The First Cellular Step for Chemotherapy Induced Peripheral Neuropathy After Vincristine Treatment May Require Binding to Neuronal Calcium Sensor‐ 1

Many chemotherapy drugs induce peripheral neuropathy (PN), a painful and irreversible side effect. Previous results reported in the literature show that Paclitaxel and vincristine, which are anti‐microtubule targeting drugs, induce PN by mechanisms that are not fully understood. The Ehrlich group has reported that in a neuronal cell line and in primary rat dorsal root ganglia, paclitaxel binds to the protein neuronal calcium sensor‐1 (NCS‐1) and triggers a calpain‐dependent decrease in intracellular calcium signaling via degradation of NCS‐1 and a subsequent decrease in NCS‐1 dependent inositol trisphosphate receptor calcium release. Other groups have previously reported disturbed calcium signaling in PN. Similar calcium effects have also been reported with vincristine. On the other hand, there are also reports showing that pretreatment with valproic acid prevents the development of PN when it is administered with cisplatin, other chemotherapy drug. We hypothesized that vincristine and valproic acid bind to NCS‐1 and produce their effects using the same cellular cascade as paclitaxel. Specifically our hypothesis was that vincristine binds to NCS‐1 and triggers calpain‐dependent processes while valproic acid binds to NCS‐1 and blocks the paclitaxel initiated calcium signaling alterations associated with PN. NCS‐1 was expressed recombinantly in a prokaryotic system. After induction and expression, NCS‐1 was extracted and purified by hydrophobic interaction chromatography, desalting, and dialysis. Interactions between NCS‐1 and both drugs were measured using isothermal titration calorimetry (ITC). First, binding of calcium to NCS‐1 was monitored to show that the NCS‐1‐calcium binding interaction matched published reports. Using ITC, we did not observe any binding reaction between Valproic acid and NCS‐1 under both calcium‐free and calcium‐saturating conditions. In the ITC study between vincristine and NCS‐1 with or without calcium, we observed that vincristine appeared to bind to NCS‐1. However, it is not conclusive if such heat changes are the result of vincristine binding to NCS‐1, the reason is that the addition of vincristine also alters the pH of the reaction buffer and this can create variations in the results. Our results suggest that the protective effects of valproic acid do not involve direct binding to NCS‐1 and it probably involves other cellular pathways. However, vincristine treatment probably does involve binding to NCS‐1 to produce the alterations in intracellular calcium signaling that produce PN. These results are important for understanding the cellular mechanism of chemotherapy induced PN and finding possible drug targets for PN. Support or Funding Information Acknowledgments: I thank the American Physiological Society, Yale University and Universidad Icesi for the economic and academic support that made possible the realization of this research project. 1NCS‐1 binding test by ITC. A) Positive control with NCS‐1 139 uM, and Calcium chloride (CaCl2) 5 mM, T: 28°C, pH: 7,2 binding between NCS‐1 and Ca +2 . B) NCS‐1: 139uM plus 5mM CaCl2 and valproic acid (VA) 6mM plus 5 mM CaCl2, T:28°C, pH: 7,3 NCS‐1 and VA do not bind. C) ) NCS‐1 100uM and vincristine (VC) 10mM, T:28°C, pH: 7,3. There may be binding between NCS‐1 and VC. D) NCS‐1 139uM plus 5mM CaCl2 and VC 4mM plus 5 mM CaCl2, T:28°C, pH: 6,6. There may be binding between NCS‐1 and VC.