Experimental and Toxicologic Pathology 67 (2015) 315–322
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Evaluation of multi-neuroprotective effects of erythropoietin using cisplatin induced peripheral neurotoxicity model Nivin Sharawy a,∗ , Laila Rashed b , Magdy Fouad Youakim c a b c
Department of Physiology, Faculty of Medicine, Cairo University, Cairo, Egypt Department of Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt Department of Anatomy, Faculty of Medicine, Cairo University, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 18 September 2014 Accepted 20 February 2015 Keywords: Cisplatin Erythropoietin Oxidative Inflammatory Apoptosis
a b s t r a c t Cisplatin (CDDP) is severely neurotoxic anti-neoplastic drug that causes peripheral neuropathies with clinical signs known as chemotherapy-induced peripheral neurotoxicity. The ameliorating effects of erythropoeitin on cisplatin-induced neuropathy, which seem to be mediated by enhancing the cell resistance to side effects of cisplatin rather than by influencing the formation or repair rates of cisplatin-induced cross-links in the nuclear DNA, had been previously reported. The main objective of our study is to investigate the roles of nitro-oxidative stress, nuclear factor kappa B (NFB) gene expressions and TNF levels on the previous reported erythropoietin anti-apoptotic neuroprotective effects during cisplatin induced neurotoxicity. The present study compared the effects of erythropoietin (50 g/kg/d thrice weekly) on cisplatin (2 mg/kg/d i.p. twice weekly for 4 weeks) induced neurophysiologic changes and the associated changes in the inflammatory mediators (TNF alpha and NFKB), oxidative stress (malondialdehyde (MDA), superoxide dismutases (SOD) and glutathione) and gene expression of both neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). In addition, sciatic nerve pro-apoptotic and anti-apoptotic indicators (Bcl, Bax, Caspase 3) were measured. We found that concomitant administration of erythropoietin significantly reversed the cisplatin induced nitro-oxidative stress – with significant increases in sciatic nerve glutathione and superoxide dismutase antioxidant enzyme levels and a significant decrease in iNOS gene expression. We conclude that erythropoietin anti-apoptotic neuro-protective effects could partially contribute to observed antioxidant effects of erthropoietin. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Cisplatin (CDDP) and the other platinum-derived drugs are among the most effective antineoplastic agents, but they are severely neurotoxic. Accumulation of cisplatin in the dorsal root ganglia (DRG) results in nerve fiber axonopathy and degeneration of large myelinated axons with signs of segmental demyelinization and remyelinization (Bianchi et al., 2006; Yoon et al., 2009). The neurotoxicity appears as a sensory-motor neuropathy [with predominant sensory manifestations such as: paresthesias, loss of vibration sense and decreased tendon reflexes] and occasionally it could be accompanied by dysfunction of the autonomic nervous system (Beijers et al., 2012). Both Siddik (2003) and Dzagnidze et al. (2007) reported that cisplatin induced reduction in sensory
∗ Corresponding author at: Kasr El-Aini, Cairo University, Al-Saray Street, 11562 Cairo, Egypt. Tel.: +20 2 01122433182; fax: +20 2 2362824. E-mail address: [email protected] (N. Sharawy). http://dx.doi.org/10.1016/j.etp.2015.02.003 0940-2993/© 2015 Elsevier GmbH. All rights reserved.
nerve conduction velocities and their corresponding amplitudes could be mediated by accumulation of persisting DNA lesions. The neurotoxicity effects of cisplatin is mediated by the formation of specific cisplatin-DNA adducts with guanine–guanine intrastrand cross-links (cisPt(NH3 )2 d(pGpG; Pt-GG), representing the majority of the DNA platination products that can interfere with replication or transcription and trigger apoptotic processes (Siddik, 2003; Yoon et al., 2009). During the past two decades, the actions of erythropoietin has shifted from a belief that the cytokine could act only on the production and differentiation of red blood cells to the knowledge that this agent could exert significant protection in certain conditions [such as sepsis, hemorrhagic shock, and ischemia/reperfusion injury (IRI)] (Patel et al., 2011). Previous studies showed that EPO prevent cisplatin-induced neurotoxicity without compromising chemotherapeutic activity. (Bianchi et al., 2006, 2007; Kassem and Yassin, 2010). Erythropoietin, through erythropoietin receptors (EpoR) in nerve axons, Schwann cells and in dorsal root ganglia (DRG) neurons, is among
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the substances with neurotrophic action-regulating nerve survive and growth-on central neurons and peripheral nerves (Beijers et al., 2012; Bianchi et al., 2006). Erythropoietin is noted to ameliorate cisplatin-induced neuropathy without interfering with the accumulation of Pt-DNA adducts in neuronal cells. Yoon et al. (2009) suggested that erythropoietin neuroprotective effects could be mediated by improving the cell resistance through activation of the phosphatidylinositol 3-kinase/Akt pathway and NFB pathway resulting in an up regulation of antiapoptotic proteins and blocking the activation of specific cell-death proteases leading to apoptosis. The aim of our study is to investigate the potency of erythropoietin to modulate cisplatin associated neuronal functional and structural changes through modulation of nitro-oxidative stress and NFKB gene expression. 2. Materials and methods 2.1. Animals After obtaining approval from the Kasr El Aini Animal Care Committee, we used 24 male albino rats from the animal house in Kasr El Aini, faculty of medicine for the experiments (body weight: 220–240 g). The animals were housed in chip-bedded cages and, prior to experiments, acclimated for 1 week in the air conditioned institutional animal care unit. They were housed under 12 h light/dark cycles, with free access to water and standard rat chow. 2.2. Experimental groups Twenty four male albino rats were randomly divided in to 3 main groups (8 per group): Placebo treated control group, cisplatin (CDDP) (Merck KGaA, Darmstadt, Germany) and CDDP plus erythropoietin (EPO) (Janssen Pharmaceuticals, Inc., Buckinghamshire, UK) treated group. CDDP was dissolved in sterile saline and rats were injected with CDDP 2 mg/kg i.p. twice weekly for 8 times using a volume of 4 mL/kg (Bianchi et al., 2006). The CDDP plus erythropoietin group was treated with the same dose of CDDP plus erythropoietin (50 g/kg i.p. thrice weekly). Control rats received i.p. injections with the sterile saline (Bianchi et al., 2006). 2.3. General protocol At the end of the treatment, thermal sensitivity tests and neurophysiologic measurements were done for each rat. Thereafter, the rats were sacrificed; the sciatic nerves were removed and frozen for further biochemical investigations. 2.3.1. Thermal sensitivity tests To analyze the somatic sensitivity, we measured the withdrawal threshold after thermal (hot and cold water) stimulation of the rat tail. The withdrawal latency was defined as the time, in seconds (s), between immersion of tail in hot/cold water (with a cut-off time of 15 s) and the time of withdrawal (Courteix et al., 2007; Sandkuhler, 2009). 2.3.2. Neurophysiologic measurements The recordings of nerve potentials were carried out using a physiological data acquisition system (PowerLab 4/SP; ADInstruments, Castle Hill, NSN, Australia). Each rat stimulated by a single square pulse stimulus (intensity 10 V; duration 0.2 ms; delivered at the rate of 1 s−1 ) from a stimulator on a PowerLab 4/SP data acquisition system. The responses were amplified with an amplifier (BIO Amp, ADInstruments) and stored on a computer. Scope software (ADInstruments) was used for data capture and analysis. The amplitude from the baseline to the peak [of sensory action potential and motor
