- Email: [email protected]
Hydrogen peroxide induces apoptosis through the mitochondrial pathway in rat Schwann cells
Neuroscience Letters 485 (2010) 60–64
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Hydrogen peroxide induces apoptosis through the mitochondrial pathway in rat Schwann cells Xinjing Luo a,b , Baoguo Chen c , Rui Zheng c , Peng Lin a , Jicheng Li b,∗ , Haixiao Chen a,∗∗ a b c
Department of Orthopaedics of Taizhou Hospital, Linhai 317000, Zhejiang, China Institute of Cell Biology of Zhejiang University, Hangzhou 310058, Zhejiang, China Department of Centralab of Taizhou Hospital, Linhai 317000, Zhejiang, China
a r t i c l e
i n f o
Article history: Received 27 June 2010 Received in revised form 11 August 2010 Accepted 23 August 2010 Keywords: Schwann cells Hydrogen peroxide Apoptosis Cytochrome c Bax Bcl-2
a b s t r a c t Oxidative stress is one of the several mechanisms that induces apoptosis in cells. It has been shown that hydrogen peroxide (H2 O2 ) induces apoptosis in several kinds of cells; however, the role of H2 O2 in the apoptosis of Schwann cells (SCs) is currently unclear. The objective of this study was to determine whether H2 O2 is capable of inducing apoptosis in SCs and whether or not such an effect is associated with the activation of mitochondrial pathway. We demonstrated that H2 O2 induces apoptosis in SCs, and is associated with increased release of cytochrome c from mitochondria and the activation of caspase-3 and -9 by up-regulation of Bax and down-regulation of Bcl-2. These results suggest a potential role for H2 O2 in SC injury by triggering apoptosis via the mitochondrial pathway under oxidative stress. © 2010 Elsevier Ireland Ltd. All rights reserved.
Apoptosis is an active mode of cell death which is encountered among normal cells, as well as tumor cells, in physiologic and pathologic situations and is distinguished from passive cell death (necrosis) [18]. The signaling events leading to apoptosis can be divided into two distinct pathways involving either mitochondria or death receptors. In the mitochondria pathway, death signals lead to changes in mitochondrial membrane permeability and the subsequent release of pro-apoptotic factors including cytochrome c from the mitochondria. Once in the cytoplasm, cytochrome c catalyzes the oligomerization of apoptotic protease activating factor-1 (Apaf-1) [8]. This promotes the activation of procaspase-9, which then initiates a caspase cascade involving the downstream executioner, procaspase-3, which in turn activates a DNase, termed caspase activated DNAse (CAD) [15,23]. In the death receptor pathway, apoptosis is triggered by cell-surface death receptors that contain death domains, such as Fas and TNF receptor. These death domains recruit adaptors and induce the activation of initiator caspase-8, followed by cleavage of downstream effector caspases and various substrates. Schwann cells (SCs), an important component of the peripheral nervous system, ensheath the axon and play an important role in axonal growth and regeneration, myelinization, and normal electrophysiological conductivity [19]. Damage to SCs is likely to induce
∗ Corresponding author. Tel.: +86 571 88208088; fax: +86 571 88208088. ∗∗ Corresponding author. Tel.: +86 576 5199666; fax: +86 576 5199669. E-mail addresses: [email protected] (J. Li), [email protected] (H. Chen). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.08.063
faulty maintenance of neurons or demyelination, and slow conductivity of the axon [10,19]. Thus, the derangement of SCs may play a role in the development of neuropathy [9]. Reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide anion, and hydroxyl radicals, are important mediators of apoptosis. Increased ROS levels have been shown to emanate from apoptotic cells, and antioxidants can block apoptosis in a variety of systems. The production of ROS has been involved in different pathologies of the peripheral nervous system. For instance, increased ROS levels have been detected in SCs in ischaemia-reperfusion injury to peripheral nerves and diabetic neuropathy [7,27]. H2 O2 is known to modulate a variety of cell functions. It has been shown that H2 O2 induces apoptosis in several kinds of cells, including neuronal cells [2,23]. However, whether H2 O2 has a potential to induce the apoptosis of SCs is currently unclear. Here we examined whether H2 O2 was capable of inducing apoptosis of SCs to determine the possible involvement of NOS in the development of neurologic complications after oxidative stress. Further, we investigated the effect of H2 O2 on the mitochondrial pathway to clarify the intracellular molecular mechanisms responsible for the H2 O2 -induced apoptosis in the SCs. A rat SC line (RSC96) was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). SC cultures were >99% pure as assessed by immunostaining with anti-S100. Cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 , until cells reached 80% confluence.
