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Oxaliplatin administration increases expression of the voltage-dependent calcium channel α2δ-1 subunit in the rat spinal cord
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Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord Q2
Ken Yamamoto a, Mayuko Tsuboi b, Toshie Kambe c, Kenji Abe e, Yoshihiko Nakatani b, Kazuyoshi Kawakami f, Iku Utsunomiya d, Kyoji Taguchi b, * a
Department of Education and Research Center for Clinical Pharmacy, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan Department of Medicinal Pharmacology, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan Department of Pharmacology, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan d Department of Developmental Education, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan e Department of Pharmacology, School of Pharmaceutical Sciences, Ohu University, 31-1 Tomitamachi, Koriyama, Fukushima 963-8611, Japan f Department of Pharmacy, Cancer Institute Hospital, 3-10-6 Ariake, Koto-Ku, Tokyo 135-8550, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2015 Received in revised form 11 January 2016 Accepted 20 January 2016 Available online xxx
Oxaliplatin is a chemotherapeutic agent that is effective against various types of cancer including colorectal cancer. Acute cold hyperalgesia is a serious side effect of oxaliplatin treatment. Although the therapeutic drug pregabalin is beneficial for preventing peripheral neuropathic pain by targeting the voltage-dependent calcium channel a2d-1 (Cava2d-1) subunit, the effect of oxaliplatin-induced acute cold hypersensitivity is uncertain. To analyze the contribution of the Cava2d-1 subunit to the development of oxaliplatin-induced acute cold hypersensitivity, Cava2d-1 subunit expression in the rat spinal cord was analyzed after oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that 6 mg/kg oxaliplatin-induced cold hypersensitivity 2 and 4 days later. Oxaliplatin-induced acute cold hypersensitivity 4 days after treatment was significantly inhibited by pregabalin (50 mg/kg, p.o.). Oxaliplatin (6 mg/kg, i.p.) treatment increased the expression level of Cava2d-1 subunit mRNA and protein in the spinal cord 2 and 4 days after treatment. Immunohistochemistry showed that oxaliplatin increased Cava2d-1 subunit protein expression in superficial layers of the spinal dorsal horn 2 and 4 days after treatment. These results suggest that oxaliplatin treatment increases Cava2d-1 subunit expression in the superficial layers of the spinal cord and may contribute to functional peripheral acute cold hypersensitivity. © 2016 Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Oxaliplatin Voltage-dependent calcium channel a2d-1 subunit Spinal cord Pregabalin Acute cold hypersensitivity
1. Introduction Oxaliplatin, a platinum-based chemotherapeutic drug, is widely used for the treatment of various types of cancer including colorectal cancer. Oxaliplatin plus capecitabine (XELOX) has been established as adjuvant therapy for stage II/III colon cancer. However, its major dose-limiting side effect is peripheral sensory neuropathy, which is characterized by paresthesia or dysesthesia
* Corresponding author. Department of Medicinal Pharmacology, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan. Tel.: þ81 42 721 1594; fax: þ81 42 721 1588. E-mail address: [email protected] (K. Taguchi). Peer review under responsibility of Japanese Pharmacological Society.
triggered by exposure to cold (1,2). Oxaliplatin-induced neurotoxicity manifests as rapid-onset neuropathic symptoms that are exacerbated by cold exposure and as chronic neuropathy that develops after several treatment cycles (3,4). The symptoms of oxaliplatin-induced acute peripheral neuropathy occur in 85e95% of patients within hours of infusion (1). Voltage-dependent calcium channels (VDCCs) have emerged as potential ion channel targets for the treatment of neuropathic pain (5,6). VDCCs are composed of a main pore-forming a1 subunit, together with auxiliary a2d, b, and g subunits. VDCC alpha-2-delta1 (Cava2d-1) subunit protein and mRNA are upregulated in dorsal root ganglia (DRGs) in models of peripheral nerve injury (7). Previous reports have suggested that peripheral nerve injury induces upregulation of Cava2d-1 subunit protein in the dorsal spinal cord
http://dx.doi.org/10.1016/j.jphs.2016.01.006 1347-8613/© 2016 Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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and contributes to the development of tactile allodynia (8). Cava2d1 subunit protein is upregulated in superficial layers of the dorsal horn after spinal nerve ligation-induced nerve injury (9,10). Moreover, Cava2d-1 subunit expression levels are significantly increased in the dorsal, but not ventral, spinal cord following spinal cord injury (11). Increased Cava2d-1 subunit expression is observed in the dorsal horn in a rat model of chemotherapy-evoked painful peripheral neuropathy (12). Increased Cava2d-1 subunit expression in the spinal cord after spinal cord injury and the neuropathic pain model is likely an important mediator of pain maintenance and development. Binding of pregabalin, a structural analog of gabapentin, to the Cava2d-1 subunit may be responsible for its efficacy in the treatment of neuropathic pain (10,13) and may play a key role in this inhibitory action in chemotherapy-induced peripheral neuropathy. Pregabalin is now commonly used to treat neuropathic pain in rodent models and patients (14,15). However, the participation of the Cava2d-1 subunit in oxaliplatin-induced acute cold hypersensitivity remains unclear. Accordingly, we hypothesized that acute cold hypersensitivity after oxaliplatin treatment is mediated by upregulation of the Cava2d-1 subunit in the spinal cord. To test this hypothesis, using a rat model of oxaliplatin-induced acute cold hypersensitivity, we investigated the effects of oxaliplatin treatment on Cava2d-1 subunit expression in the spinal cord and the mediators of pain perception, and whether pregabalin alters acute cold hypersensitivity after oxaliplatin treatment. 