Pterocarpans phaseollin and neorautenol isolated from Erythrina addisoniae induce apoptotic cell death accompanied by inhibition of ERK phosphorylation

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Toxicology 242 (2007) 71–79

Pterocarpans phaseollin and neorautenol isolated from Erythrina addisoniae induce apoptotic cell death accompanied by inhibition of ERK phosphorylation W. W¨atjen a,∗ , A. Kulawik a , A.K. Suckow-Schnitker b,1 , Y. Chovolou a , R. Rohrig a,1 , S. Ruhl a,1 , A. Kampk¨otter a , J. Addae-Kyereme c , C.W. Wright c , C.M. Passreiter b a

b

Heinrich-Heine-Universit¨at, Institute of Toxicology, P.O. Box 101007, 40001 D¨usseldorf, Germany Heinrich-Heine-Universit¨at, Institute of Pharmaceutical Biology and Biotechnology, Universit¨atsstr. 1, 40225 D¨usseldorf, Germany c The School of Pharmacy, University of Bradford, Richmond Road, West Yorkshire BD 71 DP, UK Received 3 August 2007; received in revised form 7 September 2007; accepted 10 September 2007 Available online 15 September 2007

Abstract The genus Erythrina (Leguminosae), consisting of over 100 different species, is distributed in tropical regions. In traditional medicine, Erythrina species are used to treat cancer, but little is known about the anticancer mechanisms. From the stem bark of Erythrina addisoniae Hutch. & Dalziel, six prenylated pterocarpans were isolated and analysed for pharmacological activity: While calopocarpin, cristacarpin, orientanol c, and isoneorautenol showed only a weak or moderate toxicity in H4IIE hepatoma cells (EC50 -value > 25 ␮M), the toxicity of neorautenol and phaseollin was in the low micromolar range (EC50 -value: 1 and 1.5 ␮M, respectively). We further focused on these two substances showing that both increased caspase 3/7 activity and nuclear fragmentation as markers for apoptotic cell death. Neorautenol (10 ␮M, 2 h), but not phaseollin induced the formation of DNA strand breaks (comet assay). Both substances showed no effect on NF-␬B signalling (SEAP assay: basal activity and stimulation with TNF-␣), on the other hand both pterocarpans (10 ␮M, 2 h) decreased the activation of the ERK kinase (p44/p42), an mitogen activated protein kinase which is associated with cell proliferation. We conclude that the pterocarpans phaseollin and neorautenol may be responsible for the anticarcinogenic actions of the plant extract reported in the literature. Further analysis of these substances may lead to new pharmacons to be used in cancer therapy. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Apoptosis; Caspase; Cytotoxicity; ERK; Erythrina addisoniae; NF-␬B; Prenylated pterocarpans; TNF-␣

1. Introduction Abbreviations: ERK, extracellular regulated protein kinase; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide; PBS, phosphate buffered saline; ROS, reactive oxygen species; SEAP, secreted alkaline phosphatase; TNF-␣, tumor necrosis factor ␣. ∗ Corresponding author. Tel.: +49 211 81 13003; fax: +49 211 81 14807. E-mail address: [email protected] (W. W¨atjen). 1 This work is part of the running PhD thesis.

The genus Erythrina (Leguminosae), a group of more than 100 different species, is distributed in all tropical areas of the world (Krukoff and Barneby, 1974). These plant species are widely used in folk medicine to treat diverse diseases, e.g. different kinds of infections as well as inflammation of skin and mucous membranes (Ghosal et al., 1972; Cox, 1993; Saiduh et al., 2000). They are also used due to their analgesic as well as tranquiliz-

0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2007.09.010

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Fig. 1. Structures of isolated compounds: neorautenol (1), phaseollin (2), calopocarpin (3), isoneorautenol (4), orientanol c (5), cristacarpin (6).

