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Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of cancers which lacks the expression and/or amplification of targetable biomarkers (ie, estrogen receptor, progestrogen receptor, and human epidermal growth factor receptor 2), and is often associated with the worse disease-specific outcomes than other breast cancer subtypes. Here, we report that high expression of the sortilin (SORT1) receptor correlates with the decreased survival in TNBC patients, and more importantly in those bearing lymph node metastases. By exploiting SORT1 function in ligand internalization, a new anticancer treatment strategy was designed to target SORT1-positive TNBC-derived cells both in vitro and in two in vivo tumor xenografts models. A peptide (TH19P01), which requires SORT1 for internalization and to which many anticancer drugs could be conjugated, was developed. In vitro, while the TH19P01 peptide itself did not exert any antiproliferative or apoptotic effects, the docetaxel-TH19P01 conjugate (TH1902) exerted potent antiproliferative and antimigratory activities when tested on TNBC-derived MDA-MB-231 cells. TH1902 triggered faster and more potent apoptotic cell death than did unconjugated docetaxel. The apoptotic and antimigratory effects of TH1902 were both reversed by two SORT1 ligands, neurotensin and progranulin, and on siRNA-mediated silencing of SORT1. TH1902 also altered microtubule polymerization and triggered the downregulation of the anti-apoptotic Bcl-xL biomarker. In vivo, both i.p. and i.v. administrations of TH1902 led to greater tumor regression in two MDA-MB-231 and HCC-70 murine xenograft models than did docetaxel, without inducing neutropenia. Altogether, the data demonstrates the high in vivo efficacy and safety of TH1902 against TNBC through a SORT1 receptor-mediated mechanism. This property allows for selective treatment of SORT1-positive TNBC and makes TH1902 a promising avenue for personalized therapy with the potential of improving the therapeutic window of cytotoxic anticancer drugs such as docetaxel.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Combinação de Medicamentos , Descoberta de Drogas , Feminino , Inativação Gênica , Xenoenxertos , Humanos , Metástase Linfática , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Transplante de Neoplasias , Neurotensina/farmacologia , Progranulinas/farmacologia , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , Proteína bcl-X/metabolismoRESUMO
The analgesic potency of morphine-6-glucuronide (M6G) has been shown to be 50-fold higher than morphine after intracerebral injection. However, the brain penetration of M6G is significantly lower than morphine, thus limiting its usefulness in pain management. Here, we created new entities by the conjugation of the angiopep-2 peptide (An2) that crosses the blood-brain barrier (BBB) by low-density lipoprotein receptor-related protein 1 receptor-mediated transcytosis with either morphine or M6G. We demonstrated improvement of BBB permeability of these new entities compared with that of unconjugated M6G and morphine. Intravenous or subcutaneous administration of the An2-M6G conjugate exerted greater and more sustained analgesic activity than equivalent doses of either morphine or M6G. Likewise, subcutaneous An2-morphine induced a delayed but prolonged antinociceptive effect. The effects of these conjugates on the gastrointestinal tract motility were also evaluated. An2-morphine significantly reduced the intestinal transit time, whereas An2-M6G exhibited a reduced constipation profile, as compared with an equimolar dose of morphine. In summary, we have developed new brain-penetrant opioid conjugates exhibiting improved analgesia to side effect ratios. These results thus support the use of An2-carrier peptides as an innovative BBB-targeting technology to deliver effective drugs, such as M6G, for pain management. SIGNIFICANCE STATEMENT: The metabolite morphine-6-glucuronide (M6G) does not efficiently cross the blood-brain barrier. The low-density lipoprotein receptor-related protein 1 peptide ligand angiopep-2 may serve as an effective drug delivery system to the brain. Here, we demonstrated that the coupling of M6G to angiopep-2 peptide (An2) improves its brain penetration and significantly increases its analgesic potency. The An2-M6G conjugate has a favorable side effect profile that includes reduction of developing constipation. An2-M6G exhibits a unique pharmacodynamic profile with a better therapeutic window than morphine.
