Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or...

ORIGINAL RESEARCH

Differential effects of Bcl-2 and caspases on mitochondrialpermeabilization during endogenous or exogenous reactiveoxygen species-induced cell death

A comparative study of H2O2, paraquat, t-BHP, etoposide and TNF-α-inducedcell death

Vincent Rincheval & Marie Bergeaud &

Lise Mathieu & Jacqueline Leroy &

Arnaud Guillaume & Bernard Mignotte &

Nathalie Le Floch & Jean-Luc Vayssière

Received: 30 August 2011 /Accepted: 21 March 2012 /Published online: 11 April 2012# Springer Science+Business Media B.V. 2012

Abstract In this study, we have compared several fea-tures of cell death triggered by classical inducers ofapoptotic pathways (etoposide and tumour necrosis fac-tor (TNF)-α) versus exogenous reactive oxygen species(ROS; hydrogen peroxide (H2O2), tert-butyl hydroper-oxide (t-BHP)) or a ROS generator (paraquat). Our aimwas to characterize relationships that exist betweenROS, mitochondrial perturbations, Bcl-2 and caspases,depending on source and identity of ROS. First, we havefound that these five inducers trigger oxidative stress,mitochondrial membrane permeabilization (MMP), cy-tochrome c (cyt c) release from mitochondria and celldeath. In each case, cell death could be inhibited byseveral antioxidants, showing that it is primarily ROS

dependent. Second, we have highlighted that duringetoposide or TNF-α treatments, intracellular ROS level,MMP and cell death are all regulated by caspases andBcl-2, with caspases acting early in the process. Third,we have demonstrated that H2O2-induced cell deathshares many of these characteristics with etoposide andTNF-α, whereas t-BHP induces both caspase-dependent and caspase-independent cell death. Surpris-ingly, paraquat-induced cell death, which harbours somecharacteristics of apoptosis such as cyt c release andcaspase-3 activation, is not modulated by Bcl-2 andcaspase inhibitors, suggesting that paraquat also triggersnon-apoptotic cell death signals. On the one hand, theseresults show that endogenous or exogenous ROS cantrigger multiple cell death pathways with Bcl-2 andcaspases acting differentially. On the other hand, theysuggest that H2O2 could be an important mediator ofetoposide and TNF-α-dependent cell death since theseinducers trigger similar phenotypes.

Keywords Reactive oxygen species . Cell death .

Mitochondria . Bcl-2 . Caspases . Antioxidants

AbbreviationsΔΨm Mitochondrial membrane potentialcyt c Cytochrome ceto Etoposide

Cell Biol Toxicol (2012) 28:239–253DOI 10.1007/s10565-012-9219-9

V. Rincheval (*) :M. Bergeaud : L. Mathieu : J. Leroy :A. Guillaume :B. Mignotte :N. Le Floch : J.-L. VayssièreLaboratoire de génétique et biologie cellulaire, EA4589,Université de Versailles St Quentin-en-Yvelines,Bâtiment Fermat, 45 avenue des Etats-Unis,78035 Versailles cedex, Francee-mail: [email protected]

B. MignotteLaboratoire de génétique moléculaire et physiologique,Ecole Pratique des Hautes Etudes,45 avenue des Etats-Unis,78035 Versailles cedex, France

E/TNF Emetine plus TNF-αFSC Forward scatterMMP Mitochondrial membranes permeabilizationPI Propidium iodideROS Reactive oxygen speciesSOD Superoxide dismutaset-BHP tert-Butyl hydroperoxideTNF Tumour necrosis factorVit E Vitamin E

Introduction

Reactive oxygen species (ROS) are a family of highlyreactive molecules that includes hydroxyl radical(HO.), superoxide anion (O2

.−), hydrogen peroxide(H2O2) and organic peroxide radicals. Under physio-logical conditions, the mitochondrial respiratory chainis the major site for ROS production in cells (Fleury etal. 2002). ROS are extremely transient species due totheir high chemical reactivity that is responsible fortheir destructiveness on DNA, proteins, carbohydratesand lipids. It is now admitted that ROS are importantmediators of several types of cell death such as apo-ptosis and necrosis. Apoptosis is mediated by a familyof cystein proteases known as caspases. In mammals,there are two main pathways by which caspase acti-vation is triggered: the intrinsic and extrinsic apoptoticpathways. Various signals that trigger the intrinsicpathway (such as oxidative or genotoxic stress) aremainly transduced to mitochondria which then under-go a series of biochemical events resulting in mito-chondrial membranes permeabilization (MMP)(Jourdain and Martinou 2009; Kroemer et al. 2007)and release of pro-apoptotic molecules from mito-chondria, such as cytochrome c (cyt c; Li et al.1997). The extrinsic pathway is activated by bindingof ligands (such as Fas ligand or tumour necrosisfactor (TNF)-α) to their receptors on cell surface thatcan directly activate caspases (Thorburn 2004). Thispathway may also require the involvement of mito-chondria, notably through caspase-dependent produc-tion of the pro-apoptotic protein tBid (Li et al. 1998).The mitochondrial pathway is regulated by Bcl-2 fam-ily members, including both anti-apoptotic proteins(such as Bcl-2) and pro-apoptotic proteins (such asBax) which, respectively, repress or stimulate MMP

(Desagher and Martinou 2000; Wang and Youle2009).

