Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells

Life Sciences 78 (2006) 2409 – 2417 www.elsevier.com/locate/lifescie

Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells Gardenia C.G. Milita˜o a, Ivana N.F. Dantas a, Cla´udia Pessoa a, Maria Jose´ C. Falca˜o b, Edilberto R. Silveira b, Mary Anne S. Lima b, Rui Curi c, Thaı´s Lima c, Manoel O. Moraes a, Letı´cia V. Costa-Lotufo a,* a

b

Laborato´rio de Oncologia Experimental, Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara´, P.O. Box-3157, 60430-270 Fortaleza, Ceara´, Brazil Departamento de Quı´mica Orgaˆnica e Inorgaˆnica, Universidade Federal do Ceara´, P.O. Box 12200, 60021-940, Fortaleza, Ceara´, Brazil c Departamento de Fisiologia e Biofı´sica, Universidade de Sa˜o Paulo, 05508-900, Sa˜o Paulo, Brazil Received 18 May 2005; accepted 29 September 2005

Abstract (+)-2,3,9-Trimethoxy-pterocarpan (1) (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) are cytotoxic pterocarpans isolated from the native Brazilian plant Platymiscium floribundum. The purpose of the present study was to examine whether induction of apoptosis and/or inhibition of DNA synthesis is involved in the cytotoxicity of these pterocarpans in human leukemia cells. The effect on cell viability determined using the trypan exclusion assay revealed that all compounds tested reduced the number of viable cells, while only in the presence of 3 and 4, there was an increase of nonviable cells. The analysis of membrane integrity and morphological modifications by flow cytometry in the presence of these two compounds indicated that treated cells undergo necrosis, while 1 and 2 trigger apoptosis. DNA synthesis seemed to be affected since BrdU incorporation was inhibited in a dosedependent manner in the presence of all tested compounds. Pterocarpan treatment also induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, mitochondrial depolarization and caspase- 3 activation, which indicate apoptosis induction. D 2005 Elsevier Inc. All rights reserved. Keywords: HL-60; Apoptosis; DNA synthesis

Introduction Flavonoids are a broad group of natural products found in all vascular plants, which are chemically defined as substances composed of a common 2-phenylchroman structure (C6– C3 – C6), with one or more hydroxyl groups (Harbone and Williams, 2000; Birt et al., 2001). The members of this class of compounds have been found to have many pharmacological activities, including antioxidant, anticancer, cancer chemopreventive, antiviral, antimicrobial and enzyme inhibitory effects (Harbone and Williams, 2000; Di Carlo et al., 1999; Middleton et al., * Corresponding author. Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara´, Rua Cel Nunes de Melo, 1127 Caixa Postal-3157, 60430-270 Fortaleza, Ceara´, Brazil. Tel.: +55 85 40098255; fax: +55 85 40098333. E-mail address: [email protected] (L.V. Costa-Lotufo). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.09.044

2000). According to several authors, flavonoids and isoflavonoids, which differ markedly from flavonoids by the presence of a 3-phenylchroman skeleton, are especially promising candidates for cancer prevention (Birt et al., 2001). Indeed, some flavonoids, such as quecertin, genistein and flavopiridol, have been used as chemotherapeutic agents in clinical trials (Ferry et al., 1996; Mohammad et al., 2003; Zhai et al., 2002). In our continuing search for new anticancer agents from the plant kingdom, we investigated the cytotoxic effect of nine flavonoids isolated from Platymiscium floribundum on tumor cell lines, and the pterocarpans were found to be the most active compounds (Falca˜o et al., 2005). In addition, we determined the antimitotic activity of the same pterocarpans: (+)-3,10-dihydroxy-9-methoxy-pterocarpan, (+)- 3,9-dimethoxy-pterocarpan [(+)-homopterocarpin], (+)-2,3,9-trimethoxy-pterocarpan, (+)3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] and (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin], on sea

