黏附素5胞外区克隆、转染及抗人乳腺癌细胞生长的研究

目的: 克隆和转染黏附素5胞外区，并研究其对人乳腺癌MDA-MB435细胞株生长的影响。方法: RT-PCR技术克隆黏附素5胞外区（称为CED1-4），将其插入pMSCV质粒载体，在大肠杆菌XL-blue扩增，提取和纯化pMSCV-CED1-4，酶切、电泳和测序检测CED1-4序列。CED1-4基因转染MDA-MB435细胞株，RT-PCR和Western blotting检测MDA-MB435细胞表达CED1-4。细胞增殖实验和乳腺癌裸小鼠致瘤实验检测CED1-4对MDA-MB435细胞体内外生长的影响。结果: 构建了重组体pMSCV-CED1-4，电泳显示CED1-4条带在 1 636 bp-1 018 bp区间，测序显示CED1-4基因长1 452 bp，编码484氨基酸。经PCR和Western blotting证实，CED1-4基因转染的MDA-MB435细胞在mRNA和蛋白质水平表达CED1-4。细胞增殖实验结果表明,实验组MDA-MB435细胞增殖低于实验对照组和空白对照组（P<0.05）。乳腺癌裸小鼠致瘤实验显示，实验组移植瘤平均体积和重量低于实验对照组和空白对照组（P<0.05）。结论: 黏附素5胞外区CED1-4能在体内外抑制人乳腺癌MDA-MB435细胞株的生长。     

Primary breast myoepithelial cells exert an invasion-suppressor effect on breast cancer cells via paracrine down-regulation of MMP expression in fibroblasts and tumour cells

In normal breast and ductal carcinoma in situ, myoepithelial cells form an incomplete layer separating the epithelial compartment from the stromal environment. Transition to invasive disease is marked by penetration of the myoepithelial-basement membrane (BM) interface. One mechanism involved in tumour invasion is breakdown of extracellular matrices by matrix metalloproteinases (MMPs). It was hypothesized that myoepithelial cells may modulate tumour invasion by controlling MMP gene expression, both in tumour cells and in peri-ductal fibroblasts. To investigate this, myoepithelial cells from normal breast were purified and characterized and their effect on tumour cell invasive potential was assessed. The effect on MMP gene expression of breast cancer cells cultured alone or in combination with primary normal breast fibroblasts was also analysed using RT-PCR with ELISA quantitation, with zymographic analysis to measure enzyme activity. Normal breast myoepithelial cells significantly reduced invasion by the breast cancer cell lines MCF-7, T47D, MDA-MB 231, and MDA-MB 468 when they were cultured alone or in the presence of a fibroblast population. Reduced invasion was associated with changes in MMP gene expression. In those tumour cells expressing MMP, there was a significant down-regulation of MMP-2 (MDA-MB 468, p<0.001), MMP-9 (MDA-MB 231, p=0.05; MDA-MB 468, p<0.001), and MT1-MMP (p<0.001 for both MDA-MB 231 and MDA-MB 468). Myoepithelial cells also caused a significant decrease in MMP gene expression in co-cultured fibroblasts. Furthermore, this was associated with reduced gelatinolytic activity as identified by zymography. This study demonstrates for the first time that primary myoepithelial cells from normal breast reduce breast cancer cell invasion and that this is mediated via modulation of both tumour cell and fibroblast function. This emphasizes the importance of the myoepithelial cell in controlling the breast microenvironment and focuses on the potential significance of the loss of this population with disease progression.     

Expression of most matrix metalloproteinase family members in breast cancer represents a tumor-induced host response.