compound action potential], the duration and the latency of sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) were measured. All the neurophysiologic measurements were done under standard conditions in a temperature-controlled room (25 ◦ C). 2.4. Measurement of SNCV SNCV was determined in the tail using a previously described method (Leandri et al., 2007). Briefly, stimulation was performed via two electrodes inserted at the distal end of the tail. The first set was inserted at a location named S1, with the cathode at 5 mm from the tail tip, and the anode at 2 mm; the second set was inserted at a more proximal location, called S2, with the cathode placed at 20 mm and the anode 17 mm from the tail tip. The most distal stimulating location was chosen because it could be presumed that it would be devoid of motor fibers, therefore its stimulation could be considered to activate sensory fibers only. Recording was carried out with two electrodes placed at 50 mm and 100 mm from rat tail. 2.5. Measurement of MNCV Electrodes were used to stimulate the right sciatic nerve and to record the compound muscle action potentials. For the first stimulating point, electrodes were placed proximally to the sciatic notch. The compound muscle action potentials were recorded. After stimulating at the first point, the electrodes were moved to the second stimulating point, 1 cm distal to the first point. Nerve conduction velocities (NCV) was calculated by subtracting the distal from the proximal latency (measured in milliseconds) from the stimulus artifact of the evoked potential, and the difference was divided into the distance between the two stimulating electrodes (measured in millimeters). The NCV was reported in meters per second. The amplitude of the CAP was measured from peak to peak. To determine the latency, electrical stimulation was repeated 10 times and averaged per rat. 2.5.1. Quantification of the sciatic nerve TNF level The concentration of TNF was measured by the sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA) kits (R&D syst; Minneapolis; USA) according to the manufacturer’s instructions in sciatic nerve tissue homogenate. 2.5.2. Quantification of sciatic nerve malondialdehyde (MDA) level The sciatic nerve level of MDA was estimated by colorimetry using kits purchased from BioVision, Inc.; California; USA, according to the manufacturer’s instructions. Nerve homogenate were treated with thiobarbituric acid (TBA) and sodium sulfate of 20% trichloroacetic acid, and then the mixture is heated in a boiling water bath for 30 min. The resulting chromogen is extracted with 4 ml of n. butyl alcohol and the absorbance of the organic phase is determined at the wave length of 530 nm, the determined values are expressed in terms of malondialdehyde (n mol/mg protein) used as reference standard. 2.5.3. Determination of the levels of superoxide dismutase enzyme (SOD) and glutathione in Sciatic nerve tissue For measurement of glutathione (GSH), which represents the major free thiol in most living cells, NWLSSTM Glutathione Assay kit was purchased from Northwest life science specialties (Vancouver; Canada). The general thiol reagent, 5-5 -dithiobis[2-nitrobenzoic acid] (DTNB, Ellman’s Reagent) reacts with GSH to form the 412 nm chromophore, 5-thionitrobenzoic acid (TNB) and GS-TNB. The
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GS-TNB is subsequently reduced by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH), releasing a second TMB molecule and recycling GSH; thus amplifying the response. Any oxidized GSH (GSSG) initially present in the reaction mixture or formed from the mixed disulfide reaction of GSH with GS-TNB is rapidly reduced to GSH. Superoxide dismutases (SOD) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and thus form a crucial part of cellular antioxidant defense mechanism. Cayman’s superoxide dismutase Assay Kits (Cayman’s chemical company; Ann Arbor, USA) utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to Exhibit 50% dismutation of superoxide radical. 2.5.4. Real time-polymerase chain reaction (real time-PCR) Real time-PCR (Pomega, Madison, USA) was used to measure i-NOS (inducible nitric oxide synthase), nNOS (neuronal NOS), NFKB (nuclear factor kappa-light-chain-enhancer of activated B cells) and both pro-apoptotic and anti-apoptotic markers [Bax, Bcl, Bax/Bcl, caspase3] genes expression in sciatic nerve tissue. The key equipment for real time-PCR is a specialized thermocycler with fluorescence detection modules, which is used to monitor and record the fluorescence in real time as amplification occurs. A typical workflow of real time-PCR for gene expression measurement involves RNA isolation, reverse transcription, real time-PCR assay development, real time-PCR experiment and data analysis. 2.5.5. Histological assessment of the sciatic nerve Sciatic nerves were carefully dissected, fixed in 10% formalin solution, embedded in paraffin, then sectioned and stained with hematoxylin and eosin, and examined by light microscope. 2.6. Statistical analysis The results are given as means ± standard error (SEM). Results were analyzed by using the software Prism 5 (GraphPad Software, La Jolla, CA, USA). First, data were tested for normal distribution by using the Kolmogorov–Smirnov test. If normal distribution was established, one-way analysis of variance (ANOVA) was performed. If significant differences appeared, a post hoc analysis with the Newman–Keuls multiple comparison test was conducted. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effects of erythropoietin on cisplatin induced sciatic nerve functional alterations We found significant increases in both cold and hot withdrawal latency time (p < 0.05) with cisplatin treatment in comparison to control group [cold sensitivity test (s): 6.5 ± 3.5 vs 14.9 ± 0.2; hot sensitivity test (s): 5 ± 3.5 vs 8.5 ± 3.7]. Erythropoietin treatment significantly decreased (p < 0.05) both cold and hot withdrawal latency time [cold sensitivity test (s): 4.3 ± 4.3; hot sensitivity test (s): 1.4 ± 0.6] (Table 1). In comparison to control group, we found significant decreases in SNCV, MNCV and motor potential amplitude (p < 0.05) after cisplatin treatment [SNCV (m/s): 10.2 ± 6.1 vs 0.6 ± 0.6; MNCV (m/s): 4.1 ± 2.7 vs 0.9 ± 0.4; motor potential amplitude (mV): 5.8 ± 3.5 vs 2.2 ± 1.2]. However, concomitant administration of erythropoietin significantly increased (p < 0.05) SNCV, MNCV and motor potential amplitude [SNCV (m/s): 8.1 ± 6.2; MNCV (m/s): 6.9 ± 2.9; motor potential amplitude (mV): 7.8 ± 4.2] Table 1.
