In vitro antiproliferative and antiangiogenic effects of synthetic chalcone analogues

In vitro antiproliferative and antiangiogenic effects of synthetic chalcone analogues

Toxicology in Vitro 24 (2010) 1347–1355 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxi...

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Toxicology in Vitro 24 (2010) 1347–1355

Contents lists available at ScienceDirect

Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit

In vitro antiproliferative and antiangiogenic effects of synthetic chalcone analogues Martina Pilatova a,1, Lenka Varinska a,1, Pal Perjesi b, Marek Sarissky a, Ladislav Mirossay a, Peter Solar c, Alexander Ostro d, Jan Mojzis a,* a

Department of Pharmacology, Faculty of Medicine, P.J. Safarik University, Kosice, Slovak Republic Institute of Pharmaceutical Chemistry, Faculty of Medicine, University of Pecs, Pecs, Hungary c Institute of Biology and Ecology, Faculty of Science, P.J. Safarik University, Kosice, Slovak Republic d II. Gynecology-Obstetric Clinic, Faculty of Medicine, L. Pasteur University Hospital, Kosice, Slovak Republic b

a r t i c l e

i n f o

Article history: Received 3 January 2010 Accepted 29 April 2010 Available online 5 May 2010 Keywords: Chalcones Antiproliferative Antiangiogenic

a b s t r a c t As flavonoids, chalcones possess a wide variety of biological activities including anticancer properties. In the present study we have investigated the in vitro antiproliferative and antiangiogenic effects of four synthetic chalcones. E-2-(40 -methoxybenzylidene)-1-benzosuberone (3) was the most active compound with IC50 = 107 mol l1 in Jurkat cells. In both Jurkat and HeLa chalcone 3-treated cells we found a significant increase in the proportion of cancer cells in the G2/M phase of the cell cycle as well as an increase in cells having sub-G0/G1 DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. These effects were associated with reduced expression of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax. Furthermore, chalcone 3 was selected to evaluate its effect on some angiogenic events. In non-toxic concentrations, chalcone 3 inhibited VEGF-induced migration of human umbilical vein endothelial cells. Moreover, it also decreased secretion of matrix metalloproteinase (mainly MMP-9) and vascular endothelial growth factor (VEGF). In conclusion, the present study has assessed the in vitro antiproliferative/antiangiogenic potential of chalcone 3. This results generate a rationale for in vivo efficacy studies with this compound in preclinical cancer models. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Cancer is a major disease at a worldwide level accounting for more than 7 million deaths per year. Progress made in cancer therapy has not been sufficient to significantly lower annual death rates from most epithelial tumor types resulting in an urgent need for new strategies in cancer control. Many clinically successful anticancer drugs were themselves either naturally occurring molecules or have been developed from their synthetic analogues. Great interest is currently being paid to natural products for their interesting anticancer activities. Epidemiological studies provided evidence that the high dietary intake of fruits and vegetables could be associated with lower cancer prevalence in humans. Polyphenols (mostly flavonoids) in these foods are thought to be among the constituents responsible for the reduced cancer risk (Yang

* Corresponding author. Address: Department of Pharmacology, Faculty of Medicine, P.J. Safarik University, Trieda SNP 1, 04011 Kosice, Slovak Republic. Tel./fax: +421 556428524. E-mail address: [email protected] (J. Mojzis). 1 M. Pilatova and L. Varinska contributed equally to this work and are thus considered joint first authors. 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.04.013

et al., 2001; Hoensch and Kirch, 2005; Cutler et al., 2008). A broad biological activity of flavonoids is well documented (for review see Middleton et al., 2000; Mojzis et al., 2008). Chalcones, precursors of flavonoids and isoflavonoids, are also widely distributed in edible plants and exert various biological activities including antiinflammatory, analgesic, antipyretic and antimutagenic effects (Satyanarayana and Rao, 1993; Hsieh et al., 1998; Kaur et al., 2009). Moreover, many studies and investigations using different cellular and animal models suggested that certain chalcones can inhibit tumor initiation as well as tumor progression (e.g. Dimmock et al., 1999a; Vincenzo et al., 2000; Go et al., 2005; Akihisa et al., 2006; Rozmer et al., 2006). Natural compounds achieve anticancer activities through various mechanisms by targeting different aspects of cancer progression and development and inhibition of angiogenesis is one of them (Khan and Mukhtar, 2008; Varinska et al., 2010). Angiogenesis, the development of new blood vessels from the existing vasculature, is essential in many physiological processes such as development, wound healing, reproductive cycles. Under physiological conditions, it is a highly regulated phenomenon. On the other hand, uncontrolled angiogenesis is considered a key step in tumor growth, invasion, and metastasis. Thus, suppression of

