Bax regulation

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Environmental Toxicology and Pharmacology 26 (2008) 61–67

Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation Muneo Motomura a,1 , Kyung Min Kwon b,1 , Seok-Jong Suh b , Young-Choon Lee c , Yeon-Kye Kim d , In-Seon Lee e , Myung-Sunny Kim f , Dae Young Kwon f , Ikukatsu Suzuki a,∗,2 , Cheorl-Ho Kim b,∗ b

a Department of Clinical Nutrition, Suzuka University of Medical Science, 1001-1 Kishioka, Suzuka City, Mie 510-0293, Japan Department of Biological Sciences, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Kyunggi-Do 440-746, Republic of Korea c Department of Biotechnology, Dong-A University, Hadan-dong, Saha-Gu, Busan 608-714, Republic of Korea d Laboratory of Bioactive Resources, Biotechnology Research Institute, National Fisheries Research and Development Institute, 408 Sirangri, Gijang, Haeundae, Busan 619, Republic of Korea e The Center for Traditional Microorganism Resources, Keimyung University, Daegu 704-701, Republic of Korea f Korea Food Research Institute, Songnam, Kyongki-Do 463-420, Republic of Korea

Received 18 November 2007; received in revised form 25 January 2008; accepted 28 January 2008 Available online 15 February 2008

Abstract We investigated mechanism(s) where propolis induces apoptosis in human leukemic U937 cells. Propolis inhibited the proliferation of U937 cells in a dose-dependent manner by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot analysis showed that propolis increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A, Cdk2 and Cdc2, thereby contributing to cell cycle arrest. DAPI staining assay revealed typical morphology features of apoptotic cells. Propolis-induced apoptosis was also confirmed by assays with annexin V-FITC, PI-labeling and DNA fragmentation assay. The increase in apoptosis level induced by propolis was associated with down-regulation of Bcl-2 and activation of caspase-3, but not with Bax. These results suggests that propolis-induced apoptosis is related to the selective activation of caspase-3 and induction of Bcl-2/Bax regulation. © 2008 Elsevier B.V. All rights reserved. Keywords: Propolis; Bcl-2/Bax; Caspase-3; Human leukemic U937; Apoptosis

1. Introduction A natural product, propolis, is resinous substance collected by honey bees from the buds and plant sources, and it is used in the construction and maintenance of their hives (Burdock, 1998). Use of propolis by human has a long history predated only by the discovery of honey (Burdock, 1998). Propolis has been

Abbreviations: DAPI, 4,6-diamidino-2-phenylindol; FACS, fluorescence activated cell sorter; Bcl-2, B-cell lymphoma 2; GAPDH, glyceraldehydes3-phosphate dehydrogenase; FITC, annexinV-fluorescein isothiocyanate; Bax, Bcl-2 antagonist X; PBS, phosphate buffered saline. ∗ Corresponding authors. Tel.: +82 31 290 7002; fax: +82 31 290 7015. E-mail addresses: [email protected] (I. Suzuki), [email protected] (C.-H. Kim). 1 These authors contributed equally to this work. 2 Tel.: +81 59 383 8991; fax: +81 59 383 9666. 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.01.008

reported to exert a broad spectrum of biological functions. It has long been used as a source of natural antioxidant, antibacterial, antifungal, immunomodulatory and anti-inflammatory activities (Amoros et al., 1992; Bankova et al., 1995; Banskota et al., 2001; Dobrowolski et al., 1991; Gribel and Pashinskii, 1986; Krol et al., 1990; Sforcin, 2007). It is also known for apoptosis of human carcinoma cells (Choi et al., 1999) and human leukemic HL-60 cells (Mishima et al., 2005). Biological activities of propolis mainly depend upon the presence of more than 300 compounds including flavonoides, phenolics and their esters in particular (Banskota et al., 2001; Simoes et al., 2004). Among the constituents of propolis, several compounds such as CAPE, galangin, xanthomicrol, artepillin C and chrysin are reported to have antiproliferative activity on most of the murine and human cancer cell lines (Ahn et al., 2007; Hernandez et al., 2007). Apoptosis, the genetically programmed cell death is related to the regulation of development and homeostasis. It is morpho-