A ROS-dependent cell death can be triggered byclassical apoptosis inducers such as etoposide (eto)and TNF-α, which, respectively, trigger the intrinsicand the extrinsic pathways of cell death. We havepreviously shown in HeLa cells that TNF-α- andetoposide-induced apoptosis involves early mitochon-drial ROS production that strongly accelerates theprocess of cell death (Dumay et al. 2006; Sidoti-deFraisse et al. 1998). eto is a topoisomerase II inhibitor,which triggers the intrinsic caspase activation path-way, involving mitochondrial perturbations (Karpi-nich et al. 2002), whereas TNF-α is a cytokine thattriggers the extrinsic caspase activation pathway butdoes not necessarily involve mitochondria (Thorburn2004). We have also used exogenous ROS or ROSgenerators to induce cell death in HeLa cells by incu-bating them with H2O2, paraquat and tert-butyl hydro-peroxide (t-BHP). Among ROS, H2O2 represents aparticularly important molecule because it is generatedunder nearly all oxidative stress conditions and it canparticipate in several fundamental intracellular pro-cesses. Paraquat is a non-selective herbicide whichundergoes redox cycling in vivo, being reduced byan electron donor such as NADPH, before being oxi-dized by an electron receptor such as O2 to producesuperoxide. Next, t-BHP is a short chain analogue oflipid hydroperoxides which mimics toxic effects ofperoxidized fatty acids.

In this study, we have compared several features ofcell death triggered by etoposide and TNF-α versusthe pro-oxidant molecules H2O2, t-BHP and paraquat.Our aim was to characterize relationships that existbetween ROS, mitochondrial perturbations, Bcl-2 andcaspases, depending on the origin and identity ofROS.

Materials and methods

Reagents

α-Tocopherol (vitamin E), butylated hydroxyanisole(BHA), catalase, diphenyleneiodonium chloride(DPI), emetine (E), eto, H2O2 solution, methyl violo-gen dichloride hydrate (paraquat), N-acetyl-cysteine(NAC), nordihydroguaiaretic acid (NDGA), propi-dium iodide (PI), 1,2-dihydroxybenzene-3,5-

240 Cell Biol Toxicol (2012) 28:239–253

disulfonic acid (tiron), mouse TNF-α and t-BHPsolution were from Sigma. 2′, 7′-Dichlorodihydro-fluorescein diacetate (DCFH-DA) and 3,3′-diethylox-acarbocyanine (DiOC6(3)) were from Invitrogen.z-Val-Ala-DL-Asp-Fluoromethylketone (zVAD-fmk)and N-Ac-Asp-Glu-Val-Asp-CHO (DEVD-CHO)were from Bachem. Stock solutions were prepared asfollows: NAC (50 mM), PI (1 mg/ml) and tiron (1 M)in water; catalase (105 U/ml), emetine (1 mg/ml),paraquat (100 mM) and TNF-α (1 μg/ml) in serum-free medium; BHA (200 mM), DCFH-DA (20 mM),DiOC6(3) (1 mM), NDGA (40 mM) and Vit E(100 mM) in ethanol; DPI (3 mM) and etoposide(25 mg/ml) in dimethyl sulfoxide and zVAD-fmk(50 mM) and DEVD-CHO (100 mM) in methanol.All stock solutions were stored at −20 °C.

Cell lines, cell culture and induction of cell death

HeLa and HeLa-Bcl-2 cell lines were cultured at 37 °Cin a humidified atmosphere containing 5 % CO2 inDulbecco’s modified Eagle’s medium (DMEM/F12)supplemented with 10 % foetal bovine serum togetherwith penicillin (100 μg/ml), streptomycin (100 U/ml)and glutamax (1 %, v/v) from Invitrogen. HeLa-Bcl-2cell line contains the human bcl-2 cDNA under controlof the Tet Off inducible expression system (Clontech).These cells were initially cultured in the presence oftetracycline (2 μg/ml) to inhibit expression of exoge-nous Bcl-2 and then propagated for 10 days withouttetracycline to allow accumulation of the Bcl-2 proteinprior the experiments. Cell death was induced byaddition on exponentially growing adherent cells ofH2O2 (200 μM), paraquat (5 mM), t-BHP (200 μM),etoposide (20 μg/ml) or TNF-α (2.5 ng/ml) plus em-etine (1 μg/ml). After a 24-h incubation, cells wereanalysed by flow cytometry. Antioxidants (BHA(300 μM), catalase (1,000 U/ml), DPI (2.5 μM),NAC (5 mM), NDGA (40 μM), tiron (1 mM), Vit E(100 μM)) or caspase inhibitors (DEVD-CHO(300 μM), zVAD-fmk (50 μM)) were added at thesame time than cell death inducers.

Flow cytometry

Flow cytometric measurements were performed usinga XL3C flow cytometer (Beckman-Coulter). Fluores-cence was induced by the blue line of an argon ionlaser (488 nm) at 15 mW. Green and red fluorescence

were, respectively, collected with 525 and 620 nmband-pass filters. Analyses were performed on104 cells. Mitochondrial membrane potential (ΔΨm)was assessed by retention of the green fluorescentDiOC6(3). This cationic lipophilic fluorochrome is acell permeable marker that, at low doses, specificallyaccumulates in mitochondria depending on ΔΨm(Petit et al. 1990). Intracellular ROS level was mea-sured by the production of dichlorofluorescein (DCF)derived from oxidization of DCFH. Non-fluorescentDCFH-DA is a freely permeable tracer which isdeacylated by intracellular esterases to the non-fluorescent compound DCFH and oxidized to thegreen fluorescent compound DCF by a variety ofperoxides, including hydrogen peroxide (Ubezio andCivoli 1994). The red fluorescent PI specifically pen-etrates into cells which have lost their plasma mem-brane integrity and was therefore used to characterizenecrotic cells (Darzynkiewicz et al. 1994). The de-crease in forward scatter (FSC) that reflects cellshrinkage during several types of cell death was alsoassessed (Dive et al. 1992). The percentage of non-necrotic cell death was calculated as the percentage ofcell shrinkage in the PI-negative population (Fig. 1,population h). After drug treatment, media from cul-ture dishes (containing late apoptotic cells) were kept