2. Materials and methods 2.1. Experimental animals Male Wistar rats weighing 240e330 g were used in the present study. All rats were housed individually under automatically controlled environmental conditions using a 12-h lightedark cycle (lights on from 08:00 to 20:00) with free access to food and water. All animals were quarantined in centralized animal facilities for at least 7 days upon arrival. Each animal was used only once. Experiments were carried out according to the guidelines for animal care and use published by the National Institutes of Health and the committee of Showa Pharmaceutical University. 2.2. Drugs One dose (6 mg/kg) of oxaliplatin (Elplat®) was intraperitoneally (i.p.) administered (Yakult Co., Ltd., Tokyo, Japan). Oxaliplatin was dissolved in a 5% glucose solution at a concentration of 2 mg/ml. The volume injected depended on the animal's weight to ensure i.p. injections of less than 2.5 ml. The control rats were injected by the same route with 5% glucose solution according to a volume that was adjusted to the weight of each rat. Pregabalin (SigmaeAldrich Inc., St. Louis, MO) was administered orally (p.o.; 50 mg/kg) 4 days after oxaliplatin administration. All other chemicals and materials were purchased from standard sources and were of reagent grade or higher. 2.3. Acetone spray test for acute cold hypersensitivity Observers blinded to the experimental conditions tested the rats with the acetone spray test at the same time on days 0, 2, 4, and 7 after oxaliplatin treatment. To estimate cold sensitivity of both hind paws, acetone (Kanto Chemical Co., Inc., Tokyo, Japan) was used following modification of previously described methods (16). Rats were placed in a clear plastic box (23 23 12 cm) with a wire mesh floor and allowed to habituate for 30 min prior to testing. After habituation, 50 ml fluid (acetone) was sprayed on the plantar
surface of the hind paws using a MicroSprayer® (Penn Century Inc., Philadelphia, PA). Paw withdrawal response, defined as flinching, licking, or biting of the limb, was measured within 1 min after the start of the acetone spray. The acetone spray test was performed alternately five times for each hind paw, and the paw withdrawal response was expressed as an overall percentage response. The effects of the acetone spray were repeatedly evaluated over time after oxaliplatin (6 mg/kg) or 5% glucose administration. 2.4. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) Rats were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) on days 1, 2, 4, or 7 after the start of oxaliplatin (6 mg/kg, i.p.), and the spinal cord (L4e6) was removed. Total RNA was extracted using the NucleoSpin® RNA II kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). RT-PCR was performed as follows using the PrimeScript® RT-PCR kit (Takara Bio Inc., Kusatsu, Japan) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 mg total RNA with PrimeScript RTase using an Oligo dT Primer at 42 C for 30 min. Each cDNA was subjected to PCR amplification using gene-specific primers. b-actin expression was used as an internal control in RT-PCR. The primer sequences for amplification of Cava2d-1 and b-actin were as follows: Cava2d-1 (forward) 50 -GAACTCAACTGGACAAGTGCCT-30 ; Cava2d-1 (reverse) 50 -CCATCCACTGAATAGGTCCTC-30 ; b-actin (forward) 50 -GGGTATGGAATCCTGTGGCA-30 ; b-actin (reverse) 50 -CGCAGCTCAGTAACAGTCCG-30 . Amplification conditions were: pre-denaturation for 1 min at 94 C, denaturation for 30 s at 94 C, renaturation for 30 s at 55 C, and extension for 1 min at 72 C. Aliquots of the RT-PCR products were separated on a 1% agarose gel and visualized with ethidium bromide. The PCR bands were quantified and normalized to the control band with ImageJ (National Institutes of Health Image software, version 1.35d, Bethesda, MD). 2.5. Western blot analysis On days 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) administration, rats were deeply anesthetized with pentobarbital (50 mg/kg, i.p.). Spinal cords (L4e6) were removed and homogenized in cold extraction buffer consisting of 10 mM TriseHCl buffer at pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% deoxycholate. The homogenates were centrifuged for 30 min at 15,000 g at 4 C, and the supernatant was collected. Total protein in the supernatant (30 mg) was electrophoresed on an SDS-polyacrylamide gel (7.5%), and separated proteins were transferred to polyvinylidene fluoride membranes. Anti-Cava2d-1 antibody (Alomone Labs, Jerusalem, Israel; 1:200) was used, and anti-b-actin antibody (SigmaeAldrich) was used as an internal control. Horseradish peroxidase-labeled anti-rabbit antibody (1:2000) was used as the secondary antibody (SigmaeAldrich). Specific bands were detected using enhanced chemiluminescence plus the TM Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's protocol. The intensities of immunoreactive bands were analyzed with MultiGage Ver.3 software (Fuji Film, Tokyo, Japan). 2.6. Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 20 ml potassiumfree phosphate-buffered saline (Kþ-free PBS; pH 7.4) followed by 50 ml of 4% paraformaldehyde solution. The spinal cord (L4e6) was removed, post-fixed for 3 h, cryoprotected overnight in 25% sucrose solution, and stored at 80 C until use. Spinal cords were cut at 10-
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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mm thickness, thaw-mounted on silane-coated glass slides, and airdried overnight at room temperature. Spinal cord sections were incubated with excess blocking buffer containing 2% skim milk in 0.1% Triton X-100 in Kþ-free PBS and subsequently reacted overnight at 4 C with anti-Cava2d-1 subunit antibodies (SigmaeAldrich, 1:200) in 2% bovine serum albumin/0.1% Triton X-100 in Kþ-free PBS. The sections were then incubated in fluorescein isothiocyanate-conjugated anti-rabbit IgG (SigmaeAldrich, 1:200) for 2 h at room temperature. The immunostained sections were treated with Permafluor™ (Thermo Shandon, Pittsburgh, PA), mounted on slides, covered with microslips, and observed with an Olympus laser-scanning confocal microscope (FLUOVIEW BW50, Olympus, Tokyo, Japan) at a wavelength of 495 nm. A total of five sections (90 mm apart) were randomly selected from each spinal cord. Optical density (OD) of the stained sites was determined with the ImageJ program using a computer. The OD of superficial layers of the spinal cord was calculated for day 4 after oxaliplatin treatment. 