Transient receptor potential ankyrin 1 (TRPA1) plays a critical role in a mouse model of cancer pain

Abstract

There is a major, unmet need for the treatment of cancer pain, and new targets and medicines are required. The transient receptor potential ankyrin 1 (TRPA1), a cation channel expressed by nociceptors, is activated by oxidizing substances to mediate pain-like responses in models of inflammatory and neuropathic pain. As cancer is known to increase oxidative stress, the role of TRPA1 was evaluated in a mouse model of cancer pain. Fourteen days after injection of B16-F10 murine melanoma cells into the plantar region of the right hind paw, C57BL/6 mice exhibited mechanical and thermal allodynia and thigmotaxis behavior. While heat allodynia was partially reduced in TRP vanilloid 1 (TRPV1)-deficient mice, thigmotaxis behavior and mechanical and cold allodynia were absent in TRPA1-deficient mice. Deletion of TRPA1 or TRPV1 did not affect cancer growth. Intrathecal TRPA1 antisense oligonucleotides and two different TRPA1 antagonists (HC-030031 or A967079) transiently attenuated thigmotaxis behavior and mechanical and cold allodynia. A TRPV1 antagonist (capsazepine) attenuated solely heat allodynia. NADPH oxidase activity and hydrogen peroxide levels were increased in hind paw skin 14 days after cancer cell inoculation. The antioxidant, α-lipoic acid, attenuated mechanical and cold allodynia and thigmotaxis behavior, but not heat allodynia. Whereas TRPV1, via an oxidative stress-independent pathway, contributes partially to heat hypersensitivity, oxidative stress-dependent activation of TRPA1 plays a key role in mediating thigmotaxis behavior and mechanical and cold allodynia in a cancer pain model. TRPA1 antagonists might be beneficial in the treatment of cancer pain.

Keywords: HC-030031; NADPH oxidase; allodynia; chemotherapeutic drugs; hydrogen peroxide.

Figure 1

Intraplantar (i.pl.) inoculation of cancer cells evokes mechanical and cold allodynia, heat hyperalgesia and thigmotaxis behavior in mice. (a) representative microphotographs of tumor‐bearing hind paw of C57BL/6 mice 2–16 days after inoculation (i.pl.) of B16‐F10 melanoma cells (2 × 10⁵ cells/mL, 20 μL). The drawing in the upper right panel represents the site of inoculation. Time‐dependent increase in paw thickness (b), mechanical (c), cold and heat (d) allodynia after B16‐F10 melanoma cell inoculation. (e) Thigmotaxis behavior assessed by measuring number of rearing events, time spent in the inner zone, or crossing number in the open field test at day 14 after B16‐F10 melanoma cell inoculation. Control mice were injected in the paw with PBS. BL, baseline measurement. Data are expressed as mean + S.E.M. (n = 8 mice per group). **p < 0.01, ***p < 0.001, when compared to PBS‐treated group. Two‐way ANOVA, followed by Bonferroni's post hoc test or Student's t‐test.

Figure 2

TRPA1 gene‐deletion attenuates mechanical and cold allodynia, and pain‐related behaviors in cancer pain model in mice. Time‐dependent (2–16 days) changes in (a) mechanical and cold allodynia, and heat hypersensitivity in Trpa1 ^+/+ and Trpa1 ^−/− mice. (b) Thigmotaxis behavior assessed by measuring number of rearing events, time spent in the inner zone, and crossing number in the open field test at day 14 after B16‐F10 melanoma cell inoculation or PBS injection in Trpa1 ^+/+ or Trpa1 ^−/− mice. (c) Representative real‐time PCR plot and pooled data for TRPA1 mRNA relative expression in cultured trigeminal ganglion (TG) neurons and B16‐F10 melanoma cells (n = 3 replicates from 2 independent experiments; n.d. not detectable). (d) Time‐dependent changes in paw thickness and representative microphotographs of the tumor‐bearing hind paw after B16‐F10 melanoma cell inoculation in Trpa1 ^+/+ or Trpa1 ^−/−. (e) Representative images of hematoxylin and eosin of hind paw 14 days after B16‐F10 melanoma cells inoculation in C57BL/6 mice, showing nerve trunk surrounded by tumor cells. (f) Immunofluorescence staining of TRPA1 and PGP9.5 (a specific marker for nerve fibers) in the hind paw 14 days after B16‐F10 melanoma cell inoculation in C57BL/6 mice (scale bar: 50 μm). Control mice were injected in the paw with PBS. BL, baseline measurement. Data are expressed as mean + S.E.M. (n = 8 mice per group). **p < 0.01, ***p < 0.001, when compared to Trpa1 ^+/+/PBS; ^§§§ p < 0.001, when compared to Trpa1 ^+/+/B16‐F10. Two‐way ANOVA or One‐way ANOVA, followed by Bonferroni's post hoc test.

