Hydrogen Peroxide-Mediated Cytosolic Acidification Is a ...€¦ · [CANCER RESEARCH 64,...

[CANCER RESEARCH 64, 7867–7878, November 1, 2004]

Hydrogen Peroxide-Mediated Cytosolic Acidification Is a Signal for MitochondrialTranslocation of Bax during Drug-Induced Apoptosis of Tumor Cells

Kashif A. Ahmad,1 Kartini B. Iskandar,1 Jayshree L. Hirpara,1 Marie-Veronique Clement,3,4 and Shazib Pervaiz1,2,4

1Department of Physiology, 2Oncology Research Institute, 3Department of Biochemistry, Faculty of Medicine, and 4National University of Singapore (NUS) Graduate School forIntegrative Sciences and Engineering, NUS, Singapore

ABSTRACT

Absence of the proapoptotic protein Bax renders tumor cells resistantto drug-induced apoptosis. We have shown that hydrogen peroxide(H2O2)-mediated cytosolic acidification is an effector mechanism duringdrug-induced apoptosis of tumor cells. Here, we report that Bax is criticalin determining the sensitivity of tumor cells to H2O2-induced apoptosis.More importantly, exposure of colorectal carcinoma (HCT116) and leu-kemia cells (HL60 and CEM) to H2O2 or its intracellular productionduring drug-induced apoptosis is a signal for mitochondrial translocationof Bax. Furthermore, we provide evidence that drug-induced H2O2-mediated Bax translocation in tumor cells is caspase independent butinvolves cytosolic acidification. Inhibiting cytosolic acidification preventsBax translocation, and contrarily enforced acidification of the intracellu-lar milieu results in mitochondrial recruitment of Bax, even in the absenceof a trigger. These findings provide a novel mechanism for mitochondrialtranslocation of Bax and directly implicate H2O2-mediated cytosolic acid-ification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.

INTRODUCTION

Apoptotic execution is orchestrated by intricate networking be-tween caspases and apoptogenic factors released from the mitochon-dria (1). A growing body of evidence seems to favor the involvementof intracellular reactive oxygen species at some point during apoptoticexecution (2–6). These observations become more important consid-ering the critical role of the mitochondria during apoptosis and the factthat mitochondria have been implicated directly or indirectly as theprime source of reactive oxygen species during drug-induced apo-ptosis (2, 5, 7, 8). However, it is still unclear whether reactive oxygenspecies generation is a critical initial trigger or a downstream effect ofcaspase-mediated mitochondrial damage. To that end, our findingshave highlighted the regulatory role of intracellular reactive oxygenspecies in the apoptotic pathway (6, 9–11). We showed that intracel-lular increase in H2O2 was a critical effector mechanism duringdrug-induced apoptosis of human tumor cells (5). This increase inH2O2 was responsible for early cytosolic acidification, thus creatingan environment conducive for caspase activation. Although our dataand a number of other reports describing H2O2-mediated apoptosis,e.g., the inhibitory effect of Bcl-2 overexpression on H2O2-induceddeath signaling (12–14) and the up-regulation of the proapoptoticprotein Bax in some systems (15), point to the mitochondria as thetarget organelle, the upstream events leading to the engagement ofthe mitochondria remain clouded.

The role of the proapoptotic protein Bax in the recruitment of themitochondria has been well established (16–18). A well-acceptedmodel is the death receptor-mediated activation of caspase 8 that

triggers Bid processing (19–21). Truncated Bid can then signal trans-location of cytosolic Bax to the mitochondria (19), where it can formhomo- or heterodimers with other Bcl-2 family members. The result-ant conformational change in Bax can result in channel formation thatcould mediate egress of proteins, such as cytochrome c, from themitochondrial intermembrane space (22–25), thereby activatingdownstream execution. Hence, translocation of Bax is a critical signalfor the involvement of the mitochondrial death machinery, and redis-tribution of Bax has been reported with a variety of apoptotic stimuli(22, 23, 26–28). The critical involvement of Bax during drug-inducedapoptosis is further supported by the relative insensitivity of cellslacking Bax expression to drug treatment (29) and observation thatBax�/� cells when xenografted in mice are deficient in apoptoticsignaling.

In the light of these findings, the objectives of this study were2-fold: (1) to decipher the role of Bax in H2O2-mediated apoptosis,and (2) to investigate whether the intracellular increase in H2O2 wasa signal for Bax translocation during drug-induced apoptosis of tumorcells. Here, we report that H2O2-induced apoptosis is inhibited incolorectal carcinoma cells lacking Bax (HCT116 Bax�/�), unlikeHCT116 Bax�/� cells. Exposure of HCT 116 Bax�/� or HL60human leukemia cells to H2O2 or its intracellular generation duringdrug-induced apoptosis signals translocation of Bax to the mitochon-dria, which is mediated by cytosolic acidification.

MATERIALS AND METHODS

Cell Lines. The human leukemia HL60 and CEM cell lines were purchasedfrom American Type Culture Collection (Rockville, MD) and maintained inRPMI 1640, supplemented with 10% fetal bovine serum, 1% L-glutamine, and1% S-Penicillin. HCT116 Bax�/� and Bax�/� cell lines were generous giftsfrom Dr. Vogelstein at Johns Hopkins University (Baltimore, MD). HCT116cell lines were maintained in McCoy’s 5A (Invitrogen Life Technologies, Inc.,Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, 1%L-glutamine, and 1% S-Penicillin. All cell lines were maintained in a 37°Cincubator with 5% CO2. Apoptosis was induced by exposure of cells (1 � 106/mL) to H2O2 (100–500 �mol/L) or the anticancer agent merodantoin (C1; 50�g/mL; ref. 30) for 4 to 18 hours. Cell survival was determined by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as de-scribed previously (31).