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
Cells were then replaced with H2 O2 -containing DMEM with 1% FBS. Cells were collected at the preplanned time. H2 O2 were diluted in phosphate-buffered saline (PBS) and further prepared to various concentrations in DMEM with 1% FBS. At the appropriate time after H2 O2 treatment, cells were then harvested, washed, and suspended in binding buffer containing annexin V-FITC and propidium iodide (PI) at the concentrations specified by manufacturer (MultiScience Biotech, Hangzhou, China). Cells were incubated in the dark at room temperature for 5 min. Cells were analyzed by FACScan flow cytometry (Becton Dickinson, San Jose, CA). The percentage of cell membrane phosphatidylserine residues that became labeled with annexin V was used as a measurement of apoptosis. Apoptotic cells were visualized by the TUNEL technique using an In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany), according to the manufacture’ instructions. At least 500 cells per slide were assessed for each experimental condition. At the appropriate time after H2 O2 treatment, cells were washed with PBS and lysed in radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors. After determination of the protein concentration using a bicinchoninic acid (BCA) assay, equal amounts of proteins (30–40 g) were electrophored on a SDSPAGE gel and transferred to a PVDF membrane. The membrane was blocked with 2% albumin, and then incubated with correspond-
61
ing primary antibodies. The immune complexes were visualized with a HRP-conjugated secondary antibody and a DAB assay kit (Boster Biological Technology, China), and the bands of protein were scanned and quantitated with the Gel-pro Analyzer software (Media Cybernetics). At the appropriate time after H2 O2 treatment, cells were washed and lysed in a lysis buffer (10 mM HEPES [pH 7.4], 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 1 mM PMSF, 10 g/ml pepstatin A, 10 g/ml aprotinin, and 20 g/ml leupeptin). Caspase fluorescent assay kits specific for caspase-3 and -9 (Biovision, Palo Alto, CA) were used by measuring the cleavage of a synthetic fluorescent substrate. Absorbance was read with a microplate reader at 405 nm, and the fold increase in caspase activity over those of the control was determined. At the appropriate time after H2 O2 treatment, cells were harvested, and washed with cold PBS. The methods used for the isolation of subcellular fractions were essentially the same as previously described [23]. Briefly, the cell pellet was incubated for 15 min in ice-cold extraction buffer A containing 20 mM HEPES–KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM Na–EDTA, 1 mM Na–EGTA, 1 mM DTT, and 250 mM sucrose, and cell extract centrifuged at 1000 × g for 10 min to pellet nuclei. The supernatant was collected and centrifuged at 12,000 × g for 30 min to pellet the mitochondrial. The resulting supernatant was termed as the cytosolic fraction. The
Fig. 1. Annexin V/PI and TUNEL analysis of the percentage of apoptosis induced by H2 O2 in Schwann cells. (A) Schwann cells were treated with H2 O2 for the indicated times, followed by annexin V/PI assay. Representative histograms of annexin V-FITC and PI-stained. Early apoptotic cells are annexin V-positive and PI-negative (right lower quadrant). Non-apoptotic cells are annexin/PI-negative (left lower quadrant). Late apoptotic are annexin V- and PI-positive (right upper quadrant). (a) Normal cells; (b) cells exposed to 0.8 mM H2 O2 for 16 h; (c) the graph shows the proportion of cells that are annexin V-positive. Results from three independent experiments are shown as the means ± SD. *P < 0.05 vs control group. (B) Schwann cells were treated with H2 O2 for the indicated times, followed by TUNEL analysis. TUNEL-positive cells were very darkly stained with a condense nucleus as visualized by light microscopy. (a) Normal cells; (b) 0.8 mM H2 O2 for 16 h; (c) the graph shows the proportion of cells that are TUNEL-positive. Results from three independent experiments are shown as the means ± SD. *P < 0.05 vs control group.
62
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
Fig. 2. The effect of H2 O2 on the release of cytochrome c from mitochondria. Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. Cytosolic and mitochondrial extracts were immunoblotted with anti-cytochrome c. AntiHsp60 and anti--actin were used as normalization controls for mitochondrial (Mito) proteins and cytoplasmic (Cyto) proteins, respectively. Shown in the upper panels are representative blots, and in the lower panel are presented the means ± SD of three independent experiments. *P < 0.05 vs cytoplasm of control group; # P < 0.05 vs mitochondrial of control group.
pellet was lysed, and cytochrome c in cytosolic and mitochondrial fractions was determined by Western blotting. Data are expressed as the mean ± SD. Statistical comparison between the experimental group and the control was performed using an unpaired 2-tailed Student’s t-test. P values <0.05 were considered significant. Apoptosis was measured using annexin V binding and TUNEL assay. As shown in Fig. 1A, the number of annexin V-stained cells increased after treatment with H2 O2 in a dose- and timedependent manner. The highest rate of SC apoptosis occurred after 16 h treatment with 0.8 mM of H2 O2 , which induced apoptosis in approximately 50% of SCs. Similarly, TUNEL analysis also showed that H2 O2 induces SC apoptosis. Exposure of SCs to H2 O2 induced an increase of TUNEL-positive cells in dose- and time-dependent manner (Fig. 1B). Cytochrome c release from mitochondria is a critical step in the apoptotic cascade, as this activates downstream caspases. To examine whether H2 O2 -induced apoptosis in SCs was associated with the release of cytochrome c from mitochondrial, the levels of cytochrome c in both the cytosolic and mitochondrial fractions were analyzed by Western blotting. As shown in Fig. 2, there was a significant increase in cytochrome c in the cytosol after 1–3 h treatment with H2 O2 . Simultaneously, there was a decrease in cytochrome c in the mitochondrial fraction. Caspase activation by cytochrome c is believed to be a key event during apoptosis. To confirm whether or not caspases are activated after cytochrome c release, we analyzed the changes in caspases-3 and -9 activities in SCs after H2 O2 treatment. As shown in Fig. 3A, a time-dependent increase in activity of caspase-3 and -9 was observed in H2 O2 -treated cells. There was a significant increase
Fig. 3. The effect of H2 O2 on activation of caspase-3 and -9 in Schwann cells. (A) Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. Cytosolic extracts were assayed for protease activity of caspase-3 and -9 using caspase fluorescent assay kits. Data shown are the means ± SD of three independent experiments. *P < 0.05 vs caspase-3 of control group; # P < 0.05 vs caspase-9 of control group. (B) Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. The cell lysates were immunoblotted with anti-cleavaged caspase-9 or anti-cleavaged caspase-3. Antibody against -actin served as controls. Shown in the upper panels are representative blots, and in the lower panel are presented the means ± SD of three independent experiments. *P < 0.05 vs caspase-3 of control group; # P < 0.05 vs caspase-9 of control group.
