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Direct effect of Taxol on free radical formation and mitochondrial permeability transition
Free Radical Biology & Medicine, Vol. 31, No. 4, pp. 548 –558, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(01)00616-5
Original Contribution DIRECT EFFECT OF TAXOL ON FREE RADICAL FORMATION AND MITOCHONDRIAL PERMEABILITY TRANSITION GABOR VARBIRO, BALAZS VERES, FERENC GALLYAS JR.,
and
BALAZS SUMEGI
Institute of Biochemistry, University of Pecs, Medical School, Pecs, Hungary (Received 23 January 2001; Accepted 22 May 2001)
Abstract—To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the M range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (⌬⌿) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 M cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN⫺, azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of ⌬⌿ and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity. © 2001 Elsevier Science Inc. Keywords—ROS, Permeability transition, Taxol, Apoptosis, Mitochondria, Cultured cells, Free radicals
INTRODUCTION
opening of permeability transition pore facilitate the paclitaxel-induced apoptosis in tumor cell lines [11]. These effects of Taxol were exerted in the nM–M concentration range usually after a 2–24 h exposure to the drug, with a tendency of longer exposure times for lower concentrations [4 –14]. It was also suggested that the effect of paclitaxel on the mitochondrial permeability transition pore was mediated through interaction with the cytoskeleton [15]. Mitochondrial permeability transition can lead to cell death, and can be induced by high intracellular inorganic phosphate, Ca2⫹, reactive oxygen species (ROS), or proapoptotic proteins such as Bax, whereas it is inhibited by cyclosporine A (CsA), bongkrekic acid, Bcl-2, or Bcl-xL [16]. The opening of the large inner membrane pores cause equilibration of ions within the matrix and the cytosol, dissipating the membrane potential (⌬⌿) and uncoupling the respiratory chain. The volume disregulation following the opening of the permeability transition pore results in the swelling of the matrix, leading to outer membrane disruption and the release of cytochrome c
Paclitaxel, a widely used antineoplastic agent [1–3], stabilizes tubulin dimers, thus interfering with microtubular disassembly [4], resulting in the arrest of cells in G2-M phase of the cell cycle, followed by DNA fragmentation and morphological features of apoptosis. Paclitaxel has also been shown to activate Raf-1 [5] and cause phosphorylation of Bcl-2 [6,7]. This has been claimed to inactivate Bcl-2 and deprive it of forming heterodimers with the proapoptotic Bax [8 –10], hence leading to increased free intracellular Bax level, and ultimately resulting in the activation of the caspase proteins through the Bax-induced release of cytochrome c from the mitochondrion. Mitochondrial membrane stabilizing proteins (Bcl-2 and Bcl-XL) were reported to decrease apoptotic effect of paclitaxel [7], while proteins promoting the Address correspondence to: Balazs Sumegi, Institute of Biochemistry, Medical School, University of Pecs, 12 Szigeti st., H-7624 Pecs, Hungary; Tel: ⫹36 (72) 536-000/ext. 1633; Fax: ⫹36 (72) 536-277; E-Mail: [email protected]. 548
Mitochondrial effects of Taxol
and other caspase-activating proteins into the cytosol, ultimately contributing to the apoptosis of the cell [17]. Mitochondria play a pivotal role in oxidative cell damages too, because the major source of the endogenously produced ROS is the mitochondrial respiratory chain [18 –20]. Despite numerous studies, mechanisms of mitochondrial effect of paclitaxel still remain to be elucidated. In this paper, we studied the direct mitochondrial effects of paclitaxel, namely, the induction of mitochondrial ROS production, the collapse of mitochondrial membrane potential, and the opening of mitochondrial permeability pore. These observations may provide a novel molecular mechanism for the cytotoxicity of paclitaxel that can explain its toxic effect in slowly replicating and nondividing cells. MATERIALS AND METHODS
Materials Paclitaxel was from ICN Biomedicals Inc. (Aurora, OH, USA); Taxol was from Bristol-Myers Squibb S.p.A. (Sermonetta, Italy); CsA was from Biomol Research Labs, Inc. (Plymouth Meeting, PA, USA); Rhodamine123 (Rh123), Dihydrorhodamine123 (DRh123), and N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine were from Molecular Probes (Eugene, OR, USA); anti-cytochrome c monoclonal antibody was from Pharmingen (San Diego, CA, USA); all other compounds were from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.
Animals Wistar rats were purchased from Charles River Hungary Breeding LTD. (Budapest, Hungary). The animals were kept under standardized conditions; tap water and rat chow were provided ad libitum. Animals were treated in compliance with approved institutional animal care guidelines.
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Isolation of mitochondria Liver, heart, and kidney mitochondria were prepared according to standard protocol [21] except that a Teflonglass homogenizer was used instead of a hypodermic needle for the second homogenization step. The only differences among the organs were in the primary homogenization protocol; liver and kidney was squeezed through a liver press, while heart was minced with a blender. Brain mitochondria were prepared according to Sims [22]. Isolation of mitochondria from HepG-2 and BRL 3A cells were performed exactly as described by Almeida and Medina [23] for astrocytes. All isolated mitochondria were purified by Percoll-gradient centrifuging [22] and their RCR values were determined by a Clark electrode (see below). Mitochondrial permeability transition Mitochondrial permeability transition was monitored by following the accompanying large amplitude swelling via the decrease in absorbance at 540 nm [24], measured by a Perkin-Elmer fluorimeter (London, UK) in reflectance mode. Mitochondrial membrane potential was monitored by fluorescence of Rh123, released from the mitochondria following the induction of permeability transition [13] by using a Perkin-Elmer fluorimeter at an excitation wavelength of 495 and an emission wavelength of 535 nm. Briefly, mitochondria at the concentration of 1 mg protein/ml were preincubated in the assay buffer (70 mM sucrose, 214 mM mannitol, 5 mM N-2hydroxyethyl piperasine-N⬘-2-ethanesulfonic acid, 5 mM glutamate, 0.5 mM malate, 0.5 mM phosphate) containing 1 M Rh123 and the studied substances for 60 s. Alteration of the mitochondrial membrane potential was induced by the addition of 150 M Ca2⫹ or of paclitaxel at the indicated concentration plus 2.5 M Ca2⫹. Fluorescence intensity changes were detected for 3 min. The results are demonstrated by representative original registration curves from five independent experiments, each repeated three times. Detection of cytochrome c release in vitro
Cell culture HepG-2 human hepatocellular carcinoma and BRL 3A rat liver cell lines were from American Type Culture Collection (Rockville, MD, USA). Both cell lines were grown in humidified 5% CO2 atmosphere at 37°C and maintained in culture as monolayer adherent cells in Dulbecco’s Modified Eagle’s Medium containing 1% antibiotic-antimycotic solution (Sigma) and 10% fetal calf serum. Cells were passaged at intervals of 3 d.
Detection of cytochrome c release in vitro was performed as described by Susin et al. [25] Samples from the cuvette were taken prior to, and 3 min after the induction of the permeability transition, and centrifuged at 13,000 rpm for 3 min. Supernatants were analyzed for cytochrome c by Western blotting. Enhanced chemiluminescence labeling was used for the visualization of the blots. Results are demonstrated by photomicrograph of a representative blot showing only the samples taken 3 min after the induction of the permeability transition, because
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cytochrome c content of the samples taken prior to the induction of the permeability transition were below the detection limit.
Enzymatic permeability transition assay Measuring of citrate synthase (EC 4.1.3.7) and carnitine acyltransferase (EC 2.3.1.7) activity after the induction of permeability transition [26] was performed by using a Beckman spectrophotometer (Fullerton, CA, USA) at the wavelength of 412 nm. Mitochondria at the concentration of 0.1 mg protein/ml were preincubated in the assay buffer containing the studied substances. Permeability transition was induced as above. After 4 min, 100 g/ml 5,5⬘-dithiobis-(2-nitrobenzoate), 40 nM Acetyl-Coenzyme A, and 1 mM oxaloacetate or 2.5 mM L-carnitine was added, and the alteration of the extinction was monitored for 5 min.
