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Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis
MITOCH-00744; No of Pages 6 Mitochondrion xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Mitochondrion journal homepage: www.elsevier.com/locate/mito
Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposideinduced apoptosis Björn Kruspig a, Boris Zhivotovsky a, b, Vladimir Gogvadze a, b,⁎ a b
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, Stockholm, SE-171 77 Sweden MV Lomonosov Moscow State University, 119991, Moscow, Russia
a r t i c l e
i n f o
Article history: Received 24 May 2012 Received in revised form 1 August 2012 Accepted 9 August 2012 Available online xxxx Keywords: Mitochondria Apoptosis Cancer α-Tocopheryl succinate Cisplatin Etoposide
a b s t r a c t Targeting mitochondria is a promising strategy in tumor cell elimination. D-α-tocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, is a potentially powerful tool for fighting tumors by directly affecting mitochondria. However, when used at low concentrations it can suppress apoptosis induced by the conventionally used anticancer drug cisplatin. In cells treated with cisplatin, 30 μM α-TOS prominently attenuated the manifestation of characteristic features of apoptosis — release of cytochrome c from mitochondria, caspase-3-like activity, and cleavage of poly(ADP-ribose) polymerase. In contrast, cell death induced by etoposide was not inhibited but rather stimulated by α-TOS. Thus, co-treatment with α-TOS and conventional antitumor drugs should be carried out with caution. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Mitochondria are key participants in various cell death signaling pathways. Thus, steps leading to mitochondrial destabilization and permeabilization of the outer mitochondrial membrane (OMM) with the subsequent release of pro-apoptotic proteins might be a promising strategy in tumor cell elimination. Recently, a range of compounds named mitocans (an abbreviation derived from MITOchondria and CANcer), were shown to cause cell death by targeting mitochondria (Neuzil et al., 2007). One of the mitocans, D-αtocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, was shown to induce multiple changes in tumor cells, ultimately leading to cell death. The effects of α-TOS are due to interaction with Complex II of the mitochondrial respiratory chain, causing the leakage of electrons and formation of reactive oxygen species (Dong et al., 2008). In addition, we found that α-TOS stimulated rapid entry of Ca2 + into the cytosol, compromised Ca2 + buffering capacity of the mitochondria and sensitized
Abbreviations: α-TOS, D-α-tocopheryl succinate; CCCP, carbonyl cyanide mchlorophenylhydrazone; OMM, outer mitochondrial membrane; PARP, poly(ADP-ribose) polymerase; MPT, mitochondrial permeability transition. ⁎ Corresponding author. Tel.: + 46 8 52487515; fax: + 46 8 32 90 41. E-mail address: [email protected] (V. Gogvadze).
them towards mitochondrial permeability transition (Gogvadze et al., 2010; Kruspig et al., 2012). Remarkably, α-TOS was shown to selectively kill malignant cells at concentrations that are non-toxic for normal cells. In non-malignant cells α-TOS is hydrolyzed by means of esterases. As a result, α-tocopherol is gradually released to prevent membrane oxidative damage. Indeed, α-TOS was shown to rescue cells from chemical-induced toxicity (Fariss et al., 1989), or ionizing radiation (Singh et al., 2012). However, in malignant cells the hydrolysis of α-TOS is suppressed due to lower esterase activity (Neuzil et al., 2006; Ottino and Duncan, 1997). Thus, the inability of malignant cells to cleave α-TOS determines its ability to stimulate tumor cell death. α-TOS and other vitamin E analogs, such as α-tocopheryloxyacetic acid (Dong et al., 2012), are regarded as anticancer drugs, the efficiency of which has been demonstrated in a number of studies utilizing tumor cell lines (Kruspig et al., 2012) as well as mice xenografts. Co-treatment of α-TOS and paclitaxel inhibited bladder cancer cell growth and viability in vitro and in vivo (Kanai et al., 2010). The anticancer effect of α-TOS was also demonstrated on JHU-022 solid tumor xenograft growth in immunodeficient mice (Gu et al., 2008). However, it remains unknown whether relatively low concentrations of α-TOS that does not overcome the capacity of esterases would protect cells from conventional antitumor drugs due to the cleavage of α-TOS with the formation of α-tocopherol and succinate. In the present work we analyzed the possible consequences of combined treatment of tumor cells with various concentrations of α-TOS and drugs used in
1567-7249/$ – see front matter © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved. http://dx.doi.org/10.1016/j.mito.2012.08.001
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Fig. 1. α-TOS-induced apoptosis assessed by morphological changes (A), release of cytochrome c (B), and caspase-3-like activity (C). Pan-caspase inhibitor z-VAD-FMK (10 μM) attenuated α-TOS-induced morphological changes (D). Numbers indicate percentage of cells with apoptotic morphology. Tet21N cells were incubated with 50 μM α-TOS for 17 h, and apoptosis manifestations were assessed following harvesting, as described in the Materials and methods section.
antitumor therapy — cisplatin and etoposide. We show that α-TOS should be administered with caution as a potential anticancer drug, since administered at low concentrations α-TOS together with certain drugs can rescue rather than kill cells.
