Down-regulation of c-Src/EGFR-mediated signaling activation is involved in the honokiol-induced cell cycle arrest and apoptosis in MDA-MB-231 human breast cancer cells Eun-Jung Park a , Hye-Young Min a , Hwa-Jin Chung a , Ji-Young Hong a , You-Jin Kang a , Tran Manh Hung b , Ui Joung Youn b , Yeong Shik Kim c , KiHwan Bae b , Sam Sik Kang c , Sang Kook Lee a, * a College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea b College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea c College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea article info Article history: Received 28 September 2008 Received in revised form 24 November 2008 Accepted 27 November 2008 Keywords: Honokiol MDA-MB-231 Apoptosis Src mTOR abstract Honokiol is a naturally occurring neolignan abundant in Magnoliae Cortex and has showed anti-proliferative and pro-apoptotic effects in a wide range of human cancer cells. How- ever, the molecular mechanisms on the anti-proliferative activity in cancer cells have been poorly elucidated. In this study, we evaluated the growth inhibitory activity of honokiol in cultured estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. Honok- iol exerted anti-proliferative activity with the cell cycle arrest at the G0/G1 phase and sequential induction of apoptotic cell death in a concentration-dependent manner. The honokiol-induced cell cycle arrest was well correlated with the suppressive expression of CDK4, cyclin D1, CDK2, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb) at Ser780. Apoptosis caused by honokiol was also concomitant with the cleavage of caspases (caspase-3, -8, and -9) and Bid along with the suppressive expression of Bcl- 2, but it was independent on the expression of Bax and p53. In addition, honokiol-treated cells exhibited the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmenta- tion. In the analysis of signal transduction pathway, honokiol down-regulated the expres- sion and phosphorylation of c-Src, epidermal growth factor receptor (EGFR), and Akt, and consequently led to the inactivation of mTOR and its downstream signal molecules includ- ing 4E-binding protein (4E-BP) and p70 S6 kinase. These findings suggest that honokiol- mediated inhibitory activity of cancer cell growth might be related with the cell cycle arrest and induction of apoptosis via modulating signal transduction pathways. Ó 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Breast cancer is one of the most common cause of can- cer-related death and the second leading incidence of can- cer in women [1]. Especially, estradiol-nonresponsive, estrogen receptor (ER)-negative breast cancers showed poor prognosis and less responded toward chemothera- peutic agents [1]. Therefore, continuous efforts to develop chemotherapeutic agents with novel mechanisms of action should be required for managing ER-negative breast cancers. Honokiol is a naturally occurring neolignan mainly found in Magnoliae Cortex, the bark of Magnolia obovata Thunberg (Magnoliaceae), which has been traditionally used to enhance the function of the gastrointestinal tract in Asian countries. Recently, various biological effects of honokiol have been reported including alleviation of 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.11.029 * Corresponding author. Tel.: +82 2 3277 3023; fax: +82 2 3277 2851. E-mail address: sklee@ewha.ac.kr (S.K. Lee). Cancer Letters 277 (2009) 133–140 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet