[CANCER RESEARCH 64, 1452–1459, February 15, 2004] Resveratrol Inhibits Drug-Induced Apoptosis in Human Leukemia Cells by Creating an Intracellular Milieu Nonpermissive for Death Execution Kashif Adil Ahmad,1 Marie-Veronique Clement,2 Ismail Muhamad Hanif,1 and Shazib Pervaiz1 Departments of 1Physiology and 2Biochemistry, Faculty of Medicine, National University of Singapore, Singapore ABSTRACT Efficient apoptotic signaling is a function of a permissive intracellular milieu created by a decrease in the ratio of superoxide to hydrogen peroxide and cytosolic acidification. Resveratrol (RSV) triggers apoptosis in some systems and inhibits the death signal in others. In this regard, the inhibitory effect on hydrogen peroxide-induced apoptosis is attributed to its antioxidant property. We provide evidence that exposure of human leukemia cells to low concentrations of RSV (4 – M) inhibits caspase activation, DNA fragmentation, and translocation of cytochrome c induced by hydrogen peroxide or anticancer drugs C2, vincristine, and daunorubicin. Interestingly, at these concentrations, RSV induces an increase in intracellular superoxide and inhibits drug-induced acidification. Blocking the activation of NADPH oxidase complex neutralized RSVinduced inhibition of apoptosis. Furthermore, our results implicate intracellular hydrogen peroxide as a common effector mechanism in druginduced apoptosis that is inhibited by preincubation with RSV. Interestingly, decreasing intracellular superoxide with the NADPH oxidase inhibitor diphenyliodonium reversed the inhibitory effect of RSV on drug-induced hydrogen peroxide production. These data show that low concentrations of RSV inhibit death signaling in human leukemia cells via NADPH oxidase-dependent elevation of intracellular superoxide that blocks mitochondrial hydrogen peroxide production, thereby resulting in an intracellular environment nonconducive for death execution. INTRODUCTION The effector components of apoptotic death signaling and their intricate networking have been unraveled during the past couple of decades (1–3). Consequently, it is now well established that depending on the level of activation of the initiator caspase, such as caspase-8, the death signal can recruit directly downstream effector caspases or engage the mitochondria with the resultant release of death amplification factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO (2, 4, 5). Death signaling by anticancer drugs generally relies on positive input from the mitochondria, as is evidenced by the resistance of tumor cells overexpressing the deathinhibitory protein Bcl-2 that is localized to the membranes of mitochondria, endoplasmic reticulum, and nucleus (6 – 8). Therefore, by implication, an intracellular milieu permissive for caspase activation/ activity and recruitment of mitochondria-derived amplification factors is critical for efficient apoptotic execution. To that end, we have demonstrated the critical role of cellular redox status in the regulation of death signaling (9 –12). Whereas an overwhelming accumulation of intracellular reactive oxygen species could create an oxidatively stressed environment leading to necrosis, a slight increase is a stimulus for cellular proliferation (13, 14). Pro-oxidant intracellular milieu is a hallmark of many tumor cells and is believed to endow tumor cells with a survival advantage over their normal counterparts (15, 16). Furthermore, we have demonstrated that a slightly elevated intracellular concentration of superoxide (O2Ϫ) inhibited apoptotic signaling, irrespective of the trigger (17, 18). Contrarily, our data and that of others have highlighted the critical role of intracellular H2O2 in rendering the cytosolic milieu permissive for efficient apoptotic execution (19 –21). Thenceforth, we hypothesize that a critical balance between intracellular H2O2 and O2Ϫ dictates the response of tumor cells to apoptotic stimuli (9, 10, 12), and any stimulus/signal that inhibits the ability of intracellular H2O2, triggered on drug exposure, to reduce the intracellular environment could potentially favor the acquisition of the resistant phenotype. One area of recent interest in cancer biology is the chemopreventive potential