in caspase-3 and -9 activities after 3 h treatment with H2 O2 . Similarly, immunoblot analysis with anti-cleavaged caspase-3 and -9 also showed that H2 O2 induces caspase-3 and -9 activation. Exposure of SCs to H2 O2 induced an increased level of caspase-3 and -9 cleavage (Fig. 3B). The release of cytochrome c is regulated by proteins of the Bcl-2 family, which may either inhibit (e.g., the anti-apoptotic proteins Bcl-2) or promote (e.g., the pro-apoptotic protein Bax) the process [26]. To determine the effect of H2 O2 on pro-apoptotic and antiapoptotic genes, we monitored the protein expression of Bax and Bcl-2 by immunoblot analysis. As shown in Fig. 4, H2 O2 caused an increase in Bax expression in a time-dependent manner, with maximum activation occurring at 12 h. This was accompanied by a simultaneous decrease in anti-apoptotic Bcl-2. Accumulating evidence has shown that oxidative stress plays a role in generating nerve injury during various pathologic settings; however, the precise mechanism by which ROS induces apoptotic cell death in the peripheral nerve remains unclear. In this regard, our present finding that H2 O2 -induced apoptosis in rat SCs might give novel insight into this mechanism since H2 O2 is markedly accelerated under neuropathy. In addition, we found that this phenomenon was closely related to the activation of the mitochondria pathway. ROS are important mediators of physical and chemical stresses. The exposure to ROS induces oxidation of proteins, lipids and nucleic acids. The oxidation of amino acid side chains results in protein–protein cross linkage, protein fragmentation, and height-
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
Fig. 4. The effect of H2 O2 on the level of expression of Bax and Bcl-2 in Schwann cells. Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. The cell lysates were immunoblotted with anti-Bax or anti-Bcl-2 antibody. Antibody against -actin served as the control. Shown in the upper panels are representative blots, and in the lower panel are presented the means ± SD of three independent experiments. *P < 0.05 vs Bax of control group; # P < 0.05 vs Bcl-2 of control group.
ened protein carbonyl levels causes toxicity to cells and induces cell death [22]. ROS have been implicated as mediators of apoptosis. The central role for oxidative stress in apoptosis is strongly supported by the ability of various cellular antioxidants to block apoptosis, and targeted therapies aimed at generating ROS might prove effective. Oxidative stress, characterized by overwhelming ROS, is indispensable for the development and progression of peripheral neuropathy because of the high content of phospholipids and relatively insufficient free-radical defense of peripheral nerves [25]. Oxidative stress is also one of the several mechanisms that induces apoptosis in nerve cells. In neuronal cells, ROS results in membrane lipid peroxidation, nitration of proteins, and degradation of DNA, all of which are associated with the course of apoptosis [19,23]. Increase production of ROS has been implicated in different pathologies of many neurologic disorders and brain dysfunction [2]. SCs are also the target of various oxidative stress-mediated processes [5,7,22]. A study on diabetic neuropathy showed that oxidative stress induced SC apoptosis [27]. However, involvement of ROS in the apoptosis of SCs remains unclear. Our results showed that H2 O2 could induce apoptosis of SCs in a dose- and timedependent manner (Fig. 1). Our findings indicate that H2 O2 could be an apoptotic trigger in SCs. Mitochondrial are though to play a central role in the activation of apoptosis induced by different stimuli. Mitochondria function as sentinels that receive death signals and commit cells to apoptosis by releasing cytochrome c [12]. Cytochrome c is a pro-apoptotic protein that is normally located in the mitochondria and proteolytically processed and released during apoptosis. On released into the cytosol, cytochrome c binds to Apaf, thus forming a complex, referred to as an apoptosome, which recruits and activates procaspase-9 and -3 [3,11,13], and ultimately induces nuclear DNA condensation and fragmentation. It is well known that mitochon-
63
drial are both a target and a source of ROS [21]. Accumulating evidence has shown that the production of ROS triggers the release of cytochrome c from mitochondria, intracellular Ca2+ elevation, and caspase-3 activation, all of which lead to apoptosis in neuronal cells [2]. Herein we showed that H2 O2 induced cytochrome c release from the mitochondria in SCs (Fig. 2). We also demonstrated that H2 O2 induced increased activation of caspase-3 and -9 in SCs; the increase in caspase-related proteolysis shown in Fig. 3A is supported by the increase in immunoreactive activated caspase3 and -9 shown in Fig. 3B. Taken together, our results suggest that the mitochondria pathway plays a pivotal role in H2 O2 -induced apoptosis of SCs. The cellular commitment to apoptosis is regulated by the Bcl-2 family of proteins, which includes death agonists (e.g., Bax, Bak, and Bad) and antagonists (e.g., Bcl-2 and Bcl-xL) of apoptosis [16,24]. Among these proteins, Bax and Bcl-2 play a key role in regulating apoptosis. The balance between pro-apoptotic proteins, such as Bax, and anti-apoptotic protein, such as Bcl-2, is believed to be the critical factor regulating apoptosis [4,6]. Bax and Bcl-2 are mitochondrial proteins, which have been shown to be associated with regulating mitochondrial membrane permeability [1]. Bax exerts its pro-apoptotic activity by translocating from the cytoplasm to the mitochondria, and inducing cytochrome c release from isolated mitochondria, whereas Bcl-2 exerts its anti-apoptotic activity, at least in part, by inhibiting the translocation of Bax to the mitochondria [14,17]. It has been shown that ROS induces apoptotic cell death in HeLa cells by downregulating Bcl-2 levels in SCs, and promoting the translocation of Bax from the cytosol to the mitochondria, leading to release of cytochrome c from mitochondria [20,23]. Our results also showed an increase in the expression of Bax and a simultaneous decrease in Bcl-2 in SCs in response to H2 O2 (Fig. 4). Therefore, it appears that the apoptotic effects of H2 O2 in SCs are correlated to changes in the Bcl-2 family of proteins. In conclusion, this study demonstrated that H2 O2 -induced apoptosis in SCs, which is mediated by the mitochondrial pathway. These results strongly suggest that the cytotoxicity of ROS on SCs may be associated with potential neurologic complications after oxidative stress. However, because these are in vitro experiments, we were unable to simply extrapolate our ex vivo data to in vivo animal experiments. Further in vivo studies are required to assess ROS involvement in H2 O2 -treated peripheral nerves. Acknowledgements This work was supported by Zhejiang Provincial Science and Technology Planning Project of China (No 2004C33047), Zhejiang Provincial Medical Scientific Research Foundation of China (No 2010SSA010), Zhejiang Provincial Postdoctoral Science Foundation of China, Zhejiang Provincial Natural Science Foundation of China (No Y2090233), the National Natural Science Foundation of China (NO 30950019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.08.063. References [1] J.M. Adams, S. Cory, The Bcl-2 protein family: arbiters of cell survival, Science 281 (1998) 1322–1326. [2] L. Annunziato, S. Amoroso, A. Pannaccione, M. Cataldi, G. Pignataro, A. D’Alessio, R. Sirabella, A. Secondo, L. Sibaud, G.F. Di Renzo, Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions, Toxicol. Lett. 139 (2003) 125–133. [3] J. Cai, J. Yang, D.P. Jones, Mitochondrial control of apoptosis: the role of cytochrome c, Biochim. Biophys. Acta 1366 (1998) 139–149.