2. Material and methods 2.1. Cells All cells used in these experiments were cultured in RPMI 1640 complete medium supplemented with 10% (w/v) heat-inactivated fetal calf serum and penicillin/streptomycin (100 U/ml). For Tet21N cells, 100 μg/ml hygromycin and 200 μg/ml geneticine were also added to the medium. Cells were grown in a humidified air/CO2 (5%) atmosphere at 37 °C and maintained in a logarithmic growth phase for all experiments.
2.2. Assessment of cytochrome c release Cells were permeabilized with 0.01% digitonin for 15 min and fractionated into supernatant and pellet by centrifugation for 5 min at 16,000 ×g. Samples were mixed with Laemmli's loading buffer, boiled for 5 min and subjected to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 40 mA followed by electroblotting to nitrocellulose membranes for 2 h at 120 V. Membranes were blocked for 1 h with 5% nonfat milk in phosphate-buffered saline (PBS) at room temperature and subsequently probed overnight with a mouse anticytochrome c antibody (BD Biosciences, San Jose, CA). The membranes were rinsed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000) and visualized by ECL™ (Amersham Biosciences, Buckinghamshire, UK) and X-ray film.
2.3. Measurement of caspase activity Cleavage of the fluorogenic peptide substrate (Peptide Institute, Osaka, Japan) was measured using a modified version of a fluorometric assay. Cells were pelleted and washed once with PBS. After centrifugation, cells were resuspended in PBS at a concentration of 2 × 10 6 cells/100 μl; 25 μl of the suspension was added to a microtiter plate and mixed with the appropriate peptide substrate dissolved in a standard reaction buffer (100 mM Hepes, 10% sucrose, 5 mM DTT, 0.001% NP-40 and 0.1% CHAPS, pH 7.2). Cleavage of the fluorogenic peptide substrate was monitored by AMC liberation in a Fluoroscan II plate reader (Labsystems, Stockholm, Sweden) using 355 nm excitation and 460 nm emission wavelengths. 2.4. Analysis of oxygen consumption Cellular respiration was monitored with an oxygen electrode (Hansatech Instruments, Norfolk, UK) and analyzed using OxygraphPlus software (Hansatech Instruments, Norfolk, UK). Briefly, cells were harvested and counted. Four million cells were spun down, resuspended in 300 μl of the medium in which the cells were grown, and transferred into the Oxygraph chamber. The chamber was closed and basal respiration was measured for 3–4 min. To assess the maximum capacity of the respiratory chain, mitochondria were uncoupled by 5 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). 2.5. Morphological assessment of apoptosis Cells were seeded on glass cover slips, incubated with the indicated treatment and, after staining with Hoechst (2 μg/ml) for 10 min, examined using a Zeiss LSM 510 META confocal laser scanner microscope. At least 600 cells were counted and analyzed for apoptotic morphology.
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
B. Kruspig et al. / Mitochondrion xxx (2012) xxx–xxx
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Fig. 2. α-TOS induces concentration-dependent stimulation of caspase-3-like activity (A). Tet21N cells were incubated with various concentrations of α-TOS for 17 h, and caspase-3-like activity was measured following harvesting, as described in the Materials and methods section. Results are presented as fold increase of basal activity. (B) Analysis of oxygen consumption in cells treated with high (50 μM), and low (30 μM) concentrations of α-TOS. Cells were harvested and after counting 4×106 cells were transferred into the Oxygraph chamber. The chamber was closed and basal respiration was measured for 3–4 min. To assess the maximum capacity of the respiratory chain, mitochondria were uncoupled by 5 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Trace a, control cells; trace b, cells treated with 30 μM α-TOS; trace c, cells treated with 50 μM α-TOS. (C) Phasecontrast image of control cells (left) and cells treated with 30 μM α-TOS for 17 h.
