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PubMedDate not supplied

Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: II. Effects on integrins and focal adhesions.

BACKGROUND: We have shown that inositol hexaphosphate (IP6), a natural compound and a potent anti-cancer agent, inhibited cancer cell adhesion to the extracellular matrix (ECM) proteins, thereby leading to inhibition of cell migration and invasion. Cell adhesion to ECM is mediated by specific cell surface integrins, which transduce intracellular signals through their interaction and activation of other proteins that are recruited to the focal adhesion. We hypothesize that IP6 decreases cell adhesion by suppressing the integrin receptors and their subsequent signaling pathway. MATERIALS AND METHODS: We analyzed integrin expressions of the highly invasive estrogen receptor-negative human breast cancer MDA-MB 231 cells exposed to IP6 by flow cytometry. The expression of focal adhesion proteins was investigated by immunocytochemistry and Western blotting. RESULTS: IP6 treatment caused a significant (P < 0.005) decrease in the expression of integrin heterodimers alpha 2 beta 1 (collagen receptor), alpha 5 beta 1 (fibronectin receptor) and alpha v beta 3 (vitronectin receptor); flow cytometry showed that it was the alpha 5 subunit that was down-regulated ( < 0.001). However, the expression of the alpha 2, alpha v, beta 1 and beta 3 subunits were not affected by IP6 treatment. When the expression of integrins on the cell surface was assessed, there was a dramatic 82% decrease in the expression of alpha 5 beta 1 on IP6-treated cells (P < 0.0001), indicating a decrease in cell surface expression of the heterodimers. No effect was seen when inositol hexasulfate (IS6), an analogue of IP6, was used as a control. Immunocytochemistry showed a lack of clustering of paxillin; tyrosine-phosphorylated proteins in IP6-treated cells were discontinuous and scattered around the cell periphery, whereas the patterns were more dense and localized in control cells. Consistent with these observations, focal adhesion kinase (FAK) autophosphorylation at tyrosine-397 residue was suppressed, albeit modestly, by IP6 treatment, suggesting a down-regulation in the integrin-mediated signaling pathway. CONCLUSION: The results of this study indicate that IP6-induced inhibition of cancer cell adhesion, migration and invasion may be mediated through the modulation of integrin dimerization, cell surface expression and integrin-associated signaling pathway.

Breast Neoplasms↗

PubMed2004

[Cyclooxygenase 2 and breast cancer. From biological concepts to therapeutic trials].

Cyclooxygenases (Cox) are prostaglandin synthetase enzymes which play a key role in mammary carcinogenesis. Several connections were demonstrated between Cox and a few oncogenes (v-src, v-Ha-ras, HER-2/neu, Wnt, p53 mutated), alimentary products (PUFAs), transcription factors (c-jun and c-fos), proapoptotic proteins [Bax et Bcl-x(L)] or antiapoptotic (Bcl-2), CYP19 aromatase gene, NFkappaB receptor (RANKL), angiogenesis (via VEGF, TXA2, oxid nitric synthetase, alphaVbeta3 integrin receptor), peroxisome gamma proliferator receptor (PPARgamma) and its ligand PGJ2 and with antitubuline chemotherapy drugs. No correlation of Cox2 expression with hormonal receptors was shown. In epidemiologic studies there is evidence of breast cancer risk reduction for women who take AINS for a long time. Alimentary factors like resveratrol or insaturated fat acid reduce Cox2 expression in animal and could be investigated in human studies. Clinical trials are planed with the anti Cox2 celecoxib for breast cancer prevention, in adjuvant setting, in metastatic situation combined with exemestane or antitubulin drugs or in neoadjuvant therapy.

Animals↗

PubMed2004

[Cyclooxygenase 2 and breast cancer. From biological concepts to clinical trials].