2.7. Statistical analysis All data are expressed as the mean ± SEM. In the behavioral study, the significance of the difference in the response rate between vehicle and oxaliplatin was calculated using two-way analysis of variance (ANOVA) followed by the Student's or Aspin-Welch's t-test. The significance of the difference among the vehicle, oxaliplatin, and pregabalin groups was calculated using one-way ANOVA followed by the TukeyeKramer method. The statistical significance of differences in other experiments was analyzed using one-way ANOVA followed by Dunnett's multiple comparison test. Immunohistochemical staining data are shown as the percentage of OD obtained from each oxaliplatin-treated rat (OD data on day 0 were defined as 100%). Statistical significance was accepted at P < 0.05. 3. Results 3.1. Effects of oxaliplatin on acute cold hypersensitivity We first investigated the effect of oxaliplatin (6 mg/kg, i.p., n ¼ 6) on acute cold hypersensitivity. Before the single dose of oxaliplatin (day 0), we found no significant differences in the number of withdrawal responses in any groups in the acetone spray test. The withdrawal response rate with the acetone spray test was significantly increased (F[3,40] ¼ 3.69, P < 0.05) with 6 mg/kg oxaliplatin at 2 days (day 2) and 4 days (day 4) after a single dose of oxaliplatin compared to 5% glucose treatment (Fig. 1A). This oxaliplatin-induced acute cold hypersensitivity decreased gradually up to 7 days (day 7) after the treatment. 3.2. Effect of pregabalin on oxaliplatin-induced acute cold hyperalgesia The effects of pregabalin on oxaliplatin-induced (6 mg/kg i.p., n ¼ 6) acute cold hyperalgesia are shown in Fig.1B. Administration of pregabalin (50 mg/kg p.o., n ¼ 6) 30 min before the acetone test significantly inhibited acute cold hypersensitivity on day 4 compared to oxaliplatin treatment alone (F ¼ 16.17, P < 0.01). Thus, pregabalin reversed oxaliplatin-induced acute cold hypersensitivity. 3.3. Effect of oxaliplatin on expression of Cava2d-1 subunit mRNA in the spinal cord We investigated the expression of Cava2d-1 subunit mRNA during cold hypersensitivity after oxaliplatin administration. We removed the spinal cord on days 0, 2, 4, and 7 after administration
Fig. 1. Time course of acute cold hypersensitivity after oxaliplatin administration. (A) The acetone spray test was used to measure the acute cold response in rats on days 0 (before administration), 2, 4, and 7 after oxaliplatin administration (6 mg/kg, i.p.). Data are the mean ± SEM of n ¼ 6 rats. *P < 0.05, **P < 0.01, Two-way ANOVA, followed by Student's or Aspin-Welch's t-test was used. (B) Effect of pregabalin treatment on oxaliplatin-induced acute cold hypersensitivity in rats. Behavioral responses of pregabalin (50 mg/kg, p.o.) on acute cold hypersensitivity induced by oxaliplatin treatment (6 mg/kg) on day 4. Graph shows the mean ± SEM of the response frequency to cold stimulation by the acetone spray test. Oxaliplatin-induced acute cold hypersensitivity was significantly inhibited by pretreatment with pregabalin. n ¼ 6 per group. **P < 0.01 vs. 5% glucose (vehicle), ##P < 0.01 vs. oxaliplatin. Statistical analysis was performed using one-way ANOVA with the TukeyeKramer method.
of 6 mg/kg oxaliplatin, and Cava2d-1 subunit mRNA expression was quantified with RT-PCR. As shown in Fig. 2, 6 mg/kg oxaliplatin significantly increased Cava2d-1 subunit mRNA expression (F ¼ 3.93, P < 0.05) in the spinal cord on day 2 (119.0 ± 2.9%, n ¼ 5, P < 0.05) and day 4 (138.5 ± 13.9%, n ¼ 5, P < 0.05). Cava2d-1 subunit mRNA expression was significantly increased and maximal on day 4 and returned to control levels 7 days after administration of oxaliplatin (Fig. 2). 3.4. Effect of oxaliplatin treatment on Cava2d-1 subunit protein in the spinal cord We removed the spinal cord on days 0, 2, 4, and 7 after administration of 6 mg/kg oxaliplatin treatment, and Cava2d-1 subunit protein expression was quantified by western blot analysis. As shown in Fig. 3, 6 mg/kg oxaliplatin treatment significantly increased Cava2d-1 subunit protein expression (F ¼ 6.52, P < 0.01) in the spinal cord on day 2 (184.3 ± 18.5%, n ¼ 5, P < 0.01) and day 4 (218.6 ± 26.9%, n ¼ 5, P < 0.01). A significant increase in Cava2d-1
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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labeled for the Cava2d-1 subunit protein in rats. As shown in Fig. 4B, 6 mg/kg oxaliplatin treatment significantly increased the OD percentage (F ¼ 4.01, P < 0.05) in superficial layers of the spinal dorsal horn on day 2 (130.6 ± 16.1%, n ¼ 5, P < 0.01) and day 4 (143.1 ± 12.2%, n ¼ 5, P < 0.01). Thus, oxaliplatin-induced (6 mg/kg) enhancement of Cava2d-1 subunit protein expression resulted from upregulation of expression in superficial layers of the spinal dorsal horn. 4. Discussion
Fig. 2. Oxaliplatin administration increases voltage-dependent calcium channel subunit a2d-1 (Cava2d-1) mRNA in the spinal cord. Effect of oxaliplatin (6 mg/kg, i.p.) on Cava2d-1 subunit mRNA expression in the rat spinal cord (L4e6) on days 0 (before administration), 2, 4, and 7 was measured. Histograms show the relative amount of Cava2d-1 subunit mRNA in oxaliplatin-treated rats compared with day 0. Data are the mean ± SEM. n ¼ 5 for oxaliplatin administration. *P < 0.05, versus day 0. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.
Fig. 3. Effect of oxaliplatin on Cava2d-1 subunit protein expression in the rat spinal cord (L4e6). Cava2d-1 subunit and b-actin protein in the spinal cord on days 0 (before administration), 2, 4, and 7 was measured. Cava2d-1 subunit protein expression was normalized to b-actin expression. Histograms show the relative amount of Cava2d-1 subunit protein in oxaliplatin-treated (6 mg/kg, i.p.) rats. The western blot shows representative data. Data are the mean ± SEM. n ¼ 5, **P < 0.01 versus day 0. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.
subunit protein expression was found in oxaliplatin-treated rats compared to day 0 values, and expression returned to control levels 7 days after administration of oxaliplatin (Fig. 3).