3. Results Incubation of neuroblastoma Tet21N cells with 50 μM α-TOS for 17 h resulted in the manifestation of typical features of apoptosis, including morphological changes (A), release of cytochrome c from mitochondria (B) and stimulation of caspase-3-like activity (C) (Fig. 1). The pancaspase inhibitor z-VAD-FMK attenuated α-TOS-induced morphological changes, confirming the involvement of caspases in the cell death process (Fig. 1D). Remarkably, no stimulation of caspase-3-like activity by α-TOS was found below 40 μM, whereas treatment with higher
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concentrations resulted in a sharp stimulation of caspase-3 activity (Fig. 2A). High concentrations of α-TOS prominently suppressed mitochondrial respiration, both basal and stimulated by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Fig. 2B, trace c vs trace a), whereas at low concentrations of α-TOS mitochondrial respiration was the same as that of untreated cells (trace b). Furthermore, no morphological changes were observed in cells treated with 30 μM α-TOS (Fig. 2C). In order to test whether low non-toxic concentrations of α-TOS can prevent or attenuate cell death induced by conventional anticancer drugs, cells were incubated with cisplatin in the presence or absence of α-TOS. Cisplatin stimulated apoptosis in Tet21N cells, as assessed by caspase-3 like activity (Fig. 3A). Co-incubation of Tet21N cells with cisplatin and increasing concentrations of α-TOS revealed that at low doses α-TOS gradually suppressed cisplatin-induced cell death; however further increases in α-TOS concentration resulted in the enhancement of the toxic effect of cisplatin. The suppression of cisplatin-induced apoptosis by low concentrations of α-TOS was confirmed for HCT116 colorectal carcinoma (Fig. 3B) and U1810 nonsmall cell lung cancer cells (Fig. 3C). Furthermore, the protective effect of low concentrations of α-TOS (30 μM) was also evident when Tet21N cells were co-treated with the broad-spectrum protein-kinase inhibitor staurosporine, whereas 50 μM α-TOS caused a higher sensitivity to staurosporine (Fig. 3D). When the cells were co-treated with α-TOS and another anticancer drug, etoposide, in contrast to co-treatment with cisplatin, even low doses of α-TOS enhanced the etoposide-induced stimulation of caspase-3-like activity (Fig. 3E). The contrasting effects of low concentrations of α-TOS on cisplatin- and etoposide-induced apoptosis were also evident from the analysis of the number of cells with condensed nuclei (Fig. 4A and B). Apparently, when administered at low doses α-TOS undergoes cleavage by specific esterases with the formation of α-tocopherol and succinate. In order to determine which of the two structural components of α-TOS provides protection, stimulation of cell death by cisplatin was assessed in the presence of trolox — a water-soluble form of α-tocopherol, or succinate. Succinate usually shows poor passage through the plasma membrane, but when applied at high concentrations, it can slowly penetrate into the cell (Kimmich et al., 1991). Trolox slightly attenuated the apoptotic response to cisplatin, whereas exogenous succinate markedly suppressed apoptosis, as assessed by caspase-3-like activity (Fig. 5A). Low concentrations of α-TOS successfully prevented the release of cytochrome c and cleavage of PARP upon induction of apoptosis by cisplatin (Fig. 5B), but enhanced apoptosis induced by 30 and 50 μM etoposide (Fig. 5C). Remarkably, exogenous succinate protected cells against both anticancer drugs. 4. Discussion α-TOS is regarded as an anticancer drug, the efficiency of which has been demonstrated not only in various cultured cells, but also in mice xenografts. Targeting of mitochondria with α-TOS is advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways. Indeed, we have previously found that α-TOS can kill neuroblastoma cells irrespective of their MycN-expression level and amplification (Kruspig et al., 2012). Cells in which MycN expression has been switched off are resistant to treatment with conventional anticancer drugs, such as cisplatin or doxorubicin. However, α-TOS successfully eliminated neuroblastoma cells that overexpressed MycN, as well as cells in which MycN was switched off. Thus, α-TOS-induced mitochondrial destabilization can be viewed as a promising tool for inducing apoptosis in tumor cells. Nevertheless, as shown here, this compound should be used with caution, especially in combination with certain conventionally used anticancer drugs. Thus, α-TOS applied at relatively low concentrations
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Fig. 3. Analysis of caspase-3-like activity in various cell lines treated with 10 μg/ml cisplatin and various concentrations of α-TOS, staurosporine, or etoposide for 17 h. (A) Tet21N neuroblastoma cells, (B) HCT116 colorectal carcinoma, and (C) U1810 non-small cell lung cancer cells. (D) Tet21N cells treated with 40 nM staurosporine alone, or in combination with 30 μM or 50 μM α-TOS for 17 h. (E) Effect of co-treatment with 30 μM etoposide and various doses of α-TOS on caspase-3-like activity in Tet21N cells.
potently inhibited apoptosis induced by cisplatin, a DNA-damaging agent, or staurosporine, a broad-spectrum protein-kinase inhibitor. Protection against cisplatin was observed at concentrations of α-TOS below 40 μM, when this compound was supposedly cleaved by cellular esterases. Of the two cleavage products of α-TOS, α-tocopherol and succinate, the most protective was succinate. The protective effect of succinate can be attributed to its unique capacity to “monopolize” the respiratory chain of mitochondria, as reported by Hans Krebs (Krebs et al., 1961). One of the consequences of this monopolization is an even greater reduction of mitochondrial pyridine nucleotides than that achieved by Complex I substrates (Chance and Hollunger, 1961). The redox state of mitochondrial pyridine nucleotides is involved in the regulation of various processes, such as the induction of mitochondrial permeability transition (MPT) (Costantini et al., 1996) and the detoxification of hydrogen peroxide and organic hydroperoxides via the glutathione-dependent defense system (Sies and Summer, 1975). Oxidation of succinate allows isolated mitochondria to accumulate 3–4 times more Ca 2 + before the onset of MPT compared to mitochondria oxidizing NAD-dependent substrates (Kondrashova et al., 1982), and makes them resistant to oxidative stress (Bindoli et al., 1982). In the presence, as well as in the absence, of the oxidant t-butylhydroperoxide, mitochondria retained more Ca 2 + with succinate than with beta-hydroxybutyrate as the respiratory substrate (Gogvadze et al., 1996). Such stabilization of mitochondria would make them more resistant towards OMM permeabilization and the release of pro-apoptotic proteins from the intermembrane space. Hence, upon stimulation of apoptosis, mitochondria utilizing succinate should show better retention of intermembrane space proteins and thus apoptosis will be attenuated.