of natural products. Among the compounds being evaluated for their cancer inhibiting activity is a phytoalexin, resveratrol (RSV), found in grapes and wines and known for its diverse biological activities, including antioxidant property (22–24). We reported previously that the chemopreventive activity of RSV could be the result of its ability to induce apoptotic death in human leukemia and breast carcinoma cells (25). However, depending on the cell type and the concentration used, RSV has been shown to induce or inhibit cellular proliferation and death signaling (25–29). Our present study was stimulated by a recent report that H2O2-induced apoptosis was inhibited in the presence of RSV and the implication that this could be a function of its antioxidant activity (30). Given our recent findings that drug exposure of human leukemia cells resulted in H2O2-dependent apoptosis, we set out to investigate the mechanism by which RSV inhibited apoptotic signaling triggered by exogenous H2O2 or by exposure to three anticancer agents, namely, C2, vincristine, or daunorubicin, that induce apoptotic death in cancer cells (21). MATERIALS AND METHODS Determination of Cell Viability and DNA Fragmentation. Human promyelocytic leukemia (HL60) cell line was purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% S-penicillin. In a typical survival assay, HL60 cells (1 ϫ 105 cells/well) plated in 96-well plates were preincubated with RSV (4 – M) for h and then treated with 100 M of H2O2, 50 g/ml of C2, 1.25 g/ml of vincristine, or 0.2 g/ml of daunorubicin for 18 h. Cell survival was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously or by the -galactosidase (-gal) survival assay described below (31). Propidium iodide staining was performed for analyzing DNA fragmentation as described previously (25). Stained cells were analyzed by flow cytometry (Coulter EPICS Elite ESP; Beckman Coulter, Fullerton, CA) with the excitation and emission wavelengths at 488 nm and 610 nm, respectively. At least 10,000 events were analyzed by WinMDI software. Determination of Caspase-3 and -9 Activity. Caspase-3 and -9 activity was determined using the Bio-Rad fluorescent assay kit (Hercules, CA) for the Received 8/5/03; revised 12/9/03; accepted 12/10/03. two caspases. Cells (1 ϫ 106) were preincubated for h with RSV (4 – M) Grant support: Research grants from the NMRC, Singapore (R-185-000-032-213) to and then incubated for 12 h with 100 M H2O2, 50 g/ml of C2, 1.25 g/ml S. Pervaiz and from the BMRC, Singapore (R-185-000-048-305) to S. Pervaiz and Mof vincristine, or 0.2 g/ml of daunorubicin. Fifty l of 2ϫ reaction buffer V. Clement. with 10 mM DTT and l of the conjugate substrate (DEVD-AFC for The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with caspase-3 and LEHD-AFC for caspase-9) were added to cell lysates. Caspase 18 U.S.C. Section 1734 solely to indicate this fact. activity was determined by the relative fluorescence intensity at 505 nm Requests for reprints: Shazib Pervaiz, Department of Physiology, National Univerfollowing excitation at 400 nm with a spectrofluorometer (Tecan Group, Ltd., sity of Singapore, MD9, #03-06, Singapore 117597. Phone: 65-6874-6602; Fax: 65-6778Maennedorf, Switzerland). Results are shown as fold increase (ϫ-increase) in 8161; E-mail: phssp@nus.edu.sg. 1452 APOPTOSIS INHIBITORY ACTIVITY OF RESVERATROL activity relative to untreated cells (1ϫ). In addition, cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase (PARP), was assessed by Western blot analysis using anti-PARP (clone C-2–10; PharMingen, San Diego, CA) as described previously (9). Measurements of Intracellular O2؊ and pH. Intracellular O2Ϫ was assayed by a lucigenin-based chemiluminescence assay as described previously (11). Data are shown as percentage change (% change) in the intracellular O2Ϫ concentration compared with untreated cells and are the mean Ϯ SD of three independent measurements. For measurement of cytosolic pH, cells were loaded with 10 M 2Ј,7Ј-bis(2carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM; Sigma, St. Louis, MO), and the fluorescence ratio of 525:610 nm was used to derive cytosolic pH using a standard pH calibration curve as described previously (11). Flow