Matrix Metalloprotease Inhibitor Research Articles

Abstract 1154: MicroRNA-221, -222 and -181 regulates tamoxifen resistance in breast cancer by downregulating TIMP3 and facilitating growth factor signaling

Abstract We have shown earlier that microRNA (miR)-221 and -222 are upregulated in tamoxifen (TAM) resistant breast cancer cell line MCF-7 (OHTR), and in Her2 positive primary human breast tumors compared to the Her2 negative tumors1. In addition, expression of miR-181b was 3.5-fold higher in OHTR cells compared to the MCF-7 cells and endocrine resistant primary tumors. To determine the underlying mechanism of miR-mediated regulation of TAM resistance, we focused on identification of target genes regulated by these miRs. Bioinformatics search revealed presence of seed sequences for all three miRs on 3’-UTR of TIMP3, a tissue metalloprotease inhibitor. Luciferase assay confirmed TIMP3 to be a direct target of these miRs and was found to be regulated at both RNA and protein levels. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationship between TIMP3 and miR 221/222 expression in primary human tumors, where invasive ductal carcinomas expressed high level of the miRs and normal or non-invasive cancers expressed TIMP3, but not the miRs. Cell proliferation and wound healing assay demonstrated increased sensitization of the TAM resistant cells to the drug upon ectopic expression of TIMP3. In addition, mouse mammary tumors induced with TIMP3 overexpressing cells were found to be more sensitive to TAM compared to the tumors initiated with control cells. Conversely, knocking down TIMP3 by siRNA reduced sensitivity to TAM, both in cell cultures and in mouse mammary tumors. As TIMP3 is an inhibitor of matrix metalloproteases involved in positive regulation of growth factor signaling, we speculated that reduction in TIMP3 due to high miR-221/222/181 expression could facilitate growth factor signaling in OHTR cells. Indeed, EGF-induced MAPK phosphorylation was significantly higher in OHTR cells compared to MCF-7 cells and miR-221/222 overexpressing MCF-7 cells mimicked this phenomenon. On the contrary, phosphMAPK level was reduced when TIMP3 was overexpressed in OHTR cells and increased upon knocking down of TIMP3 in MCF-7 cells. Similar differences in MAPK phosphorylation was also observed in cells treated with low level estrogen or TAM, agonists for TAM resistant cells. Based on this data we conclude that overexpression of miR-221/222 and -181b facilitates growth factor signaling in TAM resistant cells by downregulating TIMP3. 1. Miller et. al. J. Biol. Chem 2008; 283 (44): 29897 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1154. doi:10.1158/1538-7445.AM2011-1154

A One-Pot Synthesis and Biological Activity of Ageladine A and Analogues

A one-pot synthesis of ageladine A and analogues is reported. The key Pictet-Spengler reaction between 2-aminohistamine and aryl aldehydes has been successfully utilized for the synthesis of the natural product and 14 analogues. These compounds were screened for their matrix metalloprotease (MMP) and kinase inhibition to develop the first structure-activity relationship of ageladine A analogues. One compound, which showed significant kinase activity but little MMP inhibitory activity, was found to be highly active in an antiangiogenic screen, suggesting that the angiogenic activity of ageladine A is not associated with MMP inhibition but rather kinase inhibitory activity. Cytotoxicity was excluded as a mode of action by the assay of ageladine A and an analogue against 60 human cell lines.

Ancylostoma ceylanicum Excretory–Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory–secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 Å resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2′s netrin-like fold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells

miRNAs (microRNAs) have proven to play essential roles in diverse biological processes including early development, cell proliferation and cell death, and cell differentiation. However, there is only limited amount of information about their potential role in chondrogenesis. In the present study, we investigated the role of miRNA-488 in the cellular condensation, which is essential initiation for chondrogenic differentiation. We found that miRNA-488 expression is up-regulated at the precondensation stage and then down-regulated at the postcondensation stage. Blockade of miRNA-488 via the use of PNA (peanut agglutinin)-based ASOs (antisense oligonucleotides) decreased the protein level of integrins β1 and phosphorylated FAK (focal adhesion kinase) and resulted in the suppression of cell motility and migration. Moreover, in parallel with theses observation, treatment of anti-miRNA-488 oligonucleotides up-regulated the level of MMP (matrix metalloprotease)-2 activity, and co-treatment with GM6001, an MMP inhibitor, induced recovery of cellular condensation inhibited by blockade of miRNA-488. Collectively, our results suggest that miRNA-488 is one of regulator in cell to ECM (extracellular matrix) interaction through modulation of focal adhesion activity by MMP-2 during chondrogenesis of limb mesenchymal cells.

Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity

BackgroundIn the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis.Methods and ResultsWe assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression.ConclusionsTaken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression.

Identifying Chelators for Metalloprotein Inhibitors Using a Fragment-Based Approach

Fragment-based lead design (FBLD) has been used to identify new metal-binding groups for metalloenzyme inhibitors. When screened at 1 mM, a chelator fragment library (CFL-1.1) of 96 compounds produced hit rates ranging from 29% to 43% for five matrix metalloproteases (MMPs), 24% for anthrax lethal factor (LF), 49% for 5-lipoxygenase (5-LO), and 60% for tyrosinase (TY). The ligand efficiencies (LE) of the fragment hits are excellent, in the range of 0.4-0.8 kcal/mol. The MMP enzymes all generally elicit the same chelators as hits from CFL-1.1; however, the chelator fragments that inhibit structurally unrelated metalloenzymes (LF, 5-LO, TY) vary considerably. To develop more advanced hits, one hit from CFL-1.1, 8-hydroxyquinoline, was elaborated at four different positions around the ring system to generate new fragments. 8-Hydroxyquinoline fragments substituted at either the 5- or 7-positions gave potent hits against MMP-2, with IC(50) values in the low micromolar range. The 8-hydroxyquinoline represents a promising new chelator scaffold for the development of MMP inhibitors that was discovered by use of a metalloprotein-focused chelator fragment library.

Relative roles of doxycycline and cation chelation in endothelial glycan shedding and adhesion of leukocytes

Leukocyte [white blood cell (WBC)] adhesion and shedding of glycans from the endothelium [endothelial cells (ECs)] in response to the chemoattractant f-Met-Leu-Phe (fMLP) has been shown to be attenuated by topical inhibition of matrix metalloproteases (MMPs) with doxycycline (Doxy). Since Doxy also chelates divalent cations, these responses were studied to elucidate the relative roles of cation chelation and MMP inhibition. WBC-EC adhesion, WBC rolling flux, and WBC rolling velocity were studied in postcapillary venules in the rat mesentery during superfusion with the cation chelator EDTA or Doxy. Shedding and accumulation of glycans on ECs, with and without fMLP, were quantified by the surface concentration of lectin (BS-1)-coated fluorescently labeled microspheres (FLMs) during constant circulating concentration. Without fMLP, low concentrations of EDTA (1-3 mM) increased FLM-EC sequestration due to disruption of the permeability barrier with prolonged exposure. In contrast, with 0.5 μM Doxy alone, FLM adhesion remained constant (i.e., no change in glycan content) on ECs, and WBC adhesion increased with prolonged superfusion. Without fMLP, EDTA did not affect firm WBC-EC adhesion but reduced WBC rolling flux in a dose-dependent manner. With fMLP, EDTA did not inhibit WBC adhesion, whereas Doxy did during the first 20 min of superfusion. Thus, the inhibition by Doxy of glycan (FLM) shedding and WBC adhesion in response to fMLP results from MMP inhibition, in contrast to cation chelation. With either Doxy or the MMP inhibitor GM-6001, WBC rolling velocity decreased by 50%, as in the case with fMLP, suggesting that MMP inhibition reduces sheddase activity, which increases the adhesiveness of rolling WBCs. These events increase the effective leukocrit on the venular wall and increase firm WBC-EC adhesion. Thus, MMP inhibitors have both a proadhesion effect by reducing sheddase activity while exerting an antiadhesion effect by inhibiting glycocalyx shedding and subsequent exposure of adhesion molecules on the EC surface.

