-7, MMP-13, and MMP-14 and their specific inhibitor TIMP-1 in inflammatory bowel diseases and precancerous lesions of the colon, i.e., Crohn's disease and ulcerative colitis, and in adenomatous polyps (APs) for comparison. Biopsy samples of pathological and healthy tissue were obtained from 40 patients with inflammatory bowel disease (ulcerative colitis, n = 17; Crohn's disease, n = 23) and from 19 patients with APs. mRNA was measured by quantitative real-time polymerase chain reaction to study MMP and TIMP-1 gene expression in both pathological and normal mucosal specimens. For MMP-2, MMP-9, and TIMP-1, protein expression also was quantified with sandwich enzyme-linked immunosorbent assay. In biopsy specimens of Crohn's disease and ulcerative colitis, significantly increased levels of MMP-2, MMP-7, and MMP-13 mRNA were found. MMP-2 and MMP-9 showed enhanced secretion on the protein level. AP revealed an increased transcription of MMP-7 and MMP-13 genes. MMP-14 mRNA was decreased in APs. MMPs, especially MMP-7 and MMP-13, which are expressed primarily on the tumor cell surface, are elevated in inflammatory bowel disease, which may have more chance to evolve into malignancy than normal tissue. In APs, increased expression of MMP-7 and MMP-13 may serve as an early indicator for colorectal carcinogenesis.
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Affiliation(s)
- Timo Rath
- Department of Internal Medicine III, University Hospital RWTH, Aachen, Germany
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7
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Sun XF, Zhang H. Clinicopathological significance of stromal variables: angiogenesis, lymphangiogenesis, inflammatory infiltration, MMP and PINCH in colorectal carcinomas. Mol Cancer 2006; 5:43. [PMID: 17026740 PMCID: PMC1618857 DOI: 10.1186/1476-4598-5-43] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2006] [Accepted: 10/06/2006] [Indexed: 02/08/2023] Open
Abstract
Cancer research has mainly focused on alterations of genes and proteins in cancer cells themselves that result in either gain-of-function in oncogenes or loss-of-function in tumour-suppressor genes. However, stromal variables within or around tumours, including blood and lymph vessels, stromal cells and various proteins, have also important impacts on tumour development and progression. It has been shown that disruption of stromal-epithelial interactions influences cellular proliferation, differentiation, death, motility, genomic integrity, angiogenesis, and other phenotypes in various tissues. Moreover, stromal variables are also critical to therapy in cancer patients. In this review, we mainly focus on the clinicopathological significance of stromal variables including angiogenesis, lymphangiogenesis, inflammatory infiltration, matrix metalloproteinase (MMP), and the particularly interesting new cysteine-histidine rich protein (PINCH) in colorectal cancer (CRC).
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Affiliation(s)
- Xiao-Feng Sun
- Department of Oncology, Institute of Biomedicine and Surgery, University of Linköping, SE-581 85 Linköping, Sweden
| | - Hong Zhang
- Department of Dermatology, Institute of Biomedicine and Surgery, University of Linköping, SE-581 85 Linköping, Sweden
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Engwegen JYMN, Helgason HH, Cats A, Harris N, Bonfrer JMG, Schellens JHM, Beijnen JH. Identification of serum proteins discriminating colorectal cancer patients and healthy controls using surface-enhanced laser desorption ionisation-time of flight mass spectrometry. World J Gastroenterol 2006; 12:1536-44. [PMID: 16570345 PMCID: PMC4124285 DOI: 10.3748/wjg.v12.i10.1536] [Citation(s) in RCA: 117] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To detect the new serum biomarkers for colorectal cancer (CRC) by serum protein profiling with surface-enhanced laser desorption ionisation - time of flight mass spectrometry (SELDI-TOF MS).
METHODS: Two independent serum sample sets were analysed separately with the ProteinChip technology (set A: 40 CRC + 49 healthy controls; set B: 37 CRC + 31 healthy controls), using chips with a weak cation exchange moiety and buffer pH 5. Discriminative power of differentially expressed proteins was assessed with a classification tree algorithm. Sensitivities and specificities of the generated classification trees were obtained by blindly applying data from set A to the generated trees from set B and vice versa. CRC serum protein profiles were also compared with those from breast, ovarian, prostate, and non-small cell lung cancer.