Fig. 1 Effects of apoptosis inducers and exogenous ROS on amitochondrial membrane potential (ΔΨm), b cells with highintracellular ROS level and c cell death. HeLa cells were incu-bated with the following drugs: H2O2 (200 μM), paraquat(5 mM), t-BHP (200 μM), etoposide (20 μg/ml), emetine(1 μg/ml) plus TNF-α (2.5 ng/ml), during 24 h and analysedby flow cytometry (see “Materials and methods”). Typical dotplots are indicated on the right panel (control versus etoposide)for each labelling. a For DiOC6(3) fluorescence analysis, cellswith high (a) and low (b) fluorescence were distinguished. Thepercentages of cells with low ΔΨm (population b) are indicatedin the bar graph on the left panel. b Concerning intracellularROS measurements (DCF fluorescence), normal cells (d), cellswith high intracellular ROS level (c) and late dead cells whichcannot produce DCF fluorescence (e) were distinguished. Thepercentages of cells with increased intracellular ROS levels(population c) are indicated in the bar graph on the left panel.c For PI staining, normal cells (g) were distinguished from non-necrotic dead cells (h) (reduced forward scatter and low PIfluorescence) and from necrotic cells (f) (increased PI fluores-cence). Total percentages of cell death are indicated in the bargraph on the left panel as the sum of necrotic (population f) andnon-necrotic cell death (population h). These results come fromthree independent experiments. Post hoc ANOVA indicates thatthe decrease in ΔΨm, increase in ROS level and cell death(necrotic and non-necrotic) triggered by the five inducers aresignificantly different from controls

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in centrifuge tubes. Adherent cells (containing livingand early apoptotic cells) were detached using trypsinand pooled with the corresponding media, centrifugedand resuspended in complete medium at a concentra-tion of 1×106 cells/ml. Cells were then separated intwo different tubes and loaded with 0.1 μM DiOC6(3)(tube 1) or 20 μM DCFH-DA (tube 2) for 30 min at37 °C (DiOC6(3) and DCFH-DA both generate greenfluorescence and cannot be mixed together). Tube 1was also incubated with 10 μg/ml PI, 5 min prioranalysis. DiOC6(3), DCFH-DA and PI staining havebeen performed with unfixed cells. All results ana-lysed by flow cytometry come from three independentexperiments.

Western blot analysis

After treatments, cells were harvested with a scraper,washed with phosphate-buffered saline (PBS) and sus-pended in lysis buffer (50 mM Tris pH 6.8, 2 %sodium dodecyl sulphate, 50 mM DTT). Protein con-centration was determined using the Bio-Rad proteinassay. Forty micrograms per well of the boiledsamples was loaded onto a 4–12 % NuPage gel(Invitrogen). After migration, proteins were electro-transferred onto a polyvinylidene fluoride (PVDF)membrane (Millipore). Membranes were blocked withTBS-Tween 0.5 % (v/v) and 5 % (w/v) fat milk byincubation for 1 h, and proteins were incubated over-night with specific primary antibodies: 1/500 goat anti-Cu/ZnSOD (N-19, Santa Cruz), 1/500 mouse anti-GPX(B-6, Santa Cruz), 1/500 rabbit anti-MnSOD (06-984,Millipore), 1/200 rabbit anti-actin (A2066, Sigma),1/200 rabbit anti-pro-caspase-3 (H277, Santa Cruz)and 1/1,000 rabbit anti-cleaved caspase-3 (Asp175, CellSignaling). After washing with TBS-Tween, mem-branes were incubated for 1 h with horseradishperoxidase-conjugated secondary antibodies (JacksonImmunoResearch Laboratories). Proteins were detectedusing the Immobilon detection kit (Millipore) under aChemidoc imager (Bio-Rad).

Immunofluorescence

Cells were grown on glass coverslips. To follow cyt crelease from mitochondria, cells were fixed with PBS–paraformaldehyde 3.7 % for 15 min at room tempera-ture, washed with PBS and permeabilized with PBS–bovine serum albumin (BSA) 1 % (w/v)–Tween 0.5 %

(v/v) for 10 min. Cells were incubated for 2 h with the1/500 rabbit mitochondrial ISP protein (gift fromC. Godinot (Lyon, France)) and the 1/500 mouseanti-cyt c (clone 6H2.B4, BD Pharmingen) primaryantibodies in PBS-BSA-Tween, washed with PBS andthen incubated with 1/200 fluorescein isothiocyanate-conjugated anti-mouse Ig and 1/200 rhodamine-conjugated anti-rabbit Ig (Jackson ImmunoResearchLaboratories) for 1 h. Nuclei were stained by incubat-ing coverslips with To-Pro-3 (Invitrogen, 0.5 μM inTBS) for 5 min, and cells were washed with PBS.Coverslips were then mounted in a glycerol/PBS so-lution (Citifluor AF1), and cells were examined byconfocal microscopy under a Leica SPE microscopeand photographed. We counted about 300 cells fromthree independent experiments for each point to eval-uate the percentage of cells with relocalized cyt c.

Statistical analysis

Raw data were analysed by ANOVAwith Bonferroni–Holm post hoc testing after checking the assumptionof equal variance with Levene’s test. P<0.05 wasaccepted as significant.

