Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase

Abstract

Many of the widely used anticancer drugs induce dose-limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy-induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum-complexes, and proteasome-inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro-nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano-hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) without interfering with their anti-tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE-2-PyP(5+). Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of "peroxynitrite-targeted" therapeutics for CIPN.

Keywords: ATP depletion; Bortezomib; Chemotherapy-induced painful peripheral neuropathy; Mitochondrial dysfunction; Neuropathic pain; Oxaliplatin; Paclitaxel; Peripheral nerve sensory axons; Peroxynitrite; Superoxide dismutase.

Fig. 1

Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺) blocks oxaliplatin (Ox)-induced neuropathic pain in a dose-dependent manner. When compared with baseline [day (D)0], treatment with oxaliplatin (●), but not with its vehicle (Veh) (○), led to the time-dependent development of mechano-allodynia (A) and mechano-hyperalgesia (B). Daily (D0–17) injections of MnTE-2-PYP⁵⁺ (0.3 mg/kg/d, ▲; 1 mg/kg/d, ▼; 3 mg/kg/d, ■) significantly attenuated the development of oxaliplatin-induced mechano-hypersensitivity in a dose-dependent manner (A, B). Results are expressed as mean ± SD, n = 5–7 and analyzed by two-way analysis of variance with Bonferroni’s post-hoc comparisons. *P < .001 (t_day vs t_{day 0}); ^†P < .001 (Ox + MnTE-2-PyP⁵⁺ vs Ox). PWT = paw withdrawal threshold.

Fig. 2

Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺) prevents the development of bortezomib (Bort)-induced neuropathic pain. Compared with baseline [day (D)0], administration of bortezomib (black bars), but not its vehicle (Veh) (open bars), led to the development of mechano-allodynia (A) and mechano-hyperalgesia (B). Prophylactic daily injections (D0–17) of MnTE-2-PyP⁵⁺ (3 mg/kg/d; gray bars) blocked the development of both bortezomib-induced mechano-allodynia (A) and mechano-hyperalgesia (B). Hypersensitivity did not appear after drug termination up until the end of testing on D25 (A, B). Results are expressed as mean ± SD, n = 5–6, and analyzed by two-way analysis of variance with Bonferroni’s post-hoc comparisons. *P < .001 (t_day vs t_{day 0}); ^†P < .001 (Bort + MnTE-2-PyP⁵⁺ vs Bort). PWT = paw withdrawal threshold.

Fig. 3

Increased nitration and inactivation of manganese superoxide (MnSOD) in peripheral nerve sensory axons (PNSAs) with paclitaxel-treatment; prevention with Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺). Treatment with paclitaxel (Pac) (black bars), but not vehicle (Veh) (open bars), resulted in the development of mechano-allodynia (A) and mechano-hyperalgesia (B) on day (D)25 compared with D0 and was prevented by daily (D0–16) injections of 10 mg/kg/d MnTE-2-PyP⁵⁺ (gray bars) (A,B). At peak hypersensitivity, a significant increase in the nitration of MnSOD (C) and an associated decrease in its activity (D) were observed in the PNSAs of paclitaxel-treated animals compared with vehicle; treatment with MnTE-2-PyP⁵⁺ prevented this (C,D). Representative blots are shown above the quantitative bar graph corresponding to mean ± SD for an n = 4/group (C). Results are expressed as mean ± SD, n = 4–6, and analyzed by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc comparisons or two-way ANOVA with Bonferroni’s post-hoc comparisons. *P < .001 (t_day vs t_{day 0}); ^†P < .001 (Pac + MnTE vs Pac). PWT = paw withdrawal threshold.

Fig. 4

Copper zinc superoxide dismutase (CuZnSOD) activity was not altered by treatment with chemotherapeutics [paclitaxel (Pac), oxaliplatin (Ox), and bortezomib (Bort)] or Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺). Treatment with the chemotherapeutics paclitaxel (A), oxaliplatin (B), or bortezomib (C) either alone (black bars) or in combination with MnTE-2-PyP⁵⁺ (gray bars) had no effect on the activity of CuZnSOD compared with vehicle treatment (open bars). Results are expressed as mean ± SD, n = 6 and analyzed by one-way analysis of variance with Dunnett’s post-hoc comparisons. n.s. = not significant.

Fig. 5

Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺) prevents mitochondrial abnormalities and dysfunction in the peripheral nerve sensory axons (PNSAs) associated with paclitaxel (Pac)-treatment. Representative electron micrographs of the myelinated A-fiber axons of the saphenous nerve from vehicle- (A, B) and paclitaxel- (C, D) treated animals showing swollen and vacuolated mitochondria within the nerves of the paclitaxel-treated group (C, D). Paclitaxel treatment (black bars) led to an increased appearance of abnormal mitochondria (swollen and vacuolated) in both the A- (E) and C-fibers (F) compared with treatment with vehicle (Veh) (open bars). Paclitaxel-treatment also resulted in a significant impairment of adenosine triphosphate (ATP) production after stimulation in the PNSAs compared with vehicle treatment (G) on day 25. MnTE-2-PyP⁵⁺ prevented both the paclitaxel-associated increase in mitochondrial abnormalities (E, F) and dysfunction (G). Results are expressed as mean ± SD, n = 5–6, and analyzed by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc comparisons (E, F) or two-way ANOVA with Bonferroni’s post-hoc comparisons (G). *P < .05, **P < .01, ***P < .001 (Pac vs Veh); ^†P < .05, ^††P < .001 (Pac + MnTE vs Pac). CSU = Citrate Synthase Units.

Fig. 6

The oxaliplatin (Ox)-associated nitration/inactivation of manganese superoxide dismutase (MnSOD) and impaired adenosine triphosphate (ATP) production in peripheral nerve sensory axons (PNSAs) is attenuated with Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺). On day 25, PNSAs collected from oxaliplatin-treated animals (black bars) displayed increased levels of nitrated MnSOD (A), decreased MnSOD activity (B), and impaired ATP production (C) compared with tissues from vehicle(Veh)-treated animals (open bars). These alterations were prevented by administration of MnTE-2-PyP⁵⁺ (gray bars). Representative blots are shown above the quantitative bar graph corresponding to mean ± SD for an n = 6/group (A). Results are expressed as mean ± SD, n = 6 and analyzed by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc comparisons (A, B) or two-way ANOVA with Bonferroni’s post-hoc comparisons (C). *P < .001 (Ox vs Veh); ^†P < .01, ^††P < .001 (Ox + MnTE vs Ox). CSU = XXX.

Fig. 7

Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺) blocks manganese superoxide dismutase (MnSOD) nitration/inactivation and reduced adenosine triphosphate (ATP) production in the peripheral nerve sensory axons (PNSAs) of bortezomib (Bort)-treated animals. On day 25 of treatment, increased levels of nitrated MnSOD (A), decreased MnSOD activity (B), and impaired ATP production (C) were observed in the tissue of the group that received bortezomib (black bars) compared with the vehicle (Veh) group (open bars), and was prevented by administration of MnTE-2-PyP⁵⁺ (gray bars). Representative blots are shown above the quantitative bar graph corresponding to mean ± SD for an n = 6/group (A). Results are expressed as mean ± SD, n = 5–6 and analyzed by one-way analysis of variance (ANOVA) with Dunnett’s post-hoc comparisons (A, B) or two-way ANOVA with Bonferroni’s post-hoc comparisons (C). *P < .01, **P < .001 (Bort vs Veh); ^†P < .01, ^††P < .001 (Bort + MnTE vs Bort). CSU = XXX.