Cyclooxygenases (Cox) are prostaglandin synthetase enzymes which play a key role in mammary carcinogenesis. Several connections were demonstrated between Cox and a few oncogenes (v-src, v-Ha-ras, HER-2\neu, Wnt, p53 mutated), alimentary products (PUFAs), transcription factors (c-jun and c-fos), proapoptotic proteins [Bax et Bcl-x(L)] or antiapoptotic (Bcl-2), CYP19 aromatase gene, NFkappaB receptor (RANKL), angiogenesis (via VEGF, TXA2, oxid nitric synthetase, alphaVbeta3 integrin receptor), peroxisome gamma proliferator receptor (PPARgamma) and its ligand PGJ2 and with antitubuline chemotherapy drugs. No correlation of Cox2 expression with hormonal receptors was shown. In epidemiologic studies there is evidence of breast cancer risk reduction for women who take AINS for a lon time. Alimentary factors like resveratrol or insaturated fat acid reduce Cox2 expression in animal and could be investigated in human studies. Clinical trials are planed with the anti Cox2 celecoxib for breast cancer prevention, in adjuvant setting, in metastatic situation combined with exemestane or antitubulin drugs or in neoadjuvant therapy.

Animals↗

PubMed2006

Recombinant human tumor necrosis factor: an efficient agent for cancer treatment.

Recombinant human TNF (rhTNF) has a selective effect on endothelial cells in tumour angiogenic vessels. Its clinical use has been limited because of its property to induce vascular collapsus. TNF administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs was shown to be safe and efficient. When combined to the alkylating agent melphalan, a single ILP produces a very high objective response rate. ILP with TNF and melphalan provided the proof of concept that a vasculotoxic strategy combined to chemotherapy may produce a strong anti-tumour effect. The registered indication of TNF-based ILP is a regional therapy for regionally spread tumours. In soft tissue sarcomas, it is a limb sparing neoadjuvant treatment and, in melanoma in-transit metastases, a curative treatment. Despite its demonstrated regional efficiency TNF-based ILP is unlikely to have any impact on survival. High TNF dosages induce endothelial cells apoptosis, leading to vascular destruction. However, lower TNF dosage produces a very strong effect that is to increase the drug penetration into the tumour, presumably by decreasing the intratumoural hypertension resulting in better tumour uptake. TNF-ILP allowed the identification of the role of alphaVbeta3 integrin deactivation as an important mechanism of antiangiogenesis. Several recent studies have shown that TNF targeting is possible, paving the way to a new opportunity to administer TNF systemically for improving cancer drug penetration. TNF was the first agent registered for the treatment of cancer that improves drug penetration in tumours and selectively destroys angiogenic vessels.

Antineoplastic Agents↗

PubMed2007

Gonadotropin-releasing hormone agonists reduce the migratory and the invasive behavior of androgen-independent prostate cancer cells by interfering with the activity of IGF-I.

Androgen-independent prostate carcinoma is characterized by a high proliferation rate and by a strong metastatic behavior. We have previously shown that GnRH agonists exert a direct and specific inhibitory action on the proliferation of androgen-independent prostate cancer cells (DU 145). These compounds mainly act by interfering with the mitogenic activity of growth factors, such as the insulin-like growth factor-I (IGF-I). The present experiments were performed to clarify whether GnRH agonists might also affect the migratory and the invasive behavior of androgen-independent prostate cancer cells and to define their mechanism of action. First we showed that the GnRH agonist Leuprolide reduces the migration of DU 145 cells towards a chemoattractant and their ability to invade a reconstituted basement membrane. Experiments were then performed to clarify whether the GnRH agonist might act by interfering with the pro-metastatic activity of IGF-I. We found that, in androgen-independent prostate cancer cells, Leuprolide: a) interferes with the IGF-I system (receptor protein expression and tyrosine-phosphorylation); b) abrogates the IGF-I-induced phosphorylation of Akt (a kinase previously shown by us to mediate the pro-metastatic activity of IGF-I in prostate cancer cells); c) counteracts the migration and the invasive activity of the cells stimulated by IGF-I; d) abolishes the effects of IGF-I on cell morphology, on actin cytoskeleton organization and on alphavbeta3 integrin expression/cellular localization. These data indicate that GnRH agonists, in addition to their well known antiproliferative effect, can also exert a significant inhibitory activity on the migratory and invasive behavior of androgen-independent prostate cancer cells, expressing the GnRH receptor. GnRH agonists act by interfering with the pro-metastatic activity of the growth factor IGF-I.

Androgens↗

PubMed1999

A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3.

