Abstract
BackgroundPrevious study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells.MethodsThe growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (ΔΨm) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms.ResultsMethyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol.ConclusionTogether, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells.
Highlights
IntroductionWe investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells
Previous study reported that resveratrol has anti-tumor activity
Res induces apoptosis of U251 cells U251 cells were treated with Res at concentrations ranging from 0 to 300 μM for various lengths of time
Summary
We investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. Autophagy is a degradative process involving sequestration of cytoplasm and organelles into double-membrane vesicles that traffic the contents to lysosomes where recycling takes place [1,2,3]. It is a genetically programmed, evolutionarily conserved process, typically observed in hepatocytes after amino acid deprivation [4]. Persistent activation of autophagy can lead to programmed cell death [15,16]. The mechanisms by which autophagy differentially affects tumor cell survival remain to be uncovered
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