The precise mechanisms of succinate-mediated protection require further investigation. The lack of protection by low concentrations of α-TOS in the case of etoposide can be explained by the ability of etoposide to affect not only topoisomerase II, but also mitochondria (Robertson et al., 2000), causing the induction of oxidative stress and impairment of mitochondrial function (Pham and Hedley, 2001). Thus, the amount of succinate formed upon cleavage of α-TOS was not sufficient to protect against etoposide, while exogenous succinate successfully prevented apoptosis. In conclusion, a primary strategic problem in cancer therapy is how to selectively activate apoptosis in transformed cells. The ability of α-TOS to induce tumor cell death by targeting mitochondria is an important part of the anticancer strategy. However, co-treatment with conventional antitumor drugs should be carried out with caution. The data presented here highlight the role of succinate as a potential physiological modulator of the mitochondrial response to treatment. The ability of succinate to “monopolize” the respiratory chain of mitochondria might be important for the modulation of cellular physiology, including apoptotic pathways.
Acknowledgments The authors thank Prof. Marie Arsenian-Henriksson (Karolinska Institutet, Stockholm) for providing the Tet21N cell line used in the study. The work was supported by grants from the Swedish Research Council; the Swedish and the Stockholm Cancer Societies; the Swedish Childhood Cancer Foundation and the Russian Ministry of Higher Education and Science (11.G34.31.0006).
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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References Bindoli, A., Cavallini, L., Jocelyn, P., 1982. Mitochondrial lipid peroxidation by cumene hydroperoxide and its prevention by succinate. Biochim. Biophys. Acta 681, 496–503. Chance, B., Hollunger, G., 1961. The interaction of energy and electron transfer reactions in mitochondria. I. General properties and nature of the products of succinate-linked reduction of pyridine nucleotide. J. Biol. Chem. 236, 1534–1543. Costantini, P., Chernyak, B.V., Petronilli, V., Bernardi, P., 1996. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271, 6746–6751. Dong, L.F., Grant, G., Massa, H., Zobalova, R., Akporiaye, E., Neuzil, J., 2012. alphaTocopheryloxyacetic acid is superior to alpha-tocopheryl succinate in suppressing HER2-high breast carcinomas due to its higher stability. Int. J. Cancer 131, 1052–1058. Dong, L.F., Low, P., Dyason, J.C., Wang, X.F., Prochazka, L., Witting, P.K., Freeman, R., Swettenham, E., Valis, K., Liu, J., Zobalova, R., Turanek, J., Spitz, D.R., Domann, F.E., Scheffler, I.E., Ralph, S.J., Neuzil, J., 2008. Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II. Oncogene 27, 4324–4335. Fariss, M.W., Merson, M.H., O'Hara, T.M., 1989. Alpha-tocopheryl succinate protects hepatocytes from chemical-induced toxicity under physiological calcium conditions. Toxicol. Lett. 47, 61–75. Gogvadze, V., Norberg, E., Orrenius, S., Zhivotovsky, B., 2010. Involvement of Ca2 + and ROS in alpha-tocopheryl succinate-induced mitochondrial permeabilization. Int. J. Cancer 127, 1823–1832.
Fig. 5. Modulation of apoptotic response to cisplatin by trolox and succinate (A). Tet21N cells were pre-incubated for 3 h with 50 μM trolox, 30 μM α-TOS, or 20 mM succinate and treated with 10 μg/ml cisplatin for 17 h. Effect of α-TOS and succinate on cisplatin- (B) or etoposide-induced (C) release of cytochrome c and cleavage of PARP. Cells were pre-incubated for 3 h with 30 μM α-TOS or 20 mM succinate and treated with cisplatin (10 μg/ml) or 30, and 50 μM etoposide for 17 h. GAPDH serves as a loading control.
