RESUMO
Epithelial/Mesenchymal (E/M) plasticity plays a fundamental role both in embryogenesis and during tumorigenesis. The receptor for advanced glycation end products (RAGE) is a driver of cell plasticity in fibrotic diseases; however, its role and molecular mechanism in triple-negative breast cancer (TNBC) remains unclear. Here, we demonstrate that RAGE signaling maintains the mesenchymal phenotype of aggressive TNBC cells by enforcing the expression of SNAIL1. Besides, we uncover a crosstalk mechanism between the TGF-ß and RAGE pathways that is required for the acquisition of mesenchymal traits in TNBC cells. Consistently, RAGE inhibition elicits epithelial features that block migration and invasion capacities. Next, since RAGE is a sensor of the tumor microenvironment, we modeled acute acidosis in TNBC cells and showed it promotes enhanced production of RAGE ligands and the activation of RAGE-dependent invasive properties. Furthermore, acute acidosis increases SNAIL1 levels and tumor cell invasion in a RAGE-dependent manner. Finally, we demonstrate that in vivo inhibition of RAGE reduces metastasis incidence and expands survival, consistent with molecular effects that support the relevance of RAGE signaling in E/M plasticity. These results uncover new molecular insights on the regulation of E/M phenotypes in cancer metastasis and provide rationale for pharmacological intervention of this signaling axis.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Receptor para Produtos Finais de Glicação Avançada/genética , Linhagem Celular Tumoral , Transdução de Sinais , Fenótipo , Transição Epitelial-Mesenquimal , Movimento Celular , Microambiente TumoralRESUMO
Whispering gallery mode resonator lasers hold the promise of an ultralow intrinsic limit of detection. However, the widespread use of these devices for biosensing applications has been hindered by the complexity and lack of robustness of the proposed configurations. In this work, we demonstrate biosensing with an integrated microdisk laser. Al2O3doped with Yb3+ was utilized because of its low optical losses as well as its emission in the range 1020-1050 nm, outside the absorption band of water. Single-mode laser emission was obtained at a wavelength of 1024 nm with a linewidth of 250 kHz while the microdisk cavity was submerged in water. A limit of detection of 300 pM (3.6 ng/ml) of the protein rhS100A4 in urine was experimentally demonstrated, showing the potential of the proposed devices for biosensing.
Assuntos
Óxido de Alumínio/química , Técnicas Biossensoriais/instrumentação , Dispositivos Lab-On-A-Chip , Lasers , Itérbio/químicaRESUMO
Antibody-Drug Conjugates (ADCs) have been shown to produce clinical benefit in cancer patient thanks to their ability to target highly cytotoxic small molecules to tumor cells. However, the development of these complex molecules faces significant challenges due to the need to combine a large biologic drug with a small molecule drug to generate the desired bioconjugate. We describe here the use of a protein ligation methodology, based on the native chemical ligation reaction to generate site-specific Antibody-Drug Conjugates, which does not require the incorporation of unnatural modifications into the antibody. Fully native antibodies, with only the desired cytotoxic molecules attached, can be generated, thus minimizing the risk that additional modifications required for the site-specific conjugation pose a risk to the antibody activity. We demonstrate that our approach can be used to generate site-specifically modified ADCs, with potent in vitro and in vivo antitumor activity in a breast cancer tumor model.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/química , Imunoconjugados/química , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos Nus , Engenharia de Proteínas , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel conjugate between a cyclometalated platinum(II) complex with dual antiangiogenic and antitumor activity and a cyclic peptide containing the RGD sequence (-Arg-Gly-Asp-) has been synthesized by combining solid- and solution-phase methodologies. Although peptide conjugation rendered a non-cytotoxic compound in all tested tumor cell lines (± αV ß3 and αV ß5 integrin receptors), the antiangiogenic activity of the Pt-c(RGDfK) conjugate in human umbilical vein endothelial cells at sub-cytotoxic concentrations opens the way to the design of a novel class of angiogenesis inhibitors through conjugation of metallodrugs with high antiangiogenic activity to cyclic RGD-containing peptides or peptidomimetic analogues.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Oligopeptídeos/farmacologia , Compostos Organoplatínicos/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Estrutura Molecular , Neovascularização Patológica/patologia , Oligopeptídeos/síntese química , Oligopeptídeos/química , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Relação Estrutura-AtividadeRESUMO
Cilengitide is an RGD-peptide of sequence cyclo[RGDfNMeV] that was was developed as a highly active and selective ligand for the αvß3 and αvß5 integrin receptors. We describe the synthesis of three analogues of this peptide in which the N-Me group has been replaced by N-oligoethylene glycol (N-OEG) chains of increasing size: namely N-OEG2, N-OEG11, and N-OEG23, which are respectively composed of 2, 11, and 23 ethylene oxide monomer units. The different N-OEG cyclopeptides and the original peptide were compared with respect to lipophilicity and biological activity. The N-OEG2 analogue was straightforward to synthesize in solid phase using an Fmoc-N-OEG2 building block. The syntheses of the N-OEG11 and N-OEG23 cyclopeptides are hampered by the increased steric hindrance of the N-substituent, and could only be achieved by segment coupling, which takes place with epimerization and thus requires extensive product purification. All the N-OEG analogues were found to be more hydrophobic than the parent peptide, and their hydrophobicity was systematically enhanced upon increasing the length of the OEG chain. The N-OEG2 cyclopeptide displayed the same capacity as Cilengitide to inhibit the integrin-mediated adhesion of HUVEC endothelial, DAOY gliobastoma, and HT-29 colon cancer cells to their ligands vitronectin and fibrinogen. The N-OEG11 and N-OEG23 analogues also inhibited cell adhesion to these immobilized ligands, but their IC50 values dropped by 1 order of magnitude with respect to the parent peptide. These results indicate that replacement of the backbone N-Me group of Cilengitide by a short N-OEG chain provides a more lipophilic analogue with a similar biological activity. Upon increasing the size of the N-OEG chain, liophilicity is enhanced, but synthetic yields drop and the longer polymer chains may impede targeted binding.