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Amino Acid Substitution↗

PubMed1999

The integrin specificity of human recombinant osteopontin.

The ability of full-length human recombinant osteopontin (OPN) to support the adhesion of various alphav integrin-expressing cell lines was determined in order to characterize its integrin selectivity. The identity of this protein was assessed by cDNA sequence and mass spectroscopic analysis, and confirmed as full-length OPN. Neither the human embryonic kidney 293 cell line, which expresses the alphavbeta1 integrin, nor the human colonic adenocarcinoma HT-29 cell line, which expresses the alphavbeta5 integrin, were able to adhere to OPN; both of these cell lines are deficient in the beta3 subunit. In contrast, an alphavbeta3 integrin-expressing cell line, SK-MEL-24, was able to adhere to OPN in an arginine-glycine-aspartic acid dependent manner. In addition, this OPN-mediated cellular adhesion was completely blocked with an anti-alphavbeta3 integrin antibody (LM609), confirming that only the alphavbeta3 integrin mediated this cellular adhesion. These data demonstrate that, at least among the alphav integrins, only the alphavbeta3 is able to support cellular adhesion to osteopontin. This finding may have implications for the design of therapeutics targeting OPN-integrin interactions.

Cell Adhesion↗

PubMed2000

The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by alphaV integrins.

We have previously shown that oligodendrocyte progenitor cells exhibit developmental switching between alphav-associated beta integrin subunits to sequentially express alphavbeta1, alphavbeta3 and alphavbeta5 integrins during differentiation in vitro. To understand the role that alphavveta3 integrin may play in regulating oligodendrocyte progenitor cell behaviour, cells of the rat cell line, CG-4, were genetically engineered to constitutively express alphavbeta3 integrin by transfection with full-length human beta3 integrin subunit cDNA. Time-lapse videomicroscopy showed no effect of beta3 expression on cell migration but revealed enhanced proliferation on vitronectin substrata. Comparison of mitotic indices, as measured by 5-bromo-2'-deoxyuridine incorporation, confirmed that human beta3 integrin-expressing cells exhibited enhanced proliferation, as compared to both vector-only transfected, and wild-type CG-4 cells when switched to differentiation medium from growth medium, but only in cultures grown on vitronectin and not on poly-D-lysine. The effects on proliferation were inhibited by a function-blocking antibody specifically directed against the human beta3 integrin subunit. Human beta3 integrin-expressing cells also exhibited reduced differentiation. This differentiation could be reduced still further by a function-blocking monoclonal antibody against alphavbeta5 integrin, as could differentiation in the wild-type CG-4 cells. Taken together, these results suggest that alphavbeta3 integrin may regulate oligodendroglial cell proliferation and that both downregulation of alphavbeta3 integrin expression and signalling through alphavbeta5 integrin may be critical to continued differentiation in vitro.

Animals↗

PubMed2002

Relevance of extracellular matrix and its receptors in mammalian nephrogenesis revealed by metanephric organ culture system.

Mammalian nephrogenesis is modulated by a number of extracellular matrix (ECM) glycoproteins, integrins and cell adhesion molecules. We demonstrated the existence of integrins alphavbeta1, alphavbeta3, alphavbeta5 and alphavbeta6 in epithelial elements of developing nephrons. Fibrillin-1 is a putative ligand for integrin alphavbeta3, and tubulointerstitial nephritis antigen (TIN-ag) is a ligand for integrins alphavbeta3 and alpha3beta1. Fibrillin-1 and TIN-ag are also differentially expressed in the developing kidney. The inclusion of antisense oligonucleotide in a mouse kidney organ culture system indicated that the alphav-related integrins and their ligands play an important role in mammalian nephrogenesis. Recently identified modulators of cell-matrix interactions, i.e. beta-galactoside-binding mammalian lectins (galectins), are involved in cell-cell and cell-matrix interactions by cross-linking glycoconjugates located on the ECM and membrane-bound glycoproteins. We identified and cloned a new member of the galectins from embryonic kidneys, and designated it galectin-9. Since high glucose alters the expression of ECM proteins and integrins, we also investigated the influence of glucose on metanephric development. The presence of 30 mM D-glucose in metanephric organ culture induced dysmorphogenesis of the kidney accompanied by decreased expression of perlecan. Furthermore, we screened the genes differentially expressed under high glucose conditions in streptozotocin-induced newborn mouse kidneys by representational difference analysis of cDNA. We identified translocase of inner mitochondrial membrane (Tim44) and renal-specific oxido-reductase (RSOR). The roles of these molecules in glucose-induced dysmorphogenesis and the relationship with ECM-related molecules need to be addressed.