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urchin egg development (Milita˜o et al., 2005). (+)-2,3,9Trimethoxy-pterocarpan was the most active compound in both assays, suggesting that the pterocarpans could emerge as a potential class of anticancer chemicals and, moreover, that the methoxy group on C2 position is an important structural requirement for cytotoxic activity among these compounds (Falca˜o et al., 2005; Milita˜o et al., 2005). The purpose of the present study was to examine whether the cytotoxic activity of (+)-2,3,9-trimethoxy-pterocarpan (1), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) involves the induction of apoptosis and the inhibition of DNA synthesis, using HL-60 human leukemia cells as a model.

mg), m.p. 167.0 –168.7 -C (lit. 168.0 -C) (Ingham, 1976) as yellowish solids. Fraction G yielded a precipitate that was collected by removal of the supernatant liquid. Evaporation of the solvent yielded compound 3, m.p. 125.0 – 127.0 -C (lit. 123– 125 -C) (Letchier and Shirley, 1976). All structures (Fig. 1) have been determined by spectroscopy means, including one and two dimensional NMR such as COSY, HMQC, HMBC etc., physical properties and comparison with data from literature. The compounds were dissolved in DMSO at the concentration of 5 mg/mL, and added to cell cultures to final concentrations of 1.25 and 2.5 Ag/mL for compound 1 and concentrations 10 times higher (12.5 and 25 Ag/mL) for the other three compounds for 24 h. These concentrations were selected from previous data on the cytotoxicity of these compounds (Falca˜o et al., 2005).

Material and methods Cell line and cell culture Pterocarpans isolation The heartwood of P. floribundum was collected in Acarape County, Ceara´, Brazil and identified by Dr. A. G. Fernandes (Universidade Federal do Ceara´). A voucher specimen (no. 31052) has been deposited at the Herba´rio Prisco Bezerra (EAC), Departamento de Biologia, Universidade Federal do Ceara´, Ceara´, Brazil. The air dried heartwood (1.7 kg) of P. floribundum was pulverized and extracted with hexane (4000 mL) at room temperature. The solvent was removed under reduced pressure yielding a viscous brown oil (7.0 g). The marc obtained after hexane extraction, was extracted with CHCl3 (4000 mL) to yield a dark brown resinous extract (76.0 g), and later extracted with EtOH (4000 mL) to give an dark brown resinous extract (35.0 g). Part of the CHCl3 extract (50.0 g) was adsorbed onto silica gel (5.0 g) and coarsely fractionated over silica gel (150.0 g) column by elution with hexane, CH2Cl2, EtOAc and MeOH, as binary mixture with increasing polarity, yielding 10 pooled fractions after TLC analysis (solvent ratio, solvent volume, mass): A (hexane, 250 mL, 20.0 mg); B (hexane : CH2Cl2 7 : 3, 250 mL, 30.0 mg); C (hexane : CH2Cl2 1 : 1, 250 mL, 3.1 g); D (hexane : CH2Cl2 1 : 1, 500 mL, 6.9 g); E (CH2Cl2, 350 mL, 39.0 mg); F (CH2Cl2, 250 mL, 22.7 g); G (CH2Cl2 : EtOAc 1 : 1, 150 mL, 3.0 g), H (CH2Cl2 : EtOAc 1 : 1, 250 mL, 1.8 g), I (EtOAc, 350 mL, 1.2 g) and J (MeOH, 50 mL, 9.1 g). Fraction E (390.0 mg) was rechromatographed over silica gel (20.0 g) by elution with hexane, CH2Cl2, EtOAc and MeOH. Six fractions of progressive increasing polarity pooled accordingly to TLC profiles, were obtained: E 1 (hexane, 125 mL, 38.0 mg), E 2 (CH2Cl2, 125 mL, 20.0 mg), E 3 (CH2Cl2 / EtOAc 7 : 3, 100 mL, 0.19 g), E 4 (CH2Cl2 : EtOAc 1 : 1, 125 mL, 21.0 mg), E 5 (EtOAc, 25 mL, 20.0 mg) and E 6 (MeOH, 25 mL, 33.0 mg). Fraction E 1 was submitted to centrifugal chromatography (Cromatroton) using a mixture of hexane / EtOAc 1 : 1 as eluent to yield compound 2 (20.0 mg) as a yellowish solid, m.p. 87.6– 87.8 (lit. 83.0 –85.0) (McMurry et al., 1972). Fraction E 3 was further purified over Sephadex LH-20 by elution with CHCl3 / MeOH 1 : 1 to give compound 1 (30.0 mg), m.p. 123– 125 -C (lit. 122 – 124 -C) (Pueppke and VanEtten, 1975) and compound 4 (8.0