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abnormal angiogenesis may provide therapeutic strategies in the treatment of cancer disease (Ferrara and Kerbel, 2005). In the present study, we have investigated whether four synthetic chalcones possess an in vitro antiproliferative activity. The effects of selected chalcones on proliferation and cell death in the Jurkat and HeLa cancer cells were examined. Furthermore, their antiangiogenic effects on human umbilical vein endothelial cells (HUVECs) were also studied. 2. Materials and methods 2.1. Compounds tested 4-Hydroxychalcone (1), E-2-(X-benzylidene)-1-tetralones (2a, 2b) and E-2-(40 -methoxybenzylidene)-1-benzosuberone (3) were synthesized and purified as described before (Dimmock et al., 1999b, 2002). The structure and purity of the synthesized compounds were verified by spectroscopic (IR, 1H NMR) and chromatographic (TLC, GC) methods. 2.2. Cell lines Jurkat cells (human acute T-lymphoblastic leukemia) and HeLa cells (human cervical cancer) were kindly provided by M. Hajduch, MD, Ph.D. (Olomouc, Czech Republic). The cells were routinely maintained in RPMI 1640 medium with Glutamax-I supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 IU/ml) and streptomycin (100 lg/ml) (all from Invitrogen, USA), in the atmosphere of 5% CO2 in humidified air at 37 °C. Cell viability, estimated by trypan blue exclusion, was greater than 95% before each experiment. 2.3. Cytotoxicity assay The cytotoxic effects of the tested compounds were studied by using colorimetric microculture assay with the MTT end-point. In this assay, the amount of MTT reduced to formazan is proportional to the number of viable cells (Mosmann, 1983). Briefly, the cells (1  104 of Jurkat cells and 5  103 of HeLa cells per well) were plated in 96-well polystyrene microplates (Sarstedt, Germany) in the culture medium containing the tested chemicals at final concentrations 1  104–1  109 mol l1. After 72 h of incubation, 10 ll of MTT (5 mg ml1) (Sigma, Germany) were added in each well. After additional 4 h, during which insoluble formazan was produced, 100 ll of 10% sodium dodecylsulphate were added in each well and another 12 h were allowed for the formazan to be dissolved. The absorbance was measured at 540 nm using the automated MRX microplate reader (Dynatech Laboratories, UK). The absorbance of the control wells was taken as 100% and the results were expressed as a percentage of the control. 2.4. Cell cycle analysis Cell cycle distribution in cells treated with the tested agents was analyzed by propidium iodide DNA staining according Ormerod (2000). Briefly, Jurkat and HeLa cells (1  106) after drug treatment (1.0 lmol l1) for 24, 48 and 72 h were harvested and washed twice in PBS and fixed overnight in 70% ethanol at 4 °C. Then, cells were washed with PBS, incubated for 1 h with 1 mg/ ml RNase A and 10 lg/ml propidium iodide at room temperature, and analyzed for the distinct cell cycle phases on a FACS Vantage SE flow cytometer using CellQuest software (Becton Dickinson, USA). Ten thousand cells were required per analysis. PI fluorescence was detected in the pulse-processed FL3 channel (630/ 22 nm band pass filter). Results were analyzed using Win MDI soft-