RESULTS: Mass-to-charge ratios (m/z) 3.1×103, 3.3×103, 4.5×103, 6.6×103 and 28×103 were used as classifiers in the best-performing classification trees. Tree sensitivities and specificities were between 65% and 90%. Most of these discriminative m/z values were also different in the other tumour types investigated. M/z 3.3×103, main classifier in most trees, was a doubly charged form of the 6.6×103-Da protein. The latter was identified as apolipoprotein C-I. M/z 3.1×103 was identified as an N-terminal fragment of albumin, and m/z 28×103 as apolipoprotein A-I.
CONCLUSION: SELDI-TOF MS followed by classification tree pattern analysis is a suitable technique for finding new serum markers for CRC. Biomarkers can be identified and reproducibly detected in independent sample sets with high sensitivities and specificities. Although not specific for CRC, these biomarkers have a potential role in disease and treatment monitoring.
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Affiliation(s)
- Judith Y M N Engwegen
- Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute/Slotervaart Hospital, PO Box 90440, 1006 BK Amsterdam, The Netherlands.
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Baker EA, Leaper DJ, Hayter JP, Dickenson AJ. The matrix metalloproteinase system in oral squamous cell carcinoma. Br J Oral Maxillofac Surg 2005; 44:482-6. [PMID: 16338034 DOI: 10.1016/j.bjoms.2005.10.005] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2005] [Accepted: 10/13/2005] [Indexed: 11/30/2022]
Abstract
BACKGROUND The matrix metalloproteinase (MMP) system is responsible for degradation of tissue in both normal and pathological processes, including tumour invasion and metastasis. AIM To compare tissue concentrations of components of the MMP system between tumour tissue and normal tissue in patients with oral squamous cell carcinoma, and to correlate concentrations with pathological grade of tumour. METHODS Thirty-eight paired tissue samples from tumours and normal tissue were analysed by three laboratory techniques: firstly, enzyme linked immunosorbent assays (ELISA) in ng/mg protein for MMP-1, MMP-3, and tissue inhibitors of metalloproteinases (TIMPs) -1 and -2. Secondly, gelatinase activity assays to measure concentrations of total and endogenous active gelatinases, MMP-2 and MMP-9 (ng/mg protein). And thirdly to use quenched fluorescent substrate hydrolysis to measure total MMP activity (pM/min). RESULTS The concentration of all MMPs was significantly higher in tumour than in normal oral tissue (p < 0.05, Mann-Whitney U-test). Tissue concentrations of some of these factors correlated with clinical and pathological indices of aggressiveness of tumours, including T-stage, N-stage, tumour differentiation, and anatomical level of involved nodes. However, the study was not powered to show statistical significance. CONCLUSION It is the balance between proteinases and their inhibitors that controls tissue degradation at each stage of tumour invasion and metastasis. Measurement of MMPs in oral mucosal biopsy samples may establish the invasive potential of tumours at their initial presentation.
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Affiliation(s)
- E A Baker
- Professorial Unit of Surgery, University Hospital of North Tees, Stockton on Tees TS19 8PE, UK.
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Altorjay A, Paal B, Sohar N, Kiss J, Szanto I, Sohar I. Significance and prognostic value of lysosomal enzyme activities measured in surgically operated adenocarcinomas of the gastroesophageal junction and squamous cell carcinomas of the lower third of esophagus. World J Gastroenterol 2005; 11:5751-6. [PMID: 16270380 PMCID: PMC4479671 DOI: 10.3748/wjg.v11.i37.5751] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish whether there are fundamental differences in the biochemistries of adenocarcinomas of the gastroesophageal junction (GEJ) and the squamous cell carcinomas of the lower third of the esophagus (LTE).
METHODS: Between February 1, 1997 and February 1, 2000, we obtained tissue samples at the moment of resection from 54 patients for biochemical analysis. The full set of data could be comprehensively analyzed in 47 of 54 patients samples (81%). Of these, 29 were adenocarcinomas of the GEJ Siewert type I (n = 8), type II (n = 12), type III (n = 9), and 18 presented as squamous cell carcinomas of the LTE. We evaluated the mean values of 11-lysosomal enzyme and 1-cytosol protease activities of the tumorous and surrounding mucosae as well as their relative activities, measured as the ratio of activity in tumor and normal tissues from the same patient. These data were further analyzed to establish the correlation with tumor localization, TNM stage (lymph-node involvement), histological type (papillary, signet-ring cell, tubular), state of differentiation (good, moderate, poor), and survival (≤ 24 or ≥ 24 mo).
