Effect And Mechanism Of Resveratrol On Premature Senescence And Radiosensitivity In Lung Cancer Cells

BackgroudCellular senescence is a state of permanent cell cycle arrest that can be triggeredby a variety of stresses including DNA damage, telomere shortening and oxidativestress.Senescence limits the life span and proliferative capacity of cells, therefore theinduction of senescence is regarded as an important mechanism of cancer prevention.Notably, it has been suggested that therapy-induced senescence is an importantmechanism through which many anticancer agents including Ionizing radiation （IR）suppresses tumor cell growth. Moreover, our recent studies have revealed thatIR-induced tumor cell killing is largely attributed to the induction of senescence but notapoptosis in lung cancer cells, suggesting that the induction of premature senescencemay play a pivotal role in mediating the anticancer effects of chemotherapy andradiation therapy.Resveratrol (RV) is a natural component of red wine and grapes.It was reportedthat RV has many biological functions, such as antioxidant, antiviral, antibacterial,anti-inflammatory and anti platelet aggregation, therapy of cardiovascular. And it hasalso been shown to be a potential chemopreventive and anticancer agent. However, themolecular mechanisms underlying RV’s anticancer and chemopreventive effects areincompletely understood. Although previous studies have indicated that RV, at highdoses(100-200μM),can inhibit the proliferation of cancer cells by inducing apoptosis.Amajor challenge for which is that the concentration of RV required to induce apoptosisin tumor cells in vitro is too great to achieve in vivo in a clinical setting.While lowdoses (5-50μ M) RV which impact on cancer cell growth has not any definite reports.Radiotherapy is used in most of the patients during the course of cancer treatmentboth as a curative modality and for palliation. However, radioresistance is a majorobstacle to the success of radiation therapy and contributes significantly to tumor relapse and treatment failure. Therefore, there is a critical need for the development ofnovel radiosensitizers that can be used clinically to overcome tumor radioresistance andthus improve the efficacy of radiotherapy. Recent studies have indicated that treatmentwith RV can sensitize tumor cells to chemotherapeutic agents-or IR-induced cell death.However, the mechanisms by which RV increases the radiation sensitivity of cancercells remain to be determined. It is largely unknown whether and how RV treatmentmay affect IR-induced premature senescence in tumor cells.Therefore, the goal of this study was to determine whether low dose RV treatmentcould inhibit the growth of NSCLC cells through the induction of premature senescenceand whether RV treatment could sensitize NSCLC Cells to IR by increasing IR-inducedpremature senescence.Part I: Effect and mechanism of Resveratrol on PrematureSenescence in Lung Cancer Cells1. Effect of RV on the growth of lung cancer cellsIn order to determine if low dose RV treatment affects the growth of tumor cells.Both A549and H460lung cancer cells were treated with differently low doses of RV(5–100μM). MTS assay and colony formation assay were used to examine the effect ofRV treatment on the growth of NSCLC cells. The results from MTS assay indicatedthat RV at a concentration of5μM had no remarkedly inhibitory effect, compared withcontrol group（p＞0.05）, and that RV at the concentration from10to100μ M RVsignificantly inhibited lung cancer cell’s growth, which was RV dose-dependent. Andthe percentage of cell inhibition of100μM of RV was as high as98%. The results fromcolony formation assay demonstrated that even as low as10μM of RV treatment cansignificantly suppress the colony-forming activity of A549and H460cells, which wasdose-dependent. The results suggested that low dose RV inhibited the growth ofNSCLC cells in a dose-dependent manner. 