ware. Percentages of cells corresponding to G0/G1, S and G2/M phases of the cell cycle were calculated. A sub-G0/G1 fraction of cells was identified as an apoptotic population. 2.5. Apoptosis assay 2.5.1. Annexin V/PI staining The assay was performed as described previously (Kravtsov et al., 1998). Briefly, 5  105 Jurkat or HeLa cells after drug exposure (1.0 lmol l1 (3) and 10.0 lmol l1 (1, 2a 2b) for 24, 48 and 72 h) were washed twice in PBS and resuspended in 100.0 ll binding buffer (Becton Dickinson, USA). The cells were subsequently stained with annexin-V-FITC (An) and propidium iodide (PI) (Becton Dickinson, USA) according to manufacturer’s instructions. After staining, cells were resuspended in 400 ll binding buffer and 104 events were acquired immediately using the FACS Vantage SE flow cytometer. Annexin V-FITC and PI fluorescences were detected in FL1 (530/30 nm band pass filter) and FL3 (630/22 nm band pass filter) channels. Samples were acquired and analyzed using the CellQuest software (Becton Dickinson, USA). The results are represented in the form of dot plots divided into four quadrants: lower left quadrant of the dot plots shows viable, An/PI cells; lower right quadrant shows early apoptotic cells with preserved plasma membrane integrity (An+/PI); upper right quadrant shows late apoptotic/necrotic cells which have lost their plasma membrane integrity and became An+/PI+. 2.5.2. DNA fragmentation assay Chalcone-treated and untreated cells (2  106) were washed twice with 1X PBS without calcium and magnesium. Lyzation of cells was performed in lysis buffer containing 10 mmol l1 EDTA, 0.5% Triton X-100 and 10 mmol l1 TRIS (pH 8.0). Proteinase K (1 mg/ml) was added and cells were incubated for 1 h at 37 °C followed by 10 min. incubation at 70 °C. Then RNAase (200 lg/ml) was added and cells were incubated for another 1 h at 37 °C. Samples were transferred to 2% agarose gel and run with 40 V at 3 h. DNA fragments were visualized by UV illuminator. 2.6. Western blotting The cells were washed twice with ice-cold PBS and scraped into the RIPA buffer (1  PBS pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). Protease inhibitor cocktail ‘‘Complete” (Roche Diagnostics, Germany) was freshly added to RIPA buffer. Scraped lysate was transferred into the microcentrifuge tube and passed through a 21 gauge needle to shear the DNA. After the incubation of lysate on ice for 45 min and after centrifugation at 10,000g for 10 min at 4 °C, the supernatant was transferred into a new microcentrifuge tube. Protein sample was separated on 10% SDS–polyacrylamide gel, electroblotted onto Immobilon-P transfer membrane (Millipore Co., Billerica, MA, USA) and detected using anti-Bcl-2, anti-Bax (Santa Cruz Biotechnology Inc., USA), and anti-b-actin (Sigma–Aldrich, USA) primary antibodies. Then, the membranes were incubated with secondary horseradish peroxidase-conjugated antibodies (Goat anti-Rabbit IgG F(AB0 ) 2, and Goat anti-Mouse IgG F(AB0 ) 2, Pierce, USA) for 1 h and the antibody reactivity was visualized with ECL Western blotting substrate (Pierce) using Kodak Biomax film (Sigma–Aldrich). 2.7. Endothelial cells isolation and cell cultures HUVECs were isolated from freshly collected human umbilical cords by collagenase digestion of the umbilical vein interior according to the method described by Jaffe et al. (1973) and Marin et al. (2001) with minor modifications. Briefly, cords were rinsed thoroughly and flushed with cord buffer (0.14 mol l1 NaCl,

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2.8. Endothelial cell migration assay The migratory activity of HUVECs was assessed using a wound healing assay. Sub-confluent monolayers in 24-well plates were wounded with pipet tips giving acellular 1-mm-wide lane per well. After washing, cells were supplied with 1.5 ml complete medium in the absence (control) or presence of the chalcones tested. Cells were then allowed to migrate into the wound (empty space produced by the scratch) over a 24 h period. Quantitation of cell migration was performed as described by Cheung and Li (2002).

sis, the gels were renatured in 2.5% Triton X-100 (2  15 min), then incubated overnight at 37 °C in development buffer (50 mmol l1 Tris–HCl, pH 7,6; 10 mmol l1 CaCl22H2O; 50 mmol l1 NaCl; 0,05 % Brij35 [MP Biomedicals USA]). The gels were stained with 0.5% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid for 40 min at room temperature and then destained for 2 h in 50% methanol and 10% acetic acid. Proteolytic activity for MMP-2 and MMP-9 in the gel was visualized as clear white bands at 72 kDa and 92 kDa, respectively, against a dark background. The gelatinase standard (Chemicon, USA) was used as a positive control for gelatin zymography.

2.11. Statistical analysis For all experiments, mean values and standard deviations (from three experiments) were calculated using the ArcusQuickstat software package. To evaluate the statistical significance observed between groups, Student’s t-test was employed. The statistical significance was considered to be present if p < 0.05.

Cell survival (% of control)

0.00052 mol l1 Na2HPO4, 0.00015 mol l1 KH2PO4 and 0011 mol l1 glucose in distilled water, pH 7.3). The vein was then filled with 0.1% collagenase II (Gibco, USA) solution, clamped and incubated for 15 min at 37 °C. After incubation, one end of the cord was cut and the vein perfused with culture medium. The effluent with cells was collected and centrifuged. Cells were plated in 100  20 mm tissue culture dishes (Sarstedt, Germany) coated with 1.5% gelatin. Cells were grown to confluence in the Medium 199 (Gibco, USA). supplemented with 20% heat-inactivated fetal bovine serum, 100 lg/ml streptomycin, 100 IU/ml penicillin. The endothelial identity of the cells was confirmed by their ‘‘cobblestone” morphology and CD31 expression as determined by flow cytometry. The cells were stained with a CD45-FITC (BD Biosciences, USA)/CD31-PE (Caltag, USA) combination of monoclonal antibodies and analyzed using the FACS Vantage SE flow cytometer. Primary cultures were harvested at confluence with 0.05% trypsin–0.02% EDTA (Gibco, USA) and plated at a split ratio of 1:3 in tissue culture dishes. Sub-confluent cells were allowed to grow to confluence under the same conditions, harvested during the exponential cell-growth phase with trypsin–EDTA. The cells were fed with fresh medium one day before each individual experiment.

