Inhibition Of Cellular Invasion Research Articles

On the Biology of Werner's Complex

AbstractWerner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof‐of‐principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non‐covalent hydrogen‐bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound.

On the Biology of Werner's Complex.

Werner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof-of-principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non-covalent hydrogen-bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound.

P5.05 - Isolation of novel cell migration inhibitor migracins from Streptomyces and inhibition of cellular invasion in ovarian carcinoma cells

ABSTRACT Background: Bioactive metabolites that inhibit cellular migration are considered to be useful for the suppression of cancer metastasis. Then, we have looked for cancer cell migration inhibitors from microorganisms. In one hand, ovarian carcinoma is highly invasive and often metastasizes into liver, lung, and peritoneal cavity. Materials and methods: Cellular migration was assessed by wound healing assay with breast carcinoma MDA-MB-231 cells. We employed about 1500 microbial broths for random screening. From the culture filtrate with positive activity, active principle was isolated by chromatography. The structure was determined mainly by NMR and mass spectroscopy after the purification. Results: After testing the thousands of microbial culture filtrates, we have isolated novel compounds migracin A and B from Streptomyces. Migracin A and B showed similar inhibitory activity on the migration of MDA-MB-231 cells and fibrosarcoma HT-1080 cells. They also inhibited TNF-alpha and TGF-beta-induced migration of lung adenocarcinoma A549 cells. Migracin A inhibited the Matrigel invasion of clear cell ovarian carcinoma ES-2 cells without any toxicity. Since the structure of migracin is related to that of luminacin that inhibits tube formation, we have studied the effect on lumina formation. As a result, migracin A inhibited VEGF-induced limina formation in HUVEC. The mechanism of inhibition is being studied. Conclusions: We have discovered migracins as novel inhibitors of cellular migration and invasion. Migracin inhibited invasion of ovarian carcinoma cells, and is considered to be a candidate of new metastasis inhibitor.

Abstract 56: Examining the role of nm23-H1 in the metastatic profile of triple negative breast cancer (TNBC)

Abstract Background: Triple negative breast cancer makes up 10-20% of all mammary tumors. It is so named because it exhibits low or no expression of both the estrogen receptor (ER) and progesterone receptor (PR), and has low or no expression of the human epidermal growth factor (HER2) receptor. With no current molecular target identified, treatments options for TNBCs are limited to conventional chemotherapy, which has been shown to be only moderately effective. Further, diagnosis is correlated with a high rate of relapse, low survival rate five years past diagnosis, and overall poor prognosis. Nm23-H1 is the most well characterized in a class of tumor suppressor genes that have received increased attention as a potential therapeutic target in breast cancers. First identified in 1988, nm23, has been shown have low levels of mRNA expression in highly metastatic tumor cells, compared to normal or non-neoplastic tissue. This expression level coincides with not only increased metastasis but poor clinical prognosis in several type cancers, including breast cancer. Further, nm23-H1 inhibits a class of Rho small GTPases including Rac1, Rho and cdc42. These proteins mediate cell-to-cell adhesion and cytoskeletal reorganization, which ultimately moderate the metastatic profile i.e. metastasis, cellular motility, and invasion. Methods: Western blotting was used to examine the basal level of expression in a panel of mesenchymal and epithelial TNBC cell lines. Transient siRNA was used to silence nm23-H1 in MDAMB231 and HCC1806 cells to determine the effects on downstream Nm23-H1 targets Rac1, Rho and cdc42. The effects of nm23-H1 inhibition on the invasive potential of TNBC cells were assessed using Matrigel chamber assays. Wound healing assays were employed to assess the effects of nm23-H1 inhibition on the migratory potential of TNBC cells. Results: Our studies indicate that nm23-H1 is expressed in all of the TNBC cell lines that we tested but to varying degrees with mesenchymal cells expressing the protein at a higher level compared to epithelial cells. Immunoblotting suggested that silencing of nm23-H1 resulted in an increase in p-Rac. Silencing also increased the invasive ability of TNBC cells compared to control cells. Conclusion: These results demonstrate the vital role that nm23-H1 plays in the inhibition of cellular invasion in TNBCs. An increase in the expression of proteins associated with motility and invasion pathways validates the role of nm23-H1 in inhibiting the metastatic profile in TNBC. As a potential molecular marker for metastatic TNBC, nm23-H1 warrants further investigation. Citation Format: Tanisha Z. McGlothen, Rachel Tobin, Tiffanie Alcaide, LaTonia Taliaferro-Smith, Tongrui Liu, Ruth O'Regan. Examining the role of nm23-H1 in the metastatic profile of triple negative breast cancer (TNBC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 56. doi:10.1158/1538-7445.AM2014-56

