Exposure of breast cancer cells to a subcytotoxic dose of apigenin causes growth inhibition, oxidative stress, and hypophosphorylation of Akt Megan E. Harrison a , Melanie R. Power Coombs b , Leanne M. Delaney a , David W. Hoskin a,b,c, ⁎ a Department of Microbiology and Immunology, Dalhousie University, PO Box 15000, Halifax, Nova Scotia B3H 4R2, Canada b Department of Pathology, Dalhousie University, PO Box 15000, Halifax, Nova Scotia B3H 4R2, Canada c Department of Surgery, Dalhousie University, PO Box 15000, Halifax, Nova Scotia B3H 4R2, Canada abstract article info Article history: Received 11 July 2014 Accepted 11 July 2014 Available online 12 July 2014 Keywords: Apigenin Breast cancer Cell cycle Flavone Protein kinase B/Akt Reactive oxygen species Epidemiological studies show that fruit- and vegetable-rich diets are associated with a reduced risk of developing certain forms of cancer, including breast cancer. In this study we demonstrate that a subcytotoxic concentration of apigenin, which is a flavone found at high concentrations in parsley, onions, grapefruit, oranges, and chamo- mile tea, inhibited DNA synthesis in a panel of human breast cancer cell lines (MDA-MB-231, MBA-MB-468, MCF-7, SK-BR-3). Decreased proliferation of MDA-MB-468 cells in the presence of apigenin was associated with G 2 /M phase cell cycle arrest and the production of reactive oxygen species. Apigenin-treated MDA-MB- 468 cells also showed reduced phosphorylation of Akt (protein kinase B), which is an essential effector serine/ threonine kinase in the phosphatidylinositide 3-kinase pathway that promotes tumor growth and progression. However, exposure to the antioxidant reduced glutathione failed to reverse apigenin-mediated inhibition of Akt phosphorylation and cell proliferation, indicating that these effects were not due to oxidative stress. Taken together, these findings suggest that low-dose apigenin has the potential to slow or prevent breast cancer progression. © 2014 Elsevier Inc. All rights reserved. 1. Introduction Although recent years have seen tremendous progress in the detec- tion and treatment of breast cancer (Howard and Bland, 2012), this dis- ease remains the most common malignancy and the predominant cause of cancer-related death in women (Are et al., 2013). Treatment recom- mendations are currently based on whether or not a breast tumor ex- presses human epidermal growth factor receptor 2 (HER2), estrogen receptor and/or progesterone receptor; however, molecular profiling has recently revealed that breast cancer is a heterogeneous and pheno- typically diverse malignancy (Cyr and Margenthaler, 2014). Treatment options consist of surgery with adjunct radiotherapy, chemotherapy, hormone therapy, and/or targeted biologic therapy (Howard and Bland, 2012). Breast cancers that express estrogen receptor are typically treated with drugs that inhibit receptor signaling or estrogen produc- tion whereas HER2-overexpressing tumors are treated with the HER2- specific monoclonal antibody trastuzumab. Breast cancers that fail to express estrogen receptor, progesterone receptor, and HER2 (triple- negative) initially respond well to chemotherapy but have a poor prog- nosis upon recurrence due to their lack of sensitivity to hormone therapy or HER2-targeted treatment (Foulkes et al., 2010). Unfortunate- ly, breast cancers frequently become resistant to chemotherapy due to the emergence of multidrug-resistant variants (Bush and Li, 2002). In addition, current systemic breast cancer treatments also have a number of adverse side effects that include cardiac toxicity (Ades et al., 2014) and reduced quality of life due to musculoskeletal and menopausal symptoms (Henry, 2014). More effective and better tolerated adjunct treatments for breast cancer are therefore of considerable interest to both patients and clinicians. Epidemiological studies indicate that a diet rich in fruits and vegeta- bles is associated with a lower incidence of many types of cancer (Riboli and Norat, 2003), presumably because of the consumption of a multi- tude of bioactive plant compounds known as phytochemicals, many of which have potential additive or synergistic cancer-fighting activities (González-Vallinas et al., 2013; Liu, 2004; Weng and Yen, 2012). Apigenin (5,7,4′-trihydroxyflavone) is a low molecular weight polyphe- nolic flavone that is present in parsley, celery, onions, grapefruit, and or- anges; however, apigenin is most abundant in chamomile tea, which contains 0.8–1.2% apigenin by weight/volume (Shukla and Gupta, 2010). Recent studies show that systemic administration of apigenin to Sprague–Dawley rats by intraperitoneal injection protects against the development of mammary tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (Mafuvadze et al., 2011). Apigenin also preferentially induces apoptosis in HER2-overexpressing breast cancer cells by a mechanism that involves inhibition of the serine/ Experimental and Molecular Pathology 97 (2014) 211–217 ⁎ Corresponding author at: Department of Microbiology and Immunology, Dalhousie University, PO Box 15000, 5850 College Street, Halifax, Nova Scotia B3H 4R2, Canada. E-mail address: d.w.hoskin@dal.ca (D.W. Hoskin). http://dx.doi.org/10.1016/j.yexmp.2014.07.006 0014-4800/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp