2017 Volume 40 Issue 4 Pages 473-478
Paclitaxel is a chemotherapeutic agent that causes peripheral neuropathy as its major dose-limiting side effect. However, the peripheral neuropathy is difficult to manage. A study we recently conducted showed that repetitive administration of aucubin as a prophylactic inhibits paclitaxel-induced mechanical allodynia. However, the mechanisms underlying the anti-allodynic activity of aucubin, which is a major component of Plantaginis Semen, was unclear. In addition to mechanical allodynia, aucubin inhibited spontaneous and mechanical stimuli-induced firing in spinal dorsal horn neurons; however, catalpol, a metabolite of aucubin, did not show these effects. Furthermore, paclitaxel induced the expression of CCAAT/enhancer-binding protein homologous protein, a marker of endoplasmic reticulum (ER) stress, in the sciatic nerve and a Schwann cell line (LY-PPB6 cells); however, this effect was inhibited by aucubin. These results suggest that aucubin inhibits paclitaxel-induced mechanical allodynia through the inhibition of ER stress in peripheral Schwann cells.
Paclitaxel is an anti-microtubule agent originally isolated from the bark of the Pacific yew tree (Taxus brevifolia) and is wildly used to treat solid neoplasms such as breast, ovarian, and lung cancers.1,2) However, in spite of its useful anti-cancer effect, paclitaxel causes peripheral neuropathy, which is characterized by mechanical allodynia, spontaneous pain, shooting and burning pain, tingling, and numbness, with a stocking-and-glove distribution.3) The peripheral neuropathy is a major dose-limiting side effect, which disturbs cancer treatment with paclitaxel. Therefore, relief of the neuropathy is very important for improving both the QOL and cancer treatment with paclitaxel. Several drugs, including gabapentin and amifostine, have failed to relieve paclitaxel-induced peripheral neuropathy4–6); therefore, alternative therapeutic agents are need.
Goshajinkigan is a traditional herbal formulation that consists of 10 herbal medicines (Rehmanniae Radix, Achyranthis Radix, Corni Fructus, Dioscoreae Rhizoma, Plantaginis Semen, Alismatis Rhizoma, Poria, Moutan Cortex, Cinnamoni Cortex, and Processi Aconiti Radix). It has been shown to attenuate the progression of peripheral neuropathy induced by docetaxel and paclitaxel/carboplatin treatments in cancer patients.7,8) Our previous study has been shown that repetitive administration of goshajinkigan extract as a prophylactic inhibits the exacerbation of paclitaxel-induced mechanical allodynia in mice.9) We also showed that aucubin, which is an iridoid glycoside and the main component of Plantaginis Semen, plays an important role in the anti-allodynic action of goshajinkigan.10) However, the mechanisms underlying the anti-allodynic activity of aucubin are not understood completely. It was reported in a previous study that paclitaxel decreased peripheral blood flow and induced mechanical allodynia in mice.11) The study also demonstrated that a prostaglandin E1 analogue, limaprost alfadex, recovered the decreased peripheral blood flow and inhibited the exacerbation of mechanical allodynia induced by paclitaxel. This suggested that the decrease in peripheral blood flow was involved in the paclitaxel-induced mechanical allodynia.11) Since aucubin does not affect decrease of peripheral blood flow,12) it is suggested that it may inhibit the exacerbation of paclitaxel-induced mechanical allodynia through mechanisms other than the control of peripheral blood flow.
Peripheral demyelination contributes majorly to plasticity in neuropathic pain.13) However, paclitaxel promotes demyelination of the sciatic nerve.14,15) Myelin sheaths formed by Schwann cells are damaged by paclitaxel15) through the induction of endoplasmic reticulum (ER) stress.16,17) Therefore, the aim of this study was to investigate whether aucubin alleviates paclitaxel-induced nerve activity and ER stress in mice.
Male C57BL/6 NCr mice (6 weeks old) were obtained from Japan SLC Ltd. (Hamamatsu, Japan) and used for the study. The mice were housed in a room with controlled temperature (21–23°C), humidity (45–46%), and a light/dark cycle (light was from 7:00 a.m. to 7:00 p.m.). In addition, food and water were freely available to the animals. This study protocol was approved by the Committee for Animal Experiments (University of Toyama, Japan) and was conducted in accordance with the guidelines for the investigation of experimental pain in animals, which was published by the International Association for the Study of Pain.