3.5. Immunohistochemistry Immunohistochemistry revealed that most of the Cava2d-1 subunit protein in the spinal cord was localized in superficial layers of the spinal dorsal horn (Fig. 4A). Using computerized OD image analysis, we found that superficial layers of the spinal cord were positively
Our present study indicated that single administration of oxaliplatin produced acute cold hypersensitivity in rats that was attenuated by pregabalin. In previous experimental studies, single or multiple doses of oxaliplatin produce acute cold allodynia and cold hypersensitivity (17e19). Also, using the acetone spray test, our previous study showed that oxaliplatin induces acute cold hypersensitivity in a dose-dependent manner (20). Thus, the results of our study agree with these previously published findings (4,21). We first demonstrated that oxaliplatin treatment (6 mg/kg) significantly increased the expression of Cava2d-1 mRNA and protein in the rat spinal cord, and immunohistochemistry revealed that this Cava2d-1 protein expression was increased in superficial layers of the spinal cord. Increased Cava2d-1 expression in a transgenic mouse line leads to dorsal horn neuron hyperexcitability (8,22). Cava2d-1 subunit expression is upregulated in the spinal cord following an experimental nerve crush injury (23,24). This upregulation correlates with the onset of allodynia, in which the sensation of non-noxious touch causes pain-related behaviors. These data suggest that the upregulation of Cava2d-1 subunits in the spinal cord may contribute to oxaliplatin treatment-induced acute cold hypersensitivity. Pregabalin, a structural analog of gabapentin, is a ligand at the Cava2d-1 subunit, and binding at this site reduces calcium influx into nerve terminals and consequently reduces neurotransmission (25e28). Previous reports have shown that pregabalin alleviates neuropathic pain such as pain caused by peripheral nerve ligation and diabetic neuropathic pain in various models (29,30). Intrathecally administered pregabalin attenuates cold allodynia in the acetone test in a dose-dependent manner in neuropathic pain models (32). Clinically, pregabalin significantly reduces the severity of oxaliplatin-induced sensory neuropathy in patients (15). In the present study, we observed that administration of pregabalin completely reversed oxaliplatin-induced acute cold hypersensitivity. Recently, we reported that paclitaxel treatment increases VDCC current in small- and medium-diameter rat DRG neurons and upregulates Cava2d-1 subunit protein expression in DRGs (31). Cava2d-1 mRNA and protein are also increased in DRGs following paclitaxel treatment (33). Our current results show that oxaliplatin treatment-induced increased expression of Cava2d-1 subunit protein in the spinal cord. Interestingly, Cava2d-1 subunit upregulation following administration of oxaliplatin is similar to the increase in Cava2d-1 subunits in the paclitaxel-induced neuropathic pain model. Thus, this upregulation may be correlated with the onset of oxaliplatin-induced acute cold hypersensitivity. Moreover, Bauer et al. (10) reported the importance of trafficking of the Cava2d-1 subunit from DRGs to the spinal dorsal horn during the development of neuropathic pain and subsequent alleviation with pregabalin. The efficacy of pregabalin for alleviation of neuropathic pain is due to binding to Cava2d-1 subunits (13). We hypothesize that the superficial layer of the spinal cord is an important site of action for pregabalin, which may exert its antiallodynic effect mainly on the spinal cord (32). In addition, pregabalin attenuates spinal dorsal horn nociceptive responses in rat neuropathic pain models (34). The antiallodynic effect of pregabalin is mainly
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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Fig. 4. Immunohistochemistry of Cava2d-1 protein expression in the spinal cord (L4e6) after oxaliplatin treatment. (A) Spinal cord sections (10 mm thick) from days 0 (before administration), 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) treatment were stained with antibodies against Cava2d-1 subunit protein. White dashed line demarcates the border of the superficial layers of the spinal cord. (B) Optical density (OD) of Cava2d-1 subunit-stained spinal cord sections on days 0 (before administration), 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) administration. The means ± SEM of the OD for Cava2d-1 subunit protein expression are indicated by the columns and vertical bars. n ¼ 5, **P < 0.01 versus day 0. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.
mediated by spinal mechanisms, although supraspinal or peripheral mechanisms cannot be excluded. Thus, the increased expression of Cava2d-1 subunits in the spinal dorsal horn is a main target of the inhibitory action of pregabalin on oxaliplatin-induced acute cold hypersensitivity. These results indicate that pregabalin is a promising candidate drug for treatment of oxaliplatin-induced peripheral neuropathy. Thus, the anti-hyperalgesic action of pregabalin may be due to inhibition of increased Cava2d-1 subunit protein expression following oxaliplatin treatment in the spinal cord. Interestingly, pregabalin did not affect the results of the acetone test in control rats treated with 5% glucose. Pregabalin reduces the severity of oxaliplatin-induced sensory neuropathy (15), and thus, we hypothesize that pregabalin may have a therapeutic effect when the Cava2d-1 subunit protein levels are elevated in the spinal cord. On the other hand, single administration of oxaliplatin (3 mg/kg, i.p.) significantly increases expression of Cava2d-1 subunit mRNA in the mouse DRG but not in the spinal cord (35). Our result showed that Cava2d-1 subunit mRNA in the spinal cord was significantly increased 4 days after administration of oxaliplatin (6 mg/kg, i.p.). Gauchan et al. (35) studied mice 10 days after intraperitoneal administration of oxaliplatin, whereas our results were obtained 4 days after oxaliplatin treatment in rats. This may account for the reported differences, because Cava2d-1 subunit mRNA levels in neurons change over time. The different results between the two studies may also be due to different dosages (3 mg/kg or 6 mg/kg). In addition, our immunohistochemical results indicated that Cava2d-1 protein was significantly increased in superficial layers of the spinal cord 4 days after oxaliplatin treatment. In conclusion, we show that single administration of oxaliplatin increases Cava2d-1 subunit expression in superficial layers of the spinal cord. Moreover, pregabalin inhibits oxaliplatin-induced acute cold hypersensitivity.
Conflicts of interest The authors declare that there are no conflicts of interest.
Acknowledgments We thank Miss Tomomi Nakazato for technical assistance.
