Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff Antiproliferative efficacy of elderberries and elderflowers (Sambucus canadensis) on glioma and brain endothelial cells under normoxic and hypoxic conditions Sylvie Lamy 1 , Évelyne Muhire 1 , Borhane Annabi ⁎ Laboratoire d’Oncologie Moléculaire, Centre de Recherche BioMed, Université du Québec à Montréal, C.P. 8888, Succ. Centre-ville, Montréal, Québec H3C 3P8, Canada ARTICLE INFO Chemical compounds studied in this article: Cyanidin 3-O-sambubioside-5-O-glucoside chloride (PubChem CID: 44256731) Delphinidin 3-O-rutinoside chloride (PubChem CID: 44256887) Quercetin 3-O-rutinoside (PubChem CID: 5280805) Keywords: Cell proliferation Elderberry Elderflower Endothelial cells Glioma Hypoxia ABSTRACT Elderberries are among the richest sources of antioxidant anthocyanins with health-promoting properties. As low circulating plasma antioxidant levels contribute to solid tumor malignancy, we assessed the antiproliferative properties of two Canadian elderberry cultivar extracts, ‘Kent’ and ‘Scotia’, against human brain tumor-derived cell line models and brain microvascular endothelial cells under both normoxic and hypoxic culture conditions. Elderberry, concentrated elderberry and elderflower extracts inhibited cell proliferation in a dose-dependent manner with berry extracts being more efficient. The antiproliferative effects resulted in cell cycle arrest through alterations in cell cycle checkpoint protein expression and in apoptosis. Anthocyanins and or rutin contents of the extracts efficiently inhibited cell proliferation alone or in synergy. Given brain tumors are characterized by high hypoxic areas which contribute to therapy resistance, this is the first evidence suggesting that Canadian elderberry extracts could efficiently target both the cancer and vascular compartments associated to brain tumor development. 1. Introduction Malignant brain tumors are among the most feared types of cancer, not only for their poor prognosis, but also because of their direct re- percussions on quality of life and cognitive function. The World Health Organization (WHO) classification of tumors of the central nervous system establishes a grading of human tumors, and groups gliomas into 4 histological grades (Louis et al., 2007). WHO grade III and grade IV tumors are the most common type of primary malignant gliomas, of which glioblastoma multiforme is the most aggressive form. Growth of gliomas is characterized by the development of a het- erogeneous vascularization. Indeed, at an early phase of tumor growth, the vessels are small, sparsely branched, well organized and well per- fused under normoxic conditions (Hendriksen et al., 2009). However, when the tumor increases in size, the neovascularization becomes morphologically and functionally unsuitable with irregular blood flow creating hypoxic areas (Jensen, 2009). This hypoxic microenvironment is a powerful stimulus for the expression of genes involved in tumor cell proliferation and angiogenesis including hypoxia inducible factor-1α, transforming growth factor-β and vascular endothelial growth factor (VEGF) (Kaur et al., 2005), which stimulate vasculature development in order to rapidly supply oxygen to malignant cells. These tumor cells acquire resistance to treatment and successfully treating gliomas be- comes challenging. Hence, more insight into chemopreventive agent properties against tumor cell proliferation is needed. Over the past few years, there has been a growing interest in nu- traceutical intervention against different types of tumors, including brain tumors (Ramachandran, Nair, Escalon, & Melnick, 2012; Rooprai, Christidou, & Pilkington, 2003; Sheweita & Sheikh, 2011). This ap- proach uses the chemopreventive properties of naturally occurring phytochemicals, especially those from diet-derived vegetables, spices and fruits (Surh, 2003; Wiseman, 2008). Among them, anthocyanins exhibit beneficial effects against mechanisms involved in the progres- sion of various diseases (He & Giusti, 2010; Hou, 2003). These https://doi.org/10.1016/j.jff.2017.10.048 Received 24 March 2017; Received in revised form 16 October 2017; Accepted 30 October 2017 ⁎ Corresponding author. 1 These authors contributed equally to this work. E-mail addresses: lamy.sylvie@uqam.ca (S. Lamy), muhire.evelyne@courrier.uqam.ca (É. Muhire), annabi.borhane@uqam.ca (B. Annabi). Abbreviations: BBB, blood-brain barrier; Cy-3-sam-5-glu, cyanidin-3-O-sambubioside-5-O-glucoside chloride; Dp-3-rut, delphinidin 3-O-rutinoside chloride; GAPDH, glyceraldehyde 3- phosphate dehydrogenase; GLUT-1, glucose transporter-1; HBMEC, human brain microvascular endothelial cells; PI, propidium iodide; Que-3-rut, quercetin-3-O-rutinoside; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; WHO, World Health Organization Journal of Functional Foods 40 (2018) 164–179 1756-4646/ © 2017 Elsevier Ltd. All rights reserved. MARK