Matrix metalloproteinase (MMP) family members have been associated with advanced-stage cancer and contribute to tumor progression, invasion, and metastasis as determined by inhibitor studies. In situ hybridization was performed to analyze the expression and localization of all known MMPs in a series of human breast cancer biopsy specimens. Most MMPs were localized to tumor stroma, and all MMPs had very distinct expression patterns. Matrilysin was expressed by morphologically normal epithelial ducts within tumors and in tissue from reduction mammoplasties, and by epithelial-derived tumor cells. Many family members, including stromelysin-3, gelatinase A, MT-MMP, interstitial collagenase, and stromelysin-1 were localized to fibroblasts of tumor stroma of invasive cancers but in quite distinct, and generally widespread, patterns. Gelatinase B, collagenase-3, and metalloelastase expression were more focal; gelatinase B was primarily localized to endothelial cells, collagenase-3 to isolated tumor cells, and metalloelastase to cytokeratin-negative, macrophage-like cells. The MMP inhibitor, TIMP-1, was expressed in both stromal and tumor components in most tumors, and neither stromelysin-2 nor neutrophil collagenase were detected in any of the tumors. These results indicate that there is very tight and complex regulation in the expression of MMP family members in breast cancer that generally represents a host response to the tumor and emphasize the need to further evaluate differential functions for MMP family members in breast tumor progression.     

Activity of biphenyl matrix metalloproteinase inhibitor BAY 12-9566 in a human breast cancer orthotopic model

Matrix metalloproteinases (MMPs) have been implicated in the invasion, metastasis, and angiogenesis associated with human cancer by mediating the degradation of extracellular matrix components. In this paper, we report data that show that BAY 12-9566, a novel inhibitor of MMPs, inhibits angiogenesis, tumor regrowth, and the growth of lung metastases. BAY 12-9566, at 15–100 μM, inhibited tubule formation by human endothelial cells in an in vitro model, but did not prevent the proliferation of endothelial and human breast cancer cells. In the MDA-MB-435 human mammary carcinoma xenograft model, in which the primary tumor is transplanted into the murine mammary fat pad, BAY 12-9566, administered daily at a dose of 100 mg/kg/day p.o. after resection of the primary tumor, inhibited local tumor regrowth by 58% without causing any toxic effect. In addition, BAY 12-9566 treatment inhibited the number and volume of lung metastases by 57 and 88%, respectively. These effects were highly correlated with the serum concentration of BAY 12-9566 at the end of treatment. The serum of the treated animals, harvested 24 h after the last treatment, and the tumor regrown at the site of tumor transplant in the treated animals, contained less protein with MMP-9 activity (as measured in a gelatin zymography assay) than the corresponding controls. However, no difference in the activity of MMP-2 was observed. Although all clinical trials in cancer involving BAY 12-9566 have been halted, this MMP inhibitor has never been used in clinical trials in breast cancer. These results suggest that the novel MMP inhibitor BAY 12-9566 maybe a useful and safe oral treatment for breast cancer, adjunctive to surgery. This revised version was published online in July 2006 with corrections to the Cover Date.     

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri(L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.     

Endogenous angiogenesis inhibitor blocks tumor growth via direct and indirect effects on tumor microenvironment

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) belongs to a small family of endogenous proteins that inhibits a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP-dependent and -independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, and enzyme-linked immunosorbent assay (ELISA), we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intratumoral microvascular density, and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and immunohistochemical analyses of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in some endocrine organs and their tumors

Matrix metalloproteinases (MMPs) are single-chain zinc-containing metalloenzymes. The MMP gene family currently includes more than 19 endopeptidases. Both MMP-2 and 9 are widely expressed by many stromal and endothelial cells. Tissue inhibitors of metalloproteinases (TIMPs) form complexes with MMPs, which in turn inhibit active MMPs. MMP and TIMP homeostasis has been implicated in many aspects of both physiological and pathological processes. The latter include tumor invasion and metastasis. Although ductal adenocarcinomas of pancreas were immunocytochemically faintly stained for MMPs and TIMPs, normal pancreatic islets in the normal adjacent pancreas were found to be strongly stained for MMPs and TIMPs. Five kinds of islet cell tumors, including insulinomas, gastrinomas, glucagonomas, pancreatic polypeptide- (PP) omas, and nonfunctioning islet cell tumor, were stained for MMPs and TIMPs. The tumor cells were relatively weakly stained for MMPs and TIMPs compared to normal islets. Similarly, weaker staining for MMPs and TIME’s was noted for medullary thyroid carcinomas (MTCs) and pituitary adenomas. There was no correlation between immunostaining intensity of protein hormones and MMPs and TIMPs. However parathyroid hyperplasia, adenoma, and carcinoma that stained for MMPs and TIMPs were weaker, which paralleled the weaker immunostaining for parathyroid hormone and chromogranin. This weaker staining for MMPs and TIMPs in endocrine tumors may imply a less significant role of tumor invasion and metastasis by MMP and TIMP homeostasis. At present, immunocytochemical staining for MMPs and TIMPs may well be used as new markers for neuroendocrine cells and their tumors.     