The cell line used in this work was HL-60 human promyelocytic leukemia and was obtained from Children’s Mercy Hospital (Kansas City, MO, USA). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 Ag/mL streptomycin at 37 -C with 5% CO2. The cells were split on the third day and were diluted 1 day before each experiment. Trypan blue exclusion Cell viability was determined by the trypan blue dye exclusion test after incubation of HL-60 cells (3  105 cells/mL) with tested compounds. Aliquots were removed from cultures after 24 h, and cells that excluded trypan blue were counted in a Neubauer chamber. Doxorubicin (0.3 Ag/mL) was used as a positive control.

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Fig. 1. Structures of pterocarpans isolated from Platymiscium floribundum, (+)2,3,9-trimethoxy-pterocarpan (1), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4).

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Analysis of morphological changes

Cell membrane integrity

Untreated or pterocarpan-treated HL-60 cells were examined for morphological changes by light microscopy (Olympus, Tokyo, Japan). To evaluate nuclear morphology, cells were harvested, transferred to cytospin slides, fixed with methanol for 1 min, and stained with hematoxylin – eosin. Doxorubicin (0.3 Ag/mL) was used as a positive control.

The cell membrane integrity was evaluated by the exclusion of propidium iodide (2 Ag/mL). Cell fluorescence was determined by flow cytometry in a FACSCalibur cytometer (Becton, Dickinson and Company, New Jersey, USA) using the CellQuest software. Ten thousand events were evaluated per experiment and cellular debris was omitted from the analysis.

Inhibition of DNA synthesis Internucleosomal DNA fragmentation HL-60 cells (3  105 cells/mL) were plated onto 24-well tissue culture plates (2 mL/well) and treated with pterocarpans. Doxorubicin (0.3 Ag/mL) was used as a positive control. Twenty microliters of 5-bromo-2V-deoxyuridine (BrdU, 10 mM) was added to each well and incubated for 3 h at 37 -C before completing the 24 h period of drug exposure. To assay the amount of BrdU incorporated into DNA, cells were harvested, transferred to cytospin slides, and allowed to dry for 2 h at room temperature. Cells that had incorporated BrdU were labeled by direct peroxidase immunocytochemistry, utilizing the chromogen DAB. Slides were counterstained with hematoxylin, mounted, and coverslipped. Evaluation of BrdU positivity was performed by light microscopy (Olympus, Tokyo, Japan). Two hundred cells were counted per sample to determine the percentage of positive cells.

A

Based on the procedure of Nicolletti et al. (1997), HL-60 cells (1 106) were incubated at 4 -C for 24 h, in the dark, in a lysis solution containing 0.1% citrate, 0.1% Triton X-100 and 50 Ag/mL propidium iodide. Cell fluorescence was then determined by flow cytometry as described above for propidium iodide. Measurement of mitochondrial transmembrane potential Mitochondrial transmembrane potential was determined by the retention of the dye rhodamine 123 in HL-60 cells. About one million cells were washed with PBS, incubated with rhodamine 123 (5 Ag/mL) at 37 -C for 15 min in the dark, and washed twice. The cells were then incubated again in PBS at

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Fig. 2. Effect of (+)-2,3,9-trimethoxy-pterocarpan (1), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) on HL-60 cell viability determined by trypan blue staining after 24 h incubation. A—number of viable cells and B—number of non-viable cells. Negative control (C) was treated with the vehicle used for diluting the tested substances. Doxorubicin (0.3 Ag/mL) was used as positive control (D). *p < 0.05 compared to control by ANOVA followed by Student – Newman – Keuls test. Experiments were performed in triplicate.

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37 -C for 30 min in the dark and fluorescence was then measured as described above. Caspase-3 activation assay A caspase-3 colorimetric assay kit (R and D Systems, Inc., Minneapolis, MN, USA) was used to investigate caspase-3 activation in treated HL-60 leukemia cells based on the cleavage of Asp-Glu-Val-Asp (DEVD)-pNA. Briefly, cells (2  106 cells/mL) were lysed by incubation with cell lysis buffer on ice for 10 min and then centrifuged at 10,000 g for 1 min. Enzyme reactions were carried out in a 96-well flat-bottom microplate. To each reaction mixture, 50 AL cell lysate (100 –200 Ag total protein) was added. The results were expressed as specific activity (IU/mg protein) of caspase-3. Statistical analysis Data obtained from cell viability, flow cytometry and caspase-3 activity are presented as means T SEM from n experiments and evaluated by analysis of variance (ANOVA) followed by Student – Newman– Keuls test, with a signifi-

Table 1 Inhibition of 5-bromo-2V-deoxyuridine (BrdU) incorporation by HL-60 cells treated with (+)-2,3,9-trimethoxy-pterocarpan (1), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)vesticarpan] (4) Compound

Concentration (Ag/mL)

BrDU positivity (%)

T /C

Control Doxorubicin 1

– 0.3 1.25 2.5 12.5 25 12.5 25 12.5 25

73 43* 46* 45* 57* 54* 40* 15* 13* n.d.

0.59 0.63 0.62 0.78 0.74 0.54 0.20 0.18 –

2 3 4

n.d.—not determined because most cells are non-viable. Doxorubicin was used as positive control. Data are presented as percent of Brd – U positivity per 200 cells. T / C ratio was calculated using the % labeled cells: treated / control. * p < 0.05 compared by v 2 test.

cance level of 5%. For inhibition of DNA synthesis, the differences among experimental groups were compared by v 2 and the significance level was set at p < 0.05.

Fig. 3. Microscopic analysis of hematoxylin/eosin-stained HL-60 cells. Cells were untreated (A) or treated with (+)-2,3,9-trimethoxy-pterocarpan (2.5 Ag/mL, C), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (25 Ag/mL, D), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (25 Ag/mL, E) and (+)-3,4dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (25 Ag/mL, F), respectively, and analyzed by light microscopy (400). Doxorubicin (0.3 Ag/mL) was used as positive control (B). Black arrows show fragmentation of the nuclei and white arrows show vacuolization.

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Results

Analysis of morphological changes

Effects on HL60 viability

HL-60 cells treated with compound 1 at all concentrations induced chromatin condensation that became more evident at higher concentrations (Fig. 3C). The cells treated with compounds 2 and 3 at a concentration of 12.5 Ag/mL showed abundant vacuoles, reduction in cell volume and nuclear condensation. At 25 Ag/mL, compound 2 caused a reduction in cell volume, vacuolization, chromatin condensation and fragmentation of the nuclei, morphology consistent with apoptosis (Fig. 3D). In the presence of compounds 3 and 4 at 25 Ag/mL, many cells were seen with pyknotic nuclei and destabilization of the plasma membrane, indicating an increasing number of dead cells

Cell viability was determined by trypan blue exclusion assay in HL-60 cells after 24 h incubation with pterocarpans. All test compounds reduced cell viability. At a concentration of 12.5 Ag/mL, compounds 2, 3 and 4 reduced the number of viable cells by 52.5%, 57.4% and 72.1%, respectively (Fig. 2A). Compound 1 reduced the number of viable cells by 34.4% and 42.6% at 1.25 and 2.5 Ag/mL, respectively. Compounds 3 and 4 significantly increased the number of non-viable cells at both tested concentrations ( p < 0.05, Fig. 2B).

