Back
Acta Physiologica 2012; Volume 206, Supplement 693
Joint FEPS and Spanish Physiological Society Scientific Congress 2012
9/8/2012-9/11/2012
Santiago de Compostela, Spain
APOPTOTIC EFFECTS TRIGGERED BY GRAPE SEED EXTRACT ON HUMAN PROMYELOCYTIC LEUKAEMIA HL-60 CELLS
Abstract number: P104
Garrido1 M, Espino1 J, Gonzalez-Gomez2 D, Gamero-Samino3 E, Rodriguez1 AB, Pariente1 JA, Delgado4 J
1Physiology, University of Extremadura,
2Horticulture and Pomology, Technological Agri-food Institute (INTAEX),
3Enology, Technological Agri-food Institute (INTAEX),
4Microbiology, Technological Agri-food Institute (INTAEX)
Objectives:
The objective of this study was to analyse the mechanisms involved in the apoptotic effects induced by grape seed extract (GSE) on human promyelocytic leukaemia HL-60 cells.
Materials:
Grape seeds were powdered and bioactive compounds were extracted with water (70–80 ordm;C) for 3 h in Soxhlet. Caspase-3 and -9 activities were determined from cleavage of their respective specific fluorogenic substrate. Measurement of mitochondrial membrane depolarization was carried out by using the membrane-permeant, cationic dye JC-1. To detect mitochondrial permeability transition pore (mPTP) opening, cells were incubated with calcein-AM and cobalt chloride, thus resulting in mitochondrial localization of calcein fluorescence. Intracellular reactive oxygen species (ROS) generation was quantified with the non-fluorescent, cell-permeable probe dihydrorhodamine-123 (DHR-123). Propidium iodide (PI) exclusion was used to evaluate GSE-induced citotoxicity, since PI allows to discriminate between live (unstained) and dead (PI-stained) cells. Also, apoptotic cell death was analysed by redistribution of phosphatidylserine (PS) in the presence of PI.
Results:
Cleavage of its specific fluorogenic substrate showed activation of caspase-3 in GSE-treated HL-60 cells, the activation being dose-dependent and time-dependent. Moreover, activation of caspase-3 induced by GSE was accompanied by mitochondrial membrane depolarization and mPTP opening. Disruption of mitochondrial integrity caused by GSE treatment subsequently led to activation of caspase-9, and also produced a slight increase in ROS levels. Finally, cytotoxic effects elicited by GSE treatment ultimately resulted in higher rates of apoptotic cell death, as ascertained by redistribution of PS in the presence of PI.
Conclusions:
Our findings suggest that GSE induces apoptotic cell death in human leukaemic HL-60 cells, which seems to be dependent on mitochondrial damage.
Supported by MICINN-FEDER grant (BFU2010-15049). J. Espino is beneficiary of grant from MEC (AP2009-0753).
To cite this abstract, please use the following information:
Acta Physiologica 2012; Volume 206, Supplement 693 :P104