ANTICANCER RESEARCH 35: 6431-6438 (2015) Establishment of a Novel In Vitro Model for Predicting Incidence and Severity of Microtubule-targeting Agent-induced Peripheral Neuropathy YUICHI SAWAGUCHI, SATOSHI UENO, YUKIKO NISHIYMA, RYUTA YAMAZAKI and TAKESHI MATSUZAKI Yakult Central Institute for Microbiological Research, Yakult Honsha Co., Ltd., Kunitachi-shi, Tokyo, Japan Abstract. Peripheral neuropathy (PN) is a major dose- Microtubule-targeting agents (MTAs) have been clinically limiting side-effect of microtubule-targeting agents (MTAs), used since the 1960s for the effective treatment of leukemia, considered to be induced by inhibition of axonal lymphoma, and various solid tumors (1). MTAs are classified microtubules. Therefore, it was thought that a useful method roughly into the Vinca alkaloids (vincristine, vindesine, for predicting the frequencies of severe sensory-PN (FPN) vinorelbine) that de-stabilize or depolymerize microtubules, would be to evaluate the neurite-disrupting effects of MTAs. and the taxanes (paclitaxel, docetaxel) that polymerize Using neurite outgrowth from neuron-like cell lines, we microtubules, thus causing mitotic arrest. Meanwhile, MTAs comprehensively evaluated the neurite-disrupting effects of often induce neurotoxicities by having effects on neurons. several anti-cancer drugs including MTAs, and the Although it is still an open question how neurotoxicities are reversibility of the effects of MTAs. MTAs that induce PN clinically affected by MTAs, physiological and showed neurite-disrupting effects more strongly than MTAs histopathological studies in pre-clinical research showed this and anticancer drugs that do not induce PN, but the effects is due, at least in part, to the inhibition of axonal transport were not related to the FPN. On the other hand, MTAs with in neurons (2). Because of the excessive length of peripheral high FPN exhibited lower reversibility than those with low axons, MTAs are especially disruptive to axons forming FPN. These findings suggest that neurite-disrupting effects peripheral nerves and, therefore, induce peripheral are associated with the incidence of PN, and the reversibility neuropathy (PN). Generally, MTAs induce sensory PN more of the effects is associated with FPN. frequently than motor PN (3). Severe sensory-PN (grade 3/4 according to the National Cancer Institute Common Toxicity Microtubules are tubular polymers composed of α/β-tubulin Criteria scale) can be a dose-limiting factor of MTAs and heterodimers. The continuous equilibrium of microtubule significantly reduce QOL. assembly and disassembly makes the microtubules dynamic In Table I, we summarize the frequencies of severe structures that maintain cell shape, polarity, and motility; sensory-PN (FPNs) of MTAs in clinical trials. Vincristine provide a scaffold for cellular trafficking of proteins and and paclitaxel showed especially high FPNs (4-9). FPNs of organelles; and play an integral role in mitosis. Because docetaxel, vindesine, and vinorelbine are lower than those of microtubules and their dynamics are required for mitotic vincristine and paclitaxel (10-16). Indibulin, now in a phase spindle formation and chromosome separation during II clinical trial, was reported not to induce severe PN (17). mitosis, they are thought to be an important target for a These lines of clinical evidence indicate that FPNs are chemically diverse group of anticancer drugs that induce characteristic to each MTA, so it is desirable to discover mitotic arrest and cell death in vitro. MTAs with relatively low FPNs. To date, FPNs have been evaluated mainly using in vivo models in non-clinical studies (18); therefore, the establishment of an in vitro model in screening for drug discovery is anticipated. Correspondence to: Yuichi Sawaguchi, Yakult Central Institute for MTAs are considered to induce PN by interacting with Microbiological Research, Yakult Honsha Co., Ltd., Kunitachi-shi, axonal microtubules and then disrupting neurites. On the basis Tokyo, 186-8650, Japan. Tel: +81 425778960, Fax: +81 425773020, of this principle, in terms of in vitro methods for predicting e-mail: [email protected] FPNs, in order to evaluate the neurite-disrupting effects of Key Words: Peripheral neuropathy, microtubule-targeting agents, MTAs, it was thought to be useful to use neurite outgrowth neurite-distupting effects, in vitro model, PC-12 cells, SH-SY5Y evaluation from the PC-12 rat pheochromocytoma cell line cells. differentiated into neuron-like cells or the SH-SY5Y human 0250-7005/2015 $2.00+.40 6431 ANTICANCER RESEARCH 35: 6431-6438 (2015) Table I. Frequencies of peripheral neuropathy of microtubule-targeting agents in clinical trials. Agent FPNa, % Number of patients with Total number of First author grade 3/4 