Resveratrol Induces Apoptosis through ROS-Dependent Mitochondria Pathway in HT-29 Human Colorectal Carcinoma Cells M. EM ´ ILIA JUAN,* ,† UWE WENZEL, ‡,§ HANNELORE DANIEL, ‡ AND JOANA M. PLANAS † Department of Fisiologia, Facultat de Farma `cia, Universitat de Barcelona, Spain, and Molecular Nutrition Unit, Department of Food and Nutrition, Technical University of Munich, Freising, Germany trans-Resveratrol is a polyphenol found in blueberries, grapes, and wine with cancer chemopreventive properties. The low bioavailability of this compound enhances its concentration in the luminal content and becomes a potential chemopreventive agent against colon cancer. In the present study, the antiproliferative and pro-apoptotic effects on the human colorectal carcinoma HT-29 cells as well as the mechanisms underlying these effects were examined. Proliferation, cytotoxicity, and apoptosis were measured by fluorescence-based techniques. Studies of dose-dependent effects of trans- resveratrol showed antiproliferative activity with an EC 50 value of 78.9 ( 5.4 μM. Caspase-3 was activated in a dose-dependent manner after incubation for 24 h giving an EC 50 value of 276.1 ( 1.7 μM. Apoptosis was also confirmed with microscopic observation of changes in membrane permeability and detection of DNA fragmentation. The activity of trans-resveratrol on the mitochondria apoptosis pathway was evidenced by the production of superoxide anions in the mitochondria of cells undergoing apoptosis. In conclusion, trans-resveratrol inhibits cell proliferation without cytotoxicity and induces apoptosis in HT-29. Results of the present study provide evidence demonstrating the antitumor effect of trans-resveratrol via a ROS-dependent apoptosis pathway in colorectal carcinoma. KEYWORDS: Resveratrol; proliferation; apoptosis; caspase-3; ROS; colon cancer INTRODUCTION In recent years, the growing interest in the role of nutrition in the prevention of cancer has prompted the identification of specific diet constituents with chemotherapeutic properties (1). Among the dietary bioactive compounds stands out trans- resveratrol, which belongs to a class of defense molecules called phytoalexins. These are produced by several plants in response to stress, injury, UV radiation, and fungal infections. trans- Resveratrol is normally found in dietary products such as grapes (50-100 μg/g), red wine (0.1-14.3 mg/L), peanuts (0.02-1.92 μg/g), and various berries (0.07-5.8 μg/g) (2). trans-Resveratrol is gaining acceptance as a potential anti- tumor agent because of its pleitropic effects described in different experimental models of carcinogenesis (3, 4). This bioactive compound was shown to inhibit the growth of tumor cell lines derived from various human cancers (5–8). This effect has been associated with the ability of resveratrol to arrest cell cycle progression (5, 9) and to induce programmed cell death (6, 10, 11). These properties accompanied with the lack of harmful effects (12) makes trans-resveratrol an attractive chemotherapy and chemopreventive drug for cancer treatment (3). Despite the numerous studies describing the intracellular changes leading to cell cycle arrest or apoptosis in response to trans-resveratrol treatment, the effects are often cell type specific, and the underlying mechanism of action remains to be fully identified (4). Several studies have indicated that reactive oxygen species (ROS) production in mitochondria may mediate apoptosis induction (13, 14). The roles of ROS and mitochondria in trans-resveratrol induced apoptosis are still undefined. Consequently, the present study examines the anti- proliferative and pro-apoptotic effects on the human colorectal carcinoma HT-29 of trans-resveratrol and elucidates the possible involvement of ROS in the antitumoral activity of this promising bioactive compound. MATERIAL AND METHODS Chemicals and Reagents. trans-Resveratrol (Sigma, Tres Cantos, Madrid, Spain) was chemically pure. Before use, its purity was assessed by HPLC coupled to a diode-array UV detector, and a chromatogram * Corresponding author. M. Emı ´lia Juan, Departament de Fisiologia, Facultat de Farma `cia, Universitat de Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain. Tel: +34.93.402.45.05. Fax: +34.93.403.59.01. E-mail: mejuan@ub.edu. † Universitat de Barcelona. ‡ Technical University of Munich. § Present address: Molecular Nutrition Research, Interdisciplinary Research Center, Institute of Nutritional Science, Justus-Liebig- University of Giessen, Germany. J. Agric. Food Chem. 2008, 56, 4813–4818 4813 10.1021/jf800175a CCC: $40.75  2008 American Chemical Society Published on Web 06/04/2008