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3.2. Effects of erythropoietin on cisplatin induced sciatic nerve structural changes Light microscopic examination of histological section of sciatic nerves of the control group showed bundles of nerve axons, each surrounded by sheath of Schwann cell, which is surrounded by delicate connective tissue (endoneurium), and the whole bundle is surrounded a thick layer of dense connective tissue (epineurium). Meanwhile, histological examination of sciatic nerves of cisplatin treated group showed marked damage and apoptotic changes with axonal degeneration and loss of myelinated nerve fibers. Concomitant administration of erythropoietin with cisplatin was observed to successfully ameliorating the apoptotic changes with preserving myelinated nerve fibers (Fig. 1(A–C)). 3.3. Effects of erythropoietin on cisplatin induced apoptotic changes In comparison to control group, we found significant increases (p < 0.05) in pro-apoptotic markers, caspase3 [0.7 ± 0.14 vs 0.07 ± 0.01] and Bax genes expressions [1.4 ± 0.41 vs 0.19 ± 0.04], after cisplatin challenge. In addition, significant reductions (p < 0.05) in anti-apoptotic markers, Bcl gene expression [0.84 ± 0.24 vs 2.02 ± 0.46] and Bcl/Bax ratio [0.64 ± 0.37 vs 10.62 ± 3.14], after cisplatin injection were observed. Moreover, our results revealed that erythropoietin treatment significantly decreased (p < 0.05) Caspase 3 and Bax gene expressions, and significantly increased (p < 0.05) Bcl gene expression: [Caspase 3: 0.09 ± 0.04, Bax gene expression: 0.56 ± 0.25, Bcl gene expression: 1.4 ± 0.34, Bcl/Bax ratio: 3.1 ± 1.7] (Fig. 2(A–D)). 3.4. Effects of erythropoietin on cisplatin induced nitro-oxidative stress In comparison to control group, we found that cisplatin injection was associated with a significant increase (p < 0.05) in lipid peroxide marker [MDA (nmol/mg protein) [198.2 ± 29.02 vs 112.6 ± 3.2], and significant reductions (p < 0.05) of both SOD (unit/mg protein) [0.87 ± 0.19 vs 2.4 ± 0.38] and glutathione (nmol/mg protein) [25.7 ± 5.3 vs 43.01 ± 3.4]. However, co-administration of erythropoietin was found to significantly ameliorate (p < 0.05) cisplatin induced oxidative stress, with a significant decrease (p < 0.05) of MDA and significant increases (p < 0.05) of both SOD and glutathione [MDA (nmol/mg protein):122.6 ± 6.8, SOD (unit/mg protein): 1.9 ± 0.33, glutathione (nmol/mg protein): 34.19] (Fig. 3(A–C)). In addition, we noted a significant reduction (p < 0.05) in the level of nNOS gene expression [0.5 ± 0.31 vs 1.5 ± 0.31] that was associated with a significant increase (p < 0.05) of iNOS gene expression [0.1 ± 0.04 vs 0.001 ± 0.001] after cisplatin injection. We found also that erythropoietin treatment significantly reduced (p < 0.05) iNOS gene expression [0.02 ± 0.01], without significant effect on nNOS gene expression [0.72 ± 0.13] (Fig. 4(A, B)). 3.5. Effects of erythropoietin on cisplatin induced sciatic nerve NFKB gene expression and TNF level Significant increases (p < 0.05) in the TNF level (pg/ml) [97.81 ± 18.37 vs 30.4 ± 2.25] and NFKB gene expression [1.2 ± 0.54 vs 0.12 ± 0.03] were observed in cisplatin injected group in comparison to control group. Erythropoietin injection was associated with significant reduction (p < 0.05) in the level of TNF (pg/ml) [50.46 ± 9.24], without significant effect on NFKB gene expression (Fig. 5(A, B)).
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Table 1 Erythropoietin effects on cisplatin induced neuropathy. Control Cold withdraw latency (s) Hot withdraw latency (s) Sensory nerve conductive velocity (SNCV) (m/s) Sensory amplitude potential (mV) Motor nerve conductive velocity (MNCV) (m/s) Motor amplitude potential (mV)
6.5 5.0 10.2 13.1 4.1
± ± ± ± ±
Cisplatin 1.3 1.3 2.3 6.5 1.0
5.8 ± 1.3
14.9 8.5 0.6 6.5 0.9
± ± ± ± ±
0.08 $ 1.3 $ 0.2 $ 5.3$ 0.1 $
2.2 ± 0.4 $
Erythropoietin + cisplatin 4.3 1.4 8.1 2.8 6.9
± ± ± ± ±
1.3 # 0.2 # 1.9 # 1.6 1.1 #
7.8 ± 1.3 #
All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
4. Discussion Our findings further support the anti-apoptotic neuroprotective effects of erythropoietin during cisplatin induced neurotoxicity. The observed nitro-oxidative stress ameliorating effects of erythropoietin during cisplatin neurotoxicity could partially explain cells apoptotic resistance associated with erythropoietin treatment. Similar to previous studies (Bardos et al., 2003; Li et al., 2002; von Schlippe et al., 2001), significant reductions of nerve conductive velocities and compound action potential amplitude of large myelinated fibers were observed after cisplatin injection. In addition, we found that cisplatin treatment could also influence small fibers that are closely involved in thermal pathway.
In our study, the neuropathy induced by cisplatin treatment was associated with histological axonal degeneration and loss of myelinated nerve fibers. In addition, significant increases in proapoptotic markers [caspase 3, Bax gene] and significant decreases in anti-apoptotic markers [Bcl gene, Bcl/Bax] were observed after cisplatin challenge. Previous clinical and experimental studies demonstrated that the hallmarks of neuropathy and cell apoptosis is the specific cisplatin-DNA adduct lesions accumulation (Roelofs et al., 1984; Yoon et al., 2009). It was reported that cisplatin-induced genotoxic stress activates mitochondrial apoptotic pathway that is largely mediated through Bcl-2 family proteins. Bcl-2 family proteins include both pro-apoptotic members (such as Bax) that promote
Fig. 1. (A–C) Erythropoietin ameliorated cisplatin associated sciatic nerve apoptotic changes (hematoxylin and eosin, ×400).