MaterialsPaclitaxel (Sigma-Aldrich, St. Louis, MO, U.S.A.) was dissolved in physiological saline containing 10% Cremophor EL® (Sigma-Aldrich) and 10% ethanol and administered intraperitoneally (i.p.) to the mice at a dose of 0.1 mL/10 g body weight. The recommended dose of paclitaxel is 210 mg/m2 body surface area (package insert of Taxol®; Bristol-Myers Squibb, New York City, NY, U.S.A.), which corresponds to 5.9 mg/kg when the height and body weight are 170 cm and 60 kg, respectively.11) In in vitro experiments, paclitaxel was dissolved in 100% dimethyl sulfoxide (DMSO), which was diluted in the medium so that the final concentration of DMSO was 1%. Aucubin (Wako Pure Chemical Industries, Ltd., Osaka, Japan: Lot. No. ECM6048) and catalpol (Wako Pure Chemical Industries, Ltd.: Lot. No. ECG0796) were dissolved in physiological saline as well and each solution was injected i.p. at 0.1 mL/10 g body weight. They were administered once daily, starting from 24h after the paclitaxel injection.
Behavioral ExperimentsMechanical allodynia in the hind paws was assessed by punctate stimulation with a von Frey filament (North Coast Medical Inc., Morgan Hill, CA, U.S.A.) at a bending force of 0.69 mN (innocuous stimulation).11) The responses of the hind paw to the stimulation were scored as follows: 0, no response; 1, lifting of the hind paw; and 2, flinching or licking of the hind paw. The stimulation was applied six times at the same intensity to each hind paw at intervals of several seconds. The average served as the response score.
Electrophysiological RecordingThe mice were anesthetized with urethane (1.2–1.5 g/kg, i.p.), which produces a long-lasting steady level of anesthesia and does not require the administration of additional doses except in few cases. A thoracolumbar laminectomy was performed to expose the L1–L6 vertebrae, followed by placing the animal in a stereotaxic apparatus. Next, the dura was removed and the arachnoid membrane was cut to create a large window for a tungsten microelectrode. The surface of the spinal cord was irrigated with Krebs solution equilibrated with 95% O2 and 5% CO2 (10–15 mL/min) and containing 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 11 mM glucose, and 25 mM NaHCO3 at 37±1°C. Extracellular single-unit recordings of superficial dorsal horn (lamina I and II) neurons were performed as described previously.18) Recordings were obtained from the superficial dorsal horn neurons at a depth of 20–150 µm from the surface. These cells were within the superficial dorsal horn and assessed from slices obtained from the same spinal level of same-age mice. Unit signals were acquired with an amplifier (EX1; Dagan Corporation, Minneapolis, MN, U.S.A.). The data were digitized with an analog-to-digital converter (Digidata 1400A; Molecular Devices, Union City, CA, U.S.A.) stored on a personal computer with a data acquisition program (Clampex software, version 10.2, Molecular Devices) and analyzed with Clampfit software (version 10.2, Molecular Devices). We searched the area on the skin where touch (with a cotton wisp) or noxious pinch (with forceps) stimulus produced a neural response. Mechanical stimulus was applied by skin folding using a fine von Frey filament at a bending force of 0.69 mN. The stimuli were applied for 10 s to the ipsilateral hind limb at the maximal response point of the respective receptive area.
Cell CulturesA Schwann cell line, LY-PPB6, derived from a rat peripheral nerve sheath tumor was provided by RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum in 6-cm petri dishes and used for the experiments at 80–90% confluence. The medium was exchanged to serum-free DMEM supplemented with bovine serum albumin (1%). Next, the cells were pretreated with aucubin for 1 h, followed by the addition of paclitaxel (100 µM) or vehicle (1% DMSO) for 24 h. The cells were then detached and analyzed by Western blotting.