2. The relationship between RV inhibiting cell growth and apoptosis inlung cancer cellsFlowcytometric sub-G1assays show that10-50μM RV does not induce anysignificant changes in apoptosis in both A549and H460cells,while100μM RV do alittle change.In contrast, we observed a significantincrease of G1arrest in RV treatedNSCLC cells. These results suggest that RV-induced cell killing in NSCLC cells islikely apoptosis-independent. Because of the activated casepase-3and cleaved PARPare well-documented measurements of apoptosis, to confirm the induction of apoptosisin lung cancer cells, activated caspase-3and cleaved PARP was assessed byWesternblotting. The results revealed that10-50μM RV treatment did not cause anysignificant changes in the expression of activated casepase-3and cleaved PARP ineither A549or H460cells. While A549cells treated with100μM RV had the sameeffection as CPT treatment,which resulted in a pronounced increase in activatedcasepase-3and cleaved PARP in both A549and H460cells.These results stronglysuggest that10-50μM RV inhibits NSCLC cell growth via an apoptosis-independentmechanism.3. The relationship between RV inhibiting cell growth and prematuresenescence in lung cancer cellsIncreased SA-β-gal activity is a well-established biomarker of senescence.SA-β-gal staining was explored to observe the effect of low dose RV treatment onpremature senescence in A549and H460cells. The results indicatedAfter RV treatment, there were significant changes in cell morphology, with thehigher dose RV treated, there were low the number of cells decreased, and form moreand more flat, the cells gradually increased in size that the numbers of SA-β-gal-positive cell markedly increased in RV-treated versus control A549or H460cells,which was RV dose-dependent. These results suggested that low dose RV inducedpremature senescence in NSCLC cells in a dose-dependent manner.The results from BrdU incorporation assays demonstrated that the senescent lung tumor cells lost thecapacity of synthesizing DNA and incorporating BrdU in response to RV treatment.Furthermore, Western blotting data demonstrated that the expression levels of p53andp21were significantly increased in RV-treated cells both in A549and H460cells,whilethe expression of EF1A were significantly down-regulates in RV-treated cells in A549and H460cells. Together, these results suggest that low dose RV could suppress cellgrowth and cell cycle progression by inducing premature senescence in NSCLC cells.4. Effect of low dose RV on the DNA damage and ROS production inlung cancer cellsPhosphorylated H2AX(γ-H2AX) is a robust marker of DNA damage. Todetermine if DNA damage contributes to RV-induced anticancer effects, γ-H2AX fociassay was used to examine whether RV treatment causes DNA damage in lung cancercells. The result demonstrated that RV treatment resulted in a significant increase in theformation of γ-H2AX foci in both A549and H460cells. As the formation of γ-H2AXfoci is an important surrogate of DNA damage.Neutral comet assays was used tomeasure DNA-DSBs in RV treated NSCLC cells. The results demonstrated that thenucleu was formed comet tail after RV treatment. And the higher dose, the longer tail,the longer comet tail moment and Olive tail moment. These results demonstrated forthe first time that the anticancer effect of low dose RV is attributed at least in part toRV-induced DNA damage in NSCLC cells. ROS play a critical role in mediatinggenotoxic stress-induced DNA damage. Therefore, we hypothesized that RV may causeDNA DSBs via increased ROS production in NSCLC cells. To test this hypothesis,DCF-DA staining and flow cytometric assay were used to investigate the effect of lowdose RV treatment on ROS production in NSCLC cells.The results showed thatcompared with control group, the levels of ROS had no markedly inhibitory effect in10μM RV treated group, the levels of ROS were significantly increased in20or50μMRV-treated A549and H460cells. These results suggest that RV may induce NSCLCcells premature senescence via ROS mediated DNA damage. 5. Effect of NAC on the DNA damage and premature senescenceinduced by low dose RVAlthough the data have shown that RV-induced DNA DSBs is associated withincreased ROS production in NSCLC cells, it has yet to be determined if inhibition ofROS production using antioxidants can prevent RV-induced DNA damage andpremature senescence. To this end, A549or H460cells were pre-incubated with NACprior to RV treatment to determine if NAC can attenuate RV-induced DNA damage andpremature senescence in NSCLC cells. The results demonstrate that pre-treatment withNAC significantly inhibits the formation of RV-induced γ-H2AX foci in A549andH460cells. Furthermore, results from SA-β-gal staining showed that the percentage ofRV-induced premature senescent cells is substantially reduced in NAC-treated cells.Taken together, these findings strongly support that low dose RV induces NSCLC cellpremature senescence via ROS-mediated DNA damage.6．