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Fig. 2. (A–D) Erythropoietin (EPO) reversed cisplatin (CDDP) associated changes in pro-apoptotic markers, Bax (B) and Caspase3 (D), anti-apoptotic marker, Bcl (A), and Bcl/Bax ratio (C). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
mitochondrial permeability, and anti-apoptotic members (such as Bcl-2) that inhibit Bax effects, or inhibit the mitochondrial release of cytochrome c (cyt c). The release of cytochrome c from mitochondria, induced by stress, translocates Bax and truncated-Bid from cytosol to mitochondria and decreased expression of Bcl-2; which activates the caspase-9 molecules (upon cleavage of the bound zymogen procaspases-9), which in turn activate caspase-3; leading to chromatin condensation and DNA fragmentation (Bagci et al., 2006; Hong et al., 2012). Several neuroprotective strategies, which aim at a reduced accumulation of drug-induced DNA adducts and improving the survival of nerve despite of their actual damage burden have been previously investigated. However, erythropoietin seems to be the most promising approach to reduce cisplatin induced peripheral neuropathy because of its ability to increase the resistance of neuronal cell to neurotoxin effects of cisplatin (Beijers et al., 2012; Cascinu et al., 2002; Hilpert et al., 2005; Kassem and Yassin, 2010; Keswani et al., 2011; Ocean and Vahdat, 2004). Yoon et al. (2009) previously reported that erythropoeitin-through erythropoietin receptors (EpoR)-ameliorated cisplatin-induced neuropathy in mice, and that the observed neuroprotective effects seems to be mediated by enhancing the cell resistance to side effects of Cisplatin rather than by influencing the formation or repair rates of cisplatin-induced cross-links in the nuclear DNA of neuron. In our study we found that erythropoietin treatment was associated with changes in the accessible levels of two members of
the Bcl-2 family, Bax and Bcl-2, as well as Caspase 3. Erythropoietin treatment significantly decreases Bax and Caspase3 gene expression and significantly increases Bcl-2 gene induced by cisplatin. Our results could suggest that erythropoietin interfere with cisplatin activated mitochondria apoptotic pathway, an effect that could explain the observed erythropoietin neurophysiological alterations. Recently the role of reactive oxygen species (ROS) in cisplatin activated mitochondrial apoptotic pathway was suggested (Marullo et al., 2013). ROS are associated with enhancement of lipid peroxidation, depletion of the sulfhydryl groups, alteration of the signal transduction pathways, calcium homeostasis and DNA damage (Florea and Busselberg, 2011). In our study the cisplatin induced ROS effects could be partial explained by cisplatin associated effects on glutathione and SOD antioxidant enzymes. We found a significant decrease in sciatic nerve glutathione and SOD levels associated with a significant increase in MDA level. Among the antioxidant enzymes, both catalase and glutathione peroxidase play a critical role in reducing levels of hydrogen peroxide and preventing formation of ROS. In addition, superoxide dismutase (SOD) acts to prevent the accumulation of the potentially damaging superoxide anion. Therefore, activation of one or more of these enzymes would suppress accumulation of ROS and protect neurons against oxidative stress (Cheng and Mattson, 1995).
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Fig. 3. (A–C) Erythropoietin (EPO) prevents the cisplatin (CDDP) induced reduction of antioxidant enzyme, superoxide dismutase reductase (SOD) (B) and glutathione level (C), and associated increase in malondialdehyde (MDA) level (A). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
While Pratibha et al. (2006) previously reported a significant decrease in glutathione, most abundant thiol-containing antioxidant, after cisplatin therapy, both Godwin et al. (1992) and Ishikawa and Ali-Osman (1993) previously noted that cells that are resistant to cisplatin have elevated levels of glutathione
(GSH). Therefore, whether over-expression of glutathione contributes to or combats cisplatin resistance is still mater of debate. However, our results could suggest that low level of glutathione could partial contribute to cisplatin associated neurotoxic effects.
Fig. 4. (A, B) Erythropoietin (EPO) reduced the cisplatin (CDDP) induced nitric oxide synthase (iNOS) gene expression (B), and had no effects on cisplatin associated reduction in neuronal nitric oxide synthase (nNOS) gene expression (A). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
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Fig. 5. (A, B) Erythropoietin effects on cisplatin associated changes in sciatic nerve TNF level (A) and nuclear factor kappa B (NFKB) gene expression (B). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
Similar to Ognjanovic et al. (2012), who reported significant decreases in the renal activity of antioxidant defence enzymes after cisplatin injection, we found a significant reduction in sciatic nerve SOD level after cisplatin challenge. An observation that could further explain cisplatin induced ROS effects. We suggest that erythropoietin observed anti-apoptotic effects could partial explained by the noted effects of erythropoietin on the antioxidant enzyme [both SOD and glutathione were significantly increased after erythropoietin treatment]. Our observations further support Sakanaka et al. (1998), who demonstrated that erythropoietin could increase the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase in neurons. In addition, Bailey et al. (2014) reported the potent scavenging property of erythropoietin and its capacity to inhibit Fenton chemistry through catalytic iron chelation. NO has been previously commented as double-edged sword with respect to its neurotoxicity and neuroprotection (Boje, 2004). While Dawson and Dawson (2004) had found that inducible nitric oxide synthase (iNOS)-derived NO-induce neuronal apoptosis, and that could be mediated via both caspase-dependent and caspase-independent [e.g., poly (ADP-ribose) polymerase-1 mediated] mechanisms, Keswani et al. (2011) previously reported that neuronal nitric oxide synthase (nNOS)-derived NO serves as an important neuronal protection through induction of hypoxiainducible factor (HIF)-1, the transcriptional activator of several genes that potentially may be involved in the neuroprotection associated with ischemic preconditioning (Semenza, 2010; Abe et al., 2003; Keswani et al., 2011). Our study further support previous few studies that reported the role of NO in cisplatin activated mitochondria apoptotic pathway (Lee et al., 2004; Sharma et al., 2005). In our study, we found that erythropoietin significantly reduced cisplatin induced iNOS gene expression. Meanwhile, no significant effect on cisplatin associated down-regulation of nNOS was observed with concomitant erythropoietin administration. Another factor that could contribute to cisplatin activated mitochondrial apoptotic pathway is the observed significant increases in sciatic nerve NFKB gene expression and TNF level. It was reported that ionizing radiation and anticancer drugs, which primarily target DNA, may activate NFKB via mechanisms different from the conventional IKK/IB␣/NFB pathway. Yeh et al. (2004) suggested that cisplatin activated Protein phosphatase 4
(PP4; also known as protein phosphatase X), a novel Ser/Thr protein phosphatase. PP4 had been shown to activate NFKB by direct interaction with and dephosphorylation of the NFKB p65 subunit (Hu et al., 1998). Treatment with cisplatin was observed to be associated with an increase in TNF gene expression – by extracellular-signalregulated kinases (ERK) – and activation of caspase-3 that trigger cisplatin-induced apoptosis (Basu and Tu, 2005; Jo et al., 2005). In addition, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) interacts with the death receptors, DR4 and DR5, and activates caspases, leading to apoptosis via the extrinsic pathway, in contrast to the mitochondria-mediated intrinsic death signaling pathway that is activated following cytotoxic stresses (Shamimi-Noori et al., 2008; Wenger et al., 2006). In spite that previous studies suggested that the phosphatidylinositol 3-kinase/Akt and NFB could be involved in erythropoietin induced anti-apoptotic pathways (Digicaylioglu and Lipton, 2001; Kretz et al., 2005; Wang et al., 1998), we could not find significant effect of erythropoietin on cisplatin induced NFKB gene expression. This difference could be due to differences in the doses and experimental protocol. In addition, our results could suggest that TNF reduction effect of erythropoietin seems to contribute to its anti-apoptotic effects. In light of previous data we suggest that erythropoietin may exert its neuroprotective effects by its scavenging property of ROS through activation of anti-oxidant enzymes and its ability to reduce TNF. 5. Conclusion Our results further support the effectiveness of erythropoietin as a neuroprotective anti-apoptotic drugs during cisplatin induced neurotoxicity. Erythropoietin seems to ameliorate nitro-oxidative stress and the increased TNF induced by cisplatin challenge. Our results represent one step towards understanding the mechanism underlying erythropoietin anti-apoptotic property. Acknowledgment The skillful technical assistance of Afaf, Aza and Tarek is appreciated.
