The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Toxicology and Applied Pharmacology 253 (2011) 282–289

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Toxicology and Applied Pharmacology

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The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane:Ensuing energetic and oxidative stress implications

Gilberto L. Pardo-Andreu a,c,⁎, Yanier Nuñez-Figueredo b, Valeria G. Tudella c, Osmany Cuesta-Rubio d,Fernando P. Rodrigues c, Cezar R. Pestana c, Sérgio A. Uyemura e, Andreia M. Leopoldino e,Luciane C. Alberici c, Carlos Curti c

a Centro de Estudio para las Investigaciones y Evaluaciones Biológicas, Instituto de Farmacia y Alimentos, Universidad de La Habana, ave. 23 # 21425 e/214 and 222, La Coronela, La Lisa,CP 13600, Ciudad Habana, Cubab Centro para las Investigaciones y Desarrollo de Medicamentos, Ave 26, No. 1605 Boyeros y Puentes Grandes, CP 10600, Ciudad Habana, Cubac Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café s/n, 14040-903 Ribeirão Preto, SP, Brazild Departamento de Química, Instituto de Farmacia y Alimentos, Universidad de La Habana, ave. 23 # 21425 e/214 and 222, La Coronela, La Lisa, CP 13600, Ciudad Habana, Cubae Departamento de Analises Clinicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café s/n,14040-903 Ribeirão Preto, SP, Brazil

⁎ Corresponding author at: Centro de Estudio para lasBiológicas, Instituto de Farmacia y Alimentos, Univers21425 e/214 and 222, La Coronela, La Lisa, CP 13600, Ciu2736811.

E-mail address: [email protected] (G.L.

0041-008X/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.taap.2011.04.011

a b s t r a c t

Article history:Received 14 December 2010Revised 2 April 2011Accepted 18 April 2011Available online 27 April 2011

Keywords:Guttiferone-AMitochondriaMitochondrial membrane permeabilizationUncouplingOxidative stressHepG2 cellsAnti-cancer action

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro andanti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1–25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energizedmitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrialmembrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-livermitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membranepermeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca2+ efflux,ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrialmembrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolatedmitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggestthe following sequence of events: 1) GA interaction with mitochondrial membrane promoting itspermeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion dueto inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROSaccumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due toROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancerproperty of GA/structure related compounds.

Investigaciones y Evaluacionesidad de La Habana, ave. 23 #dad Habana, Cuba. Fax: +53 7

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Introduction

Guttiferone-A (GA) is a polyisoprenylated benzophenone derivative(Fig. 1) initially isolated from Symphonia globulifera roots (Gustafson etal., 1992), and recently, byour group (unpublished results), fromGarciniaaristata fresh fruits; it is a bicyclo-[3.3.1]-nonane derivative with onlyone aliphatic methyl group belonging to a bicyclo moiety. GA presentsanti-HIV (Gustafson et al., 1992), cytotoxic (Williams et al., 2003),trypanocidal, antiplasmodial (Ngouela et al., 2006) and leishmanicidal(Pereira et al., 2010) actions. In addition, structurally relatedpolyisoprenylated benzophenones isolated from plants present

cytotoxic, growth inhibiting and apoptosis inducing actions in cancercells (Baggett et al., 2005; Cao et al., 2007; Huang et al., 2009;Matsumoto et al., 2003; Merza et al., 2006; Pan et al., 2001; Sang et al.,2001; Xu et al., 2010), antibacterial activity (Chatterjee et al., 2005;Iinuma et al., 1996), as well as preventing action in rodent models ofcolorectal and tongue carcinogenesis (Tanaka et al., 2000; Yoshidaet al., 2005).

Several specific actions of GA/structurally related compoundstoward cancer cells have been reported, for example: (i) guttiferonesO and P inhibit phosphorylation of the synthetic biotinylated peptidesubstrate KKLNRTLSVA by MAPKAPK-2 (Carroll et al., 2009); (ii)xanthochymol and guttiferone E inhibit microtubule disassemblywith implications in cell replication (Roux et al., 2000); (iii) garcinolinhibits histone acetyltransferases p300, a key regulatory step in geneexpression and cell cycle (Balasubramanyam et al., 2004); (iv)oblongifolin C induces apoptosis in HeLa-C3 cells through activationof caspase 3 (Huang et al., 2009); (v) xanthochymol, guttiferone E and

O O

OH

HO

O OH

Fig. 1. Guttiferone A (GA) structure (1R,3E,5S,6R,7S)-3-[(3,4-dihydroxyphenyl)-hydroxymethylidene]-6-methyl-1,5,7-tris(3-methylbut-2-enyl)-6-(4-methylpent-3-enyl)bicyclo[3.3.1]nonane-2,4,9-trione.