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Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Barreira Hematoencefálica/metabolismo , Derivados da Morfina/química , Derivados da Morfina/metabolismo , Peptídeos/química , Administração Intravenosa , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/farmacologia , Animais , Transporte Biológico , Motilidade Gastrointestinal/efeitos dos fármacos , Masculino , Camundongos , Derivados da Morfina/administração & dosagem , Derivados da Morfina/farmacologia , Nociceptividade/efeitos dos fármacosRESUMO
Mucopolysaccharidosis type II (Hunter's disease) mouse model (IdS-KO) was investigated by both imaging mass spectrometry (IMS) and immunohistochemistry (IHC) performed on the same tissue sections. For this purpose, IdS-KO mice brain sections were coated with sublimated 1,5-diaminonaphtalene and analyzed by high spatial resolution IMS (5 µm) and anti-GM3 IHC on the same tissue sections to characterize the ganglioside monosialated ganglioside (GM) deposits found in Hunter's disease. IMS analysis have found that two species of GM3 and GM2 that are only different due to the length of their fatty acid residue (stearic or arachidic residue) were overexpressed in the IdS-KO mice compared to a control mouse. GM3 and GM2 were characterized by on-tissue exact mass and MS/MS compared to a GM3 standard. Realignment of both IMS and IHC data sets further confirmed the observed regioselective signal previously detected by providing direct correlation of the IMS image for the two GM3 overly expressed MS signals with the anti-GM3 IHC image. Furthermore, these regioselective GM MS signals were also found to have highly heterogeneous distributions within the GM3-IHC staining. Some deposits showed high content in GM3 and GM2 stearic species (r = 0.74) and others had more abundant GM3 and GM2 arachidic species (r = 0.76). Same-section analysis of Hunter's disease mouse model by both high spatial resolution IMS and IHC provides a more in-depth analysis of the composition of the GM aggregates while providing spatial distribution of the observed molecular species. Graphical Abstract Ganglioside imaging mass spectrometry followed by immunohistochemistry performed on the same tissue section.
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Encéfalo/metabolismo , Gangliosídeo G(M2)/metabolismo , Gangliosídeo G(M3)/metabolismo , Imuno-Histoquímica/métodos , Mucopolissacaridose II/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Camundongos , Camundongos KnockoutRESUMO
[This corrects the article DOI: 10.3389/fimmu.2024.1355945.].
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The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10 melanoma syngeneic tumor model demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes.
Assuntos
Antígeno B7-H1 , Nucleotidiltransferases , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Morte CelularRESUMO
Background: Breast and ovarian cancer stem cells (CSC) can contribute to the invasive and chemoresistance phenotype of tumors. TH1902, a newly developed sortilin (SORT1)-targeted peptide-docetaxel conjugate is currently in phase-1 clinical trial. Whether TH1902 impacts the chemoresistance phenotype of human triple-negative breast CSC (hTNBCSC) and ovarian CSC (hOvCSC) is unknown. Methods and Results: Immunophenotyping of hTNBCSC and hOvCSC was performed by flow cytometry and confirmed the expression of SORT1, and of CSC markers CD133, NANOG, and SOX2. Western blotting demonstrated the expression of the drug efflux pumps from the P-gp family members, ABCB1 and ABCB5. The cellular uptake of the fluorescent Alexa488-peptide from TH1902 was inhibited upon siRNA-mediated repression of SORT1 or upon competition with SORT1 ligands. In contrast to docetaxel, TH1902 inhibited in vitro migration, induced cell apoptosis and lead to G2/M cell cycle arrest of the hTNBCSC. These events were unaffected by the presence of the P-gp inhibitors cyclosporine A or PSC-833. In vivo, using immunosuppressed nude mice xenografts, TH1902 significantly inhibited the growth of hTNBCSC and hOvCSC xenografts (~80% vs. ~35% for docetaxel) when administered weekly as intravenous bolus for three cycles at 15 mg/kg, a dose equivalent to the maximal tolerated dose of docetaxel. Therapeutic efficacy was further observed when carboplatin was combined to TH1902. Conclusions: Overall, TH1902 exerts a superior anticancer activity than the unconjugated docetaxel, in part, by circumventing the CSC drug resistance phenotype that could potentially reduce cancer recurrence attributable to CSC.
