MMP-3、TIMP-3在人胃癌组织中的表达及意义

目的:探讨MMP-3和TIMP-3在人胃癌组织中的表达及其意义.方法:根据胃癌的病理大体分型将40例胃癌组织分为早期组和晚期组.其中,早期组同时不伴有淋巴结转移,晚期组伴有淋巴结转移.采用光镜、透射电镜和免疫组化方法对这两组胃癌组织的超微结构,MMP-3,TIMP-3表达和MMP-3/TIMP-3的比值进行检测.结果:MMP-3和TIMP-3主要表达于癌细胞胞浆内.早期组MMP-3阳性表达细胞较少,晚期组阳性细胞较多,二者数密度和面密度比较,具有统计学意义(P＜0.01);早期组TIMP-3阳性表达细胞较多,晚期组阳性细胞较少,二者比较,具有统计学意义(P＜0.01)MM-3/TIMP-3的比值在胃癌晚期较早期增大,具有统计学意义(P＜0.01);电镜观察显示胃癌早期淋巴细胞浸润较多,癌细胞穿基膜不明显,晚期则淋巴细胞浸润较少,癌细胞穿基膜明显.结论:MMP-3,TIMP-3的表达程度和MMP-3/TIMP-3的比值可作为判定胃癌的侵袭和转移的指标,对其预后的判断具有参考价值.     

Hyaluronan synthase 2 (HAS2) promotes breast cancer cell invasion by suppression of tissue metalloproteinase inhibitor 1 (TIMP-1)

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion.     

Clinical implications of HER-2/neu overexpression and proteolytic activity imbalance in breast cancer

The aggressive biological behavior of invasive and metastatic cancer is considered to be the most insidious and life-threatening aspect for breast cancer patients. It is mostly the result of changes in many molecular characteristics of tumor cells, including alterations in gene expression and the balance of proteolytic activity. The objective of this study was to determine the level of MMP-2, its natural inhibitor TIMP-2, their ratio and HER-2/neu as diagnostic and prognostic factors. Markers were analyzed in 240 tissue samples categorized into 96 benign breast disease and 144 breast cancer patients. Enzyme linked immunosorbent assay procedure was used to evaluate the level of MMP-2 and TIMP-2 in the cell lysate, HER-2/neu in the membrane fraction, and steroid hormone receptors (ER and PgR) in the cytosol fraction. Breast cancer patients were followed-up for three years. Receiver operating characteristic curves were used to determine the cutoff points for the investigated factors. Positive values for all investigated factors were significantly increased in breast cancer patients compared to benign ones. Mean levels for all investigated factors were significantly correlated with lymph node and hormone receptor status, while MMP-2 and TIMP-2 were correlated with tumor grade (P < 0.05). In Univariate analysis, positive MMP-2, MMP-2/TIMP-2, HER-2/neu overexpression, higher tumor grade, late clinical stages and positive lymph nodes status were significantly associated with relapse. By multivariate analysis, all aforementioned factors apart from tumor grade were independent variables. Thus, the investigated markers are constructive for biologic aggressiveness of breast cancer and MMP-2/TIMP-2 ratio might be a new significant marker in early diagnosis and estimate prognosis in breast cancer.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

Targeted disruption of the tissue inhibitor of metalloproteinases gene increases the invasive behavior of primitive mesenchymal cells derived from embryonic stem cells in vitro.

The metalloproteinase family of proteolytic enzymes can degrade extracellular matrix and facilitate invasive migration. This class of enzymes is specifically inhibited by the tissue inhibitor of metalloproteinases (TIMP-1). Using homologous recombination, we have disrupted the gene encoding TIMP-1 in pluripotent embryonic stem cells. Because the TIMP-1 gene is X linked and is hemizygous in embryonic stem cells, we have been able to study the effect of this mutation in culture. Using a basement membrane invasion assay, we found that the mutant cells, differentiated in low concentrations of serum with retinoic acid, were more invasive than their normal cell counterparts, and that this was specifically reversed by adding exogenous TIMP-1 protein. The invasive cell population had characteristics of an early population of primitive mesenchymal cells, including expression of vimentin and a transient period of invasiveness from 4-8 d after initiation of differentiation. Therefore, metalloproteinase activity can be rate limiting for cell invasion.     

