Experimental Investigations

Folia Medica, XL, 1/2008

Experimental Investigations Effects of propolis and САРЕ on proliferation and apoptosis of McCoy-Plovdiv cell line Milena N. Draganova-Filipova, Milena G. Georgieva1, Ekaterina N. Peycheva1, George A. Miloshev1, Victoria S. Sarafian, Lyudmil P. Peychev2

Department of Biology, Medical University, Plovdiv, 1Laboratory of Molecular Genetics, Institute of Molecular Biology, Bulgarian Academy of Science, Sofia, 2Department of Pharmacology and Drug Toxicology, Medical University, Plovdiv, Bulgaria Abstract The mechanisms of action of propolis can be studied in detail by comparing the effects of propolis and the effects of its constituent components. Aim: To clarify and compare the effects of Bulgarian propolis and caffeic acid phenethyl ester (CAPE, a chemically synthesized component of propolis) - by using a set of cellular, molecular-biological and immunological techniques. Material and methods: The McCoy-Plovdiv cell line was treated with propolis and САРЕ in increasing concentrations (0.01, 0.1, 1.0, 10 mg/L, and 2.5, 4, 8, 16 mg/L, respectively). The expression of the proliferating cell nuclear antigen (PCNA) and the tumour-suppressor protein p53 was studied immunocytochemically. Apoptosis was measured using a highly sensitive microgel electrophoresis technique (comet assay). Results: The results of the study showed corresponding changes in the expression of the examined proliferative antigens. PCNA was detected in all examined concentrations of the tested substances the expression being dose-dependent. Molecule localization changed from the nucleus to the cytoplasm. Treatment with САРЕ brought about gradual attenuation of PCNA expression. High propolis concentrations induced increased synthesis of p53. No p53 expression was found when cells were treated with САРЕ. The studied substances in their highest concentrations (10 mg/L propolis and 16 mg/L САРЕ) had a cytotoxic effect. The comet assay showed DNA degradation kinetics characteristic for apoptosis. Conclusions: The present study demonstrates that high concentrations of propolis and САРЕ cause apoptosis-induced cell death in McCoy-Plovdiv cells. Key words: propolis, САРЕ, apoptosis, McCoy-Plovdiv, comet assay Introduction

The life-activity products of the honey bee Apis melifera have unique nutritional and healing properties. Honey, beeswax, pollen, propolis and bee venom are widely used. Royal jelly or propolis is composed of more than 300 chemical compounds with biological properties of importance for human health.1-3 It has been proved that propolis and several of its components have an inhibiting effect on the reproduction and the realization of genetic information and induce apoptosis in different types of tumour cells.4 Propolis provokes cell death through activating caspases 8

and 9.5,6 It thus participates in the induction stages of apoptosis, triggered by intrinsic and extrinsic signals. Propolis treatment changes the expression of some adhesion molecules (β-catenins), which disrupts normal intercellular contacts and lowers the proliferative potential.7 The mechanisms by which propolis induces programmed cell death in neoplastic cells have not been fully clarified yet. The phenethyl ester of caffeic acid is one of the many flavonoids found in propolis. It is known that it is a carrier of the antiproliferative activity and the antitumour effect of propolis.8,9 In vitro САРЕ induces apoptosis through p53-dependent

Correspondence and reprint request to: M. Draganova-Filipova, Department of Biology, Medical University, Plovdiv, Bulgaria 15A Vassil Aprilov St., 4002 Plovdiv, Bulgaria Received 11 December 2007; Accepted for publication 30 January 2008

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Effects of propolis and САРЕ on proliferation and apoptosis of McCoy-Plovdiv cell line