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Sortilin (SORT1) receptor-mediated endocytosis functions were exploited for this new approach for effective and safe treatments of gynecological cancers. Here, high expression of SORT1 was found in >75% of the clinically annotated ovarian and endometrial tumors analyzed by immunohistochemistry. Therefore, the anticancer properties of the peptide-drug conjugate TH1902, a peptide that targets SORT1 and which is linked to docetaxel molecules, were investigated both in vitro using ovarian and endometrial cancer cell cultures and in vivo using xenograft models. In vitro, TH1902 inhibited cell proliferation and triggered higher SORT1-dependent cell apoptosis than unconjugated docetaxel did in ES-2 and SKOV3 ovarian cancer cell lines. The uptake of the Alexa488-TH19P01 peptide from TH1902 was reduced upon siRNA-mediated silencing of SORT1. In vivo, weekly administration of TH1902 showed better tolerability compared to equivalent docetaxel doses and inhibited tumor growth in ovarian and endometrial xenograft mice models. TH1902 as a single agent inhibited ovarian tumor growth more than either of the unconjugated taxanes or carboplatin. Furthermore, TH1902 combination with carboplatin also demonstrated better efficacy when compared to both taxanes-carboplatin combinations. Overall, TH1902 shows better in vivo efficacy, compared to that of docetaxel and even paclitaxel, against SORT1-positive ovarian and endometrial cancers and could be safely combined with carboplatin.
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Vasculogenic mimicry (VM) is defined as the formation of microvascular channels by genetically deregulated cancer cells and is often associated with high tumor grade and cancer therapy resistance. This microcirculation system, independent of endothelial cells, provides oxygen and nutrients to tumors, and contributes also in part to metastasis. VM has been observed in ovarian cancer and in triple negative breast cancer (TNBC) and shown to correlate with decreased overall cancer patient survival. Thus, strategies designed to inhibit VM may improve cancer patient treatments. In this study, sortilin (SORT1) receptor was detected in in vitro 3D capillary-like structures formed by ES-2 ovarian cancer and MDA-MB-231 TNBC-derived cells when grown on Matrigel. SORT1 gene silencing or antibodies directed against its extracellular domain inhibited capillary-like structure formation. In vitro, VM also correlated with increased gene expression of matrix metalloproteinase-9 (MMP-9) and of the cancer stem cell marker CD133. In vivo ES-2 xenograft model showed PAS+/CD31- VM structures (staining positive for both SORT1 and CD133). TH1904, a Doxorubicin-peptide conjugate that is internalized by SORT1, significantly decreased in vitro VM at low nM concentrations. In contrast, VM was unaffected by unconjugated Doxorubicin or Doxil (liposomal Doxorubicin) up to µM concentrations. TH1902, a Docetaxel-peptide conjugate, altered even more efficiently in vitro VM at pM concentrations. Overall, current data evidence for the first time that 1) SORT1 itself exerts a crucial role in both ES-2 and MDA-MB-231 VM, and that 2) VM in these cancer cell models can be efficiently inhibited by the peptide-drug conjugates TH1902/TH1904. These new findings also indicate that both peptide-drug conjugates, in addition to their reported cytotoxicity, could possibly inhibit VM in SORT1-positive TNBC and ovarian cancer patients.
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New and effective therapeutics that cross the blood-brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep-2 peptide, a sequence derived from ligands that bind to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep-2 conjugate and immunohistochemical studies of injected Angiopep-2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co-localization with the neuronal nuclei-selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [(¹²5I]-Angiopep-2 into brain endothelial cells occurred by a saturable mechanism involving LRP-1. The primary sequence and charge of Angiopep-2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Peptídeos/metabolismo , Animais , Antígenos Nucleares/análise , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Carbocianinas , Células Endoteliais/metabolismo , Proteína Glial Fibrilar Ácida/análise , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/análise , Transporte Proteico , Ensaio Radioligante , Ratos , TranscitoseRESUMO
The blood-brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain-derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep-2 transport and accumulation in brain endothelial cells were unaffected by the P-glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P-glycoprotein. However, competition studies show that activated alpha(2)-macroglobulin, a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP1) and Angiopep-2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488-Angiopep-2 co-localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep-2 transport across the BBB is, in part, mediated by LRP1.
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Barreira Hematoencefálica/fisiologia , Encéfalo/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Oligopeptídeos/fisiologia , Fragmentos de Peptídeos/metabolismo , Somatostatina/análogos & derivados , Animais , Barreira Hematoencefálica/metabolismo , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/metabolismo , Humanos , Fragmentos de Peptídeos/administração & dosagem , Peptídeos Cíclicos , Transporte Proteico/fisiologia , Ratos , Somatostatina/fisiologiaRESUMO
We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.