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logically characterized cell shrinkage, depolarization of the mitochondria, chromosomal DNA fragmentation (Wyllie, 1997). In general, it may occur through specific apoptosis signaling pathways such as death receptors and mitochondria. However, some cells proceeding to apoptosis are caused via mitochondrial pathway. Mitochondrial pathway plays a crucial role in apoptosis of mammalian cells by releasing pro-apoptotic protein including cytochrome c into the cytoplasm (Bhimani et al., 1993). Its regulation and execution of apoptosis require two major protein families, namely, Bcl-2-related proteins and caspases (Adams and Cory, 1998; Cohen, 1997). The Bcl-2 protein family contains several homologous proteins including antiapoptotic proteins (Bcl-2, Bcl-xL, Bcl-w, etc.) and pro-apoptotic proteins (Bax, Bad, Bak, Bik, Bcl-xS, etc.) (Adams and Cory, 1998). In addition, many reports have suggested that the caspase family plays an important role in apoptosis. The caspases are unique in their requirement for an Asp residue at the cleavage site in their substrates. Recently, among 14 known members of the caspase family of proteases, caspase-3 have been suggested to be a key role of the apoptotic machinery (Chen et al., 2001). Many anticancer agents induce apoptosis in human leukemia cells (Huang et al., 2005; Jin et al., 2007; Pezzuto, 1997). Among the various leukemia cells, especially human leukemia U937 cells are very sensitive to apoptosis upon anticancer agents treatment (Todisco, 2007). Previously, it was reported that propolis effectively inhibits the proliferation of human leukemic U937 cells. However, the action mechanism(s) for the propolismediated apoptosis has not been studied in human leukemic U937 cells. In this study, we investigated whether propolis induces apoptosis in human leukemia U937 cells. To determine the antiproliferative activity of propolis, we examined its effect on cell cycle arrest and apoptotic signaling. By apoptosis-related assays such as DAPI staining assay, annexin V-FITC and PI labeling assays, and DNA fragmentation assay, the apoptosis was clearly confirmed. In cellular level, the apoptosis was directly linked to down-regulation of Bcl-2 and activation of caspase-3 without any change in the Bax expression. From the present results, it was suggested that propolis induces apoptosis in U937 cells by the activation of caspase-3, and the induction of Bax and Bcl-2 regulation.

2.2. Cell viability assay Cells (1 × 104 per well) were seeded in 96-well plastic plates and incubated at 37 ◦ C in full medium containing the propolis. After 24 h, cell viability was assessed by the ability of metabolically active cells to reduce tetrazolium salt (XTT) to colored formazan compounds. The absorbance of the samples was measured with an ELISA reader (wavelength 490 nm). Each measurement was performed in triplicate.

2.3. Cell cycle analysis Cells (104 cells/well) were cultured in triplicate in 6-well plates. After 24 h of incubation, cells were treated with vehicle (0.1% DMSO), 300 and 500 ␮g/ml propolis for 24 h. Cells were harvested, fixed with 70% ethanol and stained with 50 ␮g/ml propidium iodide. Data acquisition and analysis were performed on an EPICS flow cytometer (Coulter Electronics), and data from 104 cells were collected for each data file analysis. Cell cycle analysis was performed with Multicycle software (Phoenix Flow Systems, San Diego, CA).

2.4. Nuclear staining with 4,6-diamidino-2-phenylindole (DAPI) Harvested cells were washed once with PBS, and then resuspended in phosphate buffered saline (PBS) containing 0.1% Triton X (to induce holes in the cells’ membrane increase permeability) and incubated for 10 min on ice. Spin cells down and resuspend them in 4% PBS buffered paraformaldehyde solution containing 4,6-diamidino-2-phenylindole (DAPI, Sigma). 10 ␮l of this suspension are placed on a slide glass and covered with a cover slip. The morphology of the cells’ nuclei is observed using a fluorescence microscope (Nikon Co., Japan) at excitation wavelength 350 nm.

2.5. Annexin V binding assay to detect apoptosis cells Cells (1 × 106 ) were incubated with propolis for 24 h and then harvested. Specific binding of annexin V-FITC was carried out by incubating the cells for 15 min at room temperature in a binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4) containing a saturating concentration of annexin V-FITC and PI. After incubation, the cells were pelleted and analyzed in a fluorescence activated cell sorter (FACS) analyzer (Becton–Dickinson, U.S.A.).

2.6. Agarose gel electrophoresis for DNA fragmentation

2. Materials and methods

The cells were lysed in a DNA fragmentation buffer containing 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 for 10 min on ice. The lysates were shaked and cleared by centrifugation at 13,000 × g for 10 min. The DNA in the supernatant was extracted using 25:24:1 (v/v/v) equal volume of neutral phenol:chloroform:isoamyl alcohol (Sigma) and analyzed electrophoretically on 2% agarose gels containing 0.1 mg/ml ethidium bromide (EtBr, Sigma).

