Cell lines:
- Observations suggest that MCF-7 breastt cancer cells (BCC) in culture produce both soluble and membrane-bound factor(s) which stimulate the production of pro-MMPs (including MMP1) and TIMP-1 in neighbouring stromal cells, but the factor(s) released into the medium and that associated with cell membranes are probably different. Such communication between the normal and malignant cell types may, in part, assist the cancer cells to invade and metastasise (Ito A. et al., 1995).
- Platelets were found to increase the iinvasiveness of 3 breast cancer cell (BCC) lines (MCF-7, ZR-51 and MDA-MB231) through extracellular matrix. An increase in gelatinase activity could be responsible, at least in part, for the increased invasiveness of these cells, since added TIMP-1 significantly reduced the number of cells which traversed matrigel (Belloc C. et al., 1995).
- By Western-blot, a TIMP-1-specific immmunoreactive band was observed in the supernatants of the ER-negative MDA-MB-231 and -468, and the ER-positive MCF-7 and T-47D BCC lines, but not in those of the non-malignant HBL-100 and Hs578Bst cell lines (Yoshiji H. et al., 1996).
- In MCF-7 BCC, treatment with the phorbbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) was associated with a high expression of MMP-1 mRNA, as well as an induction of the level of TIMP-1 mRNA (5- to 10-fold). In the presence of actinomycin D (AMD, 4.0 microM), an inhibitor of transcription, these stimulatory effects of TPA were abolished. Similar responses were observed when protein synthesis was inhibited by cycloheximide (CHX, 50 microM). In the presence of the cyclic AMP (cAMP) analogue N6-benzoyl (N6-Bzl)-cAMP (500 microM), the MMP-1 mRNA was unaffected and still below the level of detection, whereas a non-significant increase (< 2-fold) in TIMP-1 mRNA was observed (Ree A.H. et al., 1996).
- Calcitonin was able to prevent the indduction of TIMP-1 and -2 mRNAs by the the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (Lacroix M. and Body J.J., 1997).
- Transfection of TIMP-1 cDNA into MCF-77 breast cancer cells (BCC) resulted in up-regulation of vascular endothelial growth factor (VEGF), with a linear relationship between TIMP-1 and VEGF production in 9 cell clones examined. There was, however, no change in VEGF expression when the BCC were exposed to exogenous recombinant TIMP-1. In a comparative study involving the MDA-MB-231, -415, -435, -468, -453, BT-483, BT-20, T-47D BCC lines and the non-neoplastic HBL-100 and Hs578Bst cell lines, there was significant correlation between TIMP-1 and VEGF concentration in the serum-free culture supernatants (Yoshiji H. et al., 1998).
- Genistein is a natural flavone compounnd found in soy. Genistein was found to exert pronounced antiproliferative effects on both estrogen receptor-positive and -negative human BCC through G
2-M arrest, induction of p21WAF1/CIP1 (gene CDKN1A) expression, and apoptosis. Genistein inhibited invasion
in vitro of MCF-7 and MDA-MB-231 BCC. This inhibition was characterized by down-regulation of MMP (matrix metalloproteinase)-9 and up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1), the former of which was transcriptionally regulated at activation protein-1 sites in the MMP-9 promoter. Genistein's
in vitro effects on MMP-9 and TIMP-1 were also demonstrated in
in vivo studies in nude mouse xenografts of MDA-MB-231 and MCF-7 BCC. In these xenograft studies, genistein inhibited tumor growth, stimulated apoptosis, and upregulated p21WAF1/CIP1 expression. In the MDA-MB-231 xenograft, genistein also inhibited angiogenesis by decreasing vessel density and decreasing the levels of vascular endothelial growth factor and transforming growth factor-beta1 (Shao Z.M. et al., 1998).
- Transforming growth factor-beta (TGFbeeta1) enhances human MDA-MB-231 breast tumour cell invasion of reconstituted basement membrane
in vitro but does not inhibit proliferation of this cell line. In contrast to basal invasion, which is TIMP-1-inhibitable MMP-dependent, TGFbeta1 enhanced-invasion is dependent upon plasmin and uPA activity but does not appear to involve TIMP-1-inhibitable MMPs, as judged by inhibitor studies (Farina A.R. et al., 1998).