Determination of ROS formation For the determination of ROS-formation, mitochondria was incubated at room temperature in 20 mM tris(hydroxymethyl)-aminomethane buffer, pH 8.0 containing 250 mM sucrose, 1 mM ethylene glycol-bis(aminoethyl ether) N,N,N⬘,N⬘-tetraacetic acid (EGTA), 1mM MgCl2, 1.7 M octanoic acid, 1 M ADP, and 2 M DRh123. The fluorescence of Rh123 formed by ROS-induced oxidation of the nonfluorescent DRh123 in situ [27–29], was measured by a Perkin-Elmer fluorimeter at an excitation wavelength of 495 nm and an emission wavelength of 535 nm. Fluorescence intensity changes were recorded for 3 min. The oxidation of N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine forms N-acetyl-8-dodecyl-resorufin (resorufin), which exhibits strong red fluorescence. This product is well retained in living cells and organelles by virtue of its lipophilic tail, making it possible to detect ROS production in the lipid phase. The method is the same as described above except for using 3 M N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine instead of DRh123, and changing the excitation wavelength to 578 nm and the emission wavelength to 597 nm. Baseline ROS formation due to spontaneous oxidation of the dyes and unstimulated ROS production by the mitochondrial respiratory chain was determined from the initial slope of the registration curves (Fig. 5) and was subtracted. ROS formation was calculated from the slope of the registration curves and expressed in arbitrary units, mean ⫾ SEM of at least five independent experiments.
Mitochondrial oxygen consumption Mitochondrial oxygen consumption (mitochondrial respiration) was assessed by a Clark electrode [30]. Briefly, isolated mitochondria were suspended in 20 mM Tris buffer, pH 7.4 containing 20 mM KCl, 220 mM mannitol, 70 mM sucrose, and 1 mM EGTA. The mitochondrial respiration was stimulated with either 10 mM succinate or 10 mM pyruvate (state 4 respiration), and 3.3 mM ADP (state 3 respiration) in the absence or presence of paclitaxel and/or up to 500 M Ca2⫹ (when Ca2⫹ was used, EGTA was omitted from the buffer). Before performing experiments, RCR values of the mitochondria were determined and were found to be in the range of 3.5–5.1 for the tissues and 2.1–2.5 for the cell lines in accordance with published values [31,32].
Demonstration of permeability transition in cultured cells BRL 3A cells were seeded to glass cover slips (60 ⫻ 40 mm) at a starting density of 2500 cells/cm2. The following day, CoverWell perfusion chambers (Grace Bio-Labs, Bend, OR, USA) were mounted on to the cover slips that allowed fast replacing of the medium on the cells. Cells were loaded in culture medium with 1 g/ml of Rh123 for 30 min at 37°C. After three washes with Krebs-Ringer/HEPES (KRH) buffer [33], 20 M paclitaxel without or together with 2.5 M CsA in KRH buffer containing 5 g/ml propidium iodide (PI) was added. Incubation was continued under the microscope at room temperature, and fluorescent images were taken periodically. The fluorescence of Rh123 and PI was monitored using a Bio-Rad MRC-1024ES laser scanning confocal attachment (Herefordshire, UK) mounted on a Nikon Eclipse TE-300 inverted microscope (Kingston, UK). Cells were excited with the 488 nm line of argon ion laser. Excitation light was passed through a dichroic mirror with a dividing wavelength of 527 nm, preventing the exciting light from reaching the detectors. The emitted fluorescent light was divided by a 565 nm dichroic mirror and was measured by separate photomultipliers through a 522/35 nm and a 680/32 nm band-pass filter. Laser intensity was attenuated by 99% in order to minimize photobleaching. Fluorescence was imaged with a Plan Apo 100⫻/1.4 oil-immersion objective. Confocal images were transferred to a Compaq Prosigna 300 workstation (Houston, TX, USA) and were processed by using Bio-Rad’s LaserSharp and Photoshop (Adobe Systems, Mountain View, CA, USA) software packages. In this arrangement, green and red color represent Rh123 and PI fluorescence, respectively.
Mitochondrial effects of Taxol
Detection of ROS formation in cultured cells BRL-3A cells were seeded into 96 well plates at a starting density of 2.5 ⫻ 104 cell/well and cultured overnight in humidified 5% CO2 atmosphere at 37°C. The following day, paclitaxel at the indicated concentrations was added to the medium. One hour later, 0.5% of the water soluble mitochondrial dye, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT⫹) was added. Incubation was continued for 3 more hours, the medium was removed, and the water insoluble blue formasan dye formed stochiometrically from MTT⫹ by the endogenous ROS in situ was solubilized by acidic isopropanol. Optical densities were determined by an Anthos Labtech 2010 ELISA reader (Wien, Austria) at 550 nm wavelength. All experiments were run in at least six parallels and repeated three times. Statistical analysis Data were presented as means ⫾ SEM. For multiple comparisons of groups, ANOVA was used. Statistical difference between groups was established by paired or unpaired Student’s t-test, with Bonferroni correction. RESULTS
Effect of paclitaxel on permeability transition in isolated mitochondria In order to demonstrate direct effect of paclitaxel in inducing mitochondrial permeability transition, we monitored swelling, dissipation of ⌬⌿, opening of permeability transition pore, and cytochrome c release from isolated Percoll-gradient purified rat liver mitochondria. High-amplitude swelling of the mitochondria due to permeability transition was demonstrated by the decrease of the reflectance of 540 nm light. In a concentrationdependent manner, paclitaxel induced swelling in isolated liver mitochondrion in the presence of low concentration of Ca2⫹ (2.5 M). This amount of Ca2⫹ did not induce permeability transition by itself (data not shown). Paclitaxel at a concentration of 20 M caused maximal swelling of mitochondria (same as induced by 150 M Ca2⫹) that was prevented completely by 2.5 M CsA (Fig. 1). Twenty M paclitaxel caused the dissipation of ⌬⌿, as detected by the release of the membrane potential sensitive dye, Rh123 from liver mitochondria following the induction of permeability transition. Dissipation of ⌬⌿ was inhibited by 2.5 M CsA (Fig. 2). The opening of the permeability transition pore allows the entering of substrates (such as acetyl-coenzyme A) that are otherwise excluded by the inner mitochondrial membrane into the mitochondrial matrix. It enabled us to demonstrate the opening of the permeability pore by a simple enzyme
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assay. Citrate synthase and carnitine acyltransferase activity become measurable using externally added acetylcoenzyme A after the addition of either 150 M Ca2⫹ or 20 M paclitaxel showing the opening of the permeability pore. This effect was inhibited by 2.5 M CsA (Fig. 3). Release of cytochrome c from the mitochondrial intermembrane space following permeability transition induced by either 150 M Ca2⫹ or by 20 M paclitaxel was detected by Western blotting. Cytochrome c release was inhibited in both cases by 2.5 M CsA (Fig. 4). Effect of paclitaxel on swelling and ⌬⌿ of mitochondria isolated from different tissues and cultured cells In order to demonstrate that the direct permeability transition-inducing effect of paclitaxel is not liver specific, we monitored swelling and ⌬⌿ in Percoll purified mitochondria isolated from rat liver, kidney, heart, and brain, as well as from BRL-3A and HepG-2 cell lines. In liver, kidney, heart, and brain mitochondria, 20 M paclitaxel induced a CsA-sensitive swelling of the same amplitude as of 150 M Ca2⫹. The degree and slope of the swelling differed greatly among the different tissues, with the highest value for liver, a lower value for kidney and heart, a value close to the detection limit for brain, and a swelling below detection limit for the cell lines. There were differences in the degree and slope of the Rh123 release among the different tissues, similar to the case of swelling. However, measuring of Rh123 release was much more sensitive than of swelling, so even for cell lines, the CsA sensitivity of the Rh123 release induced by either 20 M paclitaxel or 150 M Ca2⫹ could be established (Figs. 1 and 2). Paclitaxel induced ROS formation in isolated rat liver mitochondria Because ROS formation can induce and be the consequence of mitochondrial permeability transition [11], we studied the effect of paclitaxel on ROS production in isolated Percoll-gradient purified rat liver mitochondria. ROS formation was measured by monitoring the green or red fluorescence of Rh123 or resorufin oxidized by the ROS from nonfluorescent DRh123 or N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine in situ. By virtue of its dodecyl group, resorufin was localized in membranous regions and detected ROS formation in lipid phase, while Rh123 reflected to ROS levels in aqueous phase. Paclitaxel induced ROS formation in isolated liver mitochondria in a concentration-dependent manner (Fig. 5). By using both dyes, we compared the ROS formation induced
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Fig. 1. Permeability transition induced by paclitaxel and 150 M Ca2⫹ in isolated rat liver, heart, kidney, and brain mitochondria. Mitochondria in the presence or absence of CsA was incubated at room temperature in permeability transition-buffer as described in Materials and Methods. Permeability transition-inducing agents were added at arrow. Results are demonstrated by representative original registration curves for at least five independent experiments. Line 1: no agent added; line 2: 150 M Ca2⫹; line 3: 10 M paclitaxel; line 4: 20 M paclitaxel; line 5: 20 M paclitaxel ⫹ 2.5 M CsA.