Immunolocalization of matrix metalloproteinases and their inhibitors in clinical specimens of bone metastasis from breast carcinoma

Matrix metalloproteinases (MMPs) are essential in several stages of the metastatic process, and in normal bone development and remodeling. We explored whether the interaction between tumor cells and bone leads to changes in MMP and tissue inhibitor of MMP (TIMP) expression thus affecting osteolysis in metastatic bone disease. Using immunohistochemistry we have investigated the MMP/TIMP expression in tumor cells, fibroblasts, osteoblasts and osteoclasts. Thirty one specimens of bone metastasis from breast carcinoma were stained for MMP-1, -2, -9, MT1-MMP and TIMP- 1, 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, we showed 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, suggesting that the mechanism of bone degradation by osteoclasts is not different from normal bone remodelling. This revised version was published online in July 2006 with corrections to the Cover Date.     

小鼠巨噬细胞内表达结核分枝杆菌CFP10-ESAT6融合蛋白对细胞增殖和凋亡的影响

 目的：建立培养滤液蛋白10-早期分泌性抗原靶6（CFP10-ESAT6）真核表达质粒并转染RAW2647巨噬细胞,观察胞内表达CFP10-ESAT6对细胞活性及凋亡的影响,并初步探讨其机制。方法：将CFP10-ESAT6融合基因插入真核表达质粒pEGFP-N1,构建重组质粒,转染RAW264.7巨噬细胞。采用MTT的方法测定CFP10-ESAT6融合蛋白对RAW264.7巨噬细胞活性的影响,采用结核分枝杆菌19 kD脂蛋白和十字孢碱（staurosporine）处理RAW264.7巨噬细胞,并以流式细胞术检测细胞凋亡率以及Toll样受体2（TLR2）的表达。结果：成功构建了重组质粒pEGFP-N1/CFP10-ESAT6并转染至RAW264.7巨噬细胞;与对照组相比,胞内表达CFP10-ESAT6不能影响巨噬细胞的活性,但可以明显抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡(P＜0.05),并显著抑制了TLR2的表达(P＜0.05)。结论：巨噬细胞内表达的CFP10-ESAT6融合蛋白不具有细胞毒性作用,但可以通过下调TLR2的表达来抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡。     

Establishment of red fluorescent protein-tagged HeLa tumor metastasis models: determination of DsRed2 insertion effects and comparison of metastatic patterns after subcutaneous,intraperitoneal, or intravenous injection

Metastasis is the leading cause of death in patients with cervical cancer. In this report, we establish novel fluorescent HeLa tumor metastasis models to determine whether HeLa transfected with the enhanced red fluorescent protein (DsRed2) gene in vitro and xenotransplanted through subcutaneous, intraperitoneal, or intravenous route into SCID mice would permit the detection of tumor micro-metastasis in vivo. Our results showed that DsRed2 insertions did not interfere the tumorigenic properties of HeLa cells. We also demonstrated that DsRed2-transduced HeLa cells maintained stable high-level DsRed2 expressions during their growth in vivo. DsRed2 fluorescence clearly demarcated the primary seeding place and readily allowed for the visualization of distant micro-metastasis and local invasion at the single-cell level. Lung metastasis, the major cause of cervical carcinoma related death, was found in all three models. However, intravenous injections of the HeLa-DsRed2 cells established tumor foci in the lung, while subcutaneous and intraperitoneal injections only established lung metastasis at single-cell levels. The DsRed2 tagged HeLa cancer model allowed detection and investigation of physiologically relevant patterns of cancer invasion and metastasis in vivo. This revised version was published online in July 2006 with corrections to the Cover Date.     

Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival

Up regulation of matrix metalloproteinases (MMPs), particularly collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and gelatinase A (MMP-2) is responsible for the lysis of dermal collagen and elastin fibers during chronological skin aging. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one representative of the natural MMP inhibitor family, encompassing four members. Its expression is decreased with fibroblast senescence, both ex vivo and in vivo, thus contributing to increased catabolic activity within dermis. TIMP-1 displays multiple biological functions. It inhibits most MMPs, except membrane-type MMP subfamily, with Ki in the subnanomolar range, but also interacts with the hemopexin-like (PEX) domain of pro MMP-9. Besides, it exhibits keratinocyte and fibroblast growth factor-like activity and has been described as a cell survival factor.     

Human breast cancer cells activate procollagenase-1 and invade type I collagen: invasion is inhibited by all-trans retinoic acid

Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and metastasis. These processes require the dissolution of the basement membrane and invasion of the stromal matrix (ECM), and are mediated by MMPs. Consequently, MMP inhibitors may be attractive as new anticancer agents. To examine the potential contribution of collagenase-1 (MMP-1) in invasion of stromal matrix, we used the highly invasive and metastatic breast cancer cell line MDA-MB-231 as a model system. These cells express procollagenase-1 constitutively and this expression can be repressed by all-trans retinoic acid. Invasion of these cells into a collagen type I matrix was assessed by scanning electron microscopy (SEM), and was quantitated with a computer program and confocal laser scanning microscopy (CLSM). We found that MDA-MB-231 cells freely invaded the collagen type 1 matrix, suggesting that these cells possess a mechanism for activating the latent collagenase-1. In contrast, down-regulation of collagenase-1 expression by all-trans retinoic acid caused these cells to become less invasive. To confirm a role for collagenase-1 in mediating collagen type I invasion, assays were carried out in the presence of FN-439, an inhibitor of collagenase-1 enzyme activity. We found that in the presence of the proteinase inhibitor, invasion of type I collagen by MDA-MB-231 cells was also reduced. These results indicate that collagenase-1 produced by the breast tumor cells may enhance stromal matrix degradation by enabling the tumor cells to modulate their own invasive behavior, and suggest that decreasing collagenase-1 levels may be effective in breast cancer therapy.     

Ventilator-induced lung injury upregulates and activates gelatinases and EMMPRIN: attenuation by the synthetic matrix metalloproteinase inhibitor, Prinomastat (AG3340).

Mechanical ventilation has become an indispensable therapeutic modality for patients with respiratory failure. However, a serious potential complication of MV is the newly recognized ventilator-induced acute lung injury. There is strong evidence suggesting that matrix metalloproteinases (MMPs) play an important role in the development of acute lung injury. Another factor to be considered is extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN is responsible for inducing fibroblasts to produce/secrete MMPs. In this report we sought to determine: (1) the role played by MMPs and EMMPRIN in the development of ventilator-induced lung injury (VILI) in an in vivo rat model of high volume ventilation; and (2) whether the synthetic MMP inhibitor Prinomastat (AG3340) could prevent this type of lung injury. We have demonstrated that high volume ventilation caused acute lung injury. This was accompanied by an upregulation of gelatinase A, gelatinase B, MT1-MMP, and EMMPRIN mRNA demonstrated by in situ hybridization. Pretreatment with the MMP inhibitor Prinomastat attenuated the lung injury caused by high volume ventilation. Our results suggest that MMPs play an important role in the development of VILI in rat lungs and that the MMP-inhibitor Prinomastat is effective in attenuating this type of lung injury.     