64
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
[4] A. Castorina, A. Tiralongo, S. Giunta, M.L. Carnazza, G. Rasi, V. D’Agata, PACAP and VIP prevent apoptosis in schwannoma cells, Brain Res. 1241 (2008) 29–35. [5] M. Fukunaga, S. Miyata, B.F. Liu, H. Miyazaki, Y. Hirota, S. Higo, Y. Hamada, S. Ueyama, M. Kasuga, Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells, Biochem. Biophys. Res. Commun. 320 (2004) 689–695. [6] Y. Ge, S.M. Belcher, D.R. Pierce, K.E. Light, Altered expression of Bcl2, Bad and Bax mRNA occurs in the rat cerebellum within hours after ethanol exposure on postnatal day 4 but not on postnatal day 9, Brain Res. Mol. Brain Res. 129 (2004) 124–134. [7] H. Iida, A.M. Schmeichel, Y. Wang, J.D. Schmelzer, P.A. Low, Schwann cell is a target in ischemia-reperfusion injury to peripheral nerve, Muscle Nerve 30 (2004) 761–766. [8] B. Jiang, W. Xiao, Y. Shi, M. Liu, X. Xiao, Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells, Cell Stress Chaperones 10 (2005) 252–262. [9] K. Jirsova, V. Mandys, W.H. Gispen, P.R. Bar, Cisplatin-induced apoptosis in cultures of human Schwann cells, Neurosci. Lett. 392 (2006) 22–26. [10] M.W. Kalichman, Physiologic mechanisms by which local anesthetics may cause injury to nerve and spinal cord, Reg. Anesth. 18 (1993) 448–452. [11] P. Li, D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, X. Wang, Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade, Cell 91 (1997) 479–489. [12] X. Liu, C.N. Kim, J. Yang, R. Jemmerson, X. Wang, Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c, Cell 86 (1996) 147–157. [13] B. Mignotte, J.L. Vayssiere, Mitochondria and apoptosis, Eur. J. Biochem. 252 (1998) 1–15. [14] K.M. Murphy, V. Ranganathan, M.L. Farnsworth, M. Kavallaris, R.B. Lock, Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells, Cell Death Differ. 7 (2000) 102–111. [15] S. Nagata, Apoptotic DNA fragmentation, Exp. Cell Res. 256 (2000) 12–18. [16] K. Newton, A. Strasser, The Bcl-2 family and cell death regulation, Curr. Opin. Genet. Dev. 8 (1998) 68–75.
[17] M. Nomura, S. Shimizu, T. Ito, M. Narita, H. Matsuda, Y. Tsujimoto, Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2, Cancer Res. 59 (1999) 5542–5548. [18] C. Nosseri, S. Coppola, L. Ghibelli, Possible involvement of poly(ADP-ribosyl) polymerase in triggering stress-induced apoptosis, Exp. Cell Res. 212 (1994) 367–373. [19] C.J. Park, S.A. Park, T.G. Yoon, S.J. Lee, K.W. Yum, H.J. Kim, Bupivacaine induces apoptosis via ROS in the Schwann cell line, J. Dent. Res. 84 (2005) 852– 857. [20] J.C. Reed, Double identity for proteins of the Bcl-2 family, Nature 387 (1997) 773–776. [21] C. Richter, M. Schweizer, A. Cossarizza, C. Franceschi, Control of apoptosis by the cellular ATP level, FEBS Lett. 378 (1996) 107–110. [22] G. Shokouhi, R.S. Tubbs, M.M. Shoja, L. Roshangar, M. Mesgari, A. Ghorbanihaghjo, N. Ahmadi, F. Sheikhzadeh, J.S. Rad, The effects of aerobic exercise training on the age-related lipid peroxidation, Schwann cell apoptosis and ultrastructural changes in the sciatic nerve of rats, Life Sci. 82 (2008) 840– 846. [23] M. Singh, H. Sharma, N. Singh, Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway, Mitochondrion 7 (2007) 367– 373. [24] M. Soilu-Hanninen, P. Ekert, T. Bucci, D. Syroid, P.F. Bartlett, T.J. Kilpatrick, Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a Bcl-2-independent pathway, J. Neurosci. 19 (1999) 4828–4838. [25] M.J. Stevens, I. Obrosova, X. Cao, C. Van Huysen, D.A. Greene, Effects of DL-alphalipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy, Diabetes 49 (2000) 1006–1015. [26] A. Troyano, P. Sancho, C. Fernandez, E. de Blas, P. Bernardi, P. Aller, The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells, Cell Death Differ. 10 (2003) 889–898. [27] A.M. Vincent, M. Brownlee, J.W. Russell, Oxidative stress and programmed cell death in diabetic neuropathy, Ann. N. Y. Acad. Sci. 959 (2002) 368–383.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Hydrogen peroxide induces apoptosis through the mitochondrial pathway in rat Schwann cells Xinjing Luo a,b , Baoguo Chen c , Rui Zheng c , Peng Lin a , Jicheng Li b,∗ , Haixiao Chen a,∗∗ a b c
Department of Orthopaedics of Taizhou Hospital, Linhai 317000, Zhejiang, China Institute of Cell Biology of Zhejiang University, Hangzhou 310058, Zhejiang, China Department of Centralab of Taizhou Hospital, Linhai 317000, Zhejiang, China
a r t i c l e
i n f o
Article history: Received 27 June 2010 Received in revised form 11 August 2010 Accepted 23 August 2010 Keywords: Schwann cells Hydrogen peroxide Apoptosis Cytochrome c Bax Bcl-2
a b s t r a c t Oxidative stress is one of the several mechanisms that induces apoptosis in cells. It has been shown that hydrogen peroxide (H2 O2 ) induces apoptosis in several kinds of cells; however, the role of H2 O2 in the apoptosis of Schwann cells (SCs) is currently unclear. The objective of this study was to determine whether H2 O2 is capable of inducing apoptosis in SCs and whether or not such an effect is associated with the activation of mitochondrial pathway. We demonstrated that H2 O2 induces apoptosis in SCs, and is associated with increased release of cytochrome c from mitochondria and the activation of caspase-3 and -9 by up-regulation of Bax and down-regulation of Bcl-2. These results suggest a potential role for H2 O2 in SC injury by triggering apoptosis via the mitochondrial pathway under oxidative stress. © 2010 Elsevier Ireland Ltd. All rights reserved.