Cytometric Analysis of Intracellular H2O2. Cells were exposed to the apoptotic triggers for –12 h, loaded with 5-(and-6)-chloromethyl-2Ј,7Јdichlorofluorescin diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR; M) (32) at 37°C for 15 min, and analyzed by flow cytometry (Coulter EPICS Elite ESP) using an excitation wavelength of 488 nm as described previously (21). At least 10,000 events were analyzed. Determination of Mitochondrial ⌬⌿m and Cytosolic Cytochrome c. Potential-sensitive probe 3,3Јdihexyloxacarbocyanine iodide was used to measure mitochondrial ⌬m as described previously (31). Briefly, ϫ 106 cells were incubated with 3,3Јdihexyloxacarbocyanine iodide (40 nM) for 15 at 37°C. As a positive control, cells were incubated separately with an uncoupling agent, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (100 M). At least 10,000 events were analyzed by flow cytometry with excitation set at 488 nm. Cytochrome c was detected in cytosolic extracts from 30 ϫ 106 cells by Western blot analysis using anti-cytochrome c (7H8.2C12; PharMingen) as described previously (33). Transient Transfection with pIRESRacN17 and -Gal Survival Assay. Transient transfections of CEM cells were performed using the SuperFect transfection reagents from Qiagen (Hilden, Germany), and survival of transfected cells was assessed by the -gal survival assay as described recently (34). Cell survival was calculated as [(-gal activity gϪ1 protein of transfected cells incubated with the apoptotic trigger)/(-gal activity gϪ1 protein of transfected cells incubated without the trigger)]. -Gal activity was measured using the Galacto-Star mammalian reporter kit (Applied Biosystems, Foster City, CA). Protein concentration was determined using the Coomasie Plus protein assay reagent from Pierce (Rockford, IL). Data Analysis. Data presented are mean Ϯ SD of at least three independent experiments performed in triplicate, unless otherwise indicated. Statistical significance was determined by the Student’s t test. RESULTS Low Doses of RSV Inhibit H2O2-Induced Apoptosis Upstream of the Mitochondria. Corroborating earlier findings on the deathinducing activity of H2O2 in cancer cells, exposure of HL60 cells to H2O2 (100 M) resulted in a significant decrease in cell survival (Fig. 1A). The cytotoxic activity of H2O2 was a function of activation of the apoptotic death pathway as evidenced by the significant increase in caspase activity, cleavage of the caspase-3 substrate PARP, and appearance of the sub-G1 fraction (Fig. 1, B–D). Interestingly, preincubation of cells with – M RSV for h before the addition of H2O2 for 18 h resulted in an increase in cell survival (Fig. 1A), significant inhibition of caspase-3 activity and PARP cleavage (Fig. 1, B and C), and inhibition of DNA fragmentation (Fig. 1D). Moreover, preincubation with RSV prevented H2O2-induced decrease in mitochondrial ⌬m (Fig. 2A) and significantly blocked H2O2-induced cytosolic translocation of cytochrome c from the mitochondria (Fig. 2B). One possible explanation for the observed inhibitory effect of RSV could be that RSV functioned as an efficient scavenger of H2O2. However, addition of RSV and H2O2 simultaneously to the culture medium (RSV/H2O2) had no effect on H2O2-induced cell death (Fig. 2C), whereas previous incubation with RSV for h (RSV ϩ H2O2) significantly (P Ͻ 0.05) inhibited apoptotic signaling (Fig. 2C). These data indicate that the death-inhibitory effect of RSV is not simply a function of its ability to scavenge H2O2 but involves mechanism(s) upstream of the mitochondria or signals that engage the mitochondrial death machinery. RSV Inhibits H2O2-Induced Decreases in Intracellular O2؊ and Cytosolic pH. We have shown that H2O2 induces a decrease in O2Ϫ and cytosolic acidification, thereby creating an intracellular milieu permissive for apoptotic execution (18, 34, 35). Therefore, we questioned if the inhibitory effect on H2O2-induced apoptosis was caused by the ability of RSV to create an intracellular environment noncon- Fig. 1. Preincubation with resveratrol (RSV) inhibits H2O2-induced caspase activation and DNA fragmentation in HL60 cells. A, HL60 cells (1 ϫ 106 cell/ml) were incubated with 100 M H2O2 for 18 h with or without preincubation for h with RSV (4 and M). Cell viability was determined by the MTT assay. B, caspase-9 and -3 activity was determined by fluorimetric assays, and (C) poly(ADP ribose) polymerase cleavage was assessed by Western blot analysis. D, DNA fragmentation was determined by propidium iodide staining and the appearance of sub-G1 fraction. 