RESUMEN
4-O-methylascochlorin (MAC) is a derivative of ascochlorin, a prenyl-phenol compound antibiotic isolated from the fungus Ascochyta viciae. MAC induces caspase/poly (ADP-ribose) polymerase-mediated apoptosis in leukemia cells. However, the effects of MAC on autophagy in cancer cells and the underlying molecular mechanisms remain unknown. Here, we show that MAC induces autophagy in lung cancer cells. MAC significantly induced the expression of autophagy marker proteins including LC3-II, Beclin1, and ATG7. MAC promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream signalling proteins P70S6K and 4EBP1. The AMPK activator AICAR upregulated LC3-II expression through the AMPK/mTOR pathway similar to the effects of MAC. MAC-induced LC3-II protein expression was slightly reduced in AMPK siRNA transfected cells. MAC upregulated hypoxia-inducible factor-1α (HIF-1α) and BNIP3, which are HIF-1α-dependent autophagic proteins. Treatment with CoCl2 , which mimics hypoxia, induced autophagy similar to the effect of MAC. The HIF-1α inhibitor YC-1 and HIF-1α siRNA inhibited the MAC-induced upregulation of LC3-II and BNIP3. These results suggest that MAC induces autophagy via the AMPK/mTOR signalling pathway and by upregulating HIF-1α and BNIP3 protein expression in lung cancer cells.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Terpenos/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Ascomicetos/química , Autofagia/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Asociadas a Microtúbulos/genética , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/genética , Terpenos/química , Activación Transcripcional/efectos de los fármacosRESUMEN
In this study, the plasma concentration profiles of tolterodine and its active metabolite, 5-hydroxymethyl tolterodine (5-HM tolterodine) were investigated in rats with tolterodine transdermal patches using liquid chromatography-tandem mass spectrometry. The plasma samples were extracted by a liquid-liquid extraction method, with an n-hexane/isopropyl alcohol mixture (9:1, v/v). Tiropramide was used as an internal standard (IS). Chromatographic separation was achieved using a C18 column (2.0 mm × 150 mm, 5 µm), with a mobile phase consisting of 5 mM ammonium acetate in distilled water/acetonitrile (50:50, v/v). The precursor-product ion pairs used for multiple reaction monitoring were m/z 326 â 284 (tolterodine), m/z 342 â 223 (5-HM tolterodine), and m/z 468 â 367 (IS). Subsequently, the plasma concentration levels of tolterodine and 5-HM tolterodine were measured in rat plasma after oral or transdermal administration of tolterodine and the pharmacokinetic parameters were calculated. The Cmax of the patch was less than that of the oral administration but their AUC values were comparable. The resulting data suggested that a transdermal dose of tolterodine 3 times higher (9 mg/12 cm2) could yield comparable efficacy to a 10 mg/kg oral dose in rats. These results would provide useful information on dose optimization of tolterodine transdermal patch for further clinical studies.