283G.L. Pardo-Andreu et al. / Toxicology and Applied Pharmacology 253 (2011) 282–289

guttiferone H inhibit three human colon cancer cell lines growth,HCT116, HT29 and SW480, respectively, in association with inductionof endoplasmic reticulum response (Protiva et al., 2008); (vi)guttiferone G and analogs inhibit human sirtuin type proteins 1 and2 (Gey et al., 2007); and (vii) GA inhibits cysteine/serine proteases(Martins et al., 2009).

Mitochondria are considered to be implicated in cell necrosis andapoptosis (Kroemer and Reed, 2000), so compounds lipophilicenough to reach mitochondrial membrane may promote cell deathby means of mitochondrial mechanisms. Because of a XLog P3-AAvalue of 10.4 (theoretical value) GA meets this criterion, whichrenders it with a potential ability to interact with mitochondrialmembrane. In this context, we addressed in the present work apotential involvement of mitochondria in the GA toxicity towardcancer cells by employing both hepatic carcinoma (HepG2) cells andmitochondria isolated from rat liver. The results show that energeticand oxidative stress implications resulting from direct mitochondrialmembrane permeabilization are potentially involved in GA toxicitytoward cancer cells.

Materials and methods

Compounds and reagents. All reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). All stock solutions were preparedusing glass-distilled deionized water. Stock solutions of GA wereprepared in dimethyl sulfoxide (DMSO) and added to the cell cultureor mitochondrial reaction media at 1/1000 (v/v) dilution. Controlexperiments contained DMSO at 1/1000 dilution.

GA isolation. GA was obtained from G. aristata fresh fruits through thesame procedure employed for aristophenone (Cuesta-Rubio et al.,2001). In brief, fresh fruits (2.5 kg) were extracted with n-hexane(5 l×2) for 7 days at room temperature (25 °C). A yellow residue(7.8 g) was obtained after concentration under reduced pressure(40 °C) and a portion (2 g) was purified by semi-preparative HighPressure Liquid Chromatography, HPLC (Phenomenex C-18 column,CH3OH/0.01% trifluoroacetic acid 9:1, flow rate 2.5 ml/min) in order toobtain around 0.8 g of compound 1 and 0.9 g of compound 2. Thesecompounds were initially identified as GA and aristophenone by

1H

NMR and ESI-MS spectra, respectively. Compound 1 was purified bysuccessive crystallizations from hexane solutions (purity: 99% by

HPLC) and its structure was confirmed as GA by1H NMR, COSY and

HMBC spectra employing the same spectroscopic conditions previ-ously reported (Gustafson et al., 1992).

1H NMR spectrum exhibited 9

methyl groups between δ 1.24 and 1.70, an aromatic AMX spin system[δ 7.19 (d, J=2 Hz), 6.67 (d, J=8 Hz), 6.95 (dd, J=2 and 8 Hz)], andfive vinyl protons between δ 4.8 and 5.3. Three proton signals δH 1.24(CH3-22) and δH 1.21, 1.40 (CH2-23) showed HMBC correlations tothree carbon signals at δ 68.7 (C-4), δ 51.8 (C-5,) and δ 41.0 (C-6),indicating the presence of a 3-methylbut-2-enyl moiety attached to C-5 and the existence of a bicyclo-[3.3.1]-nonane derivative. Allconnectivities established by HMBC and COSY spectra were identicalto those previously shown for guttiferone-A (Gustafson et al., 1992).In addition, the ESI-MS spectrum of GA showed a protonatedmolecule[M+H]+ atm/z 603 and its fragmentation yielded ions resulting fromsuccessive elimination of the alkyl chains from the bicyclo core(Gustafson et al., 1992). Its Log P value was determined theoreticallyusing Advanced Chemistry development (ACD/labs) software V8.19(1994–2010 ACD/Labs).

Culture of HepG2 cells. HepG2 cells were obtained from the AmericanType Culture Collection, No. HB 8065. The cell line was cultured inDulbecco's medium with 10% defined supplement fetal bovine fetalserumplus 100 IU/ml penicillinG, 100 mg/ml streptomycin and 1 μg/mlamphotericin. The cellswere seeded into12-well plates (Nunc, Roskilde,Denmark), with 1×105 cells/well in 1 ml of culture medium at 37 °C,flushedwith 5% CO2 in air for 24 h. After the incubation period, the cellswere rinsed with buffered saline solution.

