Chemopreventive and Antitumoral Properties of Resveratrol

Abstract

A great interest has grown during last decades about the use of natural dietary compounds to inhibit the onset of several human diseases, among them cancer. The study of preventive effects of phytochemicals respect to cancer incidence is called chemoprevention. A chemopreventive agent must be able to act in one or more steps of carcinogenesis blocking or suppressing the onset and progression of cancer. The stilbene resveratrol (3,5,4’-trans-hydroxystilbene) (RESV) has attracted a great interest as it could exert many potential beneficial effects in human health. In my host lab it has been previously described the capacity of RESV to re-establish Gap Junction Intercellular Communications (GJIC) in glioma cells, with a concomitant delay in the S-phase of cell cycle. Gliomas are highly malignant tumors owning a strong resistance to conventional therapies and the cells lost GJIC in the promotion/progression steps of carcinogenesis. So we wondered which kind of regulatory factors could be spread across cells after RESV treatment. During my PhD project we investigated the antiproliferative activity exerted by RESV and we demonstrated that its chemopreventive role could be in part explained through the interaction with Topoisomerase II-α (TOPO2) activity. In the first part we analysed the effects of RESV on cell cycle progression and DNA damage induction in glioma cells. At first we performed a cytofluorimetric biparametric assay in order to follow the phosphorylation of the histone H2AX (γ-H2AX) during cell cycle. γ-H2AX is a variant form of the histone H2A that is phosphorylated in response to DNA double strand breaks (DSBs) induction. For this reason it is considered a hallmark of DSBs. We found a delay in the S-phase with a concomitant induction of γ-H2AX. These data have let us to suppose a putative interaction between RESV and TOPO2. TOPO2 is an enzyme mainly expressed in proliferating cells, from the S-phase, where it is involved in DNA supercoiling removal, to the G2/M phase, where it decatenates intertwined chromatids and drives a proper chromosome segregation. Through an in vitro decatenation test we demonstrated that RESV can affect TOPO2 activity. The inhibition of TOPO2 activity can occurs through two ways: the catalytic inhibition, that inactivates the enzyme without inducing DNA DSBs and the TOPO2-poisoning, that through the stabilization of the complex DNA-TOPO2, namely the “cleavage complex”, causes DNA DSBs induction. In the second part of my PhD project we investigated whether RESV could act as a TOPO2-poison in glioma cells. We performed a Docking Simulation and we observed in silico that RESV was able to act as bridge between DNA and TOPO2 establishing several hydrogen bonds with both molecules. Starting from this preliminary data we verified the stabilization of cleavage complexes by RESV in glioma cells too, through the In Vivo Complex of Enzyme Assay (ICE). Then we analysed the consequences of the TOPO2-poisoning in terms of DNA damage induction and DNA damage response signalling pathways activation. We performed the Cytokinesis-Block Micronucleus Assay (CBMN) in order to detect the DNA DSBs induction and we found that RESV caused a significant increase of micronuclei frequency. Afterwards we analysed the activation of factors involved in the DNA damage response pathway, namely ATM/ATR and their kinase effectors Chk2 and Chk1. We found that RESV activates ATM that in turn causes the activation of Chk2. As H2AX is one of the substrates of ATM, we analysed the phosphorylation of H2AX immediately after RESV treatment and 24 hours of recovery time. Our data showed that RESV causes the induction of γ-H2AX that persists until 24 hours. Surprisingly we didn’t observe any effect in the activation of ATR and Chk1. In the last part of my PhD project we focused the attention on the consequences of TOPO2-poisoning in CHO cells, that are normal but proliferating cells. In particular we analysed the effects of RESV treatment respect to chromosome stability, namely DNA damage induction and inhibition of chromosome segregation. We found an increase of polyploidy (POL) and endoreduplicated (ENDO) cells as a consequence of chromosome segregation inhibition exerted by RESV. Endoreduplication is a phenomenon that consists in a new DNA synthesis round without a cell division. Then we performed a CBMN assay and we found a significant increase of micronuclei frequency as previously shown in glioma cells. Interestingly the co-treatment with the TOPO2 inhibitor EtBr caused a significant reduction in micronuclei frequency, indicating that both RESV and EtBr are competitor for the binding to TOPO2. Moreover a CBMN assay with an anti-kinetocore staining allowed us to find out that RESV caused the production of micronuclei positive for kinetocore indicating the presence of a whole chromosome. So RESV not only induces DSBs but also causes chromosome loss. In order to analyse the effects of the inhibition of chromosome segregation respect to mitotic progression, we analysed the frequency of mitotic figures after RESV treatment. We found an accumulation of cells in the prometa/metaphase stage at the expense of ana/telophase one. The manifestation of chromosome segregation inhibition is the presence of anaphase bridges at the ana/telophase stage. We found a significant increase of anaphase bridges after treatment with the highest dose of RESV. On the whole our data confirm the chemopreventive role of RESV in cancer cells and emphasize a new aspect of this compound not investigated in the past. Indeed we showed here for the first time that RESV is able to poisoning TOPO2, inducing in this manner DNA DSBs and activating pathways involved in DNA damage signalling and cell cycle control in cancer cells. Moreover it affects chromosome stability at various levels in CHO cells as a consequence of TOPO2 poisoning. These evidences create a new insight into RESV landscape, supporting the idea that it could be utilised as chemopreventive and/or as an adjuvant of conventional therapies against cancer.