Gogvadze, V., Schweizer, M., Richter, C., 1996. Control of the pyridine nucleotide-linked Ca2 + release from mitochondria by respiratory substrates. Cell Calcium 19, 521–526. Gu, X., Song, X., Dong, Y., Cai, H., Walters, E., Zhang, R., Pang, X., Xie, T., Guo, Y., Sridhar, R., Califano, J.A., 2008. Vitamin E succinate induces ceramide-mediated apoptosis in head and neck squamous cell carcinoma in vitro and in vivo. Clin. Cancer Res. 14, 1840–1848. Kanai, K., Kikuchi, E., Mikami, S., Suzuki, E., Uchida, Y., Kodaira, K., Miyajima, A., Ohigashi, T., Nakashima, J., Oya, M., 2010. Vitamin E succinate induced apoptosis and enhanced chemosensitivity to paclitaxel in human bladder cancer cells in vitro and in vivo. Cancer Sci. 101, 216–223. Kimmich, G.A., Randles, J., Bennett, E., 1991. Sodium-dependent succinate transport by isolated chick intestinal cells. Am. J. Physiol. 260, C1151–C1157. Kondrashova, M.N., Gogvadze, V.G., Medvedev, B.I., Babsky, A.M., 1982. Succinic acid oxidation as the only energy support of intensive Ca2 + uptake by mitochondria. Biochem. Biophys. Res. Commun. 109, 376–381. Krebs, H.A., Eggleston, L.V., D'Alessandro, A., 1961. The effect of succinate and amytal on the reduction of acetoacetate in animal tissues. Biochem. J. 79, 537–549. Kruspig, B., Nilchian, A., Bejarano, I., Orrenius, S., Zhivotovsky, B., Gogvadze, V., 2012. Targeting mitochondria by alpha-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression. Cell Mol. Life Sci. 69, 2091–2099. Neuzil, J., Dyason, J.C., Freeman, R., Dong, L.F., Prochazka, L., Wang, X.F., Scheffler, I., Ralph, S.J., 2007. Mitocans as anti-cancer agents targeting mitochondria: lessons from studies with vitamin E analogues, inhibitors of complex II. J. Bioenerg. Biomembr. 39, 65–72.
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Neuzil, J., Wang, X.F., Dong, L.F., Low, P., Ralph, S.J., 2006. Molecular mechanism of ‘mitocan’-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins. FEBS Lett. 580, 5125–5129. Ottino, P., Duncan, J.R., 1997. Effect of alpha-tocopherol succinate on free radical and lipid peroxidation levels in BL6 melanoma cells. Free Radic. Biol. Med. 22, 1145–1151. Pham, N.A., Hedley, D.W., 2001. Respiratory chain-generated oxidative stress following treatment of leukemic blasts with DNA-damaging agents. Exp. Cell Res. 264, 345–352.
Robertson, J.D., Gogvadze, V., Zhivotovsky, B., Orrenius, S., 2000. Distinct pathways for stimulation of cytochrome c release by etoposide. J. Biol. Chem. 275, 32438–32443. Sies, H., Summer, K.H., 1975. Hydroperoxide-metabolizing systems in rat liver. Eur. J. Biochem. 57, 503–512. Singh, P.K., Wise, S.Y., Ducey, E.J., Fatanmi, O.O., Elliott, T.B., Singh, V.K., 2012. alphaTocopherol succinate protects mice against radiation-induced gastrointestinal injury. Radiat. Res. 177, 133–145.
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
Contents lists available at SciVerse ScienceDirect
Mitochondrion journal homepage: www.elsevier.com/locate/mito
Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposideinduced apoptosis Björn Kruspig a, Boris Zhivotovsky a, b, Vladimir Gogvadze a, b,⁎ a b
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, Stockholm, SE-171 77 Sweden MV Lomonosov Moscow State University, 119991, Moscow, Russia
a r t i c l e
i n f o
Article history: Received 24 May 2012 Received in revised form 1 August 2012 Accepted 9 August 2012 Available online xxxx Keywords: Mitochondria Apoptosis Cancer α-Tocopheryl succinate Cisplatin Etoposide
a b s t r a c t Targeting mitochondria is a promising strategy in tumor cell elimination. D-α-tocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, is a potentially powerful tool for fighting tumors by directly affecting mitochondria. However, when used at low concentrations it can suppress apoptosis induced by the conventionally used anticancer drug cisplatin. In cells treated with cisplatin, 30 μM α-TOS prominently attenuated the manifestation of characteristic features of apoptosis — release of cytochrome c from mitochondria, caspase-3-like activity, and cleavage of poly(ADP-ribose) polymerase. In contrast, cell death induced by etoposide was not inhibited but rather stimulated by α-TOS. Thus, co-treatment with α-TOS and conventional antitumor drugs should be carried out with caution. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Mitochondria are key participants in various cell death signaling pathways. Thus, steps leading to mitochondrial destabilization and permeabilization of the outer mitochondrial membrane (OMM) with the subsequent release of pro-apoptotic proteins might be a promising strategy in tumor cell elimination. Recently, a range of compounds named mitocans (an abbreviation derived from MITOchondria and CANcer), were shown to cause cell death by targeting mitochondria (Neuzil et al., 2007). One of the mitocans, D-αtocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, was shown to induce multiple changes in tumor cells, ultimately leading to cell death. The effects of α-TOS are due to interaction with Complex II of the mitochondrial respiratory chain, causing the leakage of electrons and formation of reactive oxygen species (Dong et al., 2008). In addition, we found that α-TOS stimulated rapid entry of Ca2 + into the cytosol, compromised Ca2 + buffering capacity of the mitochondria and sensitized
Abbreviations: α-TOS, D-α-tocopheryl succinate; CCCP, carbonyl cyanide mchlorophenylhydrazone; OMM, outer mitochondrial membrane; PARP, poly(ADP-ribose) polymerase; MPT, mitochondrial permeability transition. ⁎ Corresponding author. Tel.: + 46 8 52487515; fax: + 46 8 32 90 41. E-mail address: [email protected] (V. Gogvadze).
