Melatonin limits paclitaxel-induced mitochondrial dysfunction in vitro and protects against paclitaxel-induced neuropathic pain in the rat

Abstract

Chemotherapy-induced neuropathic pain is a debilitating and common side effect of cancer treatment. Mitochondrial dysfunction associated with oxidative stress in peripheral nerves has been implicated in the underlying mechanism. We investigated the potential of melatonin, a potent antioxidant that preferentially acts within mitochondria, to reduce mitochondrial damage and neuropathic pain resulting from the chemotherapeutic drug paclitaxel. In vitro, paclitaxel caused a 50% reduction in mitochondrial membrane potential and metabolic rate, independent of concentration (20-100 μmol/L). Mitochondrial volume was increased dose-dependently by paclitaxel (200% increase at 100 μmol/L). These effects were prevented by co-treatment with 1 μmol/L melatonin. Paclitaxel cytotoxicity against cancer cells was not affected by co-exposure to 1 μmol/L melatonin of either the breast cancer cell line MCF-7 or the ovarian carcinoma cell line A2780. In a rat model of paclitaxel-induced painful peripheral neuropathy, pretreatment with oral melatonin (5/10/50 mg/kg), given as a daily bolus dose, was protective, dose-dependently limiting development of mechanical hypersensitivity (19/43/47% difference from paclitaxel control, respectively). Melatonin (10 mg/kg/day) was similarly effective when administered continuously in drinking water (39% difference). Melatonin also reduced paclitaxel-induced elevated 8-isoprostane F₂ α levels in peripheral nerves (by 22% in sciatic; 41% in saphenous) and limited paclitaxel-induced reduction in C-fibre activity-dependent slowing (by 64%). Notably, melatonin limited the development of mechanical hypersensitivity in both male and female animals (by 50/41%, respectively), and an additive effect was found when melatonin was given with the current treatment, duloxetine (75/62% difference, respectively). Melatonin is therefore a potential treatment to limit the development of painful neuropathy resulting from chemotherapy treatment.

Keywords: Paclitaxel; antioxidant; chemotherapy; melatonin; mitochondria; neuropathic pain; oxidative stress.

Figure 1

Effect of a range of concentrations of paclitaxel plus vehicle control (left) or plus 1 μmol/L melatonin (right) on (A) mitochondrial membrane potential, (B) mitochondrial metabolic activity and (C) mitochondrial volume, in a dorsal root ganglion neuronal cell line. Results are presented as percentage of data at baseline, that is vehicle control‐treated cells without paclitaxel but with melatonin treatment. Data are shown as box‐and‐whisker plots showing median, interquartile and full range (n = 6). P value is Kruskal‐Wallis. Asterisks = significantly different to without paclitaxel (*P < .05, **P < .01)

Figure 2

Mechanical hind paw withdrawal thresholds (A) of male rats receiving paclitaxel with vehicle control, paclitaxel with 5 mg/kg melatonin, paclitaxel with 10 mg/kg melatonin and paclitaxel with 50 mg/kg melatonin measured 6 hours after oral gavage melatonin/vehicle administration. AUC analysis of withdrawal thresholds (B) 2‐17 days following paclitaxel treatment, measured at 1, 6 and 24 hours after oral gavage. Behavioural data are shown as mean (SD), n = 5‐6 per treatment group. Two‐way RM ANOVA (melatonin treatment P < .0001) followed by Dunnett's multiple comparisons test used to compare all groups to paclitaxel with vehicle control. *P < .05; ***=P < .001; ****P < .0001. There was no significant effect of time of measure. (C) Serum melatonin levels from paclitaxel‐treated male rats given 10 mg oral melatonin by gavage. Individual raw data points are shown (n = 3)

Figure 3

Mechanical hind paw withdrawal thresholds (A) of male rats receiving paclitaxel with vehicle control, paclitaxel with 10 mg/kg melatonin, cremophor (paclitaxel vehicle) with vehicle control and saline with 10 mg/kg melatonin with melatonin/vehicle administered in drinking water. AUC analysis of withdrawal thresholds (B) 2‐42 days following paclitaxel treatment. AUC analysis of withdrawal thresholds (C) 2‐17 days following paclitaxel treatment with 10 mg/kg melatonin/vehicle administered in drinking water or by oral gavage. Behavioural data are shown as mean (SD), n = 5‐6 per treatment group. In (B), one‐way ANOVA followed by Tukey's multiple comparisons test ****P < .0001. In (C), 2‐way ANOVA, melatonin treatment P < .0001, interaction P = .49. (D) Serum melatonin in male rats given paclitaxel plus melatonin or vehicle in drinking water. Data are presented as box‐and‐whisker plots showing median, interquartile and full range (n = 15). *** = significantly higher than rats not given melatonin (P = .001)

Figure 4

Mechanical hind paw withdrawal thresholds (A) of male rats receiving paclitaxel with vehicle control, paclitaxel with melatonin throughout, paclitaxel with melatonin discontinued at day 18, and paclitaxel with melatonin starting day 20. 10 mg/kg melatonin/vehicle administered in drinking water. AUC analysis of withdrawal thresholds (Bi) 8‐18 days or (Bii) 20‐30 days following paclitaxel treatment. Data are shown as mean (SD), n = 6 per treatment group. In (B), one‐way ANOVA followed by Tukey's multiple comparisons test *P < .05; **P < .01; ***P < .001;****P < .0001

Figure 5

8‐Isoprostane F₂α levels in sciatic and saphenous nerve tissue isolated at day 19 of model from (A) cremophor‐ (n = 6) or paclitaxel (n = 6)‐treated male rats or (B) paclitaxel‐treated male rats administered 10 mg/kg melatonin by oral gavage (n = 6) or vehicle/no treatment (n = 11). Data expressed as % change from average control cremophor values (n = 12). Data are shown as box‐and‐whisker plots showing median, interquartile and full range. Two‐way ANOVA with P values indicating treatment significance

Figure 6

C‐fibre activity‐dependent slowing (ADS) recorded using (Ai) 2 suction electrodes to stimulate and record compound action potentials from L4/L5 dorsal roots from male rats. (Aii) Representative compound action potentials illustrating the slow C‐fibre conducting component. The ×40 traces recorded in response to 2 Hz dorsal root stimulation are shown (trace 1 black; traces 2‐39 light grey; trace 40 dark grey). Initial width (orange dashed lines) and last width (blue dashed lines) denoted. Repetitive stimulation of dorsal roots at 1 Hz (Bi) and 2 Hz (Bii) results in a progressive increase in response width. AUC analysis of width change (C) reveals that the frequency‐dependent progressive width change (2‐way ANOVA P < .0001) is reduced by paclitaxel (2‐way ANOVA, Tukey's multiple comparisons test P < .001), an effect prevented with 10 mg/kg/day melatonin treatment in drinking water (2‐way ANOVA, Tukey's multiple comparisons test P < .0001). Data are shown as mean (SD)

Figure 7

Mechanical hind paw withdrawal thresholds of (Ai) male or (Bi) female rats receiving paclitaxel, paclitaxel with melatonin, paclitaxel with duloxetine intervention treatment or paclitaxel with melatonin and duloxetine intervention treatment. 10 mg/kg/day melatonin/control administered in drinking water; 10 mg/kg/day duloxetine‐/vehicle‐injected i.p. AUC analysis of withdrawal thresholds in (Aii) males or (Bii) females during duloxetine intervention. Data are shown as mean (SD), n = 6 per treatment group. In (A/Bii), one‐way ANOVA followed by Tukey's multiple comparisons test *P < .05; **P < .01; ***P < .001; ****P < .0001