by paclitaxel to that of 50 mM H2O2. A much more intense ROS formation was detected by resorufin (61 ⫾ 6.4% that of 50 mM H2O2) than by Rh123 (21 ⫾ 4.7% that of 50 mM H2O2). When detected by Rh123, ROS formation induced by paclitaxel could be attenuated by 1 mM lipoamide, 1 mM ascorbic acid and, to a smaller extent, by 100 M tocopherol, however not by 1 mM N-acetyl-L-cysteine. ROS formation induced by paclitaxel was decreased by 1 mM lipoamide, 100 M tocopherol and, to a smaller extent, by 1 mM N-acetyl-L-cysteine, however not by 1 mM ascorbic acid when detected by resorufin (Table 1). Selective inhibitors of the respiratory complexes caused a transient fast increase of the mitochondrial ROS formation followed by a plateau where the rate of ROS formation was the same as before the addition of the given substance (data not shown). When added on
this plateau, paclitaxel induced ROS formation in the case of all inhibitors of the respiratory chain but KCN (Table 2). In addition, we found that other inhibitors of cytochrome oxidase, azide and NO, also inhibited the paclitaxel-induced ROS production (data not shown). Because inhibitors of the respiratory chain caused a transient ROS formation, we checked whether paclitaxel had an inhibitory effect on mitochondrial respiration. Oxygen consumption was detected by using a Clark electrode in isolated rat liver mitochondria. Paclitaxel up to the concentration of 20 M either alone, or in combination with up to 500 M Ca2⫹ did not inhibit the oxygen consumption stimulated by either succinate or pyruvate under our experimental conditions (data not shown). Ca2⫹ at the concentration of 150 M did not induce ROS formation (data not shown), furthermore, the ROS formation
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Fig. 2. Changes in ⌬⌿ induced by 20 M of paclitaxel and 150 M Ca2⫹ in isolated rat liver, heart, kidney, and brain mitochondria. Mitochondria in the presence or absence of CsA was incubated at room temperature in permeability transition-buffer as described in Materials and Methods. Permeability transition-inducing agents were added at arrow. Results are demonstrated by representative original registration curves for at least five independent experiments. Line 1: no agent added; line 2: 150 M Ca2⫹; line 3: 20 M paclitaxel; line 4: 20 M paclitaxel ⫹ 2.5 M CsA.
induced by either 50 M paclitaxel or 50 mM H2O2 could not be inhibited by 2.5 M CsA (Table 2). Demonstration of mitochondrial ⌬⌿ dissipation and ROS formation induced by paclitaxel in a cell line Confocal microscopy was used to monitor the changes in Rh123 and PI fluorescence images of BRL-3A cells. The cationic dye Rh123 is retained by the mitochondria and shows green fluorescence image of the mitochondria when ⌬⌿ is intact, but is released into the cytosol and shows a weak background fluorescence when ⌬⌿ is disrupted in consequence to permeability transition. On the other hand, PI is excluded by intact cell membranes, but shows red fluorescent image of the nucleus when cellular energy charge drops to zero or the integrity of the cell membrane is damaged. Representative fluorescent images taken 45 min after the addition of paclitaxel and CsA (Fig. 6) demonstrate the effect of these drugs on the cells. Green fluorescent dots of approximately the same intensity representing mitochondria were seen in each control cell (Fig. 6A). This pattern of uniform fluorescent intensity was maintained in con-
trol cells for several hours. When the cells were incubated for about 35 min in the presence of 20 M paclitaxel, mitochondria in the cells started to undergo permeability transition, which process was completed in 20 min, indicated by a complete disappearance of green fluorescence (data not shown). In Fig. 6B we demonstrated an intermediate state, when the green fluorescent intensity was decreased in some cells, completely disappeared in other cells, and red PI fluorescence of a nucleus appeared in a cell indicating that permeability transition took place in the mitochondria of some cells as a consequence of 45 min incubation with 20 M paclitaxel. CsA at a concentration of 2.5 M inhibited the permeability transition-inducing effect of paclitaxel as revealed by fluorescent images identical to control (Fig. 6C). ROS formation in BRL-3A cells was assayed quantitatively by using the water-soluble yellow mitochondrial dye MTT⫹. Cells were incubated for 1 h in the presence of paclitaxel at the indicated concentration in a CO2 thermostat, then 0.5% MTT⫹ was added to the medium and incubation was continued for 3 more hours. Means ⫾ SEM of optical densities of the blue formasan dye formed stochiometrically from MTT⫹ by the endog-
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Fig. 3. Permeability transition assay by measuring intramitochondrial enzyme activities in isolated mitochondria. Carnitine acetyl transferase and citrate synthase activities were measured as described in Materials and Methods. Data are expressed in arbitrary units mean ⫾ SEM. Open and filled bars represent carnitine acetyl transferase and citrate synthase activities, respectively. Control values represent the enzyme activities in intact mitochondria. Permeability transition was induced by either 150 M Ca2⫹ (Ca) or 20 M paclitaxel (paclitaxel) in the presence or absence of CsA. Significant difference from control is indicated by *(p ⬍ .001). Significant decrease in the enzyme activities due to the inhibition of permeability transition by CsA is indicated by **(p ⬍ .01).
enously produced ROS are presented in Fig. 7. Paclitaxel induced the formation of ROS in a concentration-dependent manner. DISCUSSION
Taxol was reported to affect mitochondrial apoptotic mechanisms via changing the phosphorylation state and expression of bcl proteins [3,12,13]. According to another report, it interfered with the mitochondrial permeability transition pore by interacting with the microtubular network in Ehrlich ascites tumor cells [15]. In order to exclude any indirect mechanism to be involved in the effect of paclitaxel on permeability transition, we used isolated mitochondria purified by Percoll-gradient centrifuging. By this purification step, we intended to re-
Fig. 4. Cytochrome c release from isolated mitochondria. Cytochrome c (cyt c) released from the intermembrane space of isolated rat liver mitochondria was detected by Western blotting. Lane 1: control; lane 2: 2.5 M CsA; lane 3: 150 M Ca2⫹; lane 4: 20 M paclitaxel; lane 5: 150 M Ca2⫹ ⫹ 2.5 M CsA; lane 6: 20 M paclitaxel ⫹ 2.5 M CsA.
move the microtubular system attached to the mitochondrial outer membrane in intact cells, and the factors necessary to phosphorylate the bcl-2 and bcl-XL proteins such as c-Raf-1, p53, and p21WAF-1 [6,14]. We observed that Taxol induced large amplitude swelling of mitochondria (Fig. 1), the dissipation of mitochondrial membrane potential (Fig. 2), opening of the permeability transition pore by enzymatic method (Fig. 3), and on cytochrome c release (Fig. 4), all of which are regarded as characteristic features of mitochondrial permeability transition [17,19]. Our results indicate that Taxol, in a concentration-dependent manner, induced a CsA-sensitive permeability transition in isolated rat liver mitochondria at a concentration of up to 20 M. This concentration range is similar to those used by other groups [6,15] for studying the prompt effects of Taxol. Because Taxol contains cremophor EL (polyoxyethylated castor oil) in order to solubilize the active substance paclitaxel, we checked whether cremophor EL could be responsible for the permeability transition-inducing effect of Taxol. To this end, we compared the effect on the mitochondrial permeability transition and Rh123 release of Taxol and paclitaxel (dissolved in dimethylsulfoxide with a final dimethylsulfoxide concentration in the reaction mixture of 50 mM). Because no difference was detected, we concluded that paclitaxel and not cremophor EL is responsible for the direct mitochondrial effect of Taxol. These results are in accord with previous findings when the effect of cremophor EL
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Table 2. Effect of Inhibitors of the Respiratory Chain and CsA on ROS Formation Induced by 20 M Paclitaxel in Isolated Rat Liver Mitochondria
Control
2 M rotenone
8 M antimycin A
5 mM KCN
8 M oligomycin
2.5 M cyclosporine A
100 ⫾ 13
98 ⫾ 15
87 ⫾ 12
0⫾0
62 ⫾ 7
100 ⫾ 11
ROS formation was determined as in Table 1. Inhibitors of the respiratory chain caused a transient increase in ROS formation followed by a plateau. Paclitaxel was added only after the ROS formation reached the plateau. Results are expressed as % of ROS formation caused by 20 M paclitaxel in the absence of the inhibitors.
