Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.     

Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells

Vascular endothelial cells play a critical role in the maintenance of endometrial homeostasis. Indeed many pathological conditions causing abnormal endometrial bleeding including progestin only contraception, hormone replacement therapy, endometrial polyps, myomas, hyperplasia and cancer are associated with aberrant angiogenesis. Critical to the process of angiogenesis is the breakdown of the surrounding tissues by matrix metalloproteases (MMPs). In addition to the cells surrounding the endometrial endothelial cells, the endothelial cells themselves produce their own panel of MMPs. We now characterize the specific MMPs that are expressed by endothelial cells derived from human endometrium. These include MMP-1, MMP-2 and MMP-10 but not MMP-3. In addition, in order to successfully carry out consistent, homogeneous and sufficient numbers of studies we investigated the in vitro expression of the MMPs with both freshly isolated, early passaged endometrial endothelial cells (HEECs) as well as with newly telomerase immortalized HEECs (T-HEECs). The latter were karyotypically normal and expressed classic endothelial cell endpoints such as tubulogenesis on matrigel and expression of the endothelial cell markers CD-31 (PECAM), von Willebrand's factor, and the Tie-2 receptors. The levels of MMP expression as well as that of the metalloprotease inhibitors TIMP-1 and TIMP-2 were similar in parent and immortalized endothelial cells.     

Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation

Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.     

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix‐rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20–30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double‐staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three‐dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF‐7 cells induced vessel‐like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up‐regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.     

Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid.

Angiogenesis, the formation of new blood vessels, is an essential feature of malignant tumour development. Gamma linolenic acid (GLA), a n-6 polyunsaturated fatty acid (PUFA), inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, pancreatic cancer and hepatoma cells and also has anti-metastatic effects on endothelial cells. In the current study, we tested whether GLA inhibited angiogenesis induced by tumour cells. A rat aortic ring assay and in vitro tube formation of human vascular endothelial cells were used to determine angiogenesis (spontaneous, angiogenic factor- and tumour cells-induced). Inclusion of GLA in this 3-D matrix culture system significantly inhibited angiogenesis from aortic rings in a concentration-dependent manner. The results from tube formation of vascular endothelial cell further confirmed that GLA suppressed angiogenesis. Furthermore, in the cell motility assay (phagokinetic assay and endothelial wounding assay), a significant reduction of the motility of vascular endothelial cells by GLA was seen. It is concluded that gamma linolenic acid inhibits angiogenic factor and tumour-induced angiogenesis in vitro at least in part via its inhibitory effect on the motility of vascular endothelial cells.     

Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers

Background

Gold nanoparticles (AuNPs) scatter light intensely at or near their surface plasmon wavelength region. Using AuNPs coupled with dynamic light scattering (DLS) detection, we developed a facile nanoparticle immunoassay for serum protein biomarker detection and analysis. A serum sample was first mixed with a citrate-protected AuNP solution. Proteins from the serum were adsorbed to the AuNPs to form a protein corona on the nanoparticle surface. An antibody solution was then added to the assay solution to analyze the target proteins of interest that are present in the protein corona. The protein corona formation and the subsequent binding of antibody to the target proteins in the protein corona were detected by DLS.

Results

Using this simple assay, we discovered multiple molecular aberrations associated with prostate cancer from both mice and human blood serum samples. From the mice serum study, we observed difference in the size of the protein corona and mouse IgG level between different mice groups (i.e., mice with aggressive or less aggressive prostate cancer, and normal healthy controls). Furthermore, it was found from both the mice model and the human serum sample study that the level of vascular endothelial growth factor (VEGF, a protein that is associated with tumor angiogenesis) adsorbed to the AuNPs is decreased in cancer samples compared to non-cancerous or less malignant cancer samples.

Conclusion

The molecular aberrations observed from this study may become new biomarkers for prostate cancer detection. The nanoparticle immunoassay reported here can be used as a convenient and general tool to screen and analyze serum proteins and to discover new biomarkers associated with cancer and other human diseases.     

The role of the tissue microenvironment in the regulation of cancer cell motility and invasion

During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion.     

