整合素与肿瘤的转移与血管生成

整合素是一类介导细胞与细胞外基质及细胞与细胞间黏附的细胞黏附分子受体,肿瘤细胞与胞外基质的相互作用对肿瘤的生成及转移有着重要的影响,整合素在肿瘤的生成、侵袭、转移以及肿瘤血管的生成过程中起着重要的作用。本文对整合素的结构、功能,以及它在肿瘤的血管生成过程中的作用,它与细胞外基质间的相互关系做了介绍。     

Enzyme-digested Fucoidan Extracts Derived from Seaweed Mozuku of Cladosiphon novae-caledoniae kylin Inhibit Invasion and Angiogenesis of Tumor Cells

Fucoidan is a uniquely-structured sulfated polysaccharide found in the cell walls of several types of brown seaweed that has recently, especially as enzyme-digested fucoidan extract, attracted a lot attention due to its anti-tumor potential. In this study, we evaluated the effects of enzyme-digested fucoidan extracts prepared from seaweed Mozuku of Cladosiphon novae-caledoniae kylin on in vitro invasion and angiogenesis abilities of human tumor cells. First, we evaluated the effect of the fucoidan extracts on oxidative stress of tumor cells, and demonstrated that intracellular H₂O₂ level and released H₂O₂ from tumor cells were both greatly repressed upon the treatment with the fucoidan extracts, suggesting that fucoidan extracts ameliorate oxidative stress of tumor cells. Next, we tested for the effects of fucoidan extracts on invasion ability of human fibrosarcoma HT1080 cells, showing that fucoidan extracts significantly inhibit their invasion, possibly via suppressing matrix metalloproteinases (MMPs) MMP-2/9 activities. Further, we investigated the effects of the fucoidan extracts on angiogenesis of human uterine carcinoma HeLa cells, and found that fucoidan extracts suppressed expression and secretion of an angiogenesis factor vascular endothelial growth factor (VEGF), resulting in suppressed vascular tubules formation of tumor cells. The results taken together clarified that enzyme-digested fucoidan extracts from Cladosiphon novae-caledoniae kylin possess inhibitory effects on invasion and angiogenesis of tumor cells. These effects might, at least partially, be elicited by the antioxidative potential of enzyme digested fucoidan extracts.     

Nicotine treatment induces expression of matrix metalloproteinases in human osteoblastic Saos-2 cells

Tobacco smoking is an important risk factor for the development of severe periodontitis.Recently,we showed that nicotine affected mineralized nodule formation,and that nicotine andlipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophagecolony-stimulating factor (M-CSF) and prostaglandin E_2 (PGE_2) by human osteoblastic Saos-2 cells.In thepresent study,we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs),tissue inhibitors of matrix metalloproteinases (TIMPs),the plasminogen activation system including thecomponent of tissue-type plasminogen activator (tPA),urokinase-type PA (uPA),and PA inhibitor type 1(PAI-1),α7 nicotine receptor,and c-fos.We also examined the effect of the nicotine antagonist D-tubocurarineon nicotine-induced expression of MMP-1.Gene expression was examined using real-time polymerase chainreaction (PCR) to estimate mRNA levels.In addition,expression of the MMP,TIMP,uPA,tPA,and PAI-1proteins was determined by Western blotting analysis.Nicotine treatment caused expression of MMP-1,2,3,and 13,but not MMP-14,to increase significantly after 5 or 10 d of culture;MMP-14 expression did notchange through day 14.Enhancement of MMP-1 expression by nicotine treatment was eliminated bysimultaneous treatment with D-tubocurarine.In the presence of nicotine,expression of uPA,PAI-1,orTIMP-1,2,3,or 4 did not change over 14 d of culture,whereas expression of tPA increased significantly byday 7.Nicotine also increased expression of the α7 nicotine receptor and c-fos genes.These results suggestthat nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1,2,3,and 13,thereby tipping the balance between bone matrix formation and resorption toward the latter process.     