Abstract 24: Role of S100A14 in proliferation and invasion of oral cancer-derived cells

Abstract Members of multifunctional S100 protein family have been reported to be differentially expressed in a variety of human cancers and are functionally linked with various aspects of carcinogenesis. S100A14 is a recently identified member of the S100 protein family. Despite reports of altered expression in different cancer types, functional significance of S100A14 in carcinogenesis is largely unknown. In this study, we aimed to investigate the functional role of S100A14 proliferation and invasion of oral squamous cell carcinoma (OSCC)-derived cell. S100A14 mRNA and protein levels were found to be down-regulated in OSCCs and OSCC-derived cells compared to the corresponding normal controls. In addition, membranous S100A14 immunoexpression was found to be gradually lost in the invasive fronts/islands compared to the central/superficial part of OSCCs. Over-expression of S100A14, by using retroviral expression construct, was found to be associated with the inhibition of cell proliferation, induction of G1-arrest and up-regulation of p21 in OSCC-derived cell lines (CaLH3 and OSCC1) with wild type p53. Similarly, over-expression of S100A14 was associated with inhibition of invasive potential of OSCC-derived cells in the in vitro Matrigel invasion assay. S100A14 mediated inhibition of cellular invasion was found to be associated with modulation of mRNA signature of genes involved in tumor invasion and metastasis, in particular MMP1, MMP9. Taken together, these data suggest that similar to other multifunctional S100 protein members, S100A14 might be involved in OSCC progression by regulating cell proliferation and invasion. Hence, further characterization of the molecular pathways/targets of S100A14 might lead to the better understanding of proliferation and invasion of OSCC and this process, in future, might be useful as a diagnostic or therapeutic approach in cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 24. doi:1538-7445.AM2012-24

Abstract B17: The small RhoGTPase RhoB as a new potential marker of EGFR-TKI resistance

Abstract Introduction: EGFR tyrosine kinase inhibitors (EGFR-TKI) are widely used in metastatic non small cell lung cancer (NSCLC). Nevertheless, the majority of patients with wild type EGFR do not respond to these treatments and, in the selected group with EGFR mutations, we some primary resistance and all the patients will ultimately develop acquired resistance. The small RHO GTPase RhoB is involved in cell proliferation, invasion, apoptosis and can act as a tumor suppressor gene in lung carcinogenesis by controlling survival and invasion through AKT pathway. Furthermore, RhoB participates in the regulation of endocytic trafficking of EGFR. Specifically, activated RhoB is able to delay the transport of internalized EGFR to lysosomes, thus modifying its downstream signaling. The objective of our study is to know if RhoB is a molecular determinant of the response to the EGFR-TKI erlotinib, and if RhoB could be a marker for erlotinib resistance. Material and Method: We used various lung cancer cell lines harboring either wild-type or mautated EGFR and K-Ras. RhoB was either overexpressed by adenovirus transfection or inhibited by interfering RNA (siRNA) in these cells. Cell proliferation after 72h of treatment with erlotinib was measured by a colorimetric assay. In the same way, we performed invasion assays and western-blot analyses of downtsream signaling pathways. Results: (1) RhoB overexpression decreases the sensitivity to erlotinib of cells presenting a wild type or mutated EGFR in a dose-dependent manner whereas the sensitivity to erlotinib of cells bearing KRas mutation was not affected by RhoB overexpression. (2) Erlotinib-induced inhibition of cellular invasion is impaired by overexpression of RhoB in wild type or mutated EGFR cell lines. (3) The phosphorylation of Akt (and not of ERK) is inhibited by erlotinib and is restored upon overexpression of RhoB invasion. Conclusions: Our results suggest that RhoB is involved in the sensitivity to erlotinib in wild-type K-Ras cells harboring either mutated or wild-type EGFR. RhoB is able to block erlotinib-induced inhibition of cell proliferation and invasion mainly through AKT pathway modulation. We plan to extend our in vitro findings in a cohort of patients treated with erlotinib.

