RESUMO
The involvement of unfolded protein response (UPR) activation in tumor survival and resistance to chemotherapies suggests a new anticancer strategy targeting UPR pathway. Arctigenin, a natural product, has been recently identified for its antitumor activity with selective toxicity against cancer cells under glucose starvation with unknown mechanism. Here we found that arctigenin specifically blocks the transcriptional induction of two potential anticancer targets, namely glucose-regulated protein-78 (GRP78) and its analog GRP94, under glucose deprivation, but not by tunicamycin. The activation of other UPR pathways, e.g., XBP-1 and ATF4, by glucose deprivation was also suppressed by arctigenin. A further transgene experiment showed that ectopic expression of GRP78 at least partially rescued arctigenin/glucose starvation-mediated cell growth inhibition, suggesting the causal role of UPR suppression in arctigenin-mediated cytotoxicity under glucose starvation. These observations bring a new insight into the mechanism of action of arctigenin and may lead to the design of new anticancer therapeutics.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Furanos/farmacologia , Glucose/metabolismo , Lignanas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 4 Ativador da Transcrição/efeitos dos fármacos , Fator 4 Ativador da Transcrição/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Tempo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-BoxAssuntos
Fratura de Colles/terapia , Manipulações Musculoesqueléticas , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.