The mechanism of ROS generation induced by low dose RV in lungcancer cellsTo better understand the mechasim by which RV induces ROS generation incancer cells, the impact of RV treatment on the expression of gene of Nox family wasobserved in NSCLC cells by Real-time RT-PCR. The results indicated that Nox1,2and5are abundantly expressed in both A549and H460cells, whereas Nox3and4are littledetected in lung cancer cells. Interestingly, our results revealed that RV treatmentselectively increases Nox5expression in both A549and H460cells, suggesting thatRV-induced ROS generation in these cells is likely attributed to increased Nox5expression.Given the important roles of antioxidant enzymes such as mitochondrialsuperoxide dismutase (SOD) and thioredoxin (TXN) in modulating intracellular ROSbalance, the effect off RV treatment on the expression of SOD and TXN wasdetermined in lung cancer cells. The real-time PCR data demonstrated that RV treatment only causes a modest increase (less than2-fold) in SOD2expression in A549cells, but has no effect on the expression of SOD1, SOD2and TXN mRNAs in H460cells. These data conformed that RV could induce ROS generation in lung cancer cellsthrough up-regulating Nox5expression.Together, these findings demonstrate that low dose RV treatment inhibits lungcancer cells growth via a previously unappreciated mechanism, namely the induction ofpremature senescence through ROS-mediated DNA damage.Summary1) Low dose （10-50μM） RV inhibits the growth of lung cancer cells via anapoptosis-independent mechanism.2) Low dose （10-50μM）RV induces premature senescence in lung cancer cells viaROS-mediated DNA damage.Part II: Effect and Mechanism of Resveratrol on radiosensitivityof lung cancer Cells1. The impact of low dose RV on the cell killing effect induced by IR inlung cancer cells.Previous studies showed that RV treatment could induce premature senescence inNSCLC cells. To investigate whether RV treatment could sensitize NSCLC cells to IR,both A549and H460cells were pre-incubated with RV (20μM) or DMSO as a vehiclecontrol for4h prior to exposure to different doses of IR treatment. Then clonogenicassays were used to determine the effect of RV treatment on IR-induced tumor cellkilling effect. The results showed that preincubation with RV significantly enhancedthe cell killing effects of IR both in A549cells and in H460cells. These results suggestthat RV increases the sensitivity of lung cancer cells to IR and is a potentialradiosensitizer.To further explore the mechanisms by which RV increases the radiation sensitivity of lung cancer cells, the effect of RV treatment on the expression ofactivated caspase-3and cleaved PARP was observed by Western blotting in irradiatedNSCLC cells. The results showed that RV treatment had little effect on the expressionof cleaved PARP and activated caspase-3in H460cells regardless of IR treatment. Incontrast, cisplatin (CPT) treatment resulted in a pronounced increase in the expressionlevels of cleaved PARP and activated caspase-3. These results indicated for the firsttime that RV enhanced IR-induced tumor cell killing effect via anapoptosis-independent mechanism.2.Effects of low dose RV on the premature senescence induced by IRin lung cancer cellsOur recent studies have shown that IR induces premature senescence in lungcancer cells in a dose-dependent manner, suggesting that the induction of senescenceplays an important role in IR-induced tumor suppression. However, it remains to bedetermined whether RV radiosensitizes lung cancer cells by augmenting IR-inducedpremature senescence. To this end, SA-β-gal staining was employed to detect senescentcells in irradiated A549and H460cells with or without RV treatment. The resultsshowed that the combination of RV and IR treatment induced more SA-β-gal-positivesenescent cells than either RV or IR treatment alone in NSCLC cells. Furthermore,Western blotting data demonstrated that there was more p53and p21expression in RVand IR-treated cells than in RV or IR-treated