RESUMO
Despite the widespread availability of immunohistochemical and other methodologies for screening and early detection of lung and breast cancer biomarkers, diagnosis of the early stage of cancers can be difficult and prone to error. The identification and validation of early biomarkers specific to lung and breast cancers, which would permit the development of more sensitive methods for detection of early disease onset, is urgently needed. In this paper, ultra-small and bright nanoprobes based on quantum dots (QDs) conjugated to single domain anti-HER2 (human epidermal growth factor receptor 2) antibodies (sdAbs) were applied for immunolabeling of breast and lung cancer cell lines, and their performance was compared to that of anti-HER2 monoclonal antibodies conjugated to conventional organic dyes Alexa Fluor 488 and Alexa Fluor 568. The sdAbs-QD conjugates achieved superior staining in a panel of lung cancer cell lines with differential HER2 expression. This shows their outstanding potential for the development of more sensitive assays for early detection of cancer biomarkers.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias Pulmonares/metabolismo , Pontos Quânticos , Receptor ErbB-2/metabolismo , Anticorpos/química , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Técnicas de Cocultura , Citometria de Fluxo , Corantes Fluorescentes/química , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Microscopia ConfocalRESUMO
A major neuropathological hallmark of Alzheimer's disease (AD) is the deposition of aggregated ß amyloid (Aß) peptide in the senile plaques. Aß is a peptide of 38-43 amino acids and its accumulation and aggregation plays a key role early in the disease. A large fraction of ß amyloid is N-terminally truncated rendering a glutamine that can subsequently be cyclized into pyroglutamate (pE). This makes the peptide more resistant to proteases, more prone to aggregation and increases its neurotoxicity. The enzyme glutaminyl cyclase (QC) catalyzes this conversion of glutamine to pE. In brains of AD patients, the expression of QC is increased in the earliest stages of pathology, which may be an important event in the pathogenesis. In this study we aimed to investigate the regulatory mechanism underlying the upregulation of QC expression in AD. Using differentiated SK-N-SH as a neuronal cell model, we found that neither the presence of Aß peptides nor the unfolded protein response, two early events in AD, leads to increased QC levels. In contrast, we demonstrated increased QC mRNA levels and enzyme activity in response to another pathogenic factor in AD, perturbed intracellular Ca(2+) homeostasis. The QC promoter contains a putative binding site for the Ca(2+) dependent transcription factors c-fos and c-jun. C-fos and c-jun are induced by the same Ca(2+)-related stimuli as QC and their upregulation precedes QC expression. We show that in the human brain QC is predominantly expressed by neurons. Interestingly, the Ca(2+)- dependent regulation of both c-fos and QC is not observed in non-neuronal cells. Our results indicate that perturbed Ca(2+) homeostasis results in upregulation of QC selectively in neuronal cells via Ca(2+)- dependent transcription factors. This suggests that disruption of Ca(2+) homeostasis may contribute to the formation of the neurotoxic pE Aß peptides in Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Aminoaciltransferases/metabolismo , Cálcio/metabolismo , Homeostase , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Aminoaciltransferases/genética , Humanos , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , Regulação para CimaRESUMO
A versatile and efficient functionalization strategy for polymeric nanoparticles (NPs) has been reported and successfully applied to PEGylated, biodegradable poly(alkyl cyanoacrylate) (PACA) nanocarriers. The relevance of this platform was demonstrated in both the fields of cancer and Alzheimer's disease (AD). Prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and subsequent self-assembly in aqueous solution of amphiphilic copolymers, the resulting functionalized polymeric NPs exhibited requisite characteristics for drug delivery purposes: (i) a biodegradable core made of poly(alkyl cyanoacrylate), (ii) a hydrophilic poly(ethylene glycol) (PEG) outer shell leading to colloidal stabilization, (iii) fluorescent properties provided by the covalent linkage of a rhodamine B-based dye to the polymer backbone, and (iv) surface functionalization with biologically active ligands that enabled specific targeting. The construction method is very versatile and was illustrated by the coupling of a small library of ligands (e.g., biotin, curcumin derivatives, and antibody), resulting in high affinity toward (i) murine lung carcinoma (M109) and human breast cancer (MCF7) cell lines, even in a coculture environment with healthy cells and (ii) the ß-amyloid peptide 1-42 (Aß(1-42)), believed to be the most representative and toxic species in AD, both under its monomeric and fibrillar forms. In the case of AD, the ligand-functionalized NPs exhibited higher affinity toward Aß(1-42) species comparatively to other kinds of colloidal systems and led to significant aggregation inhibition and toxicity rescue of Aß(1-42) at low molar ratios.
Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Nanopartículas/uso terapêutico , Neoplasias/diagnóstico , Neoplasias/terapia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular Tumoral , Coloides , Cianoacrilatos/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Microscopia Confocal , Nanopartículas/química , Nanopartículas/toxicidade , Nanotecnologia , Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Polímeros/química , Receptores de Fatores de Crescimento/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
ß-Amyloid peptide (Aß) plays a key role in the pathogenesis of Alzheimer disease (AD). Monomeric Aß undergoes aggregation, forming oligomers and fibrils, resulting in the deposition of plaques in the brain of AD patients. A widely used protocol for fibril formation in vitro is based on incubation of the peptide at low pH and ionic strength, which generates Aß fibrils several microns long. What happens to such fibrils once they are brought to physiological pH and ionic strength for biological studies is not fully understood. In this investigation, we show that these changes strongly affect the morphology of fibrils, causing their fragmentation into smaller ones followed by their aggregation into disordered structures. We show that an increase in pH is responsible for fibril fragmentation, while increased ionic strength is responsible for the aggregation of fibril fragments. This behavior was confirmed on different batches of peptide either produced by the same company or of different origin. Similar aggregates of short fibrils are obtained when monomeric peptide is incubated under physiological conditions of pH and ionic strength, suggesting that fibril morphology is independent of the fibrillation protocol but depends on the final chemical environment. This was also confirmed by experiments with cell cultures showing that the toxicity of fibrils with different initial morphology is the same after addition to the medium. This information is of fundamental importance when Aß fibrils are prepared in vitro at acidic pH and then diluted into physiological buffer for biological investigations.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Amiloide/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Concentração Osmolar , Fragmentos de Peptídeos/ultraestrutura , Estabilidade Proteica , Espalhamento de Radiação , Células Tumorais CultivadasRESUMO
We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility.
Assuntos
Actinas/química , Cortactina/química , Proteína Quinase C/química , Motivos de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Imuno-Histoquímica/métodos , Neuregulina-1/química , Fosforilação , Ligação Proteica , Família de Proteínas da Síndrome de Wiskott-Aldrich/químicaRESUMO
Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase. Using a mass spectrometric approach, we found that PKD phosphorylates cortactin on Ser 298 in the 6th cortactin repeat region and on Ser 348, right before the helical-proline rich domain of cortactin. We developed phosphospecific antibodies against these phosphorylated sequences, and used them as tools to follow the in vivo phosphorylation of cortactin by PKD. Examination of cortactin phosphorylation kinetics revealed that Ser 298 serves as a priming site for subsequent phosphorylation of Ser 348. Src, a well-known cortactin kinase, strongly potentiated the in vivo PKD mediated cortactin phosphorylation. This Src effect is neither mediated by pre-phosphorylation of cortactin nor by activation of PKD by Src. Phosphorylation of cortactin by PKD does not affect its subcellular localization, nor does it affect its translocation to podosomes or membrane ruffles. Moreover, there was no effect of PKD mediated cortactin phosphorylation on EGF receptor degradation and LPA induced migration. Taken together, these data establish cortactin as a novel PKD substrate and reveal a novel connection between Src and PKD.
Assuntos
Cortactina/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cortactina/imunologia , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Microscopia de Fluorescência , FosforilaçãoRESUMO
EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPbeta and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell-cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell-cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteína Quinase C/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Citoesqueleto , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Camundongos , Ésteres de Forbol/farmacologia , Fosforilação , Pseudópodes/metabolismo , Pseudópodes/ultraestruturaRESUMO
The protein kinase D (PKD) family is a novel group of kinases that are involved in the regulation of cell proliferation and apoptosis, and several other physiological processes. Hence, these enzymes are attractive targets for pharmacological intervention, but no specific PKD inhibitors are known. With this in mind, we have developed a high-throughput, non-radioactive enzyme-linked immunosorbent assay (ELISA) method to monitor the PKD activity with myelin basic protein (MBP) as substrate. We determined that MBP is phosphorylated by PKD on Ser-160 and that this phosphorylation can be quantified in ELISAs, by the use of phosphorylation site-specific antibodies. Antibodies were developed that are highly specific for the MBP peptide sequence surrounding the phosphorylated Ser-160. We show that our high-throughput kinase assay is useful not only for determining the cellular PKD activity but also to screen for PKD-inhibitory compounds. Our ELISA has advantages over the current radioisotope kinase assay in terms of simplicity and environmental safety.
Assuntos
Anticorpos/química , Inibidores Enzimáticos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Anticorpos/análise , Especificidade de Anticorpos , Proliferação de Células/efeitos dos fármacos , Colódio , DNA/biossíntese , Ensaio de Imunoadsorção Enzimática , Espectrometria de Massas , Membranas Artificiais , Proteína Básica da Mielina/química , Proteína Básica da Mielina/imunologia , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/isolamento & purificação , Especificidade por SubstratoRESUMO
Recent studies have demonstrated the importance of protein kinase D (PKD) in cell proliferation and apoptosis. Here, we report that in vitro cleavage of recombinant PKD1 by caspase-3 generates two alternative active PKD fragments. N-terminal sequencing of these fragments revealed two distinct caspase-3 cleavage sites located between the acidic and pleckstrin homology (PH) domains of PKD1. Moreover, we present experimental evidence that PKD1 is an in vitro substrate for both initiator and effector caspases. During doxorubicin-induced apoptosis, a zVAD-sensitive caspase induces cleavage of PKD1 at two sites, generating fragments with the same molecular masses as those determined in vitro. The in vivo caspase-dependent generation of the PKD1 fragments correlates with PKD1 kinase activation. Our results indicate that doxorubicin-mediated apoptosis induces activation of PKD1 through a novel mechanism involving the caspase-mediated proteolysis.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Doxorrubicina/farmacologia , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.