Results

The apoptosis inducers etoposide and TNF-α,the exogenous ROS H2O2 and t-BHP as wellas the ROS generator paraquat all triggerintracellular ROS level increase, mitochondrialmembrane permeabilization and cell death

We have previously shown that etoposide and TNF-αtrigger endogenous mitochondrial ROS production inHeLa cells (Dumay et al. 2006; Sidoti-de Fraisse et al.1998) whereas H2O2, paraquat and t-BHP are oftenused as inducers of oxidative stress and cell death.TNF-α used alone is generally not sufficient to killcultured cells since the interaction between TNF-αand its receptors induces antagonistic signals: the for-mation of the death inducing signalling complex(DISC) which rapidly activates caspases and expres-sion of endogenous caspase inhibitors through theactivation of NF-κB (Nagata 1997). Thus, TNF-α iscurrently used in vitro in combination with a proteinsynthesis inhibitor such as E (emetine), which is able


to inhibit the NF-κB-dependent expression of caspaseinhibitors, without interfering with DISC formation.

Since we wanted to test the influence of ROS onmitochondrial perturbations, we started to determinethe concentrations of the different compounds (eto, em-etine plus TNF-α (E/TNF), H2O2, paraquat and t-BHP)that induce a significant drop inΔΨm (see Fig. 1 legendfor concentration values). Figure 1a shows that after a24-h incubation with these compounds, approximatelyone half of the cells stained with DiOC6(3) exhibits alow ΔΨm (population “b”), indicating that they haveundergone a permeabilization of their mitochondrialmembranes (MMP). These conditions were consideredas ideal to study inhibitory or promoting effects onMMP. Next, we have measured the percentage of cellsharbouring high intracellular ROS level (population“c”), corresponding to cells undergoing oxidative stress.Figure 1b indicates that etoposide, E/TNF and paraquatcan trigger accumulation of endogenous ROS and thatexogenous H2O2 and t-BHP also provoke an efficient,but probably more direct, oxidative stress. Accumula-tion of ROSwas measured by using DCFH-DA, a probewhich becomes fluorescent after reacting with perox-ides. Since peroxides are classical by-products of oxi-dant molecules, DCFH-DA is a good probe to detectoxidative stress from different origins. However, it mustbe kept in mind that its modification requires a deace-tylation process prior oxidation, which cannot be per-formed in late dead cells with inactive esterases, such asin population “e” (see Fig. 1b). Thus, cells undergoingoxidative stress are always underestimated with thisprobe when cell death occurs. Next, we were also inter-ested in the events that could occur downstream ofmitochondrial perturbations (such as cell death), andwe have characterized two additional parameters byflow cytometry. The first parameter is the percentageof PI-positive/necrotic cells (Fig. 1c, population “f”). Inthis experiment, PI was used as a non-permeant plasmamembrane probe, which has to be used with unfixedcells. Thus, only necrotic cells, which have lost theirplasma membrane integrity, were stained with PI. Thesecond parameter is the percentage of PI-negative cellswith reduced size which indicates the percentage of non-necrotic cell death (Fig. 1c, population “h”). Apoptosisand autophagy are typical examples of non-necrotic celldeath, during which plasma membrane integrity is pre-served. Interestingly, each type of cell death is charac-terized by a more or less pronounced reduction of cellsize: cellular condensation and fragmentation during

apoptosis, ghost cells obtained after explosion of plasmamembrane during necrosis and self-digestion of cellsduring autophagy. Figure 1c shows that all inducerstrigger both necrotic and non-necrotic cell death. Thefact that specific inducers of apoptosis (etoposide,E/TNF) also trigger necrosis may appear surprising.However, it is well-known that a secondary necroticprocess provoked by the lack of engulfment of dyingcells can occur in vitro. Primary and secondary necrosiscan be distinguished by the following specificities: Pri-mary necrosis is characterized by an early loss of plasmamembrane integrity (assessed by PI labelling) which isconcomitant to MMP. This necrosis cannot be inhibitedby specific inhibitors of apoptosis such as caspase inhib-itors. Secondary necrosis occurs in vitro in late apoptoticdead cells (which are not still necrotic) because of a lackof cell engulfment by neighbouring cells or macrophages.Late apoptotic cells loose physiological regulationswhich are necessary to preserve their plasma membraneintegrity and finally become necrotic. Therefore, second-ary necrosis occurs as a consequence of apoptosis andcan be decreased in the presence of caspase inhibitors. Itmay be surprising that we have never described second-ary necrosis in previous publications concerning etopo-side and E/TNF-induced cell death, but this is simply dueto the fact that we did not probe cells with PI at this time.

Etoposide, E/TNF, H2O2, paraquat and t-BHP allinduce a ROS-dependent cell death which can beinhibited by several antioxidants

Although all inducers seemed to trigger an oxidativestress, MMP and death (see Fig. 1), we first wanted to

Fig. 2 Inhibition of etoposide, E/TNF, H2O2, paraquat and t-BHP-induced cell death by antioxidants. HeLa cells were treatedwith the following antioxidants: BHA (300 μM), catalase(1,000 U/ml), DPI (2.5 μM), NAC (5 mM), NDGA (40 μM),tiron (1 mM) and Vit E (100 μM) during 24 h. a The percentageof DCF fluorescence inhibition was calculated from mean fluo-rescence intensities obtained with cells treated and untreatedwith the antioxidants (without cell death inducers). b Flowcytometric overall cell death measurements were performed byquantifying cells that harboured small cell size (low FSC):Necrotic and non-necrotic cell death were not distinguished inthis approach. c Antioxidant treatments were combined with theincubation of the five cell death inducers: H2O2, paraquat,t-BHP, etoposide or E/TNF during 24 h, with the same concen-trations than in Fig. 1. The results come from three independentexperiments. Asterisk indicates a significant difference revealedby post hoc ANOVA, between antioxidant-treated cells andappropriate solvent-treated cells

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confirm that each cell death was ROS dependent. Forthis purpose, we have incubated cells with both celldeath inducers and antioxidants. Here, we have used

seven antioxidants with distinct properties. BHA is afood additive (E320) which has also been shown toinhibit mitochondrial respiration (Aldunate et al.