cells alone both in A549and H460cells.These results suggest that RV may radiosensitize lung cancer cells by enhancingIR-induced premature senescence.3. Effects of low dose RV on the DNA-DSBs induced by IR in lungcancer cellsGiven that DNA damage is a major cause underlying chemotherapy and ionizingradiation induced premature senescence, there was a hypothesized that RV treatment may enhance IR-induced senescence via increasing DNA damage in irradiated NSCLCcells. To test this hypothesis, γ-H2AX foci assay and neutral comet assays was used tomeasure DNA-DSBs in irradiated NSCLC cells with or without RV treatment. Theresults of γ-H2AX assay showed that both RV and IR treatment resulted in a significantincrease in the formation of γ-H2AX foci in both A549and H460cells, and thecombination of RV and IR treatment resulted in much more γ-H2AX foci formationthan either RV or IR treatment alone. The results of neutral comet assays demonstratedthat the combination of RV and IR treatment results in more DNA-DSBs than either IRor RV treatment alone. These results strongly supported that RV treatment couldpromote the induction of premature senescence in irradiated NSCLC cells by increasingIR-induced DNA-DSBs.4. Impact of RV on the phosphorylation of Akt and mTOR inirradiated lung cancer cellsIt has been shown that the inhibition of Akt activity sensitizes tumor cells toanticancer therapy. However, whether RV treatment affects the activities of Akt andmTOR remains to be determined in NSCLC cells. To address this issue, westernblotting analyses were performed to determine the expression levels of phosphorylatedAkt and phosphorylated mTOR in NSCLC cells. The results showed that RV treatmentsignificantly inhibited the expression levels of p-Akt and p-mTOR in irradiated H460cells. Moreover, the data also indicated that RV treatment resulted in a significantdecline in the phosphorylation of p70S6kinase (p-S6K, T389), a downstream target ofmTOR. Furthermore, the results showed that the combination of RV and IR treatmentlead to more markedly increases of phosphorylated p53and phosphorylated chk2levelsin irradiated NSCLC cells than in cells treated with IR or RV alone. These resultsfurther confirmed that RV treatment enhanced IR-induced DNA damage in NSCLCcells based on phosphorylation of p53and chk2are important biomarkers of DNAdamage. 5. Effect of low dose RV on ROS production in irradiated lung cancercellsPrevious studies have shown that ROS plays a critical role in modulatinggenotoxic stress-induced DNA damage and that DNA damage is able to inducepremature senescence in NSCLC cells. However, it remains to be determined whetherthe generation of ROS is involved in mediating the radiosensitization effect of RV. Toaddress this question, the levels of ROS were examined in lung cancer cells usingDCF-DA staining along with flow cytometric analyses. The results showed that RVtreatment markedly increases ROS production in irradiated NSCLC cells comparedwith those cells treated with RV or IR alone. These results suggested that ROS coludplay a pivotal role in RV-induced radiosensitization.6. Effect of NAC on the radiosensitization mediated by low dose RV inlung cancer cellsTo determine the role of ROS in RV-mediated radiosensitization, the effect ofantioxidant NAC on RV-mediated enhancement of IR-induced DNA damage andpremature senescence was investigated in NSCLC cells. Comet assays indicated thatpreincubation with NAC attenuates the enhancement effect of RV on IR-inducedDNA-DSBs. Furthermore, SA-β-gal staining assays revealed that inhibition of ROSproduction by NAC markedly diminished the enhancement effect of RV on IR-inducedsenescence in lung cancer cells.Together, these findings suggested that RV treatment could enhance IR-inducedpremature senescence via increasing ROS-mediated DNA damage in NSCLC cells.Summary1) Low dose（10-50μM） RV enhances IR-induced cell killing in lung cancer cells viaan apoptosis-independent mechanism. 2) Low dose（10-50μM） RV enhances IR-induced premature senescence via increasingROS-mediated DNA damage in lung cancer cells.Conclusion1. Low dose(10-50μM) RV induces premature senescence in NSCLC cells viaROS-mediated DNA damage.2. Low dose（10-50μM）RV sensitizes NSCLC cells to radiotherapy via increasingIR-induced premature senescence.