Fig. 5. ROS formation in isolated mitochondria. Increase of fluorescence intensities of resorufin (A) and Rh123 (B) oxidized from nonfluorescent N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine and DHRh123 by ROS was measured as described in Materials and Methods. Resorufin and Rh123 fluorescence reflected to the paclitaxel induced ROS formation in the aquous and the lipid phase, respectively. Results were calculated from the slope of the original registration curves (fluorescent intensity vs. time) and expressed as % of the ROS formation in the absence of paclitaxel. Values are mean ⫾ SEM of at least five independent experiments.
on cellular clonogenic survival and apoptosis was investigated [34]. Permeability transition-inducing effect of paclitaxel was not tissue-specific, because paclitaxel induced swelling and dissipation of ⌬⌿ was demonstrated in mitochondria isolated from not only rat liver, but heart, kidney, and brain, as well as from BRL-3A and HepG-2 cell lines. In accordance with other reports [24], the Table 1. Inhibition of ROS Formation Induced by 20 M Paclitaxel in Isolated Rat Liver Mitochondria
Control
1 mM 1 mM lipoamide ascorbic acid
Rh123 100 ⫾ 6.2 52 ⫾ 4.7 resorufin 100 ⫾ 4.3 42 ⫾ 3.2
30 ⫾ 3.1 87 ⫾ 4.0
1 mM NAC
100 M tocopherol
88 ⫾ 7.0 71 ⫾ 4.1
77 ⫾ 6.1 53 ⫾ 3.6
ROS formation was determined as described in Materials and Methods. Values representing the spontaneous oxidation of the dyes were determined from the slope of the original registration curves (fluorescent intensity vs. time) before the addition of paclitaxel and were subtracted from all calculated values. Results are expressed as the % of ROS formation caused by 20 M paclitaxel.
extent of permeability transition differed greatly in mitochondria isolated from different tissues (Fig. 1), however, 20 M paclitaxel induced permeability transition to the same extent as 150 M Ca2⫹ in all cases (data not shown). In case of mitochondria isolated from BRL-3A and HepG-2 cells, we could not detect the CsA-dependent large-amplitude swelling induced either by paclitaxel or by Ca2⫹, however, the monitoring of ⌬⌿ proved to be sensitive enough to monitor CsA-sensitive permeability transition. We investigated whether PT was induced by ROS because oxidizing agents including endogenously formed or exogenously added ROS were reported to induce permeability transition in isolated mitochondria [35]. Paclitaxel induced ROS formation in our system in a concentration-dependent manner (Fig. 5). The ROS formation induced by paclitaxel could not be inhibited by CsA (Table 2) and the substances that attenuated ROS formation (Table 1) did not affect paclitaxel-induced permeability transition (data not shown). In contrast, Ca2⫹ did not cause ROS formation even at concentrations well exceeding the ones inducing permeability transition (data not shown). These results suggest that permeability transition- and ROS-formation-inducing effect of paclitaxel in isolated Percoll-gradient purified mitochondria are mediated by separate mechanisms. Paclitaxel-induced ROS formation was about three times more intense in the lipid than in the aqueous phase, suggesting that the ROS produced in response to paclitaxel treatment was localized mainly in the mitochondrial membrane. Of the antioxidants studied, lipoamide, an agent found to diminish ROS-induced damages in isolated perfused heart [29], very effectively decreased the paclitaxel-induced ROS formation, while N-acetyl cysteine had no or moderate effect (Table 1). These results may have relevance in clinical management of the side effects of Taxol. Inhibitors of the respiratory chain affected the paclitaxel-induced mitochondrial ROS formation. In accordance with previous findings, selective inhibitors of the respiratory complexes caused a transient fast increase of
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Fig. 6. Demonstration of permeability transition in BRL-3A cell line. Confocal fluorescent microscopy images of Rh123 (green) and PI (red) fluorescence were taken periodically. Cells cultured on cover slips were loaded with Rh123, washed, and incubated in medium containing 5 g/ml PI in the absence or the presence of 20 M paclitaxel together with or without 2.5 M CsA, as described in Materials and Methods. Representative double-fluorescence photos taken after 45 min of incubation are presented. (A) no agent; (B) 20 M paclitaxel; (C) 20 M paclitaxel ⫹ 2.5 M CsA.
the mitochondrial ROS formation, followed by a plateau where the rate of ROS formation was the same as before the addition of the given substance (data not shown). When added on this plateau, 20 M paclitaxel induced ROS formation with a similar rate as in the absence of the inhibitors of the respiratory complexes except for KCN, because in the presence of KCN, paclitaxel failed to initiate mitochondrial ROS production (Table 2). It was established earlier by using inhibitors of the respiratory complexes that mitochondrial ROS formation under pathological conditions occur mainly at Complex I and Complex III [36,37]. Using similar methods, we found that paclitaxel-induced ROS formation was abolished by the inhibitors of the cytochrome oxidase (cyanide, azide, and NO), suggesting that paclitaxel interfered with the redox reactions of cytochrome oxidase, resulting in a cytochrome oxidase activity-dependent ROS production. We have also checked whether paclitaxel has any effect on the mitochondrial respiration by using a Clark electrode. We found that paclitaxel did not inhibit mitochondrial respiration stimulated either by succinate or pyruvate (data not shown), so it could not induce mitochondrial ROS formation by inhibiting the respiratory chain. Taking together all these results, we concluded that paclitaxel induces mitochondrial ROS formation in a
cytochrome oxidase-dependent way, however, not by inhibiting the respiratory complexes. Paclitaxel exerted its mitochondrial effects in vivo too. Approximately 35 min after the addition of 20 M
Fig. 7. Paclitaxel-induced ROS formation in BRL-3A cell line. Cells were cultured in 96 well plate and incubated for 1 h in medium containing paclitaxel at the indicated concentrations. MTT was added and the incubation was continued for 3 h. Acidic isopropanol was added to terminate the reaction and solubilize the blue formasan dye formed stochiometrically by ROS produced endogenously or in consequence of paclitaxel treatment. Optical densities of the formasan dye were determined and expressed as mean ⫾ SEM of three independent experiments running in at least six parallels.
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paclitaxel to BRL-3A cells, dissipation of ⌬⌿ started in some of the cells as judged by the release of the membrane-potential-sensitive Rh123 dye. It took 15–20 min for every cell in a population to undergo permeability transition. Parallel to this, the nuclei of these cells became stained by PI because the cells couldn’t exclude the dye any further due to the drop of their energy charge. This effect of paclitaxel was CsA sensitive (Fig. 6), similar to those data obtained by using isolated mitochondria. In the same cell line, we also demonstrated the concentration-dependent ROS formation-inducing effect of paclitaxel (Fig. 7). In conclusion, we demonstrated that paclitaxel has direct mitochondrial effects, namely, induction of mitochondrial permeability transition and ROS formation. We confirmed that these effects are relevant in living cells. Permeability transition, the collapse of mitochondrial membrane potential, and cytochrome c release occurs in a cyclophilin-dependent pathway, while mitochondrial ROS production is cytochrome oxidase dependent. The direct mitochondrial effects indicate a novel molecular mechanism for Taxol that are likely to contribute to the toxic effects of the drug. Acknowledgements — We wish to thank Andras Visegradi and Laszlo Grama for their help in laser scanning confocal microscopy. This work was supported by Janos Bolyai Research Fund, funds from Hungarian Science Foundation T023076, from the Ministry of Health and Welfare ETT 35/2000, and from the Ministry of Education FKFP 1393/1997.