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Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord Q2
Ken Yamamoto a, Mayuko Tsuboi b, Toshie Kambe c, Kenji Abe e, Yoshihiko Nakatani b, Kazuyoshi Kawakami f, Iku Utsunomiya d, Kyoji Taguchi b, * a
Department of Education and Research Center for Clinical Pharmacy, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan Department of Medicinal Pharmacology, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan Department of Pharmacology, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan d Department of Developmental Education, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan e Department of Pharmacology, School of Pharmaceutical Sciences, Ohu University, 31-1 Tomitamachi, Koriyama, Fukushima 963-8611, Japan f Department of Pharmacy, Cancer Institute Hospital, 3-10-6 Ariake, Koto-Ku, Tokyo 135-8550, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2015 Received in revised form 11 January 2016 Accepted 20 January 2016 Available online xxx
Oxaliplatin is a chemotherapeutic agent that is effective against various types of cancer including colorectal cancer. Acute cold hyperalgesia is a serious side effect of oxaliplatin treatment. Although the therapeutic drug pregabalin is beneficial for preventing peripheral neuropathic pain by targeting the voltage-dependent calcium channel a2d-1 (Cava2d-1) subunit, the effect of oxaliplatin-induced acute cold hypersensitivity is uncertain. To analyze the contribution of the Cava2d-1 subunit to the development of oxaliplatin-induced acute cold hypersensitivity, Cava2d-1 subunit expression in the rat spinal cord was analyzed after oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that 6 mg/kg oxaliplatin-induced cold hypersensitivity 2 and 4 days later. Oxaliplatin-induced acute cold hypersensitivity 4 days after treatment was significantly inhibited by pregabalin (50 mg/kg, p.o.). Oxaliplatin (6 mg/kg, i.p.) treatment increased the expression level of Cava2d-1 subunit mRNA and protein in the spinal cord 2 and 4 days after treatment. Immunohistochemistry showed that oxaliplatin increased Cava2d-1 subunit protein expression in superficial layers of the spinal dorsal horn 2 and 4 days after treatment. These results suggest that oxaliplatin treatment increases Cava2d-1 subunit expression in the superficial layers of the spinal cord and may contribute to functional peripheral acute cold hypersensitivity. © 2016 Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Oxaliplatin Voltage-dependent calcium channel a2d-1 subunit Spinal cord Pregabalin Acute cold hypersensitivity
1. Introduction Oxaliplatin, a platinum-based chemotherapeutic drug, is widely used for the treatment of various types of cancer including colorectal cancer. Oxaliplatin plus capecitabine (XELOX) has been established as adjuvant therapy for stage II/III colon cancer. However, its major dose-limiting side effect is peripheral sensory neuropathy, which is characterized by paresthesia or dysesthesia
* Corresponding author. Department of Medicinal Pharmacology, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan. Tel.: þ81 42 721 1594; fax: þ81 42 721 1588. E-mail address: [email protected] (K. Taguchi). Peer review under responsibility of Japanese Pharmacological Society.
triggered by exposure to cold (1,2). Oxaliplatin-induced neurotoxicity manifests as rapid-onset neuropathic symptoms that are exacerbated by cold exposure and as chronic neuropathy that develops after several treatment cycles (3,4). The symptoms of oxaliplatin-induced acute peripheral neuropathy occur in 85e95% of patients within hours of infusion (1). Voltage-dependent calcium channels (VDCCs) have emerged as potential ion channel targets for the treatment of neuropathic pain (5,6). VDCCs are composed of a main pore-forming a1 subunit, together with auxiliary a2d, b, and g subunits. VDCC alpha-2-delta1 (Cava2d-1) subunit protein and mRNA are upregulated in dorsal root ganglia (DRGs) in models of peripheral nerve injury (7). Previous reports have suggested that peripheral nerve injury induces upregulation of Cava2d-1 subunit protein in the dorsal spinal cord
http://dx.doi.org/10.1016/j.jphs.2016.01.006 1347-8613/© 2016 Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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and contributes to the development of tactile allodynia (8). Cava2d1 subunit protein is upregulated in superficial layers of the dorsal horn after spinal nerve ligation-induced nerve injury (9,10). Moreover, Cava2d-1 subunit expression levels are significantly increased in the dorsal, but not ventral, spinal cord following spinal cord injury (11). Increased Cava2d-1 subunit expression is observed in the dorsal horn in a rat model of chemotherapy-evoked painful peripheral neuropathy (12). Increased Cava2d-1 subunit expression in the spinal cord after spinal cord injury and the neuropathic pain model is likely an important mediator of pain maintenance and development. Binding of pregabalin, a structural analog of gabapentin, to the Cava2d-1 subunit may be responsible for its efficacy in the treatment of neuropathic pain (10,13) and may play a key role in this inhibitory action in chemotherapy-induced peripheral neuropathy. Pregabalin is now commonly used to treat neuropathic pain in rodent models and patients (14,15). However, the participation of the Cava2d-1 subunit in oxaliplatin-induced acute cold hypersensitivity remains unclear. Accordingly, we hypothesized that acute cold hypersensitivity after oxaliplatin treatment is mediated by upregulation of the Cava2d-1 subunit in the spinal cord. To test this hypothesis, using a rat model of oxaliplatin-induced acute cold hypersensitivity, we investigated the effects of oxaliplatin treatment on Cava2d-1 subunit expression in the spinal cord and the mediators of pain perception, and whether pregabalin alters acute cold hypersensitivity after oxaliplatin treatment. 2. Materials and methods 2.1. Experimental animals Male Wistar rats weighing 240e330 g were used in the present study. All rats were housed individually under automatically controlled environmental conditions using a 12-h lightedark cycle (lights on from 08:00 to 20:00) with free access to food and water. All animals were quarantined in centralized animal facilities for at least 7 days upon arrival. Each animal was used only once. Experiments were carried out according to the guidelines for animal care and use published by the National Institutes of Health and the committee of Showa Pharmaceutical University. 2.2. Drugs One dose (6 mg/kg) of oxaliplatin (Elplat®) was intraperitoneally (i.p.) administered (Yakult Co., Ltd., Tokyo, Japan). Oxaliplatin was dissolved in a 5% glucose solution at a concentration of 2 mg/ml. The volume injected depended on the animal's weight to ensure i.p. injections of less than 2.5 ml. The control rats were injected by the same route with 5% glucose solution according to a volume that was adjusted to the weight of each rat. Pregabalin (SigmaeAldrich Inc., St. Louis, MO) was administered orally (p.o.; 50 mg/kg) 4 days after oxaliplatin administration. All other chemicals and materials were purchased from standard sources and were of reagent grade or higher. 2.3. Acetone spray test for acute cold hypersensitivity Observers blinded to the experimental conditions tested the rats with the acetone spray test at the same time on days 0, 2, 4, and 7 after oxaliplatin treatment. To estimate cold sensitivity of both hind paws, acetone (Kanto Chemical Co., Inc., Tokyo, Japan) was used following modification of previously described methods (16). Rats were placed in a clear plastic box (23 23 12 cm) with a wire mesh floor and allowed to habituate for 30 min prior to testing. After habituation, 50 ml fluid (acetone) was sprayed on the plantar
surface of the hind paws using a MicroSprayer® (Penn Century Inc., Philadelphia, PA). Paw withdrawal response, defined as flinching, licking, or biting of the limb, was measured within 1 min after the start of the acetone spray. The acetone spray test was performed alternately five times for each hind paw, and the paw withdrawal response was expressed as an overall percentage response. The effects of the acetone spray were repeatedly evaluated over time after oxaliplatin (6 mg/kg) or 5% glucose administration. 2.4. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) Rats were deeply anesthetized with pentobarbital (50 mg/kg, i.p.) on days 1, 2, 4, or 7 after the start of oxaliplatin (6 mg/kg, i.p.), and the spinal cord (L4e6) was removed. Total RNA was extracted using the NucleoSpin® RNA II kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). RT-PCR was performed as follows using the PrimeScript® RT-PCR kit (Takara Bio Inc., Kusatsu, Japan) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 mg total RNA with PrimeScript RTase using an Oligo dT Primer at 42 C for 30 min. Each cDNA was subjected to PCR amplification using gene-specific primers. b-actin expression was used as an internal control in RT-PCR. The primer sequences for amplification of Cava2d-1 and b-actin were as follows: Cava2d-1 (forward) 50 -GAACTCAACTGGACAAGTGCCT-30 ; Cava2d-1 (reverse) 50 -CCATCCACTGAATAGGTCCTC-30 ; b-actin (forward) 50 -GGGTATGGAATCCTGTGGCA-30 ; b-actin (reverse) 50 -CGCAGCTCAGTAACAGTCCG-30 . Amplification conditions were: pre-denaturation for 1 min at 94 C, denaturation for 30 s at 94 C, renaturation for 30 s at 55 C, and extension for 1 min at 72 C. Aliquots of the RT-PCR products were separated on a 1% agarose gel and visualized with ethidium bromide. The PCR bands were quantified and normalized to the control band with ImageJ (National Institutes of Health Image software, version 1.35d, Bethesda, MD). 2.5. Western blot analysis On days 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) administration, rats were deeply anesthetized with pentobarbital (50 mg/kg, i.p.). Spinal cords (L4e6) were removed and homogenized in cold extraction buffer consisting of 10 mM TriseHCl buffer at pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% deoxycholate. The homogenates were centrifuged for 30 min at 15,000 g at 4 C, and the supernatant was collected. Total protein in the supernatant (30 mg) was electrophoresed on an SDS-polyacrylamide gel (7.5%), and separated proteins were transferred to polyvinylidene fluoride membranes. Anti-Cava2d-1 antibody (Alomone Labs, Jerusalem, Israel; 1:200) was used, and anti-b-actin antibody (SigmaeAldrich) was used as an internal control. Horseradish peroxidase-labeled anti-rabbit antibody (1:2000) was used as the secondary antibody (SigmaeAldrich). Specific bands were detected using enhanced chemiluminescence plus the TM Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's protocol. The intensities of immunoreactive bands were analyzed with MultiGage Ver.3 software (Fuji Film, Tokyo, Japan). 2.6. Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 20 ml potassiumfree phosphate-buffered saline (Kþ-free PBS; pH 7.4) followed by 50 ml of 4% paraformaldehyde solution. The spinal cord (L4e6) was removed, post-fixed for 3 h, cryoprotected overnight in 25% sucrose solution, and stored at 80 C until use. Spinal cords were cut at 10-
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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mm thickness, thaw-mounted on silane-coated glass slides, and airdried overnight at room temperature. Spinal cord sections were incubated with excess blocking buffer containing 2% skim milk in 0.1% Triton X-100 in Kþ-free PBS and subsequently reacted overnight at 4 C with anti-Cava2d-1 subunit antibodies (SigmaeAldrich, 1:200) in 2% bovine serum albumin/0.1% Triton X-100 in Kþ-free PBS. The sections were then incubated in fluorescein isothiocyanate-conjugated anti-rabbit IgG (SigmaeAldrich, 1:200) for 2 h at room temperature. The immunostained sections were treated with Permafluor™ (Thermo Shandon, Pittsburgh, PA), mounted on slides, covered with microslips, and observed with an Olympus laser-scanning confocal microscope (FLUOVIEW BW50, Olympus, Tokyo, Japan) at a wavelength of 495 nm. A total of five sections (90 mm apart) were randomly selected from each spinal cord. Optical density (OD) of the stained sites was determined with the ImageJ program using a computer. The OD of superficial layers of the spinal cord was calculated for day 4 after oxaliplatin treatment. 