RESULTS: In adenocarcinomas, the activity of α-mannosidase (AMAN), cathepsin B (CB) and dipeptidyl-peptidase I (DPP I) increased significantly as compared to the normal gastric mucosa. In squamous cell carcinomas of the esophagus, we also found a significant difference in the activity of cathepsin L and tripeptidyl-peptidase I in addition to these three. There was a statistical correlation of AMAN, CB, and DPP I activity between the level of differentiation of adenocarcinomas of the GEJ and lymph node involvement, because tumors with no lymph node metastases histologically confirmed as well-differentiated, showed a significantly lower activity. The differences in CB and DPP I activity correlated well with the differences in survival rates, since the CB and DPP I values of those who died within 24 mo following surgical intervention were significantly higher than of those who survived for 2 years or more.
CONCLUSION: Adenocarcinomas of the GEJ form a homogenous group from a tumor-biochemical aspect, and differ from the biochemical characteristics of squamous cell carcinomas of the LTE on many points. When adenocarcinomas of the GEJs are examined at the preoperative phase, the ratio of the performed AMAN, CB, and DPP I enzymatic activity of the tissue sample from the tumor and adjacent intact mucosa within2 cm of the tumor may have a prognostic value even in the preoperative examination period, and may indicate that ranking of these patients into the neo-adjuvant treatment group should be considered.
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Affiliation(s)
- Aron Altorjay
- Department of Surgery, Saint George University Teaching Hospital, Seregélyesi u. 3., Székesfehérvár, H-8000, Hungary.
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Baker EA, Leaper DJ. Profiles of matrix metalloproteinases and their tissue inhibitors in intraperitoneal drainage fluid: relationship to wound healing. Wound Repair Regen 2003; 11:268-74. [PMID: 12846914 DOI: 10.1046/j.1524-475x.2003.11406.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Matrix degradation and remodeling occurs during wound healing, thereby aiding tissue repair, angiogenesis, and cell migration. It is dependent on the balance between proteinases and their inhibitors, namely the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Acute wound fluid samples (n = 58 patients) were collected daily from the intraperitoneal drain placed after colorectal surgery from the first postoperative day until drain removal. Three laboratory techniques were performed: enzyme linked immunosorbent assays (MMP-1, MMP-3, TIMP-1, TIMP-2), gelatinase activity assays (MMP-2, MMP-9), and quenched fluorescent substrate hydrolysis (total MMP activity). Levels were correlated with each postoperative day, wound healing, and surgical outcome (p < 0.05, Spearman's correlation). Significant negative (MMP-9, MMP-3, MMP-8, TIMP-2, total MMP activity) and positive (MMP-2, TIMP-1) correlations were observed with the postoperative day, e.g., total MMP-9: day 1, median, 121 (range, 12-189) ng/ml; day 3, 46 (8-179); day 5, 31 (0-155), day 7, 20 (6-58). Differences were also observed with the type of operation, estimated blood loss, and length of operation and with postoperative complications. MMPs and TIMPs are involved in wound healing after elective colorectal cancer surgery and their levels in drain fluid may act as markers of wound healing and surgical outcome.
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Affiliation(s)
- Elizabeth A Baker
- Professional Unit of Surgery, University Hospital of North Tees, Stockton on Tees, United Kingdom.
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Underwood CK, Min D, Lyons JG, Hambley TW. The interaction of metal ions and Marimastat with matrix metalloproteinase 9. J Inorg Biochem 2003; 95:165-70. [PMID: 12763661 DOI: 10.1016/s0162-0134(03)00100-4] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The effect of a range of metal ions on the ability of Marimastat to inhibit matrix metalloproteinase 9 (MMP-9) was examined in a fluorescence based proteolytic assay. Whilst none of the metals examined significantly affected the inhibitory ability of Marimastat, several metal ions did have a significant effect on MMP-9 activity itself. In the absence of Marimastat, Zn(II) and Fe(II) significantly inhibited MMP-9 activity at metal ion concentrations of 10 and 100 microM, respectively. In both the absence and presence of Marimastat, Cd(II) significantly inhibited MMP-9 at 100 microM. In contrast, 1 mM Co(II) significantly upregulated MMP-9 proteolytic activity.