Assuntos
Etilenoglicóis/química , Venenos de Serpentes/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Etilenoglicóis/síntese química , Etilenoglicóis/farmacologia , Fibrinogênio/metabolismo , Células HT29 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Ligantes , Conformação Molecular , Venenos de Serpentes/síntese química , Venenos de Serpentes/farmacologia , Relação Estrutura-Atividade , Vitronectina/metabolismoRESUMO
A robust synthetic strategy for the introduction of the N-(4-azidobutyl) linker into peptides using standard SPPS techniques is described. Based on the example of Cilengitide it is shown that the N-(4-azidobutyl) group exerts similar conformational restraints as a backbone N-Me group and allows conjugation of a desired molecule either via click chemistry or-after azide reduction-via acylation or reductive alkylation.
Assuntos
Azidas/química , Peptídeos Cíclicos/síntese química , Acilação , Animais , Química Click , Estrutura Molecular , Peptídeos Cíclicos/química , Venenos de Serpentes/químicaRESUMO
S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Animais , Western Blotting , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Reação em Cadeia da Polimerase em Tempo Real , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/antagonistas & inibidores , Proteínas S100/imunologia , Ressonância de Plasmônio de Superfície , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel series of indolocarbazoles were synthesized and their antiproliferative activity against HUVEC, LoVo, DLD-1 and ST-486 cell lines, was investigated. Those staurosporine analogs in which a substituted dimethylaminoalkoxy chain was attached to the indolic nitrogen showed interesting activity and selectivity with respect to HUVEC proliferation. The effect on capillary tube formation in 3-dimensional matrigel matrix was studied using the most active compounds. Evaluation of their in vivo anti-angiogenic activity in a murine Lewis lung cancer model was also analyzed.
Assuntos
Inibidores da Angiogênese/síntese química , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Estaurosporina/análogos & derivados , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Capilares/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Combinação de Medicamentos , Humanos , Laminina/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Neovascularização Patológica/tratamento farmacológico , Proteoglicanas/farmacologia , Espectrofotometria Infravermelho , Estaurosporina/síntese química , Estaurosporina/química , Estaurosporina/farmacologiaRESUMO
Macrophages are key cells for HIV infection and HIV spreading inside the organism. Macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (M-CSF). In the monocyte to macrophage differentiation process with M-CSF, alphav-integrins are upregulated concomitantly with the capacity of HIV to generate a productive virus infection. In the present study we show that an anti-alphav antibody, 17E6, inhibited HIV-1 infection of primary macrophages. The effect of 17E6 on HIV-1 BaL replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (EC50) of 17+/-2 microg/ml in the absence of cytotoxicity. Similarly, a monoclonal antibody targeting the alphavbeta6 integrin (14D9.F8) also inhibited HIV-1 BaL infection in this cell type. 17E6 reduced the detection of HIV-1 BaL proviral DNA in acutely infected macrophages, but was completely ineffective against HIV-1 BaL production in chronically infected macrophages, suggesting that 17E6 inhibited HIV infection at an early stage of the virus cycle. Finally, a small molecular weight antagonist of the alphavbeta6 integrin, EMD 409849, reduced HIV replication at subtoxic concentrations. Therefore, our results suggest that alphav-containing integrins could play a role in HIV replication in macrophages and suggest that small-molecular-weight compounds might interfere with HIV replication in macrophages through the interaction with alphav integrins.
