Abstract
The induction of senescence, an irreversible growth arrest, in cancer cells is regarded as a mean to halt tumor progression. The phytoalexin resveratrol (RV) is known to possess a variety of cancer-preventive, -therapeutic, and -chemosensitizing properties. We report here that chronic treatment with RV in a subapoptotic concentration induces senescence-like growth arrest in tumor cells. In contrast to the widely accepted antioxidant property of RV, we demonstrate that one causative stimulus for senescence induction by chronic RV is an increased level of reactive oxygen species (ROS). The ROS formed upon RV exposure include hydrogen peroxide and superoxide and originate largely from mitochondria. Consistently, co-incubation with the antioxidant N-acetyl cysteine interfered with RV-mediated reactivation of the senescence program. Molecular mediators on the way from increased ROS levels to the observed growth arrest include p38 MAPK, p53, and p21. Moreover, we provide evidence that RV-initiated replication stress, apparent by activation of the ataxia telangiectasia-mutated kinase pathway, is associated with increased ROS levels and senescence induction. This is the first report linking cell cycle effects with a pro-oxidant and pro-senescent effect of RV in cancer cells.
Highlights
ataxia telangiectasia-mutated (ATM) is a protein that is well known as a central mediator of responses to DNA double strand breaks and subsequent replication stress
Tel.: 43-14277-55993; Fax: 43-1-4277-55969; E-mail: elke.heiss@univie.ac.at. 2 The abbreviations used are: ATM, ataxia telangiectasia-mutated; RV, resveratrol; ROS, reactive oxygen species; HCT, human colon tumor; SA--gal, senescence-associated -galactosidase; H2DCF-DA, 2Ј,7Ј-dichlorodihydrofluorescein diacetate; WT, wild type; Membrane Potential (MMP), mitochondrial membrane potential; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; EGF, epidermal growth factor; NAC, N-acetyl-cysteine; NOX, NAD(P)H-dependent oxygenases; ATM is a protein that is well known as a central mediator of responses to DNA double strand breaks and subsequent replication stress
RV growth-arrested cells showed a decreased response to growth factors (Fig. 1C); control cells and RV growth-arrested HCT cells were serum-starved for 16 h and stimulated with EGF for 5 min. pERK activation was taken as readout for growth factor responsiveness
Summary
Antibodies, Reagents, and Cell Lines—All chemicals and reagents including resveratrol were purchased from. Cells were washed once with 0.2% bovine serum albumin/phosphate-buffered saline and incubated with 20 M 2Ј,7Ј-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probes/Invitrogen), which is cleaved by intracellular esterases and transformed to a fluorescent dye when oxidized at 37 °C for 30 min prior to immediate analysis by flow cytometry (FACSCalibur, BD Biosciences). The mean fluorescence of 10,000 analyzed cells (corrected for autofluorescence) of each treatment group was taken as a measure for the total ROS load. For determination of superoxide levels, cells were grown in 6-well plates, treated as indicated, and exposed to 20 ng/ml dihydroethidium, a dye oxidized by superoxide, in phosphate-buffered saline, 0.2% bovine serum albumin for the last hour of incubation. After a wash in phosphate-buffered saline, 0.2% bovine serum albumin, cells were immediately analyzed by flow cytometry (FACSCalibur). Analyses of the data were performed using the software GraphPad PRISM, Version 4.0 (GraphPad Software, San Diego, CA)
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