Mitochondrion 13 (2013) 25–35

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Mitochondrion

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Edelfosine and perifosine disrupt hepatic mitochondrial oxidative phosphorylationand induce the permeability transition

Ana Burgeiro a,b,c, Cláudia V. Pereira a,c, Filipa S. Carvalho a,c, Gonçalo C. Pereira a,c,Faustino Mollinedo d, Paulo J. Oliveira a,⁎a CNC — Center for Neuroscience and Cell Biology, Department of Life Sciences, University of Coimbra, Coimbra, Portugalb BEB — PhD Programme in Experimental Biology and Biomedicine, CNC — Center for Neuroscience and Cell Biology, Department of Life Sciences, University of Coimbra, Coimbra, Portugalc Department of Life Sciences, University of Coimbra, Coimbra, Portugald Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Salamanca,Campus Miguel de Unamuno, E-37007 Salamanca, Spain

Abbreviations: AA, antimycin A;ALPs, alkylphospholipiBSA, bovine serum albumin; CsA, cyclosporin A; ER, endoplcyanide p-trifluoromethoxyphenylhydrazone; MPT, mitochRCR, respiratory control ratio; Rot, rotenone; SEM, stantetraphenylphosphonium cation; ΔΨm, mitochondrial tran⁎ Corresponding author at: Center for Neuroscience

Coimbra, 3004-517 Coimbra, Portugal. Tel.: +351 304 5E-mail address: pauloliv@ci.uc.pt (P.J. Oliveira).

1567-7249/$ – see front matter © 2012 Elsevier B.V. anhttp://dx.doi.org/10.1016/j.mito.2012.11.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 October 2012received in revised form 2 November 2012accepted 7 November 2012Available online 16 November 2012

Keywords:EdelfosinePerifosineMitochondriaToxicityLiverRat

Edelfosine and perifosine are alkylphospholipids that have been intensively studied as potential antitumor agents.Apoptotic cell death caused by these two compounds ismediated, at least in part, throughmitochondria. Addition-ally, previous works demonstrated that edelfosine induces changes inmitochondrial membrane permeability thatare somehow reduced by using cyclosporin A. Therefore, the objective of the present studywas not only to confirmmitochondrial permeability transition but also identify direct effects of both ether lipids on mitochondrial hepaticfractions, namely on mitochondrial oxidative phosphorylation and generation of hydrogen peroxide (H2O2)through the respiratory chain. Results show that edelfosine and perifosine inhibit mitochondrial respiration anddecrease transmembrane electric potential. However, despite these effects, edelfosine and perifosine were stillable to induce mitochondrial permeability transition in non-energizedmitochondria. Interestingly, edelfosine de-creased H2O2 production through the respiratory chain. In conclusion, the present work demonstrates previouslyunknown alterations of mitochondrial physiology directly induced by edelfosine and perifosine. The study is rele-vant in the understanding of mitochondrial-target effects of both compounds, as well as to acknowledge possibletoxic responses in non-tumor organs.

© 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction

Alkylphospholipids (ALPs) are a new class of antitumor agentswhich target cell membranes (Hideshima et al., 2006) but not nu-clear DNA (Vink et al., 2007). Due to their chemical structure,ALPs can act as detergents resulting in direct plasma membranedysruption (REF) (Vink et al., 2007). The plasma membrane is anactive structure that is implicated, for instance, in phospholipidturnover, lipid second messenger formation and signal transductionpathways that are crucial for life and death decisions (Vink et al.,2007). By interfering with these processes, ALPs induce cell death,although the mechanism remains controversial (Vink et al., 2007).More recently, the involvement of the mitochondrial pathway inALP-induced apoptosis was demonstrated (Cuvillier et al., 1999;

ds; ANOVA, analysis of variance;asmic reticulum; FCCP, carbonylondrial permeability transition;dard error of the mean; TPP+,smembrane electric potential.and Cell Biology, University of02 911; fax: +351 239 855789.

d Mitochondria Research Society. A

Gajate and Mollinedo, 2007; Gajate et al., 2000b). Edelfosine(1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3),an ALP ether lipid, has been widely studied as an innovative therapeuticagent for cancer treatment due to its pro-apoptotic effect on tumor cells(Gajate et al., 2012; Mollinedo et al., 1993, 1997). Edelfosine has alreadybeen tested in several tumor models, including both hematological(Alonso et al., 1997; Gajate et al., 1998, 2000b, 2009) and solid tumors(Arthur et al., 2005; Gajate et al., 2000a, 2012; Lu et al., 1993;Nieto-Miguel et al., 2006). Edelfosine targets mainly lipid rafts inhematologic malignancies and the endoplasmic reticulum (ER) in solidtumor cells (Gajate et al., 2004, 2012; Nieto-Miguel et al., 2006), there-fore showing distinct targets accordingly to the tumor cell type(Nieto-Miguel et al., 2007). The preferential ER accumulation ofedelfosine in a number of solid tumor cells (Gajate et al., 2012; Nieto-Miguel et al., 2006) culminates in downstreammitochondrial dysfunctionand cell death (Nieto-Miguel et al., 2007). Perifosine (octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate) is a second generation of ALPs(van der Luit et al., 2007), exerting a marked cytotoxic effect on humancancer cell lines, such as those derived frombreast, colon, prostate, larynx(Hilgard et al., 1997), multiple myeloma (Gajate and Mollinedo, 2007)and is being currently tested in several phase II trials for the treatmentof several human cancers (Momota et al., 2005). In addition to the

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26 A. Burgeiro et al. / Mitochondrion 13 (2013) 25–35

established role of mitochondria in energy metabolism, regulation of celldeath has emerged as another key function of these organelles (Orreniuset al., 2007). The majority of anti-carcinogenic agents have a substantialimpact on mitochondria physiology and/or morphology, since mostactivate the intrinsic, mitochondrial-dependent, apoptotic signaling(Fulda et al., 2010). Under stress conditions, increased mitochondrialoxidative stress, whichmay damagemembrane lipids, affect mitochon-drial enzyme activity, and consequently impair mitochondrial function,is often a consequence from chemotherapeutic action (Barbosa et al.,2012; Magalhaes et al., 2005; Paradies et al., 1999, 2001, 2002;Petrosillo et al., 2001).