2.7. Statistical analysis All data are expressed as the mean ± SEM. In the behavioral study, the significance of the difference in the response rate between vehicle and oxaliplatin was calculated using two-way analysis of variance (ANOVA) followed by the Student's or Aspin-Welch's t-test. The significance of the difference among the vehicle, oxaliplatin, and pregabalin groups was calculated using one-way ANOVA followed by the TukeyeKramer method. The statistical significance of differences in other experiments was analyzed using one-way ANOVA followed by Dunnett's multiple comparison test. Immunohistochemical staining data are shown as the percentage of OD obtained from each oxaliplatin-treated rat (OD data on day 0 were defined as 100%). Statistical significance was accepted at P < 0.05. 3. Results 3.1. Effects of oxaliplatin on acute cold hypersensitivity We first investigated the effect of oxaliplatin (6 mg/kg, i.p., n ¼ 6) on acute cold hypersensitivity. Before the single dose of oxaliplatin (day 0), we found no significant differences in the number of withdrawal responses in any groups in the acetone spray test. The withdrawal response rate with the acetone spray test was significantly increased (F[3,40] ¼ 3.69, P < 0.05) with 6 mg/kg oxaliplatin at 2 days (day 2) and 4 days (day 4) after a single dose of oxaliplatin compared to 5% glucose treatment (Fig. 1A). This oxaliplatin-induced acute cold hypersensitivity decreased gradually up to 7 days (day 7) after the treatment. 3.2. Effect of pregabalin on oxaliplatin-induced acute cold hyperalgesia The effects of pregabalin on oxaliplatin-induced (6 mg/kg i.p., n ¼ 6) acute cold hyperalgesia are shown in Fig.1B. Administration of pregabalin (50 mg/kg p.o., n ¼ 6) 30 min before the acetone test significantly inhibited acute cold hypersensitivity on day 4 compared to oxaliplatin treatment alone (F ¼ 16.17, P < 0.01). Thus, pregabalin reversed oxaliplatin-induced acute cold hypersensitivity. 3.3. Effect of oxaliplatin on expression of Cava2d-1 subunit mRNA in the spinal cord We investigated the expression of Cava2d-1 subunit mRNA during cold hypersensitivity after oxaliplatin administration. We removed the spinal cord on days 0, 2, 4, and 7 after administration
Fig. 1. Time course of acute cold hypersensitivity after oxaliplatin administration. (A) The acetone spray test was used to measure the acute cold response in rats on days 0 (before administration), 2, 4, and 7 after oxaliplatin administration (6 mg/kg, i.p.). Data are the mean ± SEM of n ¼ 6 rats. *P < 0.05, **P < 0.01, Two-way ANOVA, followed by Student's or Aspin-Welch's t-test was used. (B) Effect of pregabalin treatment on oxaliplatin-induced acute cold hypersensitivity in rats. Behavioral responses of pregabalin (50 mg/kg, p.o.) on acute cold hypersensitivity induced by oxaliplatin treatment (6 mg/kg) on day 4. Graph shows the mean ± SEM of the response frequency to cold stimulation by the acetone spray test. Oxaliplatin-induced acute cold hypersensitivity was significantly inhibited by pretreatment with pregabalin. n ¼ 6 per group. **P < 0.01 vs. 5% glucose (vehicle), ##P < 0.01 vs. oxaliplatin. Statistical analysis was performed using one-way ANOVA with the TukeyeKramer method.
of 6 mg/kg oxaliplatin, and Cava2d-1 subunit mRNA expression was quantified with RT-PCR. As shown in Fig. 2, 6 mg/kg oxaliplatin significantly increased Cava2d-1 subunit mRNA expression (F ¼ 3.93, P < 0.05) in the spinal cord on day 2 (119.0 ± 2.9%, n ¼ 5, P < 0.05) and day 4 (138.5 ± 13.9%, n ¼ 5, P < 0.05). Cava2d-1 subunit mRNA expression was significantly increased and maximal on day 4 and returned to control levels 7 days after administration of oxaliplatin (Fig. 2). 3.4. Effect of oxaliplatin treatment on Cava2d-1 subunit protein in the spinal cord We removed the spinal cord on days 0, 2, 4, and 7 after administration of 6 mg/kg oxaliplatin treatment, and Cava2d-1 subunit protein expression was quantified by western blot analysis. As shown in Fig. 3, 6 mg/kg oxaliplatin treatment significantly increased Cava2d-1 subunit protein expression (F ¼ 6.52, P < 0.01) in the spinal cord on day 2 (184.3 ± 18.5%, n ¼ 5, P < 0.01) and day 4 (218.6 ± 26.9%, n ¼ 5, P < 0.01). A significant increase in Cava2d-1
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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labeled for the Cava2d-1 subunit protein in rats. As shown in Fig. 4B, 6 mg/kg oxaliplatin treatment significantly increased the OD percentage (F ¼ 4.01, P < 0.05) in superficial layers of the spinal dorsal horn on day 2 (130.6 ± 16.1%, n ¼ 5, P < 0.01) and day 4 (143.1 ± 12.2%, n ¼ 5, P < 0.01). Thus, oxaliplatin-induced (6 mg/kg) enhancement of Cava2d-1 subunit protein expression resulted from upregulation of expression in superficial layers of the spinal dorsal horn. 4. Discussion
Fig. 2. Oxaliplatin administration increases voltage-dependent calcium channel subunit a2d-1 (Cava2d-1) mRNA in the spinal cord. Effect of oxaliplatin (6 mg/kg, i.p.) on Cava2d-1 subunit mRNA expression in the rat spinal cord (L4e6) on days 0 (before administration), 2, 4, and 7 was measured. Histograms show the relative amount of Cava2d-1 subunit mRNA in oxaliplatin-treated rats compared with day 0. Data are the mean ± SEM. n ¼ 5 for oxaliplatin administration. *P < 0.05, versus day 0. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.
Fig. 3. Effect of oxaliplatin on Cava2d-1 subunit protein expression in the rat spinal cord (L4e6). Cava2d-1 subunit and b-actin protein in the spinal cord on days 0 (before administration), 2, 4, and 7 was measured. Cava2d-1 subunit protein expression was normalized to b-actin expression. Histograms show the relative amount of Cava2d-1 subunit protein in oxaliplatin-treated (6 mg/kg, i.p.) rats. The western blot shows representative data. Data are the mean ± SEM. n ¼ 5, **P < 0.01 versus day 0. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.
subunit protein expression was found in oxaliplatin-treated rats compared to day 0 values, and expression returned to control levels 7 days after administration of oxaliplatin (Fig. 3).