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Evaluation of multi-neuroprotective effects of erythropoietin using cisplatin induced peripheral neurotoxicity model Nivin Sharawy a,∗ , Laila Rashed b , Magdy Fouad Youakim c a b c
Department of Physiology, Faculty of Medicine, Cairo University, Cairo, Egypt Department of Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt Department of Anatomy, Faculty of Medicine, Cairo University, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 18 September 2014 Accepted 20 February 2015 Keywords: Cisplatin Erythropoietin Oxidative Inflammatory Apoptosis
a b s t r a c t Cisplatin (CDDP) is severely neurotoxic anti-neoplastic drug that causes peripheral neuropathies with clinical signs known as chemotherapy-induced peripheral neurotoxicity. The ameliorating effects of erythropoeitin on cisplatin-induced neuropathy, which seem to be mediated by enhancing the cell resistance to side effects of cisplatin rather than by influencing the formation or repair rates of cisplatin-induced cross-links in the nuclear DNA, had been previously reported. The main objective of our study is to investigate the roles of nitro-oxidative stress, nuclear factor kappa B (NFB) gene expressions and TNF levels on the previous reported erythropoietin anti-apoptotic neuroprotective effects during cisplatin induced neurotoxicity. The present study compared the effects of erythropoietin (50 g/kg/d thrice weekly) on cisplatin (2 mg/kg/d i.p. twice weekly for 4 weeks) induced neurophysiologic changes and the associated changes in the inflammatory mediators (TNF alpha and NFKB), oxidative stress (malondialdehyde (MDA), superoxide dismutases (SOD) and glutathione) and gene expression of both neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). In addition, sciatic nerve pro-apoptotic and anti-apoptotic indicators (Bcl, Bax, Caspase 3) were measured. We found that concomitant administration of erythropoietin significantly reversed the cisplatin induced nitro-oxidative stress – with significant increases in sciatic nerve glutathione and superoxide dismutase antioxidant enzyme levels and a significant decrease in iNOS gene expression. We conclude that erythropoietin anti-apoptotic neuro-protective effects could partially contribute to observed antioxidant effects of erthropoietin. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Cisplatin (CDDP) and the other platinum-derived drugs are among the most effective antineoplastic agents, but they are severely neurotoxic. Accumulation of cisplatin in the dorsal root ganglia (DRG) results in nerve fiber axonopathy and degeneration of large myelinated axons with signs of segmental demyelinization and remyelinization (Bianchi et al., 2006; Yoon et al., 2009). The neurotoxicity appears as a sensory-motor neuropathy [with predominant sensory manifestations such as: paresthesias, loss of vibration sense and decreased tendon reflexes] and occasionally it could be accompanied by dysfunction of the autonomic nervous system (Beijers et al., 2012). Both Siddik (2003) and Dzagnidze et al. (2007) reported that cisplatin induced reduction in sensory
∗ Corresponding author at: Kasr El-Aini, Cairo University, Al-Saray Street, 11562 Cairo, Egypt. Tel.: +20 2 01122433182; fax: +20 2 2362824. E-mail address: [email protected] (N. Sharawy). http://dx.doi.org/10.1016/j.etp.2015.02.003 0940-2993/© 2015 Elsevier GmbH. All rights reserved.
nerve conduction velocities and their corresponding amplitudes could be mediated by accumulation of persisting DNA lesions. The neurotoxicity effects of cisplatin is mediated by the formation of specific cisplatin-DNA adducts with guanine–guanine intrastrand cross-links (cisPt(NH3 )2 d(pGpG; Pt-GG), representing the majority of the DNA platination products that can interfere with replication or transcription and trigger apoptotic processes (Siddik, 2003; Yoon et al., 2009). During the past two decades, the actions of erythropoietin has shifted from a belief that the cytokine could act only on the production and differentiation of red blood cells to the knowledge that this agent could exert significant protection in certain conditions [such as sepsis, hemorrhagic shock, and ischemia/reperfusion injury (IRI)] (Patel et al., 2011). Previous studies showed that EPO prevent cisplatin-induced neurotoxicity without compromising chemotherapeutic activity. (Bianchi et al., 2006, 2007; Kassem and Yassin, 2010). Erythropoietin, through erythropoietin receptors (EpoR) in nerve axons, Schwann cells and in dorsal root ganglia (DRG) neurons, is among
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the substances with neurotrophic action-regulating nerve survive and growth-on central neurons and peripheral nerves (Beijers et al., 2012; Bianchi et al., 2006). Erythropoietin is noted to ameliorate cisplatin-induced neuropathy without interfering with the accumulation of Pt-DNA adducts in neuronal cells. Yoon et al. (2009) suggested that erythropoietin neuroprotective effects could be mediated by improving the cell resistance through activation of the phosphatidylinositol 3-kinase/Akt pathway and NFB pathway resulting in an up regulation of antiapoptotic proteins and blocking the activation of specific cell-death proteases leading to apoptosis. The aim of our study is to investigate the potency of erythropoietin to modulate cisplatin associated neuronal functional and structural changes through modulation of nitro-oxidative stress and NFKB gene expression. 2. Materials and methods 2.1. Animals After obtaining approval from the Kasr El Aini Animal Care Committee, we used 24 male albino rats from the animal house in Kasr El Aini, faculty of medicine for the experiments (body weight: 220–240 g). The animals were housed in chip-bedded cages and, prior to experiments, acclimated for 1 week in the air conditioned institutional animal care unit. They were housed under 12 h light/dark cycles, with free access to water and standard rat chow. 2.2. Experimental groups Twenty four male albino rats were randomly divided in to 3 main groups (8 per group): Placebo treated control group, cisplatin (CDDP) (Merck KGaA, Darmstadt, Germany) and CDDP plus erythropoietin (EPO) (Janssen Pharmaceuticals, Inc., Buckinghamshire, UK) treated group. CDDP was dissolved in sterile saline and rats were injected with CDDP 2 mg/kg i.p. twice weekly for 8 times using a volume of 4 mL/kg (Bianchi et al., 2006). The CDDP plus erythropoietin group was treated with the same dose of CDDP plus erythropoietin (50 g/kg i.p. thrice weekly). Control rats received i.p. injections with the sterile saline (Bianchi et al., 2006). 2.3. General protocol At the end of the treatment, thermal sensitivity tests and neurophysiologic measurements were done for each rat. Thereafter, the rats were sacrificed; the sciatic nerves were removed and frozen for further biochemical investigations. 2.3.1. Thermal sensitivity tests To analyze the somatic sensitivity, we measured the withdrawal threshold after thermal (hot and cold water) stimulation of the rat tail. The withdrawal latency was defined as the time, in seconds (s), between immersion of tail in hot/cold water (with a cut-off time of 15 s) and the time of withdrawal (Courteix et al., 2007; Sandkuhler, 2009). 2.3.2. Neurophysiologic measurements The recordings of nerve potentials were carried out using a physiological data acquisition system (PowerLab 4/SP; ADInstruments, Castle Hill, NSN, Australia). Each rat stimulated by a single square pulse stimulus (intensity 10 V; duration 0.2 ms; delivered at the rate of 1 s−1 ) from a stimulator on a PowerLab 4/SP data acquisition system. The responses were amplified with an amplifier (BIO Amp, ADInstruments) and stored on a computer. Scope software (ADInstruments) was used for data capture and analysis. The amplitude from the baseline to the peak [of sensory action potential and motor
