PubMed Health⌕ Search

Biomedical subjects

S Silletti

Publications and source records attributed to S Silletti.

At least 19 recordsLinked to original sources
PubMed2002

Matrix metalloproteinase/integrin interactions as target for anti-angiogenic treatment strategies.

Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.

Allantois↗
PubMed2001

A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice.

A carcinoembryonic Ag (CEA)-based DNA vaccine encoding both CEA and CD40 ligand trimer achieved effective tumor-protective immunity against murine colon carcinoma in CEA-transgenic mice by activating both naive T cells and dendritic cells. Peripheral T cell tolerance to CEA was broken in a prophylactic model by this novel, dual-function DNA vaccine, whose efficacy was further enhanced by boosts with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). These conclusions are supported by four lines of evidence. First, a lethal challenge of MC38-CEA-KS Ag murine colon carcinoma cells was for the first time completely rejected in 100% of experimental animals treated by oral gavage of this DNA vaccine carried by attenuated Salmonella typhimurium, followed by five boosts with huKS1/4-IL-2. Second, specific activation of dendritic cells was indicated by their marked up-regulation in expression of costimulatory molecules B7.1 (CD80), B7.2 (CD86), and ICAM-1. Third, a decisive increase over control values was observed in both MHC class I Ag-restricted cytotoxicity of CTLs from successfully vaccinated mice and secretion of proinflammatory cytokines IFN-gamma and IL-12. Fourth, activation of CTLs was augmented, as indicated by up-regulation of activity markers LFA-1, CD25, CD28, and CD69. Taken together, these results suggest that a dual-function DNA vaccine encoding CEA and CD40 ligand trimer combined with tumor-targeted IL-2 may be a promising strategy for the rational development of DNA-based cancer vaccines for future clinical applications.

Animals↗
PubMed2001

Identification of a novel class of small-molecule antiangiogenic agents through the screening of combinatorial libraries which function by inhibiting the binding and localization of proteinase MMP2 to integrin alpha(V)beta(3).

The process of new blood vessel growth from existing vasculature, known as angiogenesis, is critical to several pathological conditions, most notably cancer. Both MMP2, which degrades the extracellular matrix (ECM), and integrin alpha(V)beta(3), which contributes to endothelial cell attachment to the ECM, are critically involved in this process. Recent findings have shown that MMP2 is localized in an active form on the surface of invasive endothelial cells based on its ability to directly bind integrin alpha(V)beta(3), suggesting that disrupting this protein--protein interaction may represent a new target for the development of angiogenesis inhibitors. The screening of small molecule libraries led to the identification of compounds which disrupt the MMP2--alpha(V)beta(3) interaction in an in vitro binding assay. A prototypical inhibitor was further found to prevent the degradation of the protein matrix without directly inhibiting MMP2 activity or disrupting the binding of alpha(V)beta(3) to its classical ECM ligand, vitronectin. The synthesis and screening of analogues and substructures of this lead compound allowed the identification of requisite structural features for inhibition of MMP2 binding to alpha(V)beta(3). This led to the synthesis of a more water-soluble derivative which maintains the in vitro biological properties and has potent antiangiogenic and antitumor activity in vivo, validating the target as one useful for therapeutic intervention.

Angiogenesis Inhibitors↗
PubMed2001

Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo.

Matrix metalloproteinase 2 (MMP2) can associate with integrin alpha(v)beta3 on the surface of endothelial cells, thereby promoting vascular invasion. Here, we describe an organic molecule (TSRI265) selected for its ability to bind to integrin alphav(v)beta3 and block alpha(v)beta3 interaction with MMP2. Although disrupting alpha(v)beta3/MMP2 complex formation, TSRI265 has no effect on alpha(v)beta3 binding to its extracellular matrix ligand vitronectin and does not influence MMP2 activation or catalytic activity directly. However, TSRI265 acts as a potent antiangiogenic agent and thereby blocks tumor growth in vivo. These findings suggest that activated MMP2 does not facilitate vascular invasion during angiogenesis unless it forms a complex with alpha(v)beta(3) on the endothelial cell surface. By disrupting endothelial cell invasion without broadly suppressing cell adhesion or MMP function, the use of compounds such as TSRI265 may provide a novel therapeutic approach for diseases associated with uncontrolled angiogenesis.