3.5. Immunohistochemistry Immunohistochemistry revealed that most of the Cava2d-1 subunit protein in the spinal cord was localized in superficial layers of the spinal dorsal horn (Fig. 4A). Using computerized OD image analysis, we found that superficial layers of the spinal cord were positively
Our present study indicated that single administration of oxaliplatin produced acute cold hypersensitivity in rats that was attenuated by pregabalin. In previous experimental studies, single or multiple doses of oxaliplatin produce acute cold allodynia and cold hypersensitivity (17e19). Also, using the acetone spray test, our previous study showed that oxaliplatin induces acute cold hypersensitivity in a dose-dependent manner (20). Thus, the results of our study agree with these previously published findings (4,21). We first demonstrated that oxaliplatin treatment (6 mg/kg) significantly increased the expression of Cava2d-1 mRNA and protein in the rat spinal cord, and immunohistochemistry revealed that this Cava2d-1 protein expression was increased in superficial layers of the spinal cord. Increased Cava2d-1 expression in a transgenic mouse line leads to dorsal horn neuron hyperexcitability (8,22). Cava2d-1 subunit expression is upregulated in the spinal cord following an experimental nerve crush injury (23,24). This upregulation correlates with the onset of allodynia, in which the sensation of non-noxious touch causes pain-related behaviors. These data suggest that the upregulation of Cava2d-1 subunits in the spinal cord may contribute to oxaliplatin treatment-induced acute cold hypersensitivity. Pregabalin, a structural analog of gabapentin, is a ligand at the Cava2d-1 subunit, and binding at this site reduces calcium influx into nerve terminals and consequently reduces neurotransmission (25e28). Previous reports have shown that pregabalin alleviates neuropathic pain such as pain caused by peripheral nerve ligation and diabetic neuropathic pain in various models (29,30). Intrathecally administered pregabalin attenuates cold allodynia in the acetone test in a dose-dependent manner in neuropathic pain models (32). Clinically, pregabalin significantly reduces the severity of oxaliplatin-induced sensory neuropathy in patients (15). In the present study, we observed that administration of pregabalin completely reversed oxaliplatin-induced acute cold hypersensitivity. Recently, we reported that paclitaxel treatment increases VDCC current in small- and medium-diameter rat DRG neurons and upregulates Cava2d-1 subunit protein expression in DRGs (31). Cava2d-1 mRNA and protein are also increased in DRGs following paclitaxel treatment (33). Our current results show that oxaliplatin treatment-induced increased expression of Cava2d-1 subunit protein in the spinal cord. Interestingly, Cava2d-1 subunit upregulation following administration of oxaliplatin is similar to the increase in Cava2d-1 subunits in the paclitaxel-induced neuropathic pain model. Thus, this upregulation may be correlated with the onset of oxaliplatin-induced acute cold hypersensitivity. Moreover, Bauer et al. (10) reported the importance of trafficking of the Cava2d-1 subunit from DRGs to the spinal dorsal horn during the development of neuropathic pain and subsequent alleviation with pregabalin. The efficacy of pregabalin for alleviation of neuropathic pain is due to binding to Cava2d-1 subunits (13). We hypothesize that the superficial layer of the spinal cord is an important site of action for pregabalin, which may exert its antiallodynic effect mainly on the spinal cord (32). In addition, pregabalin attenuates spinal dorsal horn nociceptive responses in rat neuropathic pain models (34). The antiallodynic effect of pregabalin is mainly
Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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Fig. 4. Immunohistochemistry of Cava2d-1 protein expression in the spinal cord (L4e6) after oxaliplatin treatment. (A) Spinal cord sections (10 mm thick) from days 0 (before administration), 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) treatment were stained with antibodies against Cava2d-1 subunit protein. White dashed line demarcates the border of the superficial layers of the spinal cord. (B) Optical density (OD) of Cava2d-1 subunit-stained spinal cord sections on days 0 (before administration), 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) administration. The means ± SEM of the OD for Cava2d-1 subunit protein expression are indicated by the columns and vertical bars. n ¼ 5, **P < 0.01 versus day 0. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.
mediated by spinal mechanisms, although supraspinal or peripheral mechanisms cannot be excluded. Thus, the increased expression of Cava2d-1 subunits in the spinal dorsal horn is a main target of the inhibitory action of pregabalin on oxaliplatin-induced acute cold hypersensitivity. These results indicate that pregabalin is a promising candidate drug for treatment of oxaliplatin-induced peripheral neuropathy. Thus, the anti-hyperalgesic action of pregabalin may be due to inhibition of increased Cava2d-1 subunit protein expression following oxaliplatin treatment in the spinal cord. Interestingly, pregabalin did not affect the results of the acetone test in control rats treated with 5% glucose. Pregabalin reduces the severity of oxaliplatin-induced sensory neuropathy (15), and thus, we hypothesize that pregabalin may have a therapeutic effect when the Cava2d-1 subunit protein levels are elevated in the spinal cord. On the other hand, single administration of oxaliplatin (3 mg/kg, i.p.) significantly increases expression of Cava2d-1 subunit mRNA in the mouse DRG but not in the spinal cord (35). Our result showed that Cava2d-1 subunit mRNA in the spinal cord was significantly increased 4 days after administration of oxaliplatin (6 mg/kg, i.p.). Gauchan et al. (35) studied mice 10 days after intraperitoneal administration of oxaliplatin, whereas our results were obtained 4 days after oxaliplatin treatment in rats. This may account for the reported differences, because Cava2d-1 subunit mRNA levels in neurons change over time. The different results between the two studies may also be due to different dosages (3 mg/kg or 6 mg/kg). In addition, our immunohistochemical results indicated that Cava2d-1 protein was significantly increased in superficial layers of the spinal cord 4 days after oxaliplatin treatment. In conclusion, we show that single administration of oxaliplatin increases Cava2d-1 subunit expression in superficial layers of the spinal cord. Moreover, pregabalin inhibits oxaliplatin-induced acute cold hypersensitivity.
Conflicts of interest The authors declare that there are no conflicts of interest.
Acknowledgments We thank Miss Tomomi Nakazato for technical assistance.
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Please cite this article in press as: Yamamoto K, et al., Oxaliplatin administration increases expression of the voltage-dependent calcium channel a2d-1 subunit in the rat spinal cord, Journal of Pharmacological Sciences (2016), http://dx.doi.org/10.1016/j.jphs.2016.01.006
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