Assessment of HepG2 cell viability by annexin-V/propidium iodidedouble-staining. Cells were seeded in a 12-well plate at a density of1×105 cells/well and incubated for 24 h in the absence (control) orpresence of GA (1–25 μM), 25 μMGA plus 1 mM isocitrate, and 25 μMCCCP. After incubations, cells were collected and washed with ice-cold PBS and binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/NaOH, 140 mM NaCl, 2.5 mMCaCl2). Cells were then incubated with FITC-conjugated Annexin-V(1:100) on ice for additional 15 min. Propidium iodide (1 μg/ml) wasadded immediately before analysis by BD FAXSCANTO™flowcytometer(BDBioscience, CA,USA). 10,000 cellswere countedper sample anddatawere analyzed by BD FACSDIVA software (BD Biosciences, CA, USA).

Mitochondrial membrane potential (ΔΨ) and ATP assays in HepG2 cells.Mitochondrial membrane potential was assessed with JC-1 probe(Molecular Probes Inc., Eugene, OR). The green-fluorescent JC-1 probeexists as a monomer at low membrane potential but forms red-fluorescent “J-aggregates” at higher potential. Cells were incubated asfor the viability assay, harvested and resuspended in DMEM mediumat a density of 1×105 cells/ml, then incubated with 1 μM JC-1 at 37 °Cfor 30 min. 10,000.00 cells were counted per samples. Relativefluorescence intensities were monitored by BD FAXSCANTO™ flowcytometer (BD Bioscience, CA, USA) and analyzed by the softwareModfit and Cell-Quest (BD Biosciences, Franklin Lakes, NJ) withsettings of FL1 (green) at 530 nm and FL2 (red) at 585 nm (Liu et al.,2007).

Cellular ATP was determined by means of the firefly luciferin–luciferase assay system. Cells (1×105) were incubated as for theviability assay and suspension was centrifuged at 50×g for 5 min at4 °C. The pellet was treated with 1 ml of ice-cold 1 M HClO4. Aftercentrifugation at 2000×g for 10 min at 4 °C, aliquots (100 μl) of thesupernatants were neutralized with 70 μl of 2 M KOH, suspended in100 mM Tris-(hydroxymethyl) aminomethane (Tris)–HCl, pH 7.8(1 ml final volume), and centrifuged again. Bioluminescence wasmeasured in the supernatant with a Sigma-Aldrich assay kit accordingto the manufacturer's instructions, using an AutoLumat LB953Luminescence photometer (Perkin-Elmer Life Sciences, Wilbad,Germany).

284 G.L. Pardo-Andreu et al. / Toxicology and Applied Pharmacology 253 (2011) 282–289

Assessment of reactive oxygen species (ROS) levels in HepG2 cells.Intracellular oxidation of dichlorodihydrofluorescein diacetate(H2DCFDA) to 2,7-dichlorofluorescein (DCF) by ROS was monitoredthrough fluorescence increase. HepG2 cells were seeded in a 12-wellplate at a density of 1×105 cells/well and incubated as for the cellviability assay. After incubations, the well plates were washed withPBS and then 100 μl/well of 10 μmol/l H2DCFDA was added to eachwell, remaining incubated at 37 °C for 45 min in a 5% CO2 incubator.Fluorescence was measured in a model F-4500 Hitachi fluorescencespectrophotometer (Tokyo, Japan) at the 503/529 nm excitation/emission wavelength pair (slits 5/10 nm) (Halliwell and Whiteman,2004).

Isolation of rat-liver mitochondria. Mitochondria were isolated bystandard differential centrifugation (Pedersen et al., 1978). MaleWistar rats weighing approximately 200 g were euthanized bydecapitation; livers (10–15 g) were immediately removed, sliced inmedium (50 ml) consisting of 250 mM sucrose, 1 mM ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and 10 mMHEPES-KOH, pH 7.2, and homogenized three times for 15 s at 1 minintervals using a Potter-Elvehjem homogenizer. Homogenates werecentrifuged (580×g, 5 min) and the resulting supernatant furthercentrifuged (10300×g, 10 min). Pellets were then suspended inmedium (10 ml) consisting of 250 mM sucrose, 0.3 mM EGTA and10 mM HEPES-KOH, pH 7.2, and centrifuged (3400×g, 15 min). Thefinal mitochondrial pellet was suspended in medium (1 ml) con-sisting of 250 mM sucrose and 10 mM HEPES-KOH, pH 7.2, and usedwithin 3 h. Mitochondrial protein contents were determined by theBiuret reaction.