Acute Hepatocyte Growth Factor Treatment Induces Long-Term Neuroprotection and Stroke Recovery via Mechanisms Involving Neural Precursor Cell Proliferation and Differentiation

Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal HGF treatment (5 μg) after a 45-minute stroke, with regard to brain injury and neurologic recovery. Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this, HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-dependent hypoxic injury in vitro. Preservation of blood-brain barrier integrity 24 hours after stroke was furthermore noticed in animals receiving HGF, which was associated with the inhibition of matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24 hours, respectively. We suggest that sustained recruitment of proliferating cells together with improved neurovascular remodeling provides an explanation for HGF-induced long-term neuroprotection.

Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.

Acute venous occlusion enhances matrix metalloprotease activity: Implications on endothelial dysfunction

Venous hypertension is associated with microvascular inflammation, restructuring, and apoptosis, but the cellular and molecular mechanisms underlying these events remain uncertain. In the present study, we tested the hypothesis that elevated venous pressure and reduction of shear stress induce elevated enzymatic activity. This activity in turn may affect endothelial surface receptors and promote their dysfunction. Using a rodent model for venous hypertension using acute venular occlusion, microzymographic techniques for enzyme detection, and immunohistochemistry for receptor labeling, we found increased activity of the matrix metalloproteases (MMPs) -1, -8, and -9 and tissue inhibitors of metalloproteases (TIMPs) -1 and -2 in both high- and low-pressure regions. In this short time frame, we also observed that elevated venule pressure led to two different fates for the vascular endothelial growth factor receptor-2 (VEGFR2); in higher-pressure upstream regions, some animals exhibited higher VEGFR2 expression, while others displayed lower levels upstream compared to their downstream counterparts with lower pressure. VEGFR2 expression was, on average, more pronounced upon application of MMP inhibitor, suggesting possible cleavage of the receptor by activated enzymes in this model. We conclude that venous pressure elevation increases enzymatic activity which may contribute to inflammation and endothelial dysfunction associated with this disease by influencing critical surface receptors.

Vascular Protection in Diabetic Stroke: Role of Matrix Metalloprotease-Dependent Vascular Remodeling

Temporary focal ischemia causes greater hemorrhagic transformation (HT) in diabetic Goto-Kakizaki (GK) rats, a model with increased cerebrovascular matrix metalloprotease (MMP) activity and tortuosity. The objective of the current study was to test the hypotheses that (1) diabetes-induced cerebrovascular remodeling is MMP dependent and (2) prevention of vascular remodeling by glucose control or MMP inhibition reduces HT in diabetic stroke. Control and GK rats were treated with vehicle, metformin, or minocycline for 4 weeks, and indices of remodeling including vascular tortuosity index, lumen diameter, number of collaterals, and middle cerebral artery (MCA) MMP activity were measured. Additional animals were subjected to 3 hours MCA occlusion/21 hours reperfusion, and infarct size and HT were evaluated as indices of neurovascular injury. All remodeling markers including MMP-9 activity were increased in diabetes. Infarct size was smaller in minocycline-treated animals. Both metformin and minocycline reduced vascular remodeling and severity of HT in diabetes. These results provide evidence that diabetes-mediated stimulation of MMP-9 activity promotes cerebrovascular remodeling, which contributes to greater HT in diabetes. Metformin and minocycline offer vascular protection, which has important clinical implications for diabetes patients who are at a fourfold to sixfold higher risk for stroke.

Wound healing and protease inhibition activity of Bacoside‐A, isolated from Bacopa monnieri wettest