Subcellular Fractionation. Subcellular fractions were obtained as de-scribed previously (5). Briefly, 20 � 106 cells were washed with ice-chilled1 � PBS at 1200 � g. Cell pellets were resuspended in 500 �L of extractionbuffer [200 mmol/L mannitol, 68 mmol/L sucrose, 50 mmol/L Pipes (pH 7.4),50 mmol/L KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L DTT, andprotease inhibitor mixture in double distilled H2O] and incubated at 4°C for 20minutes, followed by Dounce homogenization. The homogenate was centri-fuged at 150 � g for 5 minutes at 4°C. The supernatant was additionallycentrifuged at 14,000 � g for 10 minutes (fraction enriched with intactmitochondria). The supernatant from the last centrifugation was used as thecytosolic fraction. Purity of the mitochondrial fractions was confirmed byWestern blot analysis using a monoclonal antibody that recognizes the mito-chondrial-specific protein MnSOD.

Determination of Caspase 3, 8, and 9 Activities. Caspase 3, 8, and 9activities were assayed by using AFC-conjugated substrates supplied by Bio-Rad Laboratories (Hercules, CA). Cells (1 � 106/mL) were exposed to H2O2

(100–500 �mol/L) or C1 (50 �g/mL) 4 to 24 hours, washed twice with

Received 2/22/04; revised 8/23/04; accepted 8/31/04.Grant support: The National Medical Research Council (R-185-000-032-213) and

Biomedical Research Council (R-185-000-048-305), Singapore.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 with18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Shazib Pervaiz, Department of Physiology and OncologyResearch Institute, Faculty of Medicine, National University of Singapore, MD9, #03-06,Singapore 117597. Phone: 65-6874-6602; Fax: 65-6778-8161; E-mail: [email protected].

©2004 American Association for Cancer Research.

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1 � PBS, resuspended in 50 �L of chilled cell lysis buffer (provided by thesupplier), and incubated on ice for 10 minutes. Fifty �L of 2 � reaction buffer(10 mmol/L HEPES, 2 mmol/L EDTA, 10 mmol/L KCl, 1.5 mmol/L MgCl2,and 10 mmol/L DTT) and 6 �L of the fluorogenic caspase-specific substrate(DEVD-AFC for caspase 3, LETD-AFC for caspase 8, and LEHD-AFC forcaspase 9) were added to each sample and incubated at 37°C for 1 hour.Protease activity was determined by the relative fluorescence intensity at 505nm after excitation at 400 nm using a spectrofluorimeter (TECAN SpectrofluorPlus, Maennedorf, Switzerland). Results are shown as fold increase (� in-crease) in activity relative to untreated cells (1�). Caspase 3 activation wasalso assessed by staining cells with an affinity-purified antibody that recog-nizes the processed (active) form of caspase 3 (tagged with phycoerythrin; BDPharMingen) and analyzed by flow cytometry.

Propidium Iodide Staining for DNA Fragmentation. Briefly, 1 � 106

cells/mL were triggered with H2O2 or C1 for 24 hours, fixed with 70% EtOH,and stained with propidium iodide for DNA content analysis as describedelsewhere (5). Events (�10,000) were analyzed by flow cytometry with theexcitation set at 488 nm and emission at 610 nm. Data are shown as apercentage of cells with subdiploid DNA and are mean � SD of threeindependent observations.

In addition, apoptosis on exposure of cells to H2O2 or C1 was also verifiedby assaying exposure of phosphatidylserine using the Apoalert Annexin-V kit(Clontech) and analyzed by flow cytometry using excitation and emissionwavelengths at 488 and 525 nm, respectively.

Flow Cytometric Analysis of Intracellular H2O2 Concentration. Cellswere exposed to the apoptotic trigger for 15 minutes to 12 hours, loaded with5-(and-6)-chloromethyl-2�,7�-dichlorofluorescin diacetate (5 �mol/L) at 37°Cfor 15 minutes, and analyzed by flow cytometry (Coulter EPICS Elite ESP)using an excitation wavelength of 488 nm, as described previously (32).

In addition, intracellular reactive oxygen species production was assessedby a lucigenin-based chemiluminescence assay as described previously (16) orflow cytometry using the sensitive probe hydroethidine (2 �mol/L). Chemi-luminescence was monitored for 60 seconds in a TD-20/20 Luminometer(Turner Designs, Sunnyvale, CA). Lucigenin is a widely used and validatedchemiluminescent detector of intracellular O2

� in biological systems (33, 34).Data are shown as relative light units (RLU) per microgram of protein (RLU�g�1 protein) � SD from three to six independent measurements. Proteinconcentration was determined using the Coomassie Plus protein assay reagentfrom Pierce (Pierce Chemical Co., Rockford, IL).

Fig. 1. H2O2-induced apoptosis is Bax dependent. In A, lysates from HCT116 Bax�/� and Bax�/� cells (2 � 106) were subjected to 10% SDS-PAGE, and the presence or absenceof Bax was confirmed by Western blot analysis using monoclonal anti-Bax. In B, 1 � 106/mL HCT116Bax�/� (Œ) and Bax�/� (f) cells were exposed to 100 �mol/L H2O2 for4 to 24 hours, and cell survival was assessed by the MTT assay. In C, caspases 3, 8, and 9 activities were determined in HCT116Bax�/� cells using fluorogenic substrates as describedin Materials and Methods. Results are shown as fold increase (� increase) in caspase activity over the untreated cells (1�). In D, HCT116 Bax�/� cells were exposed to H2O2 for12 hours and analyzed by flow cytometry for phosphatidylserine exposure and processed caspase 3 as described in Materials and Methods. E, Western blot analysis of poly(ADP-ribose)polymerase cleavage using anti-poly(ADP-ribose) polymerase (clone C-2–10; PharMingen) in Bax�/� and Bax�/� cells after 24-hour incubation with 100 �mol/L H2O2. In F,Bax�/� and Bax�/� cells were analyzed for DNA fragmentation by PI staining after 24-hour treatment with 100 �mol/L H2O2 as described in Materials and Methods. Subdiploidpopulation (Sub-G1 fraction) is shown as mean � SD from three independent experiments. G, Western blot analysis of cytochrome c (Cyt. C) in cytosols of Bax�/� and Bax�/�cells after 12-hour treatment with 100 �mol/L H2O2 in the presence or absence of 1000 units/mL catalase. Membrane was reprobed with anti-�actin as a loading control. In H,HCT116Bax�/� cells were transiently transfected with Bax as described in Materials and Methods and exposed to 100 �mol/L H2O2 for 24 h. Cell survival was assessed by the �-galsurvival assay described elsewhere (36). All data shown are mean � SD of at least three independent experiments [MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;PI, propidium iodide].