Isolatedmitochondrial fractions are considered a very relevant in vitromodel to study direct effects of several xenobiotics on that organelle, thuscontributing to investigate drug toxicity/safety (Nadanaciva and Will,2009; Pereira et al., 2009a,b). Recently, it was reported that edelfosine in-duces swelling of isolated livermitochondria fromadult Sprague–Dawleyrats, indicating an increase in mitochondrial membrane permeabilitythrough induction of the cyclosporin-A (CsA)-sensitive mitochondrialpermeability transition (MPT) pore (Mollinedo et al., 2011). CsA, a proto-typical inhibitor of the MPT (Broekemeier and Pfeiffer, 1989) partiallyinhibited mitochondrial swelling induced by edelfosine (10 μM) on iso-lated hepatic mitochondria (Mollinedo et al., 2011). However, the phar-macological effects of CsA on the MPT were transitory for higher dosesof edelfosine and in long-term experiments (Mollinedo et al., 2011).

Theobjective of our study is to identify direct toxic effects of edelfosineand perifosine on mitochondrial hepatic fractions, specially focusing onthe alterations of mitochondrial electron transport/transmembrane elec-tric potential that may be the cause of MPT induction. The results hereobtained are relevant to understand the possible toxicity of edelfosine(as well as perifosine) on mitochondria from a non-target organ.

2. Material and methods

2.1. Materials

Edelfosine was obtained from INKEYSA (Barcelona, Spain) andperifosine fromZentaris (Frankfurt, Germany). Both drugswere preparedas previously described (Mollinedo et al., 1997). Briefly, the compoundswere dissolved in RPMI 1640 supplemented with 10% heat-inactivatedFBS, for better dissolution of the compounds. Then, solution was incubat-ed at 50 °C during 45 min, sterilized through a 0.22 μm filter and storedat 4 °C. In the experiments, vehicle was RPMI 1640 media subjected tothe same procedure previously described. The maximal volume addedwas 50 μl in a total volume of 1 ml. Calcium Green-5N was obtainedfrom Life Technologies (Paisley, United Kingdom). Horseradishperoxidase and A23187 was obtained from Sigma-Aldrich Quimica SA(Sintra, Portugal). All other compounds were of the highest grade ofpurity commercially available.

2.2. Animals

Animal handling was performed in accordance with the EuropeanConvention for the Protection of Vertebrate Animals used for Experi-mental and Other Scientific Purposes (CETS no.123) and Portugueserules (DL 129/92). The procedures were approved by the CNC Commit-tee for Animal Welfare and Protection. Male Wistar rats, Crl:WI (Han),were purchased from Charles River (France), with 8 weeks of age, andmaintained in the local animal house facility (CNC— School ofMedicine,University of Coimbra, Coimbra, Portugal). Animals were group-housedin type III-H cages (Tecniplast, Italy), 2 individuals each, with irradiatedcorn cob grit bedding (Scobis Due,Mucedola, Italy) with environmentalenrichment and under controlled environmental requirements (22 °C,45–65% humidity, 15–20 air changes/h, 12 h artificial light/dark cycle,noise levelb55 dB), free access to standard rodent food (4RF21 GLPcertificate, Mucedola, Italy) and ad libitum acidified water (at pH 2.6with HCl).

2.3. Isolation of rat liver mitochondria

Mitochondria were isolated from rat liver by conventional methodswith slight modifications (Serafim et al., 2011). Homogenization medi-um was composed of 250 mM sucrose, 10 mM HEPES, pH 7.4, 1 mMEGTA, and 0.1% fat-free bovine serum albumin (BSA). EGTA and BSAwere omitted from the final washing medium, adjusted at pH 7.2. Themitochondrial pellet was washed twice and resuspended in the wash-ing medium. Protein content was determined by the biuret method(Gornall et al., 1949) with BSA as standard. Mitochondrial preparationwas kept on ice during experiments, which were carried out after a30 min recovery and within 5 h post-isolation.

2.5. Oxygen consumption

Oxygen consumption of isolated liver mitochondria was polaro-graphically monitored with a Clark-type oxygen electrode connectedto a suitable recorder in a 1 ml thermostated, water-jacketed, closedchamber with magnetic stirring at a constant temperature of 30 °C.The standard reaction medium consisted of 125 mM sucrose, 65 mMKCl, 5 mM KH2PO4, 2.5 mM MgCl2, 10 μM EGTA and 5 mM HEPES,pH 7.2. Mitochondria were resuspended at a concentration of1.5 mg/ml in the reaction medium. Edelfosine or perifosine werepre-incubated with mitochondria for 3 min. Respiration was startedthrough the addition of 5 mM glutamate/malate or 5 mM succinateplus 3 μMrotenone. ADP (250 μM)was added to initiate state 3 respira-tion. FCCP (0.5 μM) was also added to the system to uncouple respira-tion after state 4.

