Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway

Cell Biology International 32 (2008) 100e106 www.elsevier.com/locate/cellbi

Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway Fa´bio Klamt a,*, Felipe Dal-Pizzol b, Daniel Pens Gelain a, Rodrigo Siqueira Dalmolin a, Ramatis Birnfeld de Oliveira a, Michele Bastiani c, Fabiana Horn c, Jose´ Cla´udio Fonseca Moreira a a

Center of Oxidative Stress Research, Department of Biochemistry, ICBS/Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil b Department of Medicine, University of Extremo Sul Catarinense (UNESC), Criciu´ma, Brazil c Department of Biophysics, IB/UFRGS, Porto Alegre, Brazil Received 16 March 2007; revised 30 April 2007; accepted 27 August 2007

Abstract Even though retinoids are widely used as adjuvant in chemotherapeutic interventions to improve cancer cell death, their mechanism(s) of action involves multiple overlapping pathways that remain unclear. We have previously shown that vitamin A, the natural precursor of the retinoids, induces oxidative-dependent cytochrome c release from isolated mitochondria, suggesting a putative mechanism for apoptosis activation. Using Sertoli cells in culture, we show that retinol causes mitochondrial-dependent apoptosis, involving oxidative stress. Apoptosis was evaluated by nuclear morphology, DNA fragmentation, and caspase-3/7 activity. Retinol induced oxidant- and time-dependent imbalance of several mitochondrial parameters, cytochrome c release and caspase-3/7 activation, leading cells to commit apoptosis. All parameters tested were attenuated or blocked by trolox co-administration, suggesting that retinol induces apoptosis through oxidative damage, which mitochondria plays a pivotal role. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Retinol; Mitochondria; Apoptosis; Cytochrome c; Oxidative stress

1. Introduction Retinoids are a class of natural and synthetic compounds with pleiotropic effects including antitumor activity. It has been demonstrated that retinoids inhibit growth and induce differentiation in a variety of malignancies, particularly Abbreviations: cyt c, cytochrome c; MPT, mitochondrial permeability transition; Djm, mitochondrial membrane potential; TRAP, total radical-trapping antioxidant parameter; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; SMP, sub-mitochondrial particle; MCA, 4-methylcoumaryl-7-amide; TUNEL, TdT-mediated dUTP nick end labeling; PTP, permeability transition pore. * Corresponding author at: Department de Bioquı´mica, ICBS/UFRGS, Rua Ramiro Barcelos, 2600eanexo, Porto Alegre, RS 90035-003, Brazil. Tel.: þ55 51 3308 5577; fax: þ55 51 3308 5535. E-mail address: [email protected] (F. Klamt).

epithelial cancers and leukemia (Blomhoff, 1994). In addition, high doses of retinoids have been successfully used as adjuvant for the treatment of desmoid tumors, nasopharyngeal carcinoma, and in breast and lung cancer (Leithner et al., 2000; Kuratomi et al., 1999; Windbichler et al., 1996; Pastorino et al., 1993). Vitamin A (all-trans retinol), the natural precursor of retinoids, is an essential nutrient which can be obtained either directly from the diet or by the intake and conversion of pro-vitamin A compounds. In spite of being one of the first vitamins to be discovered, the full range of biological activities mediated by vitamin A remains incomplete. Although the biological effects of retinoids are generally described as being mediated by the transcriptional regulation of target genes through nuclear receptorseretinoic acid receptors (RARa, -b, and -g) and retinoid X receptors (RXRa, -b, and -g) (Altucci and Gronemeyer, 2001)ethey have now been described as potent

