RESUMEN
Increasing preclinical and clinical studies revealed that many tumor models had resistance to anti-VEGF-A and anti-VEGF-R2 therapies. Studies have shown that simultaneously blocked DLL4-Notch and VEGF signaling pathways can synergistically inhibit density and function of tumor blood vessels and reduce tumor growth rate. We successfully developed a bispecific monoclonal antibody (named HB-32) that targeting both human DLL4 and human VEGF. HB-32 showed high binding affinity to VEGF and DLL4. Furthermore, HB-32 inhibited proliferation, migration and tube formation of HUVEC. Finally, in vivo xenograft studies demonstrated that HB-32 inhibited proliferation of breast cancer cells (MDA-MB-231) and induced tumor cell apoptosis more efficiently than an anti-VEGF antibody or anti-DLL4 antibody alone. These findings indicate that our bispecific antibody provide a potential treatment for breast cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/aislamiento & purificación , Antineoplásicos/administración & dosificación , Antineoplásicos/aislamiento & purificación , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Inyecciones Subcutáneas , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The anti-human Delta-like 4 (DLL4) monoclonal antibody MMGZ01 has a high affinity to hrDLL4 and arrests the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, promotes immature vessels, and effectively reduces breast cancer cell growth in vivo. To develop a much more effective therapy, we conjugated MMGZ01 with two small-molecule cytotoxic agents, i.e., monomethyl auristatin E (MMAE) and doxorubicin (DOX), with different linkers to generate antibody-drug conjugates (ADCs), i.e., MMGZ01-vc-MMAE (named MvM03) and MMGZ01-GMBS-DOX (named MGD03), that are more potent therapeutic agents than naked antibody therapeutic agents. The produced anti-DLL4 ADCs can be effectively directed against DLL4 and internalized. Then, the release of MMAE or DOX into the cytosol can induce G2/M or G0/G1 phase growth arrest and cell death through the induction of apoptosis. In vitro, MvM03 was highly potent and selective against DLL4 cell lines. The anti-DLL4 ADCs, particularly MvM03, showed more potent anti-tumour activity than Docetaxel, which is an inhibitor of the depolymerisation of microtubules, in two xenograft breast cancer tumour models. Our findings indicate that anti-DLL4 ADCs have promising potential as an effective therapy for breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Inmunotoxinas/farmacología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Oligopéptidos/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Oligopéptidos/administración & dosificación , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ezrin is an important membrane/actin cytoskeleton linker protein, especially in epithelia. Ezrin has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin only after a conformational activation by phosphorylation at Thr567 of ezrin. The present experiments were undertaken to investigate the detailed cellular changes in the time course of expression of ezrin-T567 mutants (nonphosphorylatable T567A and permanent phospho-mimic T567D) in parietal cells and to assess ezrin distribution and its influence on the elaborate membrane recruitment processes of these cells. T567A mutant and wild-type (WT) ezrin were consistently localized to the apical plasma membrane, even with overexpression. On the other hand, T567D went first to apical membrane at early times and low expression levels, then accumulated mainly at the basal surface after 24 h. Overexpression of WT or T567A led to incorporation of internal membranes to apical vacuoles, while overexpression of T567D led to large incorporation of apical and intracellular membranes (including H-K-ATPase) to the basal surface. Differences in polar distribution of ezrin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections, forming the basis for a novel theory for ezrin as an organizer and regulator of membrane recruitment. A model simulating the cellular distribution of ezrin and its associated membrane- and F-actin-binding forms is given to predict redistributions observed with phosphorylation and mutant overexpression, and it can easily be modified as more specific information regarding binding constants and specific sites becomes available.