2.6. Mitochondrial transmembrane electric potential

The mitochondrial transmembrane potential (ΔΨm) was indirectlyestimatedby following themitochondrial accumulation of the lipophiliccation tetraphenylphosphonium cation (TPP+), detected through theuse of a TPP+-selective electrode in combination with an Ag/AgCl-saturated reference electrode, as previously described (Oliveiraet al., 2001). Mitochondrial protein (1.5 mg) was resuspended underconstant stirring in 1 ml of reaction medium described above andsupplemented with 3 μM TPP+. Mitochondria were energized with5 mM glutamate/malate or 5 mM succinate plus 3 μM rotenone.Edelfosine or perifosine was added before mitochondrial protein. ADP(250 μM) was added to initiate phosphorylation. A matrix volume of1.1 μl/mg protein was assumed. When used, CsA (0.5 μM) was addedto the mitochondrial preparation before calcium addition in order toprevent induction of the MPT pore.

2.7. Mitochondrial calcium movements

Extramitochondrial free Ca2+ was measured by using thehexapotassium salt of the fluorescent probe Calcium Green-5N(1 μM) (Rajdev and Reynolds, 1993). In brief, liver mitochondria(0.75 mg) were resuspended in 2 ml of buffer containing 200 mMsucrose, 10 mM Tris, pH 7.4, 12 μM EGTA (to complex basal calcium)and 1 mM KH2PO4 and supplemented with rotenone (1.5 μM) andsuccinate (2.5 mM). Fluorescence was continuously recorded in awater-jacketed cuvette holder at 30 °C, under constant stirring,using a PerkinElmer LS-55 fluorescence spectrometer (PerkinElmerLife and Analytical Sciences, Boston, MA) with excitation and emis-sion wavelengths of 506 and 531 nm, respectively and excitationand emission slits of 5 nm. Edelfosine and perifosine were incubatedwith the mitochondrial preparation in the reaction buffer during1 min for the acquisition of a baseline. Then, a single pulse of calcium(27–67 nmol/mg protein) was made 1 min after the experiment hadstarted. When used, CsA (0.5 μM) was added to the mitochondrialpreparation before calcium addition in order to prevent inductionof the MPT pore.

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2.8. Mitochondrial swelling

Mitochondrial volume changes were followed by decrease in absor-bance at 540 nm using a Jasco V-560 spectrophotometer (Jasco, Tokyo,Japan). The assays were performed in 2 ml of buffer containing200 mM sucrose, 10 mM Tris, pH 7.4, 12 μM EGTA (to complexbasal calcium), and 1 mM KH2PO4. Rotenone (1.5 μM) and succinate(2.5 mM) were also added to the media to which 0.75 mg of mito-chondrial protein was added under constant stirring at 30 °C. Calcium(40–67 nmol/mg protein) was added to the preparation approximately1 min after the start of the experiment. CsA (1 μM) was added to themitochondrial preparation, before the addition of calcium, to inhibitthe MPT. Edelfosine and perifosine were added to the mitochondrialpreparation approximately 2 min after the start of the experiment. Forassays involving deenergized mitochondria, the standard media wassupplemented with 1.5 μM rotenone, 0.5 μM FCCP and 1 μM A23187,a calcium ionophore.

2.9. Detection of mitochondrial H2O2

Hydrogen peroxide release by mitochondria was determined on iso-lated liver mitochondria using a modification of the method describedby Barja (2002). Mitochondrial H2O2 was indirectly measured throughthe reaction of H2O2with homovanilic acid in the presence of horseradishperoxidase, which forms a dimer fluorescent at 312 nm excitation and420 nm emission. Mitochondria (0.4 mg/ml) were incubated at 30 °C inphosphate buffer, pH 7.4, containing 0.1 mM EGTA, 5 mM KH2PO4,3 mM MgCl2, 145 mM KCl, 30 mM HEPES, 0.1 mM homovanilic acidand 6 U/ml horseradish peroxidase. When used, rotenone (1.5 μM) andantimycin A (0.5 μg/ml) were added into incubation medium to inhibitthe activities of Complex I and Complex III, respectively. Edelfosine wasadded to the incubation medium, before the beginning of the reaction.H2O2 release frommitochondriawas initiated by using succinate or gluta-mate/malate as substrates. The reaction was performed during 15 min,with constant stirring, in a temperature-controlled water bath at 30 °C.In the end, the reaction was stopped by addition of ice-cold 2 M glycinebuffer containing 25 mMEDTA and NaOH, pH 12, followed by a centrifu-gation at 15,700 g, at 4 °C for 5 min. Then, the fluorescence of an aliquotof 100 μl of the supernatants was read in a microplate reader (Molecu-lar Devices, Spectramax Plus 384), with excitation and emission wave-lengths of 312 nm and 420 nm, respectively. The rate of peroxideproduction was calculated using a standard curve of H2O2, whichwere prepared fresh every day andwere incubated under the same con-ditions as the samples.

2.10. Statistical analysis

Data are expressed as means±SEM for the number of experimentsindicated in the legends of the figures. Differences betweenmean groupswere assessed through matched pairs one-way analysis of variance(ANOVA) followed Bonferroni's or Dunnet's post-hoc test. Significancewas accepted with p valueb0.05.

