RESUMO
Aminopeptidase N/CD13, a membrane-bound enzyme upregulated in tumor vasculature and involved in angiogenesis, can be used as a receptor for the targeted delivery of drugs to tumors through ligand-directed targeting approaches. We describe a novel peptide ligand (VGCARRYCS, called "G4") that recognizes CD13 with high affinity and selectivity. Enzymological and computational studies showed that G4 is a competitive inhibitor that binds to the catalytic pocket of CD13 through its N-terminal region. Fusing the peptide C-terminus to tumor necrosis factor-alpha (TNF) or coupling it to a biotin/avidin complex causes loss of binding and inhibitory activity against different forms of CD13, including natural or recombinant ectoenzyme and a membrane form expressed by HL60 promyelocytic leukemia cells (likely due to steric hindrance), but not binding to a membrane form of CD13 expressed by endothelial cells (ECs). Furthermore, G4-TNF systemically administered to tumor-bearing mice exerted anticancer effects through a CD13-targeting mechanism, indicating the presence of a CD13 form in tumor vessels with an accessible binding site. Biochemical studies showed that most CD13 molecules expressed on the surface of ECs are catalytically inactive. Other functional assays showed that these molecules can promote endothelial cell adhesion to plates coated with G4-avidin complexes, suggesting that the endothelial form of CD13 can exert catalytically independent biological functions. In conclusion, ECs express a catalytically inactive form of CD13 characterized by an accessible conformation that can be selectively targeted by G4-protein conjugates. This form of CD13 may represent a specific target receptor for ligand-directed targeted delivery of therapeutics to tumors.
Assuntos
Antígenos CD13 , Células Endoteliais , Leucemia Promielocítica Aguda , Animais , Camundongos , Antígenos CD13/antagonistas & inibidores , LigantesRESUMO
BACKGROUND: Early detection and removal of bladder cancer in patients is crucial to prevent tumor recurrence and progression. Because current imaging techniques may fail to detect small lesions of in situ carcinomas, patients with bladder cancer often relapse after initial diagnosis, thereby requiring frequent follow-up and treatments. RESULTS: In an attempt to obtain a sensitive and high-resolution imaging modality for bladder cancer, we have developed a photoacoustic imaging approach based on the use of PEGylated gold nanorods (GNRs) as a contrast agent, functionalized with the peptide cyclic [CphgisoDGRG] (Iso4), a selective ligand of α5ß1 integrin expressed by bladder cancer cells. This product (called GNRs@PEG-Iso4) was produced by a simple two-step procedure based on GNRs activation with lipoic acid-polyethyleneglycol(PEG-5KDa)-maleimide and functionalization with peptide Iso4. Biochemical and biological studies showed that GNRs@PEG-Iso4 can efficiently recognize purified integrin α5ß1 and α5ß1-positive bladder cancer cells. GNRs@PEG-Iso4 was stable and did not aggregate in urine or in 5% sodium chloride, or after freeze/thaw cycles or prolonged exposure to 55 °C, and, even more importantly, do not settle after instillation into the bladder. Intravesical instillation of GNRs@PEG-Iso4 into mice bearing orthotopic MB49-Luc bladder tumors, followed by photoacoustic imaging, efficiently detected small cancer lesions. The binding to tumor lesions was competed by a neutralizing anti-α5ß1 integrin antibody; furthermore, no binding was observed to healthy bladders (α5ß1-negative), pointing to a specific targeting mechanism. CONCLUSION: GNRs@PEG-Iso4 represents a simple and robust contrast agent for photoacoustic imaging and diagnosis of small bladder cancer lesions.
