Different Propolis Samples, Phenolic Content, and Breast Cancer Cell Lines: Variable Cytotoxicity Ranging from Ineffective to Potent Mehmet Fatih Seyhan 1 Eren Yılmaz 1 Özlem Timirci-Kahraman 1 Neslihan Saygılı 1 Halil _ lbrahim Kısakesen 2 Sema Gazio  glu 3 Ahmet C. Gören 4 Allison Pınar Eronat 1 A. Begüm Ceviz 1 Tülin Öztürk 5 Hülya Yılmaz-Aydo  gan 1 O guz Öztürk 1 * 1 Department of Molecular Medicine, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey 2 Department of Molecular Biology and Genetics, Faculty of Arts and Science, Istanbul Technical University, Istanbul, Turkey 3 Department of Immunology, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey 4 Chemistry Group, Organic Chemistry Laboratory, National Metrology Institute, TUBITAK UME, Kocaeli, Turkey 5 Department of Pathology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey ABSTRACT Researchers have started focusing on investigating the anticarcinogenic effects of natural products with the slightest side effects possible, because current breast cancer treatment approaches are unable to achieve absolute success especially on aggressive subtypes. Propolis is among these products with its antimicrobial, antifungal, anti-inflamma- tory, and anticancer effects. Therefore, seven different samples were collected from different regions (Argentina, China, and Istanbul-Turkey) and applied on nonaggressive breast cancer cell line (BCCL) MCF-7 and aggressive cell lines SK-BR-3, and MDA-MB-231. Initially, the phenolic/ flavonoid constituents of the propolis ethanol extracts were investi- gated by liquid chromatography-mass spectrometry–mass spectrome- try (LS-MS/MS) and high-performance liquid chromatography (HPLC) analyses. Then, the anticarcinogenic effects of the propolis samples on MCF-7, SK-BR-3, MDA-MB-231 were evaluated by WST1 analysis and only selected ones on MCF-10A and hPdLF. According to the LS-MS/ MS and HPLC analysis, Turkey originated propolis (Turkey3) were found to be richer than the other propolis samples in terms of phenolic/ flavonoid compounds. Turkey propolis significantly inhibited cell prolif- eration in both nonaggressive and aggressive BCCL (P < 0.01). There- fore, Turkey3 propolis was selected for further evaluation using Annexin V-PI apoptosis detection assays. In addition, selected com- pounds among the propolis contents such as galangin, caffeic acid, api- genin, quercetin, and ferulic acid were applied to the MCF-7 cell line to detect cytotoxic and apoptotic effects. Galangin, caffeic acid, apigenin, and quercetin remarkably induced cell proliferation inhibition at all time intervals, whereas ferulic acid was found non efficient on the MCF-7 cell line. Annexin V-PI assay clarified that all cell proliferation inhibitions were markedly apoptotic. Our findings indicated that the inhibition effect of propolis on breast cancer cell proliferation was in a propolis type-, dose- and time-dependent fashion. Turkey3 propolis showed sta- tistically significant cytotoxic effects on both the nonaggressive and aggressive BCCL. These findings were consistent with the effects of its rich phenolic and flavonoid contents, in terms of variety. © 2018 IUBMB Life, 9999(9999):1–13, 2018 Keywords: propolis; breast cancer; cell line; flavonoid; LS/MS–MS; cyto- toxicity; apoptosis Abbreviations: BCCL, breast cancer cell lines; CAPE, caffeic acid phenethyl ester; EECP, ethanol extract of Chinese propolis; ER, estrogen receptor; FBS, fetal bovine serum; HER2, human epithelial growth factor receptor 2; HPLC, high-performance liquid chromatography; IC50, the half maximal inhibitory con- centration.; LC–MS/MS, liquid chromatography-mass spectrometry–mass spectrometry; LOD, limit of detection; LOQ, limit of quantification; PI, propidium iodide; PR, progesterone receptor; WST-1, water soluble tetrazolium-1 © 2018 International Union of Biochemistry and Molecular Biology Volume 9999, Number 9999, 2018, Pages 1–13 *Address correspondence to: Prof. Oguz Ozturk, MD, PhD; Istanbul University, Institute of Experimental Medicine, Department of Molecular Medicine, Vakif Gureba Street, Capa, 34390, Istanbul, Turkey. TelFax: +90-212-444-20-00/33303. E-mail: dr.oguzozturk@gmail.com Received 19 October 2018; Accepted 29 November 2018 DOI 10.1002/iub.1996 Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com) IUBMB Life 1 Research Communication