Resveratrol induces senescence-like growth inhibition of U-2 OS cells associated with the instability of telomeric DNA and upregulation of BRCA1
Introduction
The resveratrol (trans-3,5,4′-trihydroxystilbene) has been intensely studied since the discovery of its cancer protective activity (Jang et al., 1997). Its anti-cancer role was initially associated with the prevention of oxidative damage to DNA. Later, it was observed that resveratrol could inhibit the growth of cancer cell lines arresting them at various stages of the cell cycle. The stage apparently depended on the identity of the cell line (Sgambato et al., 2001, Delmas et al., 2006 and refs. therein).
The understanding of the mechanisms by which resveratrol induces the cell cycle arrest and protects against cancer is far from complete. The resveratrol-induced cell cycle inhibition was associated with the upregulation of the well-known, cell cycle arresting proteins, e.g., p53, p21 (reviewed by Signorelli and Ghidoni, 2005) suggesting that resveratrol generated cell stress by, e.g., direct or indirect DNA damage induction. The chronic treatment of p53-positive cancer cell lines in culture with resveratrol resulted in ATM-dependent senescence that was associated with the redox stress (Heiss et al., 2007). The comet assay showed that long treatment with resveratrol induced slight DNA damage in a dose and time-dependant manner (Quincozes-Santos et al., 2007). On the other hand, resveratrol did not generate point or frameshift mutations in S. typhimurium reverse mutation assay (Matsuoka et al., 2001), however, it induced genetic instability that was manifested by increased frequency of micronuclei (Schmitt et al., 2002) and sister chromatid exchanges (Matsuoka et al., 2001). These observations appear paradoxical considering the initial report of chemo-preventive role of resveratrol against cancer (Jang et al., 1997). Moreover, recent experiments performed on mice indicated that resveratrol improved health and increased survival of the animals kept on high-calorie diet (Baur et al., 2006). These results were consistent with conclusions of previous studies showing that resveratrol extended the lifespan of unrelated species (S. cerevisiae, C. elegens, and D. melanogaster). This extension depended on the presence of a Sir2 protein that deacetylates histones and other proteins (Wood et al., 2004). Thus, part of the protective role of resveratrol may result from its ability to enhance the activity of the human homolog of Sir2 protein–SIRT1 (Howitz et al., 2003). Previously, we showed that SIRT1 overexpression induced the relocalization of the fluorescent-labeled WRN protein from nucleoli to the nucleoplasm (Vaitiekunaite et al., 2007). WRN protects against cancer and slows the aging in humans. Individuals having no functional WRN protein, due to the gene mutations, show premature aging symptoms starting from adolescence and increased risk for sarcomas and thyroid carcinomas, what results in early death (Kudlow et al., 2007).
The aforementioned observations prompted us to explore the biological and molecular consequences of the treatment of cancer cells with resveratrol. Considering our recent findings (Vaitiekunaite et al., 2007), we focused our attention on processes and molecules that are functionally related with the WRN protein.
Section snippets
Cell culture, treatment, analysis of DNA content and clonogenic assay
The U-2 OS (human osteosarcoma, ATCC) and A549 (human lung adenocarcinoma, ATCC) cells were grown at 37 °C/5% CO2 in Dulbecco's modified Eagle's medium (Sigma–Aldrich, St. Louis, MI) supplemented with 10% FBS (fetal bovine serum, Gibco-Invitrogen, Carlsbad, CA) and penicillin–streptomycin solution (Sigma–Aldrich, St. Louis, MI).
Unless otherwise mentioned, the cells were treated with 50 μM resveratrol for the indicated number of hours. The 200 mM stock solution of resveratrol (Sigma–Aldrich, St.
Results
The study was started with assessing the biological consequences of resveratrol treatment of two cancer cell lines, U-2 OS and A549. The results of the dose–response experiment measuring resveratrol's influence on cell growth are shown in Fig. 1. At 50 μM concentration resveratrol significantly inhibited the growth of both cell lines. We used this concentration in subsequent experiments. Next, we explored the cell cycle distribution of resveratrol treated cells (Fig. 2). After 24-h treatment,
Discussion
Our study, to the best of our knowledge, is the first showing that resveratrol induces aberrations of specific DNA sequences. In U-2 OS cells, the resveratrol treatment was associated with: (i) the enlargement of TRF1 nuclear bodies detected by immunocytochemical method, (ii) the amplification of telomeric DNA detected by in situ hybridization, (iii) the appearance of the micronuclei-like structures showing accumulation of telomeric DNA and (iv) the appearance of ECTR. These data indicate that
Acknowledgements
This work was supported by the Polish Ministry of Science and Higher Education grant nos. NN-401-214534 to MR and 2P05A/125/28 to DB. The technical assistance of Mrs. Iwona Matuszczyk and Dr. Magdalena Głowala is highly appreciated.
References (43)
- et al.
Ubiquitination and proteasomal degradation of the BRCA1 tumor suppressor is regulated during cell cycle progression
J. Biol. Chem.
(2004) - et al.
Chronic treatment with resveratrol induces redox stress- and ataxia telangiectasia-mutated (ATM)-dependent senescence in p53-positive cancer cells
J. Biol. Chem.
(2007) - et al.
Cellular dynamics and modulation of WRN protein is DNA damage specific
Mech. Ageing Dev.
(2005) - et al.
Sirtuin-mediated deacetylation pathway stabilizes Werner syndrome protein
FEBS Lett.
(2008) - et al.
Regulation of WRN protein cellular localization and enzymatic activities by SIRT1-mediated deacetylation
J. Biol. Chem.
(2008) - et al.
Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line
Mutat. Res.
(2001) - et al.
Homologous recombination and maintenance of genome integrity: cancer and aging through the prism of human RecQ helicases
Mech. Ageing Dev.
(2008) - et al.
The ATR pathway: fine-tuning the fork
DNA Repair (Amst)
(2007) - et al.
Resveratrol attenuates oxidative-induced DNA damage in C6 Glioma cells
Neurotoxicology
(2007) - et al.
Hormonal and genotoxic activity of resveratrol
Toxicol. Lett.
(2002)