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Fig. 4. Flow cytometric analysis of (+)-2,3,9-trimethoxy-pterocarpan (1), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) effects in HL-60 cells. A—cell viability by the exclusion of propidium iodide (2 Ag/mL), B—internucleosomal DNA fragmentation was determined by nuclear fluorescence using propidium iodide, Triton X-100 and citrate, C—mitochondrial transmembrane potential was determined by the retention of the dye rhodamine 123 (5 Ag/mL). Ten thousand events were analyzed in each experiment. Results are expressed as means and standard error obtained from 4 to 6 experiments. *p <0.05 compared to control by ANOVA followed by Student – Newman – Keuls test.

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(Fig. 3E and F). Doxorubicin 0.3 Ag/mL (Fig. 3B) also induced cell shrinkage, chromatin condensation and nuclear fragmentation. Antiproliferative effect The antiproliferative effect of pterocarpans was determined by inhibition of DNA synthesis based on BrdU incorporation into HL-60 cells. All test compounds inhibited DNA synthesis significantly at all concentrations used (Table 1). Compound 1 inhibited BrdU incorporation by 37% and 38% at concentrations of 1.25 and 2.5 Ag/mL, respectively. At 12.5 Ag/mL, compound 4 reduced BrdU incorporation by 82%. As observed for cytotoxicity, compound 3 was more active than 2, resulting in 46% and 80% inhibition of BrdU incorporation, while compound 2 caused 22% and 26% inhibition, at concentrations of 12.5 and 25 Ag/mL, respectively ( p < 0.05).

Apoptosis induction First, cell membrane integrity was determined by the exclusion of propidium iodide (Fig. 4A). Compounds 3 and 4 induced disruption of membrane integrity in a concentration-dependent manner, the former being the most potent causing 55% membrane damage at a concentration of 12.5 Ag/mL, while compound 4 caused 26% damage and compound 2 caused no significant damage at the same concentration (Fig. 4A). Compound 1 did not cause membrane damage (Fig. 4A). Furthermore, all pterocarpans caused cell shrinkage, as observed by the decrease in forward light scatter (FSC) and nuclear condensation as observed by a transient increase in side scatter (SCC), both morphological modifications compatible with apoptotic cells (Fig. 5). Internucleosomal DNA fragmentation was also examined by flow cytometry in HL-60 cells (Fig. 4B). All DNA that was

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Fig. 5. Light scattering features of HL-60 cells untreated (A) or treated with (+)-2,3,9-trimethoxy-pterocarpan (2.5 Ag/mL, B), (+)-3,9-dimethoxy-pterocarpan [(+)homopterocarpin] (25 Ag/mL, C), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (25 Ag/mL, D) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)vesticarpan] (25 Ag/mL, E) during 24 h.

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Fig. 6. DNA fragmentation assay as assessed by flow cytometry. HL-60 cells were untreated (A) or treated with (+)-2,3,9-trimethoxy-pterocarpan (2.5 Ag/mL, B), (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (12.5 Ag/mL, C), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (12.5 Ag/mL, D) and (+)-3,4dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (12.5 Ag/mL, E) for 24 h. Cells were stained with a buffer containing citrate, Triton X-100 and propidium iodide and analyzed by flow cytometry. Histograms of the DNA fluorescence of ten thousand events are shown (logarithmic scale). Units are arbitrary.

was not significant, probably because of the high number of dead cells; these results were also observed with compound 4. Compounds 1 and 2, on the other hand, led to significant mitochondrial depolarization at both concentrations. Next, caspase-3 activation was examined in the process of pterocarpan-induced apoptosis using DEVD-pNA as the substrate. All pterocarpans tested induced activation of caspase-3 in HL-60 cells (Fig. 7).

