RESUMO
MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Imunoconjugados/química , Lipossomos/química , Anticorpos de Cadeia Única/química , Animais , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Camundongos , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/uso terapêutico , Receptor ErbB-2/metabolismo , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: This phase 1 dose-escalation trial studied MM-302, a novel HER2-targeted PEGylated antibody-liposomal doxorubicin conjugate, in HER2-positive locally advanced/metastatic breast cancer. METHODS: Patients were enrolled in four cohorts: MM-302 monotherapy (8, 16, 30, 40, and 50 mg/m2 every 4 weeks [q4w]); MM-302 (30 or 40 mg/m2 q4w) plus trastuzumab (4 mg/kg q2w); MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) q3w; MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) and cyclophosphamide (450 mg/m2) q3w. RESULTS: Sixty-nine patients were treated. The most common adverse events (AEs) were fatigue and nausea. Grade 3/4 AEs of special interest included neutropenia, fatigue, mucosal inflammation, anemia, thrombocytopenia, febrile neutropenia, and palmar-plantar erythrodysesthesia. The MTD was not reached. With MM-302 ≥ 30 mg/m2, overall response rate (ORR) was 13% and median progression-free survival (mPFS) 7.4 months (95% CI: 3·5-10·9) in all arms. In 25 anthracycline-naïve patients, ORR was 28·0% and mPFS 10·9 months (95% CI: 1·8-15·3). Imaging with 64Cu-labeled MM-302 visualized tumor-drug penetrance in tumors throughout the body, including the brain. CONCLUSION: MM-302 monotherapy, in combination with trastuzumab, or trastuzumab plus cyclophosphamide, was well tolerated and showed promising efficacy. The selected phase 2 MM-302 dose was 30 mg/m2 plus 6 mg/kg trastuzumab q3w.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/administração & dosagem , Doxorrubicina/análogos & derivados , Imunoconjugados/administração & dosagem , Receptor ErbB-2/genética , Anticorpos de Cadeia Única/administração & dosagem , Trastuzumab/administração & dosagem , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Encéfalo/diagnóstico por imagem , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Ciclofosfamida/efeitos adversos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Esquema de Medicação , Feminino , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Anticorpos de Cadeia Única/efeitos adversos , Anticorpos de Cadeia Única/farmacocinética , Análise de Sobrevida , Trastuzumab/efeitos adversos , Resultado do TratamentoRESUMO
The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in Nrp2-/- mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in Nrp2-deficient mice compared to those in Nrp2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the Nrp2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, Nrp2-mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, Nrp2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema.
Assuntos
Linfedema/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-2/deficiência , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Vasos Sanguíneos/fisiopatologia , Permeabilidade Capilar , Feminino , Humanos , Inflamação/genética , Inflamação/fisiopatologia , Vasos Linfáticos/fisiopatologia , Linfedema/fisiopatologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Neuropilina-2/genética , Neuropilina-2/metabolismo , Organismos Livres de Patógenos Específicos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Effective drug delivery to tumors is a barrier to treatment with nanomedicines. Non-invasively tracking liposome biodistribution and tumor deposition in patients may provide insight into identifying patients that are well-suited for liposomal therapies. We describe a novel gradient-loadable chelator, 4-DEAP-ATSC, for incorporating (64)Cu into liposomal therapeutics for positron emission tomographic (PET). (64)Cu chelated to 4-DEAP-ATSC (>94%) was loaded into PEGylated liposomal doxorubicin (PLD) and HER2-targeted PLD (MM-302) with efficiencies >90%. (64)Cu-MM-302 was stable in human plasma for at least 48h. PET/CT imaging of xenografts injected with (64)Cu-MM-302 revealed biodistribution profiles that were quantitatively consistent with tissue-based analysis, and tumor (64)Cu positively correlated with liposomal drug deposition. This loading technique transforms liposomal therapeutics into theranostics and is currently being applied in a clinical trial (NCT01304797) to non-invasively quantify MM-302 tumor deposition, and evaluate its potential as a prognostic tool for predicting treatment outcome of nanomedicines.
Assuntos
Isótopos de Carbono/química , Quelantes/química , Doxorrubicina/análogos & derivados , Lipossomos/química , Nanomedicina/métodos , Nanopartículas/química , Animais , Linhagem Celular Tumoral , Cobre/química , Radioisótopos de Cobre/química , Doxorrubicina/química , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Transplante de Neoplasias , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios XRESUMO
Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.