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1992); catalase is a well-known enzyme which detoxi-fies H2O2 into H2O plus O2; DPI is a NADPH oxidaseinhibitor (Meier et al. 1991); NAC is a precursor toglutathione, a natural antioxidant protein; NDGA is acyclooxygenase and lipoxygenase inhibitor (VanWauwe and Goossens 1983); tiron is a superoxidescavenger (Greenstock and Miller 1975) and vitaminE is an inhibitor of lipid peroxidation (Tappel 1970).All antioxidants concentrations that were used corre-sponded to the highest concentrations that do notprovoke cellular toxicity.

We first checked antioxidant activity of the sev-en compounds by comparing average values ofDCF fluorescence intensities in control cells(corresponding to population “d” in Fig. 1b) incu-bated or not with these antioxidants. Figure 2ashows that all antioxidants significantly inhibitDCF fluorescence intensities at concentrations used,confirming their expected function (note thatFig. 1b indicates percentages of cells with highDCF fluorescence and not DCF fluorescence inten-sities values, as in Fig. 2a). Next, we have quanti-fied cell death as the percentage of cells withreduced forward scatter (see Fig. 2b) in the pres-ence or in the absence of an antioxidant and calcu-lated the percentage of cell death inhibition. Theresults presented in Fig. 2c show that the effect ofeach cell death inducer is inhibited by at least threedifferent antioxidants, demonstrating that cell deathanalysed here is tightly linked to oxidative stress.Some results were expected: Catalase stronglyinhibits H2O2-induced cell death, DPI strongly inhibitsthe effect of paraquat (mediated by NADPH oxidases),vitamin E inhibits toxicity induced by t-BHP (an ana-logue of lipid hydroperoxides) and BHA inhibitsmitochondrial-dependent production of ROS inducedby etoposide and E/TNF. Other results are more surpris-ing and will be further commented in the “Discussion”section.

Influence of Bcl-2 on MMP, intracellular ROS leveland cell death

In order to test the effect of the anti-apoptotic proteinBcl-2 on phenotypes triggered by the five inducers, wehave used a HeLa-Bcl-2 cell line which can overex-press the human bcl-2 gene under control of tetracy-cline (Tet Off system from Clontech). With thissystem, the absence of tetracycline in culture medium

leads to the accumulation of Bcl-2. The results pre-sented in Fig. 3a show that MMP is significantlyinhibited in all cases except for paraquat, demonstrat-ing that MMP triggered by paraquat is based on adistinct mechanism. Next, we can see in Fig. 3b thatBcl-2 significantly inhibits accumulation of cells withhigh ROS level with H2O2, t-BHP, etoposide andE/TNF but not with paraquat. Interestingly, it has beenshown that ROS can act upstream or downstreammitochondrial perturbations such as MMP and cyt crelease (Fleury et al. 2002). Here, it is tempting topostulate that Bcl-2 could act directly or indirectly asan antioxidant to decrease upstream ROS duringH2O2, t-BHP, etoposide and E/TNF-induced celldeath. However, we cannot reject that ROS measured24 h after beginning of treatments would rather bedownstream by-products of cell death, indirectlyinhibited via the anti-apoptotic functions of Bcl-2.This point will be further analysed in the “Discussion”section. Concerning the regulation of cell death, itappears from Fig. 3c that Bcl-2 significantly inhibitsboth necrotic and non-necrotic cell death for all celldeath inducers except for paraquat, showing thatparaquat-induced cell death is not Bcl-2 regulated.

Influence of Bcl-2 on endogenous levels of antioxidantenzymes and cytochrome c localization

Since our results suggested a possible antioxidantfunction for Bcl-2, we have checked if Bcl-2 was ableto regulate the levels of major endogenous antioxidantenzymes, such as mitochondrial Mn superoxide dis-mutase (MnSOD), glutathione peroxidase (GPX) andcytosolic Cu/Zn superoxide dismutase (Cu/ZnSOD).We have then harvested cell lysates from HeLa-Bcl-2cells, treated or not with the five cell death inducers,and performed western blot analysis to characterizethe levels of these enzymes (see “Materials andmethods”). Figure 4 shows that control cells overex-pressing or not Bcl-2 exhibit same levels of MnSOD,GPX and Cu/ZnSOD, indicating that Bcl-2 does notdirectly regulate the levels of these three enzymeswhen cell death is not induced. Nevertheless, ifMnSOD level seems to be rather stable, it can be seenthat GPX level is upregulated by H2O2 and etoposide.However, it can be noticed for both inducers that theratio between GPX and actin is stable in ± Bcl-2overexpressing cells, suggesting that GPX is not re-sponsible for the decrease in ROS level that was


previously observed (Fig. 3b). Next, it can benoticed that Cu/ZnSOD is upregulated by paraquatand E/TNF. Interestingly, Cu/ZnSOD level seemsto specifically increase in Bcl-2 overexpressingcells during E/TNF-induced cell death (compareCu/ZnSOD and actin levels). An increase inCu/ZnSOD protein level could therefore accountfor an antioxidant function indirectly involvingBcl-2 during E/TNF-induced cell death (Fig. 3b).Such an activity is not revealed for H2O2, t-BHPand etoposide-induced cell death.

