Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction
Introduction
Resveratrol (3,4′,5-trihydroxystilbene) is a well known polyphenol synthesized by a wide variety of plant species in response to injury, UV irradiation and fungal attack. Since Jang et al. [1] showed the antitumoral potential of resveratrol (RSV) in vivo, many studies have revealed a variety of resveratrol intracellular targets whose modulation gives rise to overlapping responses that lead to growth arrest and death. In particular it was found to arrest proliferation in many cancer cell models mostly in an irreversible way, leading to apoptosis [2]. Recently we showed that RSV in combined treatment with X-rays both induces a dose-dependent inhibition in cell cycle progression, particularly due to a delay during the S phase, and increases intercellular communication in human glioblastoma cells [3].
In any case, the efficacy of RSV is still debated because of the multiplicity of affected targets and contradictory effects related to dose and time of treatment and to cellular phenotype [4], [5]. One of the most interesting issues is the involvement of RSV in the maintenance of genomic stability through physical and chemical interactions with DNA, but also in influencing the redox state of cells. The DNA cleavage activity of RSV was enhanced in the presence of high levels of copper ions [6], but we showed that it does not appear to be able “per se” to induce primary DNA damage in mammalian cells [7]. On the other hand, RSV could also affect some aspects of DNA metabolism such as DNA repair, recombination and chromatin structure maintenance [8], [9] thereby indirectly modulating the integrity of genomic DNA.
Here we report that RSV treatment in human glioblastoma cells causes a dose-dependent cell cycle delay with a concomitant increase of histone H2AX phosphorylation. Being that this expression is linked to the increase of DNA double-strand breaks [10], we assayed the ability of RSV to interact with human topoisomerase II.
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Cell cultures and treatments
The human glioblastoma cell line U87-MG was purchased from the American Type Culture Collection (ATCC, USA) and was grown in DMEM cell culture medium with 10% FBS, penicillin/streptomycin and 2 mM l-glutamine, at 37 °C and 5% CO2. Cells, at exponential phase of growth, were always treated for 48 h with 20, 40 or 80 μM of resveratrol (Sigma) in immunofluorescence and flow cytometry experiments.
Immunofluorescence of H2AX nuclear foci
U87-MG were grown on μ-Slide 8 well (Ibidi) and treated with resveratrol. Immediately after treatment,
Results
To perform our experimental design, we selected three doses of RSV (20, 40 and 80 μM) on the basis of our previous study [3], where we showed that the cell survival, as measured by MTT assay, remained high (>90%) in U87 cells treated with RSV doses up to 80 μM; decreased cell survival was observed with increasing concentrations, starting from 160 μM.
Our first observation at confocal microscopy was the detection of numerous cells presenting γH2AX foci when comparing U87-MG cells treated with RSV to
Discussion
Malignant gliomas are the most common primary brain tumours in the central nervous system. Despite advances in surgical intervention and radio-treatment, the prognosis of this disease remains poor. Therefore, developing novel strategies are essential in order to improve effectiveness of treatments for this disease. In recent years, many natural compounds contained in the diet have been identified as potential chemopreventive agents and/or adjuvant in conventional cancer treatment. Among them
Conflicts of interest statement
None declared.
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These authors contributed equally to the study.