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ABBREVIATIONS
⌬⌿—mitochondrial membrane potential CsA— cyclosporine A DRh123—Dihydrorhodamine123 EGTA— ethylene glycol-bis(  -aminoethyl ether) N,N,N⬘,N⬘-tetraacetic acid KRH—Krebs-Ringer/HEPES MTT ⫹ —(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide PI—propidium iodide resorufin—N-acetyl-8-dodecyl-resorufin Rh123—Rhodamine123 ROS—reactive oxygen species
PII S0891-5849(01)00616-5
Original Contribution DIRECT EFFECT OF TAXOL ON FREE RADICAL FORMATION AND MITOCHONDRIAL PERMEABILITY TRANSITION GABOR VARBIRO, BALAZS VERES, FERENC GALLYAS JR.,
and
BALAZS SUMEGI
Institute of Biochemistry, University of Pecs, Medical School, Pecs, Hungary (Received 23 January 2001; Accepted 22 May 2001)
Abstract—To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the M range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (⌬⌿) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 M cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN⫺, azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of ⌬⌿ and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity. © 2001 Elsevier Science Inc. Keywords—ROS, Permeability transition, Taxol, Apoptosis, Mitochondria, Cultured cells, Free radicals
INTRODUCTION
opening of permeability transition pore facilitate the paclitaxel-induced apoptosis in tumor cell lines [11]. These effects of Taxol were exerted in the nM–M concentration range usually after a 2–24 h exposure to the drug, with a tendency of longer exposure times for lower concentrations [4 –14]. It was also suggested that the effect of paclitaxel on the mitochondrial permeability transition pore was mediated through interaction with the cytoskeleton [15]. Mitochondrial permeability transition can lead to cell death, and can be induced by high intracellular inorganic phosphate, Ca2⫹, reactive oxygen species (ROS), or proapoptotic proteins such as Bax, whereas it is inhibited by cyclosporine A (CsA), bongkrekic acid, Bcl-2, or Bcl-xL [16]. The opening of the large inner membrane pores cause equilibration of ions within the matrix and the cytosol, dissipating the membrane potential (⌬⌿) and uncoupling the respiratory chain. The volume disregulation following the opening of the permeability transition pore results in the swelling of the matrix, leading to outer membrane disruption and the release of cytochrome c
Paclitaxel, a widely used antineoplastic agent [1–3], stabilizes tubulin dimers, thus interfering with microtubular disassembly [4], resulting in the arrest of cells in G2-M phase of the cell cycle, followed by DNA fragmentation and morphological features of apoptosis. Paclitaxel has also been shown to activate Raf-1 [5] and cause phosphorylation of Bcl-2 [6,7]. This has been claimed to inactivate Bcl-2 and deprive it of forming heterodimers with the proapoptotic Bax [8 –10], hence leading to increased free intracellular Bax level, and ultimately resulting in the activation of the caspase proteins through the Bax-induced release of cytochrome c from the mitochondrion. Mitochondrial membrane stabilizing proteins (Bcl-2 and Bcl-XL) were reported to decrease apoptotic effect of paclitaxel [7], while proteins promoting the Address correspondence to: Balazs Sumegi, Institute of Biochemistry, Medical School, University of Pecs, 12 Szigeti st., H-7624 Pecs, Hungary; Tel: ⫹36 (72) 536-000/ext. 1633; Fax: ⫹36 (72) 536-277; E-Mail: [email protected]. 548
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and other caspase-activating proteins into the cytosol, ultimately contributing to the apoptosis of the cell [17]. Mitochondria play a pivotal role in oxidative cell damages too, because the major source of the endogenously produced ROS is the mitochondrial respiratory chain [18 –20]. Despite numerous studies, mechanisms of mitochondrial effect of paclitaxel still remain to be elucidated. In this paper, we studied the direct mitochondrial effects of paclitaxel, namely, the induction of mitochondrial ROS production, the collapse of mitochondrial membrane potential, and the opening of mitochondrial permeability pore. These observations may provide a novel molecular mechanism for the cytotoxicity of paclitaxel that can explain its toxic effect in slowly replicating and nondividing cells. MATERIALS AND METHODS
Materials Paclitaxel was from ICN Biomedicals Inc. (Aurora, OH, USA); Taxol was from Bristol-Myers Squibb S.p.A. (Sermonetta, Italy); CsA was from Biomol Research Labs, Inc. (Plymouth Meeting, PA, USA); Rhodamine123 (Rh123), Dihydrorhodamine123 (DRh123), and N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine were from Molecular Probes (Eugene, OR, USA); anti-cytochrome c monoclonal antibody was from Pharmingen (San Diego, CA, USA); all other compounds were from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.
Animals Wistar rats were purchased from Charles River Hungary Breeding LTD. (Budapest, Hungary). The animals were kept under standardized conditions; tap water and rat chow were provided ad libitum. Animals were treated in compliance with approved institutional animal care guidelines.
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Isolation of mitochondria Liver, heart, and kidney mitochondria were prepared according to standard protocol [21] except that a Teflonglass homogenizer was used instead of a hypodermic needle for the second homogenization step. The only differences among the organs were in the primary homogenization protocol; liver and kidney was squeezed through a liver press, while heart was minced with a blender. Brain mitochondria were prepared according to Sims [22]. Isolation of mitochondria from HepG-2 and BRL 3A cells were performed exactly as described by Almeida and Medina [23] for astrocytes. All isolated mitochondria were purified by Percoll-gradient centrifuging [22] and their RCR values were determined by a Clark electrode (see below). Mitochondrial permeability transition Mitochondrial permeability transition was monitored by following the accompanying large amplitude swelling via the decrease in absorbance at 540 nm [24], measured by a Perkin-Elmer fluorimeter (London, UK) in reflectance mode. Mitochondrial membrane potential was monitored by fluorescence of Rh123, released from the mitochondria following the induction of permeability transition [13] by using a Perkin-Elmer fluorimeter at an excitation wavelength of 495 and an emission wavelength of 535 nm. Briefly, mitochondria at the concentration of 1 mg protein/ml were preincubated in the assay buffer (70 mM sucrose, 214 mM mannitol, 5 mM N-2hydroxyethyl piperasine-N⬘-2-ethanesulfonic acid, 5 mM glutamate, 0.5 mM malate, 0.5 mM phosphate) containing 1 M Rh123 and the studied substances for 60 s. Alteration of the mitochondrial membrane potential was induced by the addition of 150 M Ca2⫹ or of paclitaxel at the indicated concentration plus 2.5 M Ca2⫹. Fluorescence intensity changes were detected for 3 min. The results are demonstrated by representative original registration curves from five independent experiments, each repeated three times. Detection of cytochrome c release in vitro
Cell culture HepG-2 human hepatocellular carcinoma and BRL 3A rat liver cell lines were from American Type Culture Collection (Rockville, MD, USA). Both cell lines were grown in humidified 5% CO2 atmosphere at 37°C and maintained in culture as monolayer adherent cells in Dulbecco’s Modified Eagle’s Medium containing 1% antibiotic-antimycotic solution (Sigma) and 10% fetal calf serum. Cells were passaged at intervals of 3 d.
Detection of cytochrome c release in vitro was performed as described by Susin et al. [25] Samples from the cuvette were taken prior to, and 3 min after the induction of the permeability transition, and centrifuged at 13,000 rpm for 3 min. Supernatants were analyzed for cytochrome c by Western blotting. Enhanced chemiluminescence labeling was used for the visualization of the blots. Results are demonstrated by photomicrograph of a representative blot showing only the samples taken 3 min after the induction of the permeability transition, because
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cytochrome c content of the samples taken prior to the induction of the permeability transition were below the detection limit.
Enzymatic permeability transition assay Measuring of citrate synthase (EC 4.1.3.7) and carnitine acyltransferase (EC 2.3.1.7) activity after the induction of permeability transition [26] was performed by using a Beckman spectrophotometer (Fullerton, CA, USA) at the wavelength of 412 nm. Mitochondria at the concentration of 0.1 mg protein/ml were preincubated in the assay buffer containing the studied substances. Permeability transition was induced as above. After 4 min, 100 g/ml 5,5⬘-dithiobis-(2-nitrobenzoate), 40 nM Acetyl-Coenzyme A, and 1 mM oxaloacetate or 2.5 mM L-carnitine was added, and the alteration of the extinction was monitored for 5 min.