The novel synthetic inhibitor of matrix metalloproteinases Ro 28-2653 induces apoptosis in Dunning tumor cells

The effect of synthetic inhibitors of matrix metalloproteinases (MMP) has been shown against a variety of tumors in preclinical models. Ro 28-2653, a novel synthetic MMP inhibitor, is able to reduce tumor growth in orthotopic prostatic cancer in rats (R3327 Dunning tumor). However, at present this inhibitory mechanism in tumor inhibition in vivo can only be partly explained by the inhibition of the catalytic activity of MMPs overexpressed in cancereous tissue. Using the flow cytometric method, we have investigated the effect of various concentrations of Ro 28-2653 on the Dunning tumor cells with regard to the staining of F-actin and DNA as markers of apoptosis. In combination with fluorescence microscopy we detected the loss of F-actin and the degradation of internucleosomal DNA. This effect of Ro 28-2653 on apoptosis was dose- and time-dependent increasing with concentration between 10 and 100 g/ml as well as with time of treatment between 24 and 48 h.     

Mast cells in tumor growth: Angiogenesis,tissue remodelling and immune-modulation

There is a growing acceptance that tumor-infiltrating myeloid cells play an active role in tumor growth and mast cells are one of the earliest cell types to infiltrate developing tumors. Mast cells accumulate at the boundary between healthy tissues and malignancies and are often found in close association with blood vessels within the tumor microenvironment. They express many pro-angiogenic compounds, and may play an early role in angiogenesis within developing tumors. Mast cells also remodel extracellular matrix during wound healing, and this function is subverted in tumor growth, promoting tumor spread and metastasis. In addition, mast cells modulate immune responses by dampening immune rejection or directing immune cell recruitment, depending on local stimuli. In this review, we focus on key roles for mast cells in angiogenesis, tissue remodelling and immune modulation and highlight recent findings on the integral role that mast cells play in tumor growth. New findings suggest that mast cells may serve as a novel therapeutic target for cancer treatment and that inhibiting mast cell function may lead to tumor regression.     

Angiogenesis and inflammation in skeletal muscle in response to ascites tumor in mice

This study addresses the interaction between Ehrlich ascites tumor and skeletal abdominal muscle, presenting quantitative analysis of ascites-induced angiogenesis and inflammation in this tissue of mice bearing-tumor. Time-dependent changes in the muscle (cellular activity, angiogenesis, inflammation and cytokines production) were assessed by morphometric, functional, and biochemical parameters at days 1, 4 and 8 after i.p. inoculation of Ehrlich tumor cells (2.5 x 10(7)). The number of cells stained with AgNOR technique (argyrophilic nucleolar organizer region) in the muscle, together with MTS assay used as markers of cellular activity increased progressively in parallel with the out flow rate of sodium fluorescein (blood flow index), hemoglobin content (vascular index) and VEGF production. Likewise, the inflammatory process in the muscle, as assessed by myeloperoxidase (MPO) and n-acethylglucosaminidase (NAG) activities and the levels of the chemokines, keratinocyte-derived chemokine (CXC1-3/KC) and macrophage-chemoattractant protein (CCL2/MCP-1) increased with tumor development. The combination of techniques used to describe angiogenesis and inflammation in a muscle model system has proved to be suited for quantitative measurements of microvascular changes and cellular infiltration occurring in the abdominal muscle wall of ascites-bearing mice. This study holds potential for investigating events and mechanisms associated with skeletal muscle response to neoplasic stimulus.     

Thrombospondin-2 and extracellular matrix assembly

Background

Numerous proteins and small leucine-rich proteoglycans (SLRPs) make up the composition of the extracellular matrix (ECM). Assembly of individual fibrillar components in the ECM, such as collagen, elastin, and fibronectin, is understood at the molecular level. In contrast, the incorporation of non-fibrillar components and their functions in the ECM are not fully understood.

