Background & Aims

Anastrozole, an aromatase inhibitor (AI), is a first-line drug in the endocrine treatment for postmenopausal women diagnosed with breast cancer. Anastrozole is the most frequently prescribed AI, and its use is typically recommended for several years (5-10 years) after diagnosis. However, it can cause painful musculoskeletal symptoms 1,2, which affect patients’ quality of life and may lead to therapy discontinuation3. AIs promote neurogenic inflammation (SP and CGRP release), contributing to developing and maintaining painful symptoms4. Neurogenic inflammation leads to mast cell activation and subsequent release of algogenic substances, including proteases such as tryptase5,6. Tryptase cleaves and activates PAR2 in sensory neurons and mediates several painful processes, causing a vicious cycle of peripheral sensitization and mast cell activation7,8. Thus, we demonstrated the involvement of mast cells and PAR2 in anastrozole-induced pain.

Methods

C57BL/6 mice were treated with a single anastrozole administration [0.2 mg/kg, orally (p.o.)] to induce painful symptoms characterized by mechanical allodynia and muscle strength loss, evaluated by the von Frey test and Grip Strength test, respectively. To evaluate mast cell involvement, the mice were pre-treated twice daily/4 consecutive days with compound 48/80 (mast cell depletor) or once daily/ 5 consecutive days with ketotifen fumarate (mast cell membrane stabilizer). The antinociceptive effect of tryptase inhibitors (Gabexate and Nafamostat, 10 mg/kg) or PAR2 antagonists’ (ENMD-1068, 10 mg/kg and AZ3451, 60 µg/kg) was evaluated. The PAR2 role was confirmed using global (PAR2-/-) or sensory neuron-specific (PAR2 Nav 1.8-/-) PAR2 knockout mice underwent the protocol of anastrozole-induced pain. The mechanical sensitivity and muscle strength were assessed after the treatments. The infiltration of mast cells in the plantar tissue was evaluated by histological analyses.

Results

The painful behaviours observed in the anastrozole-induced pain model were positively correlated with mast cell recruitment in the plantar tissue. The pre-treatment with compound 48/80 or ketotifen fumarate prevented anastrozole-induced mechanical allodynia and muscle strength loss. Mice treated with anastrozole presented tryptase-increased levels in the sciatic nerve and plantar tissue. The treatment with gabexate or nafamostat, both tryptase inhibitors, reversed the mechanical allodynia and the muscle strength loss induced by anastrozole treatment. ENMD-1068 and AZ3451, both PAR2 antagonists, reduced the mechanical allodynia and muscle strength loss after anastrozole treatment. The involvement of PAR2 was confirmed using genetic approaches. The anastrozole treatment could not induce painful symptoms in PAR2-/- (global knockout) or PAR2 Nav1.8-/- (sensory neuron-specific knockout) mice.

Conclusions

In this study, using histological and pharmacological tools, we demonstrated that the painful symptoms induced by anastrozole depend on the activation and recruitment of peripheral mast cells, which release tryptase (a PAR2 agonist) in peripheral tissue, contributing to these symptoms. Furthermore, we demonstrated the PAR2 involvement in the anastrozole-induced painful symptoms using pharmacological and genetic approaches. Our results indicate that mast cells are activated in the peripheral tissue, releasing tryptase after anastrozole treatment and that PAR2 activation on sensory neurons is needed to sustain painful symptoms related to anastrozole use.

References

1. Nyrop, K.A., Callahan, L.F., Rini, C., Altpeter, M., Hackney, B., DePue, A., Wilson, A., Schechter, A., Muss, H.B., 2016. Aromatase inhibitor associated arthralgia: the importance of oncology provider-patient communication about side effects and potential management through physical activity. Support. Care Cancer 24, 2643–2650. https://doi.org/10.1007/s00520-015-3065-2
2. Waks, A.G., Winer, E.P., 2019. Breast Cancer Treatment. JAMA 321, 288. https://doi.org/10.1001/jama.2018.19323
3.Bennett, M.I., Kaasa, S., Barke, A., Korwisi, B., Rief, W., Treede, R.-D., 2019. The IASP classification of chronic pain for ICD-11: chronic cancer-related pain. Pain 160, 38–44. https://doi.org/10.1097/j.pain.0000000000001363
4.Fusi, C., Materazzi, S., Benemei, S., Coppi, E., Trevisan, G., Marone, I.M., Minocci, D., De Logu, F., Tuccinardi, T., Di Tommaso, M.R., Susini, T., Moneti, G., Pieraccini, G., Geppetti, P., Nassini, R., 2014. Steroidal and non-steroidal third-generation aromatase inhibitors induce pain-like symptoms via TRPA1. Nat. Commun. 5, 5736. https://doi.org/10.1038/ncomms6736
5. Steinhoff, M., Vergnolle, N., Young, S.H., Tognetto, M., Amadesi, S., Ennes, H.S., Trevisani, M., Hollenberg, M.D., Wallace, J.L., Caughey, G.H., Mitchell, S.E., Williams, L.M., Geppetti, P., Mayer, E.A., Bunnett, N.W., 2000. Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat. Med. 6, 151–158. https://doi.org/10.1038/72247
6. Suckow, S.K., Caudle, R.M., 2008. Identification and immunohistochemical characterization of colospinal afferent neurons in the rat. Neuroscience 153, 803–813. https://doi.org/10.1016/j.neuroscience.2008.02.046
7. Oliveira, S.M., Silva, C.R., Ferreira, J., 2013. Critical role of protease-activated receptor 2 activation by mast cell tryptase in the development of postoperative pain. Anesthesiology 118, 679–690. https://doi.org/10.1097/ALN.0B013E31827D415F
8. Peach, C.J., Edgington-Mitchell, L.E., Bunnett, N.W., Schmidt, B.L., 2023a. Protease-activated receptors in health and disease. Physiol. Rev. 103, 717–785. https://doi.org/10.1152/physrev.00044.2021

Presenting Author

Maria Fernanda Pessano Fialho

Poster Authors

Maria Fialho

Msc.

Federal University of Santa Maria

Lead Author

E-mail

Evelyne Silva Brum

PhD

Federal University of Santa Maria

Lead Author

E-mail

João Pedro de Vargas Lopes

Federal University of Santa Maria

Lead Author

E-mail

Raquel Tonello

New York University

Lead Author

E-mail

Nigel Bunnett

Department of Molecular Pathobiology, Pain Research Center, NYU

Lead Author

E-mail

Sara Marchesan Oliveira

PhD

Federal University of Santa Maria

Lead Author

E-mail

Topics

  • Models: Chronic Pain - Inflammatory