Resveratrol induces senescence-like growth inhibition of U-2 OS cells associated with the instability of telomeric DNA and upregulation of BRCA1

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Abstract

Resveratrol decreases cancer risk and improves health of laboratory animals. However, it can also promote genomic instability. Part of the beneficial activity of resveratrol may result from the activation of SIRT1 deacetylase. We examined how resveratrol influenced the growth of human cancer cell lines of different origin: osteosarcoma (U-2 OS) and lung adenocarcinoma (A549) and how it modulated the expression as well as the localization of key proteins, involved in DNA repair and cell cycle regulation. Resveratrol-induced growth arrest was associated with signs of stress-induced senescence. Differential expression of BRCA1, cyclin B1, pRb and p21 in U-2 OS and A549 cells indicates that resveratrol can engage various molecular mechanisms to arrest cell cycle progression. In subset of U-2 OS cells, the upregulated BRCA1 formed foci closely associated with WRN and the telomeric protein (TRF1). Moreover, resveratrol induced telomeric instability in U-2 OS cells and the activation of DNA damage signaling in both cell lines, manifested as the phosphorylation of histone H2AX at serine 139 and of p53 at serines 15 and 37. Our data are consistent with the hypothesis that resveratrol inhibits cell growth and induces senescence by altering DNA metabolism.

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.

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