them towards mitochondrial permeability transition (Gogvadze et al., 2010; Kruspig et al., 2012). Remarkably, α-TOS was shown to selectively kill malignant cells at concentrations that are non-toxic for normal cells. In non-malignant cells α-TOS is hydrolyzed by means of esterases. As a result, α-tocopherol is gradually released to prevent membrane oxidative damage. Indeed, α-TOS was shown to rescue cells from chemical-induced toxicity (Fariss et al., 1989), or ionizing radiation (Singh et al., 2012). However, in malignant cells the hydrolysis of α-TOS is suppressed due to lower esterase activity (Neuzil et al., 2006; Ottino and Duncan, 1997). Thus, the inability of malignant cells to cleave α-TOS determines its ability to stimulate tumor cell death. α-TOS and other vitamin E analogs, such as α-tocopheryloxyacetic acid (Dong et al., 2012), are regarded as anticancer drugs, the efficiency of which has been demonstrated in a number of studies utilizing tumor cell lines (Kruspig et al., 2012) as well as mice xenografts. Co-treatment of α-TOS and paclitaxel inhibited bladder cancer cell growth and viability in vitro and in vivo (Kanai et al., 2010). The anticancer effect of α-TOS was also demonstrated on JHU-022 solid tumor xenograft growth in immunodeficient mice (Gu et al., 2008). However, it remains unknown whether relatively low concentrations of α-TOS that does not overcome the capacity of esterases would protect cells from conventional antitumor drugs due to the cleavage of α-TOS with the formation of α-tocopherol and succinate. In the present work we analyzed the possible consequences of combined treatment of tumor cells with various concentrations of α-TOS and drugs used in
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Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Fig. 1. α-TOS-induced apoptosis assessed by morphological changes (A), release of cytochrome c (B), and caspase-3-like activity (C). Pan-caspase inhibitor z-VAD-FMK (10 μM) attenuated α-TOS-induced morphological changes (D). Numbers indicate percentage of cells with apoptotic morphology. Tet21N cells were incubated with 50 μM α-TOS for 17 h, and apoptosis manifestations were assessed following harvesting, as described in the Materials and methods section.
antitumor therapy — cisplatin and etoposide. We show that α-TOS should be administered with caution as a potential anticancer drug, since administered at low concentrations α-TOS together with certain drugs can rescue rather than kill cells.
2. Material and methods 2.1. Cells All cells used in these experiments were cultured in RPMI 1640 complete medium supplemented with 10% (w/v) heat-inactivated fetal calf serum and penicillin/streptomycin (100 U/ml). For Tet21N cells, 100 μg/ml hygromycin and 200 μg/ml geneticine were also added to the medium. Cells were grown in a humidified air/CO2 (5%) atmosphere at 37 °C and maintained in a logarithmic growth phase for all experiments.
2.2. Assessment of cytochrome c release Cells were permeabilized with 0.01% digitonin for 15 min and fractionated into supernatant and pellet by centrifugation for 5 min at 16,000 ×g. Samples were mixed with Laemmli's loading buffer, boiled for 5 min and subjected to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 40 mA followed by electroblotting to nitrocellulose membranes for 2 h at 120 V. Membranes were blocked for 1 h with 5% nonfat milk in phosphate-buffered saline (PBS) at room temperature and subsequently probed overnight with a mouse anticytochrome c antibody (BD Biosciences, San Jose, CA). The membranes were rinsed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000) and visualized by ECL™ (Amersham Biosciences, Buckinghamshire, UK) and X-ray film.
2.3. Measurement of caspase activity Cleavage of the fluorogenic peptide substrate (Peptide Institute, Osaka, Japan) was measured using a modified version of a fluorometric assay. Cells were pelleted and washed once with PBS. After centrifugation, cells were resuspended in PBS at a concentration of 2 × 10 6 cells/100 μl; 25 μl of the suspension was added to a microtiter plate and mixed with the appropriate peptide substrate dissolved in a standard reaction buffer (100 mM Hepes, 10% sucrose, 5 mM DTT, 0.001% NP-40 and 0.1% CHAPS, pH 7.2). Cleavage of the fluorogenic peptide substrate was monitored by AMC liberation in a Fluoroscan II plate reader (Labsystems, Stockholm, Sweden) using 355 nm excitation and 460 nm emission wavelengths. 2.4. Analysis of oxygen consumption Cellular respiration was monitored with an oxygen electrode (Hansatech Instruments, Norfolk, UK) and analyzed using OxygraphPlus software (Hansatech Instruments, Norfolk, UK). Briefly, cells were harvested and counted. Four million cells were spun down, resuspended in 300 μl of the medium in which the cells were grown, and transferred into the Oxygraph chamber. The chamber was closed and basal respiration was measured for 3–4 min. To assess the maximum capacity of the respiratory chain, mitochondria were uncoupled by 5 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). 2.5. Morphological assessment of apoptosis Cells were seeded on glass cover slips, incubated with the indicated treatment and, after staining with Hoechst (2 μg/ml) for 10 min, examined using a Zeiss LSM 510 META confocal laser scanner microscope. At least 600 cells were counted and analyzed for apoptotic morphology.