Bacopa monnieri (L.) Wettest. (Scrophulariaceae) is a well-known medicinal herb. In the Indian system of medicine it is known as Brahmi (Sanskrit) and Indian water hyssop. Methanolic extract of Bacopa monnieri and its isolated constituent Bacoside-A were screened for wound healing activity. Bacoside-A was screened for wound healing activity by excision, incision and dead space wound on Swiss albino rats. Significant wound healing activity was observed in both extract and the Bacoside-A treated groups. The SDS-PAGE caseinolytic zymogram analysis of inhibition of matrix metalloproteases (MMPs) enzyme from the excision wound by Bacoside-A, an isolated constituent, was done with the concentrations 100 and 200 micromg/ml. In Bacoside-A treated groups, epithelialization of the excision wound was faster with a high rate (18.30 +/- 0.01 days) of wound contraction. The tensile strength of the incision wound was increased (538.47 +/- 0.14 g) in the Bacoside-A treated group. In the dead space wound model, the weight of the granuloma was also increased (89.15 +/- 0.08 g). The histological examination of the granuloma tissue of the Bacoside-A treated group showed increased cross-linking of collagen fibers and absence of monocytes. The wound healing activity of Bacoside-A was more effective in various wound models compared to the standard skin ointment Nitrofurazone.

Physiology and pathophysiology of matrix metalloproteases

Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn2+ ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with respect to substrate specificity, cellular and tissue localization, membrane binding and regulation that make this a very versatile family of enzymes with a multitude of physiological functions, many of which are still not fully understood. Essentially, all members of the MMP family have been linked to disease development, notably to cancer metastasis, chronic inflammation and the ensuing tissue damage as well as to neurological disorders. This has stimulated a flurry of studies into MMP inhibitors as therapeutic agents, as well as into measuring MMP levels as diagnostic or prognostic markers. As with most protein families, deciphering the function(s) of MMPs is difficult, as they can modify many proteins. Which of these reactions are physiologically or pathophysiologically relevant is often not clear, although studies on knockout animals, human genetic and epigenetic, as well as biochemical studies using natural or synthetic inhibitors have provided insight to a great extent. In this review, we will give an overview of 23 members of the human MMP family and describe functions, linkages to disease and structural and mechanistic features. MMPs can be grouped into soluble (including matrilysins) and membrane-anchored species. We adhere to the ‘MMP nomenclature’ and provide the reader with reference to the many, often diverse, names for this enzyme family in the introduction.

Correlated expression profile of extracellular matrix-related molecules during the inflammatory response of the teleost fish gilthead seabream

Extracellular matrix (ECM) components, in addition to their structural functions, interact with cell surface receptors and intracellular components to modulate the transduction of signals for cell growth, differentiation, migration, proliferation, polarization, apoptosis and inflammation. Our previous findings in the gilthead seabream ( Sparus aurata L.), a marine seasonal hermaphrodite teleost fish, have shown that both endocrine and immune stimuli modulate the expression of matrix metalloproteases (MMPs) and tissue inhibitors of MMP (TIMPs). In addition, collagen type I (COL1) induces the expression of some pro-inflammatory cytokines and MMPs in professional phagocytes. Consequently, in this study we use real-time RT-PCR to analyze the gene expression profile of several ECM-related molecules (MMP-2, -9 and -13, TIMP-2a, and -2b, COL1A1, and integrin β1a) in different organs of adult specimens as well as in response to innate immune challenges. Our results showed that liver had the lowest basal levels of them, although they were clearly modulated during injury and infection. In the same way, ECM-related molecules seem to participate in pro-inflammatory processes, being of particular interest COL1 which is synthesized by immune cells and is able to act as autocrine/paracrine stimulus for them. Lastly, we propose that the observed correlations between ECM-related molecules during the inflammatory response should be considered to obtain a more accurate picture of their roles in this process.

Expression profile of matrix metalloproteinase-2 and -9 and their endogenous tissue inhibitors in osteonecrotic femoral heads