1453 APOPTOSIS INHIBITORY ACTIVITY OF RESVERATROL Fig. 2. Inhibitory effect of resveratrol (RSV) on H2O2-induced death signaling is upstream of the mitochondria. A, HL60 (1 ϫ 106) cells were exposed to 100 M H2O2 for h with or without previous incubation for h with RSV (8 M), and ⌬m was determined as described in “Materials and Methods.” B, cytosolic cytochrome c was assessed by Western blot analysis. Staurosporintreated HL60 cytosolic extract was used as positive control. C, HL60 cells (1 ϫ 106/ml) were incubated simultaneously with RSV (8 M) and H2O2 (100 M) for 18 h (RSV/H2O2) or preincubated for h with RSV (8 M) before the addition of 100 M H2O2 (RSV ϩ H2O2). Cell viability was determined by MTT assay. ducive for death execution. Preincubation with RSV not only induced a slight increase in intracellular O2Ϫ but also blocked the inhibitory effect of H2O2 on intracellular O2Ϫ levels (Fig. 3A). Interestingly, preincubation with RSV before the addition of H2O2 consistently resulted in higher intracellular O2Ϫ than RSV alone. In addition, the significant decrease in cytosolic pH (P Ͻ 0.01) triggered by H2O2 was inhibited by previous incubation with RSV (Fig. 3B). RSV-Induced Inhibition of Apoptosis Can Be Reverted by Blocking NADPH Oxidase Activation. Considering the reports on the antioxidant potential of polyphenolic compounds, such as RSV, we were intrigued by our observation that low concentrations of RSV induced an increase, rather than a decrease, in intracellular O2Ϫ concentration (30, 36 –38). Therefore, we investigated the effect of blocking NADPH oxidase complex, one of the major sources of intracellular O2Ϫ, on RSV-induced increase in O2Ϫ. Pharmacologic inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) completely blocked RSV-induced increase in intracellular O2Ϫ in HL60 cells (Fig. 3C). Furthermore, incubation of cells for h with DPI before the addition of RSV significantly (P Ͻ 0.004) restored the sensitivity of HL60 and CEM leukemia cells to H2O2 (Fig. 3D; data not shown). To provide additional evidence that this was a function of NADPH oxidase activation, CEM cells were transfected transiently with a dominant negative mutant of Rac (RacN17), which inhibits NADPH oxidase-dependent increase in intracellular O2Ϫ (34). Similar to DPI, transient transfection with RacN17 completely neutralized the death-inhibitory activity of RSV and restored the sensitivity of leukemia cells to H2O2 (Fig. 3E). RSV Inhibits Apoptosis Triggered by Anticancer Drugs C2, Vincristine, and Daunorubicin. In our earlier reports, we demonstrated that exposure of human leukemia and melanoma cells to C2, a purified photoproduct of MC540, triggered mitochondrial generation of H2O2 that was responsible for the release of cytochrome c and downstream activation of the caspase cascade (21). Similar to the data obtained with H2O2, preincubation of cells with RSV before the addition of C2 resulted in an increase in cell survival compared with the cells treated with C2 alone (Fig. 4A). Whereas exposure of leukemia cells to C2 resulted in robust increases in caspase-3 and -9 activity, preincubation with RSV significantly inhibited both caspases and the cleavage of the caspase-3 substrate PARP (Fig. 4B). That this inhibition was mediated via blocking mitochondria-dependent death signaling was evidenced by the significant decrease in cytosolic translocation of cytochrome c in cells preincubated with RSV before the addition of C2 (Fig. 4C). In addition, similar to the results obtained with H2O2, previous incubation with RSV (RSV ϩ C2) was required for the inhibitory effect of RSV on C2-induced death signaling, whereas simultaneous exposure to RSV and C2 (RSV/C2) had no effect on C2 signaling (data not shown). We next questioned whether the inhibitory effect of RSV on C2induced apoptosis also was mediated by its ability to (a) create a pro-oxidant intracellular