sensory PNb patients assessed Vincristine 18.4 37 201 Sarris (4), Thomas (5), O’Brien (6) Paclitaxel 15.3 51 333 Ibrahim (7), Gradishar (8), Kim (9) Docetaxel 6.3 24 382 Bonneterre (10), Tabernero (11), Jones (12) Vindesine 4.2 6 142 Eigentler (13) Vinorelbine 1.9 3 160 Fumoleau (14), Vogel (15), Zelek (16) Indibulin 0 0 14 Kuppens (17) aFPN: Frequencies of severe sensory peripheral neuropathy=percentage of patients with grade 3/4 sensory peripheral neuropathy. With regard to the MTAs reported in several clinical trials, the percentages were calculated from the total number of patients in all reports referred to. bPN: peripheral neuropathy neuroblastoma cell line (19, 20). However, there have been no Thermo Fisher Scientific, Nepean, ON, Canada). After 24 h, the reports regarding comprehensive evaluations of various MTAs medium was changed to low-serum (1% HS) medium, and PC-12 using these methods, so it was unclear how accurately the cells were treated with 50 ng/mL nerve growth factor (NGF; Merck Millipore, Nottingham, UK) for 72 h to induce neuronal neurite-disrupting effects reflect FPN. Meanwhile, it was differentiation and neurite outgrowth. Differentiated cells were then reported that the FPNs of MTAs became lower in proportion treated for 24 h with test drugs at several concentrations in the to the dosing interval in clinical trials (3). We, therefore, presence of NGF. hypothesized that neurites disrupted by MTAs re-extended SH-SY5Y cells were seeded in 96-well plates for 24 h, followed reversibly between dosages, which contributed to the recovery by culture for 72 h with 1 μM all-trans retinoic acid (ATRA) (Wako from PN. In the present study, we first evaluated the neurite- Chem. Co.) to induce neurite outgrowth. Then, the SH-SY5Y cells disrupting effects of MTAs using known methods and then were treated for 24 h with test drugs at several concentrations in the presence of ATRA. examined how well the effects correlated with FPN. Furthermore, we established a novel in vitro model for Assay for neurite-disrupting effects and reversibility of these effects. evaluating the reversibility of the neurite-disrupting effects and For evaluation of the neurite-disrupting effects, 300 cells in examined the correlation between this reversibility and FPN. triplicate wells were randomly chosen and the proportions of neurite-forming cells (neurite length >2 × cell body length) were Materials and Methods determined. Using the same cells, cell viability was assessed with WST-8 (Kishida Chemicals, Osaka, Japan) or CellTiter 96 Aqueous Materials. Paclitaxel, vincristine, vindesine, and vinorelbine were One Solution (Promega, Madison, WI, USA). The proportions of purchased from Sigma Aldrich (St. Louis, MO, USA). Docetaxel neurite-forming cells were corrected using the following equation: was purchased from Fluka (Buchs, Switzerland). Indibulin was (Correction value for the proportion of neurite-forming cells)= purchased from Tocris Bioscience (Bristol, UK). Doxorubicin was (proportion of neurite-forming cells scored by the evaluation of purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, neurite-disrupting effects)/(cell viability). Japan). SN-38 and BI2536 were chemically synthesized by Yakult Thereafter, the concentrations of each test drug causing 50% Honsha (Tokyo, Japan). These drugs were dissolved in inhibition (IC50) of neurites were calculated from the correction dimethylsulfoxide (DMSO). The final concentration of DMSO in all values. treatments was adjusted to 0.1%. For evaluating the reversibility of the neurite-disrupting effects, after drug treatment, PC-12 and SH-SY5Y cells were cultured Cells and cell cultures. The PC-12 rat pheochromocytoma cell line without test drugs for another 48 h in DMEM containing NGF (PC- was obtained from Riken Cell Bank (Tsukuba, Japan) and cultured 12 cells) or RPMI1640 medium containing ATRA (SH-SY5Y cells). in Dulbecco’s modified Eagle’s medium (DMEM; Life After the culture, the proportions of neurite-forming cells were Technologies, Grand Island, NY, USA) with 10% (v/v) fetal bovine scored by the method mentioned above. serum (FBS) (Sigma-Aldrich) and 5% (v/v) horse serum (HS; MP Biomedicals, Aurora, OH, USA) at 37˚C in 5% CO2. HCT116 Cytotoxicity assay for cell survival. HCT116 cells were seeded in human colon cancer and SH-SY5Y human neuroblastoma cell line 96-well plates and test drugs were added to the cultures at several were both obtained from American Type Culture Collection and concentrations. After 96 h, cell viabilities were measured with WST- grown in RPMI1640 medium (Life Technologies) with 10% FBS at 8 and the concentrations of test drugs equivalent to the IC50 of cell 37˚C in 5% CO2. viabilities were calculated. Correlations between the proportion of