Scope of review

This review will focus on the role of the matricellular protein thrombospondin (TSP) 2 in ECM assembly. Based on findings in TSP2-null mice and in vitro studies, we describe the participation of TSP2 in ECM assembly, cell–ECM interactions, and modulation of the levels of matrix metalloproteinases (MMPs).

Major conclusions

Evidence summarized in this review suggests that TSP2 can influence collagen fibrillogenesis without being an integral component of fibrils. Altered ECM assembly and excessive breakdown of ECM can have both positive and negative consequences including increased angiogenesis during tissue repair and compromised cardiac tissue integrity, respectively.

General significance

Proper ECM assembly is critical for maintaining cell functions and providing structural support. Lack of TSP2 is associated with increased angiogenesis, in part, due to altered endothelial cell–ECM interactions. Therefore, minor changes in ECM composition can have profound effects on cell and tissue function. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Astroglial-Induced In Vitro Angiogenesis: Requirements for RNA and Protein Synthesis

Astrocytes are believed to affect microvascular endothelial cell differentiation in brain and retina. Bovine retinal microvessel endothelial cells formed capillary-like structures when cocultured with C₆ astroglial cells or in the absence of C₆ cells in response to the reconstituted basement membrane protein Matrigel. Using quantitative computer-assisted image analysis, the requirements for RNA and protein synthesis in these two complementary models of in vitro microvessel morphogenesis were examined. Astroglial-dependent capillary-like structure formation was inhibited by up to 87% in a dose-dependent fashion by cycloheximide (0.01–0.1 μg/ml), puromycin (0.1–0.25 μg/ml), and actinomycin D (0.01– 0.025 μg/ml). In contrast, the astroglial-independent process in response to Matrigel was not affected by these metabolic inhibitors. These findings suggest that capillary-like structures form in response to astroglial cells in two distinct sequential stages. The first consists of inductive astroglial-endothelial interactions requiring both RNA and protein synthesis. This initiates endogenous endothelial morphogenic events that do not appear to require RNA or protein synthesis, consistent with posttranslational regulatory mechanisms. The first astroglial-dependent step is relevant to the regulation of microvessel formation in brain and retina, whereas the second may represent a morphogenic pathway common to micro-vessel formation in many tissues.     

Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.     

Function of alpha3beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2).

Tumor cell migration through the three- dimensional extracellular matrix (ECM) environment is an important part of the metastatic process. We have analyzed a role played by the integrin-tetraspanin protein complexes in invasive migration by culturing MDA-MB-231 cells within Matrigel. Using time-lapse video recording, we demonstrated that the Matrigel-embedded cells remain round and exhibit only limited ability for migration by extending short, highly dynamic pseudopodia. The alpha3beta1-tetraspanin protein complexes were clustered on the thin microvilli-like protrusions extending from both the main cell body and pseudopodia. Ligation of the alpha3beta1-tetraspanin protein complexes with monoclonal antibodies specifically stimulates production of matrix metalloproteinase 2 (MMP-2) and induces formation of long invasive protrusions within Matrigel. Accordingly, treatment with the monoclonal antibodies to various tetraspanin proteins and to the alpha3 integrin subunit increases invasive potential of the MDA-MB-231 cells in the Matrigel-penetration assay. A specific inhibitor of phosphoinositide 3-kinase (PI3K), LY294002, negated the effect of the monoclonal antibodies on the morphology of the Matrigel-embedded cells and on production of MMP-2. Interestingly, broad-spectrum inhibitors of protein tyrosine kinases (genistein) and protein tyrosine phosphatases (orthovanadate), and actin filament stabilizing compound (jasplakinolide), also block protrusive activity of the Matrigel-embedded cells but have no effect on the production of MMP-2. These results indicate that alpha3beta1-tetraspanin protein complexes may control invasive migration of tumor cells by using at least two PI3K-dependent signaling mechanisms: through rearrangement of the actin cytoskeleton and by modulating the MMP-2 production.     