milieu; and (b) inhibit cytosolic acidification. Results indicate that preincubation of HL60 cells with RSV inhibited H2O2 production triggered by exposure to C2 (Fig. 5A). Similar to the results obtained with H2O2, preincubation of HL60 cells with RSV for h resulted in an increase in intracellular O2Ϫ concentration, which was even more pronounced on subsequent addition of C2 (Fig. 5B). It should be noted that a slight increase in intracellular O2Ϫ also was observed on exposure of cells to C2 alone; however, unlike RSV, this was accompanied by a surge in intracellular H2O2 production. In addition, cytosolic acidification triggered on exposure to C2 was inhibited completely on previous exposure to RSV (Fig. 5C). Furthermore, incubation of cells with DPI before the addition of RSV and C2 resulted in a decrease of intracellular O2Ϫ (data not shown) and restored death signaling in response to C2 (Fig. 5D). To gain additional insight into the death-inhibitory effect of RSV and its potential clinical implications, we next investigated the effect of RSV on apoptosis induced by two chemotherapeutic agents, vincristine and daunorubicin (39). Corroborating the results obtained with H2O2 and C2, results indicate a dose-dependent (up to M) inhibitory effect of RSV on vincristine-induced cell death (Fig. 6A) together with significant inhibition of caspase-9 and -3 activity (Fig. 6B). That the apoptosis inhibitory effect of RSV was linked to its ability to create a pro-oxidant intracellular milieu was supported additionally by the ability of DPI or transient transfection with RacN17 to revert the sensitivity of leukemia cells in the presence of RSV to vincristine-induced apoptosis (Fig. 6, C and D). The inhibitory effect of low concentrations of RSV on druginduced apoptosis was not exclusive to C2 or vincristine. Preincubation of leukemia cells to RSV for h resulted in a significant increase in cell survival (Fig. 7A), inhibition of caspase activity (Fig. 7B), and inhibition of intracellular H2O2 production (Fig. 7C) induced by exposure to daunorubicin. 1454 APOPTOSIS INHIBITORY ACTIVITY OF RESVERATROL Fig. 3. Inhibition of NADPH oxidase activation prevents resveratrol (RSV)-induced increase in intracellular O2Ϫ and overcomes its death-inhibitory effect. A, cytosolic pH was determined with the pH-sensitive probe 2Ј,7Ј-bis(2-carboxyethyl)-5,6-carboxyfluorescein. B, ϫ 106 cells were treated with RSV (8 M) for h or H2O2 (100 M) for h with or without h of previous incubation with RSV (8 M). Intracellular O2Ϫ was measured by a lucigenin-based chemiluminescence assay. C, HL60 (2 ϫ 106) cells were incubated with M RSV for h in the presence or absence of DPI (1.25 M), and intracellular O2Ϫ was measured as described previously. D, HL60 cells (1 ϫ 106/ml) were preincubated with DPI (1.25 M) or with DPI (1.25 M) ϩ RSV (8 M) for h before the addition of 100 M H2O2 for 18 h. Cell survival was assessed by the MTT assay. E, CEM cells (1 ϫ 106/ml) cotransfected with pCMV--galactosidase (-gal) and pIRES-RacN17 were exposed to 100 M H2O2 for 18 h with or without previous incubation for h with M RSV. Cell survival was assessed by the -gal survival assay. Mean of two independent transfections done in duplicate is shown. 1455 APOPTOSIS INHIBITORY ACTIVITY OF RESVERATROL NADPH oxidase complex had no effect on intracellular H2O2 increase (21). Similar to the results reported with C2, preincubation of HL60 cells with DPI did not affect intracellular H2O2 production on exposure to vincristine and daunorubicin, thus strongly suggesting mitochondria as the cellular source on drug exposure (data not shown). More importantly, decreasing intracellular O2Ϫ with DPI restored the ability of C2, vincristine, and daunorubicin to trigger intracellular H2O2 production even in the presence of RSV (Fig. 7C). DISCUSSION Fig. 4. Resveratrol (RSV) inhibits apoptosis triggered by a novel anticancer drug, C2. A, HL60 cells (1 ϫ 106/ml) were incubated simultaneously with RSV (8 M) and C2 (50 g/ml) for 18 h (RSV/C2) or preincubated for h with RSV (8 M) before the addition of C2 (RSV ϩ C2). Cell viability was determined by MTT assay. B, activity of caspase-3 and -9 was determined by fluorimetric assays. Poly(ADP