Evaluation of microtubule polimerization involved in the development of bortezomib-induced peripheral neuropathy through new in vitro and in vivo models

Bortezomib (BZ), a proteasome inhibitor, is an antineoplastic drug used in the treatment of multiple myeloma and mantle cell lymphoma. Its main clinical and dose-limiting side effect is the development of painful peripheral neuropathy. Despite BZ-induced peripheral neuropathy (BiPN) substantially reduces patients quality of life, no effective therapies have been development so far. Here we worked up in vitro and in vivo models to deeper investigate the pathogenesis of BiPN. Since BZ alters protein degradation, we supposed that proteins regulating microtubule (MT) stability may be altered after treatment. Moreover, since sensory neurons have a primary role in the development of BiPN, we treated cultured DRG sensory neurons with BZ (100nM) for 48h and 72h, and we evaluated tubulin polimerization by comparing the distribution of tubulin in polymerized (P) and soluble (S) fractions. The increase of MT polymerization following treatment of DRG sensory neurons was observed; the proportion of tubulin in polymerized fraction was increased after 48h and 72 h of BZ-treatment. To verify this mechanism also in an in vivo model, we examined MT polymerization on both acute and chronic model of BiPN. In the acute model a single-dose of 0.20 mg/kg of BZ was injected in Wistar rat, and then the polimerization was analysed in sciatic nerves after 48h and 72h. In the chronic model animals were treated with BZ 0.20 mg/kg, three times/week for eight weeks, and the neurophysiological and neuropathological damages were observed in peripheral nerves. At the end of the treatment and after 2 weeks of follow-up period, the MT polymerization in sciatic nerve was measured by western blot. In the acute schedule of treatment, the increase of tubulin in polymerized fraction was observed within 72 hours from BZ administration. Similar result was observed also at the end of chronic BZ-administration. The level of tubulin polimerization was returned to physiological levels at the end of the follow-up period. This in vitro and in vivo system can be useful for elucidate molecular mechanisms involved in BiPN and to test potential neuroprotective compound. Supported in part by a research grant from the “University of Milan-Bicocca F.A.R”