Inhibition of metalloproteinases derived from tumours: new insights in the treatment of human glioblastoma

Glioblastoma multiforme is the most commonly diagnosed malignant primary brain tumour in adults. Invasive behaviour is the pathological hallmark of malignant gliomas; consequently, its inhibition has been suggested as a therapeutic strategy. Tumour cell-derived gelatinases (matrix metalloproteinase-2, matrix metalloproteinase-9) can be considered prime factors in glioma invasiveness: their expression correlates with the progression and the degree of malignancy. Thus, broad spectrum matrix metalloproteinase inhibitors (MMP inhibitors) have been included in clinical trials. In the present study, the invasiveness, viability and progression of the human glioma cell line U87MG were investigated following treatment with N-O-isopropyl sulfonamido-based hydroxamates (compounds 1 and 2) as MMP-2 inhibitors used at nanomolar concentration. A standard broad spectrum MMP-inhibitor belonging to the classical tertiary sulfonamido-based hydroxamates family (CGS_27023A) was used too. The compounds 1 and 2 resulted in potent inhibition of cell invasiveness (P<0.0001) without affecting viability. In some clinical trials, the combined therapy of temozolomide (an alkylating agent used in glioma treatment) plus marimastat (a broad spectrum MMP inhibitor) has provided evidence of the importance of MMPs to tumor progression and invasiveness. On this basis, the effect on U87MG cells of a combined treatment with temozolomide, plus each of the two MMP inhibitors at nanomolar concentration, was investigated. The obtained data demonstrated the inhibition of cell invasiveness and viability after treatment. These results can help in developing clinical combined therapy using MMP inhibitors that, at low doses, increase the anticancer efficacy of chemotherapeutic drugs, probably without causing the side effects typical of broad-spectrum MMP inhibitors.     

Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro--cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.     

Evaluation of stromal metalloproteinases and vascular endothelial growth factors in a spontaneous metastasis model

This study aims to investigate MMP2 and MT1-MMP protein as well as VEGF-C and VEGF-D mRNA expression in tumor cells and distant organs considered to be targets for metastasis in a tumor spontaneous metastasis model previously described. Cultured tumor cells, able to express pro-MMP2, MMP2, pro-MMP9, and MT1-MMP, develop tumor growth and metastasis, mainly in the liver and spleen, when they are injected in the mammary pad gland of Wistar rats. Immunohistochemical studies of tumor masses showed small groups of tumor cells staining for MT1-MMP but not for MMP2. In the liver, tumor metastatic foci and a stromal positive staining for both MMP2 and MT1-MMP were shown. The spleen and lymph nodes, with only scattered metastatic cells, did not show MMPs immunostaining. Using RT-PCR, a significantly higher VEGF-C and VEGF-D gene expression was shown in the liver of tumor-bearing rats respect to normal rats, whereas spleen and lymph nodes did not show significant differences in mRNA VEGF-C/D levels. Taken together, our results suggest that the stroma microenvironment of target organs for metastasis has the ability to produce MMPs and VEGFs that facilitate the anchorage of tumor cells and promote tumor cell growth and angiogenesis.     

Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury. Evidence for alveolar macrophage as source of proteinases.

Matrix metalloproteinases (MMPs) have been implicated in the tissue injury seen in neutrophil-dependent models of acute lung injury. However, the role of MMPs in macrophage-dependent models of lung injury is unknown. To address this issue, the macrophage-dependent immunoglobulin A immune complex-induced lung injury model and the macrophage-dependent portion of the lipopolysaccharide-induced acute lung injury model in the rat were assessed for MMP involvement and for the source of these activities. In both models, injury was inhibited by the recombinant human tissue inhibitor of metalloproteinases-2. Bronchoalveolar lavage fluids (BALFs) from injured animals in both models showed increased levels of MMPs. Characterization of MMP production by isolated lung fibroblasts, endothelial cells, type II epithelial cells, and alveolar macrophages revealed that only the macrophage had the same spectrum of MMP activity as seen in the BALF. Further, isolated alveolar macrophages from injured lungs showed evidence of in vivo activation with the release of the same spectrum of MMP activities. Together these studies show that MMPs are produced during macrophage-dependent lung injury, that these MMPs play a role in the development of the lung injury, and that the alveolar macrophage is the likely source of these MMPs.