Abstract 85: Chloroquine potentiates the 5-fluorouracil-induced cell cycle arrest on HT-29 colon cancer cells
Authors: Sasaki Kazuhito , Tsuno Nelson H. , Sunami Eiji , Tsurita Giichiro , Okaji Yurai , Kawai Kazushige , Nishikawa Takeshi , Shuno Yasutaka , Hongo Kumiko , Kaneko Manabu , Hiyoshi Masaya , Kitayama Joji , Takahashi Koki , Nagawa Hirokazu 
Published: February 2011

Abstract
Introduction
Autophagy is a self-degradation process that is essential for cell survival, development, and homeostasis. During autophagy, cytoplasmic organelles and proteins damaged by the cellular stress, such as starvation, hypoxia and chemotherapy, are recycled and converted into more essential proteins. Thus, induction of autophagy in cancer cells may result in cell adaptation against anticancer agents, consequently leading to resistance to therapy. Chloroquine (CQ), a worldwide used anti-malarial drug, is a lysosomotropic drug, also known to inhibit autophagy by impairing the autophagic protein degradation. On the other hand, 5-Fluorouracil (5-FU) is the most common chemotherapeutic agent for colorectal cancer, which inhibits DNA synthesis and induces apoptosis and/or cell cycle arrest in colon cancer cells. In the present study, we aimed to investigate the induction of autophagy as a mechanism of resistance of colon cancer cells to 5-FU-based chemotherapy, and the role of autophagy inhibition, by CQ, in potentiating the effect of 5-FU, especially focusing on cell cycle arrest.
Procedure
The human colon cancer cell line HT-29 was used. Cells were treated with CQ and/or 5-FU at various concentrations, and the changes in the proliferative activity were investigated, especially focusing on the induction of autophagy, the changes of cell cycle, and the development of apoptosis. The proliferative ability of HT-29 was analyzed by the MTS assay. The cell cycle was evaluated by flow-cytometry after staining of cells with PI, and autophagy was quantified by the analysis of the formation of acidic vesicular organelles (AVOs) by flow-cytometry after staining of cells with acridine orange (AO), and additionally, by the analysis of the expression of LC3-II by Western blot. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. Finally, to evaluate the long-term viability of the cells, the colony formation assay was performed.
Results
Treatment of HT-29 with 5-FU resulted in significant inhibition of the proliferative activity, compared with untreated cells. Treatment of the cells with CQ prior to exposure to 5-FU, potentiated the inhibitory effect. And it was dependent on the increase of the percentage of cells in G0/G1-phase, and the simultaneous decrease of cells in G2/M-phase. Also a partial increase of apoptotic cells was observed. These effects were dependent on the decreased expression of cyclin-dependent kinase-2 and the increased expression of p21Cip1, p27Kip1, important regulators of the cell cycle progression from G1 to S phase. Therefore, pre-treatment of HT-29 with the autophagy inhibitor, CQ, resulted in potentiation of the G0/G1 phase cell cycle arrest induced by 5-FU.
Conclusion: The combination therapy using inhibitor of autophagy, such as CQ, should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 85.