ribose) polymerase (PARP) cleavage and (C) cytosolic cytochrome c were detected by Western blot analyses. Decrease in Intracellular O2؊ Overrides the Inhibitory Effect of RSV on Drug-Induced H2O2 Production. Thus far, we have shown that the inhibitory activity of RSV on drug-induced apoptosis was linked to its ability to increase NADPH oxidase-dependent intracellular O2Ϫ production. Interestingly, all of the drugs used in this study resulted in an intracellular increase in H2O2 production (Fig. 7C). In this regard, our earlier findings have implicated mitochondria in C2-induced H2O2 production because inhibition of the membrane RSV Inhibits Apoptosis by Altering Cellular Redox Status. Taken together, our data provide strong evidence that contrary to its proapoptotic activity at Ն25 M, low micromolar concentrations (4 – M) inhibit apoptotic signaling (25, 40, 41). This divergent signaling by RSV is intriguing and could be explained by our earlier observations that at concentrations of Ն32 M, apoptosis induced in HL60 cells is mediated by up-regulation of CD95 (Fas/Apo1)-CD95L interaction (25). This effect on up-regulation of the death receptor ligand is not observed at concentrations of RSV Ͻ16 M, hence the inability to trigger apoptosis in these cells (data not shown). Contrarily, at these concentrations, RSV inhibits apoptotic signaling upstream of the mitochondria, thus blocking the recruitment of mitochondrial-derived amplification factors, such as cytochrome c. We also provide evidence that RSV has a potent effect on the intracellular redox status, a critical determinant of the efficacy of the death signal (9, 42, 43). In that respect, it has been shown previously that maintaining a slightly elevated intracellular O2Ϫ promotes cellular proliferation (14, 44) and inhibits apoptotic signaling (18, 42). A pro-oxidant intracellular milieu is an invariable finding in cancer cells and has been shown to endow cancer cells with a survival advantage over their normal counterparts (15). Because a decrease in intracellular O2Ϫ and cytosolic pH is critical for efficient death execution, our results suggest that low concentrations of RSV could enable cancer cells to evade death signaling by creating a pro-oxidant intracellular milieu and inhibiting cytosolic acidification. RSV Induces Increase in O2؊ via NADPH Oxidase Activation. Considering the earlier reported ability of RSV to inhibit mitochondrial complex III-induced reactive oxygen species production, our paradoxical findings provide evidence for a pro-oxidant effect of RSV at concentrations that not trigger apoptosis (45). Such pro-oxidant activity of polyphenolics, such as RSV, has been reported recently in different systems (46, 47). In one model, reactive oxygen species generation at concentrations of RSV that triggered cell death in human cancer cells was proposed to be responsible for its cytotoxic activity (47). In addition, our results implicate the membrane NADPH oxidase complex as a potential source of O2Ϫ on incubation with low doses of RSV. Inhibition of the NADPH oxidase complex not only restored death signaling but also resulted in reverting the negative effect of RSV on drug-induced intracellular H2O2 production. This fits in well with our hypothesis that a balance between intracellular O2Ϫ and H2O2 could be a critical factor in the response of cells to apoptotic triggers, with a tilt toward the former favoring survival and a predominance of the latter facilitating death execution (10). A careful look at the data shown in Fig. 5B reveals that, similar to RSV alone, exposure of cells to the anticancer drug C2 also results in a slight elevation in intracellular O2Ϫ. However, it is important to note that C2-induced O2Ϫ is accompanied by a surge in intracellular H2O2, which is not observed with low concentrations of RSV. In addition, preincubation with RSV completely blocks C2-induced H2O2 production and maintains a significantly elevated intracellular O2Ϫ level. By implication, this suggests that an increase in intracellular O2Ϫ could inhibit downstream H2O2 production from the mitochondria, thereby 1456 APOPTOSIS INHIBITORY ACTIVITY OF RESVERATROL Fig. 5. Resveratrol (RSV) creates a pro-oxidant intracellular milieu