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Affiliation(s)
- C K Underwood
- Centre for Heavy Metals Research, School of Chemistry, University of Sydney NSW, 2006 Australia
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Baker EA, Leaper DJ. The plasminogen activator and matrix metalloproteinase systems in colorectal cancer: relationship to tumour pathology. Eur J Cancer 2003; 39:981-8. [PMID: 12706368 DOI: 10.1016/s0959-8049(03)00065-0] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The aim of this study was to determine the expression of proteinases and inhibitors from the matrix metalloproteinase (MMP) (MMPs 1, 2, 3, 9, tissue inhibitors of metalloproteinases (TIMPs) 1, 2) and plasminogen activator ((PA) urokinase (uPA), tissue type (tPA), uPAR, plasminogen activator inhibitors (PAIs) 1, 2) systems in colorectal cancer pathology by gelatin zymography, enzyme-linked immunosorbent assays (ELISAs) and quenched fluorescent substrate hydrolysis. The levels of all studied MMPs, uPA, uPAR, TIMP-1 and PAIs were significantly greater in tumour tissues than normal tissues. However, tPA and TIMP-2 were greater in normal colon (P<0.05, Mann-Whitney) e.g. PAI-1: tumour, median 14.9 (range 0.2-80.2) ng/mg total protein; normal, 2.1 (0.1-65.0). Tumour levels of several factors, in particular MMP-1 and PAI-1, correlated with pathology, i.e. Dukes' stage, differentiation, lymphatic or vascular invasion and tumour depth. The interactions between proteinase systems in colorectal cancer are complex and the balance between active proteinases and their inhibitors is important for extracellular matrix (ECM) degradation/remodelling at each stage of the metastatic cascade.
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Affiliation(s)
- E A Baker
- Professorial Unit of Surgery, University Hospital of North Tees, Stockton on Tees, TS19 8P, UK.
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Long Y, Zou L, Liu H, Lu H, Yuan X, Robertson CS, Yang K. Altered expression of randomly selected genes in mouse hippocampus after traumatic brain injury. J Neurosci Res 2003; 71:710-20. [PMID: 12584729 DOI: 10.1002/jnr.10524] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Using a cDNA microarray method, we analyzed gene expression profiles in mouse hippocampus after traumatic brain injury (TBI). Of 6,400 randomly selected arrayed genes and expressed sequence tags from a mouse cDNA library, 253 were found to be differentially expressed (106 increased and 147 decreased). Genes involved in cell homeostasis and calcium signaling were primarily up-regulated while those encoding mitochondrial enzymes, metabolic molecules, and structural proteins were predominantly down-regulated. Equal numbers of genes related to inflammatory reactions showed increased or decreased expression. Importantly, a large proportion of the dysregulated genes we identified have not been reported as differentially expressed in TBI models. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analyses of representative genes confirmed the validity of the corresponding microarray findings. Thus, our microarray-based evaluation of gene expression in traumatically injured hippocampus identified both known and novel genes that respond to TBI. Further investigation of these candidate molecules may suggest new ways to attenuate the traumatic effects of brain injury.
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Affiliation(s)
- Yan Long
- Department of Neurosurgery, Baylor College of Medicine, Houston, Texas, USA
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Albo D, Shinohara T, Tuszynski GP. Up-regulation of matrix metalloproteinase 9 by thrombospondin 1 in gastric cancer. J Surg Res 2002; 108:51-60. [PMID: 12443715 DOI: 10.1006/jsre.2002.6452] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND Matrix metalloproteinase 9 (MMP-9) plays a key role in tumor cell invasion. It was recently reported that plasma levels of MMP-9 in patients with gastric cancer correlate with the tumors' metastatic potential. We previously demonstrated that thrombospondin 1 (TSP-1) up-regulates MMP-9 expression by endothelial cells and promotes tumor cell invasion. We hypothesized that TSP-1 plays a role in the up-regulation of MMP-9 in gastric cancer. METHODS MMP-9, TSP-1, and CSVTCG-specific TSP-1 receptor expression were measured by immunohistochemical staining in 31 consecutive gastric adenocarcinomas from patients who did not undergo neoadjuvant chemotherapy or radiation therapy. Additionally, we measured TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 expression by Western blotting, zymography, and immunohistochemical staining in AGS gastric adenocarcinoma cells. We also investigated the effect of TSP-1 on MMP-9 expression by AGS cells. RESULTS TSP-1 localized to the tumor-associated extracellular matrix. CSVTCG-specific TSP-1 receptor and MMP-9 colocalized to tumor cells, fibroblasts, and tumor-associated microvessels. Intense staining for TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 correlated with markers of aggressive tumor behavior. AGS gastric adenocarcinoma cells expressed high levels of CSVTCG-specific TSP-1 receptor but not TSP-1. TSP-1 up-regulated MMP-9 expression by AGS cells. CONCLUSIONS We conclude that TSP-1 plays a role in the up-regulation of MMP-9 expression in gastric cancer. Our data also suggest a correlation between expression of TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 and the acquisition of an aggressive tumor phenotype.
