a new approach for purification of recombinant human ...

PROPOLIS INDUCED APOPTOSIS IN MC COY-PLOVDIV CELLS M. Draganova-Filipova1, M. Mourdjeva2, Z. Popova2, E. Peycheva3, G. Miloshev3, V. Sarafian1 Department of Biology, Medical Universyti-Plovdiv 1 IBIR – Bulgarian Academy of Sciences, Sofia, Bulgaria 2 IMB – Bulgarian Academy of Sciences, Sofia, Bulgaria 3 Correspondence to: M. Draganova-Filipova E-mail: [email protected]

ABSTRACT Propolis is a natural product with different biological activities. Current research is focused on its ability to suppress tumor growth by induction of apoptosis. However, its selective influence on normal and transformed cells is not fully studied yet. Our previous investigations determined the McCoy-Plovdiv cell line as a model system for in vitro examination of propolis. In the present study we analysed ethanol extracts of propolis from the region of East Rodopi mountains in final concentrations 0,001; 0,1; 1,0; 10 µg/ml. The proliferative activity was examined with MTT and NR cell vitality tests. The type of cell dead was proven via TUNEL test and by fluorescent detection of phosphatidylserine (PS) externalization. Cytotoxicity tests showed decreased cell vitality from 100 to 0% increasing with propolis concentration. The TUNEL test proved the presence of apoptotic cells. Propolis in concentrations 0,001-1,0 µg/ml induced apoptosis in a large number of cells. The results from PS externalization confirmed the same observation. Increasing propolis concentrations augment the intensity of the fluorescence signal and the number of apoptotic cells. Our results evidence that Bulgarian propolis induces apoptosis in McCoy-Plovdiv cells. Keywords: apoptosis, cell line McCoy-Plovdiv, propolis,

Introduction The application of natural products and synthetic preparations in medicine and pharmacy obligatory requires an evaluation of their influence on different cell parameters. The changes on the cell level are physiological and/or morphological. The analysis of different xenobiotics is performed in order to specify their influence on the cell and their possible toxic effect. Cytotoxicity tests are used for a quantitative evaluation. Different vital dyes reveal a specific affinity to definite cell structures. The combination of cytotoxicity tests enables us to clear the levels of influence of the tested substances and to analyze the mechanisms of the induced effects. In recent years a scientific explanation is required to determine the empirically derived various properties of propolis - antiinflammatory, antimicrobial, regenerative, antiproliferative etc. (3, 6, 9, 12). The focus of present scientific research is on its ability to inhibit the development

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of tumor formations by induction of apoptosis. A number of authors determine that propolis and some of its components (phenethyl ester of caffeic acid (CAPE), artepilin C, etc.) participate in the regulatory mechanisms of programmed cell death (11, 16). The programmed cell death is a fundamental process regulating homeostasis both in embryonic and postembryonic period. During the last decade the possibilities of apoptosis modulation find an application in the control of different diseases. One of the earliest signs for cells in an initial stage of cell death is the disturbance of the phospholipid asymmetry of the plasma membrane. During apoptosis translocation of PS occurs from the internal to the external monolayer and serves as a signal for detection of phagocytes. Annexin V has a high affinity to phosphatidylserine. The differentiation of apoptotic from necrotic and living cells is performed by combination tests of Annexin V with propidium iodide. It allows detecting the early stages of apoptosis before occurring visual morphological changes (14). Annexin V binds not only PS, but all the negatively charged

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XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY

phospholipids thus requiring a careful interpretation of the results. The application of monoclonal antibodies, directed towards PS, allows us to avoid possible nonspecific interactions. The monoclonal antibody Mab 1H6 interacts with PS, without reacting with phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and cardiolipin. (10).