Assuntos
Heterogeneidade Genética , Neoplasias/classificação , Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Análise de Célula Única/métodos , Animais , Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Análise por Conglomerados , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Padrões de Referência , Reprodutibilidade dos Testes , Neoplasias Gástricas/classificação , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Análise Serial de Tecidos , Neoplasias da Bexiga Urinária/classificação , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
Anthracycline-based regimens are a mainstay of early breast cancer therapy, however their use is limited by cardiac toxicity. The potential for cardiotoxicity is a major consideration in the design and development of combinatorial therapies incorporating anthracyclines and agents that target the HER2-mediated signaling pathway, such as trastuzumab. In this regard, HER2-targeted liposomal doxorubicin was developed to provide clinical benefit by both reducing the cardiotoxicity observed with anthracyclines and enhancing the therapeutic potential of HER2-based therapies that are currently available for HER2-overexpressing cancers. While documenting the enhanced therapeutic potential of HER2-targeted liposomal doxorubicin can be done with existing models, there has been no validated human cardiac cell-based assay system to rigorously assess the cardiotoxicity of anthracyclines. To understand if HER2-targeting of liposomal doxorubicin is possible with a favorable cardiac safety profile, we applied a human stem cell-derived cardiomyocyte platform to evaluate the doxorubicin exposure of human cardiac cells to HER2-targeted liposomal doxorubicin. To the best of our knowledge, this is the first known application of a stem cell-derived system for evaluating preclinical cardiotoxicity of an investigational agent. We demonstrate that HER2-targeted liposomal doxorubicin has little or no uptake into human cardiomyocytes, does not inhibit HER2-mediated signaling, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of liposomal doxorubicin. Taken together, this data ultimately led to the clinical decision to advance this drug to Phase I clinical testing, which is now ongoing as a single agent in HER2-expressing cancers.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/toxicidade , Neoplasias da Mama/patologia , Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Nus , Miócitos Cardíacos/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Binding of chemokines to glycosaminoglycans (GAGs) represents a crucial step in leukocyte attraction and activation. Since chemokine oligomerisation was shown to be important for GAG binding, the apparent oligomerisation constant of RANTES was determined to be 225 nM using fluorescence anisotropy. In the presence of heparan sulfate, chemokine oligomerisation was found to be promoted by the glycosaminoglycan as expressed in the increase in cooperativity and a shift towards higher melting temperatures in thermal unfolding experiments. In surface plasmon resonance investigations of RANTES-GAG binding kinetics using a heparan sulfate-coated chip, GAG-induced oligomerisation led to a bell-shaped (bi-phasic) Scatchard plot referring to cooperativity in the chemokine-GAG interaction. This was absent in the oligomerisation deficient RANTES mutants N46R and Q48K. We have further investigated the dependence of RANTES-GAG dissociation constants on oligosaccharide chain length by performing isothermal fluorescence titrations with size-defined heparin and heparan sulfate oligosaccharides as chemokine ligands. Heparin dp18 and heparan sulfate dp14 yielded the highest affinities with Kd values of 31.7 nM and 42.9 nM, respectively. Far-UV CD spectroscopy revealed a significant conformational change of RANTES upon heparan sulfate binding which is suggested to be a pre-requisite for oligomerisation and thus for optimal GPCR activation in vivo. This was shown by the impaired chemotactic activity of the RANTES N46R and Q48K mutants.
Assuntos
Quimiocina CCL5/metabolismo , Heparitina Sulfato/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL5/química , Quimiocina CCL5/genética , Quimiotaxia , Dicroísmo Circular , Escherichia coli/genética , Polarização de Fluorescência , Heparitina Sulfato/química , Humanos , Cinética , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Ressonância de Plasmônio de SuperfícieRESUMO
Increased vascular permeability and vasodilation are responses usually elicited by snake envenomation. In this report, we isolated from Macroviperalebetina venom two protein groups designated IC1 (Increasing Capillary1) and IC2 based on their activities on capillary permeability. Mass spectrometry analysis showed that IC1 contained four major proteins of 23,650, 24,306, 24,589 and 24,718Da, whereas IC2 contained three major proteins of 25,101, 25,194 and 25,298Da. N-terminal amino-acid sequencing revealed that IC1 and IC2 belong to the snake venom VEGF (svVEGF) family. IC1 and IC2 had a marked specificity for VEGFR-2, with affinities in the nanomolar range. Interestingly, they also bind to NRP1 and NRP2, with affinities in the micromolar range. This is the first report demonstrating that M. lebetina encodes several distinct svVEGFs, endowed with a capacity to interact with neuropilins. IC1 and IC2 could be valuable tools to understand the molecular properties of angiogenic factors and their receptors.