1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.08.018

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inducers of programmed cell death by an unknown receptorindependent mechanism (Clifford et al., 1999; Toma et al., 1997). By its electron transfer capacity, retinol can directly modulate serine/threonine kinases, different PKC isoforms, and cRaf activities, functioning as a tag to enable the efficient and direct redox activation of these proteins (Hoyos et al., 2000; Radominska-Pandya et al., 2000). Furthermore, others and we have demonstrated that retinol has pro-oxidant properties, suggesting a role for oxidative stress in the activation of apoptosis by retinol (Klamt et al., 2003a,b; Dal-Pizzol et al., 2000a,b; Chen et al., 1999; Murata and Kawanishi, 2000). However, no study has yet been conducted to elucidate such association. Apoptosis is regarded as an active and organized form of cell death, triggered in response to physiologic or pathologic stimuli. It is characterized by condensation and fragmentation of the chromatin accompanied by internucleosomal DNA cleavage, caspase activation, translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, and formation of the so-called ‘‘apoptotic bodies’’ (Hengartner, 2000). Apoptotic cell death, in contrast to necrotic cell death, is thought to be physiologically advantageous, because the dying cells are cleared by phagocytosis prior to cell lysis and release of potentially inflammatory mediators. Mitochondria plays a pivotal role in the initiation of apoptosis, since death factors that are usually present in the intermembrane space of this organelle are released to the cytoplasm during early stages of apoptotic cell death (Newmeyer, 2003). A common feature of the activation of the mitochondrial apoptotic pathway is the occurrence of the mitochondrial permeability transition (MPT), leading to a decrease in membrane potential (Djm) and causing mitochondria swelling (Green and Kroemer, 2004). Cytochrome c (cyt c), released from mitochondria by an increase in MPT, interacts with an adaptor moleculeeApaf-1eresulting in the activation of pro-caspase-9. Caspase-9 then cleaves and activates effector pro-caspases-3 and -7, which in turn are responsible for the biochemical and morphological changes characteristic of apoptosis (Wang et al., 2005). Using isolated mitochondria, we have recently demonstrated that retinol induces an oxidant-dependent opening of the mitochondrial permeability transition pore, leading to mitochondrial swelling and release of cyt c (Klamt et al., 2005): this, in turn, suggests a mechanism of induction of apoptosis by retinol. The experiments presented in this report were designed to investigate both if retinol supplementation is capable of activating mitochondrial-dependent programmed cell death, and if apoptosis mediated by retinol is related to the generation of free radical. Our results provide evidence for the involvement of oxidative damage to cell and mitochondria as a possible mechanism of apoptosis activation by retinol. 2. Materials and methods 2.1. Chemicals Type I collagenase, medium 199, HBSS, trolox, and all-trans retinol were purchased from Sigma, St. Louis, MO, USA. Trypsin was purchased from Difco, Detroit, MI, USA.

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2.2. Sertoli cell isolation and culture Sertoli cells from 15-day-old Wistar rats testicles were prepared and cultured as previously described (Dal-Pizzol et al., 2000b). A small percentage (3e4%) of contamination by peritubular cells, determined by histochemical demonstration of alkaline phosphatase activity, was present in these Sertoli cell preparations. After isolation, Sertoli cells were counted in a Neubauer chamber and cultivated at a plating density of 3.2  105 cells/cm2 in medium 199 (pH 7.4) supplemented with 1% fetal bovine serum (v/v) in plastic vessels. Cells were maintained at 34  C in a humidified atmosphere of 5% CO2 in air. The medium was replaced after 24 h by serum-free medium to remove unattached Sertoli and germinative cells. Control cultures received only the retinol solvent (0.1% ethanol, v/v). Previous testing has shown that ethanol alone had no effect in any of the experimental procedures (data not shown). The formation of oxidized retinol metabolites was spetrophotometrically monitored in all retinol solution before use. Cell viability was assessed by trypan blue exclusion. Trolox, a synthetic analog of vitamin E, was used as antioxidant. All results were standardized against protein content (Lowry et al., 1951).