2.1. Materials and cells

2.7. Gel electrophoresis and Western blot analysis

Raw propolis was a gift of Dr. I Suzuki, Suzuka University of Health Science, Suzuka, Mie, Japan. The sample and voucher specimens are kept in the herbarium of the Department of Biological Science, Sungkyunkwan University. Propolis which has been cleaned was preferably crushed. It was then placed in an opaque and closed container. The sample (200 g) were extracted 3 times with 500 ml of methanol at room temperature for several days (usually 8–10 days), with regular shaking the mixture at 2–3 times a day. The extracts were filtered through a 0.45 ␮m filter and lyophilized with great care. The w/w yield of the methanol extracts was about 8.5%. Human myeloid leukemia U937 cell line obtained from the American Type Culture Collection (ATCC) was grown in RPMI 1640 medium containing 100 units of penicillin–streptomycin per ml and 10% fetal bovine serum (FBS) (Welgene Inc., Korea) at 37 ◦ C in 5% CO2 incubator/humidified chamber.

The cells were harvested, lysed, and the protein concentrations were quantified using a Bio Rad protein assay (Bio-Rad Lab., Hercules, CA, U.S.A.) according to manufacturer specifications. For Western blot analysis, an equal amount of protein was subjected to electrophoresis on SDSpolyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, U.S.A.) by electroblotting. After electroblotting, the membrane was soaked in TBS–T buffer (50 mM Tris–HCl, pH 7.4, 2 M NaCl, Tween-20) containing 2% Skim milk at room temperature. The immunoblotting were probed with the desired antibodies for 6 h, incubated with the diluted enzyme-linked secondary antibody and visualized by enhanced chemiluminescence (ECL) according to the recommended procedure. The primary antibodies such as ␤-actin, p21, p27, cyclin B1, cyclin A, Cdk2 and Cdc2 antibodies were purchased from Santa Cruz

M. Motomura et al. / Environmental Toxicology and Pharmacology 26 (2008) 61–67 Biotechnology Inc. (Santa Cruz, CA, U.S.A.) and BD (Becton–Dickinson, U.S.A.). The peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled Goat anti-mouse immunoglobulin were purchased from Santa Cruz.

2.8. Statistics Results were expressed as the means ± standard of data obtained from triplicate experiment. A statistical analysis was performed with one-way analysis of variance (ANOVA) using STATISTIX analytical software. Differences at p < 0.01 are considered statistically significant.

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Table 1 Effect of propolis on cell cycle phases in U937 cells Samples of cells were stained with propidium iodide and the cellular DNA content determined by flow cytometry Cell cycle phases

G0/G1

S

G2 + M

Control (%) Propolis (300 ␮g/ml) Propolis (500 ␮g/ml)

64.2 ± 3.5 45.7 ± 2.9* 39.7 ± 2.8*

20.8 ± 2.7 21.5 ± 1.9 22.6 ± 1.8

15.0 ± 2.5 32.8 ± 2.8* 37.7 ± 2.6*

Values represent the proportions of cells in specified phases of the cell cycle. Values are the means + S.E. of 10 replicate samples. *p < 0.05 compared with control.

3. Results 3.1. Propolis inhibits the growth of U937 cells

3.3. Effect of propolis on the expression of cell cycle-related proteins in U937 cells

We investigated the effect of cell growth inhibition in the human leukemic U937 cells. Propolis treatment decreased cell viability in a concentration-dependent manner (Fig. 1). It was measured by quantitative colorimetric XTT assay after 24 h of incubation. In the range 1–1000 ␮g/ml of concentration, propolis resulted in a concentration-dependent inhibition of the growth of U937 cells. Cell viability of U937 cells was not changed at doses ranged between 1 and 100 ␮g /ml of propolis, while cell shrinkages and morphological changes of U937 cells were observed at higher doses than 100 ␮g/ml with the marked inhibition of the cell growth. These results suggest that propolis inhibits the growth of U937 cells.

Using immunoblot analysis, we examined the effect of propolis treatment on the protein expression of p21, p27, cyclin A, cyclin B1, Cdk2 and Cdc2, which are known as regulators of cell cycles. The results indicated that 300 and 500 ␮g/ml propolis increased the expression level of p21 and p27 at 24 h in U937 cells (Fig. 2). Treatment of U937 cells with 300 or 500 ␮g/ml propolis resulted in decreases in the protein expression of cyclin A and cyclin B1 in a dose-dependent manner (Fig. 2). Inhibition of propolis was also observed in the protein levels of Cdc2 and Cdk2 in the examined cell line (Fig. 2).

