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      Proapoptotic Activity of Propolis and Their Components on Human Tongue Squamous Cell Carcinoma Cell Line (CAL-27)

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          Abstract

          Propolis has been used since ancient times in folk medicine. It is a popular medicine possessing a broad spectrum of biological activities. This material is one of the richest sources of polyphenolic compounds such as flavonoids and phenolic acids. The ethanolic extract of propolis (EEP) evokes antibacterial, antiviral, antifungal and anticancer properties. Due to pharmacological properties it is used in the commercial production of nutritional supplements. In this study, gas chromatography coupled with mass spectrometry (GC-MS) was used to quantify main polyphenols in EEPs. The effect of EEPs, individual EEPs components (chrysin, galangin, pinocembrin, caffeic acid, p-coumaric acid, ferulic acid) and their mixture on viability of human tongue squamous cell carcinoma cell line (CAL-27) as well as the molecular mechanisms of the process were examined. The results of MTTs assay demonstrated that EEP, polyphenols and mixture of polyphenolic compounds were cytotoxic for CAL-27 cells in a dose dependent manner. The mechanism of cytotoxicity induced by these components undergoes through apoptosis as detected by flow cytometry. The ethanolic extracts of propolis activated caspases -3, -8, -9. Mixture of polyphenols was found as the most potent inducer of apoptosis thorough both intrinsic and extrinsic pathway. Therefore, we suggest that anticancer properties of propolis is related to synergistic activity of its main components.

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          Caspase family proteases and apoptosis.

          Ting-Jun Fan, Li-Hui Han, Ri-Shan Cong … (2005)
          Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1beta-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain, and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.
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            Propolis: is there a potential for the development of new drugs?

            José Sforcin, Vassya Bankova (2011)
            Propolis has plenty of biological and pharmacological properties and its mechanisms of action have been widely investigated in the last years, using different experimental models in vitro and in vivo. Researchers have been interested in the investigation of isolated compounds responsible for propolis action; however, there is lack of clinical research on the effects of propolis. Since propolis-containing products have been marketed and humans have used propolis for different purposes, the goal of this review is to discuss the potential of propolis for the development of new drugs, by comparing data from the literature that suggest candidate areas for the establishment of drugs against tumors, infections, allergy, diabetes, ulcers and with immunomodulatory action. The efficacy of propolis in different protocols in vitro and in vivo suggests its therapeutic properties, but before establishing a strategy using this bee product, it is necessary to study: (a) the chemical nature of the propolis sample. (b) Propolis efficacy should be compared to well-established parameters, e.g. positive or negative controls in the experiments. Moreover, possible interactions between propolis and other medicines should be investigated in humans as well. (c) Clinical investigation is needed to evaluate propolis potential in patients or healthy individuals, to understand under which conditions propolis may promote health. Data point out the importance of this research field not only for the readers and researchers in the scientific community waiting for further clarification on the potential of propolis but also for the pharmaceutical industry that looks for new drugs. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.
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              Apoptosis and necrosis: detection, discrimination and phagocytosis.

              Dmitri V. Krysko, Tom Vanden Berghe, Katharina D'Herde … (2008)
              Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.
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                Author and article information

                Contributors
                Gianfranco Pintus: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 9 June 2016
                Publication date Collection: 2016
                Volume: 11
                Issue: 6
                Electronic Location Identifier: e0157091
                Affiliations
                [1 ]Department of Pharmaceutical Analysis, Medical University of Bialystok, Bialystok, Poland
                [2 ]Department of Medicinal Chemistry, Medical University of Bialystok, Bialystok, Poland
                University of Sassari, ITALY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: UC WM. Performed the experiments: UC KS IZ. Analyzed the data: UC KS IZ WM. Contributed reagents/materials/analysis tools: UC KS IZ. Wrote the paper: UC WM.

                Article
                Publisher ID: PONE-D-16-09613
                DOI: 10.1371/journal.pone.0157091
                PMC ID: 4900600
                PubMed ID: 27281369
                SO-VID: 43f32d76-36a0-4894-9c7f-22199fa73d4e
                Copyright © © 2016 Czyżewska et al
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 6 March 2016
                Date accepted : 24 May 2016
                Page count
                Figures: 4, Tables: 2, Pages: 14
                Funding
                Funded by: Medical University of Bialystok
                Award ID: 124/KNOW/15
                Award Recipient :
                This study was conducted with the use of equipment purchased by Medical University of Bialystok as part of the OP DEP 2007-2013, Priority Axis I.3, contract No. POPW.01.03.00-20-008/09 and financially supported by the Medical University of Bialystok, Poland (KNOW Project No.124/KNOW/15). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Biology and Life Sciences
                Subject: Cell Biology
                Subject: Cell Processes
                Subject: Cell Death
                Subject: Apoptosis
                Subject: Physical Sciences
                Subject: Chemistry
                Subject: Chemical Compounds
                Subject: Phenols
                Subject: Biology and Life Sciences
                Subject: Anatomy
                Subject: Digestive System
                Subject: Mouth
                Subject: Tongue
                Subject: Medicine and Health Sciences
                Subject: Anatomy
                Subject: Digestive System
                Subject: Mouth
                Subject: Tongue
                Subject: Research and Analysis Methods
                Subject: Chromatographic Techniques
                Subject: Gas Chromatography-Mass Spectrometry
                Subject: Physical Sciences
                Subject: Chemistry
                Subject: Analytical Chemistry
                Subject: Mass Spectrometry
                Subject: Gas Chromatography-Mass Spectrometry
                Subject: Research and Analysis Methods
                Subject: Spectrum Analysis Techniques
                Subject: Mass Spectrometry
                Subject: Gas Chromatography-Mass Spectrometry
                Subject: Biology and Life Sciences
                Subject: Toxicology
                Subject: Cytotoxicity
                Subject: Medicine and Health Sciences
                Subject: Pathology and Laboratory Medicine
                Subject: Toxicology
                Subject: Cytotoxicity
                Subject: Research and analysis methods
                Subject: Bioassays and physiological analysis
                Subject: Biochemical analysis
                Subject: Colorimetric assays
                Subject: MTT assay
                Subject: Research and analysis methods
                Subject: Bioassays and physiological analysis
                Subject: Biochemical analysis
                Subject: Enzyme assays
                Subject: MTT assay
                Subject: Medicine and Health Sciences
                Subject: Oncology
                Subject: Cancers and Neoplasms
                Subject: Carcinomas
                Subject: Squamous Cell Carcinomas
                Subject: Research and Analysis Methods
                Subject: Research Design
                Subject: Quantitative Analysis
                Custom metadata
                Data Availability All relevant data are within the paper and its Supporting Information files.

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