Expression of Ets-related transcription factors and matrix metalloproteinase genes in human breast cancer cells

The expression levels of ets and MMP genes was examined in two breast cancer cell lines of differing invasive potential. The more invasive MDA-MB-231 cell line had higher levels of Ets-1, Ets-2, PEA3, ERM, Tel, Net, MMP-13 and -14 mRNA than MCF-7 cells. MMP-1, -3 and -16 mRNAs were expressed equally. TPA stimulated MMP-1, -9 and TIMP-1 mRNA expression in both cell lines. MMP-2 and MMP-7 mRNAs were not detected in either cell line. The Ets-1 protein was only detected in MDA-MB-231 cells and its level increased following TPA stimulation. TPA induced MMP-9 activity in MCF-7 cells and increased its activity in MDA-MB-231 cells, however, MMP-2 activity was not detected.     

Association of early life factors and brain tumour risk in a cohort study

Using population-based linked birth and cancer registry data, we investigated whether the risk of brain tumour in childhood (n=155) was associated with perinatal risk factors. This population-based cohort showed that being born into a larger family or to a mother with a history of miscarriage may increase childhood brain tumour risk.     

Inhibition of tumour growth by marimastat in a human xenograft model of gastric cancer: relationship with levels of circulating CEA.

Inhibition of matrix metalloproteinases (MMPs) is an attractive approach to adjuvant therapy in the treatment of cancer. Marimastat is the first orally administered, synthetic MMP inhibitor to be evaluated, in this capacity, in the clinic. Measurement of the rate of change of circulating tumour antigens was used for evaluating biological activity and defining optimum dosage in the early clinical trials of marimastat. Although tumour antigen levels have been used in the clinical management of cancer for many years, they have not been validated as markers of disease progression. In order to investigate the relationship between the effects of marimastat on tumour growth and circulating tumour antigen levels, mice bearing the human gastric tumour, MGLVA1, were treated with marimastat. The MMP inhibitor exerted a significant therapeutic effect, reducing tumour growth rate by 48% (P = 0.0005), and increasing median survival from 19 to 30 days (P = 0.0001). In addition, carcinoembryonic antigen (CEA) levels were measured in serum samples from animals sacrificed at regular intervals, and correlated with excised tumour weight. It was shown that the natural log of the CEA concentration was linearly related to the natural log of the tumour weight and that treatment was not a significant factor in this relationship (P = 0.7). In conclusion, circulating CEA levels were not directly affected by marimastat, but did reflect tumour size. These results support the use of cancer antigens as markers of biological activity in early phase trials of non-cytotoxic anticancer agents.     

Activated mesothelial cells produce heparin-binding growth factors: implications for tumour metastases

Curative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF121 and VEGF165. Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bg(c)) IL-2 receptor. Cytokine-induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum.     

Secretion of matrix metalloproteinase-2 (72 kD gelatinase/type IV collagenase = gelatinase A) by malignant human glioma cell lines: implications for the growth and cellular invasion of the extracellular matrix

Human glioma cells (T98G and A172 cell lines) were cultured on various extracellular matrix (ECM) components including type 1, IV and V collagens, fibronectin, laminin, and reconstituted basement membrane (Matrigel), and the role of matrix metalloproteinases (MMPs) in their growth and invasion was examined. T98G glioma cells grew well on these ECM components and invaded the reconstituted basement membrane. In contrast, A172 glioma cells showed growth inhibition on collagen types IV and V and Matrigel without invasion of the Matrigel. Gelatin zymography and enzyme immunoassays demonstrated that T98G glioma cells, but not A172 cells, secrete a large amount of matrix metallproteinase-2 (MMP-2, 72 kD gelatinase/type IV collagenase = gelatinase A), and this was confirmed by immunoblotting and immunohistochemistry. Of the two different tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), T98G cells produced only TIMP-1 during culture on Matrigel, whereas A172 cells secreted both. Although both human recombinant TIMP-1 and TIMP-2 stimulated T98G cell growth slightly on Matrigel, the in vitro invasiveness was significantly reduced by only recombinant TIMP-2. These results suggest that MMP-2 plays an important role in the ECM invasion of T98G human glioma cells in vitro.     

