Abstract [Background] Heat Shock Protein (HSP) 27 is a chaperone protein of small molecular weight, which protects cells in response to various stresses. Previously we elucidated that HSP27 was a key molecule in determining 5-Fluorouracil (5-FU) resistance in colorectal cancer (CRC). Thus, apatorsen, an antisense oligonucleotide that suppresses HSP27 levels intracellularly, might overcome 5-FU resistance in CRC. In this basic research, we evaluated the feasibility of adding apatorsen with 5-FU against CRC. [Methods] Human CRC cell line SW480 was treated with apatorsen (1nM, 10nM, 100nM) for 48 hours continuously. HSP27 protein level was determined by immunoblot and densitometry analysis. Next, the cells were exposed by various concentrations of 5-FU for 48 hours following treatment with apatorsen. The in vitro growth inhibition rates (IR) were assessed by MTT assay and we evaluated the change of 5-FU resistance, which was estimated as the drug concentration inducing 50% IR (IC50). [Results] Apatorsen (0, 1, 10, 100nM) successfully suppressed HSP27 protein levels (100, 86.6, 61.0, 50.5%) in a dose-dependent manner. In cell viability assays, apatorsen changed the IC50 of 26.65μg/ml in control into 15.55 (58.3%), 11.28 (42.3%), and 9.18 (34.4%) respectively. [Summaries] Apatorsen significantly enhanced 5-FU sensitivity in the CRC cell line SW480 by suppressing HSP27 levels. HSP27 may serve as a reliable target in enhancing 5-FU chemotherapy for CRC. Therefore, apatorsen is a promising treatment requiring further investigation. Citation Format: Takehiro Shimada, Masashi Tsuruta, Shingo Akimoto, Kaoru Koishikawa, Koji Okabayashi, Hirotoshi Hasegawa, Yuko Kitagawa. Apatorsen enhances 5-FU sensitivity in colon cancer cell SW480. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2015-4552