Angiogenesis Inhibitors↗
PubMed2001

Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice.

Peripheral T-cell tolerance toward human carcinoembryonic self-antigen (CEA) was broken in CEA-transgenic C57BL/6J mice by an oral CEA-based DNA vaccine. This vaccine, delivered by the live, attenuated AroA- strain of Salmonella typhimurium (SL7207), induced tumor-protective immunity mediated by MHC class I-restricted CD8+ T cells. Activation of these T cells was indicated by increased secretion of proinflammatory cytokines IFN-gamma, interleukin (IL)-12 and granulocyte/macrophage-colony stimulating factor, as well as specific tumor rejection and growth suppression in vaccinated CEA-transgenic mice after a lethal challenge with murine MC38 colon carcinoma cells. These tumor cells were double transfected with CEA and the human epithelial cell adhesion molecule (Ep-CAM)/KSA and consequently served as a docking site for a recombinant antibody-IL2 fusion protein (KS1/4-IL2) recognizing KSA. Importantly, the efficacy of the tumor-protective immune response was markedly increased by boosts with this antibody-IL2 fusion protein, resulting in more effective tumor rejection coupled with increased expression of costimulatory molecules B7.2/B7.2 and intercellular adhesion molecule 1 (ICAM-1) on dendritic cells and intensified release of proinflammatory cytokines IFN-gamma, IL-12, and granulocyte/macrophage-colony stimulating factor from T cells of successfully vaccinated CEA-transgenic C57BL/6J mice. Increased T-cell activation mediated by boosts with KS1/4-IL2 fusion protein after tumor cell challenge was further indicated by expanded expression of T-cell activation markers CD25, CD28, CD69, and LFA-1. The application of such CEA-based DNA vaccines and its further improved versions may ultimately prove useful in combination therapies directed against human carcinomas expressing CEA self-antigens.

Animals↗
PubMed2000

Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2.

Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.

Animals↗
PubMed2000

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment.

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.

Amino Acid Sequence↗
PubMed1999

A potential role for the plasmin(ogen) system in the posttranslational cleavage of the neural cell adhesion molecule L1.

L1 is a neural recognition molecule that promotes neural developmental and regenerative processes. Posttranslational cleavage of L1 is believed to be important for regulating its function in vivo, but little is known of the proteolytic systems responsible. In this study we present evidence that plasmin can regulate both L1 expression and function. The addition of plasmin to cell lines results in a dose-dependent loss of surface L1 expression, with the simultaneous appearance of soluble L1 species. The addition of plasminogen to primary neurons and melanoma cells also resulted in the generation of plasmin and the concomitant release of L1. One product of plasmin-mediated cleavage is an amino-terminal fragment of approximately 140 kDa that has been previously described as a natural posttranslational cleavage product in vivo. This fragment was confirmed to result from cleavage at two sites in the middle of the third fibronectin-like domain of L1. Cleavage at a further site, proximal to the transmembrane domain of L1, was also observed at higher plasmin concentrations. Plasmin was further confirmed to abrogate homophilic L1 interactions required for cellular aggregation. Based on these findings we propose that plasmin is likely to be an important regulator of L1-mediated processes including those documented in the nervous system.

Base Sequence↗
PubMed1998

Autocrine motility factor and the extracellular matrix. I. Coordinate regulation of melanoma cell adhesion, spreading and migration involves focal contact reorganization.