Astroglial growth factors in normal human brain and brain tumors: comparison with embryonic brain

Summary Aqueous extracts of 18-day embryonic chicken brains, 15-day embryonic and adult rat brains and human brain tumors, as well as control histologically-normal adult human brain taken from around brain tumors or around arteriovenous malformations each stimulated the growth of cultured chick astrocytes. Eight mitogenic fractions were separated reproducibly by Bio-Gel P-10 molecular seive chromatography. They had apparent molecular weights (M.W.) of 24, 17, 12, 9, 5, 2.8, 1.4 and 1.2 kD. The activity of each fraction was concentration dependent. The fractions did not appear to be artifactually derived by proteolysis from a larger mitogen since (i) protease inhibitors were added at the time of homogenization to prevent degradation, (ii) protease treatment did not produce large quantities of the lower molecular weight fractions, (iii) incubation of brain extracts for up to four hours at 30° C did not alter the activity of the various mitogenic fractions and (iv) addition of albumin to inhibit protease activity similarly did not change the profile of the factors. In contrast, treatment with protease reduced the activity of all the factors although those with M.W. of 5 and 1.2 kD were inactivated more slowly than the others. The various fractions were stable when rechromatographed. This suggested they were not chance aggregates derived artifactually during extraction but rather might have physiological and pathological roles. The activities of each mitogenic fraction were significantly higher in brain extracts from embryonic rats than in those from adult rats. In brain extracts of rat and chicken embryos the fractions of lower M.W. 5 kD to 1.2 kD were relatively abundent. In contrast in brain extracts from adult rats the predominant mitogenic fractions had apparent M.W. of 24,17 and 12 kD. In histologically normal adult human brain taken from around the tumors or around arteriovenous malformations the 5 kD fraction was present in small amounts and the fractions of lower molecular weight were present in very small amounts. In human glial brain tumors there was a preponderance of the 5 kD activity and more of the 2.8 and 1.4 kD activity fractions than in histologically normal adult human brain. But there was relatively less activity in the 24 and 17 kD fractions. The growth factor profile of human meningiomas was quite different from that of histologically normal human brain or human glial brain tumors. The fraction from meningiomas that was most mitogenic for astrocytes had a molecular weight of 12 kD. Although the higher molecular weight factors may represent already described growth factors derived from brain, the lower molecular weight factors have not been reported previously.     

Phosphatase of regenerating liver-3 promotes migration and invasion by upregulating matrix metalloproteinases-7 in human colorectal cancer cells

Phosphatase of regenerating liver (PRL)-3, a member of a subgroup of protein tyrosine phosphatases that can stimulate the degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer (CRC) relative to primary tumors. To determine whether PRL-3-induced enhancement of migration and invasion is dependent on the expression of matrix metalloproteinases (MMPs), PRL-3 was expressed in DLD-1 human CRC cells. The motility, migration and invasion characteristics of the cells were examined, and metastasis to the lung was confirmed in a nude mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the cells were inhibited by phosphatase and farnesyltransferase inhibitors. Expression of MMPs was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were partially inhibited by small interfering RNA (siRNA) knockdown of MMP-2, -13 or -14. Importantly, siRNA knockdown of MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, whereas overexpression of MMP-7 increased migration. The expression of MMP-7 was also downregulated by phosphatase and farnesyltransferase inhibitors. It was found that PRL-3 induced MMP-7 through oncogenic pathways including PI3K/AKT and ERK and that there is a relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The expression of MMP-13 and -14 was very sensitive to the inhibition of farnesyltransferase; however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3-expressing CRC cells depends primarily on the expression of MMP-7.     

