Antiproliferative Activity of Chemically Characterized Propolis from Turkey and Its Mechanisms of Action Başak Aru, a Etil Güzelmeric, b Aslı Akgül, c Gülderen Yanıkkaya Demirel, a and Hasan Kırmızıbekmez* b a Department of Immunology, Faculty of Medicine, Yeditepe University, TR-34755 Kayışdağı, İstanbul, Turkey b Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, TR-34755 Kayışdağı, İstanbul, Turkey, e-mail: hasankbekmez@yahoo.com c Faculty of Pharmacy, Yeditepe University, TR-34755 Kayışdağı, İstanbul, Turkey The aim of this study was to evaluate the ethanolic extract of propolis originated from northern Turkey for its antiproliferative, apoptotic and cell cycle arrest promoting effects on MCF7, HGC27, A549 cancer cell lines and a healthy cell line (HUVEC) in terms of DNA content, morphological features, expression of cell cycle checkpoint proteins p21, p53, Cyclin D1 and immune checkpoint protein PD-L1. The extract showed moderate antiproliferative activity against all tested cancer cell lines with IC 50 values in the range of 58.6–90.7 μg/mL in MTS assay. Further studies indicated that propolis extract exerted apoptotic effect on cancer cell lines, promoted cell cycle arrest through activation of p21 and resulted in accumulation at G0/G1 phase of cancer cells. Propolis treatment caused increased cell size, according to fluorescent imaging except for MCF7. HPTLC analysis revealed that 3-O-methylquercetin, chrysin, caffeic acid, CAPE, galangin and pinocembrin were the main components of the extract. The amounts of caffeic acid and CAPE in the extract were found to be 5.5 and 11.1 mg/g, respectively, by a validated HPLC method. Our study is the first one, revealing effect of propolis on PD-L1 expression on certain cancer cell lines. Keywords: propolis, antiproliferative activity, apoptosis, cell cycle analysis, HPTLC, HPLC, biological activity. Introduction Propolis, a resinous bee product, originates from buds or exudates of different botanical sources such as poplar, willow, birch, elm, alder, beech, and horse- chestnut trees. It has long been used in many cultures to treat ailments including colds, wounds and ulcers. Many biological activities have been performed on propolis extracts of different geographical and bota- nical origins up to now indicating their cytotoxic, anticancer, immunomodulatory, anti-inflammatory, antimicrobial and antioxidant activities. [1] Among these bioactivities, cytotoxic and anticancer activities of propolis have particularly attracted much attention in recent years. [2–5] Carcinogenesis is a multistage process; involving initiation, promotion and progression. During cell division, maintenance of tissue homeostasis, integrity of nuclear material and cell size in under control of cell cycle checkpoints. DNA replication occurs during syn- thesis or S-phase, which is followed by mitosis or M- phase where chromosome segregation is observed. These phases are divided with gap phases G1 and G2, which enables the cell to organize and integrate signals to be prepared for division. Progression of cell cycle is regulated by cyclin-dependent kinases (CDKs) and their downstream substrates. Cyclin binding allows CDK to gain active configuration, thus allows cell cycle progression. Different cyclins play roles during different phase transitions. [6] Among cyclins, cyclin D1 takes part in G1 phase by generating a complex with Cdk4; initiating DNA synthesis, thus allowing cell to enter S phase. [7] During cell division steps, cell cycle checkpoints controls cell size, ensures DNA is replicated, monitors DNA damage and chromo- some segregation. Proteins play roles in cell cycle progression control by arresting cell cycle and promot- ing apoptosis at G1/S and G2/M phases in case of an abnormality. Transcription factor p53 is a critical factor for DNA damage monitorization. Upon activation and DOI: 10.1002/cbdv.201900189 FULL PAPER Chem. Biodiversity 2019, 16, e1900189 © 2019 Wiley-VHCA AG, Zurich, Switzerland