esculetin and Leukemia--Promyelocytic--Acute

Esculetin is a polyphenolic compound with cytoprotective properties. We previously demonstrated the induction of apoptosis by esculetin in NB4 human leukemia cells, as a model, by a mechanism not well understood. To analyse the antioxidant activity of esculetin on apoptosis, we have studied the influence of co-treatments of esculetin at a concentration of 100μM with exogenous ROS donors, namely tert-butyl-hydroperoxide and hydrogen peroxide, on NB4 cells. Esculetin (100μM) exerts a protective effect on cell viability and death necrosis or late apoptosis caused by the oxidant t-BHP whereas it potentiates decrease of cell viability and cell death caused by H

Topics: Antioxidants; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Hydrogen Peroxide; Leukemia, Promyelocytic, Acute; Oxidants; Oxidation-Reduction; Superoxides; tert-Butylhydroperoxide; Umbelliferones

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.

Topics: Antioxidants; Apoptosis; Blotting, Western; Cell Survival; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Membrane Potential, Mitochondrial; Reactive Oxygen Species; Time Factors; Umbelliferones