Spectrum of matrix metalloproteinase expression in primary and metastatic colon cancer: relationship to the tissue inhibitors of metalloproteinases and membrane type-1-matrix metalloproteinase

The matrix metalloproteinases, MMP-2 and MMP-9, are capable of degrading components of the basement membrane, a vital barrier breached during the progression of colorectal cancer. The regulation of MMP-2 activation and subsequent targets is vital to understanding the metastatic process. MMP-2 was not expressed by colorectal cancer cells (C170 and C170HM(2)) in vitro but by stromal fibroblasts (46BR.1GI). There was induction of this MMP upon transwell co-cultivation of the colon cancer cells with the fibroblasts but in vivo growth did not lead to a similar increase in the metastatic tumour cells (C170HM(2)), MMP-2 again being attributed to the stromal cells. MMP-2 mRNA was overexpressed in human colorectal tumours compared to normal colorectal tissue, which correlated with Dukes' stage and immunolocalized to the stromal compartment of the tumour tissue. The active form of the MMP-2 enzyme was also present in the colorectal tumour tissue (7/8) but essentially absent in all normal colon samples examined (1/8). MMP-2 activation was not related to an increase in MT-1-MMP mRNA or a decrease in the specific inhibitor TIMP-2 in human tissue. There was however an increase in MMP-2/TIMP-2 ratio in tumour compared to normal. MMP-9, a target of active MMP-2, was present in the metastatic cell line but expression was down-regulated in the tumour cells in vivo, gelatin analysis revealed that MMP-9 was almost entirely attributable to the murine host, confirmed by PCR. There was no increase in mRNA for MMP-9 or its specific inhibitor TIMP-1 in colorectal tumour tissue compared to normal, MMP-9 protein localized to the inflammatory infiltrate. Fibroblast cells may provide malignant epithelial cells with a ready source of enzyme which is crucial to the metastatic process.     

Immunolocalization of monoclonal antibody-defined extracellular matrix antigens in human brain tumors

Summary The extracellular matrix is involved in many aspects of tumor cell biology, including tumor invasion and metastasis. 2A6 and 81C6 are murine monoclonal antibodies that identify glioma-mesenchymal extracellular matrix antigens. The 81C6 antigen is a high molecular weight glycoprotein composed of M_r 230,000 subunits. The expression of 2A6 antigen, 81C6 glycoprotein, fibronectin (FN), and laminin (LN) was examined immunohistochemically in ten malignant gliomas (MG) and four medulloblastomas (MBT). 2A6 and 81C6 were expressed in similar patterns by the neoplastic neuroepithelial cells in 9/10 MG and 1/4 MBT. The staining was typically diffuse and amorphous, without visualization of distinct cell bodies or processes. Less frequently, antigen was detected within tumor cell cytoplasm. In most tumors the staining was greatest in the perivascular regions. In two MG, 2A6 and 81 C6 were expressed only by a subpopulation of neoplastic cells. Although intense staining was also associated with hyperplastic vascular and mesenchymal cells, many small and medium size blood vessels stained weakly or not at all. In contrast, FN and LN were expressed uniformly and intensely in the tumor vasculature, but were not expressed by neoplastic neuroepithelial cells. The 2A6 antigen and 81C6 glycoprotein are immunohistochemically distinct from FN and LN. These monoclonal antibody-defined antigens are heterogeneously expressed by neoplastic neuroepithelial cells and hyperplastic vascular-mesenchymal elements in MG and MBT. The 2A6 and 81C6 monoclonal antibodies will be useful reagents in the investigation of the extracellular matrix of malignant neuroepithelial neoplasms.Presented in part at the Second International Symposium on Biology of Brain Tumours, London, October 1984.     

脑源性神经营养因子促进人脑胶质瘤细胞株生长的研究

目的:探讨脑源性神经营养因子(BDNF)在人脑胶质瘤U87-MG细胞株生长中的作用及其可能的促血管生成机制.方法:脑胶质瘤U87-MG细胞株给予BDNF蛋白刺激或BDNF抗体处理,采用real time PCR检测血管内皮生长因子(VEGF) mRNA的表达,采用Transwell小室侵袭试验检测细胞侵袭能力,采用Annexin-Ⅴ/PI双染流式细胞术分析细胞凋亡.结果:BDNF蛋白刺激后VEGF mRNA表达随着时间的延长而上调,与对照组相比,8 h时增高了2.1倍,16 h时增高了2.5倍.细胞侵袭力增加,紫色细胞计数对照组为317±16.1,8 h为419±12.5,16 h为511±10.1,差异有统计学意义,P<0.01.细胞凋亡率8 h为2.28%,16 h为2.04%,差异无统计学意义,P>0.05.BDNF抗体作用组与之相反,VEGF mRNA表达降低,8 h时降低了0.88倍,16 h时降低了0.78倍.紫色细胞计数8 h为212±18,16 h为195±17.7.细胞凋亡率8 h为22.4%,16 h为29%,差异有统计学意义,P<0.01.结论:BDNF在体外能够上调VEGF mRNA表达,促进细胞侵袭,抑制细胞凋亡.     