3. Results

3.1. Edelfosine and perifosine inhibit mitochondrial oxygen consumption

The first experimental aimwas to investigate whether the two ALPsin study altered oxygen consumption in isolated hepatic mitochondria.Concentrations of up to 20 μM of both compounds had no effect in anyparameter investigated either using glutamate/malate or succinate assubstrates (data not shown); therefore, higher concentrations wereused in the remaining study. The effects of higher concentrations onmi-tochondrial respiration sustained with substrates linked to both com-plexes are shown in Figs. 1 and 2. As can be seen in the mentionedfigures, edelfosine and perifosine induced an increase in both state 2

and state 4 respiration. However, the effects during state 2 in the pres-ence of Complex I-linked substrates were not as evident as whenComplex II-linked substrates were used. In the later case, a dose-dependent effect was observed, a pattern which was also presentwhen analyzing data from state 4 respiration. Interestingly, state 3respiration was only decreased in a dose-dependent manner whensuccinate was used. However, despite the maximal≈40% decrease inthis respiration state, in the presence of glutamate/malate, the statisticaltest used did not detect any significance (ANOVA p value=0.06). Theconcomitant decrease in state 3 followed by increased state 4 resultedin the expected decrease in RCR. The effect started as low as 50 μM,for both compounds, and at highest concentrations, RCR values wereabout half of the control (vehicle) group.

Uncoupled respiration induced by FCCP displayed a decreased pat-tern similar to that of state 3, while ADP/O ratio was unchanged whenComplex II-linked substrates were used. However, in the presence ofglutamate/malate, ADP/O was slightly decreased at highest concentra-tion (ANOVA p value=0.06), for both compounds.

3.2. Edelfosine and perifosine interfere with the generation of mitochon-drial transmembrane electric potential

Under control conditions, mitochondria developed a maximal ΔΨm

after substrate addition of −216.0 mV±7.8 in glutamate/malate- and−219.6 mV±4.8 in succinate-sustained respiration. ADP additioncaused the expected drop in the membrane potential, as ATP synthaseuses ΔΨm to phosphorylate the added ADP during state 3 respiration.After a short lag phase, during ADP phosphorylation, the ΔΨm repolar-izes to approximate initial values.

Both tested compounds decreasedΔΨm in a dose-dependent fashion,regardless of the respiratory substrate used (Figs. 3 and 4). However, thestatistical test only picked differences for the highest concentrationwhen glutamate/malate was used (both compounds, Fig. 3). Edelfosineslightly increased ADP-induced depolarization for all tested concentra-tions, but solely when Complex I-linked substrates were used. Thesame effect was detected for perifosine but only for the highest concen-tration. Regarding ΔΨ repolarization, after ADP addition, mitochondriaincubated with both ALPs were never able to recover ΔΨ to controlvalues. This was a dose-dependent effect and was observed regardlessof the respiratory substrate used. However, as in the case of ΔΨmax, thedifferences were only statistically significant for higher concentrations.The same pattern was observed for the lag phase: ALPs induced adose-dependent increase in lag phase values, for both respiratory sub-strates, with the strongest differences attained at the highest concentra-tion. No significant alterations were observed for concentrations up to20 μM of both compounds for any of the evaluated parameters (datanot shown).

3.3. Edelfosine and perifosine decrease mitochondrial calcium loadingcapacity

To confirm that both compounds induce MPT pore opening, we eval-uatedmitochondrial calcium loading capacity in the presence of an inter-mediate concentration of the tested ALPs. A typical record is depicted inFig. 5A using the fluorescence probe Calcium Green-5N. Shortly afteran increase in probe fluorescence due to calcium addition, fluorescencedecreases in all conditions tested, indicating mitochondrial calciumuptake. However, edelfosine and perifosine significantly decreasedcalcium accumulation, as the variation of fluorescence intensity in thefirst 400 s is greater in both groups when compared to vehicle control(Fig. 5B). The effects of the two compounds in the calcium-induced cal-cium release were completely inhibited when pre-incubation with CsAwas performed, indicating that loss of calcium accumulation is due toMPT pore opening (Fig. 5A and B). Data also indicates that vehicle perse does not have effect on calcium-induced calcium release (line 1 vs.line 2 on Fig. 5A).

Fig. 1. Effects of edelfosine (Edelf) and perifosine (Perif) on glutamate/malate (G/M)-energized mitochondria. Control (no additions) values for state 3, state 4 and uncoupled respirationwere 111.5±20.7, 18.3±7.2 and 111.2±37.0 natms O/min/mgprotein, respectively.Mitochondria (1.5 mg)were incubated in 1 ml of respirationmedia (seeMaterials andmethods formore details) at 30 °C. ADPwas added to induce state 3 respiration,which followedby state 4 after complete phosphorylation of added-ADP. To uncouple respiration, FCCPwas also addedto the system. RCR was calculated as the ratio between state 3 and state 4 respiration and is an indicator of the control efficiency exercised by phosphorylation on oxidation. ADP/O ratiowas calculated as the number of nanomoles of added-ADPby nanoatoms of oxygen consumedduringADP phosphorylation. Data is expressed asmeans of absolute values±SEMof four tofive different preparations. Differences between groupswere assessed through a one-wayANOVA followedby a Dunnet's test for corrections formultiple comparisons. *pb0.05 vs. vehicle(the highest volume used in the experiments). Abbreviations: Edelf — edelfosine; G/M — glutamate/malate; Perif — perifosine; V — vehicle.

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To confirm this results, calcium-induced depolarization was mea-sured by using a TPP+-sensitive electrode (Fig. 5C). A useful parameterto characterize the sensitivity to MPT pore induction is the level ofrepolarization, after a single pulse of calcium. Through this method-ology the same conclusion was made as edelfosine and perifosinenever allowed a complete repolarization of the ΔΨm. This effectwas once again abolished by pre-incubation with CsA.