120 100 80 60 40 Jurkat

20 0 -9

10

-8

10

-7

10

Matrix metalloproteinases released into conditioned media were determined by gelatinase zymography according to the method of Newcomb et al. (2005) with minor modifications. Briefly, cells (5  105) were seeded in 6-well plates in 2 ml cultivation medium for 24 h. Then, cells were washed with PBS and incubated in 1 ml of serum-free cultivation medium for additional 24 h in the absence (control) or presence of different concentrations of the chalcone 3. Medium was collected, clarified of cellular debris by centrifugation at 1000g for 20 min at 4 °C, and stored at 80 °C until analysis. Proteins were subjected to electrophoresis under non-reducing conditions on 10% polyacrylamide gels containing 1 mg/ml gelatin (Sigma–Aldrich, USA). After electrophore-

-5

10

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10

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10

-4

-5

10

-5

10

Cell survival (% of control)

120 100 80 60 40 HeLa

20 0

-9

10

-8

10

-7

10

10

-4

mol.l-1 Cell survival (% of control)

2.10. Gelatinase zymography

10

mol.l-1

2.9. VEGF quantification VEGF protein released into the conditioned medium was measured by using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, USA) according to the manufacturer’s instructions. HeLa cells (1  105) were seeded in 6-well plates in 2 ml of complete growth medium. After 24 h, the cells were washed with PBS and preconditioned for 1 h at 37 °C in 1 ml of DMEM containing 2% FBS. The preconditioned medium was replaced with 1 ml DMEM containing 2% FBS in the absence (control) or presence of compound 3 (106, 107, 108 mol l1). After 24 h incubation to allow VEGF protein secretion, medium was collected and 1 mmol l1 of phenyl methyl sulfonyl fluoride was added. The supernatant was clarified by centrifugation for 5 min at 15,000 rpm and stored at 80 °C until quantification for VEGF. The assay was repeated three times with similar results. Data from three independent experiments were pooled for statistical analysis.

-6

10

120 100 80 60 40 HUVEC

20 0

-9

10

-8

10

-7

10

10

-4

mol.l-1 Fig. 1. The cytotoxic activity of chalcone 1 (}), chalcone 2a (j), chalcone 2b (N) and chalcone 3 (s) against cancer (Jurkat and HeLa) and endothelial (HUVEC). Cells were cultured in the absence or presence of chalcones for 72 h as indicated in Section 2. The curves correspond to three experiments performed in duplicate.

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3. Results 3.1. Cytotoxic effect of chalcones The results of the cytotoxicity testing in Jurkat, HeLa and HUVEC cells are shown in Fig. 1. The data indicate that the cytotoxic effects of the studied chalcones were concentration-dependent in both cancer cell lines. Chalcones 1, 2b and 3 significantly decreased Jurkat cell survival at c = 104–106 mol l1 (p < 0.001; p < 0.01). Compound 3 was found to be the most effective of all chalcones (IC50 = 5  107 mol l1). At lower concentrations (107–109 mol l1), none of the tested compounds displayed any significant

Table 1 Flow cytometric analysis of cell cycle distribution in Jurkat and HeLa cells treated with chalcone 3 (in %). Time (h) Sub-G0/G1 G0/G1 Treatment Conc. (lmol l1) Jurkat cells Control Chalcone 3 1.0

24

50.1 ± 4.7a 26.5 ± 1.3a 10.0 ± 0.4 13.3 ± 1.6 50.9 ± 1.6a 26.4 ± 2.8a 9.0 ± 0.5 14.4 ± 0.7

a b

48 72

1.2 ± 0.1 72.7 ± 2.1 9.2 ± 1.8 49.8 ± 3.7a 7.2 ± 1.1a 4.2 ± 1.3 a

56.8 ± 1.9a 10.2 ± 0.5 4.2 ± 1.0 72.9 ± 3.6a 24.4 ± 2.1a 1.6 ± 0.1

p < 0.001 vs. untreated cells (control). p < 0.01 vs. untreated cells (control).

Treatment (lmol l1)

Conc.

T (h)

An/PI

An+/PI

An+/PI+

24

96.2 ± 4.6 90.9 ± 2.8

1.1 ± 0.3 6.2 ± 2.4

2.4 ± 0.5 2.3 ± 0.9

Jurkat cells Control Chalcone 3

1.0

48 72 24

29.9 ± 3.0a 10.3 ± 0.8a 80.4 ± 6.7

22.9 ± 2.5a 3.1 ± 0.2 15.1 ± 1.2a

46.3 ± 3.9a 85.5 ± 1.9a 3.9 ± 0.7

Chalcone 2b

10.0

48 72

59.4 ± 6.8a 28.1 ± 2.6a

12.7 ± 2.0a 3.5 ± 1.0

27.4 ± 2.3a 67.2 ± 2.9a

24

85.9 ± 7.2 61.9 ± 5.9b

5.1 ± 2.8 15.7 ± 0.9b

5.7 ± 1.8 18.0 ± 2.8b

HeLa cells Control

48 72

24

Table 2 Chalcones-induced apoptosis in Jurkat and HeLa cells measured by flow cytometry.

G2/M

2.1 ± 0.2 73.6 ± 2.8 11.6 ± 1.5 12.7 ± 0.9 30.4 ± 2.4a 25.6 ± 3.1a 10.6 ± 1.9 33.8 ± 3.0a

HeLa cells Control Chalcone 3 1.0

S

cytotoxic effect. Cytotoxic activity of chalcone 2a was the weakest among all chalcones studied displaying a significant effect only at c = 104 and 105 mol l1 (p < 0.001; p < 0.01). In HeLa cells, the maximum effect on cell survival was observed for chalcone 3 at concentrations c = 104–106 mol l1 (p < 0.001; p < 0.01). The effects of other compounds tested were weaker.

13.3 ± 0.6 25.9 ± 0.9b b

25.9 ± 1.4 1.6 ± 0.1b

Chalcone 3

1.0

48 72 24

59.7 ± 3.2a 23.9 ± 3.1a 76.3 ± 4.1

15.8 ± 2.1b 21.3 ± 1.5a 12.9 ± 2.8c

20.7 ± 1.3a 49.8 ± 2.2a 6.8.5 ± 1.7

Chalcone 2b

10.0

48 72

64.7 ± 5.1b 53.2 ± 1.8a

15.7 ± 3.0b 9.4 ± 3.9

14.8 ± 2.9b 30.6 ± 2.1a

An/PI – live cells; An+/PI – early apoptic cells; An+/PI+ – late apoptotic/necrotic cells. Cells were exposed to chalcones for 72 h. a p < 0.001 vs. control. b p < 0.01 vs. control. c p < 0.05 vs. control.

Fig. 2. Cell cycle distribution detected by flow cytometric analysis. Jurkat (A) and HeLa cells (B) were treated with chalcone 3 (1.0 lmol l1) for the indicated time. The cells were then stained with PI and their nuclei analyzed for their DNA content by flow cytometry using CellQuest software. Symbol M1, M2, M3, M4 represented sub-G0/G1 peak, G0/G1 phase, S phase and G2/M phase, respectively. Each histogram is representative of three experiments.

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In HUVECs, chalcones 1, 2a and 2b did not exert any significant cytotoxic effects. However, significant cytotoxicity was observed after HUVECs were incubated with chalcone 3 at c = 104– 106 mol l1 (p < 0.001; p < 0.01).

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3.2. Effect of chalcones on cell cycle The distribution of cells in different phases of the cell cycle is shown in Table 1 and Fig. 2. After treatment with chalcone 3 at

Fig. 3. Representative FACS analyses after staining with annexin V-FITC and PI indicating the induction of apoptosis and necrosis in Jurkat (A) and HeLa cells (B) by chalcone 3 (1.0 lmol l1) after 24, 48 and 72 h. In each plot, the lower left quadrant represents viable cells, the lower right quadrant represents early apoptotic cells, and the upper right quadrant represents necrotic or late apoptotic cells. This figure is representative of three experiments.

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c = 1 lmol l1 for 24 h, we found an increase in the proportion of Jurkat cells in the G2/M phase of the cell cycle as well as an increase in cells having sub-G0/G1 DNA content which was accompanied by a proportional decrease in G0/G1 phase cells. G2/M arrest did not persist after 48 and 72 h treatment when more than 50% of the cells were found to have sub-G0/G1 DNA content. Similar effects were observed also in HeLa cells. After 24 and 48 h of incubation, chalcone 3 induced an arrest in the G2/M phase of the cell cycle with simultaneous increase sub-G0/G1 and decrease in G0/G1 phases. Chalcone 2b possesses similar effects on both cell lines but only at c = 10 lmol l1. The remaining chalcones studied displayed only a marginal effect on the cell cycle of cancer cells (data not shown). 3.3. Chalcone-induced apoptosis 3.3.1. Annexin V/PI staining To clarify whether or not the chalcone-induced cell death involved apoptosis, flow cytometry analysis was employed. After treatment with 1.0 lmol l1 chalcone 3 for 24, 48 and 72 h, apoptotic rate were 8.5%, 69.2% and 88.6%, respectively, in comparison with control group of 3.5% in Jurkat cells. In HeLa cells, this compound increased the percentage of apoptotic cells from 10.8% to 71.1% after treatment for 72 h (p < 0.001 for both cell lines) (Table 2 and Fig. 3). After treatment with chalcone 2b (10.0 lmol l1), an increase in the proportion of An+/PI cells was observed after 24 and 48 h of treatment followed by an increase in the proportion An+/PI+ cells after 48 and 72 h of incubation in both cell lines (Table 2). For the remaining chalcones tested only negligible apoptosis-inducing activities were observed (data not shown).