ing and sedative activities (Ghosal et al., 1972; Burkill, 1995; Garin-Aguilar et al., 2000). Erythrina species are also used against cancer, e.g. stomach cancer, in folk medicine (Hartwell, 1970). In spite of this therapeutic use, little is known about the anticancer mechanisms because only few investigations on cellular level have been conducted. Erythrina addisoniae Hutch. & Dalziel occurs in tropical areas of Ghana and other West-African countries. Its stem and root bark is mainly used against dysentery, hepatitis, rheumatic disorders, and pain (Burkill, 1995). In some areas of Ghana E. addisoniae is also used against swellings and cancer (Hartwell, 1970). We previously isolated six pterocarpans (neorautenol, phaseollin, calopocarpin, isoneorautenol, orientanol c, and cristacarpin, Fig. 1) from the stem bark of Erythrina addisoniae. Here we analysed pharmacological effects (cytotoxic, pro-apoptotic effects and effects on signal transduction processes) of the isolated compounds in

H4IIE hepatoma cells to elucidate anticancer effects of the substances. 2. Materials and methods 2.1. General All chemicals were of analytical grade and were purchased from Sigma (Deisenhofen, Germany). All tissue culture reagents were purchased from PAA (Coelbe, Germany), plastic material for cell culture was obtained from Falcon (Heidelberg, Germany). Pterocarpans were isolated form the stem bark of Erythrina addisoniae. 2.2. Cell culture Metabolically active H4IIE rat hepatoma cells were grown in DMEM medium containing 4.5 g/L glucose and 2 mmol/L l-glutamine, supplemented with 10% FBS. The cell culture medium contained 100 units/mL penicillin and 100 ␮g/mL

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streptomycin and was changed twice per week. The cells were maintained in a humidified atmosphere at 37 ◦ C with 5% CO2 .

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Forty-eight hours after transfection cells were split 1:5 into 100 mm petri dishes and stably transfected cell clones (H4IIESEAP) were selected with 400 ␮g/ml hygromycin.

2.3. Determination of cytotoxicity The effect of isolated compounds on cell viability was determined using the MTT assay (Mosmann, 1983). The cells were plated on 96-multiwell plates with 10,000 cells/well. The cells were allowed to attach for 24 h and then treated with different concentrations of the pterocarpans for 24 h. After this treatment the medium was changed and the cells were incubated for 3 h under cell culture conditions with 20 ␮g/ml MTT. After this incubation the cells were lysed with 50% ethanol/49% water/1% acetic acid. The concentration of reduced MTT as a marker for cell viability was measured photometrically (560 nm). 2.4. Determination of apoptotic/necrotic cell death Caspase-3/7-activity was measured using the Apo-ONE homogeneous Caspase 3/7 assay (Promega) according to the manufacturers protocol. Briefly, 50,000 cells/well were plated on 96-multiwell plates, allowed to attach for 24 h and treated with isolated compounds for 24 h. Then, 50 ␮l of Apo-ONE Caspase-3/7-reagent was added and increase in fluorescence was measured at 37 ◦ C (excitation: 485 nm, emission: 535 nm). The increase of fluorescence was analysed for 3 h. We further investigated nuclear fragmentation (Hoechst 33342 staining) as a further feature of apoptotic cell death, as well as ethidium bromide/acridine orange staining as feature of necrotic cell death according to Michels et al. (2006). The apoptotic/necrotic index (defined as percentage of cells with fragmented nuclei/ethidium bromide staining in a randomly selected visual field) was determined by analysing three cell culture dishes for each measurement (four visual fields counted per dish) in triplicate. 2.5. Determination of DNA strand breaks For determination of DNA strand breaks the single cell gel electrophoresis (“comet”) assay was performed according to Singh et al. (1988) using alkaline conditions. H4IIE cells were seeded in a six-well (0.5 × 106 /well), incubated 24 h later with various concentrations of pterocarpans (3 h), then DNA single strand break formation was assessed (image length = head to tail distance). 2.6. Determination of NF-␬B inhibiting activity 2.6.1. Cell transfection H4IIE were stably transfected with HiFect (Amaxa) transfection reagent according to manufactures protocol. Briefly, H4IIE cells were seeded at a density of 1.5 × 105 per 35 mm petri dish and incubated overnight. Cells were transfected with 1.6 ␮g pNF-␬B-SEAP and 0.4 ␮g pTK-Hyg by using 10 ␮l HiFect transfection reagent in 1 ml serum free DME medium.