compound action potential], the duration and the latency of sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) were measured. All the neurophysiologic measurements were done under standard conditions in a temperature-controlled room (25 ◦ C). 2.4. Measurement of SNCV SNCV was determined in the tail using a previously described method (Leandri et al., 2007). Briefly, stimulation was performed via two electrodes inserted at the distal end of the tail. The first set was inserted at a location named S1, with the cathode at 5 mm from the tail tip, and the anode at 2 mm; the second set was inserted at a more proximal location, called S2, with the cathode placed at 20 mm and the anode 17 mm from the tail tip. The most distal stimulating location was chosen because it could be presumed that it would be devoid of motor fibers, therefore its stimulation could be considered to activate sensory fibers only. Recording was carried out with two electrodes placed at 50 mm and 100 mm from rat tail. 2.5. Measurement of MNCV Electrodes were used to stimulate the right sciatic nerve and to record the compound muscle action potentials. For the first stimulating point, electrodes were placed proximally to the sciatic notch. The compound muscle action potentials were recorded. After stimulating at the first point, the electrodes were moved to the second stimulating point, 1 cm distal to the first point. Nerve conduction velocities (NCV) was calculated by subtracting the distal from the proximal latency (measured in milliseconds) from the stimulus artifact of the evoked potential, and the difference was divided into the distance between the two stimulating electrodes (measured in millimeters). The NCV was reported in meters per second. The amplitude of the CAP was measured from peak to peak. To determine the latency, electrical stimulation was repeated 10 times and averaged per rat. 2.5.1. Quantification of the sciatic nerve TNF level The concentration of TNF was measured by the sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA) kits (R&D syst; Minneapolis; USA) according to the manufacturer’s instructions in sciatic nerve tissue homogenate. 2.5.2. Quantification of sciatic nerve malondialdehyde (MDA) level The sciatic nerve level of MDA was estimated by colorimetry using kits purchased from BioVision, Inc.; California; USA, according to the manufacturer’s instructions. Nerve homogenate were treated with thiobarbituric acid (TBA) and sodium sulfate of 20% trichloroacetic acid, and then the mixture is heated in a boiling water bath for 30 min. The resulting chromogen is extracted with 4 ml of n. butyl alcohol and the absorbance of the organic phase is determined at the wave length of 530 nm, the determined values are expressed in terms of malondialdehyde (n mol/mg protein) used as reference standard. 2.5.3. Determination of the levels of superoxide dismutase enzyme (SOD) and glutathione in Sciatic nerve tissue For measurement of glutathione (GSH), which represents the major free thiol in most living cells, NWLSSTM Glutathione Assay kit was purchased from Northwest life science specialties (Vancouver; Canada). The general thiol reagent, 5-5 -dithiobis[2-nitrobenzoic acid] (DTNB, Ellman’s Reagent) reacts with GSH to form the 412 nm chromophore, 5-thionitrobenzoic acid (TNB) and GS-TNB. The
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GS-TNB is subsequently reduced by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH), releasing a second TMB molecule and recycling GSH; thus amplifying the response. Any oxidized GSH (GSSG) initially present in the reaction mixture or formed from the mixed disulfide reaction of GSH with GS-TNB is rapidly reduced to GSH. Superoxide dismutases (SOD) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and thus form a crucial part of cellular antioxidant defense mechanism. Cayman’s superoxide dismutase Assay Kits (Cayman’s chemical company; Ann Arbor, USA) utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to Exhibit 50% dismutation of superoxide radical. 2.5.4. Real time-polymerase chain reaction (real time-PCR) Real time-PCR (Pomega, Madison, USA) was used to measure i-NOS (inducible nitric oxide synthase), nNOS (neuronal NOS), NFKB (nuclear factor kappa-light-chain-enhancer of activated B cells) and both pro-apoptotic and anti-apoptotic markers [Bax, Bcl, Bax/Bcl, caspase3] genes expression in sciatic nerve tissue. The key equipment for real time-PCR is a specialized thermocycler with fluorescence detection modules, which is used to monitor and record the fluorescence in real time as amplification occurs. A typical workflow of real time-PCR for gene expression measurement involves RNA isolation, reverse transcription, real time-PCR assay development, real time-PCR experiment and data analysis. 2.5.5. Histological assessment of the sciatic nerve Sciatic nerves were carefully dissected, fixed in 10% formalin solution, embedded in paraffin, then sectioned and stained with hematoxylin and eosin, and examined by light microscope. 2.6. Statistical analysis The results are given as means ± standard error (SEM). Results were analyzed by using the software Prism 5 (GraphPad Software, La Jolla, CA, USA). First, data were tested for normal distribution by using the Kolmogorov–Smirnov test. If normal distribution was established, one-way analysis of variance (ANOVA) was performed. If significant differences appeared, a post hoc analysis with the Newman–Keuls multiple comparison test was conducted. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effects of erythropoietin on cisplatin induced sciatic nerve functional alterations We found significant increases in both cold and hot withdrawal latency time (p < 0.05) with cisplatin treatment in comparison to control group [cold sensitivity test (s): 6.5 ± 3.5 vs 14.9 ± 0.2; hot sensitivity test (s): 5 ± 3.5 vs 8.5 ± 3.7]. Erythropoietin treatment significantly decreased (p < 0.05) both cold and hot withdrawal latency time [cold sensitivity test (s): 4.3 ± 4.3; hot sensitivity test (s): 1.4 ± 0.6] (Table 1). In comparison to control group, we found significant decreases in SNCV, MNCV and motor potential amplitude (p < 0.05) after cisplatin treatment [SNCV (m/s): 10.2 ± 6.1 vs 0.6 ± 0.6; MNCV (m/s): 4.1 ± 2.7 vs 0.9 ± 0.4; motor potential amplitude (mV): 5.8 ± 3.5 vs 2.2 ± 1.2]. However, concomitant administration of erythropoietin significantly increased (p < 0.05) SNCV, MNCV and motor potential amplitude [SNCV (m/s): 8.1 ± 6.2; MNCV (m/s): 6.9 ± 2.9; motor potential amplitude (mV): 7.8 ± 4.2] Table 1.