Assuntos
Nanotubos , Técnicas Fotoacústicas , Neoplasias da Bexiga Urinária , Animais , Camundongos , Meios de Contraste , Integrina alfa5beta1 , Neoplasias da Bexiga Urinária/diagnóstico por imagem , OuroRESUMO
BACKGROUND: Gold nanospheres tagged with peptides containing isoDGR (isoAsp-Gly-Arg), an αvß3 integrin binding motif, represent efficient carriers for delivering pro-inflammatory cytokines to the tumor vasculature. We prepared bi- or trifunctional nanoparticles bearing tumor necrosis factor-α (TNF) and/or interleukin-12 (IL12) plus a peptide containing isoDGR, and we tested their anti-cancer effects, alone or in combination with doxorubicin, in tumor-bearing mice. RESULTS: In vitro biochemical studies showed that both nanodrugs were monodispersed and functional in terms of binding to TNF and IL12 receptors and to αvß3. In vivo studies performed in a murine model of fibrosarcoma showed that low doses of bifunctional nanoparticles bearing isoDGR and TNF (corresponding to few nanoparticles per cell) delayed tumor growth and increased the efficacy of doxorubicin without worsening its toxicity. Similar effects were obtained using trifunctional nanoparticles loaded with isoDGR, TNF and IL12. Mechanistic studies showed that nanoparticles bearing isoDGR and TNF could increase doxorubicin penetration in tumors a few hours after injection and caused vascular damage at later time points. CONCLUSION: IsoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multi-cytokine delivery to cells of the tumor vasculature. Extremely low doses of isoDGR-coated nanodrugs functionalized with TNF or TNF plus IL12 can enhance doxorubicin anti-tumor activity.
Assuntos
Antineoplásicos/farmacologia , Citocinas , Doxorrubicina/farmacologia , Nanoestruturas/química , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Integrina alfaVbeta3 , Interleucina-12 , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/uso terapêutico , Fator de Necrose Tumoral alfaRESUMO
The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to regulation of the cardiotoxic and antitumor activities of Doxo. The effects of a physiologic dose of full-length recombinant CgA on Doxo-induced cardiotoxicity and antitumor activity were investigated in rats using in vivo and ex vivo models and in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. In vivo and ex vivo mechanistic studies showed that CgA can prevent Doxo-induced heart inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, CgA did not impair the anticancer activity of Doxo in all the murine models investigated. Furthermore, we observed that Doxo can reduce the intracardiac expression and release of CgA in the blood (i.e., an important cardioprotective agent). These findings suggest that administration of low-dose CgA to patients with low levels of endogenous CgA might represent a novel approach to prevent Doxo-induced adverse events without impairing antitumor effects.-Rocca, C., Scavello, F., Colombo, B., Gasparri, A. M., Dallatomasina, A., Granieri, M. C., Amelio, D., Pasqua, T., Cerra, M. C., Tota, B., Corti, A., Angelone, T. Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Cromogranina A/metabolismo , Doxorrubicina/efeitos adversos , Coração/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Motivos de Aminoácidos/genética , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antígenos CD13/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Estabilidade de Medicamentos , Humanos , Camundongos , Neoplasias/patologia , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Processamento de Proteína Pós-Traducional/genética , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Serina/genética , Serina/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvß3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvß3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.
Assuntos
Ouro/química , Imunoterapia/métodos , Interleucina-12/metabolismo , Nanopartículas Metálicas/química , Adenocarcinoma/terapia , Animais , Células Cultivadas , Feminino , Fibrossarcoma/terapia , Masculino , Neoplasias Mamárias Animais/terapia , CamundongosRESUMO
NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.
RESUMO
Angiogenesis, the formation of blood vessels from pre-existing vasculature, is regulated by a complex interplay of anti and proangiogenic factors. We found that physiologic levels of circulating chromogranin A (CgA), a protein secreted by the neuroendocrine system, can inhibit angiogenesis in various in vitro and in vivo experimental models. Structure-activity studies showed that a functional anti-angiogenic site is located in the C-terminal region, whereas a latent anti-angiogenic site, activated by cleavage of Q76-K77 bond, is present in the N-terminal domain. Cleavage of CgA by thrombin abrogated its anti-angiogenic activity and generated fragments (lacking the C-terminal region) endowed of potent proangiogenic activity. Hematologic studies showed that biologically relevant levels of forms of full-length CgA and CgA1-76 (anti-angiogenic) and lower levels of fragments lacking the C-terminal region (proangiogenic) are present in circulation in healthy subjects. Blood coagulation caused, in a thrombin-dependent manner, almost complete conversion of CgA into fragments lacking the C-terminal region. These results suggest that the CgA-related circulating polypeptides form a balance of anti and proangiogenic factors tightly regulated by proteolysis. Thrombin-induced alteration of this balance could provide a novel mechanism for triggering angiogenesis in pathophysiologic conditions characterized by prothrombin activation.
