Original ContributionResveratrol delays replicative senescence of human mesothelial cells via mobilization of antioxidative and DNA repair mechanisms☆
Graphical Abstract
Highlights
► Resveratrol improves proliferative capacity of human peritoneal mesothelial cells. ► Resveratrol delays the onset of senescence in human peritoneal mesothelial cells. ► Resveratrol mobilizes antioxidative and DNA repair mechanisms.
Introduction
Replicative senescence is a phenomenon that limits growth of normal somatic cells in vitro and also, probably, in vivo [1]. There is broad agreement that senescence is a cell response to extensive and usually irreparable injury to telomeric and/or nontelomeric regions of the genome [2], [3]. Although numerous internal and external stimuli have been recognized to elicit cellular senescence [4], the oxidative stress—especially in terms of a deleterious activity of reactive oxygen species (ROS)—has been found to be the major one [5]. Since a key intracellular source of ROS are mitochondria, dysfunctional metabolism of these structures has been identified as a central element driving the development of the oxidative stress in cells undergoing replicative senescence [6], [7].
Resveratrol (3,4′,5-trihydroxy-trans-stilbene; RVT) is a naturally occurring polyphenolic phytoestrogen produced by a wide range of plants, including grapes, peanuts, and mulberries. This compound is acknowledged for its cardioprotective, anti-inflammatory, and anticancer properties (see [8] for review). In recent years RVT has attracted a great deal of attention also due to its antiaging activity. It has been observed that it is able to extend the lifespan of multiple model organisms, including Saccharomyces cerevisiae [9], Drosophila melanogaster [10], and Caenorhabditis elegans [11]. There is evidence that this effect may be associated with the activation of sirtuins which belong to the family of NAD(+)-dependent histone deacetylases and play an important role in the regulation of energy homeostasis, maintenance of genetic stability, and stress response [9], [12]. In contrast to well established life-prolonging effects of RVT in vivo, the results obtained from the in vitro studies are sparse and ambiguous. It has been found that RVT improves the proliferative capacity of mesenchymal stem cells [13] and endothelial progenitor cells [14], whereas in keratinocytes it exerts the opposite effect [15]. The conflicting results have also been shown with respect to its impact on replicative cell lifespan. Namely, in fibroblasts RVT increased the number of population doublings achieved before entry into senescence [16], while in endothelial cells (HUVECs), the cell lifespan was significantly diminished [17].
Interestingly enough, there is an ongoing debate on whether RVT displays antioxidative or prooxidative activity. In a classic view, RVT is considered as a strong antioxidant due to its ability to reduce the production of ROS [18] and up-regulate the efficacy of the antioxidative systems, including reduced glutathione [19] and superoxide dismutase [20]. This picture has been blurred, however, by the plethora of studies on cancer cells in which RVT was found to disrupt cellular antioxidant activities leading to the enhanced oxidative stress and concomitant growth inhibition and/or apoptosis [21], [22]. Also in endothelial cells, their premature senescence in response to RVT has been found to proceed with an increased generation of ROS [17]. Because of this “oxidative duality,” the issue of whether and how RVT modulates cellular oxidative stress during senescence is still poorly understood.
Taking the aforementioned into account we designed a project to deeply elucidate both the ambiguities concerning the activity of RVT in vitro, namely its effect on cell growth/senescence, and the oxidative cell balance. To this end we employed human peritoneal mesothelial cells (HPMCs)—the major cell population within the peritoneal cavity with a well-characterized mechanism of senescence [23], [24], [25]—and examined parameters such as the efficiency of DNA synthesis, the number of population doublings achieved, the expression of senescence marker (SA-β-Gal), the length of telomeres, the occurrence of apoptosis, the generation of ROS, the activity of antioxidants (superoxide dismutase; SOD), the magnitude of DNA damage (8-OH-dG, γ-H2A.X), the activity of DNA repair mechanism (DNA glycosylase I; hOgg1), and the metabolism of mitochondria (biogenesis, inner membrane potential).
Section snippets
Chemicals
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The tissue culture plastics were from Nunc (Roskilde, Denmark).
Approximately 99% pure RVT was obtained from Sigma-Aldrich Corp. A stock solution was prepared in dimethyl sulfoxide (DMSO) and diluted in a culture medium to desired final concentration. The concentration of DMSO was always 0.05% (v/v). During the experiments RVT was used at concentrations up to 10 μM which, as shown in the
Resveratrol improves the proliferation of young HPMCs
In order to examine the effect of RVT (ranging from 0.1 to 10 μM) on cell proliferation in vitro, the MTT assay performed on low-density cultures exposed to this stilbene for 24 h was employed. The results showed that under these conditions RVT clearly improved the pace at which HPMCs proliferate, and this effect was evident at concentrations of 0.1, 0.5, and 1 μM. The greatest growth acceleration, specifically by 29±9% (P<0.05), was recorded in the cells exposed to RVT at 0.5 μM (Fig. 1A).
Although
Discussion
In the present study we show that resveratrol—used at a low concentration of 0.5 μM—improves proliferative capacity and delays the onset of senescence in human peritoneal mesothelial cells in vitro. Furthermore we found evidence that this beneficial effect of RVT may be linked with the mobilization of antioxidative and DNA repair mechanisms, which is preceded by hormetic-like strong but temporary increase in the generation of ROS. Importantly, such a low concentration of RVT—taking into account
Conclusions
Our findings suggest that RVT used at a low concentration of 0.5 μM, despite the stimulation of ROS release, may globally be considered as an antioxidative agent, since the temporary induction of ROS may be the way by which the stilbene mobilizes cellular antioxidative and DNA repair mechanisms. It is very likely that that such a sequence of events elicited by 0.5 μM RVT underlies the improved proliferative capacity and delayed onset of replicative senescence in the primary cultures of
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