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3.2. Effects of erythropoietin on cisplatin induced sciatic nerve structural changes Light microscopic examination of histological section of sciatic nerves of the control group showed bundles of nerve axons, each surrounded by sheath of Schwann cell, which is surrounded by delicate connective tissue (endoneurium), and the whole bundle is surrounded a thick layer of dense connective tissue (epineurium). Meanwhile, histological examination of sciatic nerves of cisplatin treated group showed marked damage and apoptotic changes with axonal degeneration and loss of myelinated nerve fibers. Concomitant administration of erythropoietin with cisplatin was observed to successfully ameliorating the apoptotic changes with preserving myelinated nerve fibers (Fig. 1(A–C)). 3.3. Effects of erythropoietin on cisplatin induced apoptotic changes In comparison to control group, we found significant increases (p < 0.05) in pro-apoptotic markers, caspase3 [0.7 ± 0.14 vs 0.07 ± 0.01] and Bax genes expressions [1.4 ± 0.41 vs 0.19 ± 0.04], after cisplatin challenge. In addition, significant reductions (p < 0.05) in anti-apoptotic markers, Bcl gene expression [0.84 ± 0.24 vs 2.02 ± 0.46] and Bcl/Bax ratio [0.64 ± 0.37 vs 10.62 ± 3.14], after cisplatin injection were observed. Moreover, our results revealed that erythropoietin treatment significantly decreased (p < 0.05) Caspase 3 and Bax gene expressions, and significantly increased (p < 0.05) Bcl gene expression: [Caspase 3: 0.09 ± 0.04, Bax gene expression: 0.56 ± 0.25, Bcl gene expression: 1.4 ± 0.34, Bcl/Bax ratio: 3.1 ± 1.7] (Fig. 2(A–D)). 3.4. Effects of erythropoietin on cisplatin induced nitro-oxidative stress In comparison to control group, we found that cisplatin injection was associated with a significant increase (p < 0.05) in lipid peroxide marker [MDA (nmol/mg protein) [198.2 ± 29.02 vs 112.6 ± 3.2], and significant reductions (p < 0.05) of both SOD (unit/mg protein) [0.87 ± 0.19 vs 2.4 ± 0.38] and glutathione (nmol/mg protein) [25.7 ± 5.3 vs 43.01 ± 3.4]. However, co-administration of erythropoietin was found to significantly ameliorate (p < 0.05) cisplatin induced oxidative stress, with a significant decrease (p < 0.05) of MDA and significant increases (p < 0.05) of both SOD and glutathione [MDA (nmol/mg protein):122.6 ± 6.8, SOD (unit/mg protein): 1.9 ± 0.33, glutathione (nmol/mg protein): 34.19] (Fig. 3(A–C)). In addition, we noted a significant reduction (p < 0.05) in the level of nNOS gene expression [0.5 ± 0.31 vs 1.5 ± 0.31] that was associated with a significant increase (p < 0.05) of iNOS gene expression [0.1 ± 0.04 vs 0.001 ± 0.001] after cisplatin injection. We found also that erythropoietin treatment significantly reduced (p < 0.05) iNOS gene expression [0.02 ± 0.01], without significant effect on nNOS gene expression [0.72 ± 0.13] (Fig. 4(A, B)). 3.5. Effects of erythropoietin on cisplatin induced sciatic nerve NFKB gene expression and TNF level Significant increases (p < 0.05) in the TNF level (pg/ml) [97.81 ± 18.37 vs 30.4 ± 2.25] and NFKB gene expression [1.2 ± 0.54 vs 0.12 ± 0.03] were observed in cisplatin injected group in comparison to control group. Erythropoietin injection was associated with significant reduction (p < 0.05) in the level of TNF (pg/ml) [50.46 ± 9.24], without significant effect on NFKB gene expression (Fig. 5(A, B)).
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Table 1 Erythropoietin effects on cisplatin induced neuropathy. Control Cold withdraw latency (s) Hot withdraw latency (s) Sensory nerve conductive velocity (SNCV) (m/s) Sensory amplitude potential (mV) Motor nerve conductive velocity (MNCV) (m/s) Motor amplitude potential (mV)
6.5 5.0 10.2 13.1 4.1
± ± ± ± ±
Cisplatin 1.3 1.3 2.3 6.5 1.0
5.8 ± 1.3
14.9 8.5 0.6 6.5 0.9
± ± ± ± ±
0.08 $ 1.3 $ 0.2 $ 5.3$ 0.1 $
2.2 ± 0.4 $
Erythropoietin + cisplatin 4.3 1.4 8.1 2.8 6.9
± ± ± ± ±
1.3 # 0.2 # 1.9 # 1.6 1.1 #
7.8 ± 1.3 #
All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
4. Discussion Our findings further support the anti-apoptotic neuroprotective effects of erythropoietin during cisplatin induced neurotoxicity. The observed nitro-oxidative stress ameliorating effects of erythropoietin during cisplatin neurotoxicity could partially explain cells apoptotic resistance associated with erythropoietin treatment. Similar to previous studies (Bardos et al., 2003; Li et al., 2002; von Schlippe et al., 2001), significant reductions of nerve conductive velocities and compound action potential amplitude of large myelinated fibers were observed after cisplatin injection. In addition, we found that cisplatin treatment could also influence small fibers that are closely involved in thermal pathway.
In our study, the neuropathy induced by cisplatin treatment was associated with histological axonal degeneration and loss of myelinated nerve fibers. In addition, significant increases in proapoptotic markers [caspase 3, Bax gene] and significant decreases in anti-apoptotic markers [Bcl gene, Bcl/Bax] were observed after cisplatin challenge. Previous clinical and experimental studies demonstrated that the hallmarks of neuropathy and cell apoptosis is the specific cisplatin-DNA adduct lesions accumulation (Roelofs et al., 1984; Yoon et al., 2009). It was reported that cisplatin-induced genotoxic stress activates mitochondrial apoptotic pathway that is largely mediated through Bcl-2 family proteins. Bcl-2 family proteins include both pro-apoptotic members (such as Bax) that promote
Fig. 1. (A–C) Erythropoietin ameliorated cisplatin associated sciatic nerve apoptotic changes (hematoxylin and eosin, ×400).