Assuntos
Cromogranina A/química , Cromogranina A/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Embrião de Galinha , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
A new cyclic peptide containing the isoDGR motif that, after coupling to albumin, selectively binds αvß3, an integrin overexpressed in the tumor vasculature. IsoDGR-tagged albumin binds tumor vessels and can be exploited as a carrier for the preparation of tumor vasculature-selective nanomedicines, such as gold nanoparticles (Au) carrying tumor necrosis factor α (TNF), a potent vascular damaging agent.
Assuntos
Integrina alfaVbeta3/química , Neoplasias/metabolismo , Ouro/química , Humanos , Integrina alfaVbeta3/administração & dosagem , Nanopartículas Metálicas/química , Fator de Necrose Tumoral alfa/químicaRESUMO
Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, neurons, cardiomyocytes, and keratinocytes, is the precursor of various peptides that regulate the carbohydrate/lipid metabolism and the cardiovascular system. We have found that CgA, locally administered to injured mice, can accelerate keratinocyte proliferation and wound healing. This biological activity was abolished by the Asp(45)Glu mutation. CgA and its N-terminal fragments, but not the corresponding Asp(45)Glu mutants, could selectively recognize the αvß6-integrin on keratinocytes (a cell-adhesion receptor that is up-regulated during wound healing) and regulate keratinocyte adhesion, proliferation, and migration. No binding was observed to other integrins such as αvß3, αvß5, αvß8, α5ß1, α1ß1, α3ß1, α6ß4, α6ß7 and α9ß1. Structure-activity studies showed that the entire CgA(39-63) region is crucial for αvß6 recognition (K(i) = 7 nM). This region contains an RGD site (residues CgA(43-45)) followed by an amphipathic α-helix (residues CgA(47-63)), both crucial for binding affinity and selectivity. These results suggest that the interaction of the RGD/α-helix motif of CgA with αvß6 regulates keratinocyte physiology in wound healing.
Assuntos
Antígenos de Neoplasias/metabolismo , Cromogranina A/metabolismo , Fibroblastos/metabolismo , Integrinas/metabolismo , Queratinócitos/metabolismo , Oligopeptídeos/metabolismo , Pele/metabolismo , Cicatrização/fisiologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Fibroblastos/citologia , Humanos , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Secretina/análogos & derivados , Secretina/metabolismo , Homologia de Sequência de Aminoácidos , Pele/citologiaRESUMO
Rationale: The αvß6- and αvß8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFß complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called "5a"), which selectively recognizes the LAP/TGFß complex-binding site of αvß6 and αvß8. Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvß6/αvß8 integrins and various αvß6/αvß8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvß8-positive prostate tumors. Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFß activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvß6/αvß8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvß6/αvß8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvß8-positive prostate tumors. Conclusions: The results indicate that 5a can home to αvß6- and/or αvß8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvß6/αvß8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFß activators.
