Activation of KCNQ Channels Prevents Paclitaxel-Induced Peripheral Neuropathy and Associated Neuropathic Pain

Abstract

Paclitaxel-induced peripheral neuropathy (PIPN) and associated neuropathic pain are the most common and serious adverse effects experienced by cancer patients receiving paclitaxel treatment. These effects adversely impact daily activities and consequently the quality of life, sometimes forcing the suspension of treatment and negatively influencing survival. Patients are usually at high risk of developing PIPN if paclitaxel induces acute pain, which strongly suggests that an acute increase in the excitability of nociceptors underlies the chronic alterations of PIPN. KCNQ/Kv7 channels are widely expressed in the primary sensory neurons to modulate their excitability. In the present study, we show that targeting KCNQ/Kv7 channels at an early stage is an effective strategy to attenuate the development of PIPN. We found that paclitaxel did not decrease the expression level of KCNQ/Kv7 channels in the primary sensory neurons as detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. However, retigabine, which is a specific KCNQ/Kv7 channel opener, attenuated significantly the development of PIPN, as shown by both morphologic and behavioral evidence. We also observed that retigabine had no obvious effect on the chemosensitivity of breast cancer cells to paclitaxel. Although retigabine has been approved by the FDA as an anticonvulsant, our study suggests that this drug can be repurposed to attenuate the development of PIPN. PERSPECTIVE: Paclitaxel-induced peripheral neuropathy and associated neuropathic pain are severe and resistant to intervention. The results of our study demonstrated that retigabine (a clinically available medicine) can be used to attenuate the development of paclitaxel-induced peripheral neuropathy.

Keywords: K(+) channels; hyperexcitability; neuropathy; paclitaxel; pain; prevention; retigabine.

Figure 1.

KCNQ channel expression in DRGs after repeated application of paclitaxel and retigabine. (A) Western blot analysis showing lack of effect of paclitaxel and retigabine on KCNQ5 protein expression in L4 and L5 DRGs. Upper panel: Bands of KCNQ5 and β-actin probed with specific antibodies. Lower panel: relative protein expression levels of KCNQ5 compared to β-actin. P = 0.089, F_(2,15) = 2.855, one-way ANOVA with repeated measures followed by Bonferroni’s post-hoc tests. Dots in columns represent one animal each. Six animals were used for each group. (B) qRT-PCR analysis showing Kcnq2, Kcnq3 and Kcnq5 mRNA levels in L4 and L5 DRGs 28 days after initial paclitaxel/retigabine treatments. All data are normalized to GAPDH. Dots in columns represent one animal each. P = 0.0669, F_(2,12) = 3.418 for KCNQ2; P = 0.2859, F_(2,12) = 1.329 for KCNQ3; P = 0.3790, F_(2,1) = 1.053 for KCNQ5. One-way ANOVA with repeated measures followed by Bonferroni’s post-hoc tests. Five animals were used for each group, and the experiments were performed twice independently. Pacli, paclitaxel; retig, retigabine.

Figure 2.

The effect of early repeated application of retigabine on the development of chronic behavioral hypersensitivity and spontaneous pain after paclitaxel. (A) Timeline for the experiments. (B) Mechanical hypersensitivity of hindpaws was measured 12, 28, and 35 days after initial paclitaxel treatment. Dots in columns represent one animal each. Repeated measures two-way ANOVA followed by Sidak’s multiple comparison test (treatment F_{(1, 9)} = 21.58, p = 0.0012; time F_{(3, 27)} = 34.16, p < 0.0001; interaction F_{(3, 27)} = 4.546, P = 0.0105. Baseline, P > 0.999; 12 days, P = 0.0024; 28 days, P = 0.0014; 35 days, P = 0.0006. (C) Conditioned place preference tests were performed 4 weeks after treatment. Dots in each column represent individual rats tested in each condition. P = 0.039, F_{(2, 16)} = 4.0, One-way ANOVA, Pacli, paclitaxel; retig, retigabine. *, p<0.05.

Figure 3.

The effect of early, repeated application of retigabine on the paclitaxel-induced activation of astrocytes in the spinal cord and degeneration of peripheral nerves. (A) Representative images showing the expression of GFAP in L4/L5 spinal cords from sham, paclitaxel with vehicle, and paclitaxel with retigabine groups. Shown is a representative experiment from 3 independent experiments. (B) Quantification of GFAP expression normalized to β-actin in L4/L5 spinal cords. N shows the number of animals tested. P = 0.02, F_(2,13) = 11.11, one-way ANOVA. (C) Representative images showing PGP9.5-stained intraepidermal nerve fibers (red) that crossed the collagen-stained dermal/epidermal junction (green) in the skin sections from paclitaxel with vehicle and paclitaxel with retigabine groups. Scale bars, 100 μm. (D) The effect of co-treatment with retigabine/vehicle on paclitaxel-induced PGP9.5-positive fiber loss in the epidermis. Dots in each column represents a section. 4–5 sections per animal were assessed. 4 sham, 5 brief vehicle-treated, and 5 brief retigabine-treated animals were used. P = 0.003, F_(2,58) = 6.567, one-way ANOVA.

Figure 4.

The effect of early, repeated application of retigabine on the morphological alteration of mitochondria in tibial nerves after paclitaxel. Representative images of swollen mitochondria (red arrows) with vacuoles and small oval mitochondria (green arrows) with intact double membranes in myelinated axons (A) and unmyelinated axons (C) of tibial nerve sections from sham (left panel), paclitaxel plus vehicle (middle panel), and paclitaxel plus retigabine (right panel) groups. Scale bars, 0.5 μm. (B) and (D) Quantification of abnormal mitochondria in both myelinated axons and unmyelinated axons. P = 0.0003, X² = 16.442 for unmyelinated fibers; P < 0.0001, X² = 32.213 for myelinated fibers; Chi-square tests. N is the number of fibers tested. Four animals for each group were used. Data were collected 28 days after initial paclitaxel treatment. *, p<0.05; **, P<0.01, ***, P<0.001.

Figure 5.

The effect of retigabine on the chemosensitivity of SKBR3 breast cancer cells to paclitaxel. (A) MTT assay of SKBR3 cells treated with 10 μM retigabine plus different concentrations of paclitaxel (0 – 100 nM) at 24 hrs, 48 hrs, and 72 hrs. (B) MTT assay of SKBR3 cells treated with 10 nM paclitaxel plus different concentration of retigabine at 72 hrs. P = 0.09, F_(3,6) = 3.487, one-way ANOVA. Dots in each column represent one independent experiment. Each experiment was performed three times.