Another hallmark of the MPT is the high-amplitude swelling in-duced by calcium. In our assays, the two ALP compounds caused adecrease in the mitochondrial suspension absorbance, typical of themitochondrial swelling induced by opening of the MPT pore. Thetotal swelling amplitude (i.e. initial absorbance minus the final ab-sorbance value) was calculated and plotted on Fig. 6 as a positivevalue. Mitochondrial swelling induced by calcium in the presence ofthe two compounds was partly inhibited by CsA (Fig. 6A), althoughthe degree of inhibition was lower thanwhen following calciummove-ments (Fig. 5). No effects were observed for concentrations lower than20 μM (data not shown).

To assesswhether the observed alterations onMPT of the tested ALPsare due to the effects in calcium transport through the uniporter or inΔΨm generation through the electron transfer chain, the same kind ofexperiments were carried on non-energized mitochondria. Since matrixcalcium accumulation is dependent on ΔΨm, a calcium ionophoreA23187 was used to assure calcium equilibrium between the extra-and intra-mitochondrial compartments. FCCP was added to completeabolish any contribution to ΔΨm from endogenous substrates. Fig. 6Bshows that both edelfosine and perifosine can still inducemitochondrialswelling, although interestingly CsA did not inhibit calcium-inducedmitochondrial swelling completely.

3.4. Edelfosine decreases hydrogen peroxide (H2O2) production on isolatedmitochondria

In order to explore the effect of ALPs in H2O2 production throughthe mitochondrial respiratory chain, which could be an explanationfor the increased sensitivity to MPT pore opening, mitochondrialH2O2 production was measured.

To pinpoint possible specific sources of free radicals in the respira-tory chain, different substrates and two specific inhibitors, rotenone(Rot) and antimycin A (AA), were used. For this particular assay,only edelfosine was used since results from both compounds werevery similar in terms of MPT induction. In vehicle group, AA andAA+Rot significantly increased H2O2 production through the respira-tory chain in the presence of glutamate/malate (G/M), as expected bythe known bioenergetics principles. However, note that Rot just by it-self was not enough to increase H2O2 production. Interestingly,edelfosine inhibited H2O2 production in all mentioned incubationstates (Fig. 7A). When succinate (Succ) was used as substrate, againAA alone and AA+Rot increased H2O2 generation in the control andvehicle groups. Interestingly, we were not able to observe massiveH2O2 production through the reverse electron transfer as the levelsof H2O2 production in the presence of Rot and with substrate alone(Succ) were not dissimilar. Again, edelfosine decreased H2O2 produc-tion in the presence of these inhibitors (Fig. 7B).

4. Discussion

Synthetic lipase-resistant analogs of lysophosphatidylcholine, col-lectively named ALPs, exert cytotoxic effects against a wide variety of

Fig. 2. Effects of edelfosine (Edelf) and perifosine (Perif) on succinate (Succ)-energized mitochondria. Control (no additions) values for state 3, state 4 and uncoupled respirationwere 107.9±24.9, 21.3±4.9 and 408.9±86.8 natms O/min/mg protein, respectively. Mitochondria (1.5 mg) were incubated in 1 ml of respiration media (see Materials andmethods section for more details) at 30 °C. ADP was added to induce state 3 respiration, which followed by state 4 after complete phosphorylation of added-ADP. To uncouple res-piration, FCCP was also added to the system. RCR was calculated as the ratio between state 3 and state 4 respiration and is an indicator of the control efficiency exercised by phos-phorylation on oxidation. ADP/O ratio was calculated as the number of nanomoles of added-ADP by nanoatoms of oxygen consumed during ADP phosphorylation. Data is expressedas means of absolute values±SEM of six different preparations. Differences between groups were assessed through a one-way ANOVA followed by a Dunnet's test for correctionsfor multiple comparisons. *pb0.05 vs. vehicle (the highest volume used in the experiments). Abbreviations: Edelf — edelfosine; Perif — perifosine; Succ — succinate; V — vehicle.

Fig. 3. Effects of edelfosine (Edelf) and perifosine (Perif) on glutamate/malate (G/M)-energized mitochondria. Maximal ΔΨm, ADP depolarization, and lag phase were measured by using aTPP+-selective electrode. Control values (no additions) for maximal ΔΨm, ADP depolarization and lag phase were −216.0±7.8 mV, −46.9±8.2 mV and 27.6±9.1 s, respectively. Data isexpressed as means of absolute values±SEM of four to five different preparations. Differences between groups were assessed through a one-way ANOVA followed by a Dunnet's test for cor-rections formultiple comparisons. *pb0.05 vs. vehicle (the highest volume used in the experiments). Abbreviations: Edelf— edelfosine;G/M— glutamate/malate; Perif— perifosine; V— vehicle.

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Fig. 4. Effects of edelfosine (Edelf) and perifosine (Perif) on succinate (Succ)-energized mitochondria. Maximal ΔΨm, ADP depolarization, and lag phase were measured by using aTPP+-selective electrode. Control values (no additions) for maximal ΔΨm, ADP depolarization and lag phase were −219.6±4.8 mV, −46.5±6.0 mV and 48.4±14.1 s, respective-ly. Data are expressed as means of absolute values±SEM of twelve different preparations. Differences between groups were assessed through a one-way ANOVA followed by aDunnet's test for connections for multiple comparisons. *pb0.05 vs. vehicle (the highest volume used in the experiments). Abbreviations: Edelf — edelfosine; Perif — perifosine;Succ — succinate; V — vehicle.

