Matrix Metalloproteinase-2 and -9 in Bile as a Marker of Liver Metastasis in Colorectal Cancer

doi:10.1006/bbrc.2001.5741

Biochemical and Biophysical Research Communications

Volume 288, Issue 1, 19 October 2001, Pages 212-216

https://doi.org/10.1006/bbrc.2001.5741 Get rights and content

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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Citation Excerpt :
However, these measurements can be challenging given the complex composition of bile, including bile salts, cholesterol, phospholipids, bilirubin, proteins, and inorganic ions (calcium, chloride, sodium, and bicarbonate) [7]. Previous studies measuring immune analytes in bile have largely been conducted in liver transplant patients where the bile was primarily collected from livers, T-tubes, or via endoscopic retrograde cholangiopancreaticography [8–16]. Also, all of these studies measured only a small number of analytes.
Gallbladder disease is highly related to inflammation, but the inflammatory processes are not well understood. Bile provides a direct substrate in assessing the local inflammatory response that develops in the gallbladder. To assess the reproducibility of measuring inflammatory markers in bile, we designed a methods study of 69 multiplexed immune-related markers measured in bile obtained from gallstone patients.
To evaluate assay performance, a total of 18 bile samples were tested twice within the same plate for each analyte, and the 18 bile samples were tested on two different days for each analyte. We used the following performance parameters: detectability, coefficient of variation (CV), intraclass correlation coefficient (ICC), and percent agreement (concordance among replicate measures above and below detection limit). Furthermore, we examined the association of analyte levels with gallstone characteristics such as type, numbers, and size.
All but 3 analytes (Stem Cell Factor, SCF; Thrombopoietin, TPO; sIL-1RI) were detectable in bile. 52 of 69 (75.4%) analytes had detectable levels for at least 50% of the subjects tested. The within-plate CVs were ⩽25% for 53 of 66 (80.3%) detectable analytes, and across-plate CVs were ⩽25% for 32 of 66 (48.5%) detectable analytes. Moreover, 64 of 66 (97.0%) analytes had ICC values of at least 0.8. Lastly, the percent agreement was high between replicates for all of the analytes (median; within plate, 97.2%; across plate, 97.2%). In exploratory analyses, we assessed analyte levels by gallstone characteristics and found that levels for several analytes decreased with increasing size of the largest gallstone per patient.
Our data suggest that multiplex assays can be used to reliably measure cytokines and chemokines in bile. In addition, gallstone size was inversely related to the levels of select analytes, which may aid in identifying critical pathways and mechanisms associated with the pathogenesis of gallbladder diseases.
Isolation and characterization of chicken bile matrix metalloproteinase
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Citation Excerpt :
The MMP are zinc-dependent endopeptidases that degrade extracellular matrix (ECM) and non-ECM proteins during tissue remodeling (Nagase et al., 2006; Iyer et al., 2012). The elevation of bile MMP has been generally linked to hepatic pathologies involving fibrosis, cancer, bile duct, and gall bladder diseases in humans and other mammalian models (Okada et al., 2001; Consolo et al., 2009; Kirimlioğlu et al., 2009; Hirashita et al., 2012; Syed et al., 2012). A recent study in fish showed that the elevation of their bile MMP levels was associated with aquatic pollution (Hauser-Davis et al., 2012).
Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.
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Oxygen free radical and matrix metalloproteinases-9 (MMP-9) play an important pathophysiological role in the development of chronic hypertension. MMP-9 activities are regulated at different levels. We hypothesize that as mediators of the expression of MMP-9 the transcription factors like nuclear factor kappa B (NF-κB), c-fos and retinoic acid receptors-α (RAR-α) with binding sites to the MMP-9 promoter are overexpressed in the spontaneously hypertensive rat (SHR) in a process that is regulated by oxygen free radicals. Transcription factor NF-κB, c-fos and RAR-α expression levels were determined by immunohistochemistry in renal, cardiac and mesentery microcirculation of the SHR and its normotensive control, the Wistar Kyoto (WKY) rat. The animals were treated with a superoxide scavenger (Tempol) for eight weeks. The elevated plasma levels of thiobarbituric acid reactive substances and MMP-9 levels in the SHR were significantly decreased by Tempol treatment (P < 0.05). The NF-κB, c-fos and RAR-α expression levels in renal glomerular, heart and mesentery microvessels were enhanced in the SHR and could also be reduced by Tempol compared to untreated animals (P < 0.05). The enhanced MMP-9 levels in SHR microvessels co-express with transcription factors. These results suggest that elevated NF-κB, c-fos and RAR-α expressions and MMP-9 activity in the SHR are superoxide-dependent.
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Citation Excerpt :
Accordingly, a chemotherapeutic agent that only possesses the ability to induce apoptosis may not be useful against highly metastatic tumors. Numerous studies indicated that inhibition of MMPs expressions or enzyme activities can provide early targets for prevention of cancer metastasis (Hsieh et al., 2007; Lu et al., 2013; Waas et al., 2003; Okada et al., 2001; Guruvayoorappan and Kuttan, 2008). After effective doses of the antimetastatic activity on osteosarcoma U2OS cells, non-toxic to the cells, were established, the determined concentrations were then used as the treatment condition for the subsequent pathway studies.
Selaginella tamariscina is a traditional medicinal plant for treatment of some advanced cancers in the Orient. However, the effect of S. tamariscina on metastasis of osteosarcoma and the underlying mechanism remain unclear. We tested the hypothesis that S. tamariscina suppresses cellular motility, invasion and migration and also investigated its signaling pathways. This study demonstrates that S. tamariscina, at a range of concentrations (from 0 to 50 μg/mL), concentration-dependently inhibited the migration/invasion capacities of three osteosarcoma cell lines without cytotoxic effects. Zymographic and western blot analyses revealed that S. tamariscina inhibited the matrix metalloproteinase (MMP)-2 and MMP-9 enzyme activity, as well as protein expression. Western blot analysis also showed that S. tamariscina inhibits phosphorylation of p38 and Akt. Furthermore, SB203580 (p38 inhibitor) and LY294002 (PI3K inhibitor) showed the similar effects as S. tamariscina in U2OS cells. In conclusion, S. tamariscina possesses an antimetastatic activity in osteosarcoma cells by down-regulating MMP-2 and MMP-9 secretions and increasing TIMP-1 and TIMP-2 expressions through p38 and Akt-dependent pathways. S. tamariscina may be a powerful candidate to develop a preventive agent for osteosarcoma metastasis.
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However, a chemotherapeutic agent that only possesses the ability to induce apoptosis may not be useful against highly metastatic tumors. Numerous studies indicate that inhibition of u-PA and MMP expression or enzyme activity can prevent cancer metastasis in its early stages (Hsieh et al., 2007; Waas et al., 2003; Okada et al., 2001; Guruvayoorappan and Kuttan, 2008). In this study, we investigated the anti-metastatic activity of P. urinaria on osteosarcoma Saos-2 cells.
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^☆: Abbreviation used: MMP, matrix metalloproteinase.