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H2O2 SIGNALS BAX TRANSLOCATION IN TUMOR CELLS

Detection of Cytochrome C Release from the Mitochondria. Cyto-chrome c release was assessed by Western blot analysis of cytosolic extractsfrom 30 � 106 cells as described previously (5) using anti-cytochrome c(7H8.2C12, PharMingen, San Diego, CA). Signal was detected by the SuperSignal Substrate Western Blotting kit (Pierce).

Determination of Mitochondrial ��m by Flow Cytometry. Potential-sensitive probe 3, 3� dihexyloxacarbocyanine iodide (DiOC6) was used tomeasure mitochondrial ��m as described previously (31). Briefly, 1 � 106

cells were incubated with 3,3�DiOC6 (40 nmol/L) for 15 minutes at 37°C andimmediately analyzed in Epic Profile flow cytometer with excitation set at 488nm. Data were analyzed for 10,000 events using the WinMDI software.

Western Blot Analyses of Poly(ADP-Ribose) Polymerase Cleavage, Bax,and Bid. For analysis of poly(ADP-ribose) polymerase cleavage, lysates from2 � 106 cells were prepared in sample buffer [50 mmol/L Tris/HCl (pH 6.8),6 mol/L urea, 3% SDS, 0.003% bromphenol blue, and 6% �-mercaptoethanol]and subjected to Western blot analysis using anti-poly(ADP-ribose) polymer-ase (clone C-2–10, PharMingen) as described (30). For Western blot analysisof Bax, cells (2 � 106) were lysed by adding 100 �L of chilled 1 � radio-immunoprecipitation assay buffer lysis buffer, and 50 �g of protein weresubjected to 15% PAGE and transferred to polyvinylidene difluoride as above.Alternatively for analysis of Bax dimerization/multimerization, cell lysateswere subjected to 10% native gel electrophoresis. Membranes were exposed to1:2,000 dilution of mouse monoclonal anti-Bax antibody (clone 6A7, BDPharmigen, San Diego, CA) at 25°C for 2 hours, followed by 1:5,000 dilutionof goat antimouse IgG-horseradish peroxidase. The anti-Bax antibody (6A7)recognizes epitopes that are in the vicinity of the dimerization domains of Bax(AA 12–24; ref. 35). Western blot analysis for Bid cleavage was performed onwhole cell lysates using a rabbit polyclonal anti-Bid IgG (Biovision ResearchProducts, Paolo Alto, CA) that recognizes the Mr 22,000 full-length Bid.Chemiluminescence was detected as described above.

Transient Transfection of HCT116 Bax�/� Cells with pcDNA3-Bax.Transient transfection of HCT116 Bax�/� cells with Bax was performedusing pcDNA3-Bax (generously provided by Prof. Stanley Korsmeyer, Boston,MA). HCT116 Bax�/� (2 � 105) cells were seeded per six-well plate in 2 mLof 10% McCoy’s Medium and incubated at 37°C for 24 hours. The mediumwas then removed, and cells were resuspended in 1.5 mL of normal growthmedium. Cells were cotransfected with a �- galactosidase-containing plasmid(p-cytomegalovirus-� galactosidase) at a ratio of 3.5 �g of pCDNA3-Bax:0.5

�g of p-cytomegalovirus-�-galactosidase as described previously (36). Lumi-nescence was recorded with a Luminometer (TD20/20; Turner Designs). Therelative light units were calculated per microgram of protein.

Measurement and Manipulation of Cytosolic pH. For measurement ofcytosolic pH, cells were loaded with 10 �mol/L BCECF-AM [2�,7�-bis(2-carboxyethyl)-5,6-carboxyfluorescein; Sigma, St. Louis, MO], and the fluo-rescence ratio of 525:610 nm was used to derive cytosolic pH using a standardpH calibration curve as described previously (5).

To manipulate cytosolic pH, cells were incubated with a known inhibitor ofthe Na�-H� antiporter, methylamiloride (10–40 �mol/L), for 4 hours beforethe measurements. Alternatively, cells were preincubated with 4 to 8 �mol/Lresveratrol for 2 hours before exposure to the apoptotic stimuli and pH. In ourrecent study (37), we have demonstrated the inhibitory effect of low doses ofresveratrol on drug-induced acidification.