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Affiliation(s)
- Daniel Albo
- Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
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Oka Y, Naito I, Manabe K, Sado Y, Matsushima H, Ninomiya Y, Mizuno M, Tsuji T. Distribution of collagen type IV alpha1-6 chains in human normal colorectum and colorectal cancer demonstrated by immunofluorescence staining using chain-specific epitope-defined monoclonal antibodies. J Gastroenterol Hepatol 2002; 17:980-6. [PMID: 12167119 DOI: 10.1046/j.1440-1746.2002.02789.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
BACKGROUND AND AIM Loss of basement membrane (BM) components, such as type IV collagen, has been demonstrated in colorectal cancer, but the fine diversity of the assembly of alpha (IV) chains, the composition of type IV collagen, and alterations in the collagen have not been fully analyzed. Here, we defined immunohistochemically the expression of alpha1-6 (IV) chains in colorectal cancer tissues and adjacent normal mucosa by the use of chain-specific monoclonal antibodies. METHODS Tissue samples of tumor and adjacent normal mucosa obtained from patients with colorectal adenocarcinoma were stained with chain-specific monoclonal antibodies raised against synthetic peptides of individual alpha (IV) chains using an indirect immunofluorescence method. RESULTS In the normal mucosa, alpha1 (IV), alpha2 (IV), alpha5 (IV), and alpha6 (IV) were found in the BM-delineating mucosal epithelium and the gland crypts, whereas alpha3 (IV) and alpha4 (IV) were limited to the BM of the luminal surface epithelium. In contrast, staining of alpha3-6 (IV) was rarely observed in the BM of cancer cells. Staining of alpha1 (IV) and alpha2 (IV) was reduced or lost from the cancer BM in relation to the degree of tumor differentiation: continuous staining in well-differentiated portions, discontinuous staining in moderately differentiated portions, and absence of staining in poorly differentiated portions. CONCLUSIONS Our findings indicate that type IV collagen expression is altered in the BM of colorectal cancer as a result of changes of alpha (IV) chain expression, particularly alpha1 (IV) and alpha2 (IV), in relation to the degree of tumor differentiation.
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Affiliation(s)
- Yuko Oka
- Department of Internal Medicine, Shigei Medical Research Institute and Hospital, Okayama, Japan
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Ornstein DL, Cohn KH. Balance between activation and inhibition of matrix metalloproteinase-2 (MMP-2) is altered in colorectal tumors compared to normal colonic epithelium. Dig Dis Sci 2002; 47:1821-30. [PMID: 12184536 DOI: 10.1023/a:1016456914723] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Matrix metalloproteinase-2 (MMP-2) is overexpressed in human cancers and facilitates tumor growth and metastasis. It is synthesized as an inactive proenzyme that is activated by membrane-type matrix metalloproteinase-1 (MT1-MMP) and inhibited by tissue inhibitor of metalloproteinase-2 (TIMP-2). We hypothesized that there is an imbalance between the expression of TIMP-2 and the expression of MMP-2 and MT1-MMP that favors activation of MMP-2 in malignant colon tumors compared to normal colonic tissue. Specimens of colon tumors and of adjacent normal mucosa were obtained from 22 patients at the time of surgical resection. MMP-2, MT1-MMP, and TIMP-2 RNA transcripts were measured in each sample using a quantitative reverse transcriptase polymerase chain reaction assay. We observed that MMP-2 RNA levels were significantly elevated in tumors compared to normal tissue (P = 0.039). In addition, the TIMP-2:MMP-2 ratio was twofold lower (P = 0.001) and the TIMP-2:MT1-MMP ratio was 1.5-fold lower (P = 0.003) in tumors compared to normal mucosa. These results suggest that the balance between genes that activate and inhibit MMP-2 is shifted toward activation in colon tumors. The abnormal expression of gene products that regulate MMP-2 activity may be an important early step in the malignant transformation of colon cancer and may provide a useful target for new chemoprevention and adjuvant treatment strategies.