¹: Present address: Faculty of Human Life and Environmental Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.

²: To whom correspondence should be addressed. Fax: +81-3-5689-2704. E-mail: [email protected].

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Regular ArticleMatrix Metalloproteinase-2 and -9 in Bile as a Marker of Liver Metastasis in Colorectal Cancer☆

Abstract

Eur. J. Cancer

J. Biol. Chem.

J. Biol. Chem.

J. Surg. Res.

Anal. Biochem.

Anal. Biochem.

Matrix metalloproteinases

Br. J. Surg.

Matrix metalloproteinases: Biologic activity and clinical implications

J. Clin. Oncol.

Metalloproteinases and cancer invasion

Semin. Cancer Biol.

Expression and localization of the matrix metalloproteinase pump-1 (MMP-7) in human gastric and colon carcinomas

Mol. Carcinog.

Expression of MMP-7 (PUMP-1) mRNA in human colorectal cancers

Int. J. Cancer

Messenger RNA for two type IV collagenases is located in stromal cells in human colon cancer

Am. J. Pathol.

Colocalisation of matrix metalloproteinase-9 mRNA and protein in human colorectal cancer stromal cells

Br. J. Cancer

Prediction of colorectal cancer relapse and survival via tissue RNA levels of matrix metalloproteinase-9

J. Clin. Oncol.

Stromal expression of 72kDa type IV collagenase (MMP-2) and TIMP-2 mRNAs in colorectal neoplasia

Am. J. Pathol.

Regular Article
Matrix Metalloproteinase-2 and -9 in Bile as a Marker of Liver Metastasis in Colorectal Cancer☆