Novel Approach for Quantitative Measurement of Matrix Metalloprotease-1 (MMP1) in Human Breast Cancer Cells Using Mass Spectrometry (original) (raw)
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Journal of Chromatography A, 2008
Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC-MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC-MS analysis of 500 L crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations.
Matrix metalloproteinase localisation by in situ-RT-PCR in archival human breast biopsy material
Molecular and Cellular Probes, 2008
Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix metalloproteinases (MMPs) using in situ-RT-PCR (IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC) and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets. r
International Journal of Advanced Research (IJAR), 2019
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide. Matrix metalloproteinases (MMPs) are a family of enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularization. The current study, was aimed to determine the effect of gelatinizes in breast cancer progression by the measurement the levels of MMP-2 and MMP-9 in serum of breast tumor by ELISA. The present study involved 80 samples including 60 patients of breast tumor, which were selected from Medical Centre for Oncology, and 20 as control(normal subject),which select fromsouthern technical university from south region of Iraq in Basrah city during the period from October 2017 to February 2018.The results showed that measurements of MMP-2 and MMP-9 in the serum of the women with malignant tumor were highly significant (P≤0.01) compared with benign and control, regarding the grade of cancer subjects, stage and lymph node metastasis.This conclude the present of a correlation of the serological measurement of MMP-2 and MMP-9 in patients with breast cancer and metastasis.
Expression of matrix metalloproteinases inhuman breast cancer tissues
BACKGROUND: Breast cancer is the most common cancer affecting women in the world today. Matrix metalloproteinases (MMPs) are a family of endopeptidases that can degrade extracellular matrix proteins and promote cell invasion and metastasis. MMPs are differentially expressed and their expressions are often associated with a poor prognosis for patients. OBJECTIVE: The aim of this study is to investigate and compare the expression of MMPs in different grades of human breast cancer tissues with normal breast tissues. PATIENTS AND METHODS: We collected 39 breast cancer samples (24 grade II and 15 grade III) along with 16 normal breast tissues from outside the tumor margin during cancer removal surgery. The samples were analysed for the expression of all known MMPs using real-time quantitative PCR.
Folia Histochemica et Cytobiologica, 2010
Different types of matrix metalloproteinases, including membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) can be easily detected in biological fluids and therefore may be contemplated as putative tumor markers. Although increased activity of MT1-MMP/MMP-14 have already been found in breast cancer, little is known about its circulating levels. The aim of the present study was therefore to evaluate serum levels of active form of membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14). A novel type of activity enzyme-linked immunosorbent assay was used to detect serum levels of MT1-MMP/MMP-14 in 18 patients with invasive ductal breast cancer and 11 healthy controls. In the breast cancer group of patients MT1-MMP/MMP-14 mean (±SD) concentration was 16.91±5.87 ng/ml which was significantly higher (p<0.0001) than the mean values obtained for the control i.e. 8.55±1.66 ng/ml. Conclusions: Higher levels of soluble form of MT1-MMP/MMP-14 could play a role in invasiveness and metastasis of breast cancer. Whether or not it has a potential as biochemical marker remains to be determined.
Assay of matrix metalloproteases types 8 and 9 by ELISA in human breast cancer
British Journal of Cancer, 1995
Smmary Results from model tumour systems suggest that either increased levels of certain metalloproteases (MMPs) or decreased levels of their inhibitors correlate with metastatic potential. In this study, levels of two MMPs, i.e. MMP-8 and-9, and their inhibitor tissue inhibitor of metalloprotease type I (TIMP-1) were measured by enzyme-linked immunosorbent assay in human breast tumours. Levels of MMP-8 and-9 correlated significantly with each other, but neither MMP correlated with urokinase plasminogen activator. Levels of both MMP-8 and-9 were also significantly related to levels of TIMP-1. In contrast. neither MMP correlated with plasminogen activator inhibitor. No relationship was found between MMP-8, MMP-9 or TIMP-l and either tumour size or metastasis to axillary nodes. MMP-8 and-9 levels were inversely related to levels of oestrogen receptors. MMP-8 but not MMP-9 levels were also inversely correlated with progesterone receptor levels. It is concluded that the assay for MMP-8 and-9 described here will permit the evaluation of these proteases as prognostic markers in cancer.
Molecular Biotechnology, 2014
Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed 'form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol. have contributed equally to this work.