- Bcl-2 overexpression was found to induuce TIMP-1 expression in breast epithelial cell lines (MCF10A, MCF10AneoT.TG3B, and MCF-7), whereas it had no effect on TIMP-2 expression. It was demonstrated that TIMP-1 inhibits cell death induced by hydrogen peroxide, Adriamycin, or X-ray irradiation (Li G. et al., 1999).
- It was examined whether TIMP-1 was ablle to exert a growth factor-like effect on two clonal cell lines (BC-3A and BC-61), isolated from a parental line of human BCC (8701-BC), and endowed with different growth and invasive behaviour
in vitro and in nude mouse. Only the more tumorigenic clonal cell line (BC-61) was responsive to TIMP-1 treatment by increasing its proliferative rate in a dose-dependent manner. treatment of BC-61 cells with a proliferation-promoting concentration of hTIMP-1 was able to stimulate tyrosine-targeted phosphorylation (Luparello C. et al., 1999).
- Analysis of MMP expression by RT-PCR sshowed expression of MMP-1, MMP-3, and MMP-13 in highly invasive MDA-MB-231 BCC, but not in slightly invasive MCF-7, T-47D, and BT-20 cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516 (Balduyck M et al., 2000).
- MCF7 breast tumor cells overexpressingg human c-Jun exhibit a transformed phenotype characterized not only by increased tumorigenicity but also by enhanced motility and invasion. The cellular phenotypic response to c-Jun overexpression is likely due, at least in part, to altered patterns of gene expression. By comparative hybridization, a total of 21 upregulated or downregulated gene targets responsive to c-Jun overexpression were identified. Interestingly, 8 of these genes (the down-regulated GADD153 and p21(cip1/waf1); the up-regulated vimentin, TIMP-1, SPARC, GSTp, PAI-1, and fibronectin) have been previously found associated with c-Jun or AP-1 activity and therefore provide internal validation for this approach to target gene discovery (Rinehart-Kim J. et al., 2000).
- Two invasive BCC (MDA-MB-231 and BT-5449) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive BCC lines (MCF-7, T47D and BT-483). TIMP-1 and TIMP-2 were able to attenuate the migration of MDA-MB-231 cells through bone marrow fibroblast monolayers (Saad S. et al., 2000).
- Whether changes in oxygen levels may aaffect TIMP-1 expression by cultured breast cancer cells was examined. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h (Canning M.T. et al., 2001).
Tumors:
- Expression of mRNAs encoding MMP1, MMPP2 and MMP3, and of TIMP1 were studied in human mammary pathology by
in situ hybridization and Northern blot analysis. Out of 6 benign lesions, 2 expressed MMP2 mRNAs. mRNAs encoding MMP1 and MMP3 were detectable in occasional stromal and tumor cells in 2 out of 17 carcinomas. Thirteen out of 17 cancers expressed MMP2 mRNA throughout the tumor in stromal cells close to noninvasive tumor clusters and well-differentiated invasive cancer cells. TIMP1 mRNA expression was detected in noninvasive and well-differentiated invasive tumor cells (Polette M. et al., 1993).
- TIMP-1 mRNA expression appeared signifficantly higher (p<0.01) in malignant tumor tissues than in their paired non-malignant counterparts. Similar differences were observed in the level of TIMP-1 protein expression in the paired breast samples examined (Yoshiji H. et al., 1996).
- TIMP-1 was found to be expressed in booth stromal and tumor components in most breast tumors (Heppner K.J. et al., 1996).
- In a series of 34 breast cancer patiennts, high levels of TIMP-1 mRNA in primary carcinomas correlated with the presence of lymph node metastases (P=0.0067), development of distant metastases (P=0.014), and early death of the disease (P=0.020). No correlations were found with patient age, tumor size, grade of anaplasia, steroid receptor status, MMP-2 and MMP-9 mRNA levels (Ree A.H. et al., 1997).
- In malignant breast tissue, high levells of TIMP-1 mRNA were found in all areas where tissue remodeling was evident, particularly in cells at the tumor-stromal interface. Small blood vessels in both the tumor and stroma were often outlined by high levels of TIMP-1 mRNA (Lindsay C.K. et al., 1997).