Assuntos
Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Venenos de Víboras/química , Viperidae/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Purpose: Therapeutic nanoparticles are designed to deliver their drug payloads through enhanced permeability and retention (EPR) in solid tumors. The extent of EPR and its variability in human tumors is highly debated and has been proposed as an explanation for variable responses to therapeutic nanoparticles in clinical studies.Experimental Design: We assessed the EPR effect in patients using a 64Cu-labeled nanoparticle, 64Cu-MM-302 (64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin), and imaging by PET/CT. Nineteen patients with HER2-positive metastatic breast cancer underwent 2 to 3 PET/CT scans postadministration of 64Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797).Results: Significant background uptake of 64Cu-MM-302 was observed in liver and spleen. Tumor accumulation of 64Cu-MM-302 at 24 to 48 hours varied 35-fold (0.52-18.5 %ID/kg), including deposition in bone and brain lesions, and was independent of systemic plasma exposure. Computational analysis quantified rates of deposition and washout, indicating peak liposome deposition at 24 to 48 hours. Patients were classified on the basis of 64Cu-MM-302 lesion deposition using a cut-off point that is comparable with a response threshold in preclinical studies. In a retrospective exploratory analysis of patient outcomes relating to drug levels in tumor lesions, high 64Cu-MM-302 deposition was associated with more favorable treatment outcomes (HR = 0.42).Conclusions: These findings provide important evidence and quantification of the EPR effect in human metastatic tumors and support imaging nanoparticle deposition in tumors as a potential means to identify patients well suited for treatment with therapeutic nanoparticles. Clin Cancer Res; 23(15); 4190-202. ©2017 AACR.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Nanopartículas/administração & dosagem , Adolescente , Adulto , Idoso , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos da radiação , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/química , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Feminino , Humanos , Fígado/diagnóstico por imagem , Fígado/efeitos dos fármacos , Pessoa de Meia-Idade , Nanopartículas/química , Metástase Neoplásica , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Receptor ErbB-2/sangue , Baço/diagnóstico por imagem , Baço/patologia , Trastuzumab/administração & dosagemRESUMO
Trastuzumab is the standard of care for HER2-positive breast cancer patients, markedly improving disease-free and overall survival. Combined with chemotherapy, it enhances patient outcomes, but cardiotoxicity due to the trastuzumab treatment poses a serious adverse effect. MM-302 is a HER2-targeted PEGylated liposome that encapsulates doxorubicin to facilitate its delivery to HER2-overexpressing tumor cells while limiting exposure to nontarget tissues, including the heart. In this study, we evaluated the feasibility and preclinical activity of combining MM-302 with trastuzumab. MM-302 and trastuzumab target different domains of the HER2 receptor and thus could simultaneously bind HER2-overexpressing tumor cells in vitro and in vivo. Furthermore, trastuzumab did not disrupt the mechanism of action of MM-302 in delivering doxorubicin to the n0ucleus and inducing DNA damage. Reciprocally, MM-302 did not interfere with the ability of trastuzumab to block prosurvival p-Akt signaling. Interestingly, coadministration of the two agents acutely increased the deposition of MM-302 in human xenograft tumors and subsequently increased the expression of the DNA damage marker p-p53. Finally, the combination of MM-302 and trastuzumab induced synergistic antitumor activity in HER2-overexpressing xenograft models of breast and gastric cancer. Collectively, our findings highlight a novel combination therapy that efficiently targets HER2-overexpressing cells through multiple mechanisms and support the ongoing investigation of combined MM-302/trastuzumab therapy for HER2-positive metastatic breast cancer in a randomized phase II clinical trial.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/genética , Neoplasias Gástricas/tratamento farmacológico , Antibióticos Antineoplásicos/administração & dosagem , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Sinergismo Farmacológico , Feminino , Humanos , Polietilenoglicóis/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Trastuzumab/administração & dosagem , Proteína Supressora de Tumor p53/metabolismoRESUMO
Neuropilin-1 (NRP1) has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.
RESUMO
Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody-liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into nontarget cells. In this work, we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance antitumor activity in preclinical models without negatively affecting nontarget tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Furthermore, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure, and increasing vascular perfusion. The novel dosing strategy for cyclophosphamide described herein is readily translatable to standard clinical regimens, represents a potentially significant advance in addressing the drug delivery challenge, and may have broad applicability for nanomedicines. This work formed the basis for clinical evaluation of cyclophosphamide for improving liposome deposition as part of an ongoing phase I clinical trial of MM-302 in HER2-positive metastatic breast cancer.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Ciclofosfamida/farmacologia , Doxorrubicina/análogos & derivados , Receptor ErbB-2/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Ciclofosfamida/administração & dosagem , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Feminino , Humanos , Ifosfamida/administração & dosagem , Ifosfamida/farmacologia , Camundongos , Polietilenoglicóis/administração & dosagem , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) >> IL-8(R68A) > IL-8(K67A) >> IL-8(K20A) > IL-8(R60A) >> IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.
Assuntos
Heparina/química , Heparina/metabolismo , Interleucina-8/química , Interleucina-8/metabolismo , Modelos Moleculares , Substituição de Aminoácidos , Sítios de Ligação , Simulação por Computador , Dissacarídeos/química , Dissacarídeos/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fluorescência , Interleucina-8/genética , Mutagênese Sítio-Dirigida , Mutação/genética , Termodinâmica , TitulometriaRESUMO
IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Interleucina-8/farmacologia , Substituição de Aminoácidos , Animais , Anti-Inflamatórios/química , Artrite Reumatoide/tratamento farmacológico , Sítios de Ligação , Bovinos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Guanidina/química , Heparitina Sulfato/química , Humanos , Interleucina-8/química , Interleucina-8/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Ligação Proteica , Desnaturação Proteica , Receptores de Interleucina-8A/químicaRESUMO
Neuropilins (NRP1 and NRP2) are coreceptors for vascular endothelial growth factor (VEGF) and mediate angiogenesis and tumor progression. VEGF binds to the NRP1 and NRP2 B domains. Previously, it was shown that mutagenesis of the soluble NRP2 B domain (MutB-NRP2) increased affinity to VEGF by 8-fold. Here, we show that MutB-NRP2 inhibited (125)I-VEGF binding to NRP1, NRP2, and VEGFR-2. It antagonized VEGF-induced VEGFR-2/NRP2 complex formation and inhibited VEGF-induced activation of AKT, a mediator of cell survival, without affecting activation of VEGFR-2. In three-dimensional embryoid bodies, a model of VEGF-induced angiogenesis, MutB-NRP2 inhibited VEGF-induced sprouting. When overexpressed in human melanoma cells, MutB-NRP2 inhibited tumor growth compared with control tumors. Avastin (bevacizumab), a monoclonal antibody to VEGF, inhibited VEGF interactions with VEGFR-2, but not with NRPs. The combination of MutB-NRP2 and Avastin resulted in an enhanced inhibition of human melanoma tumor growth compared with MutB-NRP2 treatment only or Avastin treatment only. In conclusion, these results indicate that MutB-NRP2 is a novel antagonist of VEGF bioactivity and tumor progression.
Assuntos
Melanoma/prevenção & controle , Mutação/genética , Neuropilina-2/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Bevacizumab , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Imunofluorescência , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Neuropilina-1/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Suínos , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neuropilins (NRP) play a central role in neuronal and blood vessel development as receptors for two ligand types, the semaphorin (SEMA) family of axon guidance modulators and the VEGF family of angiogenesis stimulators. The role of NRPs in axon guidance is well documented but a role in blood vessel development is less so. NRPs mediate normal developmental angiogenesis as shown in mouse and zebrafish models, and pathological angiogenesis in tumors and retinal disease. The ability of two disparate ligand families to bind to the same receptor is unusual but may be explainable by analysis of neuropilin structure. There are two NRP genes, nrp1 and nrp2. The NRPs have a relatively large extracellular domain consisting of sub domains, which are ligand binding sites. VEGF(165) binds to the b1b2 subdomain, SEMA3A and SEMA3F also bind to b1b2 but to a1a2 as well. Mutagenesis studies have identified NRP amino acids that bind VEGF(165) but not SEMA3F. These NRP structural elements might dictate differential SEMA and VEGF(165) binding properties, which in turn regulate angiogenesis. This article reviews the latest information of NRP structure and how structure influences angiogenesis. In addition, the role of NRPs in human cancer is addressed.
Assuntos
Neovascularização Fisiológica/fisiologia , Neuropilinas/química , Neuropilinas/fisiologia , Semaforinas/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Humanos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/fisiopatologia , Ligação Proteica/fisiologiaRESUMO
It is now well established that neuropilins (NRP1 and NRP2), first described as mediators of neuronal guidance, are also mediators of angiogenesis and tumor progression. NRPs are receptors for the class-3 semaphorin (SEMA) family of axon guidance molecules and also for the vascular endothelial growth factor (VEGF) family of angiogenic factors. VEGF-NRP interactions promote developmental angiogenesis as shown in mouse knockout and zebrafish knockdown studies. There is also evidence that NRPs mediate tumor progression. For example, overexpression of NRP1 enhances tumor growth whereas NRP1 antagonists, such as soluble NRP1 and anti-NRP1 antibodies, inhibit tumor growth. Furthermore, some class-3 SEMAs acting via NRPs inhibit tumor angiogenesis, progression and metastasis. Clinical data suggest that high NRP levels correlate with poor prognosis and survival in a variety of cancer types. Taken together, these results suggest that NRPs are potentially valuable targets for new anti-cancer therapies. We analyze here the current knowledge on NRPs and their role in angiogenesis and tumor progression and enumerate strategies for targeting these receptors.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neuropilinas/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neuropilinas/antagonistas & inibidores , Neuropilinas/genética , Semaforinas/metabolismoRESUMO
Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280-290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.
Assuntos
Substituição de Aminoácidos , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-2/metabolismo , Mutação Puntual , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Humanos , Ligantes , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Neuropilina-1/genética , Neuropilina-1/metabolismo , Neuropilina-2/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Suínos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.