Extracellular influences on tumour angiogenesis in the aged host

Whether tumours are epithelial or non-epithelial in origin, it is generally accepted that once they reach a certain size all solid tumours are dependent upon a vascular supply to provide nutrients. Accordingly, there is great interest in how the extracellular environment enhances or inhibits vascular growth. In this minireview, we will examine key extracellular components, their changes with ageing, and discuss how these alterations may influence the subsequent development of tumour vasculature in the aged host. Because of the tight correlation between advanced age and development of prostate cancer, we will use prostate cancer as the model throughout this review.     

Monoclonal antibodies to growth factors and growth factor receptors: their diagnostic and therapeutic potential in brain tumors

Journal of Neuro-Oncology -     

Glioma-produced extracellular matrix influences brain tumor tropism of human neural stem cells

Summary A major obstacle in the treatment of gliomas is the invasive capacity of the tumor cells. Previous studies have demonstrated the capability of neural stem cells (NSCs) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. Less is known about the factors involved in brain tumor tropism of NSCs and their interactions within the tumor environment. As gliomas progress and invade, an extensive modulation of the extracellular matrix (ECM) occurs. Tumor-ECM derived from six glioblastoma cell lines, ECM produced by normal human astrocytes and purified ECM compounds known to be upregulated in the glioma environment were analyzed for their effects on NSCs motility in vitro. We found that tumor-produced ECM was highly permissive for NSC migration. Laminin was the most permissive substrate for human NSC migration, and tenascin-C the strongest inducer of a directed human NSC migration (haptotaxis). A positive correlation between the degree of adhesion and migration of NSCs on different ECM compounds exists, as for glioma cells. Our in vitro data suggest that the ECM of malignant gliomas is a modulator of NSC migration. ECM proteins preferentially expressed in areas of glioma cell invasion may provide a permissive environment for NSC tropism to disseminated tumor cells. ^† These authors contributed equally to this work.     

Expression of matrix metalloproteinases in human glioma cell lines in the presence of IL-10

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.     

白藜芦醇对宫颈癌HeLa细胞基质金属蛋白酶及其组织抑制剂的影响

目的探讨白藜芦醇对宫颈癌侵袭和转移的影响及其初步作用机制。方法选用Transwell侵袭系统,观察白藜芦醇对宫颈癌细胞侵袭转移的影响;用明胶酶谱法、RT-PCR和Western blot法了解白藜芦醇对宫颈癌细胞MMP-2、MMP-9、TIMP-1、TIMP-2的活性、mRNA及蛋白表达的影响。结果白藜芦醇1、0.5mmol/L两组平均穿过的细胞数分别为9.20±1.924,10.20±2.049与空白对照组(39.80±2.387)相比,明显减少,P<0.05。同时,白藜芦醇能够明显降低宫颈癌细胞MMP-2、MMP-9的活性、mRNA及蛋白表达水平(P<0.05),上调TIMP-1、TIMP-2的活性(F=636.354,P<0.05,F=87.516,P<0.05)、mRNA(F=9.000,P<0.05,F=10.288,P<0.05)及蛋白表达水平(F=39.329,P<0.05,F=12.148,P<0.05),并使得MMP-2/TIMP-2、MMP-9/TIMP-1减小,P<0.05。结论白藜芦醇能有效抑制宫颈癌侵袭和转移。其作用机制可能与降低宫颈癌细胞运动能力、抑制MMP-2、MMP-9的活性、mRNA及蛋白表达水平,上调TIMP-1、TIMP-2的活性、mRNA及蛋白表达水平,并使得MMP-2/TIMP-2、MMP-9/TIMP-1减小,MMP-2/TIMP-2、MMP-9/TIMP-1的平衡能力相应提高有关。     

3-Bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate inhibits cancer cell invasion in vitro and tumour growth in vivo