Western BlottingThe protein of mouse sciatic nerve or LY-PPB6 cells was extracted using lysis buffer (20 mM Tris–HCl (pH 7.5), 137 mM NaCl, 1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL aprotinin, and 1 µg/mL leupeptin), followed by the measurement of protein concentration by the Bradford assay (Bio-Rad, Hercules, CA, U.S.A.). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, the membrane was incubated with mouse anti-CCA AT/enhancer-binding protein (CHOP) monoclonal antibody (1 : 2000; Cell Signaling Technology, Danvers, MA, U.S.A.) or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (1 : 10000; Novus Biologicals LLC, Littleton, CO, U.S.A.) at 4°C overnight, and then with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) (1 : 2000; GE Healthcare Bio-Sciences Co., Piscataway, NJ, U.S.A.) for 1 h at room temperature. Signals were visualized by chemiluminescence reaction (GE Healthcare Bio-Sciences Co.) using X-ray films and analyzed using the ImageJ program (National Institutes of Health, Bethesda, MD, U.S.A.). Signal intensity was normalized to that of GAPDH.
Statistical AnalysisAll the data are presented as the mean and standard error of the mean. Statistical significance was analyzed using one-way ANOVA or two-way repeated measures ANOVA followed by a post hoc Holm–Šidák test (three or more groups). A p value <0.05 was considered statistically significant.
A single i.p. injection of paclitaxel (5 mg/kg) induced mechanical allodynia, with a peak effect occurring at day 14 after the injection; however, it almost completely subsided by day 28 (Fig. 1A). The administration of aucubin (50 mg/kg) inhibited exacerbation of paclitaxel-induced mechanical allodynia; however, this effect was not demonstrated by catalpol (50 mg/kg) (Fig. 1B). This result is similar to that obtained in our previous study, in which aucubin and catalpol were administered to mice at doses of 5–50 mg/kg and 50 mg/kg, respectively.10)
(A) Development of mechanical allodynia after a single injection of paclitaxel. At day 0, PTX (5 mg/kg) or vehicle (VH1) was injected intraperitoneally to male C57BL/6 NCr mice. Data are presented as mean and standard error of the mean (n=6). Main effect of PTX treatment, F1,10=316.3 (p<0.001). Interaction between PTX treatment and time, F19,190=9.2 (p<0.001). * Indicates p<0.05 when compared with VH1 (Holm–Šidák test). (B) Effects of ACB and CTP on PTX-induced mechanical allodynia. ACB (50 mg/kg), CTP (50 mg/kg), or their vehicle (VH2) was then administered once daily as an intraperitoneal injection to the mice, starting from the day after the PTX injection was administered. The data on day 14 after PTX injection was shown. Data are presented as mean and standard error of the mean (n=8). * Indicates p<0.05 when compared with the VH1 plus VH2 treatment, whereas # indicates p<0.05 when compared with PTX plus VH2 (Holm–Šidák test).
Spontaneous firing in superficial dorsal horn neurons was significantly increased in mice treated with paclitaxel but not in those treated with the vehicle (Fig. 2). However, this effect was significantly inhibited by the repetitive administration of aucubin (50 mg/kg) (Fig. 2). On the other hand, the catalpol treatment (50 mg/kg) did not affect the spontaneous firing in the neurons (Fig. 2).
At day 0, PTX (5 mg/kg) or vehicle (VH1) was injected intraperitoneally to male C57BL/6 NCr mice. Next, ACB (50 mg/kg), CTP (50 mg/kg), or vehicle (VH2) was administered once daily as an intraperitoneal injection to the mice, starting from the day after the PTX injection was administered. At day 14, extracellular single-unit recordings were obtained from superficial dorsal horn neurons at a depth of 20–150 µm from the surface. Upper panel: Image is representative of the spontaneous firing in the spinal dorsal horn neurons. Data are presented as mean and standard error of the mean (n=12) (the total number of neurons was 38–61). * Indicates p<0.05 when compared with VH1 plus VH2, whereas # indicates p<0.05 when compared with PTX plus VH2 (Holm–Šidák test).
The mechanical stimuli induced with the von Frey filament evoked transient firing in the superficial dorsal horn neurons of the vehicle-treated mice after detachment of the filaments from their skins (Fig. 3). However, in the paclitaxel-treated mice, sustained and severe firing in the superficial dorsal horn neurons was induced during application of the mechanical stimuli (Fig. 3). The firing evoked by the mechanical stimuli was also inhibited by aucubin (50 mg/kg) (Fig. 3). However, the catalpol treatment (50 mg/kg) had no impact on the mechanical stimuli-evoked firing in the paclitaxel-treated mice (Fig. 3).