RESULTS

H2O2-Induced Apoptosis Is Blocked in Bax�/� Cells. The sen-sitivity of human colon carcinoma HCT116 Bax�/� and HCT116Bax�/� cells (Fig. 1A) to H2O2 (100 �mol/L) for 4 to 24 hours wasdetermined. Unlike HCT116 Bax�/� cells, Bax�/� cells were re-sistant to the effect of H2O2 (Fig. 1B). H2O2 triggered significantincreases in caspases 9 and 3 activities (but no caspase 8 activation orcleavage of Bid; Fig. 1C), increase in membrane phosphatidylserineexposure and processing of caspase 3 (Fig. 1D), cleavage of caspase3 substrate poly(ADP-ribose) polymerase (Fig. 1E), and increase insubdiploid DNA fraction (Fig. 1E) in Bax�/� cells, whereas littleeffects on caspase activation (Fig. 1, C and E), and DNA fragmenta-tion (Fig. 1F) were observed in Bax�/� cells. H2O2 also inducedcytosolic translocation of cytochrome c in HCT 116 Bax�/� cells(inhibited by catalase) but not the Bax�/� variant (Fig. 1G). Fur-thermore, transient transfection of Bax�/� cells with a vector con-taining full-length Bax (pcDNA3-Bax) restored their sensitivity toH2O2 (Fig. 1H), thus highlighting the importance of Bax in H2O2-induced apoptosis.

Fig. 1 Continued.

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H2O2 Influences Subcellular Localization of Bax. H2O2 hasbeen shown to induce up-regulation of Bax and a shift in the intra-cellular Bax:Bcl-2 ratio in some systems (38). Although exposure ofHCT116 Bax�/� cells to H2O2 did not significantly change theBax:Bcl-2 ratio (Fig. 2A), analysis of subcellular fractions clearlyshowed translocation of Bax from the cytosol to the mitochondria(Fig. 2B). Mitochondrial localization of Bax could be detected 6 hoursafter exposure to H2O2 (data not shown) and was maximal at 12 hoursafter treatment (Fig. 2B). Importantly, Bax translocation was inhibitedby the H2O2 scavenger catalase (1000 units/mL), additionally sup-porting the role of H2O2 in mitochondrial translocation of Bax.Contrarily, preincubation with the general caspase inhibitor ZVAD-fmk (50 �mol/L) did not inhibit H2O2-induced Bax translocation (Fig.2C). Similar to HCT116 cells, HL60 and CEM leukemia cells under-went apoptosis on exposure to H2O2, as indicated by the appearanceof subdiploid DNA, increases in caspase 3 and 9 activities, processingof caspase 3, and decrease in cell survival (Fig. 3, A–D). In addition,the involvement of the mitochondrial death pathway was evident bythe drop in ��m of cells exposed to H2O2 (Fig. 4A) and cytosolictranslocation of cytochrome c from the mitochondria (Fig. 4B) thatwas significantly inhibited by catalase. More importantly, cytosolicBax was redistributed to the mitochondrial fractions in HL60 andCEM cells, which could be inhibited by catalase (Fig. 4, C and D) butnot ZVAD-fmk (Fig. 4C). These data suggest a critical role for H2O2

in signaling Bax to the mitochondria but argue against the involve-ment of the caspase family in this pathway.

H2O2 Is a Signal for Bax Translocation during Drug-InducedApoptosis. Having demonstrated that exogenously added H2O2 trig-gered translocation of Bax in tumor cells, we next asked whether thiswas also the mechanism used by anticancer drugs that trigger apo-ptosis via intracellular H2O2 production. HL60 cells were exposed tothe novel experimental anticancer agent C1, a small (Mr � 242)synthetic compound, first isolated and purified on photo-oxidation ofmerocyanine 540. The chemical structure of this imidazole compoundis N,N�-Dibutyl-thio-4,5-imidazolindion, and its apoptosis-inducingactivity was reported previously by our group (30).

Exposure of HL60 cells to 50 �g/mL C1 for 4 hours resulted in asignificant increase in intracellular H2O2 (Fig. 5A, panel a). Theintracellular generation of reactive oxygen species was additionallyconfirmed by a lucigenin-based chemiluminescence assay and flowcytometry on loading cells with hydroethidine. Results clearly indi-cate that exposure of cells to C1 for 2 to 8 hours resulted in asignificant increase (1.4–2�) in intracellular reactive oxygen speciescompared with the cells treated with the carrier solvent (Fig. 5, panelb). Similarly, a right shift in the fluorescence of C1-treated cellsloaded with hydroethidine (Fig. 5, panel c; mean fluorescence inten-sity shifted from 2.9 to 3.25) provided additional proof for drug-induced increases in intracellular reactive oxygen species generation.In addition, exposure to C1 resulted in a significant decrease in cellsurvival (Fig. 5B), H2O2-dependent increase in subdiploid DNA frac-tion, increased processing of caspase 3, and cleavage of the caspase 3substrate poly(ADP-ribose) polymerase (Fig. 5, C–E). In addition, ourdata clearly provide evidence that C1 induced apoptosis by targetingthe mitochondria, as demonstrated by the drop in ��m of cells,cytosolic translocation of cytochrome c, and increase in caspase 9activity (Fig. 6, A–C). More importantly, Bax translocated from thecytosol to the mitochondria on exposure to C1, which could besignificantly inhibited by catalase but not the general caspase inhibitorZVAD-fmk (Fig. 6D). In addition, native gel electrophoresis revealedthat C1, similar to pure H2O2, triggered dimerization and multimer-ization of Bax (Fig. 6E). A careful look at the blots in Fig. 6, D andE, showed that the intensity of the bands was much weaker in thecatalase pretreated lanes in both the cases. This was additionallyreinforced by a stronger inhibition of the oligomeric form by catalase(Fig. 6E), thus corroborating the involvement of H2O2 in C1-inducedBax activation. The fact that preincubation of cells with H2O2 scav-enger catalase significantly blocked all mitochondrial changes trig-gered by C1 in tumor cells (Fig. 6) underscores the critical role ofH2O2 as the mediator of mitochondrial recruitment and apoptosistriggered by C1.