Cellular interactions with the extracellular matrix are complex and are involved in numerous biological processes. These interactions may be modulated by cytokines such as tumor cell autocrine motility factor, a secreted molecule that regulates cellular growth and migration by a receptor-mediated pathway. In this report we provide evidence suggesting that high- and low-metastatic K1735 melanoma cells coordinate their attachment, spreading and migratory responses to autocrine motility factor and the extracellular substratum through alterations in focal adhesion plaque architecture that are dependent on both inherent cellular metastatic phenotype and the composition of the supporting matrix. Thus, since activation of the autocrine motility factor receptor has previously been shown to enhance the experimental metastasis of the high- but not the low-metastatic K1735 cells, differences in melanoma cell malignancy in this system may be due in part to a coordinated interplay between cytokine-mediated responses and extracellular matrix-directed regulation of cellular adhesion, spreading and motility.

Animals↗
PubMed1998

Autocrine motility factor and the extracellular matrix. II. Degradation or remodeling of substratum components directs the motile response of tumor cells.

Autocrine motility factor is a tumor-secreted cytokine which regulates cellular growth and motility by a receptor-mediated pathway. In the accompanying report (Part I of II), it was demonstrated that high (K1735-M1) and low (K1735-C1.11) metastatic murine melanoma cells display distinct adhesion and spreading characteristics which correlate with their differential spontaneous and stimulated migrations on the extracellular matrix components fibronectin, laminin and collagen IV. These parameters were further related to discrete profiles of focal adhesion plaque integrity and reorganization. Here we describe unique migration patterns observed in these murine melanoma cells which reflect differences in degradation and/or remodeling of the cellular substratum. These profiles of matrix interaction were influenced distinctly by autocrine motility factor and dictated by both substrate composition and cellular phenotype. Since activation of the autocrine motility factor receptor stimulates invasion of a reconstituted basement membrane and enhances experimental metastasis by high- but not low-metastatic K1735 cells, differences in the invasive phenotypes of these cells may be due in part to their differential responses to external stimuli coupled with internal propensities toward either matrix degradation and migration (high-metastatic cells) or matrix remodeling and stasis (low-metastatic cells).

Animals↗
PubMed1998

Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity.

Angiogenesis depends on both cell adhesion and proteolytic mechanisms. In fact, matrix metalloproteinase 2 (MMP-2) and integrin alphavbeta3 are functionally associated on the surface of angiogenic blood vessels. A fragment of MMP-2, which comprises the C-terminal hemopexin-like domain, termed PEX, prevents this enzyme binding to alphavbeta3 and blocks cell surface collagenolytic activity. PEX blocks MMP-2 activity on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth. Importantly, a naturally occurring form of PEX can be detected in vivo in conjunction with alphavbeta3 expression in tumors and during developmental retinal neovascularization. Levels of PEX in these vascularized tissues suggest that it interacts with endothelial cell alphavbeta3 where it serves as a natural inhibitor of MMP-2 activity, thereby regulating the invasive behavior of new blood vessels.

Allantois↗
PubMed1997

A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins.

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. The interaction between L1-Ig6 and alphaIIbbeta3 was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with alphavbeta3 and alphavbeta1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. Whereas beta3 integrin binding to L1-Ig6 was evident in the presence of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the beta1 integrins was only observed in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by alpha5beta1 and alphavbeta1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that alphavbeta3 or active alphaIIbbeta3 > alphavbeta1 > alpha5beta1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.

Adult↗
PubMed1996

Autocrine motility factor signals integrin-mediated metastatic melanoma cell adhesion and invasion.

The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N-benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C.

Animals↗
PubMed1996

Tumor autocrine motility factor responses are mediated through cell contact and focal adhesion rearrangement in the absence of new tyrosine phosphorylation in metastatic cells.