Materials and methods Propolis from the East Rodopes, extracted in 96% ethanol with concentration of 0,001; 0, 01; 0, 1; 1,0; 10,0 μg/ml is examined. The final concentration of ethanol in culture medium does not exceed 0,01%. Cell line and cell culturing McCoy-Plovdiv is a serum-free cell line based on the standard fibroblast cell line McCoy. It is stored and cultured in an entirely defined culture medium without serum (7). Ham’s F-12 (Sigma-Aldrich) and DMEM (Sigma-Aldrich) at ratio 1:1 are used. Penicillin of 100 U/ml and 100 mg/ml streptomycin are added to the medium. The cells are successfully cultured in 96 well-plates and on coated slides. They are grown in an incubator Heraeus (Germany) at 37 °C, at 5 % CO2 and high humidity. NR and MTT tests Following the NR and MTT tests, the McCoy Plovdiv cells are cultured in flat 96 well-plates (Greiner) with density 2x105/ml and cultured for 24 hours, there after the medium is replaced with a fresh one containing propolis in the tested concentrations. After 24, 48, 72, 96 hours of incubation NRand MTT tests are performed. In each plate it is added 100 ml of the vital dye in final concentrations -50μg/ml NR and 500 μg/ml MTT. After two hours incubation in a thermostat at 37°C the dyes are removed and the cells are subjected to PBS wash. The extracting agents for NR is 1 % acetic acid ethanol solution and DMSO for MTT. The results are spectrophotometrically reported in wave length 405 and 540nm (NR) and 540 and 690 (MTT) (17). Vitality of cells is measured in % by detecting the absorption of treated cells / control cells x 100. Each experiment is repeated at least 6 times and the results are calculated as a mean value. Immunofluorescence (Externalization of PS) McCoy-Plovdiv cells are cultivated in DMEM/Ham`s in concentration 2x105/cm2. The cells are treated with propolis in concentration 0,001-10 μg/ml. After 24 h cells are fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, and incubated with Mab 1H6 (hybridoma supernatant) overnight at RT. The cells are washed with PBS XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY

and then incubated with anti-mouse IgG serum labeled with FITC (SAPU, Lanarkshire, Scotland) diluted 1:50 in PBS for 1 h at room temperature. Monoclonal antibodies of irrelevant specificity are used as negative controls. After the incubation periods the cells are washed extensively in PBS mounted with Mowiol (Aldrich, Germany) and observed under an epifluorescent microscope Leitz (Germany). TUNEL – test The cells are cultured on adhesive treated slides with density 2x105/cm2. There is 24 hours cultivation, and then the medium is changed by a new one containing propolis alcohol extract in the tested concentrations. The cells are treated for 24 hours. They are fixed at room temperature with 4% formaldehyde/PBS for 25 minutes. Permeabilization with 0, 2 % Triton X-100/PBS occurs, thereupon the TUNEL test is performed according to the protocols recommended by the manufacturing company (DeadEndTM ColorimetricTUNEL System; Promega Cat. N: G7130). The apoptotic cells are brown colored by diaminobenzidine (DAB). Cells treated with 0,01% of ethanol are used as a negative control. The cells of the positive control are treated with 20 mM H2O2 for 3 hours, while cells treated with DNA-ase are used as an internal control. The number of apoptotic cells is detected under a light microscope in 500 counted cells for each concentration. The percentage ratio of the normal to the apoptotic cells is calculated.

Results and Discussion The cytotoxic tests reveal the negative effect of the increased concentrations of propolis on McCoy –Plovdiv cells survival. The percentage of survived cells decreases from 100 to 0% during 72 hours treatment. The observed effect depends on time and concentration. Both test results reveal a concentration dependent percentage decrease of cell vitality. The data from the two tests are statistically comparable. TUNEL-test The TUNEL- test shows a trend toward a percentage increase of cell death after propolis treatment. The number of cells in apoptosis increases at concentration of 001-1,0 μg/ml propolis. At the lowest concentration the percentage of apoptotic cells is 3,09%, at 0,01μg/ml - 5,2%; 0,1 μg/ml of propolis leads to 6,59% apoptotic cells, at 1μg/ml - 7,72%. The cells shrink, their size decreases, the chromatin condenses, fragmentation of the nucleus occurs. Apoptotic bodies are formed. The values are average from the number of apoptotic cells

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detected in totally 500 counted cells for each concentration.

apoptotic cells,%

10 8 6 4 2 0 0,001

0,01

0,1

1

concentration of propolis,μg/ml Fig. 1. TUNEL-test on McCoy-Plovdiv cell line, treated with propolis. The statistically reliable differences are evaluated at p< 0,05 for n=500.