In search for new anticancer agents, we have evaluated the antiinvasive and antimigrative properties of recently developed synthetic coumarin derivatives among which two compounds revealed important activity: 3-chlorophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate and 3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate. Both drugs were able to inhibit cell invasion markedly in a Boyden chamber assay, the bromo derivative being more potent than the reference matrix metalloprotease (MMP) inhibitor GI 129471. In vivo, tumour growth was reduced when nude mice grafted with HT1080 or MDA-MB231 cells were treated i.p. 3 days week(-1) with the bromo coumarin derivative. These effects were not associated with the inhibition of urokinase, plasmin, MMP-2 or MMP-9. The mechanism of action of the drugs remains to be elucidated. However, these two coumarin derivatives may serve as new lead compounds of an original class of antitumour agents.     

结缔组织生长因子转染对人乳腺癌细胞系生物学行为的影响

背景与目的:结缔组织生长因子(CTGF)是CCN家族成员之一,参与体内多种生理和病理生理过程。本研究探讨CTGF对人乳腺癌细胞生物学行为的影响。方法:构建含有CTGF开放读码框的真核表达质粒,通过脂质体介导的方法将正义质粒转染MCF-7细胞,反义质粒转染MDA-MB-231细胞。观察CTGF不同表达水平与乳腺癌细胞增殖、凋亡、周期分布和体外侵袭等生物学行为的关系。结果:转染正义质粒使MCF-7细胞CTGF表达上调,抑制细胞增殖,促进细胞凋亡,细胞发生G0G1期阻滞,体外侵袭能力下降。转染反义质粒使MDA-MB-231细胞CTGF表达下调,促进细胞增殖,减少细胞凋亡,增强细胞体外侵袭能力,但对细胞周期无影响。结论:CTGF具有抑制乳腺癌细胞生长和侵袭的功能。促进细胞凋亡是CTGF抑制乳腺癌细胞体外生长的机制之一。CTGF可能对细胞周期有一定的影响。     

Differential regulation of matrix metalloproteinases in human cancer cells by exogenous factors

Matrix degradation by cancer cells is an important attribute of malignant behaviour. We have postulated that during invasion and metastasis, matrix metalloproteinase (MMP) production by cancer cells is modulated by microenvironmental factors. To determine this, we screened human cancer cell lines of diverse histogenesis for their secreted MMP profile following exposure to a diversity of soluble and insoluble agents. Enzymatic changes were observed in the presence of phorbol ester (TPA) and hydrogen peroxide (H2O2). More specifically, we observed: (a) increased secretion and/or activation of gelatinases A (MMP-2) and B (MMP-9) after exposure of 8 cell lines to 10(-6) M TPA; (b) increased activation of interstitial collagenase (MMP-1) caseinolysis after stimulation of 3 cancer cell lines with 10(-7) M TPA; and (c) increased activation of MMP-2 after exposure of 2 cell lines to 0.5 mM H3O2. In functional assays, exposure of lung Calu-6 carcinoma cells to 0.5 mM H2O2 and PC-3 cells to TPA, increased their ability to degrade radiolabelled endothelial cell-derived and osteoblast-like-derived basement membranes, respectively. RNA hybridization studies confirmed and further documented these observations. We conclude that modulation of MMP activity in human cancer cells is a common phenomenon regardless of lineage. Modulatory mechanisms such as increased de novo pro-enzyme synthesis and/or release, and enhanced proenzyme activation may be involved. Furthermore, our results provide evidence that extracellular matrix degradation by human cancer cells during invasion and metastasis may be stimulated by soluble matrix-derived factors in a cell type-specific fashion.     

Insulin-like growth factors in human breast cancer

Several protooncogenes and suppressor genes and a variety of growth factors and their receptors have been shown to be mutated, deleted, or activated in human breast cancer. These changes may account for the unregulated growth of breast carcinoma cells. Insulin-like growth factors I and II (IGF-I, IGF-II) belong to a family of polypeptides with growth promoting properties and structural homology to insulin. They exert their mitogenic effects by binding to the IGF-I receptor and activating its tyrosine protein kinase. Other proteins that specifically bind the IGFs include the plasma membrane IGF-II receptor, which also binds lysosomal hydrolases, and several IGF-binding proteins which may serve to modulate IGF interactions with receptors.