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Waas ET, Lomme RMLM, DeGroot J, Wobbes T, Hendriks T. Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer. Br J Cancer 2002; 86:1876-83. [PMID: 12085179 PMCID: PMC2375422 DOI: 10.1038/sj.bjc.6600366] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2002] [Revised: 04/05/2002] [Accepted: 04/11/2002] [Indexed: 01/02/2023] Open
Abstract
The bioactivity of matrix metalloproteinases was studied in tissues from colorectal cancer patients by means of both quantitative gelatin zymography and a fluorometric activity assay. Next to paired samples of tumour tissue and distant normal mucosa (n=73), transitional tissue was analysed from a limited (n=33) number of patients. Broad-spectrum matrix metalloproteinase activity and both the active and latent forms of the gelatinases matrix metalloproteinase-2 and -9 were higher in tumour than in normal mucosa. The ratio's between active and latent forms of matrix metalloproteinase-2 and -9 were highest in tumour tissue and normal mucosa, respectively. Matrix metalloproteinase-2 levels, both active and latent forms, correlated inversely with stage of disease, the tumours without synchronous distant metastases containing significantly (P=0.005) more active matrix metalloproteinase-2 than the others. At much lower levels of activity, the same trend was observed in distant normal mucosa. The level of latent form of matrix metalloproteinase-9 in tumour depended on tumour location. Neither the active form of matrix metalloproteinase-9 nor broad-spectrum matrix metalloproteinase activity in tumour tissue did correlate with any of the clinicopathological parameters investigated. The results demonstrate explicit differences between the activity of matrix metalloproteinase-2 and -9, indicating different roles for both gelatinases in tumour progression. Such data are necessary in order to develop rational anti-cancer therapies based on inhibition of specific matrix metalloproteinases.
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Affiliation(s)
- E T Waas
- Department of Surgery, University Medical Centre St Radboud, Nijmegen PO Box 9101, 6500 HB, The Netherlands
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Guo M, Mathieu PA, Linebaugh B, Sloane BF, Reiners JJ. Phorbol ester activation of a proteolytic cascade capable of activating latent transforming growth factor-betaL a process initiated by the exocytosis of cathepsin B. J Biol Chem 2002; 277:14829-37. [PMID: 11815600 DOI: 10.1074/jbc.m108180200] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-beta in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and beta-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-beta antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-beta.
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Affiliation(s)
- Meng Guo
- Institute of Environmental Health Sciences, Wayne State University and the Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA
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Abstract
Matrix metalloproteinases (MMPs) appear to play a key role in the development and progression of human malignancies. MMPs mediate the destruction of the extracellular matrix, which is an important early step in tumor invasion and metastasis. Growing evidence suggests that MMPs also have angiogenic activity and participate in the early stages of tumorigenesis and primary tumor growth. Investigations in experimental animal models have confirmed the importance of MMPs in the pathogenesis of colorectal cancer, and studies in humans show a direct association between increased MMP expression and tumor invasiveness, development of metastases, and shortened survival. In this review, the physiologic role of MMPs in normal tissues is examined and data supporting the role of MMPs in the pathogenesis of colorectal cancer are reviewed. The results of clinical trials with MMP inhibitors in colorectal cancer and promising areas for future investigation are also discussed.
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Affiliation(s)
- Angela G Mysliwiec
- Department of Hematology/Oncology, Brooke Army Medical Center, San Antonio, TX, USA
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Baker EA, Leaper DJ. Measuring gelatinase activity in colorectal cancer. EUROPEAN JOURNAL OF SURGICAL ONCOLOGY 2002; 28:24-9. [PMID: 11869009 DOI: 10.1053/ejso.2001.1179] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
AIMS Gelatinases (MMP-2, -9) are the most extensively studied MMPs in cancer. The aim of the study was to determine the levels of active and latent forms of the gelatinases in paired colorectal tumour and normal tissue ( n=77) and correlate these with pathological stage. METHODS Gelatinase levels were compared following the techniques of gelatin zymography (active and latent) and the novel gelatinase activity assays (total and endogenous/active). RESULTS Both latent and active MMP-2 and MMP-9 lysis bands (zymography) and both total (active and latent) MMP-9 and endogenous (active) MMP-9 and MMP-2 levels (activity assays) were greater in tumour than normal colorectal tissue. Total MMP-2 and MMP-9 levels as determined by activity assays correlated with both the Dukes staging (e.g. total MMP-9 in tumours: adenoma, 1.0 (0.3--3.6); Dukes A, 9.6 (2.4--35.4); Dukes B, 14.7 (1.5--103.9); Dukes C, 22.3 (2.2--57.9) and Dukes D, 37.4 (2.1--47.0) ng/mg protein) and with lymphatic invasion (e.g. total MMP-9; in tumours which had undergone lymphatic invasion, 22.7 (2.1--57.9) and those with no lymphatic invasion, 14.0 (1.5--103.9) ng/mg protein). CONCLUSIONS Both gelatinases were upregulated in tumour tissue, however total and not endogenous active levels correlated with the pathological stage of the tumour. Therefore gelatinases may only be activated when required for tumour invasion and metastasis.