Interstitial collagenase, gelatinases A and B and their endogenous inhibitors in squamous cell cervical carcinomas

Interstitial collagenase and gelatinases are matrix metalloproteinases (MMP), which play the key role in tumor invasion and metastasis determining tumor malignancy. The aim of this study was to elucidate peculiarities of expression of interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9) and their endogenous tissue inhibitors TIMP-1 and TIMP-2 as invasive factors of squamous cell carcinomas (SCC) of human cervical cancer. The study was carried out using 24 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to tumor. All carcinoma specimens expressed the E7 HPV-16 gene. Results obtained indicate that the increased expression of MMP-1 and MMP-9 and low expression of TIMP-1 and TIMP-2 make the main contribution to the destructive (invasive) potential of SCC. Changes in MMP-2 expression are less important. In the specimens of morphologically normal tissue adjacent to the tumor, substantial expression of MMP-1, MMP-2 and MMP-9 was registered. This expression appears to make additional contribution to the tumor destructive potential.     

PTEN蛋白表达和明胶酶及其抑制因子在乳腺癌侵袭转移中的关系

目的研究乳腺癌中明胶酶(MMP-2、MMP-9)及其抑制物(TIMP-1)与抑癌基因PTEN产物PTEN蛋白的表达在肿瘤侵袭转移中的关系.方法应用免疫组织化学S-P法检测98例浸润性乳腺癌组织MMP-2、MMP-9及TIMP-1和PTEN蛋白的表达.结果乳腺癌MMP-2、MMP-9与PTEN表达呈显著负相关,而TIMP-1与PTEN蛋白表达呈显著正相关(P<0.05).在PTEN低表达组中,MMP-2、MMP-9的表达与腋窝淋巴结受累呈正相关;在PTEN高表达组中,MMP-2、MMP-9的表达与腋窝淋巴结受累呈正相关,TIMP-1与腋窝淋巴结受累呈负相关.结论在乳腺癌侵袭转移中MMP-2、MMP-9、TIMP-1与PTEN蛋白表达显著相关.MMP-2、MMP-9和TIMP-1对肿瘤细胞侵袭转移的作用可能在一定程度上受到PTEN的调控.     

Enhanced immunoreactivity of TIMP-2 in the stromal compartment of tumor as a marker of favorable prognosis in ovarian cancer patients

Degradation of the extracellular matrix and basement membrane is a critical step in tumor progression. Matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP 2) act in a coordinated manner to form an integrated system involved in ovarian cancer (OC) progression. In this study, the authors describe the expression of TIMP-2 detected by immunohistochemistry in 6 OC cell lines and in 43 malignant epithelial ovarian tumors (in tumor and stromal compartments) in sections originating from primary laparotomies. No significant correlations between overall and progression-free survival and TIMP-2 expression in tumor compartment were observed. The analysis demonstrated a significant association between enhanced stromal expression of TIMP-2 and better clinical response to cisplatin- and paclitaxel-based chemotherapy. Increased expression of TIMP-2 in the stromal compartment and simultaneous overexpression in both stromal and tumor compartments strongly correlated with increased survival. No significant correlations were found in vitro between resistance to cisplatin, paclitaxel, or topotecan and the expression of TIMP-2 in the OC cell lines, suggesting stromal influences on tumor chemoresistance in the physiological environment. This study supports the concept of TIMP-2 expression in the stromal compartment of OC as a promising marker of prognosis and response to cisplatin- and paclitaxel-based chemotherapy in OC patients.     

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

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.     