Figure 3

TRPV1 gene‐deletion reduced the heat hyperalgesia in a cancer pain model in mice. Time course (2–16 days) of (a) paw thickness, (b) mechanical and cold allodynia and (c) heat allodynia in Trpv1 ^+/+ andTrpv1 ^−/− mice. (d) Mechanical, cold and heat allodynia in capsazepine (CPZ, 4 mg/kg, i.p.) treated mice at day 14 after B16‐F10 melanoma cells (2 × 10⁵ cells/mL, 20 μL) intraplantar (i.pl.) inoculation. Control mice were injected in the paw with PBS. BL, baseline measurement. Data are expressed as mean + S.E.M. (n = 8 mice per group). ***p < 0.001, when compared to Trpv1 ^+/+/PBS or PBS‐treated group; ^§ p < 0.05, ^§§§ p < 0.001, when compared to Trpv1 ^+/+/B16‐F10 or VehCPZ/B16‐F10. Two‐way ANOVA followed by Bonferroni's post hoc test.

Figure 4

TRPA1 antisense oligonucleotide or TRPA1 antagonists administration decreased pain‐related responses in a cancer pain model in mice. (a) Capsaicin‐ and AITC‐evoked acute nociception (eye wiping response) after intrathecal (i.th.) antisense/mismatch (AS/MM) oligonucleotides (ODN). (b) Mechanical and cold allodynia after AS/MM ODN administration. (c) Thigmotaxis behavior assessed by measuring number of rearing events or the time spent in the inner zone in the open field test. (d) Mechanical and cold allodynia after intragastric (i.g.) HC‐030031 (HC03, 300 mg/kg) administration; (e) Mechanical and cold allodynia after A967079 (A96, 100 mg/kg, i.g.) administration; (f) Thigmotaxis behavior assessed by measuring number of rearing events or the time spent in the inner zone in the open field test, measured 1 h after HC03 (300 mg/kg, i.g.) administration. All the tests were performed at day 14 after B16‐F10 melanoma cells (2 × 10⁵ cells/mL, 20 μL) intraplantar (i.pl.) inoculation. Control mice were injected in the paw with PBS. BL, baseline measurement. Veh is the vehicle of different treatments. Data are expressed as mean + S.E.M. (n = 8 mice per group). **p < 0.01; ***p < 0.001 when compared to PBS; ^§ p < 0.05, ^§§§ p < 0.001, when compared to B16‐F10 or Veh/B16‐F10. Two‐way ANOVA followed by Bonferroni's post hoc test.

Figure 5

Melanoma cell inoculation increased the oxidative stress markers and α‐lipoic acid attenuates pain‐related responses. (a) H₂O₂ levels or (b) NADPH oxidase activity measured in the hind paw skin tissue after B16‐F10 intraplantar (i.pl.) inoculation of melanoma cells (2 × 10⁵ cells/mL, 20 μL). (c) Mechanical and cold allodynia and heat hypersensitivity after intragastric (i.g.) administration of the antioxidant α‐lipoic acid (αLA, 100 mg/kg). (d) Thigmotaxis behavior assessed by measuring number of rearing events, or the time spent in the inner zone in the open field test, measured 1 h after αLA (100 mg/kg, i.g.) administration. All tests were performed at day 14 after B16‐F10 melanoma cell (2 × 10⁵ cells/mL, 20 μL) inoculation (i.pl.). Control mice were injected in the paw with PBS. BL, baseline measurement. Veh is the vehicle of αLA. Data are expressed as mean + S.E.M. (n = 8 mice per group). *p < 0.05, ***p < 0.001 when compared to PBS; ^§ p < 0.05, ^§§§ p < 0.001, when compared to veh αLA/B16‐F10. Student's t‐test, two‐way ANOVA, or one‐way ANOVA followed by Bonferroni's post hoc test.