Animals↗

PubMed2001

Tamoxifen and estrogen effects on TGF-beta formation: role of thrombospondin-1, alphavbeta3, and integrin-associated protein.

We have found that the enhanced activation of latent TGF-beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavbeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-beta activation.

Adenocarcinoma↗

PubMed2003

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.

Animals↗

PubMed2005

The integrin alpha5beta1 regulates alphavbeta3-mediated extracellular signal-regulated kinase activation.

BACKGROUND: Integrin-mediated cell migration is essential for wound repair. Previous studies have shown that the interaction between integrins and the extracellular matrix (ECM) can initiate intracellular signaling pathways to regulate cell movement. Both the focal adhesion kinase (FAK) and the extracellular signal-regulated kinase/activated mitogen-activated protein kinase (ERK/MAPK) signaling pathways are required for efficient cell migration. Our previous work has shown that co-expression of the integrin alpha5beta1 inhibits alphavbeta3-mediated cell migration. We hypothesized that alpha5beta1 may regulate cell migration by modulating these alphavbeta3-mediated intracellular signaling events. METHODS: CHO B3 (alphavbeta3+) and B3C5 (alphavbeta3+/alpha5beta1+) cells were monitored by flow cytometry to determine integrin expression. Cells were allowed to migrate on fibrinogen (FBG)-coated transwells, with or without PD98059, an inhibitor of the ERK activator, mitogen-activated protein kinase kinase (MEK). Fixation, staining, and cell counting were used to quantify cell migration. Cells adherent to FBG were lysed and analyzed for FAK and ERK/MAPK activation by immunoblotting followed by image analysis densitometry. All experiments were repeated in triplicate. RESULTS: Treatment with PD98059 significantly decreased alphavbeta3-mediated cell migration on FBG (P = 0.0001) to a level comparable to untreated B3C5 cells. Following adhesion to FBG, B3 cells demonstrated a marked increase in ERK/MAPK activation compared to B3C5 cells. However, no significant difference was detected in FAK activation. CONCLUSION: Signaling through the ERK/MAPK pathway is required for efficient alphavbeta3-mediated migration on FBG. Inhibition of alphavbeta3-mediated migration by the integrin alpha5beta1 correlates with altered intensity and duration of ERK/MAPK activation, but not FAK activation, in response to adhesion. This suggests a mechanism for the regulatory effect of alpha5beta1 on alphavbeta3-mediated cell migration.

Animals↗

PubMed2002

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.

Animals↗

PubMed2004

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells.

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.

Animals↗

PubMed2006

Expression of integrins in cerebral arteriovenous and cavernous malformations.

OBJECTIVE: To assess and compare levels and patterns of expression for integrins alphavbeta1, alphavbeta3, and alphavbeta5 in arteriovenous malformations (AVMs) and cavernous malformations (CCMs) of the brain. MATERIALS AND METHODS: Specimens from 10 AVM and 10 CCM lesions were selected from 112 patients with AVMs and 97 patients with CCMs who were treated microsurgically in the Department of Neurosurgery, Marmara University, Istanbul, Turkey. Sections were immunohistochemically stained with antibodies for integrins alphavbeta1, alphavbeta3, and alphavbeta5. Separate histological layers of the vascular wall were evaluated, and levels of expression were graded using a four-tier system. RESULTS: Integrin alphavbeta1 was more strongly expressed in AVMs than in CCMs. This difference was most pronounced in the endothelium and subendothelium/media. Integrin alphavbeta3 was more strongly expressed in CCM endothelium than in AVM endothelium (average grades, 0.9 and 0.4, respectively). All 10 of the CCM lesions expressed integrin alphavbeta5 in the endothelium, whereas only five of the AVMs showed minimal expression of this molecule in the endothelium. CONCLUSION: Current scientific understanding of the roles integrins play in angiogenesis is far from complete. The levels and patterns of expression for these molecules in the histological layers of the vascular walls of AVMs and CCMs provide some clues about the complex biological activities of integrins in these lesions. If one accepts the premise that immunohistochemistry has its inherent methodological problems, integrins alphavbeta1, alphavbeta3, and alphavbeta5 are expressed in AVMs and CCMs in different ways that may be linked to stages of angiogenic maturation. Integrin alphavbeta1 is expressed more strongly in endothelium and subendothelium/media of AVMs than in the corresponding layers of CCMs. Integrins alphavbeta3 and alphavbeta5 are expressed more strongly in CCM endothelium than in AVM endothelium. In addition, integrin alphavbeta5 staining was stronger in CCM subendothelium than AVM subendothelium/media.

