Resveratrol Sequentially Induces Replication And Oxidative Stresses To Drive P53-CXCR2 Mediated Cellular Senescence In Cancer Cells

Resveratrol(RSV)is mainly found in grapes and red wine,also in some other plants and fruits,such as peanuts,cranberries,pistachios,blueberries and bilberries.Resveratrol provides a wide range of health benefits,including antiaging,protection of platelets,reduction of blood glucose and anticancer activities.In the recent years,these properties have been widely studied in cell and animal models.The antiaging effects of resveratrol has attracted extensive attention.The antioxidant property and the ability of activating silencing information regulator(SIRT1)may mediate the antisenescence effect of RSV.Antiaging effects have been reported in organisms ranging from yeast,nematodes to mammals.However,in recent years,there were also studies showing that resveratrol can induce senescence of tumor cells via increasing ROS and inducing DNA damage.Therefore,the exact effects and the underlying mechanisms of RSV remains to be fully understood.There are conflicting reports on whether RSV displays antioxidative or prooxidative activity.RSV is mainly considered as a strong antioxidant due to its ability to reduce the production of ROS and to up-regulate the efficacy of the antioxidative systems,including reduced glutathione and superoxide dismutase.This notion,however,was challenged by numerous studies on cancer cells in which RSV was found to increase oxidative stress.RSV was also shown to induce premature senescence in endothelial cells,which was accompanied by increased generation of ROS.What regulates this "oxidative duality," of RSV remains to be clarified.In addition,many studies have shown that resveratrol play an anti－inflammatory role by inhibiting the activity of NF-κB and NLRP3.However,RSV was also reported to promote type II collagen and COX-2 expression via the ERK,p38 and Akt signaling pathways in rabbit articular chondrocytes.It is unclear what dictates RSV to exhibit pro-inflammatory or anti-inflammatory effect.Inflammation and oxidative stress are closely related and tightly linked pathophysiological processes.However,whether the proinflammation effect of RSV is dependent on its pro-oxidative effect is not clear.Therefore,resveratrol may exhibit anti-senescence,antioxidant and anti-inflammatory effect in some contexts,but exert opposite effects in others.This study addressed the senescence-inducing effect of RSV on cancer cells and the underlying mechanisms.The results are presented in the following five parts:PART ONERSV induces S phase arrest,DNA damage response and cellular senescenceRSV has been shown to inhibit the proliferation of a variety of cancer cells.We first subjected human osteosarcoma cells U20S to various concentrations of RSV for 48h and determined their effect on cell viability.RSV inhibited the proliferation of U20S cells in a dose-dependent manner.Analysis of cell cycle distribution by flow cytometry indicated that RSV dose-dependently induced S-phase arrest.There was a remarkable increase in the amount of γ-H2AX,a marker for DNA double-strand breaks,in RSV-treated cells,as revealed by immunofluorescence.Meanwhile,ATM,a master regulator of DNA damage response,was activated by RSV.In addition,typically of cells experiencing replication stress,the level of phosphorylated Chk1 level was increased.It appeared that the cells arrested at S-phase could progress to senescence,as the majority of cells remained in S-phase when they became senescent.PART TWOResveratrol sequentially induces replication and oxidative stressesRSV was reported to function as an inhibitor of ribonucleotide reductase and to be more potent than hydroxyurea,a commonly used inducer of replication stress,in inhibiting the activity of mouse ribonucleotide reductase.RSV was indeed capable of inducing S phase arrest.Because oncogene-induced senescence is mediated by replication stress and can be rescued by exogenous nucleosides.We speculated that if cellular senescence induced by RSV was mediated by replication stress,nucleosides should also be able to rescue the senescence.We tested this on U20S cells and indeed found that induction of DNA damage by RSV was significantly attenuated by nucleosides.Furthermore,nucleosides greatly rescued the stalled replication,as shown by the increased EdU incorporation.In keeping with the reduction in DNA damage,p53 activation was attenuated by nucleosides.Importantly,RSV-induced senescence was greatly reduced by nucleosides.We tested the inhibitory effect of nucleosides on RSV-induced senescence in two additional cell lines HT1080(fibrosarcoma)and A549(lung cancer)and obtained similar results.These results suggest that replication stress mediates RSV-induced senescence and the senescence can be effectively rescued by exogenous nucleosides.Oxidative stress was also shown to be a key mediator of RSV-induced senescence in HCT116 colon cancer cells and human endothelial cells in previous studies.We found the ROS levels were