H2O2-Induced Cytosolic Acidification Signals MitochondrialRecruitment of Bax. We have demonstrated previously that H2O2

added exogenously or triggered endogenously during drug-inducedapoptosis is a stimulus for cytosolic acidification, thereby creating apermissive intracellular milieu for death execution (5, 6). In agree-ment with our earlier studies, here we show that C1 or H2O2 induceda significant drop in cytosolic pH, which could be inhibited bycatalase (Fig. 7A). Therefore, we questioned whether H2O2-dependentcytosolic acidification could be the stimulus for Bax translocationduring drug-induced apoptosis. To prove that, we exploited the abilityof low doses of the polyphenolic compound resveratrol to block H2O2

and drug-induced pH drops in human leukemia cells (37). Corrobo-rating our recent findings, preincubation of HL60 cells for 2 hourswith 4 to 8 �mol/L resveratrol inhibited acidification induced by C1or H2O2 (Fig. 7B). More interestingly, resveratrol exposure blockedmitochondrial translocation of Bax induced by either of the stimuli(Fig. 7, C and D). The effect of resveratrol on C1-induced Bax

Fig. 2. H2O2 triggers mitochondrial translocation of Bax. In A, lysates ofHCT116Bax�/� cells (5 � 106) after exposure to H2O2 for 0–24 hours were subjectedto Western blot analysis using anti-Bax or anti-Bcl-2 antibodies as described in Materialsand Methods. In B, HCT116Bax�/� cells (1 � 107) were treated with H2O2 for 12 hoursin the presence or absence of either 1,000 units/mL catalase or 100 �mol/L ZVAD-fmk(C), and cytosolic and mitochondrial fractions were subjected to Western blot analysisusing monoclonal anti-Bax. Mitochondrial purity was verified by blotting with a mono-clonal antibody against the mitochondrial protein MnSOD.

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activation appears to be a function of its inhibitory effect on Baxdimerization (data not shown). It should be pointed out that resveratroltreatment for 0 to 24 hours did not alter the overall expression ofeither Bax or Bcl-2 (Fig. 7E). Furthermore, we have reported previ-ously that resveratrol did not elicit H2O2 scavenging activity at theselow concentrations.

Stimulated by these findings and to verify that drop in cytosolicpH was a signal for Bax translocation, we assessed the effect ofenforced intracellular acidification on Bax translocation. To thatend, we made use of the pharmacological inhibitor of the pHregulator Na�/H� antiporter, methylamiloride. As expected,methylamiloride (10 – 40 �mol/L) induced a significant drop incytosolic pH (Fig. 8A). Most interestingly, exposure of tumor cellsto methylamiloride alone resulted in significant translocation ofBax to the mitochondria (Fig. 8B), which was followed by celldeath (Fig. 8C). It should be pointed out that exposure of cells tomethylamiloride for 0 to 12 hours did not alter the intracellularexpression of Bax or Bcl-2 (Fig. 8D). Indeed, enforced acidifica-tion of the milieu was a much stronger stimulus for translocation ofBax as shown in Fig. 8B (virtually all Bax translocated as com-pared with 60% in case of C1), additionally consolidating the

direct effect of a pH drop on mitochondrial recruitment of Bax.These data provide strong evidence to support cytosolic acidifica-tion, downstream of H2O2 production, as an effector mechanismfor Bax translocation during drug-induced apoptosis of tumor cells.

Translocation of Bax Triggers Additional Increase in Mito-chondrial H2O2 Production. Current opinion holds that the prin-cipal source of reactive oxygen species, including H2O2, duringapoptotic signaling is the mitochondria (2, 5, 12). How does onereconcile with these findings given our observations that H2O2 actsupstream of the mitochondria to signal mitochondrial localizationof Bax? To address this, we asked whether mitochondrial translo-cation of Bax resulted in a second wave of intracellular H2O2.HCT116 Bax�/� and Bax�/� cells were exposed to 100 �mol/LH2O2, and the intracellular level of H2O2 was determined at 30minutes, 1, 6, and 12 hours. The initial increase in fluorescenceobserved in both Bax�/� and Bax�/� cells is indicative ofcellular uptake of exogenously added H2O2 (Fig. 9A, panel a).However, by 1 hour, intracellular H2O2 returned to the baseline inBax�/� cells and remained unchanged for �12 hours (Fig. 9A,panels b and c). On the contrary, Bax�/� cells showed a subse-quent increase in intracellular H2O2 starting at 6 hours and signif-

Fig. 3. H2O2 induces apoptosis and in human leukemia cells. HL60 and CEM leukemia cells (1 � 106/mL) were incubated with 100 �mol/L H2O2 for 12 hours in the presenceor absence of 1,000 units/mL catalase. In A, DNA fragmentation was assessed by PI staining; in B, activities of caspases 3 and 9 were determined by fluorogenic assays as describedin Materials and Methods. Data are shown as fold increase (� increase) in activity over untreated cells (1�) and mean � SD of three independent experiments. In C, active caspase3 was assayed by staining cells with an antibody that recognizes the processed form of caspase 3 tagged with PE. Cells were then analyzed by flow cytometry. In D, cell survival onH2O2 treatment (100 �mol/L for 18 hours) was assessed by MTT assay as described in Materials and Methods [PI, propidium iodide; PE, phycoerythrin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].