Standard incubation procedure. Mitochondria were energized with5 mM potassium succinate (plus 2.5 μM rotenone) in a standardincubation medium consisting of 125 mM sucrose, 65 mM KCl, 2 mMinorganic phosphate and 10 mM HEPES-KOH pH 7.4 at 30 °C.

Continuous-monitoring mitochondrial assays. Mitochondrial respira-tion was monitored polarographically by an oxygraph equipped witha Clark-type oxygen electrode (Hansatech instruments, oxythermelectrode unit, UK), and the mitochondrial membrane potential wasdetermined spectrofluorimetrically using 10 μM safranine O as aprobe (Zanotti and Azzone, 1980) in a Model F-4500 Hitachifluorescence spectrophotometer (Tokyo, Japan) at the 495/586 nmexcitation/emission wavelength pair; these assays were performed inthe presence of 0.1 mM EGTA and 2 mM K2HPO4. Ca2+ efflux wasmonitored spectrofluorimetrically using 150 nM Calcium Green 5N(Molecular Probes, OR, USA) as a probe, at the 506/531 nm excitation/emission wavelength pair (Rajdev and Reynolds, 1993). Mitochon-drial swelling was estimated spectrophotometrically from thedecrease in apparent absorbance at 540 nm, using a Model U-2910Hitachi spectrophotometer (Japan). The oxidation of mitochondrialNAD(P)H (NADPH+NADH) was monitored in a F-4010 Hitachifluorescence spectrophotometer at the 366/450 nm excitation/emission wavelength pair (Fagian et al., 1990). ROS were monitoredspectrofluorimetrically using 1 μMAmplex red (Molecular Probes, OR,USA) and 1 UI/ml horseradish peroxidase at the 563/587 nmexcitation/emission wavelength pair in a Model F-4500 Hitachifluorescence spectrophotometer (Votyakova and Reynolds, 2001).

ATP assay in mitochondria. Mitochondrial ATP was determined bymeans of the firefly luciferin–luciferase assay system (Lemasters andHackenbrock, 1976). After a 10-min treatment with GA, themitochondrial suspension (1 mg protein/ml) was centrifuged at9000×g for 5 min at 4 °C, and the pellet was treated with 1 ml ofice-cold 1 MHClO4. After centrifugation at 14,000×g for 5 min at 4 °C,100-μl aliquots of the supernatants were neutralized with 70 μl of 2 MKOH, suspended in 100 mM Tris–HCl, pH 7.8 (1 ml final volume) and

centrifuged at 15,000×g for 15 min. Bioluminescence was measured inthe supernatant with a Sigma/Aldrich assay kit according to themanufacturer's instructions, using an AutoLumat LB953 Luminescencephotometer (Perkin-Elmer Life Sciences, Wilbad, Germany).

Evaluation of mitochondrial membrane fluidity. Mitochondrialmembrane fluidity was evaluated by fluorescence anisotropy (r).Mitochondria (0.4 mg protein) were incubated in 2 ml (final volume)of standard reaction medium containing 0.5 μM 1,6-diphenyl-1,3,5-hexatriene (DPH) for 30 min, at 37 °C, in the presence of GA.Fluorescence was measured in a Model F-4500 Hitachi fluorescencespectrophotometer equipped with polarizer system (Hitachi, Tokyo,Japan) at the 362/432 nm excitation/emission wavelength pair.Fluorescence anisotropy data were calculated using the formular= lΠ− I⊥/IΠ+2I⊥, where lΠ and I⊥ refer to the intensity of thefluorescence light emission measured parallel and perpendicularly tothe polarization plane of the excitation beam, respectively (Praetet al., 1986; Martins et al., 2008).

Statistical analysis. Statistical analysis was performed using two-wayANOVA, assuming equality of variance with Student–Newman–Keulspost-hoc test for pairwise comparisons. Results with Pb0.05 wereconsidered to be statistically significant.