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Affiliation(s)
- E A Baker
- Professional Unit of Surgery, University Hospital of North Tees, Stockton on Tees, UK.
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Roeb E, Dietrich CG, Winograd R, Arndt M, Breuer B, Fass J, Schumpelick V, Matern S. Activity and cellular origin of gelatinases in patients with colon and rectal carcinoma differential activity of matrix metalloproteinase-9. Cancer 2001; 92:2680-91. [PMID: 11745204 DOI: 10.1002/1097-0142(20011115)92:10<2680::aid-cncr1622>3.0.co;2-7] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
BACKGROUND Expression and enzymatic activity of gelatinases were examined in biopsy specimens from patients with colon and rectal neoplasms. The objective of this study was to determine whether the activity of these enzymes is altered between tumor areas compared with areas of noninvolved, normal mucosa and between colon and rectal carcinoma. METHODS Matrix metalloproteinase (MMP) production was analyzed by Western immunoblot analysis and gelatin zymography. mRNA was determined by quantitative, real-time polymerase chain reaction analysis. RESULTS Patients with colon carcinoma (n = 20 patients) showed a significant increase in levels of MMP-9 (92 kDa and 88 kDa) and MMP-2 (72 kDa and 62 kDa) in tumor areas compared with noninvolved regions. In contrast, patients with rectal carcinoma (n = 10 patients) had revealed the same high activity of MMP-9 in tumor regions and corresponding healthy tissue. Confirming activity measurements, in colon tumors, but not in rectal tumors, there was significant up-regulation of MMP-9 transcription compared with healthy tissue in the same patients. There were no significant changes in the tissue inhibitor of metalloproteinase-1 protein when colon and rectal tumor tissues were compared with the corresponding noninvolved regions. Cell culture experiments revealed fibroblasts as the cellular origin of MMPs. The findings showed that the secretion and activation of gelatinases depend on soluble factors secreted by tumor cells and are influenced by extracellular matrix components. CONCLUSIONS This is the first report showing differences in MMP-9 activity between rectal carcinoma and colon carcinoma. Previous results indicating an active involvement of stromal cells in the generation of MMPs during tumor invasion are extended. Because the abundance of gelatinases increases in colorectal carcinoma, inhibitors of these proteases may be of therapeutic value.
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Affiliation(s)
- E Roeb
- Department of Internal Medicine III, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany.
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Okada N, Ishida H, Murata N, Hashimoto D, Seyama Y, Kubota S. Matrix metalloproteinase-2 and -9 in bile as a marker of liver metastasis in colorectal cancer. Biochem Biophys Res Commun 2001; 288:212-6. [PMID: 11594775 DOI: 10.1006/bbrc.2001.5741] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Matrix metallproteinases (MMP)-2 and -9 are associated with cancer invasion and metastasis. MMP-2 and MMP-9 activities have never been assayed in bile. In the present study we investigated whether MMP-2 and -9 activities in the bile could be a marker for evaluation of liver metastasis in colorectal cancer. Fifty-three patients underwent colorectal resection for histologically verified adenocarcinoma. Twenty-six patients had colorectal cancer without liver metastasis and 27 patients had metastatic liver tumor. Six patients were studied as carcinoma-free control. MMP-2 and MMP-9 activities were assayed in bile using gelatin zymography and quantitated. Active MMP-2 activity of colorectal cancer with liver metastasis group (24.1 +/- 2.5 pixel count) was significantly higher than that of colorectal cancer without liver metastasis group (11.4 +/- 1.3 pixel count) (P < 0.001) or of control group (6.4 +/- 1.0 pixel count) (P < 0.001). Active MMP-9 was not detected in bile. ProMMP-9 activity of colorectal cancer with liver metastasis group (530.3 +/- 127.5 pixel count) was significantly higher than that of colorectal cancer without liver metastasis group (213.9 +/- 33.2 pixel count) (P = 0.008). This is the first report showing that the levels of active MMP-2 and proMMP-9 in bile were significantly higher in liver metastasis of colorectal cancer than in metastasis-free colorectal cancer. The results suggest that activities of active MMP-2 and proMMP-9 in the bile may be useful markers for predicting liver metastasis in colorectal cancer.