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icantly detectable even after 12 hours of the initial exposure toH2O2 (Fig. 9A, panels b and c). Indeed, a summary of threeindependent experiments showing change (percentage of controlcells) in mean fluorescence intensity of dichlorofluorescin clearlydemonstrates a significant increase in mean fluorescence intensityat 6 and 12 hours after exposure to H2O2 in Bax�/� cells, whereasno detectable difference is seen in Bax�/� cells (Fig. 9B). Intra-cellular reactive oxygen species production was further verified inBax�/� and Bax�/� cell lines by hydroethidine staining andflow cytometry (Fig. 9C). It is important to point out that thetranslocation of Bax to the mitochondria is also detected first at 6hours after incubation with H2O2 with a maximum shift observedat 12-hour postincubation (Fig. 9D). Furthermore, to confirm thatintracellular reactive oxygen species generation on exposure to theapoptosis-inducing agent C1 was, indeed, upstream of Bax,HCT116Bax�/� and Bax�/� cells were exposed to C1, andreactive oxygen species production was assessed by loading cellswith DCHF-DA or hydroethidine and analyzed by flow cytometry.Results clearly indicate that C1 triggered reactive oxygen speciesproduction in both cell lines as early as 15 minutes after exposure(Fig. 10); however, similar to H2O2, the second increase in intra-cellular reactive oxygen species was only observed in Bax�/�cells (data not shown). These results suggest a scenario whereexogenous H2O2 or intracellular H2O2 produced by drug exposuretriggers Bax translocation (via inducing cytosolic acidification),which then acts as a signal for the second wave of H2O2 productionfrom the mitochondria, thereby reinforcing the death signal.

DISCUSSION

H2O2: Signal for Mitochondrial Targeting of Bax. Our recentfindings and many related studies strongly argue in favor of a signal-ing role for reactive oxygen species, such as H2O2 (5, 10, 11, 38, 39).Aside from the up-regulation of Bax on exposure of some cell typesto H2O2, the inhibitory effect of Bcl-2 overexpression (12) supportsthe involvement of the mitochondrial death pathway in H2O2-inducedapoptosis. However, as the mitochondria are a major source of intra-cellular reactive oxygen species, it is tempting to speculate thatreactive oxygen species, such as H2O2, may function both upstreamand downstream of the mitochondria.

Tumor cells lacking Bax (Bax�/�) are resistant to the effect of someanti-cancer drugs (29). In agreement with that, we show here thatHCT116 Bax(�/�) cells are resistant to death induced by apoptoticconcentrations of H2O2. Furthermore, transient transfection of Bax inHCT116 Bax(�/�) cells restores their sensitivity to H2O2, thus under-scoring the critical role of Bax in H2O2-induced apoptosis. One probablemechanism underlying the differential sensitivity of Bax�/� andBax�/� cells to H2O2 could be that H2O2 increases the expression ofBax or alters the cellular Bax:Bcl-2 ratio in Bax�/� cells, as suggestedby some studies. However, our results show that the Bax:Bcl-2 ratio is notsignificantly altered on 4- to 24-hour exposure to H2O2. Interestingly,analysis of subcellular distribution of Bax (in HCT116, HL60, and CEMcells) revealed that Bax redistributed to the mitochondrial fraction fromthe cytosol on exposure to H2O2, which could be significantly blocked bythe H2O2 scavenger catalase.

Fig. 4. H2O2 triggers caspase-independent translocation of Bax to the mitochondria. In A, HL60 cells (1 � 106/mL) were incubated with H2O2 (100 �mol/L for 6 hours) or CICCP(100 �mol/L for 1 hour), and the change in ��m was assessed by flow cytometry using DiOC6 as described in Materials and Methods. In B, cytosolic fractions from HL60 cells(2.5 � 107) treated with 100 �mol/L H2O2 for 12 hours in the presence or absence of 1,000 units/mL catalase were subjected to Western blot analysis for cytochrome c. Anti-� actinwas used as a loading control. In C, mitochondrial and cytosolic fractions from HL60 cells (2.5 � 107), incubated with 100 �mol/L H2O2 for 12 hours in the presence or absence ofcatalase or ZVAD-fmk, were subjected to Western blot analysis using anti-Bax. Mitochondrial purity was confirmed by probing with anti-MnSOD. D, Western blot analysis usinganti-Bax on fractions obtained from CEM cells after exposure to 100 �mol/L H2O2 with or without catalase.

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One could always argue that the changes elicited on exposure ofcells to exogenous H2O2 may not have physiologic relevance, becausesuch high levels of intracellular H2O2 are rarely observed in livingcells. Therefore, to present a more real-life situation, we exploited theability of certain anticancer drugs to increase intracellular productionof reactive oxygen species, specifically H2O2 (5). Indeed, exposure ofHCT116 Bax�/� or HL60 cells to a novel anticancer compound C1resulted in an increase in intracellular H2O2 and translocation of Baxto the mitochondria. This translocation of Bax was inhibited bycatalase, thus establishing the critical role of intracellular H2O2 in mito-chondrial recruitment during drug-induced apoptosis of tumor cells.

Recruitment of Bax to the mitochondria during apoptotic signalinghas been linked to the activation of upstream caspase 8 and caspase8-mediated cleavage of the proapoptotic protein Bid (40). This isparticularly true on ligation of death receptors, such as CD95 (Apo1/Fas). Incidentally, H2O2 and anticancer drugs have been shown toup-regulate the expression of the CD95 receptor or its ligand (CD95L)in some systems (41, 42). In these systems, blocking receptor signal-ing or downstream caspase activation could abrogate Bax transloca-tion and, consequently, the death signaling circuitry downstream ofthe mitochondria. Interestingly, in our study, neither of the stimulitriggered up-regulation of the CD95 receptor (data not shown), andinhibition of caspase activation had no effect on the mitochondrialtranslocation of Bax. In addition, the relative lack of caspase 8

activation and absence of downstream Bid cleavage provide addi-tional evidence in favor of a mechanism for signaling Bax to themitochondria that is either parallel to or independent of death receptorand/or caspase activation. A similar mechanism of caspase-independ-ent conformational change of Bax on triggering apoptosis has beenreported previously (43).