Results

Effects of GA on viability, mitochondrial membrane potential, ATP andROS levels in HepG2 cells

The incubation of HepG2 cells with GA for 24 h promoted cellviability decrease (Fig. 2A), as assessed by Annexin-V/PI double-staining (flow cytometry). At 25 μM, GA promoted around 50% celldeath, an effect close similar to the effect of 25 μM CCCP. Isocitrate(1 mM), in turn, partly prevented cell death induced by 25 μMGA. Theeffect of GA on HepG2 cell mitochondrial membrane potential wasestimated with the mitochondrion-specific dye, JC-1. As shown inFig. 2B, GA promoted an extensivemitochondrial membrane potentialdissipation in HepG2 cells. Unlike cell viability, this effect was notprevented by isocitrate. GA also induced ATP depletion in HepG2 cellsafter 24 h incubation (Fig. 2C), as well as ROS levels increase (Fig. 2D),both effects partly prevented by isocitrate. The concentration–response pattern for all above GA effects was closely similar,suggesting a correlation between them; interesting, they were largelypotentiated in HepG2 cells exposed to low glucose levels (results notshown), denoting energetic implications. We therefore performedstudies on the GA effects in isolated rat-liver mitochondria, a classicalmodel for studies on mitochondrial mechanisms.

Effects of GA on respiration of isolated rat-liver mitochondria

Fig. 3A shows concentration–response traces for the effects of GAon respiration of mitochondria isolated from rat liver. State 4respiration rate supported by 5 mM succinate plus rotenone (V4)was increased by GA, denoting a mitochondrial uncoupling action(Fig. 3B). On the other hand, mitochondrial state 3 respiration rate(V3) was not affected by GA, denoting lack of respiratory chain or ATPsynthase inhibition (Figs. 3A and B). As expected, the V4 increase ledto a decrease of the mitochondrial respiratory control ratio (Fig. 3C).

Effects of GA on mitochondrial membrane potential and Ca2+ uptake/release

Fig. 4A shows that GA promoted dissipation of mitochondrialmembrane potential (lines b, c, d, e versus line a), consistently with theobserved increase of V4. This effect was not inhibited by either theclassical mitochondrial permeability transition inhibitor cyclosporine A,ruthenium red or EGTA (lines f, g and h, respectively). The fluorescence

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Fig. 2. Effects of GA on HepG2 cell viability (A), mitochondrial membrane potential (B),ATP levels (C) and ROS levels (D). HepG2 cells (1×105) were treated with GA (1, 5, 10and 25 μM), 25 μM GA plus 1 mM isocitrate, and 25 μM CCCP. Control cells onlycontained GA vehicle (DMSO 1/1000 v/v).The assay conditions are described inMaterials and methods. The bars are mean±SEM of three different experiments. ⁎, #, $

Significantly different from control, consecutive GA concentrations or isocitrate treatmentat Pb0.05, respectively.

285G.L. Pardo-Andreu et al. / Toxicology and Applied Pharmacology 253 (2011) 282–289

units (means±SEM at 250 s) were: 51.60±2.31 (line a), 56.51±1.91(line b), 97.62±4.73 (line c), 111.68±5.22 (line d), 204.53±6.52(line e), 114.8±5.72 (line f), 103.4±4.69 (line g), 100.7±5.25 (line h);differences statistically significant were found between (line a) andthe other lines, at Pb0.05. Fig. 4B shows that GA inducedmitochondrialCa2+ release, also in a way not prevented by cyclosporine A, butpartially prevented by the Ca2+-uniporter blocker, ruthenium red. Thefluorescence units (means±SEM at 250 s) were: 41.90±3.86 (line a),

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Fig. 3. Effects of GA on respiration of isolated rat-liver mitochondria energized withsuccinate. Representative recordings (A), phosphorylating (V3) and basal (V4)respiration rates (B), and respiratory control ratio, RCR (C). Mitochondria (0.5 mgprotein/ml) were incubated without (controls) or with GA in the standard mediumsupplemented with 0.1 mM EGTA, under the conditions described in Materials andmethods. The additions in panel A were: (a) control, (b) 5, (c) 10, (d) 15, (e) 20,(f) 25 μM GA and (g) 1 μM CCCP. For V3 determination, 100 μM ADP was added. V4

was determined after ADP exhaustion. The bars represent mean±SEM (n=3).⁎ Significantly different from controls at Pb0.05.

286 G.L. Pardo-Andreu et al. / Toxicology and Applied Pharmacology 253 (2011) 282–289

58.00±4.38 (line b), 142.30±5.82 (line c), 133.42±7.43 (line d), and91.62±6.83 (line e); differences statistically significant were foundbetween (line a) and the other lines, at Pb0.05. Fig. 4C shows that GAprevented mitochondrial Ca2+ uptake when the compound was addedto the medium prior to energized mitochondria. The fluorescence units(means±SEM at 250 s) were: 39.69±4.41 (line a), 48.90±3.72(line b), 123.55±6.53 (line c), and 172.96±7.56 (line d); differencesstatistically significant were found between (line a) and the other lines,at Pb0.05.