or independent pathways, which makes it a potent antiproliferative agent.10 It is well known that the content of different chemical components in propolis varies according to the geographical region it is obtained from. Gardjeva P, et al. have provided evidence that the quantity of САРЕ in Bulgarian propolis equals that of Brazilian and Argentinian propolis.11 Information about the mechanisms of the processes of cell proliferation and cell death can be obtained by tracing out the changes in the expression of basic regulatory molecules. PCNA is a key factor, participating in the replication and the reparation of nuclear DNA. It is found in the S and G2 phases of the cell cycle as part of the DNA-synthetase complex.12 The regulation of PCNA expression is accomplished at transcriptional and post-translational level. The basic transcriptional factor of the PCNA-gene is the p53 protein. The latter takes part in arresting the cell cycle in G1 phase and allows the cell to repair the lesions in DNA. If repair is impossible, p53 induces apoptosis.13 Despite the advances in synthetic chemistry which keeps creating new drugs to treat different diseases, the interest in using natural products for treatment has increased in the recent years. The rich chemical composition of propolis is responsible for its application as a therapeutic and prophylactic means in infectious, tumour and immunodeficient conditions. Progressive tumour growth is often the result of suppressed apoptosis. In this respect, it is of great importance to elucidate the molecular and cellular mechanisms via which propolis itself and its components influence cell proliferation and cell death. The lack of comparative studies on Bulgarian propolis and some of its isolated components in vitro makes it necessary to clarify and compare their mechanisms of action. The aim of this study was to investigate the effects of Bulgarian propolis and to compare them with those of САРЕ. Material and methods

Materials We used propolis produced in the Eastern Rhodope mountains, extracted in 96% ethanol in final concentrations of 0.001; 0.01; 0.1; 1.0; 10 mg/L; САРЕ (Sigma, Cat. N:С8221-1G) – in concentrations of 2.5; 4; 8; 16 mg/L. The amount of ethanol in the final working concentrations did not exceed 0.1%.

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Methods Cell cultivation. The McCoy-Plovdiv cell line is a fibroblast synovial cell line, obtained from McCoy cell line and adapted for cultivation in serum-free conditions.14 It was cultivated in Ham’s 12/DMEM culture medium (Sigma-Aldrich Cat. N:4388) in 1:1 ratio. 100 IU/ml penicillin-G and 100 µg/ml streptomycin were added to the medium. The cells were incubated at 37°С, 5% СО2 and high humidity. After formation of a confluent monolayer the culture medium was removed and a fresh stock, containing different concentrations of the examined substances was added. The cells were treated for 24 and 48 hours with propolis and for 24 hours with САРЕ. They were fixed in ice-cold acetone. Cells cultivated in propolis and САРЕ-free medium were used as controls. Immunocytochemistry. The expressions of the proliferative antigen PCNA and of the tumour-suppressor protein p53 was examined using the biotinstreptavidin peroxidase method with the universal DАКО kit (Cat.N:K0672). Monoclonal antibodies against PCNA (Cat. N:1529) and p53 (Cat. N: 1581) were used as primary antibodies. Visualization of the reaction was performed by aminoethylcarbasol, while hematoxylin was used to contrast the nuclei. The expression was assessed by the presence of red-brown granular staining. The reaction intensity was measured according to the following semi-quantitative scale: «+» – slight staining in single cells, «++» – presence of expression in 50% of the 100 counted cells, «+++» – highly intensive homogenous expression in 100% of the cells. Comet assay. The comet assay or gel electrophoresis of single cells is a sensitive method for assessing DNA fragmentation. The method was used according to the protocol of Matassov et al.15 Propolis- and САРЕ-treated cells were included in low melting agarosis and were spread as microgel on coverslips. Electrophoresis was performed after cell lysis and DNA denaturation. The DNA-lesions typical for apoptosis were observed under a fluorescent microscope. The DNA-binding dye SYBR Green 1 was used (Molecular Probes, Cat. N: S-7567). Statistical methods. The analysis of data was performed with Statistica 4.5 (StatSoft, Inc. Microsoft). Differences were considered statistically significant at P < 0.05. Results were presented as mean ± SE. The reliability of the comparison was

Folia Medica, XL, 1/2008

Figure 1. Immunocytochemical detection of PCNA and p53 in McCoy-Plovdiv cells treated with propolis and САРЕ. A. PCNA expression in untreated cells at 48 hours. Homogeneous high intensity cytoplasmic expression - «+++». × 40. B. PCNA expression in cells treated with propolis (0.01 mg/L) at 48 hours. The marker is positive on the membranes of cells with high intensity of expression. - «+++». × 40. C. p53 expression in untreated cells at 48 hours. The marker is not expressed. × 40. D. p53 expression in cells treated with propolis (0.01 mg/L) at 48 hours. The antigen is expressed in the cytoplasm with «++» intensity. × 40. E. PCNA expression in cells treated with CAPE (2.5 mg/L) at 24 hours. Nuclear localization in single cells with «+» intensity. × 40. F. PCNA expression in cells treated with CAPE (4 mg/L) at 24 hours. Homogeneous expression in the nuclei and in the cytoplasm of all cells with «+++» intensity. × 40.