Assuntos
Carcinoma , Neoplasias Pancreáticas , Neoplasias da Próstata , Masculino , Animais , Camundongos , Humanos , Cromogranina A/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Distribuição Tecidual , Peptídeos/química , Integrinas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Vasostatin-1 (VS-1), the N-terminal fragment of chromogranin A (CgA), decreases the permeability of endothelial cells in vitro and in vivo. AIMS: Here, we investigated whether a similar effect could be observed also on intestinal epithelial cells (IECs) in vitro and whether VS-1 could have favorable effects on animal models of acute or chronic colitis, which are characterized by increased permeability of the intestinal epithelium. METHODS: In vitro, VS-1 was tested on IEC monolayers showing increased permeability, on mechanically injured IEC monolayers, and on the production of the chemokine IL-8/KC by lipopolysaccharide (LPS)-stimulated IECs. In vivo, VS-1 was tested in animal models of dextran sodium salt (DSS)-induced acute or chronic colitis. RESULTS: In vitro, VS-1 inhibited increased permeability of IECs induced by interferon-γ and tumor necrosis factor-α. Moreover, VS-1 promoted healing of mechanically injured IEC monolayers, most likely through stimulation of cell migration, rather than cell proliferation. Eventually, VS-1 inhibited LPS-induced production of IL-8. In vivo, VS-1 exerted protective effects in animal models of acute or chronic colitis upon oral, but not systemic administration. CONCLUSIONS: VS-1 is therapeutically active in animal models of acute or chronic, DSS-induced colitis. The mechanisms underlying this effect are likely to be multiple, and may include inhibition of enhanced intestinal permeability, repair of injured intestinal mucosae, and inhibition of the production of IL-8/KC and possibly other inflammatory cytokines.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Cromogranina A , Colite , Colo/metabolismo , Células Epiteliais/metabolismo , Fragmentos de Peptídeos , Administração Oral , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cromogranina A/administração & dosagem , Cromogranina A/farmacocinética , Doença Crônica , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-8/antagonistas & inibidores , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacocinética , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacocinética , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of alphavbeta3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alphavbeta3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alphavbeta3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (alphavbeta5, alphavbeta6, alphavbeta8, and alpha5beta1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.
Assuntos
Antígenos CD13/metabolismo , Integrinas/metabolismo , Oligopeptídeos/química , Animais , Antineoplásicos/farmacologia , Adesão Celular , Dissulfetos/química , Células Endoteliais/citologia , Humanos , Ácido Isoaspártico/química , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
Fibroblast adhesion can be modulated by proteins released by neuroendocrine cells and neurons, such as chromogranin A (CgA) and its N-terminal fragment vasostatin-1 (VS-1, CgA(1-78)). We have investigated the mechanisms of the interaction of VS-1 with fibroblasts and of its pro-adhesive activity and have found that the proadhesive activity of VS-1 relies on its interaction with the fibroblast membrane via a phospholipid-binding amphipathic alpha-helix located within residues 47-66, as well as on the interaction of the adjacent C-terminal region 67-78, which is structurally similar to ezrin-radixin-moesin-binding phosphoprotein 50 (a membrane-cytoskeleton adapter protein), with other cellular components critical for the regulation of cell cytoskeleton.
Assuntos
Adesão Celular , Cromogranina A/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/metabolismoRESUMO
Gold nanoparticles functionalized with isoDGR, a tripeptide motif that recognizes αvß3 integrin overexpressed in tumor vessels, have been used as nano-vectors for the delivery of cytokines to tumors. Functionalization of nanogold with this peptide has been achieved by coating nanoparticles with a peptide-albumin conjugate consisting of heterogeneous molecules with a variable number of linkers and peptides. To reduce nanodrug heterogeneity we have designed, produced and preclinically evaluated a homogeneous and well-defined reagent for nanogold functionalization, consisting of a head-to-tail cyclized CGisoDGRG peptide (iso1) coupled via its thiol group to maleimide-PEG11-lipoamide (LPA). The resulting iso1-PEG11-LPA compound can react with nanogold via lipoamide to form a stable bond. In vitro studies have shown that iso1, after coupling to nanogold, maintains its capability to bind purified αvß3 and αvß3-expressing cells. Nanogold functionalized with this peptide can also be loaded with bioactive tumor necrosis factor-α (TNF) to form a bi-functional nanodrug that can be stored for three days at 37°C or >1 year at low temperatures with no loss αvß3-binding properties and TNF-cytolytic activity. Nanoparticles functionalized with both iso1 and TNF induced tumor eradication in WEHI-164 fibrosarcoma-bearing mice more efficiently than nanoparticles lacking the iso1 targeting moiety. These results suggest that iso1-PEG11-LPA is an efficient and well-defined reagent that can be used to produce robust and more homogeneous nano-vectors for the delivery of TNF and other cytokines to αvß3 positive cells.
