title: Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism.
abstract: Acute promyelocytic leukemia (APL) is a type of cancer, in which immature cells called promyelocytes proliferate abnormally. Human NB4 cell line appears to be a suitable in vitro model to express the characteristics of APL. In this work, we have investigated the effects of esculetin, a coumarin derivative with antioxidant properties, on the viability, the induction of apoptosis and the expression of apoptotic factors in NB4 cells. Cells treated with esculetin at several concentrations (20-500 μM) and for different times (5-24 h) showed a concentration- and time-dependent viability decrease with increased subdiploid DNA production. Esculetin inhibited cell cycle progression and induced DNA fragmentation. Moreover, annexin-V-FITC cytometry assays suggested that increased toxicity is due to both early and late apoptosis. This apoptosis process is be mediated by activation of caspase-3 and caspase-9. Treatments with progressively increasing concentrations (from 100 μM to 500 μM) of esculetin produced a reduction of Bcl2/Bax ratio in NB4 cells at 19 h, without affecting p53 levels. Proapoptotic action of esculetin involves the ERK MAP kinase cascade since increased levels of phosphorylated ERK were observed after those treatments. Increments in the levels of phosphorylated-Akt were also observed. Additionally, esculetin induced the loss of mitochondrial membrane potential with a release of cytochrome c into the cytosol which starts at 6 h of treatment with esculetin and increases up to 24 h. Esculetin induced an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an observed increase at 2-4 h of treatment. No significant changes in NO production was observed. Esculetin reduced the GSH levels in a time-dependent manner. In summary, the present work shows the cytotoxic action of esculetin as an efficient tool to study apoptosis mechanism induction on NB4 cell line used as a relevant model of APL disease.
authors.affiliation: Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Rubio
authors.affiliation: Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Calviño
authors.affiliation: Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: García-Pérez
authors.affiliation: Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain.
authors.forename: Angel
authors.initials: A
authors.lastname: Herráez
authors.affiliation: Unidad de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Facultad de Medicina y Ciencias de la Salud, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid), Spain. Electronic address: josecarlos.diez@uah.es.
authors.forename: José C
authors.initials: JC
authors.lastname: Diez
mesh_terms: D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D017382:Reactive Oxygen Species; D013997:Time Factors; D014468:Umbelliferones / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species; D014468:Umbelliferones; C007628:esculetin
keywords: Apoptosis; Caspases; Intracellular kinases; Leukemia; Peroxides; Superoxides