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determined by the Student’s t-test with separate variance estimates and by one-way analysis of variance (ANOVA). Results

Immunocytochemistry PCNA was found in the cytoplasm one hour after the start of cell cultivation. The intensity of the reaction increased thereafter reaching its maximum at 72 hours when the nuclei and the perinuclear zone became positive (Fig. 1A). After that the PCNA expression began to attenuate. The marker was not detected after the formation of a confluent cell monolayer. In the propolis-treated cells the marker expression was found in concentrations from 0.001 to 1.0 mg/L and the localization changed from the nucleus to the cytoplasm. The intensity of the reaction increased with the elevation of propolis concentration. After a 48-hour propolis treatment a clear staining of the plasma membrane was observed. (Fig. 1B). After 48 hours of propolis treatment (1 mg/L) the characteristic red-brown staining was seen in fewer cells most of them being rounded. PCNA outlined the cell membrane and showed lower intensity compared with smaller concentrations. The p53 marker was not detected in the control cells (Fig. 1C). After treatment with 0.01 mg/L of propolis a positive reaction with homogenous cytoplasmic localization was established. The number of positive cells increased in the subsequent concentrations (Fig. 1D). The most intensive reaction was observed 48 hours after treating the cells with 1 mg/L of propolis. The level of expression and the localization of PCNA and p53 markers changed depending on the concentration and treatment duration. In prolonged exposures the intensity of the expression increased. There was a correlation between the two markers. Intense proliferative activity was observed in the presence of PCNA and in the absence of p53. Enhanced expression of p53 was accompanied by a slight expression of PCNA. CAPE treatment upregulated the PCNA expression and the localization changed from the nucleus towards the cytoplasm. At 2.5 mg/L the antigen was detected in the cell nuclei (Fig. 1E). At 4 mg/L the reaction was the most intensive and homogeneously distributed in the cytoplasm (Fig. 1F). In the subsequent concentrations the marker gradually decreased its expression and at the highest concentration (16 mg/L) was not expressed at all. The lowest proliferation level was also observed 56

at this point. There was no expression of p53 in any of the studied concentrations. Comet assay The comet assay showed that DNA was significantly damaged when the McCoy-Plovdiv cells were treated with propolis and САРЕ. The DNA lesions were typical of apoptosis. As the method is highly sensitive, comets were seen even at the lowest concentrations of the examined substances. With the increase of concentration there was a natural rise in the percentage of apoptotic cells. The lowest propolis concentration (0.01 mg/L) induced apoptosis in 22% of the cells, while in the highest concentration (10 mg/L) the apoptotic cells were as high as 46% of all cells (Fig. 2A). In treatment with 2.5 mg/L САРЕ apoptosis was detected in 30% of the cells reaching 72% in the highest concentration – 16 mg/L (Fig. 2B). A lower percentage of comets was observed in propolis-treated cells than in САРЕ treated cells. The chemically pure propolis component exhibits higher cytotoxicity than the product as a whole. The data were obtained by calculating the percentage of apoptotic cells in counting 1500 cells (nuclei and comets) for each concentration of the examined substances. Discussion

In a number of studies on tumour and normal cells propolis and some of its components have been demonstrated to affect proliferation and lower the number of surviving cells by inducing apoptosis.16 The natural honey-bee product affects cell homeostasis because it participates in the apoptosis-inducing process via two mechanisms – by external and internal signals. The extrinsic apoptotic pathway is initiated by specific propolis compounds which bind with cell membrane receptors and activate the apoptotic process.4,5 The intrinsic pathway is activated at mitochondrial level.5,9 In case of non-repairable DNA damage the p53 protein changes the expression of genes from the Bcl-2 family. The disrupted balance between the pro- and antiapoptotic members of this family induces a change in mitochondrial membrane permeability, release of cytochrome C from the mitochondria into the cytosol and cascade activation of caspases. Propolis from different geographic regions has different components depending on the flora, the bee species, the season of pollen collection and the method of extraction. Because of insufficient data