RESUMO
Chromogranin A (CgA), a secretory protein released in the blood by the neuroendocrine system, consists of a mixture of full-length molecules and fragments endowed of vasoregulatory activity. The extent and the role of CgA fragmentation were investigated in patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC, n=172). Multivariate analysis showed that full-length CgA was associated with better progression free and overall survival, whereas CgA C-terminal fragmentation was associated with worse prognosis. In vitro studies showed that PDAC cells can promote the cleavage of CgA C-terminal region by activating plasminogen to plasmin. Limited digestion of full-length CgA with plasmin abolished its anti-angiogenic activity and generated pro-angiogenic molecules. The fragmentation of CgA C-terminal region was increased also in murine models of PDAC. In these models, the inhibition of CgA fragmentation with aprotinin, an inhibitor of plasmin and other serine proteases, or the blockade of pro-angiogenic fragments with specific antibodies inhibited the growth of PDAC implanted subcutaneously in mice. Finally, administration of full-length CgA to mice bearing orthotopic PDAC reduced tumor perfusion, as measured by contrast-enhanced ultrasound. These findings suggest that PDAC can promote the cleavage of circulating CgA C-terminal region to generate fragments that regulate the tumor vascular biology and that may represent new potential therapeutic targets.
RESUMO
Targeted delivery of IFNgamma to tumors has been achieved by fusing this cytokine with GCNGRC, a tumor neovasculature homing peptide. Although the therapeutic efficacy of this protein (called IFNgamma-NGR) in animal models is greater than that of IFNgamma, frequent administrations of IFNgamma-NGR may result in lower efficacy and tumor resistance. We investigated the role of indoleamine 2,3-dioxygenase (IDO), an IFNgamma-inducible enzyme that may down-regulate T cells by affecting local tryptophan catabolism in tumor resistance to repeated treatments with IFNgamma-NGR. The study was carried out in immunocompetent mice and in nu/nu mice bearing RMA lymphoma, B16F melanoma, or WEHI-164 fibrosarcoma and in vitro using cultured tumor cells. IDO activity was increased in lymphoma homogenates after multiple treatments with IFNgamma-NGR but not after a single treatment. Coadministration of 1-methyl-tryptophan, an inhibitor of IDO, increased tumor responses to multiple treatments in the lymphoma, melanoma, and fibrosarcoma models. No synergism between IFNgamma-NGR and 1-methyl-tryptophan was observed in vitro in tumor cell proliferation assays or in nu/nu tumor-bearing mice, suggesting that the antitumor effect was host mediated. We conclude that IDO is critically involved in tumor resistance to repeated treatments with IFNgamma-NGR, likely causing excessive stimulation of tryptophan catabolism and inhibiting antitumor immune mechanisms. Coadministration of IFNgamma-NGR with IDO inhibitors could represent a new strategy for increasing its antitumor activity.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Interferon gama/fisiologia , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Interferon gama/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/patologia , Proteínas Recombinantes de Fusão/química , Triptofano/análogos & derivados , Triptofano/farmacologiaRESUMO
The unbalanced production of pro- and antiangiogenic factors in tumors can lead to aberrant vasculature morphology, angiogenesis, and disease progression. In this study, we report that disease progression in various murine models of solid tumors is associated with increased cleavage of full-length chromogranin A (CgA), a circulating vasoregulatory neurosecretory protein. Cleavage of CgA led to the exposure of the highly conserved PGPQLR site, which corresponds to residues 368-373 of human CgA1-373, a fragment that has proangiogenic activity. Antibodies against this site, unable to bind full-length CgA, inhibited angiogenesis and reduced tumor perfusion and growth. The PGPQLR sequence of the fragment, but not of the precursor, bound the VEGF-binding site of neuropilin-1; the C-terminal arginine (R373) of the sequence was crucial for binding. The proangiogenic activity of the CgA1-373 was blocked by anti-neuropilin-1 antibodies as well as by nicotinic acetylcholine receptor antagonists, suggesting that these receptors, in addition to neuropilin-1, play a role in the proangiogenic activity of CgA1-373. The R373 residue was enzymatically removed in plasma, causing loss of neuropilin-1 binding and gain of antiangiogenic activity. These results suggest that cleavage of the R373R374 site of circulating human CgA in tumors and the subsequent removal of R373 in the blood represent an important "on/off" switch for the spatiotemporal regulation of tumor angiogenesis and may serve as a novel therapeutic target. SIGNIFICANCE: This work reveals that the interaction between fragmented chromogranin A and neuropilin-1 is required for tumor growth and represents a novel potential therapeutic target.