- In patients with primary breast carcinnomas, TIMP-1 concentrations were found to correlate positively with tumor size (p=0.0067, n=126) and inversely with estrogen receptor levels (p=0.004, n=138). No significant relationship was found between TIMP-1 concentrations and number of nodal metastases. The levels of TIMP-1 correlated significantly with MMP1 (p=0.0178, n=137), proMMP2 (p=0.0001, n=115), active MMP2 activity (p=0.0012, n=115), MMP3 (p=0.012, n=130), and proMMP9 (p=0.0063, n=115). No significant relationship was found between TIMP-1 levels and active MMP9 activities (p=0.4963, n=115). Finally, using an optimum cutoff point, patients with tumors containing high concentrations of TIMP-1 had both a shorter disease-free interval and overall survival than patients with low levels of the inhibitor (McCarthy K. et al., 1999).
- By Western blotting, TIMP-1 and
-2 were measured in paired tumour and normal tissue samples from 43 breast cancer patients. TIMP-1 was found in 82% breast tumours and 50% normal breast samples. For TIMP-2, the corresponding values were 100% and 80%. The amounts of TIMP-1 and TIMP-2 were higher in tumour samples than in normal tissue samples (Garbett E.A. et al., 1999).
- The expression patterns of matrix-metaalloproteinases (MMP)-1, -2, and -3 and of the tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were determined by in situ hybridization with isotopically labeled RNA probes in normal breast tissue (n=6), fibrocystic disease (n=20), five cases of which contained radial scars, lobular carcinoma in situ (CLIS; n=5), ductal carcinoma in situ (DCIS; n=9) and invasive carcinomas (n=24). Only a few cells displayed MMP-1- and MMP-2-specific labeling in normal breast tissue and fibrocystic disease. Noninvasive ductal carcinomas showed elevated MMP-2 transcript levels in peritumor stromal cells in the absence of significant MMP-1 specific signals. In general, compared with adjacent normal breast tissue, a gradual increase of MMP-2 was found in noninvasive to invasive cancers. Invasive ductal and lobular carcinomas displayed co-expression of MMP-1 and MMP-2 by stromal cells, mainly of the invasion front, with high signal intensity particularly in high-grade invasive carcinomas. Tumor cells and peritumor stroma showed low MMP-3 transcript levels, especially in medullary carcinomas. TIMP-1 and -2 transcript levels were increased in invasive carcinomas correlating with the histological grade (Brummer O. et al., 1999).
- In a co-culture system, secreted TIMP--1-EGFP, having the enhanced green fluorescent protein of the jelly fish Aequorea victoria fused to the carboxyl-terminus of TIMP-1, could be visualized binding to the surface of MCF-7 BCC but not non-neoplastic HBL-100 breast epithelial cells. TIMP-1-EGFP localized to the nucleus of MCF-7 cells after 72 hrs in co-culture. These findings suggest that TIMP-1 may preferentially bind to and be taken up by malignant breast epithelial cells and that TIMP-1 may play a yet unidentified role in nuclear functions (Ritter L.M. et al., 1999).
- It has been proposed that plasma measuurements of TIMP-1 by ELISA may have value in the management of breast cancer patients (Holten-Andersen M.N. et al., 1999).
- Using an immunohistochemical approach (polyclonal antibody) on a series of 79 infiltrating ductal carcinomas (IDCs), 8 tubular carcinomas, and 27 infiltrating lobular carcinomas (ILCs), staining for TIMP-1 was seen in the stroma and also in relation to small blood vessels, with more than 90 per cent of tumours showing this staining pattern (Jones J.L. et al., 1999).
- In a series of breast tumors, TIMP-2 llevels were found to be correlated significantly with those for TIMP-1 (Remacle A. et al., 2000).
- Thirty one specimens of bone metastasiis from breast carcinoma were stained for MMP1, 2, 9, 14 (MT1-MMP) and TIMP1, and 2 and compared with staining in normal breast tissue, primary breast carcinoma and normal bone. Specimens came from patients in three clinical scenarios: from open biopsies without or with pathological fracture, or bone marrow biopsies containing tumor from patients with pancytopenia but without clinical evidence of osteolysis. By bone histomorphometry the latter group showed a heavy tumor load not different from the open biopsy groups but displayed little active bone resorption and low numbers of osteoclasts. Cell type-specific MMP/TIMP expression was observed and the staining patterns were comparable between the three groups of patients. Though no major differences in the MMP/TIMP staining of tumor cells and fibroblasts were observed between bone metastasis and primary tumor, it was shown that tumor cells do express MMPs capable of degrading bone matrix collagen. The number and activity of osteoclasts and osteoblasts was increased dramatically in bone metastases, their MMP/TIMP profiles, however, were not different from normal bone (Lhotak S. et al., 2000).
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