Published: February 07, 2011 r2011 American Chemical Society 1996 dx.doi.org/10.1021/jf103656v | J. Agric. Food Chem. 2011, 59, 1996–2003 ARTICLE pubs.acs.org/JAFC Gallic Acid Induces G2/M Phase Arrest of Breast Cancer Cell MCF-7 through Stabilization of p27 Kip1 Attributed to Disruption of p27 Kip1 /Skp2 Complex Jeng-Dong Hsu, †,‡,§ Shao-Hsuan Kao, ||,§ Ting-Tsz Ou, || Yu-Jen Chen, || Yi-Ju Li, †,|| and Chau-Jong Wang* ,|| † Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan ‡ Department of Pathology, School of Medicine, and ) Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan ABSTRACT: Gallic acid (GA), 3,4,5-trihydroxybenzoic acid, is a natural polyphenolic acid and widely found in gallnuts, tea leaves and various fruits. Previous studies have shown that GA possesses anti-inflammatory, antiallergic and anticarcinogenic activity. In the present study, we aim to investigate the antitumor effects of GA on breast cancer cell. Our results revealed that GA treatment significantly reduced the cell growth of human breast cancer cell MCF-7 in a dose-dependent manner. Further flow cytometric analysis showed that GA induced significant G2/M phase arrest but slightly affected the population of sub-G1MCF-7 cells. Therefore, levels of cyclins, cyclin-dependent kinases (CDKs), and their regulatory proteins involved in S-G2/M transition were investigated. Our findings revealed that levels of cyclin A, CDK2, cyclin B1 and cdc2/CDK1 were diminished; in contrast, levels of the negative regulators p27 Kip1 and p21 Cip1 were increased by GA treatment. Additionally, Skp2, a specific ubiquitin E3 ligase for polyubiquitination of p27 Kip1 was reduced by GA treatment. Further investigation showed that GA attenuated Skp2-p27 Kip1 association and diminished polyubiquitination of p27 Kip1 in MCF-7 cells. Moreover, knockdown of p27 Kip1 but not p21 Cip1 significantly alleviated GA-induced accumulation of G2/M phase. These findings indicate that GA may upregulate p27 Kip1 level via disruption of p27 Kip1 /Skp2 association and the consequent degradation of p27 Kip1 by proteosome, leading to G2/M phase arrest of MCF-7 cell. It is suggested that GA should be beneficial to treatment of breast cancer and p27 Kip1 -deficient carcinomas. KEYWORDS: gallic acid, cell cycle arrest, MCF-7, p27 Kip1 , p21 Cip1 , Skp2 ’ INTRODUCTION Gallic acid (GA), known as 3,4,5-trihydroxylbenzoic acid, is a polyhydroxyl phenolic compound and is ubiquitously present in fruits, gallnuts, green tea and red wine. 1-5 GA has been demon- strated to have a broad spectrum of biological activities, including antimicrobial, 6-8 and anti-inflammatory 9 activities. GA has been investigated as a potential anticancer agent in various human cancer cell lines, such as TE-2 (esophageal cancer), MKN-28 (gastric cancer), HT-29 and Colo201 (colon cancer), MCF-7 (breast cancer) and CaSki (cervix cancer). 10 Additionally, previous studies have shown that GA induced apoptosis in cancer cells in association with oxidative stresses derived from reactive oxygen species (ROS), mitochondrial dysfunction and an increase in intracellular Ca 2þ level. 11,12 However, GA also exhibits antiapoptotic potential in normal human lymphocytes 13 and acts as strong natural antioxidant scavenging ROS. 13-16 Molecular mechanisms underlying induction of GA in the aspect of cell fate remains sketchy. Breast cancer is the commonest cancer in most countries in Asia, and the incidence is increasing at a more rapid rate than in Western countries, which may be due to changes in the lifestyle and diet. 17 Hormone replacement therapy such as prolonged exposure to estrogen and/or progesterone and reproductive history are the most major risk factors for breast cancer. 18 Additionally, the impor- tance of estrogen in breast cancer development is also supported by studies demonstrating the occurrence of marked changes in estro- gen signaling and in the expression of the two estrogen receptors (ERs), ER alpha and ER beta, during breast tumorigenesis and progression. 19-21 To date, a universal surgical resection followed by radiotherapy and/or chemotherapy are the most common treat- ments for breast cancer to prevent its metastasis and recurrence. 22 Therefore, the means that intervenes in the proliferation and pro- gression of breast cancer is important prior to the clinical treatments. The significance of cell cycle mediators involved in carcino- genesis is now well documented. Critical genes that regulate cell cycle checkpoints have been demonstrated to be lost and/or aberrant in a variety of cancers in human. 23 Cells progress through the various phases of the cell cycle through the interactions of different cyclins with their speci fic kinases, cyclin-dependent kinases (CDKs), 24 which can be negatively regulated by two classes of CDK inhibitors, inhibitors of CDK4 (INK4) and kinase inhibitor proteins (KIPs). The latter include p21 Cip1 , 25 p27 Kip1 26 and p57 Kip2 . 27,28 In the present study, we focused on the effects of GA on cell proliferation and the cell cycle of breast cancer cells with emphasis on the underlying mechanism. To investigate the alteration of cell proliferation and cell cycle distribution induced by GA, MTT assay and flow cytometric analysis were performed. The expression level of important cell cycle regulators was determined by immunoblot- ting. Additionally, the protein-protein interactions between the regulators and GA were also demonstrated by immunoprecipitation and immunoblotting. Received: September 23, 2010 Accepted: January 25, 2011 Revised: January 16, 2011