Asunto(s)
Comunicación Celular/fisiología , Polaridad Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Actinas/metabolismo , Actinas/fisiología , Animales , Línea Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/fisiología , Humanos , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/fisiología , Conejos , Factores de TiempoRESUMEN
In its dormant state, the membrane cytoskeletal linker protein ezrin takes on a NH(2) terminal-to-COOH terminal (N-C) binding conformation. In vitro evidence suggests that eliminating the N-C binding conformation by Thr(567) phosphorylation leads to ezrin activation. Here, we found for resting gastric parietal cells that the levels of ezrin phosphorylation on Thr(567) are low and can be increased to a small extent ( approximately 40%) by stimulating secretion via the cAMP pathway. Treatment of cells with protein phosphatase inhibitors led to a rapid, dramatic increase in Thr(567) phosphorylation by 400% over resting levels, prompting the hypothesis that ezrin activity is regulated by turnover of phosphorylation on Thr(567). In vitro and in vivo fluorescence resonance energy transfer analysis demonstrated that Thr(567) phosphorylation opens the N-C interaction. However, even in the closed conformation, ezrin localizes to membranes by an exposed NH(2) terminal binding site. Importantly, the opened phosphorylated form of ezrin more readily cosediments with F-actin and binds more tightly to membrane than the closed forms. Furthermore, fluorescence recovery after photobleaching analysis in live cells showed that the Thr567Asp mutant had longer recovery times than the wild type or the Thr567Ala mutant, indicating the Thr(567)-phosphorylated form of ezrin is tightly associated with F-actin and the membrane, restricting normal activity. These data demonstrate and emphasize the functional importance of reversible phosphorylation of ezrin on F-actin binding. A novel model is proposed whereby ezrin and closely associated kinase and phosphatase proteins represent a motor complex to maintain a dynamic relationship between the varying membrane surface area and filamentous actin length.
Asunto(s)
Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Mucosa Gástrica/citología , Células Parietales Gástricas/metabolismo , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Transferencia Resonante de Energía de Fluorescencia , Técnicas In Vitro , Fosforilación , Conformación Proteica , ConejosRESUMEN
ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF(4) and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis-resistant ARF6(Q67L) resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Movimiento Celular/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Membranas Intracelulares/metabolismo , Factor 6 de Ribosilación del ADP , Actinas/metabolismo , Compuestos de Aluminio/química , Sustitución de Aminoácidos , Membrana Celular/metabolismo , Citoesqueleto/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Fluoruros/química , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , ProteómicaRESUMEN
Phosphorylation of the membrane-cytoskeleton linker protein ezrin has been functionally linked to acid secretion and vesicle recruitment to the apical secretory membrane in gastric parietal cells. Phosphorylation of the conserved T567 residue of ezrin has been shown to alter the N/C oligomerization of ezrin and promote the formation of actin-rich surface projections in other cells. To test the importance of T567 as a regulatory site for ezrin in parietal cell activation, we incorporated wild-type (WT) and mutant forms of ezrin, including the nonphosphorylatable T567A mutation and a mutant mimicking permanent phosphorylation, T567D. All ezrin constructs included C-terminal cyan-fluorescent protein (CFP) and were incorporated into adenoviral constructs for efficient introduction into cultured parietal cells from rabbit stomach. Fluorescence microscopy was used to localize CFP-ezrin and monitor morphological responses. Accumulation of a weak base (aminopyrine) was used to monitor receptor-mediated acid secretory response of the cultured cells. Similar to endogenous ezrin, WT and T567A CFP-ezrin localized heavily to apical membrane vacuoles with considerably lower levels associated with the surrounding basolateral membrane. Interestingly, H,K-ATPase within cytoplasmic tubulovesicles was incorporated into the apical vacuoles along with WT and T567A mutant ezrin. In these parietal cells secretagogue stimulation produced a striking vacuolar expansion associated with HCl secretion and the secretory phenotype. Expression of T567D CFP-ezrin was quite different, being rarely associated with apical vacuoles. T567D was more typically localized to the basolateral membrane, often associated with long spikes and fingerlike projections. Moreover, the cells did not display secretagogue-dependent morphological changes and, to our surprise, H,K-ATPase was recruited to the T567D CFP-ezrin-enriched basolateral projections. We conclude that T567 phosphorylation, which is probably regulated through Rho signaling pathway, may direct ezrin to membrane-cytoskeletal activity at the basolateral membrane and away from apical secretory activity. The large basolateral expansion is predicted to recruit membranes from sources not normally targeted to that surface.