2.6.2. Reporter gene assay H4IIE-SEAP cells were seeded at a density of 2 × 105 cells per 12-well plates and incubated for 48 h. Cells were preincubated with 0.5 ␮M neorautenol and phaseollin for 1 h and then stimulated with 5 ng/ml TNF-␣ for 24 h without medium change. Activity of the reporter enzyme (SEAP) in the medium was measured using a chemiluminescence based detection method. In brief, 30 ␮l conditioned cell culture medium was mixed with 30 ␮l of 1 × dilution buffer (50 mM Tris, 150 mM NaCl, pH 7.4) and incubated for 30 min at 65 ◦ C to heat inactivate endogenous alkaline phosphatase activity. Samples were mixed with 30 ␮l assay buffer (2 M diethanolamine, 28 mM l-homoarginine) and 30 ␮l CSPD substrate. After 15 min incubation at dark, SEAP activity was measured in a plate luminometer (Victor 1420, Wallac). In each experiment it was verified that inhibition of SEAP activity was not due to cytotoxic effects (MTT assay). 2.7. Determination of ERK phosphorylation Cells were seeded in a six-well (0.5 × 106 /well) for 48 h then cells were incubated with various pterocarpans (10 ␮M) for 2 h. Cells were washed three times with PBS, detached from the plates and harvested by centrifugation (12,000 rpm, 15 min, 4 ◦ C). Cell lysis was performed in RIPA buffer including a phosphatase inhibitor cocktail (sodium vanadate, sodium fluoride, sodium molybdate, imidazole, sodium tartrate, okadaic acid) followed by three freeze/thaw cycles. Proteins were collected from supernatant and determined by Bradford method (1976). After heating, (95 ◦ C, 10 min) 8 ␮g of protein (detection of P-ERK) and 1 ␮g of protein (detection of ERK) were separated on a discontinuous 10% and 4.5% PAGE gel and then the proteins were transferred to a PVDF membrane at 200 mA for 1 h. The membrane was blocked with 3% non-fat dried milk (Sigma) in TBS buffer (10 mM Tris–HCl, 150 mM NaCl, pH 7.5) for 1 h, and then incubated with the p-ERK antibody sc7383 (1:1000 Santa Cruz Biotechnology, Santa Cruz, CA) or ERK antibody sc-94 (1:400 Santa Cruz Biotechnology, Santa Cruz, CA) at 4 ◦ C overnight. The membrane was washed 3 times with TBST buffer (TBS + 0.1% Tween20) and incubated with the corresponding HRP-conjugated secondary antibody (1:5000 and 1:2000, respectively) at room temperature for 1 h. The target protein was detected by BM Chemiluminescence Blotting Substrate (Roche, Mannheim) using X-ray film. 2.8. Statistics Data are given as mean ± S.E.M of at least three independent experiments. The significance of changes in the test responses was assessed using a one-way ANOVA followed by LSD post hoc test (Analyse-it, Leeds, UK), differences were considered to be significant at P < 0.05.

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3. Results 3.1. Toxicity of the pterocarpans in hepatoma cells The toxic potential of the isolated pterocarpans was analysed in H4IIE rat hepatoma cells using the MTT reduction assay (Fig. 2): The pterocarpans isoneorautenol, orientanol c, cristacarpin, and calopocarpin showed a moderate toxicity in H4IIE cells (EC50 -values (24 h): 25, 40, 75 and >100 ␮M, respectively). On the other hand, the toxicity of neorautenol and phaseollin exerted a prominent toxicity with EC50 -values in the low micromolar range (1 and 1.5 ␮M, respectively). Analysing the time-course of cytotoxicity we found that first effects occurred 3 h after incubation with neorautenol (1 ␮M) or phaseollin (2 ␮M) (Fig. 3A). 3.2. Formation of DNA strand breaks We used the comet assay to investigate if the toxicity of neorautenol and phaseollin in H4IIE cells was