2.3. ATP content, mitochondrial activity and anion superoxide generation Intracellular ATP levels were determined using luciferin/luciferase assay (Spragg et al., 1985). Mitochondrial activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It is widely assumed that MTT is reduced by active mitochondria (Liu et al., 1997) Superoxide production was determined in washed sub-mitochondrial particles (SMP), isolated from Sertoli cells by differential centrifugation as previously described (Klamt et al., 2005), using a spectrophotometric assay based on the superoxide-dependent oxidation of epinephrine to adrenochrome at 37  C (E480nm ¼ 4.0 mM/cm) (Boveris, 1984). The reaction medium consisted of 230 mM mannitol, 70 mM sucrose, 20 mM TriseHCl (pH 7.4), SMP (1.0 mg protein/mL), 0.1 mM catalase (E.C. 1.11.6.1), and 1 mM epinephrine. Succinate (7 mM) was used as substrate. Superoxide dismutase (E.C. 1.15.1.1) was used at 0.1e0.3 mM final concentration to give assay specificity.

2.4. Western-blot analysis For detection of cyt c release, cells were incubated with ice-cold digitonin lysis buffer (75 mM KCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, 190 mg/ml digitonin, pH 7.4) for 5 min. The buffer (cytosolic proteins from permeabilized cells) was aspirated gently and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes as described previously (Gottlieb and Granville, 2002). After blocking with 5% non-fat dry milk, membranes were incubated with anti-cyt c monoclonal antibody (BD Biosciences, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies (Dakocytomation, USA). Bands were visualized by chemiluminescence using the ECL kit from NEN (Boston, MA, USA). Densitometric analysis of bands were performed using ImageJ 1.36b (National Institutes of Health, USA) software.

2.5. Apoptosis assays For caspase activity, cells were lysed and cell extracts were incubated for 30 min at 37  C with 100 mM acetyl-DEVD-(4-methyl-coumaryl-7-amide) (MCA) peptide substrate (Peptide Institute, Japan) in a total volume of 200 mL adjusted with ICE buffer (50 mM Hepes buffer, pH 7.5, 10% sucrose, 0.1% Triton X-100), and DTT (10 mM) was added to the reaction mixture. Substrate hydrolysis was monitored for 2 h at 370 nm excitation/460 nm emission in a microplate fluorescence reader (Molecular Devices Corporation) equipped with a software module for kinetic analysis (SOFTMax PRO, Molecular Devices, Sunnyvale, USA); substrate hydrolysis was quantified by comparison with a standard curve of MCA (Lee and Shacter, 2000). Lowmolecular-weight apoptotic DNA fragments were isolated by the procedure of Herrmann et al. (1994). Internucleosomal DNA fragments (equivalent to w3  106 cells) were separated by 2% agarose electrophoresis at 80 V for

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2 h. Apoptotic cell death was also examined by TdT-mediated dUTP nick end labeling (TUNEL) technique. Treated cells were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature. According to the method of a TUNEL staining kit (in situ Cell Death Detection Kit; Roche Applied Science, Germany), cells were treated with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min at 4  C and labeled with terminal deoxyribonucleotidyl transferase (TdT) for 30 min at 37  C, and analyzed using fluorescence microscopy.

2.6. Statistical analysis Results are expressed as the mean  SEM. Data were analyzed by one-way analysis of variance (ANOVA), using a NeumaneKeuls test to compare mean values across groups. Differences were considered to be significant when p < 0.05.

