RESUMEN
Autophagy defects lead to the buildup of damaged proteins and organelles, reduced survival during starvation and infections, hypersensitivity to stress and toxic substances, and progressive neurodegeneration. Here we show that, surprisingly, Drosophila mutants lacking the core autophagy gene Atg16 are not only defective in autophagy but also exhibit increased resistance to the sedative effects of ethanol, unlike Atg7 or Atg3 null mutant flies. This mutant phenotype is rescued by the re-expression of Atg16 in Corazonin (Crz)-producing neurosecretory cells that are known to promote the sedation response during ethanol exposure, and RNAi knockdown of Atg16 specifically in these cells also delays the onset of ethanol-induced coma. We find that Atg16 and Crz colocalize within these neurosecretory cells, and both Crz protein and mRNA levels are decreased in Atg16 mutant flies. Thus, Atg16 promotes Crz production to ensure a proper organismal sedation response to ethanol.
Asunto(s)
Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Etanol/farmacología , Neuropéptidos/genética , Sistemas Neurosecretores/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Proteína 7 Relacionada con la Autofagia/deficiencia , Proteína 7 Relacionada con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/deficiencia , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Hipnóticos y Sedantes/farmacología , Neuropéptidos/metabolismo , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/metabolismo , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Transducción de Señal , Factores de TiempoRESUMEN
We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy.