RESUMO
The rapid expansion of molecular screening libraries in size and complexity in the last decade has outpaced the discovery rate of cost-effective strategies to single out reagents with sought-after cellular activities. In addition to representing high-priority therapeutic targets, intensely studied cell signaling systems encapsulate robust reference points for mapping novel chemical activities given our deep understanding of the molecular mechanisms that support their activity. In this chapter, we describe strategies for using transcriptional reporters of several well-interrogated signal transduction pathways coupled with high-throughput biochemical assays to fingerprint novel compounds for drug target identification agendas.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Genes Reporter , Ensaios de Triagem em Larga Escala , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Linhagem Celular , Células Cultivadas , Humanos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , TransfecçãoRESUMO
A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-ß1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-ß1 and T(reg) cell frequency as well as peripheral blood TGF-ß1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-ß1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3ß and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-ß1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-ß1 secretion as well as via inhibition of T cells' response to TGF-ß1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.
Assuntos
Flavanonas/farmacologia , Extratos Vegetais/farmacologia , Scutellaria/química , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Flavanonas/imunologia , Glioma/tratamento farmacológico , Glioma/imunologia , Glioma/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/imunologia , Glicogênio Sintase Quinase 3 beta , Interleucina-10/antagonistas & inibidores , Interleucina-10/imunologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Extratos Vegetais/imunologia , Folhas de Planta/química , Ratos , Transdução de Sinais , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Two immunocompromised patients with 2009 H1N1 influenza pneumonia had viral shedding for over 5 weeks despite therapy with oseltamivir. Declining or persistently low cycle threshold values noted on serial qualitative real-time reverse transcriptase PCR (rRT-PCR) of respiratory specimens implied increasing viral load and probable drug resistance. Oseltamivir resistance was later confirmed by pyrosequencing.