ANTICANCER RESEARCH 35: 6431-6438 (2015)

Establishment of a Novel In Vitro Model for Predicting Incidence and Severity of -targeting Agent-induced

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 , considered to be induced by inhibition of axonal , and various solid tumors (1). MTAs are classified . Therefore, it was thought that a useful method roughly into the (, , for predicting the frequencies of severe sensory-PN (FPN) ) that de-stabilize or depolymerize microtubules, would be to evaluate the neurite-disrupting effects of MTAs. and the (, ) 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- 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 separation during II , 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 cell line cells. differentiated into neuron-like cells or the SH-SY5Y human

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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

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). 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 neurite-reextending cells Induction of neurite outgrowth and drug treatment. PC-12 cells were after drug treatment and FPN were determined by calculating r2 as seeded in 96-well plates coated with collagen (2,000 cells/well, a measure of the goodness of fit for their linear regression.

6432 Sawaguchi et al: A Novel In Vitro Model for Severity and Incidence Prediction of MTA-induced PN

Figure 1. Microscopic images of NGF-differentiated PC-12 cells treated with test drugs. PC-12 cells were seeded in collagen-coated 96-well plates and treated with NGF for 72 h. The differentiated cells were cultured for another 24 h with DMSO (control), 100 nM vincristine, 100 nM paclitaxel, 100 nM docetaxel, 100 nM vindesine, 100 nM vinorelbine, 100 nM indibulin, 10 μM SN-38, 10 μM doxorubicin, or 10 μM BI2536 in the presence of NGF, and were then observed under a microscope.

Results is generally necessary to evaluate their active and toxic concentrations. Therefore, we measured the anti-proliferative Neurite-disrupting effects. We evaluated the neurite-disrupting effects of each test drug on HCT116 cells as an index of their effects of MTAs (vincristine, paclitaxel, docetaxel, vindesine, anti-cancer activities (Table II). From the IC50 for (a) neurites vinorelbine and indibulin) using NGF-differentiated PC-12 rat of PC-12 cells and (b) cell proliferation of HCT116 cells, pheochromocytoma cells. As a negative control, we values of a/b (mentioned as index values of neuropathy: IVNs) additionally evaluated the following anticancer drugs, not were calculated. Drugs with low IVN are considered to show affecting microtubule dynamics directly: SN-38 (topoisomerase both anticancer activities and neurite-disrupting effects, and I inhibitor), doxorubicin (topoisomerase II inhibitor), and therefore to have a narrow safety margin. Vincristine, BI2536 (polo-like kinase I inhibitor). Microscopic images of paclitaxel, docetaxel, vindesine, and vinorelbine all showed the PC-12 cells after drug treatment are shown in Figure 1. similar IVNs regardless of their different FPNs. Indibulin, a Radio et al. reported that neurites are disrupted as a result of MTA not inducing severe PN, showed clearly higher IVN than not only microtubule destruction induced by MTAs, but also the other MTAs. SN-38, doxorubicin, and BI2536 showed even cytotoxicity induced by some drugs (21). In order to exclude higher IVNs than indibulin, although these could not be such non-specific effects, we corrected the proportions of calculated because of the lack of their neurite-disrupting neurite-forming cells using cell viability after drug treatment effects. We similarly evaluated neurite-disrupting effects using (Figure 2). Vincristine, paclitaxel, docetaxel, vindesine, SH-SY5Y human neuroblastoma cells using the IC50 for vinorelbine, and indibulin almost completely disrupted neurites neurites, and the IVNs of each test drug evaluated are shown in at 10 to 1,000 nM. On the other hand, SN-38, doxorubicin, and Table III. Index values of neuropathy of vincristine, paclitaxel, BI2536 tended to decrease the proportions of neurite-forming docetaxel, vindesine, and vinorelbine were lower than those of cells concentration-dependently, but did not disrupt neurites doxorubicin and BI2536, which agreed with the results with completely even at 10 μM, the highest concentration tested in PC-12 cells. On the other hand, among MTAs, vincristine, this experiment. When discussing the safety margin of drugs, it paclitaxel, and vinorelbine showed relatively high IVNs.

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Figure 2. Effects of test drugs on neurites and cell viability of differentiated PC-12 cells. Differentiated PC-12 cells were evaluated for both neurites and cell viability following 24 h of exposure to DMSO (control), vincristine, paclitaxel, docetaxel, vindesine, vinorelbine, indibulin, SN-38, doxorubicin, or BI2536 at several concentrations. Proportion of neurite-forming cells were corrected as described in Materials and Methods. Data are expressed as percentages of controls, and are presented as means±SD from triplicate wells.

6434 Sawaguchi et al: A Novel In Vitro Model for Severity and Incidence Prediction of MTA-induced PN

Figure 3. Neurite-reextending PC-12 cells after treatment of MTAs. Differentiated PC-12 cells were treated with DMSO (control), 10 nM vincristine, 1000 nM paclitaxel, 10 nM docetaxel, 10 nM vindesine, or 10 nM vinorelbine for 24 h followed by withdrawal of the MTAs from the culture medium for 48 h, and then the rates of neurite-forming cells were evaluated. Data are expressed as percentages of the control, and are presented as means±SD from triplicate wells (*p<0.05, **p<0.01). Significantly different from 10 nM vincristine-treated group.

Figure 4. Neurite-reextending SH-SY5Y cells after treatment with MTAs. SH-SY5Y cells were cultured with ATRA for 72 h following exposure to DMSO (control), 1 μM vincristine, 10 μM paclitaxel, 1 μM docetaxel, 0.1 μM vindesine, or 0.1 μM vinorelbine for 24 h. Then, the MTAs were removed from the culture medium for 48 h and the proportions of neurite-forming cells were evaluated. Data are expressed as percentages of the control, and are presented as means±SD from triplicate wells (**p<0.01). Significantly different from 1 μM vincristine-treated group.