Apoptosis is an active mode of cell death which is encountered among normal cells, as well as tumor cells, in physiologic and pathologic situations and is distinguished from passive cell death (necrosis) [18]. The signaling events leading to apoptosis can be divided into two distinct pathways involving either mitochondria or death receptors. In the mitochondria pathway, death signals lead to changes in mitochondrial membrane permeability and the subsequent release of pro-apoptotic factors including cytochrome c from the mitochondria. Once in the cytoplasm, cytochrome c catalyzes the oligomerization of apoptotic protease activating factor-1 (Apaf-1) [8]. This promotes the activation of procaspase-9, which then initiates a caspase cascade involving the downstream executioner, procaspase-3, which in turn activates a DNase, termed caspase activated DNAse (CAD) [15,23]. In the death receptor pathway, apoptosis is triggered by cell-surface death receptors that contain death domains, such as Fas and TNF receptor. These death domains recruit adaptors and induce the activation of initiator caspase-8, followed by cleavage of downstream effector caspases and various substrates. Schwann cells (SCs), an important component of the peripheral nervous system, ensheath the axon and play an important role in axonal growth and regeneration, myelinization, and normal electrophysiological conductivity [19]. Damage to SCs is likely to induce
∗ Corresponding author. Tel.: +86 571 88208088; fax: +86 571 88208088. ∗∗ Corresponding author. Tel.: +86 576 5199666; fax: +86 576 5199669. E-mail addresses: [email protected] (J. Li), [email protected] (H. Chen). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.08.063
faulty maintenance of neurons or demyelination, and slow conductivity of the axon [10,19]. Thus, the derangement of SCs may play a role in the development of neuropathy [9]. Reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide anion, and hydroxyl radicals, are important mediators of apoptosis. Increased ROS levels have been shown to emanate from apoptotic cells, and antioxidants can block apoptosis in a variety of systems. The production of ROS has been involved in different pathologies of the peripheral nervous system. For instance, increased ROS levels have been detected in SCs in ischaemia-reperfusion injury to peripheral nerves and diabetic neuropathy [7,27]. H2 O2 is known to modulate a variety of cell functions. It has been shown that H2 O2 induces apoptosis in several kinds of cells, including neuronal cells [2,23]. However, whether H2 O2 has a potential to induce the apoptosis of SCs is currently unclear. Here we examined whether H2 O2 was capable of inducing apoptosis of SCs to determine the possible involvement of NOS in the development of neurologic complications after oxidative stress. Further, we investigated the effect of H2 O2 on the mitochondrial pathway to clarify the intracellular molecular mechanisms responsible for the H2 O2 -induced apoptosis in the SCs. A rat SC line (RSC96) was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). SC cultures were >99% pure as assessed by immunostaining with anti-S100. Cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 , until cells reached 80% confluence.
