Abstract 990: Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms

Purpose: Four 5-fluorouracil (5-FU)-resistant human gastric cancer cell lines were established by the long-term exposure of parent cell lines (MKN45, MKN-74, KATO-III, and NCI-N87) to 5-FU. The cytotoxicity and mechanisms of several chemotherapeutic agents were then investigated using 5-FU-resistant gastric cancer cell lines. Method: 5-FU-resistant cell lines were established by continuous exposure to escalating concentrations (1-5 μM) of 5-FU over a 1-year period. The sensitivities of the cell lines to chemotherapeutic agents (paclitaxel, docetaxel, cisplatin, oxaliplatin, epirubicin, and SN-38) were then evaluated using the WST-8 colorimetric assay. To elucidate the mechanisms of resistance, the gene expressions and copy numbers were measured using an Agilent Technologies whole human genome oligo DNA microarray and a CGH microarray. The protein and mRNA expressions of a target enzyme of 5-FU, thymidylate synthase (TS), were also investigated using western blotting and RT-PCR, respectively. Results: The IC50 ratios of the 5-FU-resistant cell lines were 2.6 to 15.6 times higher than those of the parent cell lines in vitro. MKN-45/5-FU showed cross-resistance to cisplatin and oxaliplatin and KATO-III/5-FU showed cross-resistance to SN-38, while MKN-74/5-FU showed collateral sensitivities to docetaxel, cisplatin, and oxaliplatin, and NCI-N87/5-FU showed collateral sensitivities to cisplatin, oxaliplatin, SN-38, and epirubicin. RT-PCR showed that TS mRNA expression was higher in the parent cell lines than in the 5-FU-resistant cell lines, and the TS protein expression level was also higher in all the resistant cell lines. For MKN-45/5-FU and KATO-III/5-FU, not only the mRNA expression level but also the TS gene copy number was increased. Interestingly, the expression of MAP3K15, which is an apoptosis-facilitating factor, increased in NCI-N87/5-FU, which showed collateral sensitivity to cisplatin, oxaliplatin, SN-38, and epirubicin. Conclusion: Analyzing the molecular mechanisms of 5-FU-resistant cell lines may be useful for the development of effective chemotherapy regimens for relapsed gastric cancer after 5-FU-based chemotherapy. Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Ayako Nakamura, Kazuhiko Hayashi, Teiji Takechi. Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-