3.3.2. DNA fragmentation assay Among the characteristics of apoptosis, one of the most prominent biochemical features is the formation of DNA fragments of approximately 180 bp also known as DNA laddering. As shown in Fig. 4A, treatment of Jurkat cells with chalcone 3 resulted in the formation of definite fragments that could be seen via electrophoretic examination as a characteristic ladder pattern (100 lmol l1 for 24, 48 and 72 h, lanes 3, 4, and 7, respectively; 10 lmol l1 for 72 h, lane 6 and 1 lmol l1 for 72 h lane 5). Similar results were also obtained with chalcone 2b (100 lmol l1 for 24, 48 and 72 h, lanes 8, 9, and 12, respectively; 10 lmol l1 for 72 h, lane 11 and 1 lmol l1 for 72 h, lane 10). In HeLa cells (Fig. 4B) both chalcones produced similar effects except the lowest concentration (1 lmol l1) in which no fragments were observed. The remained compounds induced DNA fragmentation only at high concentration (100 lmol l1) and after prolonged incubation period (48 and 72 h) (data not shown). 3.3.3. Effect of chalcone on apoptosis-related proteins Bcl family proteins such as Bcl-2 and Bax are believed to mediate the apoptotic pathway. To clarify the mechanism of chalcone 3induced apoptotic cell death, we examined the contribution of Bcl family proteins using Western blot analysis. Bcl family proteins, consisting of pro-apoptotic (such as Bax, Bak, Bid, Bad) and anti-apoptotic (such as Bcl-2, Bclx L, Bcl-w) members, serve as critical regulators of the death signal. Bax and Bcl-2 are important regulators of cytochrome c release from mitochondria. As shown in Fig. 5, chalcone 3 up-regulated the expression of Bax and down-regulated the expression of Bcl-2, thereby increasing Bax:Bcl-2 ratio by 1.6–2.3-fold after 12–24 h of incubation, respectively. Thus it seems that chalcone 3-induced apoptosis may involve Bax/Bcl-2 signal transduction. 3.4. Effect of chalcones on endothelial cell migration For the determination of potential anti-migratory activities, we tested the compounds in a wound closure assay. After wounding with a pipet tip, solvent controls reformed a confluent monolayer within 24 h of incubation. Chalcones were added at concentrations of 105–108 mol l1 (concentrations of 107 and 108 mol l1 were non-toxic). In the presence of compound 3 a potent dosedependent inhibition of endothelial cell migration was observed at all concentrations tested (Fig. 6). Significantly weaker effects on endothelial cell migration were observed for compounds 1, 2a and 2b (data not shown). 3.5. Suppression of VEGF secretion by chalcone 3 Treatment of HeLa cells with chalcone 3 at concentrations of 106–108 mol l1 resulted in a dose-dependent suppression of

C

12

24 Bax

Fig. 4. DNA fragmentation of Jurkat (A) and HeLa cells (B) after 24, 48 and 72 h incubation with chalcones 3 (Ch3) and 2a (Ch2a) at concentrations 104– 106 mol l1. Apoptotic DNA fragmentation was qualitatively analyzed by DNA gel electrophoresis. The extracted DNA was loaded on 2% agarose gel and was stained with ethidium bromide. Lanes indicate different treatments: 1 – negative control, 2 – positive control (etoposide 50 ll ml1 for 48 h), 3 – Ch3 – 104 24 h, 4 – Ch3 – 104 48 h, 5 – Ch3 – 106 72 h, 6 – Ch3–105 72 h, 7 – Ch3–104 72 h, 8 – Ch2a – 104 24 h, 9 – Ch2a – 104 48 h, 10 – Ch2a – 106 72 h, 11 – Ch2a – 105 72 h, 12 – Ch2a – 104 72 h.

Bcl-2 β-actin Fig. 5. Effect of chalcone 3 on the levels of Bcl-2 and Bax proteins after 12 and 24 h incubation. Western blot analyses were performed as described in Section 2. bActin was used as internal control.