Autocrine motility factor (AMF) is a tumor-secreted cytokine that acts as a motogen as well as a mitogen via a receptor-mediated signaling pathway(s). Expression of the AMF receptor (AMF-R) in normal cells is regulated by cell contact whereas in transformed cells AMF-R is constitutively expressed irrespective of cell density. Here we have analyzed the regulation of AMF-R expression in a BALB/c angiosarcoma tumor system that allows investigation of cellular characteristics associated with tumor progression. The metastatic cell variant (A31-M) displayed a higher rate of unstimulated motility and responded to tumor-derived AMF locomotory stimulus as compared with the nonmetastatic cell variants (A31-TR and A31-TU) and, although a similar level of receptor expression was detected in cellular extracts from subconfluent cultures of these sublines, surface localization differed and cell contact down-regulated AMF-R expression in the normal but not the transformed cell counterparts. AMF promoted marked rearrangement of focal adhesion plaque proteins in the AMF migration-responsive cells exclusively. Reorganization of vinculin after AMF stimulation was paralleled by morphological redistribution of tyrosine-phosphorylated proteins and the tyrosine kinase pp125FAK in the migration-responsive cells; however, we did not observe a concomitant change in the pp125FAK phosphorylation state or the general level of cellular tyrosine phosphorylation in response to treatment, suggesting that the induction of cellular migration by AMF is independent of tyrosine phosphorylation events at the focal contacts and may therefore represent a novel pathway of cytokine-induced migration regulation.

Animals↗
PubMed1995

Loss of cell-contact regulation and altered responses to autocrine motility factor correlate with increased malignancy in prostate cancer cells.

Tumor cell migration and proliferation in new organ environments are critical steps in cancer progression and can be modulated by tumor- and host-secreted molecules. Autocrine motility factor (AMF) is a tumor-secreted cytokine which regulates growth and motility by a receptor-mediated pathway. The AMF receptor, a 78-kDa cell surface glycoprotein (gp78), is regulated by cell contact in normal fibroblastic and bladder cells; however, this mechanism is disrupted during tumor progression. A prostatic carcinoma cell line which is low- to non-metastatic in nude mice (PC-3) and a derived metastatic variant (PC-3M) were examined to determine if gp78 cell density regulation is involved in prostate cancer progression. Both cell lines expressed gp78 and, although the basal migration of the parental PC-3 cells was higher than that of the metastatic variant, only the PC-3M cells were capable of responding to tumor-derived AMF with increased motility. Furthermore, these cells exhibited differential patterns of wound closure in an experimental system whereby the low-metastatic PC-3 cells migrated primarily along the wound edge while individual high-metastatic PC-3M cells entered the cell-free wound area directly. Cell surface gp78 distribution distinguished the cell populations with a markedly concentrated display of gp78 in polarized capped regions on the surface of the metastatic cells. Cell-cell contact down-regulated gp78 expression in the parental, but not the metastatic, cells, and mitogenic responses to exogenous AMF differed between these cell lines as well. In this model, metastasis appears to be associated with aberrant regulation of gp78 expression and distribution, coupled with enhanced exploitation of AMF's locomotory and proliferative effects.

Cell Movement↗
PubMed1994

Autocrine motility factor induces differential 12-lipoxygenase expression and activity in high- and low-metastatic K1735 melanoma cell variants.

A M(r) 55,000 tumor cell-secreted cytokine has been described which influenced the migration of the producing cells and was called autocrine motility factor (AMF). Activation of the cell surface receptor for AMF (gp78) was shown to stimulate production of a 12-lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE], in highly metastatic murine melanoma cells. AMF stimulated the motility of the high-metastatic (K1735-M1) but not the low-metastatic variant (K1735-Cl.11) of the K1735 murine melanoma and increased expression of the 12-lipoxygenase enzyme predominantly in the high-metastatic counterpart. The K1735-M1 cells responded to motile stimulation with increased endogenous 12-(S)-HETE production, and, reciprocally, exogenous 12-(S)-HETE up-regulated surface gp78 and caused gp78 translocation from an intracellular perinuclear pool to tubulovesicles which extended to the cell periphery in the K1735-M1 cells exclusively. These results suggest that differences in AMF responses may be due to alterations in the capacity of low-metastatic cells to transduce signals through 12-lipoxygenase or to involve downstream effector(s) of 12-(S)-HETE after gp78 activation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