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1.
FASEB J ; 26(8): 3188-98, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22532440

RESUMO

Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR-3, CAOV-3; patient samples malignant 1 and 2) and resistant (DOV-13, SKOV-3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70-fold with malignant 2, 43-fold with OVCAR-3). Using agonists, antagonists, and shRNA-mediated knockdown of dsRNA receptors, we show that TLR3, RIG-I, and mda5 coordinated a caspase 8/9- and interferon-dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5- to 7.5-fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4-0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI=1.5-2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA-sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , RNA de Cadeia Dupla/uso terapêutico , Biomarcadores Tumorais/análise , Caspases/metabolismo , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Humanos , Interferon beta/metabolismo , NF-kappa B/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , RNA de Cadeia Dupla/efeitos dos fármacos , RNA Mensageiro/metabolismo
2.
Anal Biochem ; 407(2): 278-80, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20732291

RESUMO

We have demonstrated that an approach using guanidine hydrochloride at low concentrations to progressively disrupt protein-protein interactions can be quantitated using dynamic light scattering. This approach is sensitive enough to detect ligand-induced changes of subunit-subunit interactions for homo-hexameric glutamate dehydrogenase, allowing ΔΔG of reversible subunit dissociation to be calculated. The use of dynamic light scattering makes this approach generally applicable to soluble proteins to monitor the relative strength of protein-protein interactions with a particular emphasis on assessing the impact of ligand binding on such interfaces.


Assuntos
Glutamato Desidrogenase/química , Luz , Espalhamento de Radiação , Guanidina/química , Ligantes , Ligação Proteica , Desnaturação Proteica , Subunidades Proteicas/química
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