骨关节炎患者血清中基质金属蛋白酶-2、-9的明胶酶谱分析

为探讨基质金属蛋白酶-2、-9在骨关节炎发病中的作用,应用明胶酶谱分析方法研究其在骨关节炎患者血清中的表达水平。实验对象为27例膝骨关节炎患者,对照组为7例外伤骨折患者。结果发现病例组血清中基质金属蛋白酶-2和-9的表达水平均明显高于对照组(P<0.05)。该结果显示基质金属蛋白酶-2和-9可能在骨关节炎的发病过程中均起着重要的作用。     

Retrospective analysis of a secondary structure prediction: the catalytic domain of matrix metalloproteinases.

Secondary structure prediction of the catalytic domain of matrix metalloproteinases is evaluated in the light of recently published experimentally determined structures. The prediction was made by combining conformational propensity, surface probability, and residue conservation calculated for an alignment of 19 sequences. The position of each observed secondary structure element was correctly predicted with a high degree of accuracy, with a single beta-strand falsely predicted. The domain fold was also anticipated from the prediction by analogy with the structural elements found in the distantly related metalloproteinases thermolysin, astacin, and adamalysin.     

The tumor microenvironment: a potential arbitrator of the tumor suppressive and promoting actions of TGFbeta

Transforming growth factor beta (TGFbeta) members are secreted in biologically inactive complexes that must be activated in order to enable binding to their cell surface receptors. Interestingly, many of the proteins that can activate TGFbeta have been implicated in either suppressing or promoting tumorigenesis. Included among these are matrix proteins (thrombospondin-1), receptors (integrins alphanubeta6 and alphanubeta8) and proteases (matrix metalloproteases and plasmin). These proteins cannot only activate TGFbeta, but can also modulate cell responsiveness to TGFbeta. In this section, we review data highlighting the complexity and bidirectionality of TGFbeta matrix interactions within the tumor microenvironment, and propose that these dynamic interactions are a critical spatial and temporal determinant of the effects of TGFbeta on tumorigenesis.     

Distinct modes of collagen type I proteolysis by matrix metalloproteinase (MMP) 2 and membrane type I MMP during the migration of a tip endothelial cell: insights from a computational model

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the proteolytic processing of extracellular matrix (ECM) structural proteins under physiological and pathological conditions. During sprouting angiogenesis, the MMPs expressed by a single "tip" endothelial cell exhibit proteolytic activity that allows the cells of the sprouting vessel bud to migrate into the ECM. Membrane type I matrix metalloproteinase (MT1-MMP) and the diffusible matrix metalloproteinase MMP2, in the presence of the tissue inhibitor of metalloproteinases TIMP2, constitute a system of proteins that play an important role during the proteolysis of collagen type I matrices. Here, we have formulated a computational model to investigate the proteolytic potential of such a tip endothelial cell. The cell expresses MMP2 in its proenzyme form, pro-MMP2, as well as MT1-MMP and TIMP2. The interactions of the proteins are described by a biochemically detailed reaction network. Assuming that the rate-limiting step of the migration is the ability of the tip cell to carry out proteolysis, we have estimated cell velocities for matrices of different collagen content. The estimated velocities of a few microns per hour are in agreement with experimental data. At high collagen content, proteolysis was carried out primarily by MT1-MMP and localized to the cell leading edge, whereas at lower concentrations, MT1-MMP and MMP2 were found to act in parallel, causing proteolysis in the vicinity of the leading edge. TIMP2 is a regulator of the proteolysis localization because it can shift the activity of MT1-MMP from its enzymatic toward its activatory mode, suggesting a tight mechanosensitive regulation of the enzymes and inhibitor expression. The model described here provides a foundation for quantitative studies of angiogenesis in extracellular matrices of different compositions, both in vitro and in vivo. It also identifies critical parameters whose values are not presently available and which should be determined in future experiments.     

