American Association for Cancer Research
15357163mct140462-sup-132430_1_supp_2658236_nbtknt.pdf (3.14 MB)

Supplementary Methods, Figures 1 - 9 from Tivantinib (ARQ 197) Exhibits Antitumor Activity by Directly Interacting with Tubulin and Overcomes ABC Transporter–Mediated Drug Resistance

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journal contribution
posted on 2023-04-03, 14:06 authored by Aki Aoyama, Ryohei Katayama, Tomoko Oh-hara, Shigeo Sato, Yasushi Okuno, Naoya Fujita

Supplementary method and Figures. Figure S1 contains expression levels of c-MET in the cells used in this paper. Figure S2 contains the photographs taken after treatment with microtubule inhibitor. Figure S3 contains Immunohistochemistry of tubulin after tivantinib treatment. Figure S4 contains the photographs indicated the effect of tivantinib on tubulin. Figure S5 contains immunoblot data of various cells treated with various microtubule inhibitors. Figure S6 contains mouse body weight change and immunoblot data of xenografts. Figure S7 contains the data of tubulin competition assay using various inhibitors. Figure S8 contains the result of docking simulation using GOLD docking program. Figure S9 contains the data of apoptotic cell after drug treatment.



Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However, as recently reported by us and another group, tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules, we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report, tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin, we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin, we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore, tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast, other microtubule-targeting drugs, such as vincristine, paclitaxel, and colchicine, could not suppress the growth of cells overexpressing ABC transporters. Moreover, the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter–mediated multidrug-resistant tumor cells and is potentially useful against various tumors. Mol Cancer Ther; 13(12); 2978–90. ©2014 AACR.