Recent findings also implicate ceramide production in response toactivation of the membrane sphingomyelinase in signaling Bax to themitochondria (44, 45). Ceramide production is observed in a varietyof apoptotic models, including drug-induced apoptosis (45, 46), and inresponse to H2O2 in tumor cells (3). However, inhibiting ceramidesynthesis in our system did not affect the subcellular localization ofBax triggered by C1 or H2O2 (data not shown). Collectively, thesedata indicate that Bax translocation triggered in tumor cells duringdrug (C1)-induced apoptosis was a direct result of intracellular H2O2

production, independent of the upstream caspase 8 or ceramide path-ways. Corroborating these findings is a recent study suggestingglutathione-dependent activation of Bax by oxidative stress in HeLacells stably transfected with the cystic fibrosis transmembrane con-ductance regulator (47).

Drug-Induced Bax Translocation Is Dependent on H2O2-Medi-ated Cytosolic Acidification. Cytosolic acidification is an earlyevent in apoptosis and provides an intracellular milieu permissive forefficient death execution. In this regard, exposure of cells to H2O2 or

Fig. 5. Apoptosis and Bax translocation induced by C1 are mediated by intracellular H2O2. HL60 cells (1 � 106/mL) were exposed to 50 �g/mL C1 in the presence or absenceof 1000 units/mL catalase for 2 to 8 hours (for intracellular reactive oxygen species), 12 hours (for caspase activation and cytochrome c translocation), or 18 hours (for DNAfragmentation and cell survival). In A, intracellular reactive oxygen species was detected by flow cytometry using H2O2-specific probe DCHF-DA (a), lucigenin-based chemilumi-nescence assay (b), or flow cytometry using the fluorescent probe hydroethidine (c) as described in Materials and Methods. In B, cell survival was assessed by the MTT assay, andthe mean � SD of three independent experiments is shown. Statistical analysis of data was performed using the paired t test. In C, PI staining was used to assess subdiploid DNA (sub-G1fraction). In D, processed caspase 3 was assayed in C1-treated cells by staining with antiactive caspase 3 and analyzed by flow cytometry. In E, poly(ADP-ribose) polymerase cleavagein lysates was determined by Western blot using anti-poly(ADP-ribose) polymerase as described in Materials and Methods [MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; PI, propidium iodide].

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drugs that trigger intracellular increase in H2O2 results in a significantdrop in cytosolic pH (5). Accordingly, signals that inhibit apoptoticacidification impede death signaling as demonstrated in our recentstudy (37). Our results provide strong evidence that the link betweenH2O2 and Bax translocation could be the drop in cytosolic pH broughtabout by exposure of cells to exogenous H2O2 or endogenous pro-duction of H2O2 on drug exposure. Not only did inhibition of acidi-fication triggered by H2O2 or C1 result in a significant reduction inmitochondrial localization of Bax, but also, most interestingly, clamp-ing cytosolic pH to a more acidic range (7.1) directly induced Baxtranslocation in tumor cells. Because caspase inhibitors had no effectin our system, these results point to a direct effect of an acidicintracellular milieu in trafficking Bax from the cytosol to the mito-chondria. It is interesting to note that one of the first studies to reporta relationship between cytosolic pH and Bax translocation had con-cluded to the contrary, i.e., pH increase was shown to facilitate Baxtranslocation (48). However, in our system, the absence of any initialincrease in cytosolic pH downstream of H2O2 or C1 even as early as30 minutes after treatment (data not shown), fact that inhibition of pHdrop abrogated Bax translocation, and direct effect of acidic intracel-lular milieu on Bax translocation provide strong evidence that acidicpH is a direct signal for Bax translocation even in the absence of a

trigger. Furthermore, this phenomenon of H2O2-mediated, acidifica-tion-induced Bax translocation may not be exclusive to C1, because inour earlier study, we demonstrated the ability of commonly usedchemotherapeutic drugs vincristine and daunorubicin to trigger anearly increase in intracellular H2O2 (37). Interestingly, these com-pounds also induce cytosolic acidification and Bax-dependent apo-ptosis in tumor cells.5 Given these observations, it is highly likely thatacidification-induced Bax activation may be a common mechanismduring drug-induced apoptosis in tumor cells. Indeed, our findingslinking an acidic milieu to Bax translocation are in agreement with arecent study demonstrating a relationship between cytosolic acidifi-cation and Bax translocation during staurosporine and tumor necrosisfactor-induced apoptosis (49). Changes in pH affect the conformationof Bax, and a change in conformation facilitates Bax translocation tothe mitochondria. In this regard, both positively and negativelycharged residues contribute to the pH dependence of Bax conforma-tion (48). It is plausible that similar to the effect of an alkaline pH, aconsiderable acidic shift in the pH could also induce a conformationalchange in Bax, thus making it more amenable for membrane insertion.

5 S. Pervaiz et al., unpublished data.

Fig. 6. H2O2-dependent Bax translocation and activation during drug-induced apoptosis in tumor cells. In A, HL60 cells (1 � 106/mL) were exposed to 50 �g/mL C1 for 4 and6 hours or CICCP (100 �mol/L for 1 hour), and the change in ��m was assessed by flow cytometry using DiOC6 as described in Materials and Methods. In B, cytosolic fractions fromcells after 12-hour incubation with C1 in the presence or absence of catalase were subjected to Western blot analysis for cytochrome c translocation. In C, caspase 9 activity was assayedusing a fluorogenic substrate (LEHD-fmk) and shown as fold increase (� increase) over that of untreated cells. Data are mean � SD of three independent experiments, and thesignificance was calculated by the paired t test. D, mitochondrial and cytosolic fractions from HL60 cells (2.5 � 107) after exposure to 50 �g/mL C1 for 12 hours in the presence orabsence of 1,000 units/mL catalase or ZVAD (100 �mol/L) were subjected to Western blot analysis using anti-Bax as described in Materials and Methods. In E, Baxdimerization/multimerization in HL60 cell lysates was determined by 10% native gel electrophoresis and Western blot analysis using anti-Bax (6A7). � and ��, dimers and multimersof Bax.