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
Cells were then replaced with H2 O2 -containing DMEM with 1% FBS. Cells were collected at the preplanned time. H2 O2 were diluted in phosphate-buffered saline (PBS) and further prepared to various concentrations in DMEM with 1% FBS. At the appropriate time after H2 O2 treatment, cells were then harvested, washed, and suspended in binding buffer containing annexin V-FITC and propidium iodide (PI) at the concentrations specified by manufacturer (MultiScience Biotech, Hangzhou, China). Cells were incubated in the dark at room temperature for 5 min. Cells were analyzed by FACScan flow cytometry (Becton Dickinson, San Jose, CA). The percentage of cell membrane phosphatidylserine residues that became labeled with annexin V was used as a measurement of apoptosis. Apoptotic cells were visualized by the TUNEL technique using an In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany), according to the manufacture’ instructions. At least 500 cells per slide were assessed for each experimental condition. At the appropriate time after H2 O2 treatment, cells were washed with PBS and lysed in radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors. After determination of the protein concentration using a bicinchoninic acid (BCA) assay, equal amounts of proteins (30–40 g) were electrophored on a SDSPAGE gel and transferred to a PVDF membrane. The membrane was blocked with 2% albumin, and then incubated with correspond-
61
ing primary antibodies. The immune complexes were visualized with a HRP-conjugated secondary antibody and a DAB assay kit (Boster Biological Technology, China), and the bands of protein were scanned and quantitated with the Gel-pro Analyzer software (Media Cybernetics). At the appropriate time after H2 O2 treatment, cells were washed and lysed in a lysis buffer (10 mM HEPES [pH 7.4], 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 1 mM PMSF, 10 g/ml pepstatin A, 10 g/ml aprotinin, and 20 g/ml leupeptin). Caspase fluorescent assay kits specific for caspase-3 and -9 (Biovision, Palo Alto, CA) were used by measuring the cleavage of a synthetic fluorescent substrate. Absorbance was read with a microplate reader at 405 nm, and the fold increase in caspase activity over those of the control was determined. At the appropriate time after H2 O2 treatment, cells were harvested, and washed with cold PBS. The methods used for the isolation of subcellular fractions were essentially the same as previously described [23]. Briefly, the cell pellet was incubated for 15 min in ice-cold extraction buffer A containing 20 mM HEPES–KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM Na–EDTA, 1 mM Na–EGTA, 1 mM DTT, and 250 mM sucrose, and cell extract centrifuged at 1000 × g for 10 min to pellet nuclei. The supernatant was collected and centrifuged at 12,000 × g for 30 min to pellet the mitochondrial. The resulting supernatant was termed as the cytosolic fraction. The
Fig. 1. Annexin V/PI and TUNEL analysis of the percentage of apoptosis induced by H2 O2 in Schwann cells. (A) Schwann cells were treated with H2 O2 for the indicated times, followed by annexin V/PI assay. Representative histograms of annexin V-FITC and PI-stained. Early apoptotic cells are annexin V-positive and PI-negative (right lower quadrant). Non-apoptotic cells are annexin/PI-negative (left lower quadrant). Late apoptotic are annexin V- and PI-positive (right upper quadrant). (a) Normal cells; (b) cells exposed to 0.8 mM H2 O2 for 16 h; (c) the graph shows the proportion of cells that are annexin V-positive. Results from three independent experiments are shown as the means ± SD. *P < 0.05 vs control group. (B) Schwann cells were treated with H2 O2 for the indicated times, followed by TUNEL analysis. TUNEL-positive cells were very darkly stained with a condense nucleus as visualized by light microscopy. (a) Normal cells; (b) 0.8 mM H2 O2 for 16 h; (c) the graph shows the proportion of cells that are TUNEL-positive. Results from three independent experiments are shown as the means ± SD. *P < 0.05 vs control group.
62
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
Fig. 2. The effect of H2 O2 on the release of cytochrome c from mitochondria. Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. Cytosolic and mitochondrial extracts were immunoblotted with anti-cytochrome c. AntiHsp60 and anti--actin were used as normalization controls for mitochondrial (Mito) proteins and cytoplasmic (Cyto) proteins, respectively. Shown in the upper panels are representative blots, and in the lower panel are presented the means ± SD of three independent experiments. *P < 0.05 vs cytoplasm of control group; # P < 0.05 vs mitochondrial of control group.
pellet was lysed, and cytochrome c in cytosolic and mitochondrial fractions was determined by Western blotting. Data are expressed as the mean ± SD. Statistical comparison between the experimental group and the control was performed using an unpaired 2-tailed Student’s t-test. P values <0.05 were considered significant. Apoptosis was measured using annexin V binding and TUNEL assay. As shown in Fig. 1A, the number of annexin V-stained cells increased after treatment with H2 O2 in a dose- and timedependent manner. The highest rate of SC apoptosis occurred after 16 h treatment with 0.8 mM of H2 O2 , which induced apoptosis in approximately 50% of SCs. Similarly, TUNEL analysis also showed that H2 O2 induces SC apoptosis. Exposure of SCs to H2 O2 induced an increase of TUNEL-positive cells in dose- and time-dependent manner (Fig. 1B). Cytochrome c release from mitochondria is a critical step in the apoptotic cascade, as this activates downstream caspases. To examine whether H2 O2 -induced apoptosis in SCs was associated with the release of cytochrome c from mitochondrial, the levels of cytochrome c in both the cytosolic and mitochondrial fractions were analyzed by Western blotting. As shown in Fig. 2, there was a significant increase in cytochrome c in the cytosol after 1–3 h treatment with H2 O2 . Simultaneously, there was a decrease in cytochrome c in the mitochondrial fraction. Caspase activation by cytochrome c is believed to be a key event during apoptosis. To confirm whether or not caspases are activated after cytochrome c release, we analyzed the changes in caspases-3 and -9 activities in SCs after H2 O2 treatment. As shown in Fig. 3A, a time-dependent increase in activity of caspase-3 and -9 was observed in H2 O2 -treated cells. There was a significant increase
Fig. 3. The effect of H2 O2 on activation of caspase-3 and -9 in Schwann cells. (A) Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. Cytosolic extracts were assayed for protease activity of caspase-3 and -9 using caspase fluorescent assay kits. Data shown are the means ± SD of three independent experiments. *P < 0.05 vs caspase-3 of control group; # P < 0.05 vs caspase-9 of control group. (B) Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. The cell lysates were immunoblotted with anti-cleavaged caspase-9 or anti-cleavaged caspase-3. Antibody against -actin served as controls. Shown in the upper panels are representative blots, and in the lower panel are presented the means ± SD of three independent experiments. *P < 0.05 vs caspase-3 of control group; # P < 0.05 vs caspase-9 of control group.