sub-diploid in size was considered fragmented. All test compounds caused significant DNA fragmentation ( p < 0.05). Compound 1 led to 49.5% fragmentation at all concentrations tested. Compound 3 induced 46.2% and compound 4, 44.2% DNA fragmentation at a concentration of 12.5 Ag/mL, while compound 2 caused 37% fragmentation at the same concentration. Compound 1 caused cell cycle arrest at G2/M at all concentrations and compound 2 also caused G2/M arrest at 12.5 Ag/mL (Fig. 6). The compounds tested induced mitochondrial depolarization in HL-60 cells, as measured by incorporation of rhodamine-123 (5 Ag/mL) using flow cytometry (Fig. 4C). Compound 3 induced a significant effect at a concentration of 12.5 Ag/mL, while at the highest concentration its effect

Discussion In recent years, considerable attention has been focused on identifying naturally occurring chemopreventive substances capable of inhibiting, retarding, or reversing the process of

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Fig. 7. Colorimetric assay of caspase-3 activation in HL-60 after treatment with (+)-2,3,9-trimethoxy-pterocarpan (1), (+)-3,9-dimethoxy-pterocarpan [(+)homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4). The results are expressed as specific activity. Doxorubicin (D, 0.3 Ag/mL) was used as positive control. Experiments were performed in duplicate.

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multistage carcinogenesis. In this context, flavonoids are of special interest since they appear to exert a beneficial effect by interfering with several key mechanisms involved in the pathogenesis of cancer (Le Marchand, 2002; Matsui et al., 2005). Moreover, they generally have a relatively low toxicity, and they are particularly abundant in vegetables and fruits (Birt et al., 2001). Many flavonoids have shown growth-inhibitory effects in several human cancer cell lines (Le Marchand, 2002; Matsui et al., 2005). Cultured mammalian cells provide an important tool for determining the cytotoxicity of compounds with therapeutic activity (Pailard et al., 1999). Herein, we determined the cytotoxicity of four pterocarpans using two different methods with leukemia cells: the trypan blue exclusion test and flow cytometry, both assessing cell membrane integrity (Renzi et al., 1993; Darzynkiewicz et al., 1992). Trypan exclusion test is useful in determining cell viability. Compound 1 reduced the number of viable cells without increasing the number of non-viable cells. This compound probably blocks cell division or induces cell death without membrane damage. In fact, cell membrane integrity, assessed by flow cytometry, was not lost after treatment with compound 1. Compound 2 also reduced viable cell number without increasing the number of dead cells at 12.5 and 25 Ag/mL. At both concentrations tested, compounds 3 and 4 reduced the number of viable cells and increased non-viable cell number. Membrane damage was also seen by flow cytometry after treatment with these compounds, showing that cells underwent necrosis. Such antiproliferative effects were further investigated to determine the mechanism of cytotoxic action exhibited by these pterocarpans. DNA synthesis was affected after treatment with compound 1 at all concentrations, resulting in a lower number of cell divisions that corroborated with trypan blue exclusion. BrdU incorporation was inhibited in a dosedependent manner in cells treated with compounds 2, 3 and 4. This effect was observed at the lowest concentration tested for these compounds; however, compound 4 was much more active. At 25 Ag/mL, compound 4 caused cell destruction making quantification impossible, suggesting that treated cells underwent necrosis. The treatment of HL-60 cells with the compounds studied induced morphological alterations typical of the apoptotic process, including reduction in cell volume, vacuolization, chromatin condensation and DNA fragmentation (Kerr et al., 1972; Hu and Kavanagh, 2003). In fact, Birt et al. (2001) have pointed out that the observed antiproliferative activity of isoflavonoids suggests that these compounds may inhibit the cell cycle or induce apoptosis. Induction of apoptosis in tumor cells is of generous benefit for cancer chemotherapy (Los et al., 2003). Apoptosis is programmed cell death that involves genetically controlled morphological and biochemical events, including phosphatidylserine externalization, cytochrome c leakage from the mitochondria, caspase activation, reduction of cell and nuclear volume, chromatin condensation, and internucleosomal DNA fragmentation (Schultz and Harrington, 2003). The develop-