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Fig. 2. (A–D) Erythropoietin (EPO) reversed cisplatin (CDDP) associated changes in pro-apoptotic markers, Bax (B) and Caspase3 (D), anti-apoptotic marker, Bcl (A), and Bcl/Bax ratio (C). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
mitochondrial permeability, and anti-apoptotic members (such as Bcl-2) that inhibit Bax effects, or inhibit the mitochondrial release of cytochrome c (cyt c). The release of cytochrome c from mitochondria, induced by stress, translocates Bax and truncated-Bid from cytosol to mitochondria and decreased expression of Bcl-2; which activates the caspase-9 molecules (upon cleavage of the bound zymogen procaspases-9), which in turn activate caspase-3; leading to chromatin condensation and DNA fragmentation (Bagci et al., 2006; Hong et al., 2012). Several neuroprotective strategies, which aim at a reduced accumulation of drug-induced DNA adducts and improving the survival of nerve despite of their actual damage burden have been previously investigated. However, erythropoietin seems to be the most promising approach to reduce cisplatin induced peripheral neuropathy because of its ability to increase the resistance of neuronal cell to neurotoxin effects of cisplatin (Beijers et al., 2012; Cascinu et al., 2002; Hilpert et al., 2005; Kassem and Yassin, 2010; Keswani et al., 2011; Ocean and Vahdat, 2004). Yoon et al. (2009) previously reported that erythropoeitin-through erythropoietin receptors (EpoR)-ameliorated cisplatin-induced neuropathy in mice, and that the observed neuroprotective effects seems to be mediated by enhancing the cell resistance to side effects of Cisplatin rather than by influencing the formation or repair rates of cisplatin-induced cross-links in the nuclear DNA of neuron. In our study we found that erythropoietin treatment was associated with changes in the accessible levels of two members of
the Bcl-2 family, Bax and Bcl-2, as well as Caspase 3. Erythropoietin treatment significantly decreases Bax and Caspase3 gene expression and significantly increases Bcl-2 gene induced by cisplatin. Our results could suggest that erythropoietin interfere with cisplatin activated mitochondria apoptotic pathway, an effect that could explain the observed erythropoietin neurophysiological alterations. Recently the role of reactive oxygen species (ROS) in cisplatin activated mitochondrial apoptotic pathway was suggested (Marullo et al., 2013). ROS are associated with enhancement of lipid peroxidation, depletion of the sulfhydryl groups, alteration of the signal transduction pathways, calcium homeostasis and DNA damage (Florea and Busselberg, 2011). In our study the cisplatin induced ROS effects could be partial explained by cisplatin associated effects on glutathione and SOD antioxidant enzymes. We found a significant decrease in sciatic nerve glutathione and SOD levels associated with a significant increase in MDA level. Among the antioxidant enzymes, both catalase and glutathione peroxidase play a critical role in reducing levels of hydrogen peroxide and preventing formation of ROS. In addition, superoxide dismutase (SOD) acts to prevent the accumulation of the potentially damaging superoxide anion. Therefore, activation of one or more of these enzymes would suppress accumulation of ROS and protect neurons against oxidative stress (Cheng and Mattson, 1995).
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Fig. 3. (A–C) Erythropoietin (EPO) prevents the cisplatin (CDDP) induced reduction of antioxidant enzyme, superoxide dismutase reductase (SOD) (B) and glutathione level (C), and associated increase in malondialdehyde (MDA) level (A). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
While Pratibha et al. (2006) previously reported a significant decrease in glutathione, most abundant thiol-containing antioxidant, after cisplatin therapy, both Godwin et al. (1992) and Ishikawa and Ali-Osman (1993) previously noted that cells that are resistant to cisplatin have elevated levels of glutathione
(GSH). Therefore, whether over-expression of glutathione contributes to or combats cisplatin resistance is still mater of debate. However, our results could suggest that low level of glutathione could partial contribute to cisplatin associated neurotoxic effects.
Fig. 4. (A, B) Erythropoietin (EPO) reduced the cisplatin (CDDP) induced nitric oxide synthase (iNOS) gene expression (B), and had no effects on cisplatin associated reduction in neuronal nitric oxide synthase (nNOS) gene expression (A). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
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Fig. 5. (A, B) Erythropoietin effects on cisplatin associated changes in sciatic nerve TNF level (A) and nuclear factor kappa B (NFKB) gene expression (B). All values are expressed in means ± SEM. $ p < 0.05 vs control, # p < 0.05 vs CDDP treated group (8 rats per group).
Similar to Ognjanovic et al. (2012), who reported significant decreases in the renal activity of antioxidant defence enzymes after cisplatin injection, we found a significant reduction in sciatic nerve SOD level after cisplatin challenge. An observation that could further explain cisplatin induced ROS effects. We suggest that erythropoietin observed anti-apoptotic effects could partial explained by the noted effects of erythropoietin on the antioxidant enzyme [both SOD and glutathione were significantly increased after erythropoietin treatment]. Our observations further support Sakanaka et al. (1998), who demonstrated that erythropoietin could increase the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase in neurons. In addition, Bailey et al. (2014) reported the potent scavenging property of erythropoietin and its capacity to inhibit Fenton chemistry through catalytic iron chelation. NO has been previously commented as double-edged sword with respect to its neurotoxicity and neuroprotection (Boje, 2004). While Dawson and Dawson (2004) had found that inducible nitric oxide synthase (iNOS)-derived NO-induce neuronal apoptosis, and that could be mediated via both caspase-dependent and caspase-independent [e.g., poly (ADP-ribose) polymerase-1 mediated] mechanisms, Keswani et al. (2011) previously reported that neuronal nitric oxide synthase (nNOS)-derived NO serves as an important neuronal protection through induction of hypoxiainducible factor (HIF)-1, the transcriptional activator of several genes that potentially may be involved in the neuroprotection associated with ischemic preconditioning (Semenza, 2010; Abe et al., 2003; Keswani et al., 2011). Our study further support previous few studies that reported the role of NO in cisplatin activated mitochondria apoptotic pathway (Lee et al., 2004; Sharma et al., 2005). In our study, we found that erythropoietin significantly reduced cisplatin induced iNOS gene expression. Meanwhile, no significant effect on cisplatin associated down-regulation of nNOS was observed with concomitant erythropoietin administration. Another factor that could contribute to cisplatin activated mitochondrial apoptotic pathway is the observed significant increases in sciatic nerve NFKB gene expression and TNF level. It was reported that ionizing radiation and anticancer drugs, which primarily target DNA, may activate NFKB via mechanisms different from the conventional IKK/IB␣/NFB pathway. Yeh et al. (2004) suggested that cisplatin activated Protein phosphatase 4
(PP4; also known as protein phosphatase X), a novel Ser/Thr protein phosphatase. PP4 had been shown to activate NFKB by direct interaction with and dephosphorylation of the NFKB p65 subunit (Hu et al., 1998). Treatment with cisplatin was observed to be associated with an increase in TNF gene expression – by extracellular-signalregulated kinases (ERK) – and activation of caspase-3 that trigger cisplatin-induced apoptosis (Basu and Tu, 2005; Jo et al., 2005). In addition, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) interacts with the death receptors, DR4 and DR5, and activates caspases, leading to apoptosis via the extrinsic pathway, in contrast to the mitochondria-mediated intrinsic death signaling pathway that is activated following cytotoxic stresses (Shamimi-Noori et al., 2008; Wenger et al., 2006). In spite that previous studies suggested that the phosphatidylinositol 3-kinase/Akt and NFB could be involved in erythropoietin induced anti-apoptotic pathways (Digicaylioglu and Lipton, 2001; Kretz et al., 2005; Wang et al., 1998), we could not find significant effect of erythropoietin on cisplatin induced NFKB gene expression. This difference could be due to differences in the doses and experimental protocol. In addition, our results could suggest that TNF reduction effect of erythropoietin seems to contribute to its anti-apoptotic effects. In light of previous data we suggest that erythropoietin may exert its neuroprotective effects by its scavenging property of ROS through activation of anti-oxidant enzymes and its ability to reduce TNF. 5. Conclusion Our results further support the effectiveness of erythropoietin as a neuroprotective anti-apoptotic drugs during cisplatin induced neurotoxicity. Erythropoietin seems to ameliorate nitro-oxidative stress and the increased TNF induced by cisplatin challenge. Our results represent one step towards understanding the mechanism underlying erythropoietin anti-apoptotic property. Acknowledgment The skillful technical assistance of Afaf, Aza and Tarek is appreciated.
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