3.2. Effects of propolis on cell cycle distribution and apoptosis induction in U937 cells

Morphological changes of apoptotic cells such as nuclear apoptotic bodies were analyzed by fluorescence microscopy with DAPI. Human leukemic U937 cells were treated with 100, 300 and 500 ␮g/ml of propolis for 24 h. A few apoptotic cells were observed at 100 ␮g/ml of propolis. The percentage of apoptotic cells was increased in a concentration-dependent manner. However, 300 and 500 ␮g/ml of propolis-induced nuclei damage of human leukemic U937 cells (Fig. 3A).

To examine the mechanism responsible for propolismediated cell proliferation inhibition, cell cycle distribution was evaluated using flow cytometric analysis. The results showed that the addition of propolis caused an accumulation of cells in the G2/M phase in U937 cells at 24 h. Compared with the control, 300 and 500 ␮g/ml propolis increased the population of the G2/M phase from 15% to 32.8% and 37.7% in U937 cells, respectively (Table 1).

Fig. 1. Propolis inhibits the growth of U937 cells. Cell were treated with propolis 1–1000 ␮g/ml of propolis for 24 h. Data is expressed as the means ± standard of data obtained from triplicate experiment and significantly different by oneway ANOVA followed least significant difference (LSD) at the p < 0.01 level of alpha.

3.4. Nuclear staining with DAPI

Fig. 2. The effect of propolis on cell cycle-related factors. The cells were treated with 300 and 500 ␮g/ml of propolis, respectively, for 24 h. Total cell lysates were prepared, and proteins were subjected to Western blotting analysis with monoclonal antibodies. ␤-Actin was used as a loading control.

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Fig. 3. Induction of apoptosis by propolis in human leukemic U937 cells. (A) Fluorescent microscopic analysis of cells stained with DAPI. 24 h after propolis treatment the cells were fixed, stained with DAPI and analyzed for morphological characteristics associated with apoptosis. Apoptotic bodies are indicated by white arrows. (B) 1 × 106 cells were treated with propolis (0, 100, 300 or 500) for 24 h. The cells were then permeablized, stained for Annexin V, and stored on ice until analyzed by FACS. Increased Annexin V staining was seen in U937 cells in the presence of propolis. In all panels, cells in the lower left quadrant are alive, cells in the lower right quadrant are in early apoptosis, in the upper right are in late apoptosis, and cells in the upper left quadrant are dead. Percentage of total signal within the quadrant is indicated. (C) After 24 h, the genomic DNA was extracted, and analyzed on a 2.0% agarose gel.

3.5. Assessment of apoptotic cells Apoptosis was determined by staining cells with annexin V-FITC and PI labeling, because annexin V can identify the externalization of phosphatidylserine during the progression of apoptosis. A dose-dependent increase in apoptosis rate of U937 cells were demonstrated by annexin V and PI staining (Fig. 3B). After 24 h incubation, a positive staining with annexin V was observed in 4% of U937 cells treated with 100 ␮g/ml of propolis. It seems to be slightly different with the control. Annexin V was bound in 9.85% or 14.44% of U937 cells incubated for 24 h with 300 and 500 ␮g/ml of propolis, respectively. These results demonstrated the involvement of an apoptotic process induced by propolis. 3.6. Propolis induces DNA fragmentation Apoptosis is morphological change of cells, such as chromatin condensation and fragmentation of nuclei, and formation of apoptotic bodies were observed in U937 cells. We investigated

whether propolis induces DNA fragmentation in U937 cells. As shown in Fig. 3C, increasing concentrations of propolis induced the progressive accumulation of fragmented DNA. 100 ␮g/ml of propolis did not cause significant DNA fragmentation. However, 300 and 500 ␮g/ml of propolis allowed appearance as atypical ladder pattern of DNA fragmentation due to internucleosomal cleavage associated with apoptosis. These results also indicate that propolis induces the apoptosis in U937 cells. 3.7. Propolis induces apoptosis through Bcl-2/Bax regulation Levels of anti-apoptotic and apoptotic proteins following propolis treatment were examined by Western blotting in order to determine whether propolis induces U937 cells death by the regulation between Bax and Bcl-2. Treatment of U937 cells with propolis did not change the expression of Bax. However, propolis significantly decreases the expression levels of Bcl2 in a concentration-dependent manner (Fig. 4). These results suggest that propolis regulates Bax/Bcl-2 level in U937 cells.