催乳素对佐剂关节炎大鼠滑膜组织MMP-9表达的影响

目的：探讨催乳素（PRL）对佐剂关节炎（AA）大鼠滑膜组织基质金属蛋白酶-9（MMP-9）表达的影响,为进一步阐明PRL在类风湿性关节炎（RA）发病过程中的作用机制及为RA的治疗提供实验依据。方法：雄性Wistar大鼠32只分为4组（n=10）：①正常对照组（A组）（n=10）;②佐剂关节炎对照组（B组）（n=10）;③高催乳素血症佐剂关节炎组（C组）（n=10）;④低催乳素血症佐剂关节炎组（D组）。放射免疫法测定各组血清PRL含量;明胶酶谱法测定MMP-9的活性;免疫组织化学方法检测各组滑膜组织MMP-9免疫反应变化;Western blot测定各组滑膜组织MMP-9的表达。结果：B组滑膜组织MMP-9的活性及表达明显高于A组,C组MMP-9的活性及表达最高,D组较B组有所减少,但仍高于A组。结论：PRL影响滑膜组织MMP-9分泌是其影响RA发病过程的一种作用机制。     

Influence of organosulphur compounds from garlic on the secretion of matrix metalloproteinases and their inhibitor TIMP-1 by cultured HUVEC cells

Organosulphur compounds from garlic, especially diallyl disulphide (DADS) at non-toxic concentrations, affected production and secretion of some matrix metalloproteinases (MMPs) and of tissue inhibitor of metalloproteinase-1 (TIMP-1), one of their inhibitors, by human umbilical vein endothelial cells. Addition of DADS to the culture medium resulted in a concentration-dependent reduction of secreted MMP-2 protein and activity as well as TIMP-1 protein. In the presence of inducers (phorbol 12-myristate 13-acetate, forskolin and tumor necrosis factor alpha) addition of DADS caused a distinct concentration-dependent decrease of MMP-9 and TIMP-1 secretion, while not affecting MMP-9 mRNA levels. Intracellular protein levels remained low and were not affected. Other organosulphur compounds like allyl mercaptan and S-allylcysteine showed no or less clear effects on MMP-secretion or TIMP-1-secretion. These results suggest that DADS may mediate some of the biological effects ascribed to garlic preparations through affecting MMP-TIMP balance.     

Anti-cancer therapies targeting the tumor stroma

For anti-tumor therapy different strategies have been employed, e.g., radiotherapy, chemotherapy, or immunotherapy. Notably, these approaches do not only address the tumor cells themselves, but also the tumor stroma cells, e.g., endothelial cells, fibroblasts, and macrophages. This is of advantage, since these cells actively contribute to the proliferative and invasive behavior of the tumor cells via secretion of growth factors, angiogenic factors, cytokines, and proteolytic enzymes. In addition, tumor stroma cells take part in immune evasion mechanisms of cancer. Thus, approaches targeting the tumor stroma attract increasing attention as anti-cancer therapy. Several molecules including growth factors (e.g., VEGF, CTGF), growth factor receptors (CD105, VEGFRs), adhesion molecules (alphavbeta3 integrin), and enzymes (CAIX, FAPalpha, MMPs, PSMA, uPA) are induced or upregulated in the tumor microenvironment which are otherwise characterized by a restricted expression pattern in differentiated tissues. Consequently, these molecules can be targeted by inhibitors as well as by active and passive immunotherapy to treat cancer. Here we discuss the results of these approaches tested in preclinical models and clinical trials.     

基质衍生的血管生成抑制剂研究进展

血管的生成与肿瘤密切相关,抑制肿瘤血管生成可以调节肿瘤的生长。体内存在着内源性的促血管生成因子和抑制因子的平衡,当促血管形成因子增强就会产生新生血管供肿瘤生长,而当抑制因子增强则会抑制肿瘤的生长。本文即对细胞外基质衍生的内源性血管生成抑制因子TSP、内皮他丁、Arresten;Canstatin、Endorepellin、Fibulin、Tumstatin等的特性、应用和作用机制等作一总结。     

Crystal structure of the catalytic domain of human matrix metalloproteinase 10

The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.     

Effects of diosgenin on myometrial matrix metalloproteinase-2 and -9 activity and expression in ovariectomized rats

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.