Saikosaponin C induces endothelial cells growth, migration and capillary tube formation

Saikosaponin C is one of the saikosaponins that are consisted in a Chinese herb, Radix Bupleuri. Recently, saikosaponins have been reported to have properties of cell growth inhibition, inducing cancer cells differentiation and apoptosis. However, saikosaponin C had no correlation with cell growth inhibition. In this study, we investigated the role of saikosaponin C on the growth of endothelial cells and angiogenesis. We found that saikosaponin C yielded a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. In addition to inducing endothelial cells growth, saikosaponin C also induced endothelial cells migration and capillary tube formation. The gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and the p42/p44 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells growth, migration and angiogenesis were also induced by saikosaponin C. From these results, we suggest that saikosaponin C may have the potential for therapeutic angiogenesis but is not suitable for cancer therapy.     

Rho GTPase function in tumorigenesis

Malignant tumor cells display uncontrolled proliferation, loss of epithelial cell polarity, altered interactions with neighboring cells and the surrounding extracellular matrix, and enhanced migratory properties. Proteins of the Rho GTPase family regulate all these processes in cell culture and, for that reason, Rho GTPases, their regulators, and their effectors have been suggested to control tumor formation and progression in humans. However, while the tumor-relevant functions of Rho GTPases are very well documented in vitro, we are only now beginning to assess their contribution to cancer in human patients and in animal models. This review will give a very brief overview of Rho GTPase function in general and then focus on in vivo evidence for a role of Rho GTPases in malignant tumors, both in human patients and in genetically modified mice.     

Human Tumor Cells Induce Angiogenesis through Positive Feedback between CD147 and Insulin-Like Growth Factor-I

Tumor angiogenesis is a complex process based upon a sequence of interactions between tumor cells and endothelial cells. Previous studies have shown that CD147 was correlated with tumor angiogenesis through increasing tumor cell secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In this study, we made a three-dimensional (3D) tumor angiogenesis model using a co-culture system of human hepatocellular carcinoma cells SMMC-7721 and humanumbilical vein endothelial cells (HUVECs) in vitro. We found that CD147-expressing cancer cells could promote HUVECs to form net-like structures resembling the neo-vasculature, whereas the ability of proliferation, migration and tube formation of HUVECs was significantly decreased in tumor conditioned medium (TCM) of SMMC-7721 cells transfected with specific CD147-siRNA. Furthermore, by assaying the change of pro-angiogenic factors in TCM, we found that the inhibition of CD147 expression led to significant decrease of VEGF and insulin-like growth factor-I (IGF-I) secretion. Interestingly, we also found that IGF-I up-regulated the expression of CD147 in both tumor cells and HUVECs. These findings suggest that there is a positive feedback between CD147 and IGF-I at the tumor-endothelial interface and CD147 initiates the formation of an angiogenesis niche.     

Chaotic neovascularization induced by aggressive fibrosarcoma cells overexpressing S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines essential for cell proliferation. Overexpression of S-adenosylmethionine decarboxylase in rodent fibroblasts led to aggressive transformants (Amdc-s cells) that had unforeseen high invasive capacity in nude mice, invading rapidly from the subcutaneous injection site into the peritoneal cavity and its organs. In vitro, these cells were much more invasive than Ras-oncogene-transformed fibroblasts, or human HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells. In immunohistological characterization, Amdc-s-induced tumors showed chaotic neovascularization, with abundant pleomorphic vessel-like structures that had noncontiguous or totally missing laminin (basement membrane) and CD31 (endothelial cell) immunoreactivity. Gene expression and protein analyses of Amdc-s cells showed them to overexpress several pro-angiogenic molecules, including vascular endothelial growth factor (VEGF-A), and to exhibit profound down-regulation of the anti-angiogenic thrombospondin-1 (TSP-1). By reintroduction of TSP-1 into Amdc-s cells, the high invasiveness was efficiently inhibited in vitro. Interestingly, Amdc-s cells showed up-regulation of hepatocyte growth factor (HGF) and also expressed the MET receptor, creating thus an autocrine loop able to regulate VEGF-A and TSP-1 levels. Further, we found Amdc-s cells to express increased amounts of matrix metalloproteinase-2 (MMP-2) and the large isoform of tenascin-C (TN-C), which may also contribute to the angiogenic switch and invasiveness. Consequently, Amdc-s cells offer an excellent model to sort out the key molecules of aggressive tumor growth, and thereby help in designing rational, novel anti-vascular and other cancer therapies.     

Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro

The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized-reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary-like tubes (CLT) formed by endothelial cells was reduced by up to 10-fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP-2 and MMP-9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25-70% of the capillary-like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF-blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast-conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary-like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three-dimensional cell organization.     

Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells

We have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of three-dimensional collagen gels to invade the underlying matrix as capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary-like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel-like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.     

Regulation of in vitro capillary tube formation by anti-integrin antibodies

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti- integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.     

N-cadherin mediates angiogenesis by regulating monocyte chemoattractant protein-1 expression via PI3K/Akt signaling in prostate cancer cells

Over the past decade, evidence continues to mount showing that N-cadherin is a critical protein in cancer progression and metastasis. In the present study, we evaluated the expression of N-cadherin in human prostate cancer tissue specimens and cell lines. Enhanced expression of N-cadherin was observed in both the malignant and bone-metastasized prostate tissue specimens compared to the healthy prostate tissues. Consistent with the tissue array data, N-cadherin was highly expressed in PC3, but not in Du145 and LNCaP human prostate cell lines. Based on cell to cell binding assay, we found that N-cadherin expression facilitates homotypic interaction between human prostate cancer cells and human microvascular endothelial cells (HMEC). Human angiogenesis antibody array and in vitro angiogenesis assay showed that siRNA-mediated knockdown of N-cadherin reduced the secretion of monocyte chemoattractant protein-1 (MCP-1), which played a potential role in stimulating capillary network formation of HMEC. Additionally, culture supernatant of Du145 cells transfected with full-length N-cadherin expressing plasmid showed increased MCP-1 expression and chemoattractant ability compared to normal Du145 cells. Further, we noticed that blocking PI3K activity inhibited N-cadherin mediated MCP-1 expression. Our data demonstrated that N-cadherin in prostate cancer cell mediates cell–cell adhesion and regulates MCP-1 expression via the PI3K/Akt signaling pathway.     

Inhibition of angiogenesis by S-adenosylmethionine

Metastasis is a leading cause of mortality and morbidity in cancer. One of the steps in metastasis process is the formation of new blood vessels. Aberrant DNA methylation patterns are common in cancer cells. In recent studies, S-adenosylmethionine (SAM), which is a DNA methylating agent, has been found to have inhibitory effects on some carcinoma cells in vivo and in vitro. In the present study, we have used SAM to investigate whether it is effective against angiogenesis in vitro. Our results have shown that SAM can reduce the formation and organization of capillary-like structures of endothelial cells in tumoral environment. Besides, we have found SAM can block endothelial cell proliferation and the migration of cells towards growth factors-rich media. In conclusion, our study suggests that SAM may be used against angiogenesis as a natural bio-product.     

Hypoxia-reoxygenation increase invasiveness of PANC-1 cells through Rac1/MMP-2

Pancreatic cancer is an aggressive malignancy with proclivity to early metastasis. High expression and activation of the collagenase matrix metalloproteinase-2 (MMP-2) have been found in human pancreatic cancer tissues, being these increased levels of active MMP-2 correlated to tumor invasion and metastasis. Hypoxia and reoxygenation (H-R) are critical pathophysiological conditions during ischemia-reperfusion injury, which has been shown to enhance both invasion and metastasis. In the present study, we investigated the effects of H-R on MMP-2 levels and the invasiveness properties of human pancreatic cancer cells PANC-1. Using specific inhibitors, we found that H-R treatment of these tumor cells induced secretion and activation of MMP-2, which was required for H-R-stimulated basement membrane degradation and cell invasion. Our results also indicate that signaling events involved in H-R-enhanced PANC-1 invasiveness comprehend PI3K-dependent activation of Rac1, which mediated the formation of NADPH-generated reactive oxygen species responsible for MMP-2 secretion and activation.