Assuntos
Neoplasias da Mama/prevenção & controle , Carcinoma Pulmonar de Lewis/prevenção & controle , Cromogranina A/metabolismo , Melanoma/prevenção & controle , Neovascularização Patológica/prevenção & controle , Neuropilina-1/metabolismo , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Proliferação de Células , Feminino , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Análise Espaço-Temporal , Células Tumorais CultivadasRESUMO
Endothelial cell damage and platelet activation contribute to sustained vasculopathy, which is a key clinical characteristic of systemic sclerosis (SSc), also known as scleroderma. Microparticles released from activated platelets in the blood of SSc patients (SSc-microparticles) are abundant and express the damage-associated molecular pattern (DAMP) HMGB1. SSc-microparticles interacted with neutrophils in vitro and in immunocompromised mice and promoted neutrophil autophagy, which was characterized by mobilization of their granule content, enhanced proteolytic activity, prolonged survival, and generation of neutrophil extracellular traps (NETs). Neutrophils migrated within the mouse lung, with collagen accumulation in the interstitial space and the release of soluble E-selectin by the vascular endothelium. Microparticle-neutrophil interaction, neutrophil autophagy and survival, and generation of NETs abated in the presence of BoxA, a competitive inhibitor of HMGB1. Consistent with these results, neutrophils in the blood of SSc patients were autophagic and NET by-products were abundant. Our findings implicate neutrophils in SSc vasculopathy and suggest that platelet-derived, microparticle-associated HMGB1 may be a potential indicator of disease and target for novel therapeutics.
Assuntos
Neutrófilos/metabolismo , Escleroderma Sistêmico/metabolismo , Adulto , Idoso , Animais , Autofagia/fisiologia , Micropartículas Derivadas de Células/metabolismo , Feminino , Proteína HMGB1/antagonistas & inibidores , Proteína HMGB1/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Ativação Plaquetária/fisiologia , Escleroderma Sistêmico/imunologiaRESUMO
PURPOSE: Subnanogram doses of NGR-tumor necrosis factor (TNF), a TNF-alpha derivative able to target tumor neovessels, can enhance the antitumor activity of doxorubicin and melphalan in murine models. We have examined the antitumor activity of NGR-TNF in combination with various chemotherapeutic drugs acting via different mechanisms, including, besides doxorubicin and melphalan, cisplatin, paclitaxel, and gemcitabine. EXPERIMENTAL DESIGN: Chemotherapeutic drugs were tested alone and in combination with NGR-TNF (0.1 ng) in murine lymphoma, fibrosarcoma, and mammary adenocarcinoma models. Different administration schedules have been tested and the effects on tumor growth, animal weight, tumor perfusion, and cell cytotoxicity have been investigated. RESULTS: Pretreatment with NGR-TNF enhanced the response to all these drugs although to a different extent. The increased efficacy was not accompanied by increased toxicity at least as judged from the loss of animal weight. The synergistic effect was transient, maximal synergism being observed with a 2-hour delay between NGR-TNF and drug administrations in all models and with all drugs tested. NGR-TNF did not increase the in vitro cytotoxicity of chemotherapeutic drugs against tumor cells, suggesting that the in vivo synergism depends on NGR-TNF effects on host cells rather than on tumor cells. CONCLUSIONS: Targeted delivery of low doses of NGR-TNF to the tumor vasculature can increase the efficacy of various drugs acting via different mechanisms. Optimal administration schedule requires 2 hours of pretreatment with NGR-TNF independently from the mechanism of drug cytotoxicity. This work could provide important information for designing clinical studies with NGR-TNF in combination with chemotherapeutic drugs.