Chlorogenic Acid Induces Apoptotic Cell Death in U937 Leukemia Cells through Caspase- and Mitochondria-dependent Pathways

Chlorogenic acid exists widely in edible and medicinal plants and acts as an antioxidant. It is known to exert antitumor activity via induction of apoptosis in many human cancer cells. However, its signaling pathway in human leukemia cells still remains unclear. Therefore, we investigated the roles of reactive oxygen species (ROS), mitochondria and caspases during chlorogenic acid-induced apoptosis of U937 human leukemia cells. Chlorogenic acid exhibited a strong cytotoxicity and induced apoptosis in U937 cells, as determined by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Chlorogenic acid induced apoptosis by promoting ROS production and reduced the mitochondrial membrane potential (ΔΨm), as assayed by flow cytometry. Furthermore, the activity of caspase-3 was evaluated and results indicated that chlorogenic acid promoted caspase-3 activity in U937 cells. Results from western blot analysis showed that chlorogenic acid promoted expression of caspase-3, -7, -8 and -9 in U937 cells. Taken together, these results suggest that chlorogenic acid may induce apoptosis by reducing the levels of ΔΨm and by increasing the activation of caspase-3 pathways in human leukemia U937 cells in vitro. Development of drug resistance in tumor cells and sideeffects in patients have led to limitations to current chemotherapy in patients with leukemia (1, 2). In clinical practice, camptothecin from Camptotheca acuminata and paclitaxel from Taxus brevifolia, originating from natural products, are currently used as chemotherapeutic agents (3); both compounds can induce cell cytotoxic effects, including the induction of cell-cycle arrest and apoptosis (4, 5). It is well-known that caspases (a group of cysteine proteases) play important roles in apoptosis. After injury to mitochondria, cytochrome c and other apoptotic-inducing factors can be released from mitochondria and then also activate caspase-3, -7 or -9 signals (6, 7). Thus, agents which can induce caspase activation may lead to the induction of apoptosis and it has been recognized that induction of cancer 971 Correspondence to: Dr. Chao-Lin Kuo, School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, R.O.C. Tel: +886 422053366 ext. 5202, Fax: +886 22070439, e-mail: clkuo@mail.cmu.edu.tw