ment of cytofluorimetric approaches to monitor these multiple cell alterations permits a precise and reliable quantification of apoptosis (Lecoeur et al., 1997). Apoptotic cells exhibit some morphological modifications that are readily detected by flow cytometry according to their light scatter properties (FSC/SCC) (Lecoeur et al., 1997; Petit et al., 1995). Cell shrinkage and increased granularity can be analyzed following the FSC and SCC criteria, respectively. Thus, the morphological alterations induced by the pterocarpans tested are compatible with the presence of an increasing number of apoptotic cells. The pterocarpan treatment of HL60 leukemic cells induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, as expected for apoptotic cells. Moreover, histograms of the DNA fluorescence indicated that compounds 1 and 2 caused cell cycle arrest at G2/M. The leakage of cytochrome c is also a common feature of apoptosis triggered by different stimuli. This leakage is closely associated with mitochondrial depolarization and decrease in ATP synthesis (Pedersen, 1999). Mitochondrial depolarization was assessed by flow cytometry using the rhodamine 123 dye, showing that this parameter was also altered by treatment with pterocarpans, which also indicated apoptosis induction. These findings from flow cytometry measurements were corroborated by the activation of caspase-3 observed in pterocarpan-treated HL60 cells. This enzyme is a main executor of apoptosis, playing a central role in its biological processing (Earnshaw et al., 1999). In conclusion, the present data point to the importance of pterocarpans as an emerging potential class of anticancer chemicals, exhibiting an antiproliferative effect on HL-60 by inhibiting DNA synthesis and triggering apoptosis. It is worthwhile to mention that the methoxy group on C2 position is an important pharmacophore for pterocarpans, where compound 1 is the most promising of the compounds studied. Moreover, the increasing number of hydroxyl groups seems to increase the nonspecific toxicity of pterocarpans, since compounds with this chemical group cause cell death by necrosis and compounds with methoxy groups cause cell cycle arrest followed by apoptosis. Acknowledgements We wish to thank CNPq, CAPES, FUNCAP, FINEP, BNB/ FUNDECI, FAPESP and PRONEX for the financial support in the form of grants and fellowship awards. We are grateful to Silvana Franc¸a dos Santos and Maria de Fa´tima Texeira for technical assistance. We are also indebted to Dr. Albert Leyva for reading the manuscript. References Birt, D.F., Hendrich, S., Wang, W., 2001. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacology and Therapeutics 90, 157 – 177. Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M.A., Lassota, P., Traganos, F., 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795 – 808.