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Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Encéfalo/irrigação sanguínea , Caveolina 1/metabolismo , Movimento Celular , Células Endoteliais/fisiologia , Neovascularização Fisiológica , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Caveolina 1/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Ciclosporina/farmacologia , Ciclosporinas/farmacologia , Cães , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , RNA Interferente Pequeno/genética , RatosRESUMO
By controlling access to the brain, the blood-brain barrier (BBB) restricts the entry of proteins and potential drugs to cerebral tissues. We demonstrate here the transcytosis ability of aprotinin and peptides derived from Kunitz domains using an in vitro model of the BBB and in situ brain perfusion. Aprotinin transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 10-fold greater than that of holo-transferrin. Sucrose permeability was unaffected by high concentrations of aprotinin, indicating that transcytosis of aprotinin was unrelated to changes in the BBCEC monolayer integrity. Alignment of the amino acid sequence of aprotinin with the Kunitz domains of human proteins allowed the identification and design of a family of peptides, named Angiopeps. These peptides, and in particular Angiopep-2, exhibit higher transcytosis capacity and parenchyma accumulation than aprotinin. Overall, these results suggest that these Kunitz-derived peptides could be advantageously used as a new brain delivery system for pharmacological agents that do not readily enter the brain.
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Encéfalo/metabolismo , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos/métodos , Peptídeos/administração & dosagem , Peptídeos/genética , Sequência de Aminoácidos/genética , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos/farmacocinética , RatosRESUMO
Tissue plasminogen activator (t-PA) is an extracellular serine protease that converts the proenzyme plasminogen into the broad-spectrum substrate serine protease, plasmin. Plasmin, one of the most potent pro-angiogenic factors, is a key element in fibrinolysis, cell migration, tissue remodeling and tumor invasion. In the present investigation, we assessed the impact of the truncated form of soluble melanotransferrin (sMTf) on plasminogen activation by t-PA and subsequent endothelial cell detachment. Co-treatment of human endothelial microvessel cells with plasminogen, t-PA and sMTf significantly increased plasmin formation and activity in the culture medium. Plasmin generated in the presence of sMTf also led to a 30% reduction in fibronectin detection within cell lysates and to a 9-fold increase within the corresponding cell medium. Moreover, the presence of sMTf increases EC detachment by 6-fold compared to cells treated only with plasminogen and t-PA. Although the addition of alpha(2)-antiplasmin completely prevented plasmin formation and EC detachment, epigallocatechin gallate, GM6001 and a specific antibody directed against MMP-2 prevented cellular detachment without interfering with plasminogen activation. Overall, these data suggest that the anti-angiogenic properties of sMTf may result from local overstimulation of plasminogen activation by t-PA, thus leading to subsequent degradation of the Fn matrix and EC detachment.
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Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Neoplasias/fisiologia , Ativadores de Plasminogênio/fisiologia , Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/fisiologia , Antígenos de Neoplasias , Adesão Celular/fisiologia , Linhagem Celular , Endotélio Vascular/citologia , Ativação Enzimática/fisiologia , Humanos , Antígenos Específicos de Melanoma , SolubilidadeRESUMO
We have previously demonstrated that human recombinant soluble melanotransferrin (hr-sMTf) interacts with the single-chain zymogen pro urokinase-type plasminogen activator (scu-PA) and plasminogen. In the present work, the impact of exogenous hr-sMTf on endothelial cells (EC) migration and morphogenic differentiation into capillary-like structures (tubulogenesis) was assessed. hr-sMTF at 10 nM inhibited by 50% the migration and tubulogenesis of human microvessel EC (HMEC-1). In addition, in hr-sMTf-treated HMEC-1, the expression of both urokinase-type plasminogen activator receptor (u-PAR) and low-density lipoprotein receptor-related protein (LRP) are down-regulated. However, fluorescence-activated cell sorting analysis revealed a 25% increase in cell surface u-PAR in hr-sMTf-treated HMEC-1, whereas the binding of the urokinase-type plasminogen activator (u-PA)*plasminogen activator inhibitor-1 (PAI-1) complex is decreased. This reduced u-PA-PAI-1 binding is correlated with a strong inhibition of the HMEC-1 plasminolytic activity, indicating that exogenous hr-sMTf treatment alters the internalization and recycling processes of free and active u-PAR at the cellular surface. Overall, these results demonstrate that exogenous hr-sMTf affects plasminogen activation at the cell surface, thus leading to the inhibition of EC movement and tubulogenesis. These results are the first to consider the potential use of hr-sMTf as a possible therapeutic agent in angiogenesis-related pathologies.