The pathogenetic mechanisms of osteonecrosis of the femoral head are unresolved to date. This study analyzed the matrix metalloprotease (MMP) and tissue inhibitor of matrix metalloprotease (TIMP) expression and activity which might add to the impaired bone matrix repair capacity affecting the balance between bone resorption and de novo bone formation in osteonecrosis of the femoral head (ONFH). Cancellous bone biopsies were taken at the femoral head and neck of patients with advanced ONFH and patients with primary osteoarthritis (OA) who were undergoing total hip arthroplasty. We assessed the gene expression levels of MMP-2 and -9, TIMP-1 and -2, Il-1beta, Il-6 and TNF-alpha in both biopsies. These data were corroborated by MMP activity screening, collagen profiling and ELISAs for determination of MMP, TIMP, Il-1beta, Il-6 and TNF-alpha concentrations. Gene expression rates of MMP-2 and TIMP-1 were higher in ONFH biopsies whereas TIMP-2 and MMP-9 gene expression was not significantly altered. MMP protein profile shifted towards increased biosynthesis of both, active and pro-MMP-2 in ONFH bone lysates and decreased pro-MMP-9 production. Expression profiles of pro-inflammatory cytokines and collagens in OA and ONFH bone lysates were highly similar. Increased biosynthesis and activation of MMP-2 in ONFH samples may add to shifting the bone matrix turnover balance towards resorption of bone macromolecules thereby counter-acting new bone matrix production. OA- and ONFH-affected bone exhibits a similar pro-inflammatory cytokine and collagen expression profile suggesting a relationship on the molecular level of inflammation and collagenous matrix composition between both diseases.

Abstract 4115: Pituitary adenylate cyclase activating polypeptide causes the transactivation of epidermal growth factor receptors in lung cancer cells

Abstract Pituitary adenylate cyclase activating polypeptide (PACAP) is an autocrine growth factor for some lung cancer cells. The activated PAC1 receptor (R) causes phosphatidylinositol turnover, elevates cyclic AMP and increases proliferation of lung cancer cells (Zia et al., Cancer Res. 55:4886 (1995)). PAC1R mRNA is present in NSCLC cell line NCI-H838, which has epidermal growth factor (EGF)R. PACAP or EGF stimulated the proliferation of NCI-H838 cells whereas PACAP(6-38) or gefitinib inhibited growth. To investigate the cellular basis of these actions, the ability of PACAP to alter EGFR function was investigated using NSCLC cells. PACAP, but not the closely related vasoactive intestinal peptide (VIP) significantly increased EGFR tyrosine phosphorylation (4-fold) 2 min after addition to NCI-H838 cells. The ability of PACAP to increase EGFR or ERK tyrosine phosphorylation was inhibited by PACAP(6-38) (PAC1R antagonist), gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), or antibody to transforming growth factor (TGF) α. These results suggest that PACAP may increase the rate of release of TGFα from NCI-H838 cells, resulting in elevated EGFR tyrosine phosphorylation. PACAP stimulation of EGFR tyrosine phosphorylation was inhibited by addition of N-acetylcysteine (anti-oxidant), tiron (superoxide scavenger), DPI (NADPH oxidase inhibitor) or U-73122 (Phospholipase C inhibitor) but not H89 (protein kinase A inhibitor) to NCI-H838 cells. These results indicate that PACAP, a growth factor for NSCLC, causes transactivation of the EGFR in an oxygen-dependent manner that involves phospholipase C but not protein kinase A. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4115.

Abstract 1333: Upregulation of chemokines including RANTES/CCL5 upon Toll-like receptor 3 activation in breast cancer: Association with leukocyte infiltration

Abstract The association of leukocytes with the microenvironment of different tumors and their role in disease progression is well-documented. However, the mechanism of recruitment of leukocytes to the tumor microenvironment remains unclear. In particular, we have previously demonstrated the capability of tumor-associated dendritic cells to promote neovascularization. In this light, toll-like receptors (TLR) have recently been identified as potential players in different cancers such as malignant melanomas, thyroid cancer and pancreatic cancer. These are a family of pattern-recognition receptors that, when activated by a ligand, trigger signaling pathways involved chiefly in the inflammation process of the innate immune system. Here, we investigated the role of TLR-3 in the capability of the tumor cells to generate chemokines capable of attracting leukocytes to the tumor microenvironment. For this purpose we used the 4T1 model of breast cancer in BALB/c mouse. First, we were able to determine the expression of TLR-3 in these cells by PCR analysis. Then, 4T1 mouse breast cancer cells were treated with poly(I:C), a well-known ligand for TLR-3, and the supernatants were checked for the presence of different chemokines (RANTES, SDF-1α, MIP-3α, MIP-3β) by sandwich ELISA. These molecules were selected taking into account that their receptors are present in dendritic cells. Further, a reverse transcription quantitative PCR was performed to detect the RNA expression levels of these chemokines. In both cases, a significant increase in RANTES levels upon TLR-3 activation was observed. Further, a chemokine PCR array showed upregulation of 10 chemokines, RANTES among them, on 4T1 cells stimulated with poly(I:C). A subsequent protein array analysis of different mouse cytokines in poly(I:C)-treated 4T1 cells revealed also an increase in RANTES, IL-6 and MIP-2 and a decrease in the matrix metalloprotease inhibitor TIMP-1. Interestingly, IL-6 upregulation was further confirmed by ELISA. These results are supportive of our hypothesis that TLR-3 activation could lead to the release of chemokines that assist in leukocyte recruitment. Further in vivo studies are required to observe the effect of knocking down TLR-3 in cancer cells on the capability of the tumor to promote leukocyte infiltration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1333.

Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells

Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr(1068) of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)alpha antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species.

Adipocyte-Derived Microvesicles Are Associated with Multiple Angiogenic Factors and Induce Angiogenesis in Vivo and in Vitro

We previously reported that 3T3-L1 and rat primary adipocytes secreted microvesicles, known as adipocyte-derived microvesicles (ADMs). In the present study, we further characterized the 3T3-L1 ADMs and found that they exhibited angiogenic activity in vivo. Antibody arrays and gelatin zymography analyses revealed that several angiogenic and antiangiogenic proteins, including leptin, TNFalpha, acidic fibroblast growth factor (FGFa), interferon-gamma, and matrix metalloprotease (MMP)-2 and MMP-9, were present in the ADMs. Gene expression of most of these angiogenic factors was induced in the adipose tissue of diet-induced obese mice. Furthermore, leptin, TNFalpha, and MMP-2 were up-regulated at the protein level in the adipocyte fractions prepared from epididymal adipose tissues of high-fat-diet-induced obese mice. ADMs induced cell migration and tube formation of human umbilical vein endothelial cells, which were partially suppressed by neutralizing antibodies to leptin, TNFalpha, or FGFa but not to interferon-gamma. Supporting these data, a mixture of leptin, TNFalpha, and FGFa induced tube formation. ADMs also promoted cell invasion of human umbilical vein endothelial cells through Matrigel, which was suppressed by the addition of the MMP inhibitor 1,10'-phenanthroline and a neutralizing antibody to MMP-2 but not to MMP-9. These results suggest that ADMs are associated with multiple angiogenic factors and play a role in angiogenesis in adipose tissue.

Multimodal therapy for synergic inhibition of tumour cell invasion and tumour-induced angiogenesis

BackgroundSquamous cell carcinoma of the head and neck (SCCHN) are highly invasive tumours with frequent local and distant recurrence. Metastasis formation requires degradation of the extracellular matrix, which is fulfilled by membrane-associated proteases such as the urokinase plasminogen activator (uPA). WX-UK1 is a competitive active site inhibitor of the protease function of uPA that impairs on the capacity of tumour cells to invade in vitro.MethodsIn the present study, effects of combinations of WX-UK1 with matrix metalloprotease inhibitors (MMP, galardin®) and cyclooxygenase-2 (COX-2, celecoxib®) inhibitors on tumour cell proliferation, invasion, and angiogenesis induction were evaluated. Matrigel invasion chambers and a spheroid co-cultivation model with human fibroblast served to determine the invasive potential of both FaDu (SCCHN) and HeLa (cervical carcinoma) cells, each treated with combinations of Celecoxib®, Galardin®, and WX-UK1.ResultsBlocking of single protease systems resulted in a significant 50% reduction of tumour cell invasion using WX-UK1, while the triple combination was even more effective with 80% reduction of invasion. Additionally, a sprouting assay with HUVEC was used to test the anti-angiogenetic potential of the triple combination, resulting in a 40% decrease in the sprouting rate.ConclusionsA combined approach targeting different families of proteases and cyclooxygenases represents a promising adjuvant therapy.