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tumors (Mollinedo et al., 1997; Unger et al., 1989) and represents anovel class of antitumor agents, once they target cell membranes(Hideshima et al., 2006), rather than nuclear DNA (Vink et al.,2007). The prototype of these compounds, edelfosine, structurally re-sembles lysophosphatidylcholine since it has the same polar headgroup and a single long apolar hydrocarbon chain which allows aneasy and stable insertion into plasma membranes, interfering withlipid-based signal transduction and often resulting in apoptosis oftumor cells (van der Luit et al., 2007). A second generation of ALPs in-cludes single-chain alkylphosphocholines and their derivatives (van derLuit et al., 2007), such as perifosine. This compound has received increas-ing attention as an anticancer agent, especially in combinationwith otherpharmacologic drugs (Dasmahapatra et al., 2004; Hideshima et al., 2006;Li et al., 2006; Momota et al., 2005; Nyakern et al., 2006; Rahmani et al.,2005), as well as with radiotherapy (Vink et al., 2006a,b, 2007).

Mollinedo et al. (2011) reported that edelfosine increases mem-brane permeability, whichmight explain the involvement of mitochon-dria in edelfosine-mediated apoptosis in tumor cells. In the study, theauthors used isolated liver mitochondria from adult Sprague–Dawleyrats, demonstrating that edelfosine concentrations between 5 μM and50 μM caused an increase of membrane permeability, whichwas partlyinhibited by CsA (Mollinedo et al., 2011). One of the main objectives ofthe presentworkwas to verify if increasedMPT, that is usually observedafter in vitro treatment of isolated mitochondria with ALPs (Mollinedoet al., 2011), occurred in the presence of alterations in mitochondrialoxidative phosphorylation andH2O2 generation through the respiratorychain. Additionally, perifosine, a second generation ALP, was used to

observe if direct mitochondrial effects were similar to edelfosine.Despite data showing effects on the plasma membrane level (Gajateand Mollinedo, 2011; Mollinedo et al., 2010, 2011) it is tentative tospeculate that direct mitochondrial effects can also contribute to celldeath in tumor cells. In fact, mitochondria, and in particular the MPT,has been involved in the induction of cell death in many tumors(Barbosa et al., 2012). It is also noteworthy to point that drug-inducedtoxicity in non-target organs is often related to MPT induction (Konet al., 2004; Pereira et al., 2011; Suski et al., 2011). Hence, the presentstudy is relevant as it supplies clues about mitochondrial perturbationswhich occur during edelfosine and perifosine treatment and which canbe translated into effects in tumor and non-tumor cells.

Based on the data presented throughout this paper, it is reasonable tothink that edelfosine and perifosine, once accumulated in mitochondrialmembranes affect oxidative phosphorylation efficiency and, therefore,compromise normal cell behavior. Data from mitochondrial respirationand membrane potential (Figs. 1 to 4) demonstrate that the two testedcompounds similarly affect mitochondrial bioenergetics in a dose-dependent manner. Inhibition of uncoupled respiration, as well as in-creased state 4 and state 2 respiration and decreased ΔΨm, state 3 andRCR make it difficult to ascertain whether the two compounds affectthe respiratory chain or the phosphorylative system.

However, the increased state 2 and state 4 clearly shows the possibil-ity of increased proton leak that would be not only responsible for thedecreased ΔΨm, as well as the decrease in state 3 and RCR but withoutsignificant changes in ADP/O. Moreover, the fact that the toxicity ofedelfosine and perifosine is mostly similar regardless of the substrate

Fig. 5. Mitochondrial calcium-loading capacity. (A) Representative recording of mitochondrial calcium-loading capacity in the presence of edelfosine (Edelf) or perifosine (Perif). Line 1,untreated control; line 2, vehicle (the highest volume used in the experiments); line 3, cyclosporin A (CsA); line 4, edelfosine 75 μM; line 5, edelfosine 75 μM+CsA; line 6, perifosine75 μM; line 7, perifosine 75 μM+CsA. The fluorescent probe Calcium Green-5N was used to evaluate the levels of extramitochondrial calcium as described under Materials andmethods section. (B) Assessment of mitochondrial calcium-loading capacity in the presence of edelfosine or perifosine. ΔI was calculated as the difference between absorbance at 50 s(before calcium addition) and the absorbance at 400 s (end of the experiment). 75 μM edelfosine (Edelf) or 75 μM perifosine (Perif) was incubated with mitochondria (0.75 mg). 27to 67 nmol calcium/mg protein was used to induce the MPT. Data is expressed as means of absolute values±SEM of three different preparations. Differences between groups wereassessed through a one-way ANOVA followed by a Bonferroni's test for corrections for multiple comparisons. *pb0.05 vs. vehicle; #pb0.05 vs. Edelf+CsA or Perif+CsA. (C) Edelfosineand perifosine stimulate calcium-induced depolarization. A single pulse of calcium was added to energized mitochondria in order to initiate time-dependent transmembrane potentialalterations, which were followed by a TPP+-sensitive electrode. Values correspond to ΔΨmax attained after 5 min of calcium addition. Data is expressed as means of absolute values±SEM of four different preparations. Differences between groups were assessed through a one-way ANOVA followed by a Bonferroni's test for corrections for multiple comparisons.*pb0.05 vs. vehicle; #pb0.05 vs. Edelf+CsA or Perif+CsA. Abbreviations: CsA — cyclosporin A; Edelf — edelfosine; Perif — perifosine; V — vehicle.

