RESUMO
ß-Lapachone (ß-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with ß-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in ß-lap treated cells. ß-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitochondrial NADH dehydrogenase Nde2p was found to be resistant to ß-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by ß-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in ß-lap treated cells, suggesting that ß-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, ß-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to ß-lap and to elicit checkpoint responses. Remarkably, ß-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of ß-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.
Assuntos
Antineoplásicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Naftoquinonas/farmacologia , eIF-2 Quinase/metabolismo , Antineoplásicos/metabolismo , Dicumarol/farmacologia , Humanos , Immunoblotting , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales , Sais de Tetrazólio , Tiazóis , TranscriptomaRESUMO
The eIF2α kinases integrate translation initiation rates with nutrient availability, thus allowing cells to adapt to nutrient scarcity. Recent evidence has uncovered new functions of these kinases in tumour cell biology, ranging from regulation of cell cycle progression, maintenance of genome stability, control of apoptosis, and cell survival under nutrient stress and hypoxia. Accordingly, active eIF2α kinases modulate the antineoplasic activity of several antitumour drugs, either by exacerbating their cytotoxic effect or by promoting chemoresistance. Understanding of eIF2α kinases molecular roles may provide mechanistic insights into how tumour cells sense and adapt to nutrient restriction, thus helping to implement more effective approaches for cancer chemotherapy.