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Células Endoteliais/efeitos dos fármacos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Neoplasias/farmacologia , Receptores de Superfície Celular/metabolismo , Antígenos de Neoplasias , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Células Endoteliais/fisiologia , Humanos , Antígenos Específicos de Melanoma , Microtúbulos/fisiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 microg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CH(R)C5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CH(R)C5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.
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Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Camellia sinensis , Catequina/farmacologia , Resistência a Múltiplos Medicamentos , Flavonoides , Fenóis/farmacologia , Polímeros/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Células CACO-2 , Catequina/análogos & derivados , Divisão Celular/efeitos dos fármacos , Cricetinae , Humanos , Ligação Proteica/efeitos dos fármacos , Rodamina 123/farmacologia , Vimblastina/farmacologiaRESUMO
PURPOSE: Overexpression of P-glycoprotein (Pgp) encoded by the MDR1 gene is one of the major obstacles to successful cancer chemotherapy. The goal of this study was to evaluate if, among other natural coumarins, cnidiadin, a furanocoumarin present in traditional Chinese medications and in a spice commonly used in Greek food, inhibits Pgp transport activity and has the potential to reverse MDR1 multidrug resistance. METHODS: Using MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells as a model of cells expressing the human MDR1 phenotype, and verapamil or CsA or both as positive control, we tested the capacity of six natural coumarins (umbelliferone, esculin, esculetin, cnidiadin, angelicin and psoralen) to induce the accumulation of rhodamine-123 (R-123) and [3H]-vinblastine ([3H]-VBL) and to modulate the photolabeling of Pgp by SDZ 212-122, a diazirin cyclosporin A. The growth-inhibitory effect of cnidiadin and its capacity to enhance the cell toxicity of vinblastine (VBL) or vincristine (VCR) was then evaluated by the WST-1 assay in two cell lines overexpressing Pgp (MDCK-MDR1 and vincristine-resistant KB/VCR). RESULTS: Cnidiadin was the only tested coumarin capable of significantly accumulating R-123 and [3H]-VBL and inhibiting Pgp photolabeling in MDCK-MDR1 cells. The dose-dependent increase in [3H]-VBL uptake (IC50 26.5 microM) induced by cnidiadin in the dose range 1-100 microM correlated with inhibition of Pgp photolabeling. At 10 microM cnidiadin inhibited photolabeling by 59% and sensitized both MDCK-MDR1 and KB/VCR cells to vinca alkaloids. CONCLUSION: Cnidiadin is a cytotoxic agent capable in vitro of competitively inhibiting the binding and efflux of drug by Pgp and of enhancing the cell toxicity of vinca alkaloids in two cell lines (MDCK-MDR1 and mutant human carcinoma KB/VCR) overexpressing Pgp. This suggests that diet or traditional preparation containing cnidiadin may contribute to the reversal of MDR1 multidrug resistance and may affect the bioavailability of Pgp substrates orally administered. However, due to its cell toxicity, clinical interest in cnidiadin as a chemosensitizer appears to be limited.
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Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Resistência a Múltiplos Medicamentos , Furocumarinas/farmacologia , Genes MDR , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Administração Oral , Animais , Antineoplásicos Fitogênicos/farmacologia , Disponibilidade Biológica , Cumarínicos/farmacologia , Cães , Interações Medicamentosas , Vimblastina/farmacologia , Vincristina/farmacologiaRESUMO
Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G-Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.
