Dual inhibition of αV integrins and Src kinase activity as a combination therapy strategy for colorectal cancer. Jia, J; Starodub, A; Cushman, I; Liu, Y; Marshall, DJ; Hurwitz, HI; Nixon, AB Anti-cancer drugs
24
237-50
2013
Show Abstract
Both Src and αV integrins are important for tumor growth and angiogenesis. They are interconnected and responsible for important features of the tumor phenotype including invasiveness, metastasis, angiogenesis, and resistance to apoptosis. This study examines whether combinational inhibition of both integrin and Src pathways would exert greater antiangiogenesis and antitumor effects than either pathway alone. Using in-vitro cell culture systems, the activity of CNTO95 (Intetumumab), an αV integrin inhibitor, and dasatinib, an Src inhibitor, on proliferation, adhesion, and migration was evaluated in colon cancer cell lines, HCT-116 and RKO, as well as HUVEC cells. The antiangiogenic effect of this combinatory regimen was also tested using an in-vitro tubular network formation assay. The effects of CNTO95 and dasatinib on the activation of Src and integrin pathway signal transduction were also determined by western blotting. The combination of CNTO95 plus dasatinib inhibited adhesion, migration, and paxillin phosphorylation in both HCT-116 and RKO cells. CNTO95 and dasatinib also led to increased apoptosis of HCT-116 cells; however, similar effects were not observed in RKO cells. In addition, dual treatment of CNTO95 and dasatinib exerted enhanced effects on HUVEC cell proliferation, invasion, tubular network formation, and paxillin phosphorylation. In conclusion, our results suggest that concurrent inhibition of both the integrin and the Src pathways exert more pronounced antiangiogenic and antitumor effects than with either pathway being inhibited alone. | 23275294
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Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion. Swanson KV, Griffiss JM, Edwards VL, Stein DC, Song W. Cellular microbiology
13
1078-90
2011
Show Abstract
Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC-1-B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB-EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands. | 21501367
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Neisseria meningitidis adhesin NadA targets beta1 integrins: functional similarity to Yersinia invasin. Nägele, V; Heesemann, J; Schielke, S; Jiménez-Soto, LF; Kurzai, O; Ackermann, N The Journal of biological chemistry
286
20536-46
2011
Show Abstract
Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a β1 integrin-dependent adherence with subsequent internalization of yersiniae by a β1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine β1 integrin-expressing cell lines we could demonstrate the role of the β1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human β1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that β1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human β1 integrins. | 21471204
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Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices. Stratman, AN; Saunders, WB; Sacharidou, A; Koh, W; Fisher, KE; Zawieja, DC; Davis, MJ; Davis, GE Blood
114
237-47
2009
Show Abstract
Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly. Full Text Article | 19339693
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Design and activity of multifunctional fibrils using receptor-specific small peptides. Y Ohga, F Katagiri, K Takeyama, K Hozumi, Y Kikkawa, N Nishi, M Nomizu Biomaterials
30
6731-8
2009
Show Abstract
We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin alphavbeta3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-alphav integrin antibody. Further, cell attachment to B133 was inhibited by anti-alpha2 and anti-beta1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-alphav and anti-beta1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin alphavbeta3 and that RGD-B133 interacts with both integrin alphavbeta3 and integrin beta1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering. | 19765823
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PCI-enhanced adenoviral transduction employs the known uptake mechanism of adenoviral particles. Engesaeter, BØ; Bonsted, A; Berg, K; Høgset, A; Engebråten, O; Fodstad, Ø; Curiel, DT; Maelandsmo, GM Cancer gene therapy
12
439-48
2005
Show Abstract
The development of methods for efficient and specific delivery of therapeutic genes into target tissues is an important issue for further development of in vivo gene therapy. In the present study, the physical targeting technique, photochemical internalization (PCI), has been used together with adenovirus. The combination of PCI and adenoviral transduction has previously been shown to be favorable compared to adenovirus used alone, and the aim of this study was to verify the role of the adenoviral receptors and identify the uptake pathway used by adenoviral particles in photochemically treated cells. All examined cell lines showed augmented transduction efficiency after PCI-treatment, with a maximum of 13-fold increase in transgene expression compared to conventionally infected cells. Blocking of CAR induced a complete inhibition of PCI-enhanced transgene expression. However, photochemical