mediated via an induction of DNA strand breaks. Incubation with 10 ␮M neorautenol for 2 h strongly increases the formation of DNA strand breaks (Fig. 3B) with an average image length of 26.8 ± 4.6 ␮m, while phaseollin showed no effects (same image length as control cells without DNA strand breaks: 16.5 ± 0.7 ␮m corresponding to the diameter of the nucleus). The genotoxic potential of neorautenol was found to be in the same order of magnitude as that of 500 ␮M H2 O2 (used as positive control) which caused an average image length of 23.4 ± 2.1 ␮m. 3.3. Modulation of NF-κB activation and ERK phosphorylation NF-␬B is a transcription factor generally associated with cellular survival due to induction of enzymes like MnSOD. We analysed if neorautenol and phaseollin interfered with the activation of NF-␬B by the cytokine TNF-␣ (H4IIE cells stably transfected with pNF-␬B-SEAP reporter plasmid). Incubation with TNF-

Fig. 2. Cytotoxicity in H4IIE cells. H4IIE cells were incubated with pterocarpans for 24 h, then MTT reduction as a marker of cell viability was measured (absorbance at 560 nm). Results are expressed as viable cells in percent of control (absorbance value of DMSO control: 0.217 ± 0.005, 0.205 ± 0.012, 0.217 ± 0.04, 0.24 ± 0.08, 0.205 ± 0.038 and 0.208 ± 0.039, respectively). Data are means ± S.E.M. (n = 3), *p < 0.05 vs. corresponding control (DMSO).

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Fig. 3. Toxic effects of phaseollin and neorautenol. (A) H4IIE cells were incubated with neorautenol and phaseollin for different time points, then MTT reduction as a marker of cell viability was measured (absorbance at 560 nm). Results are expressed as viable cells in percent of control (absorbance values of DMSO controls of 0.1, 0.5, 1 and 2 ␮M neorautenol and phaseollin: 0.488 ± 0.016, 0.458 ± 0.027, 0.465 ± 0.024, 0.479 ± 0.011 and 0.462 ± 0.015, 0.421 ± 0.023, 0.457 ± 0.027, 0.463 ± 0.023). Data are means ± S.E.M. (n = 3), *p < 0.05 vs. corresponding control (DMSO). (B) H4IIE cells were incubated with the pterocarpans for 2 h, then the formation of DNA strand breaks was detected using the comet assay. Results are expressed as average image length (␮m). Data are means ± S.D. (n = 3), *p < 0.05 vs. corresponding control (DMSO). Representative pictures are of the comet assay: (a) control, (b) 10 ␮M neorautenol, (c) 10 ␮M phaseollin. (C) H4IIE cells stably transfected with pNF-␬B-SEAP reporter plasmid were preincubated with neorautenol and phaseollin (0.5 ␮M, 1 h) and then stimulated with 5 ng/ml TNF-␣ for 24 h without medium change. Cell culture supernatants were assayed for SEAP activity, results are expressed as fold activity of the TNF-␣ stimulated cells (means ± S.E.M., n = 4). (D) Effect of neorautenol and phaseollin (10 ␮M, 2 h) on ERK phosphorylation in H4IIE cells. A representative blot is shown (n = 3 with essentially the same results).

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Fig. 4. Induction of apoptosis/necrosis in H4IIE cells. (A) H4IIE cells were incubated with neorautenol a phaseollin for 24 h, then caspase 3/7 activity was measured using homogeneous Apo-ONE assay (Promega). Results are expressed as increase in relative fluorescence units (rfu) for 3 h ± S.D. (n = 3), *p < 0.05 vs. control (DMSO). (B) Apoptosis is further confirmed by nuclear fragmentation (Hoechst staining): (a) control cells, (b) incubation with 2 ␮M neorautenol (24 h), (c) incubation with 2 ␮M phaseollin (24 h), (d–f) corresponding pictures showing nuclei (Hoechst staining), (g and h) apoptotic and necrotic indices after incubation of H4IIE cells with neorautenol and phaseollin (24 h). Data are means ± S.E.M. (n = 3), *p < 0.05 vs. corresponding control (DMSO).