The five cell death inducers that we have used inthis article trigger MMP, a process that can be accom-panied by the release of pro-apoptotic proteins from

mitochondria, such as cyt c. Figure 5a shows repre-sentative pictures of both cyt c and mitochondria la-belling, which were taken to follow a potential releaseof cyt c. Nuclei staining was also realized to charac-terize nuclear morphology, which can be indicative ofapoptotic cell death. These experiments were per-formed in HeLa-Bcl-2 cells (± Bcl-2), with or withoutinduction of cell death. In each picture of treated cells,an arrow shows an example of a cell harbouring dif-fuse/cytosolic cyt c labelling, which is different fromthe punctated aspect of mitochondrial labelling.Figure 5a shows that cells harbouring released cyt ccan be observed with the five cell death inducers. Acount of these events indicates that the percentage of

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- + - + - + - + - + - + :Bcl-2 Control H2O2 Paraquat t-BHP eto E/TNF



Fig. 3 Regulation of etopo-side, E/TNF, H2O2, paraquatand t-BHP-induced celldeath by Bcl-2. HeLa-Bcl-2cells overexpressing (+) ornot (−) the anti-apoptoticprotein Bcl-2 were used inthis study (see “Materialsand methods”). Cells weretreated during 24 h with thesame concentrations ofinducers than in Fig. 1 andwere analysed by flowcytometry. The bar graphsindicate the percentages ofcells with low ΔΨm (a),high intracellular ROS level(b) as well as necrotic andnon-necrotic cell death (c).These results come fromthree independent experi-ments. Asterisk indicates asignificant differencerevealed by post hocANOVA between cellsoverexpressing (+) or not(−) Bcl-2. In c, doubleasterisks indicate a signifi-cant difference for both ne-crotic and non-necrotic celldeath


cells with released cyt c (Fig. 5b) is almost the samefor H2O2, paraquat, t-BHP and etoposide (approxi-mately 45 % after a 24-h treatment) whereas there isa smaller percentage for E/TNF (35 %). Figure 5b alsoshows that Bcl-2 significantly inhibits cyt c releasewith each cell death inducer, but with different inten-sities. We have calculated the percentage of Bcl-2-dependent inhibition of cyt c release and shown inFig. 5c that Bcl-2 is less efficient to inhibit this phe-nomenon during paraquat-induced cell death than forthe four other inducers. Another particularity ofparaquat-induced cell death is the nuclear morphologywhich does not show fragmented, apoptotic-like nu-clei, but only condensed chromatin, suggesting thatcells treated with paraquat do not undergo apoptosis.These results show once again that paraquat-inducedcell death regulation is different from H2O2, etoposideand E/TNF. The situation for t-BHP seemed to beintermediate, since condensed and fragmented chro-matin could both be observed.

Influence of caspase inhibition on MMP, intracellularROS level and cell death

Here we have used two chemical inhibitors to determinethe role of initiator and executive caspases: zVAD (Z)which is an inhibitor of both initiator and executivecaspases as well as DEVD (D) which is a specific

inhibitor of the executive caspases −3 and −7. We havefirst tested the efficiency of both inhibitors on caspase-3processing and activation. Caspase-3 is synthesized as a32-kDa proenzyme which can be cleaved into an inter-mediate form of 19 kDa and the 17-kDa subunit (p17) offully active caspase-3. A lack of p17 attests for anincomplete processing and the absence of fully activecaspase-3. Figure 6a shows that p17 is absent in controlcells whereas it is produced after incubation with eachone of the five inducers. This result may seem logical, ifwe take into account that released cyt c, which wasobserved for all inducers, can trigger the activation ofa caspase cascade. Interestingly, Fig. 6a shows that bothzVAD and DEVD are active at used concentrationsbecause they efficiently inhibit production of p17. Next,Fig. 6b shows that zVAD significantly decreases MMPduring H2O2, etoposide and E/TNF-induced cell death,indicating that caspases regulate MMP, acting upstreamof or at mitochondria. The effect of DEVD on etoposideand E/TNFwas weaker than the effect of zVAD and wasnot significant with post hoc ANOVA. Concerning para-quat and t-BHP-induced cell death, it appears that MMPis not influenced by the presence of zVAD or DEVD,suggesting that this process is caspase-independent anddistinct from the processes observed for H2O2, etopo-side and E/TNF. Caspase inhibitors were also found toinfluence accumulation of cells with high intracellularROS level (Fig. 6c) for H2O2, etoposide and E/TNF.zVAD strongly decreases ROS level with etoposide andE/TNF, suggesting that endogenous ROS productionfirst requires the activation of caspases. The mildereffect of DEVD was not found to be significant foretoposide and E/TNF. In contrast to H2O2, etoposide

GPX

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Fig. 4 Regulation of MnSOD, GPX and Cu/ZnSOD proteinlevels after treatment with H2O2, t-BHP, paraquat, etoposide orE/TNF in HeLa-Bcl-2 cell line. HeLa-Bcl-2 cells overexpress-ing (+) or not (−) Bcl-2 were treated during 24 h with the sameconcentrations of inducers than in Fig. 1, and cellular extractswere analysed by western blot (see “Materials and methods”) tocharacterize protein levels of MnSOD, GPX and Cu/ZnSOD.Actin was used as a loading control. Representative picturestaken from one of three independent experiments are shown

Fig. 5 Analysis of cytochrome c release from mitochondriaafter treatment with H2O2, t-BHP, paraquat, etoposide orE/TNF in HeLa-Bcl-2 cell line. HeLa-Bcl-2 cells overexpress-ing or not Bcl-2 were grown on glass coverslips and treatedduring 24 h with the same concentrations of cell death inducersthan in Fig. 1. Cells were then fixed, permeabilized, stained forcyt c, mitochondria (anti-mitochondrial ISP protein antibody)and nuclei (see “Materials and methods”) and analysed byconfocal microscopy (a). In each picture (excepting controls),an arrow indicates one example of a cell harbouring cytosoliccyt c. Cells harbouring released cyt c were counted, and thepercentage of cells with released cyt c is indicated (b). Thepercentage of Bcl-2-dependent inhibition of cyt c release foreach cell death inducer is also shown (c). Asterisk indicates asignificant difference revealed by post hoc ANOVA betweencells overexpressing or not Bcl-2 for a specific inducer in b anda significant difference between distinct inducers in c. Theseresults come from three independent experiments