Assuntos
Doença de Alzheimer/sangue , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/sangue , Doença de Alzheimer/diagnóstico , Animais , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias , Soluções Tampão , Cátions Bivalentes/química , Linhagem Celular , Precipitação Química , Cricetinae , Eletroforese em Gel Bidimensional , Glicosilação , Humanos , Imunoglobulinas/metabolismo , Lectinas/química , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/química , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/urina , Proteínas do Tecido Nervoso/metabolismo , Lectinas de Plantas/química , Conformação Proteica/efeitos dos fármacos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/sangue , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Processamento de Proteína Pós-Traducional , Proteínas e Peptídeos Salivares/química , Sensibilidade e Especificidade , Proteína Estafilocócica A/metabolismoRESUMO
Anti-HER2 monoclonal antibodies (mAb) have been shown to reduce tumor size and increase survival in patients with breast cancer, but they are ineffective against brain metastases due to poor brain penetration. In previous studies, we identified a peptide, known as Angiopep-2 (An2), which crosses the blood-brain barrier (BBB) efficiently via receptor-mediated transcytosis, and, when conjugated, endows small molecules and peptides with this property. Extending this strategy to higher molecular weight biologics, we now demonstrate that a conjugate between An2 and an anti-HER2 mAb results in a new chemical entity, ANG4043, which retains in vitro binding affinity for the HER2 receptor and antiproliferative potency against HER2-positive BT-474 breast ductal carcinoma cells. Unlike the native mAb, ANG4043 binds LRP1 clusters and is taken up by LRP1-expressing cells. Measuring brain exposure after intracarotid delivery, we demonstrate that the new An2-mAb conjugate penetrates the BBB with a rate of brain entry (Kin) of 1.6 × 10(-3) mL/g/s. Finally, in mice with intracranially implanted BT-474 xenografts, systemically administered ANG4043 increases survival. Overall, this study demonstrates that the incorporation of An2 to the anti-HER2 mAb confers properties of increased uptake in brain endothelial cells as well as BBB permeability. These characteristics of ANG4043 result in higher exposure levels in BT-474 brain tumors and prolonged survival following systemic treatment. Moreover, the data further validate the An2-drug conjugation strategy as a way to create brain-penetrant biologics for neuro-oncology and other CNS indications.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Neoplasias Encefálicas/tratamento farmacológico , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Antineoplásicos/síntese química , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Feminino , Células MCF-7 , Camundongos , Camundongos Nus , Peptídeos/síntese química , Peptídeos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The blood-brain barrier contributes to brain homeostasis by controlling the access of nutrients and toxic substances to the central nervous system (CNS). The acquired brain endothelial cells phenotype results from their sustained interactions with their microenvironment. The endothelial component is involved in the development and progression of most CNS diseases such as brain tumors, Alzheimer's disease, or stroke, for which efficient treatments remain to be discovered. The endothelium constitutes an attractive therapeutical target, particularly in the case of brain tumors, because of the high level of angiogenesis associated with this disease. Drug development based on targeting differential protein expression in the vasculature associated with normal tissues or with disease states holds great potential. This article highlights some of the growing body of evidence showing molecular differences between the vascular bed phenotype of normal and pathological endothelium, with a particular focus on brain tumor endothelium targets, which may play crucial roles in the development of brain cancers. Finally, an overview is presented of the emerging therapies for brain tumors that take the endothelial component into consideration.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/citologia , Sistemas de Liberação de Medicamentos/métodos , Endotélio Vascular/citologia , Inibidores da Angiogênese/administração & dosagem , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , HumanosRESUMO
The concept of cancer prevention by use of naturally occuring substances that could be included in the diet is under investigation as a practical approach towards reducing cancer incidence, and therefore the mortality and morbidity associated with this disease. Tea, which is the most popularly consumed beverage aside from water, has been particularly associated with decreased risk of various proliferative diseases such as cancer and atherosclerosis in humans. Various studies have provided evidence that polyphenols are the strongest biologically active agents in green tea. Green tea polyphenols (GTPs) mainly consist of catechins (3-flavanols), of which (-)-epigallocatechin gallate is the most abundant and the most extensively studied. Recent observations have raised the possibility that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance. This article will review the effects and the biological activities of green tea catechins in relation to these mechanisms, each of which plays a crucial role in the development of cancer in humans. The extraction of polyphenols from green tea, as well as their bioavailability, are also discussed since these two important parameters affect blood and tissue levels of the GTPs and consequently their biological activities. In addition, general perspectives on the application of dietary GTPs as novel antiangiogenic and antitumor compounds are also presented.