At day 0, PTX (5 mg/kg) or vehicle (VH1) was injected intraperitoneally to male C57BL/6 NCr mice. Next, ACB (50 mg/kg), CTP (50 mg/kg), or vehicle (VH2) was administered once daily as an intraperitoneal injection to the mice, starting from the day after the PTX injection was administered. At day 14, extracellular single-unit recordings were obtained from superficial dorsal horn neurons at a depth of 20–150 µm from the surface. Upper panel: Image is representative of von Frey filament (0.69 mN)-evoked firing in the spinal dorsal horn neurons. Data are presented as mean and standard error of the mean (n=12) (the total number of neurons was 38–61). * Indicates p<0.05 when compared with VH1 plus VH2, whereas # indicates p<0.05 when compared with PTX plus VH2 (Holm–Šidák test).
The transcription factor CHOP is a marker of ER stress.19) It has been reported that paclitaxel induces CHOP expression.17) In the present study, paclitaxel increased (p<0.05) CHOP expression in the sciatic nerves of the mice more than the vehicle did (Fig. 4). As a result, the mean expression level of CHOP in the sciatic nerves of the paclitaxel-treated mice was more than 1.4 times higher than that in the sciatic nerves of the vehicle-treated mice (Fig. 4). Furthermore, aucubin (50 mg/kg) significantly inhibited the paclitaxel-induced CHOP expression more than the vehicle did (Fig. 4).
At day 0, PTX (5 mg/kg) or vehicle (VH1) was injected intraperitoneally to male C57BL/6 NCr mice. Next, ACB (50 mg/kg) or vehicle (VH2) was administered once daily as an intraperitoneal injection to the mice, starting from the day after the PTX injection was administered. At day 14, proteins were extracted from the sciatic nerve and CHOP expression was analyzed by Western blotting. Upper panel: Representative immunoblots of CHOP and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The graph shows CHOP/GAPDH ratios that have been normalized to the CHOP/GAPDH ratio in the mice treated with VH1 and VH2. Data are presented as mean and standard error of the mean (n=6). * Indicates p<0.05 when compared to VH1 plus VH2, whereas # indicates p<0.05 when compared to PTX plus VH2 (Holm–Šidák test).
It was observed that treatment of LY-PPB6 cells with paclitaxel (100 µM) resulted in a significant increase in CHOP expression (Fig. 5). The expression level of CHOP in the paclitaxel-treated cells was >30 times higher than that in the vehicle-treated cells (Fig. 5). The results also indicated that pretreatment of the cells with aucubin (50–500 µM) significantly inhibited paclitaxel-induced CHOP expression (Fig. 5).
LY-PPB6 cells were treated with PTX (100 µM) or vehicle (1% DMSO). The cells were treated with ACB for 1 h prior to the PTX treatment. Proteins in the cells were extracted 24 h after the PTX treatment, after which CHOP expression was analyzed by Western blotting. Upper panel: Representative immunoblots of CHOP and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The graph shows CHOP/GAPDH ratios normalized to the CHOP/GAPDH ratio in the vehicle-treated cells. Data are presented as mean and standard error of the mean (n=4). * Indicates p<0.05 when compared to the vehicle, whereas # indicates p<0.05 when compared to PTX (Holm–Šidák test).
It was observed that a single injection of paclitaxel resulted in mechanical allodynia, which was of its highest intensity at day 14 after the injection. In our preliminary experiments, a single administration of aucubin did not alleviate paclitaxel-induced allodynia at day 14 after the paclitaxel injection (data not shown), which was similar to the effect observed with goshajinkigan.9) This suggests that aucubin requires repetitive administration to produce an anti-allodynic effect. In addition, the results of the present study show that repetitive administration of aucubin, but not its metabolite catalpol, inhibits the exacerbation of paclitaxel-induced mechanical allodynia, which complements the results in our previous report.10) Therefore, the data suggest that aucubin inhibits the development of mechanical allodynia.