significantly elevated by RSV and RSV-induced senescence was significantly attenuated by antioxidant NAC.These results indicate that oxidative stress mediates RSV-induced senescence.Since both replication and oxidative stresses mediate RSV-induced senescence,we next examined the kinetics of two types of stress following RSV treatment.While S-phase arrest was noticeable at 12h,elevation of ROS was detected only at 36 h after RSV treatment.Furthermore,co-treatment with NAC for 24h was unable to rescue the S-phase arrest,though a noticeable rescuing effect was detected at 72h.We get similar results In the HT1080 cells.These results indicate that S-phase arrest occurs before the increase in ROS.Upregulation of NADPH oxidases 1 and 4 by RSV was reported to be responsible for senescence in human endothelial cells.We treated the cells with NOX1/4 inhibitor GKT137831(GKT).GKT significantly attenuated the induction of ROS by RSV,but did not completely abolish the pro-oxidant effect of RSV.We next determined the expression of genes encoding various NADPH oxidases by quantitative PCR and observed a significant increase in NOX1 expression at 48h,but not at 24h.In addition,NOX2,NOX3 and NOX5 appeared to be upregulated by RSV,especially at 48h.NOX4 remained steady during the duration of RSV treatment.Together,these results suggest that oxidative stress as a mediator of cellular senescence occurs after cells already experience replication stress.We then tested whether RSV-induced oxidative stress is dependent on S-phase arrest.we arrested NHFs at G0/G1 phase by serum starvation for 48h and then applied RSV.The ROS levels were elevated by RSV in serum-starved cells,indicating that the ROS increase by RSV is not specific to S-phase arrested cells.There was also a significant increase in DNA damage,as reflected the increased percentage of y-H2AX positive cells.We also detected DNA damage response,as measured by ATM and Chk1 phosphorylation.The partial rescue of S-phase arrest by prolonged treatment of NAC also suggests that while the ROS increase is a relatively delayed response when compared to S-phase arrest,it may still function to reinforce or maintain replication stress in RSV-treated cells.When NHF and U20S cells were stressed by H2O2,RSV could actually reduce the level of ROS.These results suggest that RSV may either act as an antioxidant or pro-oxidant depending on experimental settings.PART THREEp53-mediated CXCR2 upregulation contributes to cellular senescenceWe previously showed that the upregulation of p53-CXCR2 axis plays a key role in stress-induced senescence in normal human fibroblasts and U20S cells.We found that CXCR2 expression was significantly increased by RSV in a dose-and time-dependent manner.However,after CXCR2 expression reached its peak level at day 5,it began to subside in the following days,p21 followed a similar pattern.We confirmed that the upregulation of CXCR2 following RSV treatment was dependent on p53,since the expression of CXCR2,like that of p21,in response to RSV was greatly reduced when p53 was depleted.Importantly,depletion of CXCR2 greatly attenuated RSV-induced senescence.Together,these results indicate that the p53-CXCR2 axis also functions to drive RSV-induced senescence.We also observed that H2O2-induced CXCR2 expression can be attenuated by RSV,indicating that while CXCR2 mediates RSV-induced cellular senescence at higher doses,its expression was attenuated by RSV under a different condition.We found that the percentage of apoptotic cells was significantly higher in CXCR2-depleted(shCXCR2)cells than that in control(shNeg)cells when treated with RSV.The induction of anti-apoptotic protein BCL2 by RSV was attenuated with the depletion of CXCR2.Depletion of CXCR2 by siRNA similarly caused more U20S cells to undergo apoptosis when compared to control.CXCR2 knockdown also rendered U20S cells and normal human fibroblasts more prone to apoptosis induced by hydrogen peroxide.Together,these results indicate that by driving cellular senescence,CXCR2 effectively protects cells from apoptosis.PART FOURNF-kB activation inhibits apoptosis and promotes cellular senescenceRSV has been reported to possess both anti-inflammatory and pro-inflammatory effects.We found that RSV(50 μM)increased the expression of IL-6,IL-8,IL-1β and COX-2 in U20S cells in a time-dependent manner,indicative of pro-inflammatory effect of RSV.We also found that NAC could attenuate resveratrol-induced expression of IL-8 and IL-1β in U20S cells,suggesting that oxidative stress mediates resveratrol-induced inflammation.In addition,RSV could activate NF-κB in a concentration-dependent manner.NF-κB inhibitor could attenuate the expression of inflammatory factors induced by RSV.We also tested the role of NF-κB in RSV-induced cellular senescence and found that the upregulation of BCL-2 and BCL-xL was attenuated when NF-κB was inhibited,which was accompanied by increased apoptosis.These results indicate that NF-κB may play an important role in mediating cellular senescence induced by RSV.