Asunto(s)
Polaridad Celular , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Fosfoproteínas/metabolismo , Treonina/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Extensiones de la Superficie Celular/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fenotipo , Fosfoproteínas/genética , Fosforilación , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vacuolas/metabolismoRESUMEN
The ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to regulated HCl secretion in gastric parietal cells. Here, we show that the integrity of ezrin is essential for parietal cell activation and provide the first evidence that ezrin interacts with PALS1, an evolutionarily conserved PDZ and SH3 domain-containing protein. Our biochemical study verifies that ezrin binds to PALS1 via its N terminus and is co-localized with PALS1 to the apical membrane of gastric parietal cells. Furthermore, our study shows that PALS1 is essential for the apical localization of ezrin, as either suppression of PALS1 protein accumulation or deletion of the PALS1-binding domain of ezrin eliminated the apical localization of ezrin. Finally, our study demonstrates the essential role of ezrin-PALS1 interaction in the apical membrane remodeling associated with parietal cell secretion. Taken together, these results define a novel molecular mechanism linking ezrin to the conserved apical polarity complexes and their roles in polarized epithelial secretion of gastric parietal cells.
Asunto(s)
Membrana Celular/metabolismo , Polaridad Celular , Proteínas de la Membrana/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Fosfoproteínas/metabolismo , Animales , Células Cultivadas , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Humanos , Proteínas de la Membrana/genética , Nucleósido-Fosfato Quinasa/genética , Fosfoproteínas/genética , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Gastric ezrin was initially identified as a phosphoprotein associated with parietal cell activation. To explore the nature of ezrin phosphorylation, proteins from resting and secreting gastric glands were subjected to two-dimensional SDS-PAGE. Histamine triggers acid secretion and a series of acidic isoforms of ezrin on two-dimensional SDS-PAGE. Mass spectrometric analysis of these acidic ezrin spots induced by stimulation suggests that Ser66 is phosphorylated. To determine whether Ser66 is a substrate of protein kinase A (PKA), recombinant proteins of ezrin, both wild type and S66A mutant, were incubated with the catalytic subunit of PKA and [32P]ATP. Incorporation of 32P into wild type but not the mutant ezrin verified that Ser66 is a substrate of PKA. In addition, expression of S66A mutant ezrin in cultured parietal cells attenuates the dilation of apical vacuolar membrane associated with stimulation by histamine, indicating that PKA-mediated phosphorylation of ezrin is necessary for acid secretion. In fact, expression of phosphorylation-like S66D mutant in parietal cells mimics histamine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution revealed a blockade of stimulation-induced proton pump mobilization in S66A but not S66D ezrin-expressing parietal cells. These data suggest that PKA-mediated phosphorylation of ezrin plays an important role in mediating the remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Parietales Gástricas/metabolismo , Fosfoproteínas/metabolismo , Adenosina Trifosfato/metabolismo , Aminopirina/farmacocinética , Animales , Transporte Biológico , Clonación Molecular , Proteínas del Citoesqueleto , Electroforesis en Gel Bidimensional , Liberación de Histamina , Células Parietales Gástricas/efectos de los fármacos , Fosforilación , Fosfoserina/metabolismo , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Serina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation.
Asunto(s)
Diacetil/análogos & derivados , Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Células Parietales Gástricas/citología , Células Parietales Gástricas/metabolismo , Seudópodos/metabolismo , Aminopirina/farmacocinética , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Células Cultivadas , Reactivadores de la Colinesterasa/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Citoesqueleto/fisiología , Diacetil/farmacología , Ácido Gástrico/metabolismo , ConejosRESUMEN
Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Polaridad Celular , Células Parietales Gástricas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Activadoras de ras GTPasa , Animales , Membrana Celular/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Humanos , Células Parietales Gástricas/citología , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP cdc42/genéticaRESUMEN
Gastric gland stimulation triggers H(+),K(+)-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLO-permeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H(+),K(+)-ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H(+),K(+)-ATPase-containing tubulovesicles to apical plasma membrane.