3. Results 3.1. Retinol causes alteration of the cellular redox status that damages mitochondria The cytotoxic effect of retinol over mitochondrial physiology was studied. We found that retinol treatment causes a time- and oxidant-dependent disturb of mitochondrial metabolism, observed as a reversible drop in mitochondrial activity (Fig. 1A) and in the cellular ATP levels (Fig. 1B), and as a sustained increase in the steady state production of anion superoxide by the mitochondrial electron transport chain (Fig. 1C). Interestingly, ATP levels and mitochondria activity were restored to control (untreated values) at 12 h and maintained until 24 h of retinol treatment (Fig. 1). All these effects of retinol were prevented by trolox co-administration (Fig. 1), demonstrating the dependence of the generation of oxidants by retinol treatment. 3.2. Retinol induces oxidant-dependent mitochondrial cyt c release and caspase-3/7 activation Classically, one of the landmarks of the activation of the intrinsic (mitochondrial) pathway of apoptosis is the release of death factors (i.e. cyt c) from the inner mitochondrial membrane to the cytosol. Cyt c was detected in the cytosol at 1 h of treatment (Fig. 2A) and there was no detection of cyt c in untreated cells. Densitometric analysis showed a sustained increase in the amount of cyt c released to cytosol in treated cells (Fig. 2B). At 4 h of treatment we found a 4-fold increase in caspase-3/7 activity (Fig. 2B). Furthermore, our data suggest an association with the release of cyt c from mitochondria, activation of caspase-3/7 (Figs. 2A,B) and anion superoxide production (Fig. 1C) (i.e. cyc c release and increased anion superoxide production before caspase-3/7 activation). These effects of retinol were dependent, at least in part, to the generation of free radicals, since trolox co-administration caused a dramatic decrease in the amount of cyt c released from mitochondria and in caspase-3/7 activity (Fig. 2). 3.3. Retinol induces oxidant-dependent apoptosis When cultured Sertoli cells were treated with 7 mM of retinol for 24 h, we observed a decrease in cell viability, as

Fig. 1. Effects of retinol treatment on mitochondrial activity, ATP content and anion superoxide generation. Sertoli cells were treated with: (-), no treatment; (B), retinol 7 mM; (A), retinol 7 mM þ trolox 0.1 mM by different times and A) MTT reduction (mitochondrial activity), B) ATP content and C) mitochondrial anion superoxide generation was determined as described under Section 2. Data represent mean  SEM from 3 independent experiments carried out in triplicates. **Different from control cells (P < 0.01), *(P < 0.05) (one-way analysis of variance).

visualized by an increase in the percentage of non-attached cells (Fig. 3A), in agreement with previous results showing trypan blue positive cells (Klamt et al., 2003b). Fig. 3B shows that treatment with 7 mM of retinol for 12 h resulted in

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induced by retinol involves free radical generation and oxidative damage to mitochondria.

4. Discussion

Fig. 2. Time course of cyt c release and caspase-3/7 activation in Sertoli cells treated with retinol. A) Cells were treated and, at the indicated times, were digitonin-permeabilized and cytosolic cyt c was analysed by Western blot. Similar results were obtained in two independent experiments. Representative data of the densitometric analysis of the amount of cyt c released is present in B). C) Cells were treated with: (-), no treatment; (B), retinol 7 mM; (A), retinol 7 mM þ trolox 0.1 mM, and at the indicated times, cell lysates were prepared and assayed for caspase-3/7 activity using DEVD-MCA as substrate. The data represent means  SEM from 3 independent experiments carried out in triplicates. **Different from control cells (P < 0.01) (one-way analysis of variance).

internucleossomal DNA fragmentation (ladder pattern), which is considered an earlier fingerprint of the apoptotic process. No DNA fragmentation was observed in untreated cells (Fig. 3B), or in Sertoli cells treated with vehicle (ethanol 0.1%) alone (data not shown). Using the TUNEL technique, we observed positive nuclear staining with the morphological features of apoptosis in treated cells (i.e. DNA fragmentation, formation and release of apoptotic bodies) (Fig. 3C, white arrow). For best data analysis, TUNEL positive cells were counted and expressed as percentage of total cells (Fig. 3D). Trolox (0.1 mM) co-administration attenuated the apoptotic parameters tested. Altogether, these results strongly support that apoptosis

