Abstract 5370: The therapeutic strategy using Nek2 siRNA and 5FU for cholangiocarcinoma cell
Authors: Kokuryo Toshio , Yokoyama Yukihiro , Yamaguchi Junpei , Nagino Masato 
Published: August 2015

Abstract
Introduction: Nek2 (NIMA-related kinase 2) is a member of the serine/threonine kinase family, which is involved in cell division and centrosome splitting. We reported that Nek2 suppression causes growth inhibition in several cancers, such as cholangiocarcinoma, breast cancer and colon cancer. In this study, we investigated a therapeutic strategy using Nek2 siRNA combined with the cytotoxic chemotherapy drug 5FU.
Methods: HuCCT1 cells (cholangiocarcinoma cell line) were first transfected with Nek2 siRNA or Control siRNA, and then treated with 5FU (0, 1, 5, 10, 50 and 100μM) 24h after transfection. Cell death was determined using the Trypan blue dye exclusion test. Floating and attached cells were collected 72 h after transfection and stained with 1% Trypan blue. Viable cells and Dead cells were counted under microscope. Cell proliferation was analyzed using a MTT assay, which is based on the cleavage of tetrazolium salt by mitochondrial dehydrogenases. Absorbance of the formazan dye was evaluated at 1.5 h after adding the reagent. Antitumor efficacy was assessed using Nek2 siRNA in a xenograft nude mouse model. HuCCT1 cells were inoculated subcutaneously into the femoral area of the mice. The siRNAs were injected around the tumor, and then 5FU was administered orally at a dose of 4 mg/kg body weight for each mouse from 7 days after tumor inoculation. The siRNA and 5FU was administered once a week for a total of three weeks. Estimated tumor volume (mm3) was calculated by the following equation; (L×W2)/2, where L is the tumor length and W is the tumor width (in mm).
Result and discussion: 5FU alone induced the cell death of HuCCT1 in dose-dependent manner. The number of dead cells treated with Nek2 siRNA and 5FU was significantly higher than that of cells treated with Control siRNA and 5FU. Combined treatment of Nek2 siRNA with 5FU also suppressed proliferation of HuCCT1 cells compared to Control siRNA with 5FU. The combination of Nek2 siRNA and 5FU was more effective than the administration of Nek2 siRNA alone. Furthermore, the combined treatment suppressed the tumor formation in a xenograft nude mouse model. There was no complication related to combined chemotherapeutic treatments of Nek2 siRNA and 5FU. Nek2 siRNA can be used in combination with conventional chemotherapeutic drugs for cancer treatment.
Conclusion: The therapeutic strategy using Nek2 siRNA and 5FU may be an effective treatment option for cholangiocarcinoma. Further investigation is necessary to clarify the detailed mechanism regulating cell growth and death by a combination of Nek2 siRNA and 5FU.
Note: This abstract was not presented at the meeting.
Citation Format: Toshio Kokuryo, Yukihiro Yokoyama, Junpei Yamaguchi, Masato Nagino. The therapeutic strategy using Nek2 siRNA and 5FU for cholangiocarcinoma cell. [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 5370. doi:10.1158/1538-7445.AM2015-5370