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used, suggests that both compounds may have a similar mitochondrialtarget.

The presentwork also confirms that edelfosine, aswell as perifosine,enhances Ca2+-dependent MPT, which also results into an increase ofcalcium-induced calcium release (Fig. 5) and mitochondrial swelling(Fig. 6). These events play an important role in some modes of celldeath (Ott et al., 2007), as MPT induction causes drastic changes in mi-tochondrial ultrastructure and functional activity (Orrenius et al., 2003).

However, in our experimental setup, we found intriguingly thatCsA did not always abolished MPT induction when pre-incubated

with the mitochondrial preparation. In fact, the degree of protectiondepended on the type of technique used, for example, there was notan overall protection against mitochondrial swelling (Fig. 6A), althoughcalcium-induced calcium release or calcium-induced depolarizationwas totally inhibited (Fig. 5). While the reason for the difference is dif-ficult to explain, it is possible that the interaction of the test compoundswith mitochondrial membranes cause an increase in ion permeabilitythat is not inhibited by CsA but which results inmitochondrial swelling.Alternatively, the interaction between the two compounds and mito-chondrial lipid rafts (Mollinedo et al., 2011) may cause morphology

Fig. 6. Effects of edelfosine (Edelf) and perifosine (Perif) on mitochondrial swelling. Swelling was followed at 540 nm and its total amplitude (as a positive value) was calculated as thedifference between basal line absorbance (first 110 s) and the absorbance at 500 s (for further details of the method, see Materials and methods section). A single dose of 75 μM ofedelfosine (Edelf) or perifosine (Perif) was incubated with mitochondria and in specific cases both compounds were also incubated in the presence of CsA. (A) Mitochondrial swellingafter induction of the MPT in succinate-energized mitochondria. Data is expressed as means of absolute values±SEM of five different preparations. Differences between groups wereassessed through a one-way ANOVA followed by a Bonferroni's test for corrections for multiple comparisons. *pb0.05 vs. vehicle (the highest volume used in the experiments);#pb0.05 vs. Perif+CsA. (B)Mitochondrial swelling after induction of theMPT in non-energizedmitochondria. Mitochondria were incubated in standard reactionmedium supplementedwith 1.5 μM rotenone, 0.5 μM FCCP and 1 μM A23187 (calcium ionophore). In A) and B), a single pulse of 40–67 nmol calcium/mg protein was applied to induce the MPT. Data isexpressed as means of absolute values±SEM of three different preparations. Differences between groups were assessed through a one-way ANOVA followed by a Bonferroni's test forcorrections for multiple comparisons. *pb0.05 vs. vehicle. Abbreviations: CsA — cyclosporin A; Edelf — edelfosine; Perif — perifosine; V — vehicle.

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alterations in the presence of calcium which are not dependent on theMPT, although this ismuch difficult to demonstrate. It is also interestingthat CsA was also shown to have variable activity against edelfosine-inducedMPT in a previous study. Cyclosporin-Awas ineffective for higheredelfosine concentrations and in long-term experiments (Mollinedoet al., 2011). Although these previous results confirm that CsA may beof limited utility to completely inhibit the increase in inner membranepermeability caused by the ALPs in study, differences existed betweenthe work by Mollinedo et al. and our present study. First, Mollinedo etal. used a higher concentration of CsA (10 μM) and a longer incubationperiod (25 min) when compared with the present study (1 μM and2–3 min). Other differences also existed in terms of composition of themitochondrial isolation and reaction media, specially the inclusion ofmannitol by Mollinedo et al. The incomplete protection afforded by CsAin swelling experiments (this work and Mollinedo et al., 2011) togetherwith increased state 2 and state 4 respiration, strengthen the idea of in-creased non-specific membrane permeability and/or ion/proton leak.

Since MPT induction in the presence of the ALPs occurred in thepresence of alterations in mitochondrial oxidative phosphorylation, in-cluding decreased ΔΨm, we used non-energized mitochondria in thepresence of a calcium ionophore and FCCP to exclude that the ALPsmay induce the MPT through a voltage-regulated mechanism. Again,both edelfosine and perifosine were able to induce the MPT, althoughCsA had a marginal protective effect. The results demonstrate a poten-tial direct effect of the two ALPs on a pore component (directly orthrough a membrane-mediated effect), excluding MPT induction

following mitochondrial ΔΨm variations. The insensitivity to CsA maybe explained by one of the possibilities pointed above, plus the factthat the MPT in non-energized mitochondria may not be as sensitiveto the action of CsA,which has been proposed to bemediated by alteredcyclophilin D role in pore regulation (Doczi et al., 2011).