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Fig. 4. Activation of caspase-3 and regulation of Bax/Bcl-2 level in U937 cells. After 24 h incubation with propolis, the cells were lysed and the cellular proteins were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Bcl-2, 26 kDa; Bax, 23 kDa; pro-caspase-3, 32 kDa; cleaved caspase-3, 17/19 kDa. GAPDH was used as a loading control.

In addition, these results indicate that apoptotic signaling triggered by propolis is mainly related to mitochondrial pathway. Next, we determined whether caspase-3 might be activated during the induction of apoptosis by propolis because caspase-3 is known to play an essential role as an executor in apoptosis. Then, we observed that a 32 kDa precursor (procaspase-3) was disappeared in a dose-dependent manner, indicating that caspase-3 was activated by propolis. A small amount of cleaved caspase-3 was detected in U937 cells treated with 100 ␮g/ml of propolis. Activation of caspase-3 was observed in a dosedependent manner in U937 cells, as evidenced by detecting the 17/19 kDa cleaved caspase-3 proteins (Fig. 4). These results demonstrate conclusively that propolis induces apoptosis in U937 cells, accompanying by the loss of mitochondrial membrane potential, Bcl-2 down-regulation and triggered caspase-3 activation. 4. Discussion Cancer is the most common cause of morbidity and mortality worldwide. The target of research has been on the discovery of natural and synthetic compounds that can be used in the prevention or treatment of cancer. Natural products of either plant or animal origin that exhibited anti-tumor activity have been discovered (Pezzuto, 1997). As a candidate, propolis has recently gained popularity as a health food in various parts of the world, including Taiwan, Japan, Brazil, U.S.A. and Europe. The composition of propolis depends on local flora, phenology of the plants and the vegetation at the site of collection. Due to the geographical difference, propolis samples from Asia, Europe and North and South America contain different chemical substances (Marcucci, 1995). The major components of propolis are flavonoids, phenolic acid esters, terpenoids and prenylated derivatives of coumaric acids (Marcucci and Bankova, 1999;

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Tazawa et al., 1999). Propolis has been reported to possess therapeutic or preventive effects in exert of biological functions (Banskota et al., 2001). It is well known that propolis effectively induces inhibition of growth and apoptotic effects in human melanoma A2058 cells, human breast cancer cells (MCF-7), human neuroblastoma cells (IMR-32), and rat glioma cells (C6), human leukemia cells (HL-60) (Chen et al., 2007). For leukemic cells, Aso et al. (2004) reported that propolis effectively inhibits the proliferation of human leukemic U937 cells by inhibiting DNA, RNA and proteins synthesis. However, the action mechanism(s) for the propolis-mediated apoptosis has not been studied in human leukemic U937 cells. The aim of our study was, therefore, to determine the capacity of propolis to induce apoptosis and identify the biochemical mechanisms of the induction on a human leukemic U937 cells. We have provided evidences that propolis induces inhibition of growth (Fig. 1) when U937 cells were treated with various concentrations of propolis (1–1000 ␮g/ml) for 24 h. Cell viability of U937 cells was not changed at doses ranged between 1 and 100 ␮g/ml of propolis. However, the cell growth was markedly suppressed at concentrations higher than 100 ␮g/ml. Eukaryotic cell cycle progression involves sequential activation of Cdks, whose activation is dependent upon their association with cyclins (Sancar et al., 2004). A complex formed by the association of Cdc2 (also known as Cdk1 or p34Cdc2) and cyclin B1 plays a major role at entry into mitosis (Sancar et al., 2004). The phosphorylation of Tyr15 of Cdc2 suppresses activity of Cdk1/cyclin B1 kinase complex. Dephosphorylation of Tyr15 of Cdc2 is catalyzed by Cdc25 phosphatases, and this reaction is believed to be the rate-limiting step for entry into mitosis (De Souza et al., 2000). Cell cycle progression is also regulated by the relative balance between the cellular concentration of cyclin-dependent kinase inhibitors (CKIs), such as members of the cyclin-dependent kinase-interacting protein/cyclin-dependent kinase inhibitory protein (CIP/KIP) and inhibitor of cyclin-dependent kinase (INK) families, and that of cyclin–CDK complexes. The Cip/Kip family, including CIP/p21, and KIP/p27, bind to cyclin–CDK complexes and prevent kinase activation and subsequently blocking the progression of the cell cycle at the G0/G1 or G2/M phases (Sancar et al., 2004; Di Gennaro et al., 2003). In the present results, we have found that propolis not only causes a significant reduction in the expression of cyclin A and cyclin B of U937 cells, but also decreases the expression of Cdc2. In addition, propolis increased the expression of p21 and p27. Thus, it was suggested that propolis cause cell cycle arrest by reducing the complex of Cdc2/cyclin B due to down-regulation of multiple G2/M regulating proteins. Apoptosis is a cell death process characterized by morphological and biochemical features (Koopman et al., 1994), and certain that activation of caspase-3 is common in two major pathways, such as death receptors and the mitochondrial pathway that both lead to the activation of caspases-3 (Hengartner, 2000). The death receptors pathway is triggered by activation of death receptor at the plasma membrane level and results in the activation of caspase-8 (Hengartner, 2000). The mitochondrial pathway is triggered by various apoptotic proteins such as Bcl-2 family of proteins and anti-apoptotic proteins, Bcl-2, inhibits