G.C.G. Milita˜o et al. / Life Sciences 78 (2006) 2409 – 2417 Di Carlo, G., Mascolo, N., Izzo, A.A., Capasso, F., 1999. Flavonoids: old and new aspects of a class of natural therapeutic drugs. Life Sciences 65, 337 – 353. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and function during apoptosis. Annual Review of Biochemistry 6, 383 – 424. Falca˜o, M.J.C., Pouliquem, Y.B.M., Lima, M.A.S., Gramosa, N.V., CostaLotufo, L.V., Milita˜o, G.C.G., Pessoa, C., Moraes, M.O., Silveira, E.R., 2005. Cytotoxic flavonoids from Platymiscium floribundum. Journal of Natural Products 68, 423 – 426. Ferry, D.R., Smith, A., Malkhandi, J., Fyfe, D.W., deTakats, P.G., Anderson, D., Baker, J., Kerr, D.J., 1996. Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition. Clinical Cancer Research 2, 659 – 668. Harbone, J.B., Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry 55, 481 – 504. Hu, W., Kavanagh, J.J., 2003. Anticancer therapy targeting the apoptotic pathway. The Lancet Oncology 4, 721 – 729. Ingham, L.J., 1976. Fungal modification of pterocarpan phytoalexins from Melilotus alba and Trifolium pratense. Phytochemistry 15, 1489 – 1495. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with a wide-ranging implications in tissue kinetics. British Journal of Cancer 26, 239 – 257. Le Marchand, L., 2002. Cancer preventive effects of flavonoids—\a review. Biomedicine and Pharmacotherapy 56, 296 – 301. Lecoeur, H., Ledru, E., Pre´vost, M.C., Gougeon, M.L., 1997. Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods. Journal of Immunological Methods 209, 111 – 123. Letchier, M.R., Shirley, M.I., 1976. Phenolic compounds from the heartwood of Dalbergia nitidula. Phytochemistry 15, 354 – 355. Los, M., Burek, C.J., Stroh, C., Benedyk, K., Hug, H., Mackiewicz, A., 2003. Anticancer drugs of tomorrow: apoptotic pathways as target for drug design. Drug Discovery Today 8, 67 – 77. Matsui, J., Kiyokawa, N., Takenouchi, H., Taguchi, T., Suzuki, K., Shiozawa, N., Saito, M., Tang, W.-R., Katagiri, Y.U., Okita, H., Fugimito, J., 2005. Dietary bioflavonoids induce apoptosis in human leukemia cells. Leukemia Research 29, 573 – 581.

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McMurry, T.B.H., Martin, E., Donnely, D.M.X., Thompson, J.C., 1972. 3hydroxy-9-methoxy and 3-methoxy-9-hydroxy-pterocarpans. Phytochemistry 11, 3283 – 3286. Middleton, E., Kandaswami, C., Theoharides, T.C., 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacological Reviews 52, 673 – 751. Milita˜o G.C.G., Jimenez P.C., Wilke D.V., Pessoa C., Falca˜o M.J.C., Lima M.A.S., Siveira E.R., Moraes M.O., Costa-Lotufo L.V., 2005. Antimitotic properties of pterocarpans isolated from Platymiscium floribundum on sea urchin eggs. Planta Medica 71, 683-685. Mohammad, R.M., Al-katib, A., Aboukammel, A., Doerge, D.R., Sarkar, F., Kucuk, O., 2003. Genistein sensitizes diffuse large cell lymphoma to CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy. Molecular Cancer Therapeutics 2, 1361 – 1368. Nicolletti, I., Magliorati, G., Pagliacci, M.C., Grignani, F., Riccardi, C., 1997. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. Journal of Immunological Methods 139, 217 – 279. Pailard, F., Finot, F., Mouche, I., Prenez, A., Vericat, J.A., 1999. Use of primary cultures of rat hepatocytes to predict toxicity in the early development of new entities. Toxicology In Vitro 13, 693 – 700. Pedersen, P.L., 1999. Mitochondrial events in the life and death of animal cells: a brief overview. Journal of Bioenergetics Biomembranes 31, 291 – 304. Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., Gougeon, M.L., 1995. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. The Journal of Cell Biology 130, 157 – 167. Pueppke, S.G., VanEtten, H.D., 1975. Identification of three new pterocarpans (6a,11a-dihydro-6H-benzo-furo[3,2-c][1]benzopyrans) from Pisum sativum infected with Fusarium solani f. sp. pisi. The Journal of Chemical Society Perkin 1, 946 – 948. Renzi, D., Valtolina, M., Foster, R., 1993. The evaluation of a multi-endpoint cytotoxicity assay system. Alternatives to Laboratory Animals 21, 89 – 96. Schultz, D.R., Harrington, W.J., 2003. Apoptosis: programmed cell death at molecular level. Semininars in Arthritis and Rheumatism 32, 345 – 369. Zhai, S., Senderowicz, A., Sausville, E., Figg, W., 2002. Flavopiridol, a novel cyclin-dependent kinase inhibitior, in clinical development. The Annals of Pharmacotherapy 36, 905 – 911.