treatment managed to enhance the transduction efficiency of the retargeted virus AdRGD-GFP showing also that the virus-CAR interaction is not vital for obtaining a photochemical effect on adenoviral transduction. Blocking the alpha(V)-integrins reduced the gene expression significantly in photochemically treated cells. Subjecting HeLa cells expressing negative mutant-dynamin to light treatment after infection gave no significant increase in gene transfer, while the gene transfer were enhanced seven-fold in cells with wild-type dynamin. Furthermore, chlorpromazine inhibited photochemical transduction in a dose-dependent manner, whereas Filipin III had no effect on the gene transfer. In summary, the data presented imply that adenoviral receptor binding is important and clathrin-mediated endocytosis is the predominant uptake mechanism for adenoviral particles in photochemically treated cells. | 15678152
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Novel compound enables high-level adenovirus transduction in the absence of an adenovirus-specific receptor. Christine M Fouletier-Dilling, Pablo Bosch, Alan R Davis, Jessica A Shafer, Steven L Stice, Zbigniew Gugala, Francis H Gannon, Elizabeth A Olmsted-Davis Human gene therapy
16
1287-97
2005
Show Abstract
Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus. | 16259562
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The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. Besson, A; Wilson, TL; Yong, VW The Journal of biological chemistry
277
22073-84
2002
Show Abstract
Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility. | 11934885
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Mutations in the heparin binding domain of fibronectin in cooperation with the V region induce decreases in pp125(FAK) levels plus proteoglycan-mediated apoptosis via caspases. Kapila, Y L, et al. J. Biol. Chem., 274: 30906-13 (1999)
1999
Show Abstract
Intact fibronectin (FN) protects cells from apoptosis. When FN is fragmented, specific domains induce proteinase expression in fibroblasts. However, it is not known whether specific domains of FN can also regulate apoptosis. We exposed fibroblasts to four recombinant FN fragments and then assayed for apoptosis using criteria of cellular shape change, condensed nuclear morphology, and DNA fragmentation. The fragments extended from the RGD-containing repeat III10 to III15; they included (V(+)) or excluded (V(-)) the alternatively spliced V region and contained either a mutated (H(-)) or an unmutated (H(+)) heparin binding domain. Only the V(+)H(-) fragment triggered decreases in pp125(FAK) levels and apoptosis, which was rescued by intact FN and inhibitors of caspase-1 and caspase-3. This apoptotic mechanism was mediated by a chondroitin sulfate proteoglycan, since treating cells with chondroitin sulfate or chondroitinase reversed the apoptotic cell shape changes. The alpha4 integrin receptor may also be involved, since using a blocking antibody to alpha4 alone induced apoptotic cell shape changes, whereas co-treatment with this antibody plus V(+)H(+) reversed these effects. These results demonstrate that the V and heparin binding domains of FN modulate pp125(FAK) levels and regulate apoptosis through a chondroitin sulfate proteoglycan- and possibly alpha4 integrin-mediated pathway, which triggers a caspase cascade. | 10521484
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Role of the integrin alpha v beta 6 in cell attachment to fibronectin. Heterologous expression of intact and secreted forms of the receptor. Weinacker, A, et al. J. Biol. Chem., 269: 6940-8 (1994)
1994
Show Abstract
The integrin alpha v beta 6 has been shown to be a fibronectin-binding protein. To determine whether the cytoplasmic and transmembrane domains of alpha v beta 6 are necessary for binding to fibronectin, a truncated, secreted form of the integrin lacking these domains was engineered and expressed in Chinese hamster ovary cells. Fibronectin affinity chromatography demonstrated that the secreted integrin, like its full-length counterpart, was capable of binding fibronectin. Monoclonal antibodies were made to secreted alpha v beta 6 and to beta 6-transfected NIH 3T3 cells. In experiments designed to determine whether alpha v beta 6 can mediate cell attachment to fibronectin, full-length human beta 6 was expressed in Chinese hamster ovary cells and in the human colon carcinoma cell line SW480. beta 6-expressing cells were identified by alpha v beta 6-specific antibodies, and the beta 6-transfectants were used in cell-adhesion assays. In Chinese hamster ovary cells, human beta 6 associated with hamster alpha v but was incapable of mediating cell attachment to fibronectin. However, expression of beta 6 in these cells had the dominant negative effect of decreasing alpha v beta 5-dependent adhesion to vitronectin. In SW480 cells, beta 6 expression conferred the ability to bind to fibronectin even in the presence of inhibitory antibodies against beta 1 integrins. In such cells, fibronectin binding ability could be blocked by an antibody to alpha v integrins. These results constitute the first direct evidence that alpha v beta 6 mediates cell attachment to fibronectin. | 8120056
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