Determination of ROS formation For the determination of ROS-formation, mitochondria was incubated at room temperature in 20 mM tris(hydroxymethyl)-aminomethane buffer, pH 8.0 containing 250 mM sucrose, 1 mM ethylene glycol-bis(aminoethyl ether) N,N,N⬘,N⬘-tetraacetic acid (EGTA), 1mM MgCl2, 1.7 M octanoic acid, 1 M ADP, and 2 M DRh123. The fluorescence of Rh123 formed by ROS-induced oxidation of the nonfluorescent DRh123 in situ [27–29], was measured by a Perkin-Elmer fluorimeter at an excitation wavelength of 495 nm and an emission wavelength of 535 nm. Fluorescence intensity changes were recorded for 3 min. The oxidation of N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine forms N-acetyl-8-dodecyl-resorufin (resorufin), which exhibits strong red fluorescence. This product is well retained in living cells and organelles by virtue of its lipophilic tail, making it possible to detect ROS production in the lipid phase. The method is the same as described above except for using 3 M N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine instead of DRh123, and changing the excitation wavelength to 578 nm and the emission wavelength to 597 nm. Baseline ROS formation due to spontaneous oxidation of the dyes and unstimulated ROS production by the mitochondrial respiratory chain was determined from the initial slope of the registration curves (Fig. 5) and was subtracted. ROS formation was calculated from the slope of the registration curves and expressed in arbitrary units, mean ⫾ SEM of at least five independent experiments.
Mitochondrial oxygen consumption Mitochondrial oxygen consumption (mitochondrial respiration) was assessed by a Clark electrode [30]. Briefly, isolated mitochondria were suspended in 20 mM Tris buffer, pH 7.4 containing 20 mM KCl, 220 mM mannitol, 70 mM sucrose, and 1 mM EGTA. The mitochondrial respiration was stimulated with either 10 mM succinate or 10 mM pyruvate (state 4 respiration), and 3.3 mM ADP (state 3 respiration) in the absence or presence of paclitaxel and/or up to 500 M Ca2⫹ (when Ca2⫹ was used, EGTA was omitted from the buffer). Before performing experiments, RCR values of the mitochondria were determined and were found to be in the range of 3.5–5.1 for the tissues and 2.1–2.5 for the cell lines in accordance with published values [31,32].
Demonstration of permeability transition in cultured cells BRL 3A cells were seeded to glass cover slips (60 ⫻ 40 mm) at a starting density of 2500 cells/cm2. The following day, CoverWell perfusion chambers (Grace Bio-Labs, Bend, OR, USA) were mounted on to the cover slips that allowed fast replacing of the medium on the cells. Cells were loaded in culture medium with 1 g/ml of Rh123 for 30 min at 37°C. After three washes with Krebs-Ringer/HEPES (KRH) buffer [33], 20 M paclitaxel without or together with 2.5 M CsA in KRH buffer containing 5 g/ml propidium iodide (PI) was added. Incubation was continued under the microscope at room temperature, and fluorescent images were taken periodically. The fluorescence of Rh123 and PI was monitored using a Bio-Rad MRC-1024ES laser scanning confocal attachment (Herefordshire, UK) mounted on a Nikon Eclipse TE-300 inverted microscope (Kingston, UK). Cells were excited with the 488 nm line of argon ion laser. Excitation light was passed through a dichroic mirror with a dividing wavelength of 527 nm, preventing the exciting light from reaching the detectors. The emitted fluorescent light was divided by a 565 nm dichroic mirror and was measured by separate photomultipliers through a 522/35 nm and a 680/32 nm band-pass filter. Laser intensity was attenuated by 99% in order to minimize photobleaching. Fluorescence was imaged with a Plan Apo 100⫻/1.4 oil-immersion objective. Confocal images were transferred to a Compaq Prosigna 300 workstation (Houston, TX, USA) and were processed by using Bio-Rad’s LaserSharp and Photoshop (Adobe Systems, Mountain View, CA, USA) software packages. In this arrangement, green and red color represent Rh123 and PI fluorescence, respectively.
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Detection of ROS formation in cultured cells BRL-3A cells were seeded into 96 well plates at a starting density of 2.5 ⫻ 104 cell/well and cultured overnight in humidified 5% CO2 atmosphere at 37°C. The following day, paclitaxel at the indicated concentrations was added to the medium. One hour later, 0.5% of the water soluble mitochondrial dye, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT⫹) was added. Incubation was continued for 3 more hours, the medium was removed, and the water insoluble blue formasan dye formed stochiometrically from MTT⫹ by the endogenous ROS in situ was solubilized by acidic isopropanol. Optical densities were determined by an Anthos Labtech 2010 ELISA reader (Wien, Austria) at 550 nm wavelength. All experiments were run in at least six parallels and repeated three times. Statistical analysis Data were presented as means ⫾ SEM. For multiple comparisons of groups, ANOVA was used. Statistical difference between groups was established by paired or unpaired Student’s t-test, with Bonferroni correction. RESULTS
Effect of paclitaxel on permeability transition in isolated mitochondria In order to demonstrate direct effect of paclitaxel in inducing mitochondrial permeability transition, we monitored swelling, dissipation of ⌬⌿, opening of permeability transition pore, and cytochrome c release from isolated Percoll-gradient purified rat liver mitochondria. High-amplitude swelling of the mitochondria due to permeability transition was demonstrated by the decrease of the reflectance of 540 nm light. In a concentrationdependent manner, paclitaxel induced swelling in isolated liver mitochondrion in the presence of low concentration of Ca2⫹ (2.5 M). This amount of Ca2⫹ did not induce permeability transition by itself (data not shown). Paclitaxel at a concentration of 20 M caused maximal swelling of mitochondria (same as induced by 150 M Ca2⫹) that was prevented completely by 2.5 M CsA (Fig. 1). Twenty M paclitaxel caused the dissipation of ⌬⌿, as detected by the release of the membrane potential sensitive dye, Rh123 from liver mitochondria following the induction of permeability transition. Dissipation of ⌬⌿ was inhibited by 2.5 M CsA (Fig. 2). The opening of the permeability transition pore allows the entering of substrates (such as acetyl-coenzyme A) that are otherwise excluded by the inner mitochondrial membrane into the mitochondrial matrix. It enabled us to demonstrate the opening of the permeability pore by a simple enzyme
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assay. Citrate synthase and carnitine acyltransferase activity become measurable using externally added acetylcoenzyme A after the addition of either 150 M Ca2⫹ or 20 M paclitaxel showing the opening of the permeability pore. This effect was inhibited by 2.5 M CsA (Fig. 3). Release of cytochrome c from the mitochondrial intermembrane space following permeability transition induced by either 150 M Ca2⫹ or by 20 M paclitaxel was detected by Western blotting. Cytochrome c release was inhibited in both cases by 2.5 M CsA (Fig. 4). Effect of paclitaxel on swelling and ⌬⌿ of mitochondria isolated from different tissues and cultured cells In order to demonstrate that the direct permeability transition-inducing effect of paclitaxel is not liver specific, we monitored swelling and ⌬⌿ in Percoll purified mitochondria isolated from rat liver, kidney, heart, and brain, as well as from BRL-3A and HepG-2 cell lines. In liver, kidney, heart, and brain mitochondria, 20 M paclitaxel induced a CsA-sensitive swelling of the same amplitude as of 150 M Ca2⫹. The degree and slope of the swelling differed greatly among the different tissues, with the highest value for liver, a lower value for kidney and heart, a value close to the detection limit for brain, and a swelling below detection limit for the cell lines. There were differences in the degree and slope of the Rh123 release among the different tissues, similar to the case of swelling. However, measuring of Rh123 release was much more sensitive than of swelling, so even for cell lines, the CsA sensitivity of the Rh123 release induced by either 20 M paclitaxel or 150 M Ca2⫹ could be established (Figs. 1 and 2). Paclitaxel induced ROS formation in isolated rat liver mitochondria Because ROS formation can induce and be the consequence of mitochondrial permeability transition [11], we studied the effect of paclitaxel on ROS production in isolated Percoll-gradient purified rat liver mitochondria. ROS formation was measured by monitoring the green or red fluorescence of Rh123 or resorufin oxidized by the ROS from nonfluorescent DRh123 or N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine in situ. By virtue of its dodecyl group, resorufin was localized in membranous regions and detected ROS formation in lipid phase, while Rh123 reflected to ROS levels in aqueous phase. Paclitaxel induced ROS formation in isolated liver mitochondria in a concentration-dependent manner (Fig. 5). By using both dyes, we compared the ROS formation induced
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Fig. 1. Permeability transition induced by paclitaxel and 150 M Ca2⫹ in isolated rat liver, heart, kidney, and brain mitochondria. Mitochondria in the presence or absence of CsA was incubated at room temperature in permeability transition-buffer as described in Materials and Methods. Permeability transition-inducing agents were added at arrow. Results are demonstrated by representative original registration curves for at least five independent experiments. Line 1: no agent added; line 2: 150 M Ca2⫹; line 3: 10 M paclitaxel; line 4: 20 M paclitaxel; line 5: 20 M paclitaxel ⫹ 2.5 M CsA.