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Fig. 2. α-TOS induces concentration-dependent stimulation of caspase-3-like activity (A). Tet21N cells were incubated with various concentrations of α-TOS for 17 h, and caspase-3-like activity was measured following harvesting, as described in the Materials and methods section. Results are presented as fold increase of basal activity. (B) Analysis of oxygen consumption in cells treated with high (50 μM), and low (30 μM) concentrations of α-TOS. Cells were harvested and after counting 4×106 cells were transferred into the Oxygraph chamber. The chamber was closed and basal respiration was measured for 3–4 min. To assess the maximum capacity of the respiratory chain, mitochondria were uncoupled by 5 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Trace a, control cells; trace b, cells treated with 30 μM α-TOS; trace c, cells treated with 50 μM α-TOS. (C) Phasecontrast image of control cells (left) and cells treated with 30 μM α-TOS for 17 h.
3. Results Incubation of neuroblastoma Tet21N cells with 50 μM α-TOS for 17 h resulted in the manifestation of typical features of apoptosis, including morphological changes (A), release of cytochrome c from mitochondria (B) and stimulation of caspase-3-like activity (C) (Fig. 1). The pancaspase inhibitor z-VAD-FMK attenuated α-TOS-induced morphological changes, confirming the involvement of caspases in the cell death process (Fig. 1D). Remarkably, no stimulation of caspase-3-like activity by α-TOS was found below 40 μM, whereas treatment with higher
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concentrations resulted in a sharp stimulation of caspase-3 activity (Fig. 2A). High concentrations of α-TOS prominently suppressed mitochondrial respiration, both basal and stimulated by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Fig. 2B, trace c vs trace a), whereas at low concentrations of α-TOS mitochondrial respiration was the same as that of untreated cells (trace b). Furthermore, no morphological changes were observed in cells treated with 30 μM α-TOS (Fig. 2C). In order to test whether low non-toxic concentrations of α-TOS can prevent or attenuate cell death induced by conventional anticancer drugs, cells were incubated with cisplatin in the presence or absence of α-TOS. Cisplatin stimulated apoptosis in Tet21N cells, as assessed by caspase-3 like activity (Fig. 3A). Co-incubation of Tet21N cells with cisplatin and increasing concentrations of α-TOS revealed that at low doses α-TOS gradually suppressed cisplatin-induced cell death; however further increases in α-TOS concentration resulted in the enhancement of the toxic effect of cisplatin. The suppression of cisplatin-induced apoptosis by low concentrations of α-TOS was confirmed for HCT116 colorectal carcinoma (Fig. 3B) and U1810 nonsmall cell lung cancer cells (Fig. 3C). Furthermore, the protective effect of low concentrations of α-TOS (30 μM) was also evident when Tet21N cells were co-treated with the broad-spectrum protein-kinase inhibitor staurosporine, whereas 50 μM α-TOS caused a higher sensitivity to staurosporine (Fig. 3D). When the cells were co-treated with α-TOS and another anticancer drug, etoposide, in contrast to co-treatment with cisplatin, even low doses of α-TOS enhanced the etoposide-induced stimulation of caspase-3-like activity (Fig. 3E). The contrasting effects of low concentrations of α-TOS on cisplatin- and etoposide-induced apoptosis were also evident from the analysis of the number of cells with condensed nuclei (Fig. 4A and B). Apparently, when administered at low doses α-TOS undergoes cleavage by specific esterases with the formation of α-tocopherol and succinate. In order to determine which of the two structural components of α-TOS provides protection, stimulation of cell death by cisplatin was assessed in the presence of trolox — a water-soluble form of α-tocopherol, or succinate. Succinate usually shows poor passage through the plasma membrane, but when applied at high concentrations, it can slowly penetrate into the cell (Kimmich et al., 1991). Trolox slightly attenuated the apoptotic response to cisplatin, whereas exogenous succinate markedly suppressed apoptosis, as assessed by caspase-3-like activity (Fig. 5A). Low concentrations of α-TOS successfully prevented the release of cytochrome c and cleavage of PARP upon induction of apoptosis by cisplatin (Fig. 5B), but enhanced apoptosis induced by 30 and 50 μM etoposide (Fig. 5C). Remarkably, exogenous succinate protected cells against both anticancer drugs. 4. Discussion α-TOS is regarded as an anticancer drug, the efficiency of which has been demonstrated not only in various cultured cells, but also in mice xenografts. Targeting of mitochondria with α-TOS is advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways. Indeed, we have previously found that α-TOS can kill neuroblastoma cells irrespective of their MycN-expression level and amplification (Kruspig et al., 2012). Cells in which MycN expression has been switched off are resistant to treatment with conventional anticancer drugs, such as cisplatin or doxorubicin. However, α-TOS successfully eliminated neuroblastoma cells that overexpressed MycN, as well as cells in which MycN was switched off. Thus, α-TOS-induced mitochondrial destabilization can be viewed as a promising tool for inducing apoptosis in tumor cells. Nevertheless, as shown here, this compound should be used with caution, especially in combination with certain conventionally used anticancer drugs. Thus, α-TOS applied at relatively low concentrations
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Fig. 3. Analysis of caspase-3-like activity in various cell lines treated with 10 μg/ml cisplatin and various concentrations of α-TOS, staurosporine, or etoposide for 17 h. (A) Tet21N neuroblastoma cells, (B) HCT116 colorectal carcinoma, and (C) U1810 non-small cell lung cancer cells. (D) Tet21N cells treated with 40 nM staurosporine alone, or in combination with 30 μM or 50 μM α-TOS for 17 h. (E) Effect of co-treatment with 30 μM etoposide and various doses of α-TOS on caspase-3-like activity in Tet21N cells.
