Cannabinoid type-1 receptor reduces pain and neurotoxicity produced by chemotherapy

Abstract

Painful peripheral neuropathy is a dose-limiting complication of chemotherapy. Cisplatin produces a cumulative toxic effect on peripheral nerves, and 30-40% of cancer patients receiving this agent experience pain. By modeling cisplatin-induced hyperalgesia in mice with daily injections of cisplatin (1 mg/kg, i.p.) for 7 d, we investigated the anti-hyperalgesic effects of anandamide (AEA) and cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), an inhibitor of AEA hydrolysis. Cisplatin-induced mechanical and heat hyperalgesia were accompanied by a decrease in the level of AEA in plantar paw skin. No changes in motor activity were observed after seven injections of cisplatin. Intraplantar injection of AEA (10 μg/10 μl) or URB597 (9 μg/10 μl) transiently attenuated hyperalgesia through activation of peripheral CB₁ receptors. Co-injections of URB597 (0.3 mg/kg daily, i.p.) with cisplatin decreased and delayed the development of mechanical and heat hyperalgesia. The effect of URB597 was mediated by CB₁ receptors since AM281 (0.33 mg/kg daily, i.p.) blocked the effect of URB597. Co-injection of URB597 also normalized the cisplatin-induced decrease in conduction velocity of Aα/Aβ-fibers and reduced the increase of ATF-3 and TRPV1 immunoreactivity in dorsal root ganglion (DRG) neurons. Since DRGs are a primary site of toxicity by cisplatin, effects of cisplatin were studied on cultured DRG neurons. Incubation of DRG neurons with cisplatin (4 μg/ml) for 24 h decreased the total length of neurites. URB597 (100 nM) attenuated these changes through activation of CB₁ receptors. Collectively, these results suggest that pharmacological facilitation of AEA signaling is a promising strategy for attenuating cisplatin-associated sensory neuropathy.

Figure 1.

Assessment of a model of cisplatin-induced mechanical hyperalgesia. A, Weight loss was observed only in mice given the highest daily dose of cisplatin (3 mg/kg). At doses of 0.5 or 1 mg/kg, cisplatin did not produce weight loss (B, baseline). B, Mechanical hyperalgesia occurred following repeated injections of cisplatin (1 and 3 mg/kg of body weight, daily for 7 d, i.p.). The withdrawal frequency to a mechanical stimulus (3.7 mN) increased with the increased cumulative dose compared with responses before the first injection of cisplatin. C, The latency to withdrawal from a radiant heat stimulus was shorter following 7 d of treatment with cisplatin (1 mg/kg). D, In the rotarod test, mice that received cisplatin (1 mg/kg) performed as well as the vehicle-treated group. ^#Significantly different from baseline in the group treated with 3 mg/kg cisplatin; *significantly different from baseline in the group treated with 1 mg/kg cisplatin (p < 0.05, two-way ANOVA with Bonferroni's test; n = 6–15 mice/treatment). Error bars indicate SEM.

Figure 2.

Administration of AEA or URB597 decreased mechanical hyperalgesia produced by cisplatin. A, A single intraplantar injection of AEA (10 μg) into a hindpaw decreased withdrawal responses to a mechanical stimulus ipsilateral to the injection in cisplatin-treated mice (PD, predrug). Co-injection of AM281 (10 μg, i.pl.), the CB₁R antagonist, but not AM630 (4 μg, i.pl.), the CB₂R antagonist, blocked the effect of AEA. Neither antagonist had an effect when injected alone. B, Intraplantar injection of URB597 (9 μg) into a hindpaw attenuated mechanical hyperalgesia ipsilateral to the injection in cisplatin-treated mice. The effect of URB597 was blocked by co-injection of the CB₁R antagonist AM281 (10 μg, i.pl.); AM630 (4 μg, i.pl.) had no effect. *Significantly different from vehicle; ^#significantly different from AEA or URB597 (p < 0.05, two-way ANOVA with Bonferroni's test; n = 4–8 mice/treatment). Error bars indicate SEM.

Figure 3.