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mitochondrial membrane permeabilization (Costantini et al., 2000; Debatin et al., 2002; Kroemer and Reed, 2000). In addition, Bcl-2 has been reported to directly inhibit members of the caspase family, including caspase-3 and caspase-9 (Deveraux and Reed, 1999; Park et al., 2007). Whereas pro-apoptotic protein, Bax protein enhances mitochondrial perturbations, Bax (Bcl-2 antagonist X) was identified as a Bcl-2-interacting protein that opposed Bcl-2 and promoted apoptotic cell death (Oh et al., 2006; Paris et al., 2007; Reed, 2006). In the present study, chromatin condensation and DNA fragmentation, which are typical morphologic properties observed in apoptotic cells, were clearly caused by the treatment of propolis (Fig. 3A and C). Consistent with the observation, propolis was shown to induce a dose-dependent increase in end-stage of apoptotic event (Fig. 3B). In addition, propolis-induced apoptosis via decrease in Bcl-2/Bax regulation (Fig. 4). In general, Bcl-2/Bax regulation is regarded to be a key factor in apoptosis induction. There are three different kinds of the Bcl-2/Bax regulation: (1) as a case for low levels of Bcl-2 and Bcl-2/Bax ratio, the Bcl-2 level and Bcl-2/Bax ratio were decreased during H2 O2 -mediated apoptosis of SCC-25 cells (Kowaltowski et al., 2001); (2) as a case for low level of Bcl-2 and high level of Bax, as miltefosineinduced apoptosis in U937 cells (Paris et al., 2007); (3) as our case, propolis did not alter the expression levels of Bax in U937 cells, but selectively down-regulated the expression of Bcl-2. As a similar report to the present case, ␤-sitosterol decreased Bcl-2 level without any alteration in Bax level in U937 cells (Park et al., 2007). In summary, propolis induces growth inhibition and apoptosis in human leukemic U937 cells. The selective activation of caspase-3 and down-regulation of Bcl-2 by propolis may mediate apoptosis in U937 cells. These results indicate that propolis can be used for cancer preventive agent. Acknowledgment This work was in part supported by The KOSEF, Ministry of Science and Technology, Korea. References Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Ahn, M.R., Kunimasa, K., Ohta, T., Kumazawa, S., Kamihira, M., Kaji, K., Uto, Y., Hori, H., Nagasawa, H., Nakayama, T., 2007. Suppression of tumorinduced angiogenesis by Brazilian propolis: major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation. Cancer Lett. 252 (2), 235–243. Amoros, M., Simoes, C.M., Girre, L., Sauvager, F., Cormier, M., 1992. Synergistic effect of flavones and flavonols against herpes simplex virus type 1 in cell culture. Comparison with the antiviral activity of propolis. J. Nat. Prod. 55, 1732–1740. Aso, K., Kanno, S., Tadano, T., Satoh, S., Ishikawa, M., 2004. Inhibitory effect of propolis on the growth of human leukemia U937. Biol. Pharm. Bull. 27, 727–730. Bankova, V., Christov, R., Kujumgiev, A., Marcucci, M.C., Popov, S., 1995. Chemical composition and antibacterial activity of Brazilian propolis. Z. Naturforsch. C, J. Biosci. C 50, 167–172. Banskota, A.H., Tezuka, Y., Kadota, S., 2001. Recent progress in pharmacological research of propolis. Phytother. Res. 15, 561–571.

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