potently inhibited apoptosis induced by cisplatin, a DNA-damaging agent, or staurosporine, a broad-spectrum protein-kinase inhibitor. Protection against cisplatin was observed at concentrations of α-TOS below 40 μM, when this compound was supposedly cleaved by cellular esterases. Of the two cleavage products of α-TOS, α-tocopherol and succinate, the most protective was succinate. The protective effect of succinate can be attributed to its unique capacity to “monopolize” the respiratory chain of mitochondria, as reported by Hans Krebs (Krebs et al., 1961). One of the consequences of this monopolization is an even greater reduction of mitochondrial pyridine nucleotides than that achieved by Complex I substrates (Chance and Hollunger, 1961). The redox state of mitochondrial pyridine nucleotides is involved in the regulation of various processes, such as the induction of mitochondrial permeability transition (MPT) (Costantini et al., 1996) and the detoxification of hydrogen peroxide and organic hydroperoxides via the glutathione-dependent defense system (Sies and Summer, 1975). Oxidation of succinate allows isolated mitochondria to accumulate 3–4 times more Ca 2 + before the onset of MPT compared to mitochondria oxidizing NAD-dependent substrates (Kondrashova et al., 1982), and makes them resistant to oxidative stress (Bindoli et al., 1982). In the presence, as well as in the absence, of the oxidant t-butylhydroperoxide, mitochondria retained more Ca 2 + with succinate than with beta-hydroxybutyrate as the respiratory substrate (Gogvadze et al., 1996). Such stabilization of mitochondria would make them more resistant towards OMM permeabilization and the release of pro-apoptotic proteins from the intermembrane space. Hence, upon stimulation of apoptosis, mitochondria utilizing succinate should show better retention of intermembrane space proteins and thus apoptosis will be attenuated.
The precise mechanisms of succinate-mediated protection require further investigation. The lack of protection by low concentrations of α-TOS in the case of etoposide can be explained by the ability of etoposide to affect not only topoisomerase II, but also mitochondria (Robertson et al., 2000), causing the induction of oxidative stress and impairment of mitochondrial function (Pham and Hedley, 2001). Thus, the amount of succinate formed upon cleavage of α-TOS was not sufficient to protect against etoposide, while exogenous succinate successfully prevented apoptosis. In conclusion, a primary strategic problem in cancer therapy is how to selectively activate apoptosis in transformed cells. The ability of α-TOS to induce tumor cell death by targeting mitochondria is an important part of the anticancer strategy. However, co-treatment with conventional antitumor drugs should be carried out with caution. The data presented here highlight the role of succinate as a potential physiological modulator of the mitochondrial response to treatment. The ability of succinate to “monopolize” the respiratory chain of mitochondria might be important for the modulation of cellular physiology, including apoptotic pathways.
Acknowledgments The authors thank Prof. Marie Arsenian-Henriksson (Karolinska Institutet, Stockholm) for providing the Tet21N cell line used in the study. The work was supported by grants from the Swedish Research Council; the Swedish and the Stockholm Cancer Societies; the Swedish Childhood Cancer Foundation and the Russian Ministry of Higher Education and Science (11.G34.31.0006).
Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001
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Fig. 5. Modulation of apoptotic response to cisplatin by trolox and succinate (A). Tet21N cells were pre-incubated for 3 h with 50 μM trolox, 30 μM α-TOS, or 20 mM succinate and treated with 10 μg/ml cisplatin for 17 h. Effect of α-TOS and succinate on cisplatin- (B) or etoposide-induced (C) release of cytochrome c and cleavage of PARP. Cells were pre-incubated for 3 h with 30 μM α-TOS or 20 mM succinate and treated with cisplatin (10 μg/ml) or 30, and 50 μM etoposide for 17 h. GAPDH serves as a loading control.
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Please cite this article as: Kruspig, B., et al., Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis, Mitochondrion (2012), http://dx.doi.org/10.1016/j.mito.2012.08.001