Alleviation of cisplatin-induced neuropathic pain, neuronal apoptosis, and systemic inflammation in mice by rapamycin

Abstract

Platinum-based chemotherapeutic treatment of cancer patients is associated with debilitating adverse effects. Several adverse effects have been well investigated, and can be managed satisfactorily, but chemotherapy-induced peripheral neuropathy (CIPN) remains poorly treated. Our primary aim in this study was to investigate the neuroprotective effect of the immunomodulatory drug rapamycin in the mitigation of cisplatin-induced neurotoxicity. Pain assays were performed in vivo to determine whether rapamycin would prevent or significantly decrease cisplatin-induced neurotoxicity in adult male Balb/c mice. Neuropathic pain induced by both chronic and acute exposure to cisplatin was measured by hot plate assay, cold plate assay, tail-flick test, and plantar test. Rapamycin co-treatment resulted in significant reduction in cisplatin-induced nociceptive-like symptoms. To understand the underlying mechanisms behind rapamycin-mediated neuroprotection, we investigated its effect on certain inflammatory mediators implicated in the propagation of chemotherapy-induced neurotoxicity. Interestingly, cisplatin was found to significantly increase peripheral IL-17A expression and CD8- T cells, which were remarkably reversed by the pre-treatment of mice with rapamycin. In addition, rapamycin reduced the cisplatin-induced neuronal apoptosis marked by decreased neuronal caspase-3 activity. The rapamycin neuroprotective effect was also associated with reversal of the changes in protein expression of p21^Cip1, p53, and PUMA. Collectively, rapamycin alleviated some features of cisplatin-induced neurotoxicity in mice and can be further investigated for the treatment of cisplatin-induced peripheral neuropathy.

Keywords: CIPN; IL-17A; cisplatin; p21; rapamycin.

FIGURE 1

Effect of rapamycin on thermal hypersensitivity induced by chronic exposure to cisplatin. Balb/c mice were treated with cisplatin 3 mg/kg (i.p.) with or without rapamycin 3 mg/kg (s.c.) every other day for 12 days. (A) Impact of rapamycin on cisplatin using cold plate test. (B) Impact of rapamycin on cisplatin using hot plate test. *p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001 indicates statistical significance between treatment groups (two-way ANOVA followed by Tukey’s post-hoc test; n = 8 mice). ns, non-significant.

FIGURE 2

Effect of rapamycin on thermal hypersensitivity induced by acute exposure to cisplatin. Balb/c mice were treated with a single dose cisplatin 10 mg/kg (i.p.) with or without rapamycin 3 mg/kg (s.c.) for 24 h. (A,C) Impact of rapamycin on cisplatin using hot plate test 4 and 24 h post-treatment, respectively. (B,D) Impact of rapamycin on cisplatin using tail flick test 4 and 24 h post-treatment, respectively. *p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001 indicates statistical significance between treatment groups (two-way ANOVA followed by Tukey’s post-hoc test; n = 8 mice).

FIGURE 3

Rapamycin reduces the systemic inflammation. Balb/c mice were treated with a single dose cisplatin 3 mg/kg (i.p.) with or without rapamycin 3 mg/kg (s.c.) for 12 days. (A) Impact of rapamycin on cisplatin-induced effect on IL-17A expression in CD8 + T cells. (B) Impact of rapamycin on cisplatin-induced effect on IL-17A expression in CD8- T cells. IL-17A expression was evaluated in the blood by flow cytometry. *p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001 indicates statistical significance between treatment groups. (two-way ANOVA followed by Tukey’s post-hoc test; n = 4 mice). (C) Representative flow cytometry plots for evaluation of intracellular IL-17A levels in CD4 + and CD8 + T cells from blood in treated mice. ns, non-significant.

FIGURE 4

Assessment of senescence and apoptosis in neurons. Neurons were further incubated in culture medium containing or not (control) 10 μM Cisplatin (Cis), either in the absence or presence of 100 nM Rapamycin (Rap) for 24 h. When indicated, neurons were pre-treated with 100 nM Rapamycin for 3 (-3 h) hours before Cisplatin exposure. (A) Fluorometric detection of caspase-3 activity was performed as an index of neuronal apoptosis (n = 5). (B–D) Neurons were treated with indicated treatment as mention previously for 24 h. Thereafter, p53, p21, and PUMA proteins expression levels were determined by Western blot analysis. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicates statistical significance between treatment groups (two-way ANOVA followed by Tukey’s post-hoc test; n = 3). (E) Representative western blot showing protein level of p53, p21, and PUMA in neuronal cultures. ns, non-significant.