and inhibits C2-mediated decrease in cytosolic pH and cell death. A, HL60 (1 ϫ 106) cells were treated with 50 g/ml of C2 for h with or without previous incubation with M RSV, and intracellular H2O2 was determined. B, ϫ 106 cells (in ml) were treated with RSV (8 M) for h or 50 g/ml of C2 for h with or without h of previous incubation with RSV (8 M), and intracellular O2Ϫ was determined. C, cells were treated as in (A), and cytosolic pH was assessed with 2Ј,7Ј-bis(2-carboxyethyl)-5,6-carboxyfluorescein. D, HL60 cells (1 ϫ 106/ml) were preincubated with DPI (1.25 M) or with DPI (1.25 M) ϩ RSV (8 M) for h before the addition of 50 g/ml C2 for 18 h. Cell survival was assessed by the MTT assay. inhibiting the decrease in intracellular pH. It is intriguing as to how an increase in intracellular O2Ϫ blocks mitochondrial-derived H2O2. One possibility could be inhibition of upstream caspase activation, as has been shown previously, or other proapoptotic factors, such as deathpromoting Bcl-2 family members, required for the engagement of the mitochondrial pathway (48). Impeding these upstream pathways could account for the inhibition of drug-induced mitochondrial H2O2 production and downstream cytochrome c release observed in cells preincubated with RSV. This in turn fails to amplify downstream caspase cascade (caspase-9 and -3) and leads to a substantial decrease in the sensitivity of cells to apoptotic stimuli that require mitochondrial amplification factors. Taken together, these results indicate that the increase in intracellular O2Ϫ on exposure of leukemia cells to RSV was mediated through the activity of NADPH oxidase complex and linked directly or indirectly to the apoptosis-inhibitory activity of RSV. In addition, data presented here not only implicate mitochondrial H2O2 production as a critical effector mechanism during druginduced apoptosis but also demonstrate the ability of an increase in intracellular O2Ϫ to prevent H2O2 production and thereby impede the recruitment of the mitochondrial death pathway. Fig. 6. The inhibitory effect of resveratrol (RSV) on vincristine-induced apoptosis can be neutralized by inhibition of the NADPH oxidase complex. A, HL60 cells (1 ϫ 106 cell/ml) were preincubated with RSV (2, 4, or M) for h before treatment with 1.25 g/ml of vincristine for 18 h. Cell viability was determined by the MTT assay. B, caspase-9 and -3 activity was determined by fluorimetric assays. C, HL60 cells (1 ϫ 106/ml) were preincubated with DPI (1.25 M or 2.5 M) ϩ RSV (8 M) for h before the addition of 1.25 g/ml of vincristine for 18 h. Cell survival was assessed by the MTT assay. D, CEM leukemia cells (1 ϫ 106/ml) were cotransfected with pCMV--galactosidase (gal) and pIRES-RacN17 and exposed to 1.25 g/ml of vincristine for 18 h with or without previous incubation for h with M RSV. Cell survival was assessed by the -gal assay. 1457 APOPTOSIS INHIBITORY ACTIVITY OF RESVERATROL Fig. 7. Resveratrol (RSV) inhibits daunorubicininduced apoptosis, and preincubation with DPI reverts the effect of RSV on drug-induced H2O2 production. A, ϫ 106 cell/ml were incubated with RSV (8 M) for h, followed by 18 h of exposure to 0.2 g/ml of daunorubicin. Cell viability was determined by the MTT assay. B, caspase-9 and -3 activity was determined by fluorimetric assays. C, HL60 (1 ϫ 106) cells were preincubated with 1.25 M DPI before the addition of RSV (DPI/RSV), followed by treatment with C2 (50 g/ml), daunorubicin (0.2 g/ml), or vincristine (1.25 g/ml) for h. Intracellular H2O2 was determined by flow cytometry. Potential Implications. These findings could be potentially important in light of the recent interest in the biological activity of flavonoids or flavonoid-like molecules, such as RSV, for their possible use in combination chemotherapy regimens. Although in vitro exposure of tumor cells to RSV at relatively high concentrations results in apoptotic cell death, because of the low bioavailability of RSV, plasma levels as high as 50 –100 M may not be physiologically attainable (49, 50). Our data suggest death-inhibitory and/or prosurvival activity of RSV in leukemia cells at doses that may be relevant physiologically (49, 50). 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