Folia Medica, XL, 1/2008 60 *

comets, %

50 40

*

30

*

20 10

A

0 control

0.01

1,0

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comets, %

concentration of propolis, mg/L

80 70 60 50 40 30 20 10 0

**

**

** *

B

control

2,5

4

8

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concentration of CAPE, mg/L Figure 2. Results from the comet assay. A. Comet assay in propolis treatment; B. Comet assay in CAPE treatment. The data are significantly different from the controls at *p < 0.05, n = 9; **p < 0.01, n = 9

from in vitro studies on cell cultures of Bulgarian propolis, the choice of treatment time and the applied concentrations were based on other authors’ research. The latter vary according to the type of the cell lines used and the research design.16,17 McCoy-Plovdiv is a highly sensitive test-system for the detection of biologically active substances.14,18 In our study the highest concentrations (10 mg/L propolis and 16 mg/L САРЕ) had a pronounced cytotoxic effect. When compared with the controls there were significant differences (р < 0.05, р < 0.01 when n = 9) (Fig. 2). The comet assay showed the DNA degradation kinetics characteristic of apoptosis. Apoptosis in McCoy-Plovdiv cells is induced by both the whole compound and САРЕ, but the pathways of activating cell death differ. Most probably this effect is due to the presence of specific components in propolis whose individual actions either complement or neutralize each other. The changes that occur in the expressions of PCNA and p53 are an indication of subtle alterations

in the complex mechanisms of cell proliferation. Propolis upregulates the p53 expression which is evidence of initial DNA damage, but the simultaneous detection of PCNA suggests that the mechanisms controlling the DNA repair are also activated. This is supported by the comet assay which showed fewer apoptotic cells. The fact that p53 is expressed in propolis-treated cells implicates this protein in the induction of cell death. Propolis is composed of many compounds which synergetically use different mechanisms of action from those of its ingredient compounds acting independently. Propolis induces apoptosis in the McCoy-Plovdiv cells activating a p53-dependent pathway, while САРЕ’s action is most probably due to activation of external apoptosis triggering mechanisms. There is no correlation between the two markers when cells are treated with САРЕ. Only PCNA is positively expressed and the reaction intensity changes in a proportional dose-effect dependent manner. The fact that there was no p53 expression

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in any of the studied concentrations suggests that САРЕ induces apoptosis in McCoy-Plovdiv cells via p53-independent pathways. Nomura M, et al. use murine epidermal JB6 Cl 41 cells and prove that in vitro САРЕ triggers apoptosis through p53-dependent and independent pathways. This fact determines this protein as a potent antitumour agent.10 Our results suggest that САРЕ interacts with surface death receptors and activates cell death through an extrinsic pathway. ConclusionS

The present study reports the first ever data about the proliferative action of Bulgarian propolis and its compounds in vitro. On this basis the effects of propolis and CAPE on normal and tumour cell lines could be further compared showing the potential of Bulgarian propolis to be used independently or in combination with chemotherapeutic drugs to manage carcinogenic conditions. Acknowledgments

The authors wish to extend their gratitude to Assoc. Professor M. Draganov for placing the McCoy-Plovdiv cell line at their disposal and for his invaluable methodological help. The study was funded under project no. MU-L-1408 from the Ministry of Education and Science and project no. 01/2005; 06/2003 of the Medical University, Plovdiv. References

1. Almeida EC de, Menezes H. Anti-inflammatory activity of propolis extracts: a review. J Venom Anim Toxins 2002; 8:191-212. 2. Peychev L, Sarafian V, Murdjeva M. Therapeutic use of Bulgarian propolis: a review. Union of Scientists research, Plovdiv, series General Medicine, Pharmacy and Dentistry, Medicine and Stomatology 2006; vol.VІІ:52-8. 3. Borrelli F, Izzo AA, Di Carlo G, et al. Effect of propolis extract and caffeic acid phenethyl ester on formation of aberrant crypt foci and tumor in the rat colon. Fitoterapia 2002; 73:38-43. 4. Kimoto T, Arai S, Kohguchi M, et al. Apoptosis and suppression of tumor growth by Artepillin C extracted from Brazilian propolis. Cancer Detect Prev 1998;22:506-15. 5. Chen CN, Wu CL, Lin JK. Propolin C from propolis induces apoptosis through activating caspases, Bid