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Affiliation(s)
- N Okada
- Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Abstract
Wound healing is a complex process involving the interactions of many different cell types, matrix components and biological factors, including proteinases and cytokines. This study compared the levels of proteinases (matrix metalloproteinases and plasminogen activators), proteinase inhibitors (tissue inhibitors of metalloproteinases and plasminogen activator inhibitors), inflammatory cytokines and growth factors in acute wound fluid samples collected from the surgical drains of elective breast (n = 24) and colorectal (n = 26) patients on the first postoperative day. Gelatin zymography was used to determine matrix metalloproteinase-2 and -9 levels, quenched fluorescence substrate hydrolysis was applied for total MMP activity and enzyme-linked immunoassays were used to quantitate other factors. Colorectal wound fluid samples showed significantly (p < 0.05) greater levels of the matrix metalloproteinases (MMP-1, 2, 3, and 9), tissue inhibitor of metalloproteinases-1, urokinase plasminogen activator receptor and the inflammatory cytokines (interleukin-1beta, -6, and tumor necrosis factor-alpha); e.g., matrix metalloproteinase-3 colon; median 275 (range 11-2.530) ng/ml; breast; 530-400. However, tissue plasminogen activator and growth factor levels (epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor-beta1) were significantly greater in breast samples; e.g., epidermal growth factor breast 468 (103-1, 444) pg/ml; colon 57(1-573). There was no difference in the levels of urokinase type plasminogen activator, plasminogen activator inhibitor-1 and -2, tissue inhibitor of metalloproteinases -2 or vascular endothelial growth factor. Acute wound fluid from different surgical wounds showed different profiles of proteinases, proteinase inhibitors, and cytokines. This may lead to differences in the rate of tissue remodeling and therefore healing in these two wounds in vivo.
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Affiliation(s)
- E A Baker
- Professorial Unit of Surgery, North Tees General Hospital, Stockton on Tees, United Kingdom.
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Baker EA, Bergin FG, Leaper DJ. Matrix metalloproteinases, their tissue inhibitors and colorectal cancer staging. Br J Surg 2000; 87:1215-21. [PMID: 10971431 DOI: 10.1046/j.1365-2168.2000.01531.x] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
BACKGROUND Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in tumour invasion and metastasis. The levels of MMPs, TIMPs and total MMP activity were compared in paired colorectal tumour (n = 50) and normal tissue (n = 49) samples and correlated with clinical and pathological staging. METHODS Gelatin zymography (MMP-2 and MMP-9), enzyme-linked immunosorbent assays (MMP-1, MMP-3, TIMP-1 and TIMP-2) and quenched fluorescent substrate hydrolysis (total MMP activity) were employed in resection specimens from 50 patients, four with adenomas and 46 with colorectal cancer. RESULTS The levels of active MMP-2 and MMP-9 and total MMP-1, MMP-3 and TIMP-1 were significantly greater in tumour tissue than in normal colon (e.g. TIMP-1 tumour median 72 (range 25-351) versus normal 26 (4-107) ng per mg total protein content; P<0.05); however, TIMP-2 levels were significantly greater in normal tissue (P<0.05). Total MMP activity was significantly greater in tumour than in normal tissue (15 025 (1750-174 400) versus 7250 (750-354 650) pmol l-1 min-1 mg protein-1; P<0.05). Correlations were found between both MMP and TIMP levels and pathological tumour staging. MMP-1 appeared to be most important as its concentration correlated positively with Dukes staging, tumour differentiation and lymphatic invasion. CONCLUSION The levels of the studied MMPs and total MMP activity were upregulated in colorectal tumours. MMP-1 is important in colorectal cancer progression.
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Affiliation(s)
- E A Baker
- Professorial Unit of Surgery, North Tees General Hospital, Stockton on Tees TS19 8PE, UK
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