In the present study, we recoded the activity of superficial dorsal horn neurons using the extracellular electrophysiological recording technique. In the paclitaxel-treated mice, firing of superficial dorsal horn neurons was increased by mechanical stimuli in the same way as mechanical allodynia was. Interestingly, spontaneous firing in the superficial dorsal horn neurons was increased in the paclitaxel-treated mice, which suggests that the spontaneous firing may be related to spontaneous pain or numbness. However, the mechanisms underlying the increased activity of superficial dorsal horn neurons in the paclitaxel-treated mice remain unclear. Yadav et al.20) reported that paclitaxel induces the expression of γ-aminobutyric acid (GABA) transporter-1 and increases the uptake of GABA. This results in decreases in GABA levels in the synaptic clefts of spinal dorsal horn neurons. In addition, it has been demonstrated that paclitaxel increases the expression of Na+–K+–2Cl− cotransporter-1 (NKCC1), which contributes to diminished synaptic inhibition in the spinal cord.21) These findings suggest that paclitaxel treatment increases the activity of spinal dorsal horn neurons through the diminishing of synaptic inhibition. Another proposed mechanism is demyelination of sensory neurons, which increases the nerve activity.14,15) Demyelination of sensory neurons leads to the formation of ephapses and sprouting between sensory neurons,13) which suggests that demyelination of sensory neurons may be involved in the induction of abnormal nerve firing that causes neuropathic pain.
In addition to inhibition of mechanical allodynia, the repetitive administration of aucubin, but not catalpol, attenuated both mechanical stimuli-induced and spontaneous firing in the superficial dorsal horn neurons in the paclitaxel-treated mice. These findings suggest that aucubin has effects on both the spinal cord and the peripheral nervous system. Systemic administration of iridoid glycosides containing aucubin has been shown to increase GABA content in the brain.22) Mechanical allodynia is primary mediated by myelinated A-fiber, but not non-myelinated C-fiber, sensory neurons.23) It has been reported that in sensory neurons, paclitaxel damages Schwann cells, which form myelin sheaths15) around the neurons during ER stress.16,17) In the present study, paclitaxel induced the expression of CHOP, an ER stress marker,19) in the sciatic nerves of the mice and in the LY-PPB6 cells; however, this effect was inhibited by aucubin. The comparison of the inhibitory action of aucubin in in vivo and in vitro studies is difficult. However, the concentration of aucubin (50–500 µM) used in the LY-PPB6 cells may be lower than that in the mouse. In the mouse, a dose of aucubin used was 50 mg/kg of body weight administered by intraperitoneal injection. Since it is considered that the circulating blood volume of mouse is estimated to be 7.3% of the body weight,24) the blood volume is approximately 1.8 mL in mouse (e.g., 25 g of body weight). The concentration of aucubin is approximately 2 mM. Hence, it is considered that these concentrations of aucubin used in in vitro study are reasonable. Taken together, these results suggest that aucubin may inhibit demyelination. However, the mechanisms underlying the inhibition of ER stress by aucubin remains unclear. It has been reported that paclitaxel causes ER stress, which results from the stimulation of an increase in intracellular Ca2+ levels by paclitaxel16); however, this is inhibited by aucubin.25) Therefore, aucubin may have protected against paclitaxel-induced ER stress in the mice sciatic nerves and LY-PPB6 cells via inhibition of increase in intracellular Ca2+ levels. It has also been demonstrated that paclitaxel shows cytotoxicity by inducing oxidative stress.26) This indicates that an antioxidant activity by aucubin27–29) may attenuate paclitaxel-induced cytotoxicity that is mediated through oxidative stress.
In the present study, no changes in the number of peripheral axons were observed in the paclitaxel-treated mice (data not shown), which indicates that a single injection of paclitaxel may not cause severe damage to peripheral neurons. Therefore, we focused on the responses of Schwann cells to the aucubin treatment. However, paclitaxel induces neurotoxicity in neuronal cells by inducing ER stress,17) which suggests that aucubin acts on both Schwann cells and neurons. However, future studies are necessary to investigate the neuronal effects of aucubin on the peripheral nervous system.
It has been demonstrated that repetitive administration of goshajinkigan extract to breast-cancer-bearing mice prevents paclitaxel-induced peripheral neuropathy without interfering with the anti-cancer effect of paclitaxel.30) Therefore, since goshajinkigan contains Plantaginis Semen, which contains aucubin as its main component, the clinical use of aucubin may not affect the anti-cancer effect of paclitaxel. However, the effects of aucubin on paclitaxel-induced dysesthesia, cancer-induced pain, and tumors will be evaluated in tumor-bearing mice in future studies.
In conclusion, repetitive administration of aucubin as a prophylactic attenuates the exacerbation of paclitaxel-induced mechanical allodynia and spinal neuronal activation through the inhibition of ER stress.
This study was supported in part by a Grant-in-Aid for the Cooperative Research Project from Institute of Natural Medicine, University of Toyama in 2016.
The authors declare no conflict of interest.