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Fig. 4. Effects of GA onmembrane potential (A), Ca2+ release (B) and Ca2+ uptake (C) inmitochondria (0.5 mg protein/ml) energized with succinate, expressed as ArbitraryFluorescence Units (AFU), as described in Materials and methods. For panel A, theadditions were: (a) control, (b) 5, (c) 10, (d) 15, (e) 20 μM GA, (f) 20 μM GA plus 1 μMruthenium red, (g) 20 μM GA plus 1 μM cyclosporine A and (h) 20 μM GA plus 200 μMEGTA. For panel B: (a) control, (b) 5, (c) 10 μMGA, (d) 10 μMGA plus 1 μM cyclosporineA and (e) 10 μM GA plus 1 μM ruthenium red. For panel C: (a) control, (b) 5, (c) 10 and(d) 20 μM GA. CCCP (1 μM) additions are indicated. Results are representative of threeexperiments with different mitochondrial preparations.

Effects of GA on mitochondrial ATP levels

After 10-min incubation GA induced decrease in the ATP levels ofisolated rat-livermitochondria by around 45% and 65% at 5 and 25 μM,respectively (Fig. 5). It denotes energetic impairment and, like forHepG2 cells, it was probably a consequence of the GA-promoteddissipation of the mitochondrial membrane potential.

Effects of GA on mitochondrial swelling

Fig. 6 shows that GA induced non-specific mitochondrial mem-brane permeabilization in isotonic sucrose-based medium, monitoredas mitochondrial swelling assessed by absorbance decrease (lines b, c,d, and e). This effect was not inhibited by cyclosporine A (line f), EGTA(line g) or the antioxidant enzyme catalase (line h), excluding any linkwith the mitochondrial permeability transition process. The presenceof isocitrate, a NAD(P)H regenerating substrate (line i), partlyprevented the GA-induced mitochondrial swelling. The absorbancevalues (means±SEM at 250 s) were: 1.660±0.019 (line a), 1.163±0.017 (line b), 0.742±0.021 (line c), 0.674±0.014 (line d), 0.626±0.015, (line e), 1.184±0.017 (line f), 1.385±0.023 (line g), 1.40±0.024 (line h), and 1.650±0.025 (line i); differences statisticallysignificant were found between (line a) and the other lines, except for(line i), at Pb0.05.

Effects of GA on mitochondrial ROS levels

In order to examine the influence of GA on mitochondrial ROSlevels we assessed H2O2 released to the medium by means of theAmplex Red assay, in the absence of Ca2+ (100 μMEGTA). Fig. 7 showsthat at around the same concentration range in which the othereffects were observed, GA increased ROS levels in isolated rat-livermitochondria (lines b, c, and d). The H2O2 concentrations released tothe medium (means±SEM at 400 s) were: 6.20±0.12, 7.22±0. 14,9.11±0.14 and 10.9±0.16 nmol/ml for lines a, b, c, and d, respec-tively. Differences statistically significant were found between (line a)and the other lines, at Pb0.05.

Effects of GA on mitochondrial NAD(P)H levels

NADPH is the major source of reducing equivalents for theantioxidant systems glutathione peroxidase/reductase and thioredox-ine peroxidase/reductase; its reduced state in mitochondrial matrix iscontrolled by the membrane potential-sensitive NADP+ transhydro-genase (Hoek and Rydstrom, 1988). We assessed the influence of GAon mitochondrial NAD(P)H levels under the same experimental

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Fig. 5. Effects of GA on ATP levels of mitochondria (1 mg protein/ml) energized withsuccinate, 10 min after incubation in the standard medium, as described in Materialsand methods. The bars represent mean±SEM (n=3). ⁎ Significantly different fromcontrol at Pb0.05.

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Fig. 6. Effects of GA on mitochondrial swelling in mitochondria energized withsuccinate. Mitochondria (0.5 mg protein/ml) were incubated in standard mediumsupplemented with 20 μM Ca2+, under the conditions described in Materials andmethods. The additions were: (a) control, (b) 5, (c) 10, (d) 15, (e) 20 μM GA, (f) 5 μMGA plus 1 μM cyclosporine A, (g) 5 μM GA plus 200 μM EGTA, (h) 5 μM GA plus 3 μMcatalase, and (i) 5 μM GA plus 1 mM isocitrate. Results are representative of threeexperiments with different mitochondrial preparations.