Reversibility of neurite-disrupting effects. In order to evaluate caused by cytotoxicity. Vincristine and paclitaxel showed the reversibility of the neurite-disrupting effects, we measured similar reversibility. Docetaxel, vindesine, and vinorelbine the proportions of neurite-reextending PC-12 cells after showed about 2-fold higher reversibility than vincristine. We treatments of some MTAs, followed by their withdrawal similarly evaluated the reversibility of some MTAs using SH- (Figure 3). The MTAs were applied at the lowest SY5Y cells (Figure 4). Vincristine and paclitaxel showed concentration at which they disrupted the neurites of more similar reversibility, whereas the reversibility of docetaxel, than 95% of the cells, so as to reduce the non-specific effects vindesine, and vinorelbine was approximately 2- to 2.5-fold

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Table II. Test drugs evaluated for effects on neurite of PC-12 cells and proliferation of HCT116 cells.

a Agent IC50 for the IC50 for the IVN neurite-inhibiting proliferation- effects on inhibiting effects on PC-12 cells (nM) HCT116 cells (nM)

Vincristine 1.7 1.4 1.2 Paclitaxel 12.0 7.3 1.7 Docetaxel 2.8 1.4 1.9 0.2 1.7 0.1 Vindesine 0.6 1.4 0.4 Vinorelbine 1.5 2.0 0.7 Indibulin 491.9 5.4 90.4 SN-38 >10000 2.1 >4762 Doxorubicin >10000 1.4 >7143 BI2536 >10000 17.5 >571 a IVN; Index Values of Neuropathy=ratio of IC50 for the neurite- Figure 5. Correlations between reversibility of neurite-disrupting effects inhibiting effect on PC-12 cells to IC50 for the proliferation-inhibiting and FPN across MTAs in PC-12 and SH-SY5Y cells. The figure shows effect on HCT116 cells. bivariate correlations between FPN and the proportion of neurite- reextending cells after drug withdrawal across MTAs, in PC-12 (filled square and solid line) and SH-SY5Y cells (open square and broken line). The correlation coefficient (r2) and significance (p) are indicated. Table III. Test compounds evaluated for effects on neurites on SH-SY5Y cells.

a Agent IC50 for the IVN neurite-inhibiting effect and then examined the correlations between FPN and on SH-SY5Y cells (nM) neurite-disrupting effects, or reversibility. Using NGF-differentiated PC-12 rat pheochromocytoma Vincristine 59.4 43.7 cells, we measured the neurite-disrupting effects of various Paclitaxel 906.0 125.0 Docetaxel 12.4 8.7 MTAs (vincristine and paclitaxel, which show relatively high Vindesine 4.6 3.3 FPNs (4-9); docetaxel, vindesine, and vinorelbine, which show Vinorelbine 273.5 133.6 relatively low FPNs (10-16); and indibulin, which does not Doxorubicin >10000 >7143 induce severe PN (17)) and anticancer drugs not directly BI2536 >10000 >571 affecting microtubule dynamics (SN-38, doxorubicin, and a BI2536) by calculating IVN as an index of the safety margin. IVN; Index Values of Neuropathy=ratio of IC50 for the neurite- inhibiting effect on SH-SY5Y cells to IC50 for the proliferation- The neurite-disrupting effect of indibulin was markedly lower inhibiting effect on HCT116 cells (Table II). than those of the other MTAs, which could induce PN. In addition, SN-38, doxorubicin, and BI2536 did not show neurite-disrupting effects at all. These results indicated that the neurite-disrupting effects against PC-12 cells reflect the higher than that of vincristine. These results showed that the incidence of PN. However, the neurite-disrupting effects were reversibility of the neurite-disrupting effects was similar using considered not to be associated with FPN, since the MTAs that either PC-12 or SH-SY5Y cells. By surveying FPN and the could induce PN showed equivalent neurite-disrupting effects reversibility of the neurite-disrupting effects across MTAs, we to each other. We similarly evaluated the neurite-disrupting uncovered negative correlations between them in both PC-12 effects using SH-SY5Y human neuroblastoma cells. The and SH-SY5Y cells (Figure 5; correlation coefficients r2=0.79 neurite-disrupting effects of vincristine, paclitaxel, docetaxel, and 0.93, p=0.093 and 0.044, respectively). vindesine, and vinorelbine were not correlated with their FPNs, but were clearly lower than those of doxorubicin and Discussion BI2536. Therefore, it was suggested that the neurite-disrupting effects observed in not only rat but also human neuroblastoma In the present study, with the aim of establishing an in vitro cell lines reflect the incidence of PN, but not FPN model for predicting the incidence and severity of MTA- We established a novel in vitro model to evaluate the induced PN, we evaluated neurite-disrupting effects and the reversibility of the neurite-disrupting effects of MTAs using reversibility of the effects induced by clinically used MTAs, PC-12 or SH-SY5Y cells, and examined the correlations

6436 Sawaguchi et al: A Novel In Vitro Model for Severity and Incidence Prediction of MTA-induced PN between the reversibility measured with these models and 10 Bonneterre J, Roché H, Monnier A, Guastalla JP, Namer M, FPN. As a result of evaluations with both PC-12 and SH- Fargeot P, Assadourian S: Docetaxel vs. 5- plus SY5Y cells, there were strong negative correlations between vinorelbine in metastatic after therapy failure. Br J Cancer 87: 1210-1215, 2002. the reversibility of the neurite-disrupting effects and the FPN 11 Tabernero J, Climent MA, Lluch A, Albanell J, Vermorken JB, of each MTA. 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