in caspase-3 and -9 activities after 3 h treatment with H2 O2 . Similarly, immunoblot analysis with anti-cleavaged caspase-3 and -9 also showed that H2 O2 induces caspase-3 and -9 activation. Exposure of SCs to H2 O2 induced an increased level of caspase-3 and -9 cleavage (Fig. 3B). The release of cytochrome c is regulated by proteins of the Bcl-2 family, which may either inhibit (e.g., the anti-apoptotic proteins Bcl-2) or promote (e.g., the pro-apoptotic protein Bax) the process [26]. To determine the effect of H2 O2 on pro-apoptotic and antiapoptotic genes, we monitored the protein expression of Bax and Bcl-2 by immunoblot analysis. As shown in Fig. 4, H2 O2 caused an increase in Bax expression in a time-dependent manner, with maximum activation occurring at 12 h. This was accompanied by a simultaneous decrease in anti-apoptotic Bcl-2. Accumulating evidence has shown that oxidative stress plays a role in generating nerve injury during various pathologic settings; however, the precise mechanism by which ROS induces apoptotic cell death in the peripheral nerve remains unclear. In this regard, our present finding that H2 O2 -induced apoptosis in rat SCs might give novel insight into this mechanism since H2 O2 is markedly accelerated under neuropathy. In addition, we found that this phenomenon was closely related to the activation of the mitochondria pathway. ROS are important mediators of physical and chemical stresses. The exposure to ROS induces oxidation of proteins, lipids and nucleic acids. The oxidation of amino acid side chains results in protein–protein cross linkage, protein fragmentation, and height-
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
Fig. 4. The effect of H2 O2 on the level of expression of Bax and Bcl-2 in Schwann cells. Schwann cells were treated with H2 O2 (0.8 mM) for the indicated times. The cell lysates were immunoblotted with anti-Bax or anti-Bcl-2 antibody. Antibody against -actin served as the control. Shown in the upper panels are representative blots, and in the lower panel are presented the means ± SD of three independent experiments. *P < 0.05 vs Bax of control group; # P < 0.05 vs Bcl-2 of control group.
ened protein carbonyl levels causes toxicity to cells and induces cell death [22]. ROS have been implicated as mediators of apoptosis. The central role for oxidative stress in apoptosis is strongly supported by the ability of various cellular antioxidants to block apoptosis, and targeted therapies aimed at generating ROS might prove effective. Oxidative stress, characterized by overwhelming ROS, is indispensable for the development and progression of peripheral neuropathy because of the high content of phospholipids and relatively insufficient free-radical defense of peripheral nerves [25]. Oxidative stress is also one of the several mechanisms that induces apoptosis in nerve cells. In neuronal cells, ROS results in membrane lipid peroxidation, nitration of proteins, and degradation of DNA, all of which are associated with the course of apoptosis [19,23]. Increase production of ROS has been implicated in different pathologies of many neurologic disorders and brain dysfunction [2]. SCs are also the target of various oxidative stress-mediated processes [5,7,22]. A study on diabetic neuropathy showed that oxidative stress induced SC apoptosis [27]. However, involvement of ROS in the apoptosis of SCs remains unclear. Our results showed that H2 O2 could induce apoptosis of SCs in a dose- and timedependent manner (Fig. 1). Our findings indicate that H2 O2 could be an apoptotic trigger in SCs. Mitochondrial are though to play a central role in the activation of apoptosis induced by different stimuli. Mitochondria function as sentinels that receive death signals and commit cells to apoptosis by releasing cytochrome c [12]. Cytochrome c is a pro-apoptotic protein that is normally located in the mitochondria and proteolytically processed and released during apoptosis. On released into the cytosol, cytochrome c binds to Apaf, thus forming a complex, referred to as an apoptosome, which recruits and activates procaspase-9 and -3 [3,11,13], and ultimately induces nuclear DNA condensation and fragmentation. It is well known that mitochon-
63
drial are both a target and a source of ROS [21]. Accumulating evidence has shown that the production of ROS triggers the release of cytochrome c from mitochondria, intracellular Ca2+ elevation, and caspase-3 activation, all of which lead to apoptosis in neuronal cells [2]. Herein we showed that H2 O2 induced cytochrome c release from the mitochondria in SCs (Fig. 2). We also demonstrated that H2 O2 induced increased activation of caspase-3 and -9 in SCs; the increase in caspase-related proteolysis shown in Fig. 3A is supported by the increase in immunoreactive activated caspase3 and -9 shown in Fig. 3B. Taken together, our results suggest that the mitochondria pathway plays a pivotal role in H2 O2 -induced apoptosis of SCs. The cellular commitment to apoptosis is regulated by the Bcl-2 family of proteins, which includes death agonists (e.g., Bax, Bak, and Bad) and antagonists (e.g., Bcl-2 and Bcl-xL) of apoptosis [16,24]. Among these proteins, Bax and Bcl-2 play a key role in regulating apoptosis. The balance between pro-apoptotic proteins, such as Bax, and anti-apoptotic protein, such as Bcl-2, is believed to be the critical factor regulating apoptosis [4,6]. Bax and Bcl-2 are mitochondrial proteins, which have been shown to be associated with regulating mitochondrial membrane permeability [1]. Bax exerts its pro-apoptotic activity by translocating from the cytoplasm to the mitochondria, and inducing cytochrome c release from isolated mitochondria, whereas Bcl-2 exerts its anti-apoptotic activity, at least in part, by inhibiting the translocation of Bax to the mitochondria [14,17]. It has been shown that ROS induces apoptotic cell death in HeLa cells by downregulating Bcl-2 levels in SCs, and promoting the translocation of Bax from the cytosol to the mitochondria, leading to release of cytochrome c from mitochondria [20,23]. Our results also showed an increase in the expression of Bax and a simultaneous decrease in Bcl-2 in SCs in response to H2 O2 (Fig. 4). Therefore, it appears that the apoptotic effects of H2 O2 in SCs are correlated to changes in the Bcl-2 family of proteins. In conclusion, this study demonstrated that H2 O2 -induced apoptosis in SCs, which is mediated by the mitochondrial pathway. These results strongly suggest that the cytotoxicity of ROS on SCs may be associated with potential neurologic complications after oxidative stress. However, because these are in vitro experiments, we were unable to simply extrapolate our ex vivo data to in vivo animal experiments. Further in vivo studies are required to assess ROS involvement in H2 O2 -treated peripheral nerves. Acknowledgements This work was supported by Zhejiang Provincial Science and Technology Planning Project of China (No 2004C33047), Zhejiang Provincial Medical Scientific Research Foundation of China (No 2010SSA010), Zhejiang Provincial Postdoctoral Science Foundation of China, Zhejiang Provincial Natural Science Foundation of China (No Y2090233), the National Natural Science Foundation of China (NO 30950019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.08.063. References [1] J.M. Adams, S. Cory, The Bcl-2 protein family: arbiters of cell survival, Science 281 (1998) 1322–1326. [2] L. Annunziato, S. Amoroso, A. Pannaccione, M. Cataldi, G. Pignataro, A. D’Alessio, R. Sirabella, A. Secondo, L. Sibaud, G.F. Di Renzo, Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions, Toxicol. Lett. 139 (2003) 125–133. [3] J. Cai, J. Yang, D.P. Jones, Mitochondrial control of apoptosis: the role of cytochrome c, Biochim. Biophys. Acta 1366 (1998) 139–149.