Externalization of phosphatidylserine The analysis of the externalization results of PS reveals a dependence on propolis concentration. The higher the concentration, the bigger the percentage of fluorescent cells positive for PS is observed. The intensity of fluorescence is also augmented in a concentration dependent manner.

A.

B.

C.

Fig. 2. Immunofluorescence of phosphatidylserine in McCoy –Plovdiv cells treated with propolis: A) 0, 01μg/ml; B) 1, 0 μg/ml; C) 10μg/ml; magnification х 400

It is proved that propolis has a cytotoxic influence in vitro on tumor cells (5, 8, 13). Morphological changes, impairment in the genetic apparatus and in protein synthesis occur. Changes in the regulation of the cell cycle and damaged cell interactions are observed. Apoptosis occurs as a result of different mechanisms due to the altered expression of specific BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE

receptors and adhesion molecules. The antiproliferative activity is detected in different tumor cells and defines its possible role as an antitumor agent (1, 2, 4, 15). The antiproliferative effect of propolis is detected by the changes in McCoy-Plovdiv cells after treatment with increasing propolis concentrations. The decrease of proliferative capacity is proved by vitality tests, as well as by the morphological changes in the cell. NR and MTT tests show the decrease of percentage living cells with the increase of propolis concentration and presume the induction of cell death. The specificity of the used vital dyes towards definite cell components proposes that disturbances affect both lysosomes and mitochondria. There are a number of confirmations of the fact that cell death caused by propolis is as a result of induced apoptosis. Propolis reduces cancerogenesis in vitro by inhibition of DNA-synthesis, transcription of RNA and protein synthesis in lymphoma cells (2, 4). The whole compound, CAPE and artepillin C induce apoptosis in different tumor cell lines (HL-60, Colon cancer cell line, SW480, U937), by setting external and internal apoptotic ways (1, 15, 16). Our research proposes detection of the induced type of cell death by two different methods - PS and TUNEL-tests. Both the exposition of PS and the intranucleosomic fragmentation of DNA are indicators of apoptosis. PS is a membrane component, functioning as a “eat-me” signal especially for receptors on the surface of macrophages, which eliminate apoptotic cells without causing inflammation. The externalization of PS precedes the fragmentation of DNA and the loss of membrane integrity. Our results record the increase of percentage apoptotic cells with the elevation of propolis concentration. The treated with propolis McCoyPlovdiv cells show high levels of DNA damage typical for apoptosis. Nevertheless, at low concentrations (0,001-0,01μg/ml) the cells preserve their morphology and physiological activity and the percentage of living cells is commensurable with the controls (NR- and MTT- tests). PS externalization is a sign of induced apoptosis. Presumably still the low concentrations induce changes which the cells cannot repair and thus activate mechanisms for a cell suicide. The high concentrations of propolis are toxic for them. DNA fragmentations proved with the TUNEL–technique confirm that the cell death induced by propolis in the McCoy-Plovdiv cells is as a result of apoptosis. The lack of detailed research on the biological properties

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of Bulgarian propolis requires a study of its effects and participation in the regulation of cell division and death. The analysis of its mechanisms of action reveals new approaches for the application of Bulgarian propolis as a preventive and therapeutic agent.

Acknowledgments The authors thank Assoc. Profs. M. Draganov and L. Peychev for providing the McCoy-Plovdiv cell line and propolis extracts. The research is partly supported by grant NO-5/1999 from MU-Plovdiv.

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