Assuntos
Anticorpos Monoclonais/imunologia , HIV-1/fisiologia , Integrina alfaV/imunologia , Integrinas/antagonistas & inibidores , Macrófagos/virologia , Antígenos de Neoplasias/imunologia , Células Cultivadas , DNA Viral/análise , Humanos , Integrinas/imunologia , Leucócitos Mononucleares , Macrófagos/imunologia , Provírus/genética , Receptores de HIV/metabolismo , Replicação ViralRESUMO
Tumour cell adhesion within the microvasculature of host organs, its stabilisation and cell invasion into the host organs, appear to be important steps in the formation of distant metastases. Intravital fluorescence-video microscopy was used to investigate the early steps in metastasis formation of colon carcinoma cells within the liver, which is the main target organ of colorectal carcinomas. The involvement of alphav-integrins was analysed in vivo using HT-29 cells after treatment with different function-blocking antibodies [pan-alphav (n=9 animals), specific alphavbeta3 (n=8 animals) and alphavbeta5 (n=8 animals)] or linear Arg-Gly-Asp (RGD)-containing peptides (RGD-peptides) (n=6 animals). Treatment with anti-alphav and anti-alphavbeta5 antibodies resulted in significantly (P<0.001) decreased tumour cell adhesion in vivo within the hepatic microvasculature. Cells treated with anti-alphavbeta3 antibodies or unspecific immunoglobulin-G (IgG) did not show significant changes in their adhesive properties. Furthermore, inhibition of cell adhesion was achieved by linear RGD-peptides in a dose-dependent manner. Relative numbers of migrated cells were not affected by any of the treatments. These results suggest that alphav-integrins, especially alphavbeta5, can influence the ability of circulating tumour cells to adhere within the hepatic microvessels. In contrast, migration of adherent cells into the liver parenchyma was not affected by alphav-integrin inhibition. Our findings support the hypothesis that specific interactions between circulating tumour cells and host organs are required for organ-specific tumour cell arrest.
Assuntos
Neoplasias do Colo , Integrinas/fisiologia , Neoplasias Hepáticas/secundário , Receptores de Vitronectina/fisiologia , Adesão Celular , Ensaio de Imunoadsorção Enzimática , Células HT29 , Humanos , Integrinas/antagonistas & inibidores , Invasividade Neoplásica/fisiopatologia , Oligopeptídeos/fisiologia , Receptores de Vitronectina/antagonistas & inibidoresRESUMO
The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.
Assuntos
Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Neovascularização Patológica/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Apoptose/efeitos dos fármacos , Técnicas de Cultura de Células , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/biossíntese , Oligonucleotídeos Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , SurvivinaRESUMO
Recent evidence has established different functions for the tumor suppressor protein, p16(INK4A) aside from controlling the cell cycle. Here we report that cdk4/6 inhibition blocked both human umbilical vein endothelial cells (HUVEC) spreading on a vitronectin matrix and HUVEC migration on vitronectin. p16 can also act as an anti-angiogenic molecule in vitro since HUVEC and HMEC cells transfected with Ad-p16 or treated with Antennapedia p16 peptides are unable to differentiate on a Matrigel matrix. Both, p16, cyclin D1, cdk4 and cdk6 were immuno-colocalized with Ezrin, Rac, Vinculin, alphav-integrin, and FAK proteins in the ruffles and lamellipodia of migratory cells. Our results indicate that p16 is a key component of a new cytoplasmic pathway controlling angiogenesis of endothelial cells via the alphavbeta3-integrin-mediated migration.
Assuntos
Movimento Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Endotélio Vascular/citologia , Neovascularização Fisiológica/fisiologia , Pseudópodes/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismo , Vinculina/metabolismo , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
BACKGROUND & AIMS: A deficient leukocyte recruitment has been suggested in tumor vasculature, but little is known about the underlying molecular mechanism. To characterize leukocyte-endothelium interaction in experimental colon cancer, quantify the main endothelial cell adhesion molecules (CAMs), and evaluate the effect of tumor-derived products. METHODS: Leukocyte recruitment was assessed by intravital videomicroscopy in mice bearing HT29-derived tumors. Endothelial CAMs were measured using the dual-radiolabeled antibody technique. The role of molecules mediating leukocyte rolling (P-, E-, and L-selectin) or adhesion (intercellular adhesion molecule 1 [ICAM-1] and vascular cell adhesion molecule 1 [VCAM-1]) carcinoembryonic antigen (CEA), and transforming growth factor (TGF) beta1 was assessed through immunoblockade, whereas participation of nitric oxide (NO) and cyclooxygenase (COX) metabolites were evaluated by means of nonselective and selective inhibition. RESULTS: Basal and lipopolysaccharide-stimulated leukocyte rolling and adhesion were markedly reduced in tumor vasculature. ICAM-1 immunoblockade prevented leukocyte adhesion in both tumor and nontumor microvessels. Neither baseline nor LPS-induced endothelial ICAM-1, P-, and E-selectin expression in tumors were reduced with respect to nontumor vasculature. Although VCAM-1 expression was reduced in tumor endothelium, immunoneutralization of VCAM-1 failed to reverse LPS-induced leukocyte recruitment in this setting. CEA immunoblockade and COX inhibition did not modify the deficient leukocyte rolling. Nonselective NO inhibition partially reversed the defective adhesion response in tumor microvessels. Finally, TGF-beta1 immunoblockade partially and selectively restored impaired leukocyte rolling and adhesion in tumor microvessels. CONCLUSIONS: Impaired leukocyte recruitment in tumor vasculature cannot be attributed to a depressed expression of the main CAMs. Selective restoration after NO inhibition and TGF-beta1 immunoblockade suggests involvement of both molecules in this phenomenon.