Abstract 4141: Notch signaling: A new potential target in the treatment of uveal melanoma

Abstract Uveal melanoma is the most common primary intra-ocular malignancy in adults, and causes significant mortality due to its propensity to metastasize. Our aim is to investigate the role of Notch signaling in promoting uveal melanoma proliferation and invasion. We examined five established uveal melanoma cell lines, and found using qPCR that the Notch 1-2-3 receptors, Jag1-2 ligands and the pathway target Hes1 were expressed to varying degrees in all lines. We then blocked Notch signaling using the gamma-secretase inhibitor (GSI) MRK003, and found that only three of the lines (OCM1, OCM3, OCM8) had their growth inhibited by the drug. Interestingly, these three lines had significantly higher levels of Hes1 mRNA as compared to the two lines resistant to GSI treatment (Mel 285, Mel 290). GSI treatment induced a dose-dependent reduction of anchorage-independent clonogenic growth, with 50% inhibition in OCM1, 70% in OCM3, and 40% in OCM8, as well as a significant reduction in Hes1 mRNA and protein levels. Apoptosis, as measured by cleavage and activation of caspase-9, was also induced. Finally, we observed inhibition of cellular invasion in GSI treated cultures using transwell migration and scratch assays. Preliminary studies of snap-frozen primary tumors indicate that Notch pathway components are expressed in primary uveal melanoma in vivo at levels as high or higher than those in our cell line models. In addition, oligonucleotide microarray analysis showed a significant increase in Jag2 and Notch3 expression levels in primary uveal melanomas that metastasized as compared to those that did not. Our findings suggest that the Notch pathway plays an important role in inducing cellular proliferation and invasion in uveal melanoma, and that inhibiting this pathway using pharmacological agents may be effective in preventing tumor growth and metastasis. 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 4141.

The FN13 peptide inhibits human tumor cells invasion through the modulation of αvβ3 integrins organization and the inactivation of ILK pathway

We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized αvβ1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized αvβ3 integrins, whereas SK-OV-3 organized αvβ5 dimers. The functional block of αvβ5 integrins, with an inactivating anti-αvβ5 antibody, led to the induction of αvβ3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of αvβ1 dimers, leading to ILK pathway inactivation, only when the organization of αvβ3 integrins is induced in the plasma membrane.

Deguelin, an Akt Inhibitor, Suppresses IκBα Kinase Activation Leading to Suppression of NF-κB-Regulated Gene Expression, Potentiation of Apoptosis, and Inhibition of Cellular Invasion

Deguelin, a constituent of the bark of the African plant Mundulea sericea (Leguminosae), exhibits antiproliferative and anticarcinogenic activities through a mechanism that is not well understood. Because various steps in carcinogenesis are regulated by NF-kappaB, we postulated that the activity of deguelin is mediated through this transcription factor. We found that deguelin suppressed NF-kappaB activation induced by carcinogens, tumor promoters, growth factors, and inflammatory stimuli. This suppression was not cell-type specific, because NF-kappaB activation was suppressed in both lymphoid and epithelial cells. Moreover, constitutive NF-kappaB activation was also blocked by deguelin. The suppression of TNF-induced NF-kappaB activation by deguelin occurred through the inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Deguelin also suppressed the NF-kappaB reporter activity induced by TNFR1, TNFR-associated death domain, TNFR-associated factor 2, and IkappaBalpha kinase, but not that induced by p65. The inhibition of NF-kappaB activation thereby led to the down-regulation of gene products involved in cell survival, proliferation, and invasion. Suppression of these gene products by deguelin enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results demonstrate that deguelin inhibits the NF-kappaB activation pathway, which may explain its role in the suppression of carcinogenesis and cellular proliferation.

Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response

Rickettsia rickettsii, a Gram-negative and obligate intracellular bacterium, preferentially infects the vascular endothelium during human infections leading to inflammation and dysfunction. The aim of this study was to determine whether R. rickettsii infection of endothelial cells (EC) activates p38 and/or c-jun N-terminal kinases (JNK) mitogen-activated protein (MAP) kinase, key regulatory proteins that control the response to inflammatory stimuli. We show that infection of cultured human EC results in the dose-dependent activation of p38, as assessed by increased phosphorylation and activity, without affecting the status of JNK. Rickettsia inactivation by heat or formaldehyde abolished the activation of p38 kinase and inhibition of cellular invasion by infection at low temperature, pre-treatment of host EC with cytochalasin D, or pre-incubation of rickettsiae with an irreversible phospholipase inhibitor led to a diminished p38 phosphorylation, suggesting requirement of invasion by viable rickettsiae for this host cell response. SB 203580, a p38-specific inhibitor, had no effect on infection-induced activation of the ubiquitous transcriptional regulator nuclear factor-kappa B, but effectively reduced the expression and secretion of important chemoattractant cytokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 by R. rickettsii-infected EC. Selective inhibition of p38 activity may be exploited as an anti-inflammatory target to prevent rickettsial vasculitis and to develop new and improved chemotherapeutic agents.

Acidic Sphingomyelinase Mediates Entry of N. gonorrhoeae into Nonphagocytic Cells

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae–induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.