by paclitaxel to that of 50 mM H2O2. A much more intense ROS formation was detected by resorufin (61 ⫾ 6.4% that of 50 mM H2O2) than by Rh123 (21 ⫾ 4.7% that of 50 mM H2O2). When detected by Rh123, ROS formation induced by paclitaxel could be attenuated by 1 mM lipoamide, 1 mM ascorbic acid and, to a smaller extent, by 100 M tocopherol, however not by 1 mM N-acetyl-L-cysteine. ROS formation induced by paclitaxel was decreased by 1 mM lipoamide, 100 M tocopherol and, to a smaller extent, by 1 mM N-acetyl-L-cysteine, however not by 1 mM ascorbic acid when detected by resorufin (Table 1). Selective inhibitors of the respiratory complexes caused a transient fast increase of the mitochondrial ROS formation followed by a plateau where the rate of ROS formation was the same as before the addition of the given substance (data not shown). When added on
this plateau, paclitaxel induced ROS formation in the case of all inhibitors of the respiratory chain but KCN (Table 2). In addition, we found that other inhibitors of cytochrome oxidase, azide and NO, also inhibited the paclitaxel-induced ROS production (data not shown). Because inhibitors of the respiratory chain caused a transient ROS formation, we checked whether paclitaxel had an inhibitory effect on mitochondrial respiration. Oxygen consumption was detected by using a Clark electrode in isolated rat liver mitochondria. Paclitaxel up to the concentration of 20 M either alone, or in combination with up to 500 M Ca2⫹ did not inhibit the oxygen consumption stimulated by either succinate or pyruvate under our experimental conditions (data not shown). Ca2⫹ at the concentration of 150 M did not induce ROS formation (data not shown), furthermore, the ROS formation
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Fig. 2. Changes in ⌬⌿ induced by 20 M of paclitaxel and 150 M Ca2⫹ in isolated rat liver, heart, kidney, and brain mitochondria. Mitochondria in the presence or absence of CsA was incubated at room temperature in permeability transition-buffer as described in Materials and Methods. Permeability transition-inducing agents were added at arrow. Results are demonstrated by representative original registration curves for at least five independent experiments. Line 1: no agent added; line 2: 150 M Ca2⫹; line 3: 20 M paclitaxel; line 4: 20 M paclitaxel ⫹ 2.5 M CsA.
induced by either 50 M paclitaxel or 50 mM H2O2 could not be inhibited by 2.5 M CsA (Table 2). Demonstration of mitochondrial ⌬⌿ dissipation and ROS formation induced by paclitaxel in a cell line Confocal microscopy was used to monitor the changes in Rh123 and PI fluorescence images of BRL-3A cells. The cationic dye Rh123 is retained by the mitochondria and shows green fluorescence image of the mitochondria when ⌬⌿ is intact, but is released into the cytosol and shows a weak background fluorescence when ⌬⌿ is disrupted in consequence to permeability transition. On the other hand, PI is excluded by intact cell membranes, but shows red fluorescent image of the nucleus when cellular energy charge drops to zero or the integrity of the cell membrane is damaged. Representative fluorescent images taken 45 min after the addition of paclitaxel and CsA (Fig. 6) demonstrate the effect of these drugs on the cells. Green fluorescent dots of approximately the same intensity representing mitochondria were seen in each control cell (Fig. 6A). This pattern of uniform fluorescent intensity was maintained in con-
trol cells for several hours. When the cells were incubated for about 35 min in the presence of 20 M paclitaxel, mitochondria in the cells started to undergo permeability transition, which process was completed in 20 min, indicated by a complete disappearance of green fluorescence (data not shown). In Fig. 6B we demonstrated an intermediate state, when the green fluorescent intensity was decreased in some cells, completely disappeared in other cells, and red PI fluorescence of a nucleus appeared in a cell indicating that permeability transition took place in the mitochondria of some cells as a consequence of 45 min incubation with 20 M paclitaxel. CsA at a concentration of 2.5 M inhibited the permeability transition-inducing effect of paclitaxel as revealed by fluorescent images identical to control (Fig. 6C). ROS formation in BRL-3A cells was assayed quantitatively by using the water-soluble yellow mitochondrial dye MTT⫹. Cells were incubated for 1 h in the presence of paclitaxel at the indicated concentration in a CO2 thermostat, then 0.5% MTT⫹ was added to the medium and incubation was continued for 3 more hours. Means ⫾ SEM of optical densities of the blue formasan dye formed stochiometrically from MTT⫹ by the endog-
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Fig. 3. Permeability transition assay by measuring intramitochondrial enzyme activities in isolated mitochondria. Carnitine acetyl transferase and citrate synthase activities were measured as described in Materials and Methods. Data are expressed in arbitrary units mean ⫾ SEM. Open and filled bars represent carnitine acetyl transferase and citrate synthase activities, respectively. Control values represent the enzyme activities in intact mitochondria. Permeability transition was induced by either 150 M Ca2⫹ (Ca) or 20 M paclitaxel (paclitaxel) in the presence or absence of CsA. Significant difference from control is indicated by *(p ⬍ .001). Significant decrease in the enzyme activities due to the inhibition of permeability transition by CsA is indicated by **(p ⬍ .01).
enously produced ROS are presented in Fig. 7. Paclitaxel induced the formation of ROS in a concentration-dependent manner. DISCUSSION
Taxol was reported to affect mitochondrial apoptotic mechanisms via changing the phosphorylation state and expression of bcl proteins [3,12,13]. According to another report, it interfered with the mitochondrial permeability transition pore by interacting with the microtubular network in Ehrlich ascites tumor cells [15]. In order to exclude any indirect mechanism to be involved in the effect of paclitaxel on permeability transition, we used isolated mitochondria purified by Percoll-gradient centrifuging. By this purification step, we intended to re-
Fig. 4. Cytochrome c release from isolated mitochondria. Cytochrome c (cyt c) released from the intermembrane space of isolated rat liver mitochondria was detected by Western blotting. Lane 1: control; lane 2: 2.5 M CsA; lane 3: 150 M Ca2⫹; lane 4: 20 M paclitaxel; lane 5: 150 M Ca2⫹ ⫹ 2.5 M CsA; lane 6: 20 M paclitaxel ⫹ 2.5 M CsA.
move the microtubular system attached to the mitochondrial outer membrane in intact cells, and the factors necessary to phosphorylate the bcl-2 and bcl-XL proteins such as c-Raf-1, p53, and p21WAF-1 [6,14]. We observed that Taxol induced large amplitude swelling of mitochondria (Fig. 1), the dissipation of mitochondrial membrane potential (Fig. 2), opening of the permeability transition pore by enzymatic method (Fig. 3), and on cytochrome c release (Fig. 4), all of which are regarded as characteristic features of mitochondrial permeability transition [17,19]. Our results indicate that Taxol, in a concentration-dependent manner, induced a CsA-sensitive permeability transition in isolated rat liver mitochondria at a concentration of up to 20 M. This concentration range is similar to those used by other groups [6,15] for studying the prompt effects of Taxol. Because Taxol contains cremophor EL (polyoxyethylated castor oil) in order to solubilize the active substance paclitaxel, we checked whether cremophor EL could be responsible for the permeability transition-inducing effect of Taxol. To this end, we compared the effect on the mitochondrial permeability transition and Rh123 release of Taxol and paclitaxel (dissolved in dimethylsulfoxide with a final dimethylsulfoxide concentration in the reaction mixture of 50 mM). Because no difference was detected, we concluded that paclitaxel and not cremophor EL is responsible for the direct mitochondrial effect of Taxol. These results are in accord with previous findings when the effect of cremophor EL
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Table 2. Effect of Inhibitors of the Respiratory Chain and CsA on ROS Formation Induced by 20 M Paclitaxel in Isolated Rat Liver Mitochondria
Control
2 M rotenone
8 M antimycin A
5 mM KCN
8 M oligomycin
2.5 M cyclosporine A
100 ⫾ 13
98 ⫾ 15
87 ⫾ 12
0⫾0
62 ⫾ 7
100 ⫾ 11
ROS formation was determined as in Table 1. Inhibitors of the respiratory chain caused a transient increase in ROS formation followed by a plateau. Paclitaxel was added only after the ROS formation reached the plateau. Results are expressed as % of ROS formation caused by 20 M paclitaxel in the absence of the inhibitors.