Mitochondria are well known to be one source of free radicals as aconsequence of mitochondrial respiration (Sastre et al., 2000). TheseROS can then interact directly with mitochondrial proteins, lipids andDNA, obscuring their functions and operation of the organelle, whichmay directly influence cell viability and trigger cell death (Ott et al.,2007). There is growing evidence that most of O2

• generated by intactmammalian mitochondria in vitro is produced at Complex I level; how-ever, in addition to Complex I, Complex III is regarded as another impor-tant site for O2

• production (Turrens and Boveris, 1980). Data regardingH2O2 production through the respiratory chain revealed that edelfosine,the single ALP tested, decreases H2O2 production (Fig. 7). Interestingly,edelfosine inhibited H2O2 production using substrates for both Com-plex I and II (Fig. 7), which is per se interesting, having in mind theinducing effects on the transition pore on isolated mitochondriaand the pro-apoptotic effect on intact cells. One interesting possibilityis that edelfosine may act as a Complex III inhibitor, inhibiting ROS pro-duction by that complex, similarly to myxothiazol (Zmijewski et al.,2009). However, another explanation for the decreased H2O2 productionmay be due to the lower ΔΨm observed in the presence of these ALPs.High proton-motive force is a requirement in the mechanism of electronescape from the respiratory chain tomolecular oxygen (Huttemann et al.,

Fig. 7. H2O2 production by liver mitochondria oxidizing Complex I- (A) and Complex II-linked (B) substrates, measured as described in theMaterials andmethods section. In both panels,black bars represent control group (vehicle; the highest volume used in the experiments) and white bars the treated group (edelfosine). The labels below each bar represent the assaysconditions used, namely respiratory substrates (G/M and Succ) and inhibitors (Rot and AA). Further details about the procedure can be found in theMaterials andmethods section. Data isexpressed as means of absolute values±SEM of four to five different preparations. Differences between groups were assessed through a one-way ANOVA followed by a Bonferroni's testfor corrections for multiple comparisons. *pb0.05 vs. subst (vehicle control — black bars); #pb0.05 vs. subst+AA (vehicle control — black bars); Φpb0.05 vs. subst+Rot+AA (vehiclecontrol — black bars). Abbreviations: AA— antimycin A; G/M — glutamate/malate; Rot — rotenone; Subst— substrates (glutamate/malate or succinate); Succ — succinate; V — vehicle.

33A. Burgeiro et al. / Mitochondrion 13 (2013) 25–35

2008). Both interferences can derive from edelfosine membrane interac-tions. The results confirmed the data byMollinedo et al. (Mollinedo et al.,2011), which demonstrated that theMPT caused by edelfosine is unlikelyto directly result from increased oxidative stress.

It has been demonstrated that edelfosine co-localizes with plasmamembrane lipid rafts, reorganizing membrane rafts and inducing apo-ptosis in human leukemic cells (Gajate and Mollinedo, 2001). In fact,Fas association with lipid rafts is required for edelfosine-induced apo-ptosis (Gajate and Mollinedo, 2001). However, lipid rafts are not onlypresent at the plasmamembrane, but also in cell organelles such as en-doplasmic reticulum, Golgi apparatus and even mitochondria (Malorniet al., 2007), although some controversy exists about the later (Zhenget al., 2009). Taking this into account, one interesting possibility isthat edelfosine may be accumulated in mitochondrial lipid rafts inter-fering with mitochondrial activity.

The present work shows that on isolated hepatic mitochondrialfractions, both edelfosine and perifosine are toxic at concentrationsabove 50 μM. Mollinedo et al. (2011)observed mitochondrial mem-brane permeabilization for concentrations as low as 5 μM, but differ-ences exist in the experimental protocol and animal strain used. Onemajor limitation of the present study (as in many others) is the lack ofknowledge of the real concentration of edelfosine or perifosine thatreaches mitochondria in intact cells. In fact, and depending of tumorcell type, the majority of reports used 10–20 μM edelfosine to havepro-apoptotic effects (Gajate et al., 1998, 2000b, 2012; Mollinedoet al., 2010; Nieto-Miguel et al., 2006, 2007). Moreover, at high

concentrations, ALPs are often cytotoxic due to their detergent-likecharacter causing normal cell lysis (Girault et al., 2011). However,we do not see a widespread detergent-type effect in the mitochondri-al preparations used. The lack of effect of CsA could be explained bysuch effect, but the total inhibition of calcium-induced calcium releaseand depolarization, makes this an unlikely cause. Other possibilitiescan be selective outermitochondrialmembrane permeabilization or se-lective permeability to some ions, with calcium exception.

In conclusion, the present work demonstrates that the ether phos-pholipids edelfosine and perifosine induce the MPT and disturb mito-chondrial oxidative phosphorylation. Induction of the MPT by bothALPs appears not to be dependent on ΔΨm alterations and it is unlikelyto result from increased hydroperoxides generated by the respiratorychain. The results are relevant when understanding the mechanismsbehind the pro-apoptotic activity of the two compounds and also interms of toxicity to non-target organs. The fact that it was recently dem-onstrated that exogenous ether lipids predominantly targetmitochondria(Kuerschner et al., 2012), makes this study a valuable tool to understandchemical–biological interactions of this class of compounds.

Acknowledgments

This work was supported by a research grant from Fundação para aCiência e Tecnologia (FCT) (Ministério da Ciência, Tecnologia e EnsinoSuperior de Portugal, co-funded by FEDER/Compete/National funds)(PTDC/QUI-QUI/101409/2008) to P.J.O., Ministerio de Ciencia e

34 A. Burgeiro et al. / Mitochondrion 13 (2013) 25–35

Innovación of Spain (SAF2008-02251, and RD06/0020/1037 fromRed Temática de Investigación Cooperativa en Cáncer, Instituto de SaludCarlos III, cofunded by the Fondo Europeo de Desarrollo Regional of theEuropean Union) to F.M., Junta de Castilla y León (GR15-ExperimentalTherapeutics and Translational Oncology Program, and BiomedicineProject 2009) to A.B., G.C.P., C.V.P. and F.S.C. were recipients of Ph.D.fellowships from the Portuguese FCT (SFRH/BD/32943/2006, SFRH/BD/36938/2007, SFRH/BD/48029/2008 and SFRH/BD/64694/2009,respectively).

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