b


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and E/TNF, caspase inhibitors do not modify intra-cellular ROS level during paraquat or t-BHP treat-ments, indicating that these processes are alsocaspase independent. Next, Fig. 6d shows that cas-pase inhibitors attenuate necrotic cell death forH2O2, etoposide and E/TNF, but not for paraquatand t-BHP, pointing out that a secondary necrosisprocess occurs with these first three inducers. Thecaspase inhibitors also diminish non-necrotic celldeath for all the inducers except paraquat. In

conclusion, these results indicate that paraquat-induced cell death is caspase independent and thatt-BHP induces both apoptosis and primary necrosis,in agreement with nuclear morphology observed inFig. 5a. The fact that caspase-3 is activated duringparaquat-induced cell death which is caspase inde-pendent is surprising. However, this could beexplained by the fact that paraquat could also triggernon-apoptotic cell death signals which do not re-quire caspase activity.

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Fig. 6 Regulation of etopo-side, E/TNF, H2O2, paraquatand t-BHP-induced celldeath by caspase inhibitors.HeLa cells incubated withthe five inducers were trea-ted at the same time withcaspase inhibitors: zVAD-fmk (Z) which inhibits bothinitiator and executive cas-pases or DEVD-CHO (D)which inhibits executivecaspases, or no treatment(Ø). zVAD-fmk and DEVD-CHO were, respectively,used at 50 and 300 μM. Af-ter 24 h, flow cytometryanalysis was performed andcell lysates were harvested.a Western blot was per-formed to analyse process-ing and activation ofcaspase-3 (see “Materialsand methods”). The bargraphs indicate the percen-tages of cells with lowΔΨm(b), high intracellular ROSlevel (c) as well as cell death(d). These results come fromthree independent experi-ments. Asterisk indicates asignificant differencerevealed by post hocANOVA between controlsand zVAD or DEVD. In d,double asterisks indicate asignificant difference forboth necrotic and non-necrotic cell death whereassingle asterisk indicates asignificant difference fornon-necrotic cell death butnot for necrosis


Discussion

In this study, we have compared several features ofcell death triggered by classical inducers of apoptoticpathways (etoposide and TNF-α) versus exogenousROS (H2O2, t-BHP) or a ROS generator (paraquat).Etoposide and E/TNF induce the most similar pheno-types despite the fact that they have quite differenttargets. H2O2-induced cell death shares many charac-teristics with etoposide and TNF-α, with caspasesacting upstream MMP and ROS production whereast-BHP induces both caspase-dependent and caspase-independent cell death. Paraquat seemed to be themost particular one, with MMP, ROS production andcell death which were apparently not regulated by Bcl-2 and caspases.

We have shown that cell death triggered by the fiveinducers could be inhibited by several antioxidants(Fig. 2), demonstrating that all these processes areprimarily ROS dependent. Some results were expectedwhereas others results were more surprising. For in-stance, it was astonishing that NAC had differentialeffects on etoposide and E/TNF whereas we havepreviously shown that these inducers both trigger mi-tochondrial production of ROS (Sidoti-de Fraisse et al.1998 and unpublished data). It is also surprising thatetoposide and E/TNF-induced cell death are notinhibited by catalase. Besides, the fact that tiron doesnot inhibit paraquat-induced cell death was most un-expected and had been the object of numerousattempts. We propose that these surprising resultsmight be the consequence of at least two typesof events, such as differential interconversion ofROS and undescribed side effects of chemical com-pounds (e.g. antioxidants). In the first case (intercon-version), similar ROS could be primarily produced butrapidly transformed into distinct new species, trigger-ing differential responses to specific antioxidants.Concerning the lack of effect of catalase on E/TNFand etoposide, it is possible that both superoxide andH2O2 are too rapidly converted into hydroxyl radicalto be detoxified. In the second case (side effects),which may concern tiron, we have tested this antiox-idant coming from different suppliers, with concentra-tions ranging from 1 μM to 10 mM with no protectiveeffect on paraquat-induced cell death. We have postu-lated that conversion of superoxide generated byNADPH oxidase into H2O2 was too rapid to beinhibited by tiron, and we have tested the effect of

tiron in the presence of a Cu/Zn superoxide dismutaseinhibitor (DDC 500 μM), but still with no success.Finally, we tried to find publications describing aninhibition of paraquat-induced cell death by tiron andnoticed that such data seemed to be missing from theliterature. During our numerous experimental trials,we have observed that sub-toxic concentrations ofparaquat combined with sub-toxic concentrations oftiron could provoke massive cell death (data notshown), suggesting that tiron could react directly orindirectly with paraquat to trigger an uncharacterizedtoxic effect, possibly masking its known function onsuperoxide. Despite this unexplained result, the factthat paraquat-induced cell death is strongly inhibitedby the NADPH oxidase inhibitor DPI (and by otherantioxidants) is, however, a result which strongly sug-gests that paraquat exerts its normal functions in ourexperimental conditions.