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Remaining scratched area (%)

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1

120

2

3

4

5

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7 MMP-9 MMP-2

100 80 ***

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*** ***

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*

20 0 C 0h

C 24h

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mol.l-1

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Fig. 8. MMP-9 and MMP-2 protein secreted from HUVECs. Results show that chalcone 3 downregulates secretion of MMP-2 and MMP-9 in a dose-dependent manner. Cells were incubated in the absence (control; Lane 1) or presence (Lanes 2– 6) of different concentrations of chalcone 3 and the level of MMP-2 and MMP-9 protein secreted into the medium was measured by gelatin zymography. This is a representative gel picture of one of three separate experiments with similar results. 1 – C, control; chalcone 3 treatment (concentrations in mol l1) 2 – 108; 3 – 107; 4 – 106; 5 – 105; 6 – 104; 7 – standard MMP-9 and -2.

mol.l-1

VEGF concentration (pg/ml)

Fig. 6. Inhibitory effect of chalcone 3 on HUVECs migration in wound migration assays after 24 h of incubation. Percentage of remaining scratched area was calculated after being marked and quantified by the histogram function of the Adobe Photoshop 5.5 program. ***p < 0.001 (C 24 h vs. C 0 h and chalcone 3 vs. C 24 h), *p < 0.05 (chalcone 3 vs. C 24 h).

500.00 400.00 *

300.00

*

200.00 100.00 0.00 C

10

-6

-7

10

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10

mol.l-1 Fig. 7. Effect of chalcone 3 treatment on VEGF production in HeLa cells. The culture media was collected and VEGF was assayed using ELISA kit as described in Section 2. C – control; *p < 0.05.

VEGF protein secretion (Fig 7). Our results showed that the amount of VEGF secreted was significantly decreased by chalcone 3 at c = 106 and 107 mol l1 (p < 0.05).

3.6. Suppression of MMP-9 activity by chalcone 3 To investigate the effect of chalcone 3 on MMP-2 and MMP-9 activity, gelatin zymography was performed. Gelatin zymography revealed that chalcone 3 reduced MMP-9 activity in HUVECs in a concentration-dependent manner. The active bands of MMP-9 gradually diminished when HUVECs were treated with different doses of chalcone 3 (from 108 to 104 mol l1). Inhibitory effect of chalcone 3 on MMP-2 activity was observed only at the highest concentration (Fig. 8).

4. Discussion Edible plant matter contain many microconstituents that are now recognized as being biologically active. Epidemiological studies provided evidence that the high dietary intake of flavonoids with plant foods could be associated with lower cancer prevalence in humans (Neuhouser, 2004; Gerhauser, 2005). Although there is no relevant study focused to cancer-preventive effect of chalcones in diet today, their chemico-biological similarity with flavonoids allow to speculate on the role of chalcones in cancer-preventive effect of polyphenols. Chalcones and chalcone derivatives have been shown to exhibit cytotoxic activity against cancer cells and may

have potential applications in cancer treatment (Akihisa et al., 2006; Modzelewska et al., 2006; Ye et al., 2005). In the present study we have investigated the inhibitory effects of chalcones on cancer as well as endothelial cell functions. Our results clearly show that some chalcones possess a cytotoxic activity against Jurkat and HeLa cells. We have shown that among four chalcones examined compound 3, the chalcone with benzosuberone structure, exhibited significantly stronger growth inhibitory effect in cancer cells than other compounds tested. However, the exact mechanisms by which chalcone compounds exert their cytotoxic effects in cancer cells remains unclear. It has been documented that various naturally occuring chalcones induce cell cycle arrest and apoptosis in different cancer cell lines (Lou et al., 2009; Motani et al., 2008; Ding et al., 2009). In this study we have shown that the decrease in cell viability by compound 3 was associated with induction of the cell cycle arrest at the G2/M phase followed by an increase in the fraction of cells with sub-G0/G1 DNA content which is considered a marker of apoptotic cell death. Similar increase in the proportion of cells in the G2/M phase of the cell cycle accompanied by a decrease in the G0/G1 phase cells and increase of the sub-G0/G1 peak after exposure of Jurkat cells to E-2-(40 -methoxybenzylidene)-1-benzosuberone (chalcone 3) was documented also by Rozmer et al. (2006). We have also observed that treatment with chalcone 3 resulted in the induction of apoptosis in both cell lines, suggesting that the inhibition of cell viability might be, at least in part, mediated by this mechanism. Apoptosis is modulated by anti-apoptotic and pro-apoptotic effectors that involve a large number of proteins. Therefore, to gain insight into how chalcones may affects mechanisms controlling apoptosis, we investigated the effect of chalcone 3 on anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. Bcl-2 protein functions as a suppressor of apoptosis (Miyashita and Reed, 1993). In contrast, Bax is a pro-apoptotic protein and its predominance over Bcl-2 promotes apoptosis (Oltvai et al., 1993). In the present study, we have found that the treatment of Jurkat cells with chalcone 3 cells resulted in the reduction of Bcl-2 protein expression, with concomitant increase in the Bax indicating that the increased ratio of Bax:Bcl-2 may play an important role in the induction of apoptosis by chalcone 3. The presence of apoptosis induced by chalcone 3 was further evidenced by annexin V/PI staining as well as by nuclear DNA fragmentation which is a classical feature of apoptotic cell death. It has been documented that many natural health products inhibit angiogenesis. Among the known ‘‘natural” angiogenesis inhibitors, polyphenols seem to play an important role (Kanadaswami et al., 2005; Mojzis et al., 2008). Because the formation of new blood vessels from endothelial cells is a prerequisite for solid tumor growth and proliferation (Ruegg and Mutter, 2007), we have also examined the effects of chalcones on some of the processes involved in angiogenesis. In a series of complementary assay systems, we investigated the effects of chalcones on endothelial cell