64
X. Luo et al. / Neuroscience Letters 485 (2010) 60–64
[4] A. Castorina, A. Tiralongo, S. Giunta, M.L. Carnazza, G. Rasi, V. D’Agata, PACAP and VIP prevent apoptosis in schwannoma cells, Brain Res. 1241 (2008) 29–35. [5] M. Fukunaga, S. Miyata, B.F. Liu, H. Miyazaki, Y. Hirota, S. Higo, Y. Hamada, S. Ueyama, M. Kasuga, Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells, Biochem. Biophys. Res. Commun. 320 (2004) 689–695. [6] Y. Ge, S.M. Belcher, D.R. Pierce, K.E. Light, Altered expression of Bcl2, Bad and Bax mRNA occurs in the rat cerebellum within hours after ethanol exposure on postnatal day 4 but not on postnatal day 9, Brain Res. Mol. Brain Res. 129 (2004) 124–134. [7] H. Iida, A.M. Schmeichel, Y. Wang, J.D. Schmelzer, P.A. Low, Schwann cell is a target in ischemia-reperfusion injury to peripheral nerve, Muscle Nerve 30 (2004) 761–766. [8] B. Jiang, W. Xiao, Y. Shi, M. Liu, X. Xiao, Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells, Cell Stress Chaperones 10 (2005) 252–262. [9] K. Jirsova, V. Mandys, W.H. Gispen, P.R. Bar, Cisplatin-induced apoptosis in cultures of human Schwann cells, Neurosci. Lett. 392 (2006) 22–26. [10] M.W. Kalichman, Physiologic mechanisms by which local anesthetics may cause injury to nerve and spinal cord, Reg. Anesth. 18 (1993) 448–452. [11] P. Li, D. Nijhawan, I. Budihardjo, S.M. Srinivasula, M. Ahmad, E.S. Alnemri, X. Wang, Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade, Cell 91 (1997) 479–489. [12] X. Liu, C.N. Kim, J. Yang, R. Jemmerson, X. Wang, Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c, Cell 86 (1996) 147–157. [13] B. Mignotte, J.L. Vayssiere, Mitochondria and apoptosis, Eur. J. Biochem. 252 (1998) 1–15. [14] K.M. Murphy, V. Ranganathan, M.L. Farnsworth, M. Kavallaris, R.B. Lock, Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells, Cell Death Differ. 7 (2000) 102–111. [15] S. Nagata, Apoptotic DNA fragmentation, Exp. Cell Res. 256 (2000) 12–18. [16] K. Newton, A. Strasser, The Bcl-2 family and cell death regulation, Curr. Opin. Genet. Dev. 8 (1998) 68–75.
[17] M. Nomura, S. Shimizu, T. Ito, M. Narita, H. Matsuda, Y. Tsujimoto, Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2, Cancer Res. 59 (1999) 5542–5548. [18] C. Nosseri, S. Coppola, L. Ghibelli, Possible involvement of poly(ADP-ribosyl) polymerase in triggering stress-induced apoptosis, Exp. Cell Res. 212 (1994) 367–373. [19] C.J. Park, S.A. Park, T.G. Yoon, S.J. Lee, K.W. Yum, H.J. Kim, Bupivacaine induces apoptosis via ROS in the Schwann cell line, J. Dent. Res. 84 (2005) 852– 857. [20] J.C. Reed, Double identity for proteins of the Bcl-2 family, Nature 387 (1997) 773–776. [21] C. Richter, M. Schweizer, A. Cossarizza, C. Franceschi, Control of apoptosis by the cellular ATP level, FEBS Lett. 378 (1996) 107–110. [22] G. Shokouhi, R.S. Tubbs, M.M. Shoja, L. Roshangar, M. Mesgari, A. Ghorbanihaghjo, N. Ahmadi, F. Sheikhzadeh, J.S. Rad, The effects of aerobic exercise training on the age-related lipid peroxidation, Schwann cell apoptosis and ultrastructural changes in the sciatic nerve of rats, Life Sci. 82 (2008) 840– 846. [23] M. Singh, H. Sharma, N. Singh, Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway, Mitochondrion 7 (2007) 367– 373. [24] M. Soilu-Hanninen, P. Ekert, T. Bucci, D. Syroid, P.F. Bartlett, T.J. Kilpatrick, Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a Bcl-2-independent pathway, J. Neurosci. 19 (1999) 4828–4838. [25] M.J. Stevens, I. Obrosova, X. Cao, C. Van Huysen, D.A. Greene, Effects of DL-alphalipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy, Diabetes 49 (2000) 1006–1015. [26] A. Troyano, P. Sancho, C. Fernandez, E. de Blas, P. Bernardi, P. Aller, The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells, Cell Death Differ. 10 (2003) 889–898. [27] A.M. Vincent, M. Brownlee, J.W. Russell, Oxidative stress and programmed cell death in diabetic neuropathy, Ann. N. Y. Acad. Sci. 959 (2002) 368–383.