Study of matrix metalloproteinases and their inhibitors in breast cancer
British Journal of Cancer, 2007
BACKGROUND: Extracellular matrix metalloproteases (MMPs) have raised an extraordinary interest in cancer research because of their potential role in basal membrane and extracellular matrix degradation, consequently facilitating tumour invasion and metastases development. METHODS: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs 1, 2, 7, 9, 11, 13, 14, and their tissue inhibitors, TIMPs 1, 2 and 3. More than 2600 determinations on cancer specimens from 133 patients with clinically localised prostate carcinoma, 20 patients with prostatic intraepithelial neoplasia and 50 patients with benign prostate hyperplasia and controls, were performed. RESULTS: When compared with benign pathologies, prostate carcinomas had higher expression of all MMPs and TIMPs. Dendogram shows a first-order division of tumours into two distinct MMPs/TIMPs molecular profiles, one of them with high MMPs/TIMs expression profile (n ¼ 70; 52.6%). Tumours with high expression of MMP-11 or -13, or cluster thereof, were significantly associated with higher probability of biochemical recurrence. CONCLUSION: The expression of MMPs and TIMPs seems to have an important role in the molecular biology of prostate carcinomas, and their expression by tumours may be of clinical interest to used as indicators of tumour aggressiveness.
Mammographic Density and Matrix Metalloproteinases in Breast Tissue
Cancer Microenvironment, 2009
Mammographic density is a strong risk factor for breast cancer, yet the underlying histopathologic correlates are not clear. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play important roles in multiple stages of tumorigenesis. This study examined the association between mammographic density and expression of MMPs 1, 3, 9, and 12 and TIMP3 in benign and malignant breast tissue of 277 women with mainly Caucasian and Japanese ancestry. Tissue microarrays with up to 4 benign and 4 malignant cores per woman were stained immunohistochemically and evaluated. Digitized prediagnostic mammograms were assessed for densities using a computer-assisted method. General linear models adjusted for known confounders were applied to estimate mean densities by staining category. Strong expression of all MMPs was about twice as frequent in malignant as in benign tissue, while TIMP3 expression in stromal tissue was higher in benign than malignant cores. For MMP3 and 9, less than 10% of cores stained positive; thus, they were not further analyzed. None of the markers showed a statistically significant association with breast density in the entire study population and ethnic-specific results were conflicting and difficult to explain. Although not statistically significant, mean density was consistently lower with more extensive TIMP3 expression in stromal and epithelial tissue. These findings indicate that the higher breast cancer risk in women with dense breasts may be influenced by lower TIMP3 expression. However, future investigations into activities and ratios of additional proteases and their inhibitors as well as other pathways, such as inflammation, are needed.
International Journal of Molecular Medicine, 2006
MT1-MMP (membrane type-1 matrix metalloproteinase), otherwise known as MMP14 is a proteolytic enzyme known to be involved in degradating extracellular matrix and assist progression of cancer invasion and progression. We investigated the impact of targeting the expression of MT1-MMP in breast cancer and its clinical relevance. Human breast cancer cell line MDA-MB-231 was used. Expression of MT1-MMP in the breast cancer cell line was manipulated by way of retroviral ribozyme transgene. The in vitro invasion, growth and cell migration were determined on cell lines transfected with either the transgene or control plasmid. Protein and message levels of MMP14 was also assessed using immunohistochemistry and real-time quantitative analysis, and correlated with clinical and pathological information of the patients. Retroviral ribozyme transgene to human MT1-MMP successfully knocked down the levels of MT1-MMP mRNA from MDA-MB-231 cells. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus, HGF, compared with control and wild-type cells. The invasion index for MT1-MMP knockdown cells were 13±3.1 (without HGF) and 16.4±2.3 (with HGF, p=0.14), and the index for transfection control cells 25.3±4.3 (without HGF) and 40.4±4.1 (with HGF, p=0.0049). Transfection with the transgenes did not change the rate of cell growth. In clinical breast cancer, MT1-MMP staining was both membranous and cytoplasmic. Tumour cells displayed stronger staining compared with normal mammary epithelial cells. Tumour tissues had a marginally higher levels of the MMP14 transcript (8.6±1.9), compared with normal tissues (4.7±1.4), p=0.13. No significant difference was observed between node positive and node negative tumours (9.0±2.2 vs 8.7±3.1, p=0.24). Marginally higher levels of the MMP14 transcript were seen in tumours which developed metastasis and local recurrence. However, tumours from patients who died of breast cancer related causes had significantly higher levels of the transcript, compared with tumours from patients who remained disease-free 10 years after initial surgery (12.2±2.5 vs 6.3±1.2, p=0.0091). MT1-MMP is a proteolytic enzyme that is pivotal in controlling the invasiveness of breast cancer cells. It is highly expressed in aggressive breast tumours and is associated with clinical outcome. The enzyme is a potential therapeutic target in breast cancer.