Next, we have studied the effect of Bcl-2 andshown that this protein inhibits MMP, cyt c releaseand overall cell death with all the inducers that wereused, except for paraquat. An intriguing resultconcerning Bcl-2 is its ability to decrease the percent-age of cells with high intracellular ROS level (Fig. 3b).Since etoposide and E/TNF trigger production of en-dogenous mitochondrial ROS, this suggests that Bcl-2could act on mitochondrial production of ROS. Fur-thermore, the fact that Bcl-2 inhibits oxidative stressafter exposition to H2O2 and t-BH (which are exoge-nous ROS) suggests that Bcl-2 could also directly orindirectly act as an antioxidant. Nevertheless, we can-not reject that ROS measured 24 h after beginning oftreatments would rather be downstream by-products ofcell death, indirectly inhibited via the anti-apoptoticfunctions of Bcl-2. Kinetic studies of ROS productionwould probably help to understand how Bcl-2 actuallyexerts this function. The use of HeLa ρ0 cells and/orantioxidants specifically inhibiting mitochondrialROS production, such as BHA (Sidoti-de Fraisse etal. 1998), would probably also be very informative.Next, it appears from Fig. 4 that particular stresses canincrease the level of specific antioxidant proteins.H2O2 and etoposide increase GPX level whereas para-quat and E/TNF increase Cu/ZnSOD level. The resultsshown in this figure finally suggest that Bcl-2 wouldhave at least an indirect antioxidant function duringE/TNF-induced cell death, since Bcl-2 would allow animportant accumulation of Cu/ZnSOD with thisinducer.


During our study, we have also characterized therelease of cyt c from mitochondria in HeLa cells over-expressing or not Bcl-2, and several observationscould be made from Fig. 5. First, we observed thatcyt c is released after an incubation with each celldeath inducer. This was one rare parameter, withMMP, that paraquat seems to share with otherinducers, suggesting that MMP and cyt c release areoften tightly linked. Second, paraquat seemed to bedissimilar to other inducers since its induction of cyt crelease is less inhibited by Bcl-2 (Fig. 5c). We did notfurther examine the possible causes of this phenome-non, notably because the formation of mitochondrialpores are still a matter of debate and are very difficultto characterize. However, it is possible that paraquatinduces the formation of distinct mitochondrial poresfrom the other inducers. In this case, Bcl-2 mightpartially or selectively close these pores, enough toinhibit cyt c release but insufficiently to inhibit protonleakage and MMP.

Immunofluorescence experiments also gave us theopportunity to follow the nuclear morphology duringcell death induced by the five inducers (Fig. 5a).While it is obvious that H2O2, etoposide and E/TNFtrigger a nuclear fragmentation, which is typical ofapoptosis, paraquat only triggers nuclear condensa-tion, confirming that paraquat-induced cell death isdifferent from the last three inducers. Besides, thenuclear phenotype induced by t-BHP seemed to beintermediate, with both condensed paraquat-like nu-clei and apoptotic fragmented nuclei, suggesting thatdifferent types of cell death could simultaneouslyoccur with this inducer.

Apart from nuclear morphology, caspase activity isanother criterion which is well admitted to be typicalof apoptotic cell death. By checking the activity ofzVAD and DEVD, we have observed that activecaspase-3 was produced in cells treated with the fiveinducers (Fig. 6a). While this observation wasexpected for H2O2, etoposide and E/TNF, this resultwas more surprising for paraquat, since several resultssuggested that paraquat did not trigger apoptosis. Fur-ther analysis of zVAD and DEVD treatments onparaquat-induced cell death indicated that zVAD andDEVD had no effect on MMP, ROS production andcell death, demonstrating that paraquat does not in-duce apoptosis in our system. This interesting resultled us to postulate that caspases could also be activat-ed in non-apoptotic cell death, suggesting that other

molecules or processes would mediate the executionof cell death. We then hypothesized that the release ofcyt c from mitochondria during paraquat treatmentwas sufficient to trigger a caspase cascade leading tocaspase-3 activation.

Another aspect of our study was to characterizeboth necrotic and non-necrotic cell death. It is impor-tant to note that two types of necrotic cell death canoccur in cultured cells: primary necrosis and second-ary necrosis. Primary necrosis is characterized by anearly loss of plasma membrane integrity which isconcomitant to MMP and cannot be inhibited by spe-cific inhibitors of apoptosis such as caspase inhibitors.Secondary necrosis occurs in vitro in late apoptoticcells which loose physiological regulations to preservetheir plasma membrane integrity and finally becomenecrotic. Secondary necrosis is inhibited by specificinhibitors of apoptosis, such as caspase inhibitors. Inter-estingly, we have observed after a 72-h treatment withthe five inducers that 100 % of the cells were necrotic,harbouring low forward scatter and high PI fluorescence(data not shown). This indicates that a final necroticprocess is inevitable for dying cells in culture. Theresults obtained in Fig. 6 provide precious informationconcerning the types of cell death that were inducedwith the five inducers. They showed that H2O2, etopo-side and E/TNF triggered both apoptosis (confirmed bynuclear morphology) and secondary necrosis, since thislast process is inhibited by zVAD and DEVD (Fig. 6d).They also showed that paraquat induces primary necro-sis (which is not inhibited by caspase zVAD and DEVD)and caspase-independent non-necrotic cell death, whichcould be autophagy or perhaps an aborted apoptosis.Finally, t-BHP seems to trigger both apoptotic cell death(inhibited by zVAD and DEVD) and primary necrosiswhich would be in agreement with both fragmented andcondensed nuclei observed in Fig. 5a.

In conclusion, this study allowed us to distinguishbetween three categories of ROS-dependent cell deathinducers: first, H2O2, etoposide and E/TNF whichtrigger an apoptotic process characterized by a Bcl-2and caspase-regulated MMP; second, paraquat whichtriggers a caspase-independent cell death that remainsto be identified, as well as primary necrosis and third,t-BHP which induces Bcl-2 and caspase-regulated ap-optosis as well as primary necrosis. On the one hand,these results show that endogenous or exogenous ROScan trigger multiple cell death pathways with Bcl-2and caspases acting differentially. On the other hand,


they suggest that H2O2 could be an important mediatorof etoposide and TNF-α since these inducers triggersimilar phenotypes.

Acknowledgments This work was supported in part by grantsfrom the Ligue Nationale Contre le Cancer (comité des Yvelines).

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