Regulation of MMP-2 expression and activity by β-1,3-<Emphasis Type="Italic">N</Emphasis>-acetylglucosaminyltransferase-8 in AGS gastric cancer cells

β-1,3-N-acetylglucosaminyltransferase-8(β3Gn-T8) catalyzes the transfer of GlcNAc to the non-reducing terminus of the Galβ1-4GlcNAc of tetraantennary N-glycan in vitro. It has been reported to be involved in malignant tumors, but a comprehensive understanding of how the glycolsyltransferase correlates with the invasive potential of human gastric cancer is not currently available. Therefore, we investigated the ability and possible mechanism involved with β3Gn-T8 in modulating matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in AGS gastric cancer cells. Here, we found out that siRNA-mediated suppression of the β3Gn-T8 could directly reduce the MMP-2 expression and activity as observed in RT-PCR, western blot and gelatin zymography analysis. Meanwhile, TIMP-2 expression had been increased. Cell invasion assay using matrigel matrix-coated transwell inserts showed that the invasive property was greatly suppressed in β3Gn-T8 siRNA transfected cells. Furthermore, cells overexpressing β3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene, but TIMP-2 expression had been inhibited. The invasive ability of these cells was also enhanced. Protein–protein interaction analysis using STRING database showed that β3Gn-T8 and MMP-2 may have related signal pathway. In summary, our results reveal a new mechanism by which β3Gn-T8 can regulate MMP-2 and TIMP-2. We suggest that β3Gn-T8 can be used as a novel therapeutic target for human gastric treatment.     

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.     

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Acute exercise induced changes in rat skeletal muscle mRNAs and proteins regulating type IV collagen content

This experiment tested the hypothesis that running-induced damage to rat skeletal muscle causes changes in synthesis and degradation of basement membrane type IV collagen and to proteins regulating its degradation. Samples from soleus muscle and red and white parts of quadriceps femoris muscle (MQF) were collected 6 h or 1, 2, 4, or 7 days after downhill running. Increased muscle beta-glucuronidase activity indicated greater muscle damage in the red part of MQF than in the white part of MQF or soleus. In the red part of MQF, type IV collagen expression was upregulated at the pretranslational level and the protein concentration decreased, whereas matrix metalloproteinase-2 (MMP-2), a protein that degrades type IV collagen, and tissue inhibitor of metalloproteinase-2 (TIMP-2), a protein that inhibits degradation, were increased in parallel both at mRNA and protein levels. Type IV collagen mRNA level increased in the white part of MQF and soleus muscle. The protein concentration increased in the white part of MQF and was unchanged in soleus muscle. MMP-2 and TIMP-2 changed only slightly in the white part of MQF and soleus muscle. The changes seem to depend on the severity of myofiber injury and thus probably reflect reorganization of basement membrane compounds.     

Activation of pro-gelatinase B by endometase/matrilysin-2 promotes invasion of human prostate cancer cells

This work has explored a putative biochemical mechanism by which endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) may promote human prostate cancer cell invasion. Here, we showed that the levels of MMP-26 protein in human prostate carcinomas from multiple patients were significantly higher than those in prostatitis, benign prostate hyperplasia, and normal prostate glandular tissues. The role of MMP-26 in prostate cancer progression is unknown. MMP-26 was capable of activating pro-MMP-9 by cleavage at the Ala(93)-Met(94) site of the prepro-enzyme. This activation proceeded in a time- and dose-dependent manner, facilitating the efficient cleavage of fibronectin by MMP-9. The activated MMP-9 products generated by MMP-26 appeared more stable than those cleaved by MMP-7 under the conditions tested. To investigate the contribution of MMP-26 to cancer cell invasion via the activation of MMP-9, highly invasive and metastatic human prostate carcinoma cells, androgen-repressed prostate cancer (ARCaP) cells were selected as a working model. ARCaP cells express both MMP-26 and MMP-9. Specific anti-MMP-26 and anti-MMP-9 functional blocking antibodies both reduced the invasiveness of ARCaP cells across fibronectin or type IV collagen. Furthermore, the introduction of MMP-26 antisense cDNA into ARCaP cells significantly reduced the MMP-26 protein level in these cells and strongly suppressed the invasiveness of ARCaP cells. Double immunofluorescence staining and confocal laser scanning microscopic images revealed that MMP-26 and MMP-9 were co-localized in parental and MMP-26 sense-transfected ARCaP cells. Moreover, MMP-26 and MMP-9 proteins were both expressed in the same human prostate carcinoma tissue samples examined. These results indicate that MMP-26 may be a physiological and pathological activator of pro-MMP-9.