Repeated systemic injections of URB597 (0.3 mg/kg daily, i.p.) reduced the development of cisplatin-evoked hyperalgesia in cisplatin-treated mice. A, When cisplatin was co-injected with URB597, the development of mechanical hyperalgesia was delayed, and it was reduced in magnitude (B, baseline). The effect of URB597 was blocked by daily co-injection of the CB₁ receptor antagonist AM281 (0.33 mg/kg, i.p.). B, Cotreatment with URB597 blocked the development of heat hyperalgesia. C, Acute treatment with AM281 (10 μg, i.p.) reversed the anti-hyperalgesic effect of repeated URB597 administration. The y-axis in C is the same as in B. D, Repeated administration of URB597 for 7 d had no effect on motor function as measured by the rotarod assay. *Significantly different from vehicle; ^#significantly different from URB597 (p < 0.05, two-way ANOVA with Bonferroni's test; n = 4–8 mice/treatment). Error bars indicate SEM.

Figure 4.

URB597 attenuated effects of cisplatin on protein expression in DRGs. A, TRPV1- and ATF-3-ir were detected by immunofluorescence in L3–L5 DRGs from mice treated with vehicle, cisplatin, or cisplatin plus URB597. Cisplatin (1 mg/kg of body weight, daily for 7 d, i.p.) increased the occurrence of TRPV1- and ATF3-ir in neurons. Co-injection of URB597 (0.3 mg/kg daily, i.p.) with cisplatin attenuated the effect cisplatin on protein-ir. Scale bars: 10 μm (for images within each antigen). B, Quantitative summary of the effect of treatments on TRPV1-ir in neurons. Data are expressed as the mean ± SEM. ^aSignificantly different from each other group (p < 0.05, one-way ANOVA with Student–Newman–Keuls test; n = 4 mice/treatment). C, Quantitative summary of the effect of treatments on ATF-ir in neurons. Data are expressed as the median and 25th and 75th percentile range. *Significantly different from vehicle control and cisplatin plus URB597 groups (n = 6 mice/treatment; p < 0.001, Kruskal–Wallis ANOVA on ranks test).

Figure 5.

URB597 attenuated effects of cisplatin in nerve. A, Representative examples of Aα/Aβ components of the CAP from mice treated with vehicle, cisplatin, and cisplatin plus URB597. Electrical stimuli are indicated by arrows, and latencies of spike initiations are shown by dashed lines. Latencies corresponded to conductance velocities of 37.2 m/s (vehicle), 26.9 m/s (cisplatin), and 36.1 m/s (cisplatin plus URB597). B, Quantitative summary showing that treatment with cisplatin (1 mg/kg, daily for 7 d, i.p.) reduced the conduction velocity of Aα/Aβ-fibers. Cotreatment with URB597 (0.3 mg/kg daily, i.p.) blocked the effect of cisplatin. C, Western blots of p-NF and actin protein in tibial nerves of mice treated with (1) vehicle, (2) cisplatin alone, or (3) cisplatin plus URB597. D, Quantitative summary of the amount of p-NF protein defined as the ratio of RT97-ir to actin-ir within the same sample. The decrease in p-NF was protected by coadministration of URB597. *Significantly different (p < 0.05, one-way ANOVA with Bonferroni's test). Values inside the bars represent the sample size. Error bars indicate SEM.

Figure 6.

Cytotoxicity of cisplatin in vitro was confirmed on cultured fibrosarcoma cells. In comparison to the vehicle-treated culture (A), treatment with cisplatin (4 μg/ml for 24 h; B) decreased the number of viable fibrosarcoma cells. Cultures were incubated in acridine orange (0.67 μm for 5 min) to visualize live cells (Bank, 1987).

Figure 7.

Adult DRG neurons treated with cisplatin in vitro exhibited reduced neurite growth, which was prevented by cotreatment with URB597. A, Dissociated DRG neurons cultured in the control conditions exhibited long, branched neurites with strong tubulin-ir. B, DRG neurons treated with cisplatin (4 μg/ml) for 24 h had fewer and shorter tubulin-ir neurites compared with neurons in the control condition. C, Cotreatment with URB597 (100 nm) reversed the effect of cisplatin. Scale bar, 50 μm. All images are the same magnification. D, Quantitative summary of the effect of treatments on neurite length. Data are expressed as the median and 25th and 75th percentile range. The effect of URB597 was blocked by cotreatment with the CB₁R antagonist AM281 (1 μm). The CB₂R antagonist AM630 (1 μm) did not block the effect of URB597. Neither antagonist alone had effect on cisplatin-treated DRG neurons. URB597 had no effect on the length of tubulin-ir neurites in the control conditions. *Significantly different from vehicle in the control condition. ^#Significantly different from vehicle, URB597 plus AM281, or URB597 plus AM630 groups in cisplatin-treated conditions (n = 143–191 neurons/treatment in 3 different culture preparations; p < 0.001, Kruskal–Wallis ANOVA on ranks).