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Fig. 8. Effects of GA on NAD(P)H depletion/oxidation. Mitochondria (1 mg protein/ml)were incubated in standard medium supplemented with 100 μM EGTA and 5 μMrotenone, under the conditions described in Materials and methods. The additionswere: (a) control, (b) 5, (c) 10, (d) 20 μM GA, (e) 20 μM GA plus 3 μM catalase. t-BHP(t-butyl hydroperoxide, 250 μM) addition is indicated. Results are representative of threeexperiments with different mitochondrial preparations.

30

AMit

287G.L. Pardo-Andreu et al. / Toxicology and Applied Pharmacology 253 (2011) 282–289

condition for the ROS assay. Fig. 8 shows a decrease of fluorescence ofmitochondria exposed to GA (lines b, c and d) compared to controlorganelles (line a), denoting NAD(P)H depletion/oxidation; catalasedid not prevent this effect (line e). The fluorescence units at 400 swere: 25.17±0.46 (line a), 23.58±0.37 (line b), 22.12±0.21 (line c)19.82±0.36 (line d), and 19.22±0.42 (line e); differences statisticallysignificant were found between (line a) and the other lines, at Pb0.05.Both NAD(P)H depletion/oxidation (Fig. 9A) and ROS levels increase(Fig. 9B) by GA (lines b) were partially prevented by the replacementof NADPH by isocitrate (lines c). These results suggest that GA-induced mitochondrial membrane potential dissipation renderstranshydrogenase unable to sustain the reduced state of NADPH andallows mitochondria to accumulate ROS.

20a

10

c

b

AFU

0

Interaction of GA with mitochondrial membrane

A decrease in fluorescence anisotropy (r) reflects increase in DPHmobility intomembranes and decrease in membrane structural order/increase of membrane fluidity. Fig. 10 shows that GA interacts withmitochondrial membrane increasing its fluidity.

12

10

8

d

c

b

6

4

2

a

Mit

0 100 200 300 400Time (s)

H2O

2 (nm

o1/m

1)

Fig. 7. Effects of GA on ROS levels in mitochondria energized with succinate.Mitochondria (1 mg protein) were incubated in the standard medium supplementedwith 0.1 mM EGTA, 1 μM Amplex Red and 1 IU/ml horseradish peroxidase, at a finalvolume of 2 ml, as described in Materials and methods. The additions were: (a) control,(b) 5, (c) 10, (d) 20 μM GA. Results are representative of three experiments withdifferent mitochondrial preparations.

Discussion

The effects of GA in the present study were compared with theeffects of the classicmitochondrial uncoupler CCCP since GA displayeduncoupling action both in HepG2 cells and rat liver isolatedmitochondria. It is worthy to consider that most of these effectshave been frequently associated with mitochondria-mediated celldeath (Kroemer et al., 1995; Skulachev, 2006; Xia et al., 2009). Indeed,

0 100 200 300 400 500 600

Time (s)

10

12B

b

2

4

6

8 c

a

0 100 200 300 4000

2

Time (s)

H2O

2 (nm

o1/m

1)

Fig. 9. Effects of isocitrate on NAD(P)H depletion/oxidation (A) and ROS levels increase(B) promoted by GA. Mitochondria were incubated under the conditions described inFig. 7 for Panel A or Fig. 8 for Panel B. The additions were: (a) control, (b) 20 μM GA,(c) 20 μMGA plus 1 mM isocitrate. Results are representative of three experiments withdifferent mitochondrial preparations.