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Fig. 8. Enforced acidification triggers Bax trans-location. HL60 cells were treated with 0–40�mol/L methylamiloride for 4 (A), 12 (B), or 18(C) hours. Cytosolic pH was determined byBCECF-AM loading, Bax translocation by Westernblotting, and cell survival by the MTT assay, asdescribed in Materials and Methods. In D, HL60cells were treated with 25 �mol/L methylamiloridefor 0 to 12 hours, and lysates were analyzed byWestern blotting using anti-Bcl-2 or anti-Bax anti-bodies [MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].

Fig. 7. Drug-induced Bax translocation is dependent on H2O2-mediated intracellular acidification. In A, HL60 cells (1 � 106) were exposed to 50 �g/mL or C1 or 100 �mol/L H2O2

for 6 hours in the presence or absence of 1000 units/mL catalase, and cytosolic pH was measured with BCECF-AM as described in Materials and Methods. In C, cells were preincubatedfor 2 hours with RSV (4 and 8 �mol/L), followed by exposure to C1 and H2O2 for 6 hours, and cytosolic pH was determined as described above. In C, mitochondrial and cytosolicfractions from HL60 (2.5 � 107) or CEM (D) cells after exposure to 50 �g/mL of C1 or 100 �M H2O2 for 12 hours with or without preincubation with RSV (4–8 �mol/L) weresubjected to Western blot analysis using anti-Bax as described in Materials and Methods. In E, HL60 cells were exposed to 8 �mol/L RSV for 6–24 hours, and lysates were analyzedby Western blotting using anti-Bcl-2 or anti-Bax antibodies. (RSV, resveratrol)

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Indeed, dimerization/multimerization of Bax in cells treated with C1or H2O2 provide evidence to support conformational change in Bax inresponse to these apoptotic stimuli. Furthermore, considering theability of drugs such as C1 to trigger early increases in intracellularH2O2, it is possible that the presence of scavenger catalase may have

a more longer and sustained inhibitory effect on drug-induced Baxtranslocation.

H2O2 Mitochondrial Bax- H2O2: Reactive Oxygen Species-Dependent Reactive Oxygen Species Production. Our data providea novel mechanism for drug-induced translocation of Bax and argue in

Fig. 9. Mitochondrial translocation of Bax results in additional increases in intracellular H2O2. In A, HCT116Bax�/� and Bax�/� cells (1 � 106) were exposed to H2O2 (100�mol/L), and intracellular levels of H2O2 were measured at 30 minutes, 1, 6, and 12 hours as described in Materials and Methods. In B, mean fluorescence intensity indicative of themean intracellular H2O2 levels from three independent experiments is plotted to demonstrate the increase in intracellular H2O2 in Bax�/� cells. In C, HCT116 Bax�/� and Bax�/�cells were incubated with H2O2 for 15 minutes and loaded with the fluorescent probe hydroethidine. Events (�10,000) were analyzed by flow cytometry. In D, Bax�/� cells (1 � 107)were incubated with 100 �mol/L H2O2 for 1, 6, and 12 hours, followed by cell fractionation and Western blot analysis using anti-Bax as described in Materials and Methods.

Fig. 10. Intracellular H2O2 production by C1 isupstream of Bax.HCT116 Bax�/� (A). In B,Bax�/� cells were incubated with 50 �g/mL for 15minutes, followed by loading of cells separatelywith DCHF-DA or hydroethidine for intracellularreactive oxygen species detection. Cells were ana-lyzed by flow cytometry as described in Materialsand Methods.

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favor of an upstream role for H2O2-mediated cytosolic acidification.Earlier studies have implicated superoxide anion and H2O2 as prob-able species responsible for mitochondrial damage, such as lipidperoxidation of the mitochondrial membranes and induction of mito-chondrial permeability transition (MPT) (50), changes that couldexplain the increase in the outer membrane permeability and leakageof pro-apoptotic proteins to the cytosol. However, most of thesestudies seem to imply that the mitochondrial burst of H2O2 is likely tobe a downstream effector mechanism for the execution signal. Ourdata argue strongly in favor of a two-hit mechanism for drug-inducedincreases in intracellular H2O2. The initial increase (first hit) inintracellular H2O2 on drug treatment of tumor cells leads to thetargeting of Bax to the mitochondria in a caspase-independent man-ner, thus resulting in the recruitment of the mitochondrial deathpathway. Bax translocation can then bring about mitochondrialchanges, such as induction of MPT, formation of a functional channelin the outer membrane, and as our data suggest, serve as a stimulus forthe next burst of H2O2 (second hit) from the mitochondria. This couldlead to peroxidative damage of mitochondrial lipids, such as cardio-lipin, and facilitate the egress of cytochrome c, Smac/Diablo, orapoptosis inducing factor (AIF). This phenomenon of reactive oxygenspecies-induced reactive oxygen species production has been de-scribed before as a probable mechanism for the induction of MPT, andmore recently, in a model of nerve growth factor-deprived neuronalcell death, Bax insertion has been proposed to induce reactive oxygenspecies burst, strong enough to trigger the release of cytochrome c(51). It should be pointed out that the initial early increase (within 15minutes of exposure) in intracellular H2O2 occurs independent of Bax(both Bax�/� and Bax�/� cells); however, more importantly, thesubsequent (second) increase is entirely dependent on Bax and itstranslocation to the mitochondria. One probable mechanism for Bax-induced H2O2 production could be that Bax translocation leads tocytochrome c release, which depletes the mitochondrial electron trans-port chain with the resultant leakage of electrons to molecular oxygento form O2

�, which is then dismutated by the mitochondrial super-oxide dismutase to H2O2.

Taken together, our data demonstrate a novel mechanism for mi-tochondrial recruitment of the proapoptotic protein Bax and provide amechanistic explanation to connect extramitochondrial death circuitryto the mitochondria, the point of convergence for effective drug-induced death signals.

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