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and cytochrome C release in human melanoma cells. Biochem Pharmacol 2004;67:53-66. 6. Lee YJ, Kuo HC, Chu CY, et al. Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells. Biochem Pharmacol 2003;66:2281-9. 7. Wang D, Xiang DB, He YJ, et al. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro. World J Gastroenterol 2005;11:4008-12. 8. Orsolic N, Terzic S, Mihaljevic Z, et al. Effects of local administration of propolis and its polyphenolic compounds on tumor formation and growth. Basic Biol Pharm Bull 2005;28:1928-33. 9. Woo KJ, Jeong YJ, Park JW, et al. Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells. Biochem Biophys Res Commun 2004;325:1215-22. 10. Nomura M, Kaji A, Ma W, et al. Suppression of cell transformation and induction of apoptosis by caffeic acid phenethyl ester. Mol Carcinogen 2001;31:83‑9. 11. Gardjeva P, Alexandrov A, Dimitrova S, et al. A study on chemical composition and antifungal activity of Bulgarian propolis. Union of Scientist research, Plovdiv, series General Medicine, Pharmacy and Dentistry, Medicine and Stomatology 2006;7:201‑5. 12. Ivanova V, Karaivanov M, Marinov E. Assessment of cell proliferation in clinical pathology. Clin Аpplic Immunol 2002;1:28-33. 13. Macdonald F, Ford CHJ. Molecular biology of cancer. BIOS Scientific Publishers;1997:53. 14. Kamberov E, Draganov M, Murdjeva M. Development of a new serum-free cell culture system, McCoy-Plovdiv. In Vitro Cell&Dev Biol - Animal 2000;36:284-6. 15. Matassov D, Kagan T, Leblanc J, et al. Measurement of apoptosis by DNA fragmentation. Methods Mol Biol 2004;282:1-17. 16. C hen MF, Wu CT, Chen YJ, et al. Cell killing and radiosensitization by caffeic acid phenethyl ester (CAPE) in lung cancer cells. J Radiat Res 2004;45:253-60. 17. S cifo C, Cardile V, Russo A, et al. Resveratrol and propolis as necrosis or apoptosis inducers in human prostate carcinoma cells. Oncol Res 2004;14:415‑26. 18. Ganchevska P, Draganov M, Sarafian V. Characterization of the proliferative activity of the serumfree cell line McCoy-Plovdiv. Trakia J Sci 2004; 2:12‑5.

Folia Medica, XL, 1/2008 Эффекты прополиса и САРЕ на пролиферацию и апоптоз клеточной линии МсСоу-Plovdiv М. Драганова-Филипова, М. Георгиева, Е. Пейчева, Г. Милошев, В. Сарафян, Л. Пейчев

Резюме Сравнительное рассматривание эффектов прополиса и его отдельных составных частей позволяет детально изучить механизмы его действия. Цель: Применить набор клеточных, молекулярнобиологических и иммунологических техник в целях выяснения и сравнения эффектов болгарского прополиса и химически синтезированного прополисного компонента САРЕ. Материалы и mетоды: Клеточная линия МсСоуPlovdiv третирована нарастающими концентрациями прополиса (0.01; 0.1; 1.0; 10 mg/L) и САРЕ (2.5; 4; 8; 16 mg/L). Экспрессия пролиферативного антигена PCNA и опухолево-супрессорного белка р53 исследована иммуноцитохимично. Для детекции

апоптоза использован высокочувствительный метод – микрогель электрофорез (Кометный тест). Результаты: Результаты исследования показывают закономерное изменение в экспрессии исследуемых пролиферативных антигенов. PCNA устанавливается при всех исследованных концентрациях тестированных веществ, при чем экспрессия является доза-зависимой. Локализация молекулы изменяется от ядра к цитоплазме. При третировании САРЕ проявляется тенденция к постепенному затиханию экспрессии PCNA. Высокие концентрации прополиса индуцируют повышенный синтез р53. При третировании САРЕ отсутствует экспрессия р53. Самые высокие применяемые концентрации (10 mg/L прополиса и 16 mg/L САРЕ) имеют цитотоксический эффект. При Кометном тесте наблюдается характерная для апоптоза кинетика расщепления ДНК. Выводы: Исследование доказывает, что высокие концентрации прополиса и САРЕ приводят к клеточной смерти вследствие апоптоза в клетках МсСоуPlovdiv.

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