288 G.L. Pardo-Andreu et al. / Toxicology and Applied Pharmacology 253 (2011) 282–289

GA and other structurally related compounds present severalproposed biological actions (Gustafson et al., 1992; Ngouela et al.,2006; Pereira et al., 2010; Williams et al., 2003), including well-documented toxicity toward cancer cells (Baggett et al., 2005; Cao etal., 2007; Huang et al., 2009; Matsumoto et al., 2003; Merza et al.,2006; Pan et al., 2001; Sang et al., 2001;Williams et al., 2003; Xu et al.,2010). In addition to these previously proposed actions, we hereprovided for the first time evidence that GA may interact withmitochondrial membrane and presents both energetic and oxidativestress implications: a cyclosporine A/EGTA-insensitive mitochondrialmembrane permeabilization, mitochondrial uncoupling (membranepotential dissipation/state 4 respiration rate increase), Ca2+ efflux,ATP depletion, mitochondrial NAD(P)H depletion/oxidation and ROSlevels increase. NAD(P)H depletion/oxidation probably resulted fromthe mitochondrial membrane potential dissipation, a condition inwhich the NADP+ transhydrogenase is unable to sustain the reducedstate of mitochondrial matrix NADPH. NADP+ transhydrogenase is amitochondrial membrane enzyme responsible for driving the reduc-tion of NADP+ by NADH coupled to the protonmotive force (chemicaland electrical potential energy) (Hatefi and Yamaguchi, 1996; Hoekand Rydstrom, 1988). NADPH, in turn, is implicated in cell antioxidantdefenses involving glutathione (GSH) and thioredoxin (TRX), eitherreductants themselves or cofactor for GSH and TRX peroxidases; GSHand TRX reduced species are regenerated by NADPH-requiringreductases (Carmel-Harel and Storz, 2000). Therefore, whetherNADPH is oxidized/depleted, ROS tend to accumulate.

We recently demonstrated that nemorosone, a natural occurringcompound structurally related to GA, elicits a potent protonophoricuncoupling action onmitochondria (Pardo-Andreu et al., 2011). In thepresence of the Ca2+-uniporter blocker ruthenium red, nemorosoneinduced mitochondrial swelling in a way sensitive to the classicmitochondrial permeability transition (MPT) inhibitor cyclosporine A.Unlike nemorosone, GA uncoupled mitochondria through a non-protonophoric mechanism (result not shown). In addition, mitochon-drial swelling elicited by GA was not inhibited by cyclosporine A orEGTA and, therefore, it does not correspond to the MPT process(Zoratti and Szabò, 1995). Rather, the evidence that GA increasedmitochondrial membrane fluidity suggests that a direct interactionwith mitochondrial membrane, whose major structural lipids arecardiolipins, accounts for its permeabilizing action on the organelle.

The evidence that isocitrate partly prevented GA-induced NADPHoxidation/depletion and mitochondrial swelling in isolated mito-chondria, as well as cell viability decrease, ATP depletion and ROSlevels increase in HepG2 cells, suggests that NADPH oxidation/depletion is at least partly involved in the GA permeabilizing actionon mitochondria and its consequence on cells. Isocitrate is the

6

8

10

*

2

4*#

0

Guttiferone A (µM)

25105Control

*#

r

Fig. 10. Effects of GA on mitochondrial membrane fluidity evaluated with thefluorescent probe DPH. Experimental conditions are described in Materials andmethods. Data are expressed as fluorescence anisotropy (r). ⁎, # Significantly differentfrom control or consecutive GA concentrations at Pb0.001, respectively.

substrate of NADP+-dependent isocitrate dehydrogenase, a majorNADPH source in mitochondria with a key role in cellular defenseagainst ROS (Jo et al., 2001). In citosol, NADPH is provided primarilyby the pentose phosphate pathway, including glucose-6-phosphatedehydrogenase and 6-phosphogluconate dehydrogenase. In thisregard, the fact that HepG2 cells incubated in medium with lowglucose levels were more sensitive to GA-induced death, mitochon-drial membrane potential dissipation, ATP depletion and ROS levelsincrease reinforces the proposed GA toxicity mechanism. Low glucoseimpairs NADPH re-generation in citosol and may potentiate mito-chondria-mediated cytotoxic actions.

In conclusion, the present results suggest the following sequenceof events for the GA action on mitochondria: 1) GA interaction withmitochondrial membrane increasing its fluidity and promoting itspermeabilization; 2) mitochondrial membrane potential dissipation;3) NAD(P)H oxidation/depletion due to inability of membranepotential-sensitive NADP+ transhydrogenase of sustaining its re-duced state; 4) ROS accumulation inside mitochondria and cells; 5)additional mitochondrial membrane permeabilization due to ROS;and 6) ATP depletion. The evidence that Ca2+ efflux was only partiallyprevented by the Ca2+-uniporter blocker ruthenium red in isolatedmitochondria and the inability of isocitrate to prevent mitochondrialmembrane potential dissipation in HepG2 cells suggest that this latteris an early event associated to the GA action on mitochondria, whichcould ultimately, via energetic and oxidative stress implications,result in cell ATP depletion.

Here we demonstrated that GA promotes mitochondrial energeticimpairment/oxidative stress in apparent association with mitochon-drial membrane permeabilization and this action is potentiallyimplicated in the well-documented anti-cancer property of GA/structure related compounds.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Acknowledgments

This work was partially supported by CAPES-Brazil/MES-Cuba(064/09).

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