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proliferation and migration, MMP activity as well as on VEGF secretion in in vitro conditions. The toxic effects of chalcones were significantly weaker in HUVECs compared with the toxicity in cancer cells. Only chalcone 3 showed similar toxicity against both HUVECs and cancer cells. However, at non-toxic concentrations (107 and 108 mol l1) this chalcone appears to affect several important angiogenesis pathways that may potentially lead to its efficient inhibition of angiogenesis. A critical step in the invasion and metastasis is the breakdown of biological barriers such as the basement membrane, what requires the activation of proteolytic enzymes (Geiger and Peeper, 2009). Matrix metalloproteinases, particularly MMP-2 (72-kDa gelatinase A) and MMP-9 (92-kDa gelatinase B), play a crucial role in tumor invasion and angiogenesis (Zhang et al., 2004). They are enzymes that break down extracellular matrix proteins to allow further differentiation and spread of endothelial cells during angiogenesis. Several reports indicate that MMPs play a major regulatory role in the initiation of angiogenesis (Folkman, 2006). In this report, we have demonstrated that chalcone 3 inhibited the activity of MMP-9. This result indicates that the decreased activity of MMP-9 may contribute to the decrease in the migration of chalcone 3-treated HUVECs. Our results are consistent with other studies showing the ability of chalcones to inhibit different MMPs (Kwon et al., 2009). As it was mentioned above, the formation of tumor vasculature is a crucial event supporting tumor growth (Folkman, 2002). Vascular endothelial growth factor is one of the most potent angiogenic factors. Inhibiting its production may present a promising strategy for the treatment of cancer (Cao, 2008). Here, we have demonstrated that chalcone 3 inhibited VEGF production in HeLa cells. Similar effect of chalcones on VEGF secretion were also documented by Dell’Eva et al. (2007) and Kwon et al. (2009). In conclusion, the present study has assessed the in vitro antiproliferative efficacy of E-2-(40 -methoxybenzylidene)-1-benzosuberone (chalcone 3). Moreover, for the first time, the in vitro antiangiogenic potential of this compound was documented. Taken together, antiangiogenic and apoptosis-inducing potential of E-2(40 -methoxybenzylidene)-1-benzosuberone generate a rationale for in vivo efficacy studies with this compound in preclinical cancer models. Acknowledgements This work was supported by the Slovak Research and Development Agency under the Contract No. APVV-0325-07 and by the Slovak Grant Agency for Science (Grant No. 1/4236/07). Work in our laboratory is also supported by the LABMED A.S. We declare that we have no competing financial interests. References Akihisa, T., Tokuda, H., Hasegawa, D., Ukiya, M., Kimura, Y., Enjo, F., Suzuki, T., Nishino, H., 2006. Chalcones and other compounds from the exudates of Angelica keiskei and their cancer chemopreventive effects. Journal of Natural Products 69, 38–42. Cao, Y., 2008. Molecular mechanisms and therapeutic development of angiogenesis inhibitors. Advances in Cancer Research 100, 113–131. Cutler, G.J., Nettleton, J.A., Ross, J.A., Harnack, L.J., Jacobs Jr., D.R., Scrafford, C.G., Barraj, L.M., Mink, P.J., Robien, K., 2008. Dietary flavonoid intake and risk of cancer in postmenopausal women: the Iowa Women’s Health Study. International Journal of Cancer 123, 664–671. Dell’Eva, R., Ambrosini, C., Vannini, N., Piaggio, G., Albini, A., Ferrari, N., 2007. AKT/ NF-kappaB inhibitor xanthohumol targets cell growth and angiogenesis in hematologic malignancies. Cancer 110, 2007–2011. Dimmock, J.R., Elias, D.W., Beazely, M.A., Kandepu, N.M., 1999a. Bioactivities of chalcones. Current Medicinal Chemistry 12, 1125–1149. Dimmock, J.R., Kandepu, N.M., Nazarali, A.J., Kowalchuk, T.P., Motaganahalli, N., Quail, J.W., Mykytiuk, P.A., Audette, G.F., Prasad, L., Perjési, P., Allen, T.M., Santos, C.L., Szydlowski, J., De Clercq, E., Balzarini, J., 1999b. Conformational and

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