In earlier reports, our research group demonstrated that 7 mM retinol (vitamin A) treatment leads to many changes in Sertoli cell metabolism, such as upregulation of antioxidant enzyme activities, increase in damage to biomolecules, abnormal cellular division, pre-neoplasic transformation, and cytoskeleton conformational changes. These effects were observed to be dependent on the production of reactive oxygen species (ROS), suggesting extra-nuclear (non-genomic) effects of retinol metabolism (Klamt et al., 2003a,b; Dal-Pizzol et al., 2000a,b; Chen et al., 1999; Murata and Kawanishi, 2000). Recently, using isolated rat liver mitochondria as a model, we found that retinol induces oxidative-dependent mitochondrial permeability transition (MPT) and cyt c release that was dependent on the opening of the permeability transition pore (PTP), since MPT mediated by retinol was blocked by ciclosporin A (CsA) co-administration (Klamt et al., 2005). In this report, we examined the role of free radical generation and mitochondria damage in the induction of programmed cell death by retinol. Overall, it is shown, for the first time, that vitamin A induces an oxidant-dependent apoptosis that involves the activation of the intrinsicemitochondrialeapoptotic pathway. The pro-oxidative effect of retinol decreases mitochondrial activity, ATP content, and leads to cyt c release. Cyt c is loosely bound to cardiolipin in the inner mitochondria membrane and cardiolipin is the main target of lipid peroxidation in mitochondria (Kagan et al., 2004). Cardiolipin oxidation induces cyt c release to the inner mitochondria membrane space (Ott et al., 2002). In most current models, cyt c release from mitochondria to cytosol is mediated by the opening of PTP complex. PTP is thought to be formed by the interaction of a voltage-dependent anion channel (VDAC, in the outer mitochondria membrane), with the adenine nucleotide transporter (ANT, in the inner mitochondria membrane), in a process regulated by cyclophilin (CyP-D, in the mitochondrial matrix) (Halestrap et al., 2002). It has long been known that PTP is modulated by the redox status of mitochondria (i.e. directly by glutathione or thioredoxin, and indirectly by NAD(P)H status) (Kowaltowski et al., 2001), and data suggested that the ANT protein is the component of PTP with high sensitivity to oxidative stress (Halestrap et al., 2002; Costantini et al., 2000). The direct interaction of retinoids with ANT was demonstrated, but the concentrations used in this study were far higher from physiological (Notario et al., 2003). The present and previous results suggest that retinol directly induces oxidative damage to mitochondria, leading to cyt c release through the opening of mitochondrial PTP. Based on the lipophilic nature of retinoids, the cellular treatment with retinol would lead to an increased intracellular concentration of these compounds, mainly in the mitochondrial fraction (Chew et al., 1991). Further studies should characterize the

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Fig. 3. Effects of retinol treatment on apoptotic parameters and cellular viability. A) Sertoli cells received no treatment (1) or were treated with retinol 7 mM (2) or retinol 7 mM þ trolox 0.1 mM (3) for 24 h, and cell morphology were analyzed by light microscopy (magnification 200) (Bar size: 10 mm). B) Sertoli cells were treated with retinol 7 mM for 12 h, and DNA fragmentation was analyzed by conventional agarose gel eletrophoresis as described under Section 2. M.S.: molecular standard. C) Sertoli cells received no treatment (1) or were treated with retinol 7 mM (2) or retinol 7 mM þ trolox 0.1 mM (3) for 24 h, TUNEL stained, and examined by fluorescence microscopy as described under Section 2. (Magnification 400). D) TUNEL positive cells were counted and expressed as % of control. The data represent means  SEM from 3 independent experiments carried out in triplicates. *Different from control cells (P < 0.01), #different from retinol treated cells (P < 0.05) (one-way analysis of variance).