Fig. 5. ROS formation in isolated mitochondria. Increase of fluorescence intensities of resorufin (A) and Rh123 (B) oxidized from nonfluorescent N-acetyl-8-dodecyl-3,7-dihydroxyphenoxazine and DHRh123 by ROS was measured as described in Materials and Methods. Resorufin and Rh123 fluorescence reflected to the paclitaxel induced ROS formation in the aquous and the lipid phase, respectively. Results were calculated from the slope of the original registration curves (fluorescent intensity vs. time) and expressed as % of the ROS formation in the absence of paclitaxel. Values are mean ⫾ SEM of at least five independent experiments.
on cellular clonogenic survival and apoptosis was investigated [34]. Permeability transition-inducing effect of paclitaxel was not tissue-specific, because paclitaxel induced swelling and dissipation of ⌬⌿ was demonstrated in mitochondria isolated from not only rat liver, but heart, kidney, and brain, as well as from BRL-3A and HepG-2 cell lines. In accordance with other reports [24], the Table 1. Inhibition of ROS Formation Induced by 20 M Paclitaxel in Isolated Rat Liver Mitochondria
Control
1 mM 1 mM lipoamide ascorbic acid
Rh123 100 ⫾ 6.2 52 ⫾ 4.7 resorufin 100 ⫾ 4.3 42 ⫾ 3.2
30 ⫾ 3.1 87 ⫾ 4.0
1 mM NAC
100 M tocopherol
88 ⫾ 7.0 71 ⫾ 4.1
77 ⫾ 6.1 53 ⫾ 3.6
ROS formation was determined as described in Materials and Methods. Values representing the spontaneous oxidation of the dyes were determined from the slope of the original registration curves (fluorescent intensity vs. time) before the addition of paclitaxel and were subtracted from all calculated values. Results are expressed as the % of ROS formation caused by 20 M paclitaxel.
extent of permeability transition differed greatly in mitochondria isolated from different tissues (Fig. 1), however, 20 M paclitaxel induced permeability transition to the same extent as 150 M Ca2⫹ in all cases (data not shown). In case of mitochondria isolated from BRL-3A and HepG-2 cells, we could not detect the CsA-dependent large-amplitude swelling induced either by paclitaxel or by Ca2⫹, however, the monitoring of ⌬⌿ proved to be sensitive enough to monitor CsA-sensitive permeability transition. We investigated whether PT was induced by ROS because oxidizing agents including endogenously formed or exogenously added ROS were reported to induce permeability transition in isolated mitochondria [35]. Paclitaxel induced ROS formation in our system in a concentration-dependent manner (Fig. 5). The ROS formation induced by paclitaxel could not be inhibited by CsA (Table 2) and the substances that attenuated ROS formation (Table 1) did not affect paclitaxel-induced permeability transition (data not shown). In contrast, Ca2⫹ did not cause ROS formation even at concentrations well exceeding the ones inducing permeability transition (data not shown). These results suggest that permeability transition- and ROS-formation-inducing effect of paclitaxel in isolated Percoll-gradient purified mitochondria are mediated by separate mechanisms. Paclitaxel-induced ROS formation was about three times more intense in the lipid than in the aqueous phase, suggesting that the ROS produced in response to paclitaxel treatment was localized mainly in the mitochondrial membrane. Of the antioxidants studied, lipoamide, an agent found to diminish ROS-induced damages in isolated perfused heart [29], very effectively decreased the paclitaxel-induced ROS formation, while N-acetyl cysteine had no or moderate effect (Table 1). These results may have relevance in clinical management of the side effects of Taxol. Inhibitors of the respiratory chain affected the paclitaxel-induced mitochondrial ROS formation. In accordance with previous findings, selective inhibitors of the respiratory complexes caused a transient fast increase of
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Fig. 6. Demonstration of permeability transition in BRL-3A cell line. Confocal fluorescent microscopy images of Rh123 (green) and PI (red) fluorescence were taken periodically. Cells cultured on cover slips were loaded with Rh123, washed, and incubated in medium containing 5 g/ml PI in the absence or the presence of 20 M paclitaxel together with or without 2.5 M CsA, as described in Materials and Methods. Representative double-fluorescence photos taken after 45 min of incubation are presented. (A) no agent; (B) 20 M paclitaxel; (C) 20 M paclitaxel ⫹ 2.5 M CsA.
the mitochondrial ROS formation, followed by a plateau where the rate of ROS formation was the same as before the addition of the given substance (data not shown). When added on this plateau, 20 M paclitaxel induced ROS formation with a similar rate as in the absence of the inhibitors of the respiratory complexes except for KCN, because in the presence of KCN, paclitaxel failed to initiate mitochondrial ROS production (Table 2). It was established earlier by using inhibitors of the respiratory complexes that mitochondrial ROS formation under pathological conditions occur mainly at Complex I and Complex III [36,37]. Using similar methods, we found that paclitaxel-induced ROS formation was abolished by the inhibitors of the cytochrome oxidase (cyanide, azide, and NO), suggesting that paclitaxel interfered with the redox reactions of cytochrome oxidase, resulting in a cytochrome oxidase activity-dependent ROS production. We have also checked whether paclitaxel has any effect on the mitochondrial respiration by using a Clark electrode. We found that paclitaxel did not inhibit mitochondrial respiration stimulated either by succinate or pyruvate (data not shown), so it could not induce mitochondrial ROS formation by inhibiting the respiratory chain. Taking together all these results, we concluded that paclitaxel induces mitochondrial ROS formation in a
cytochrome oxidase-dependent way, however, not by inhibiting the respiratory complexes. Paclitaxel exerted its mitochondrial effects in vivo too. Approximately 35 min after the addition of 20 M
Fig. 7. Paclitaxel-induced ROS formation in BRL-3A cell line. Cells were cultured in 96 well plate and incubated for 1 h in medium containing paclitaxel at the indicated concentrations. MTT was added and the incubation was continued for 3 h. Acidic isopropanol was added to terminate the reaction and solubilize the blue formasan dye formed stochiometrically by ROS produced endogenously or in consequence of paclitaxel treatment. Optical densities of the formasan dye were determined and expressed as mean ⫾ SEM of three independent experiments running in at least six parallels.
Mitochondrial effects of Taxol
paclitaxel to BRL-3A cells, dissipation of ⌬⌿ started in some of the cells as judged by the release of the membrane-potential-sensitive Rh123 dye. It took 15–20 min for every cell in a population to undergo permeability transition. Parallel to this, the nuclei of these cells became stained by PI because the cells couldn’t exclude the dye any further due to the drop of their energy charge. This effect of paclitaxel was CsA sensitive (Fig. 6), similar to those data obtained by using isolated mitochondria. In the same cell line, we also demonstrated the concentration-dependent ROS formation-inducing effect of paclitaxel (Fig. 7). In conclusion, we demonstrated that paclitaxel has direct mitochondrial effects, namely, induction of mitochondrial permeability transition and ROS formation. We confirmed that these effects are relevant in living cells. Permeability transition, the collapse of mitochondrial membrane potential, and cytochrome c release occurs in a cyclophilin-dependent pathway, while mitochondrial ROS production is cytochrome oxidase dependent. The direct mitochondrial effects indicate a novel molecular mechanism for Taxol that are likely to contribute to the toxic effects of the drug. Acknowledgements — We wish to thank Andras Visegradi and Laszlo Grama for their help in laser scanning confocal microscopy. This work was supported by Janos Bolyai Research Fund, funds from Hungarian Science Foundation T023076, from the Ministry of Health and Welfare ETT 35/2000, and from the Ministry of Education FKFP 1393/1997.
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ABBREVIATIONS
⌬⌿—mitochondrial membrane potential CsA— cyclosporine A DRh123—Dihydrorhodamine123 EGTA— ethylene glycol-bis(  -aminoethyl ether) N,N,N⬘,N⬘-tetraacetic acid KRH—Krebs-Ringer/HEPES MTT ⫹ —(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide PI—propidium iodide resorufin—N-acetyl-8-dodecyl-resorufin Rh123—Rhodamine123 ROS—reactive oxygen species