Adult↗

PubMedDate not supplied

MicroPET imaging of breast cancer alphav-integrin expression with 64Cu-labeled dimeric RGD peptides.

PURPOSE: Alphavbeta3 and alphavbeta5 integrins are cell adhesion molecules that play a vital role in tumor angiogenesis and metastasis. The ability to visualize and quantify integrin expression in vivo will foster our understanding of the role of integrins alphavbeta3 and alphavbeta5 in tumor angiogenesis and allow for direct assessment of anti-angiogenic treatment efficacy based on integrin antagonists. This study compared the tumor targeting characteristics of two dimeric 64Cu-labeled RGD peptide agonists of alphavbeta3 integrin. PROCEDURES: Dimeric RGD peptides E[c(RGDyK)]2 and E[c(RGDfK)]2 were conjugated with 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with positron emitter 64Cu(t(1/2)=12.8 h, beta+=19%). Both 64Cu-DOTA-E[c(RGDyK)]2 and 64Cu-DOTA-E[c(RGDfK)]2 were used in biodistribution, microPET imaging and whole-body autoradiography studies in athymic female nude mice with orthotopically growing MDA-MB-435 breast carcinoma xenografts. RESULTS: At all time points, activity accumulation of 64)Cu-DOTA-E[c(RGDyK)]2 in tumors was significantly higher compared to the D-Phe analog. Liver uptake of the D-Tyr derivative was lower than the D-Phe derivative at early time points but the difference became marginal with time. Overall, 64Cu-DOTA-E[c(RGDyK)]2 yielded better position emission tomography (PET) images in orthotopic MDA-MB-435 bearing mice than did 64Cu-DOTA-E[c(RGDfK)]2. Both radiotracers had alphav-integrin specific tumor activity accumulation, as demonstrated by significant reduction of uptake with a coinjected blocking dose of c(RGDyK). CONCLUSIONS: The radiolabeled dimeric RGD peptides 64Cu-DOTA-E[c(RGDyK)]2 and 64Cu-DOTA-E[c(RGDfK)]2 have high and specific tumor uptake in a human breast cancer tumor xenograft, with the D-Tyr derivative showing better in vivo kinetics than the D-Phe derivative, most likely due to the increased hydrophilicity of the D-Tyr. Both dimeric peptides showed better tumor retention than the previously tested monomeric RGD counterparts, presumably because of bivalency and increase in apparent molecular size.

Animals↗

PubMed2004

Chemical adaptor immunotherapy: design, synthesis, and evaluation of novel integrin-targeting devices.

A series of beta-diketone derivatives of RGD peptidomimetics that selectively bind to alphavbeta3 and alphavbeta5 integrins were synthesized and covalently docked to the reactive lysine residues of monoclonal aldolase antibody 38C2. The resulting targeting devices strongly and selectively bound to human cancer cells expressing integrins alphavbeta3 and alphavbeta5 as analyzed by flow cytometry. In vitro and in vivo studies revealed that these novel integrin-targeting devices efficiently inhibit tumor growth. Thus, the combination of beta-diketone derivatives of RGD peptidomimetics that target cell surface integrins alphavbeta3 and alphavbeta5 with monoclonal aldolase antibodies through formation of a covalent bond of defined stoichiometry holds promise as a new approach to cancer therapy.

Antibodies, Monoclonal↗

PubMed2001

Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

1-Phosphatidylinositol 4-Kinase↗