molecular events associated with retinol-mediated oxidation of the permeability transition pore complex. The release of cyt c to the extra-mitochondrial environment by retinol administration may induce two processes: i) increase in mitochondrial superoxide radical production (Cai and Jones, 1998); and ii) activation of the ‘‘apoptosome’’ complex and the effector caspase-3/7. Even though we haven’t analyzed caspase-9 activation by retinol treatment, the temporal association of cyt c release and caspase-3/7 activation found here implicates that this might have occurred. Furthermore, the role of the mitochondria on the activation of the apoptotic machinery may be limited by the cellular redox environment

(Pervaiz and Cle´ment, 2002) and maintenance of ATP levels (Lee and Shacter, 1999). Because of the presence of critical cysteine residues in the active site of the enzyme, caspase activities are sensitive to oxidation (Lee and Shacter, 2000). In fact, high oxidative environment changes the pattern of cellular death from apoptosis to necrosis, mainly by caspase inactivation (Lee and Shacter, 1999). Even the other limiting situation to activate apoptotic machinery, the availability of intracellular ATP, was temporarily dropped but then restored to control levels in retinol-treated cells. Our results suggest that, in contrast to stronger oxidants (i.e. HOCl; HO ; high concentration of H2O2) that kill cells mainly by necrotic death, retinol 

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in the concentration used in this study is able to cause a milder oxidation that lead cells to enter apoptosis. The mechanism which retinol causes oxidative stress is still not clear, but the presence of the polyene conjugated double bounds characteristic of all natural retinoids could be the direct source for the pro-oxidant properties of the molecule. Others have demonstrated that retinol directly induces anion superoxide generation (Murata and Kawanishi, 2000) and here we have shown that cells treated with retinol have an increased production of O2 by mitochondrial electron transport chain. In addition, long time exposure to retinol causes an increase in iron uptake and storage, mainly in the mitochondria and nuclear fractions (Dal-Pizzol et al., 2000a). Based on our previous studies that demonstrated an increased formation of 8-oxo-dGua in retinol-treated cells, the formation of high reactive HO by iron-mediated Fenton chemistry also explain the pro-oxidant properties of vitamin A in biological systems (Klamt et al., 2003a). Sertoli cells are epithelial in origin and, like other epithelial cells (i.e. skin and respiratory tract), are responsive to retinol. Normal retinol concentration in human serum and liver is around 4.1 mmol/g of tissue (1e4 mM) and higher concentrations can be achieved (Schmidt et al., 2003). In the testes, the metabolic interconversion and long term storage of vitamin A mainly take place in Sertoli cells, were it reaches concentrations as high as 5 mM (Livrea and Packer, 1993). The World Health Organization (WHO) recommended a dietary retinol intake of 300 UI (approximately 1 mM), and considered a safe level of intake 600 UI. It is important to note that the intake of retinol 7 mM could readily be achieved with oral supplementation (Olson, 1994). Therapeutic dose of retinol varies from the orally administration of 300,000 U (314.19 mM)/day for the treatment of lung cancer to 50,000 U (52.36 mM)/day administrated intramuscularly for the treatment of nasopharyngeal carcinoma (Kuratomi et al., 1999; Pastorino et al., 1993). Spermatogenesis is completely dependent on the availability of retinol; absence or excess of retinol leads to testicular lesions and spermatogenesis disorders. Our data also suggest that perturbation of testicular functions by retinol supplementation could be related to oxidative stress-mediated apoptosis of Sertoli cells. In the view of the present results, we conclude that retinol is able to activate the apoptosis machinery in a pro-oxidant, mitochondrial-dependent manner, inducing Sertoli cells to die by the activation of the intrinsic programmed cell death pathway. These results suggest that, in addition to the use of combined therapies based on both maturation of neoplasic cells by retinoids and cell death induced by others chemotherapeutic drugs, new therapies exploring the direct influence of retinol in the activation of apoptotic machinery in tumor cells could be developed as well. 



Acknowledgments We thank Dr Elena Aida Bernard for critical discussions and we acknowledge the Brazilians funds CNPq, CAPES, FAPERGS, PROPESQ/UFRGS.

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