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␣ (5 ng/ml, 24 h) increased SEAP activity 3.3-fold. Preincubation with 0.5 ␮M neorautenol and phaseollin for 1 h had no significant effect on NF-␬B, neither on stimulation by TNF-␣ nor were basal NF-␬B levels reduced (Fig. 3C). As positive control the NF-␬B inhibitor CAPE (caffeic acid ethylphenylester) was used: Incubation of H4IIE cells with 40 ␮M CAPE (1 h) inhibited both basal NF-␬B dependent transcriptional activity (0.23 ± 0.023 vs. 0.31 ± 0.014-fold of TNF-␣ stimulated value) as well as it completely blocks TNF-␣ induced SEAP activity to control levels (0.31 ± 0.018-fold of TNF-␣ stimulated value). Since in many tumours a constitutive activation of the mitogen activated (MAP kinase) signalling pathway occurs, we analysed the effect of neorautenol and phaseollin on MAP kinase signalling pathways in our cellular model. Incubation of H4IIE cells with neorautenol and phaseollin (10 ␮M, 2 h) strongly diminished the phosphorylation (=activation) of the ERK MAP kinase (p44/p42). This enzyme is constitutively active in our model system since the cells were maintained in 10% FBS containing of a variety of growth factors which are strong activators of this pathway (Fig. 3D). 3.4. Induction of apoptotic/necrotic cell death We further focused on these two substances and showed that both induced apoptotic cell death in H4IIE cells detected via increased caspase 3/7 activity (ApoONE assay): A significant increase in enzyme activity was found for neorautenol and phaseollin at 1 and 2 ␮M, respectively (Fig. 4A). We further investigated the induction of apoptotic cell death by analysing the amount of fragmented nuclei. Simultaneous the cells were stained with ethidium bromide to analyse the amount of necrotic cells (cells with disrupted cell membrane). Beside the amount of apoptotic cells seen after incubation with 2 ␮M of the pterocarpans, there is also an amount of cells with disrupted cell membrane so it has to be concluded that the cell death by neorautenol and phaseollin is not exclusively apoptotic, but also necrotic (Fig. 4B). 4. Discussion The main compounds found in the stem bark, roots and seeds of Erythrina species are alkaloids (Chawla and Kapoor, 1995), prenylated flavonoids (El-Masry et al., 2002), isoflavonoids (El-Masry et al., 2002), lectins (Konozy et al., 2003) and pterocarpans (Dagne et al., 1993). Since extracts of E. addisoniae are used in traditional medicine against cancer, we were interested in the constituents of the extract as well as in cytotoxic

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and proapoptotic potential of the isolated compounds. From this plant several prenylated isoflavonoids (e.g. orientanol E, senegalensin, warangalone, warangalone 4 -methyl ether and 2,3-dihydroauriculatin) were previously isolated (Bae et al., 2006). Pterocarpans are a class of natural products which show interesting pharmacological activities. Prenylation additionally enhances pharmacological activity as shown e.g. for flavonoids (W¨atjen et al., 2007): Hedge et al. (1997) reported an inhibition of phospholipase A2 by erycrystagallin isolated from Samoan Erythrina species (IC50 : low micromolar range). Atricarpan A, atricarpan B, atricarpan C, and atricarpan D inhibited butyrylcholinesterase with IC50 values between 12.5 and 65 ␮M. Atricarpan A and atricarpan D inhibit lipoxygenase and acetylcholine esterase enzymes with IC50 -values of 13.5 and 20.5 ␮M, respectively (Ahmad et al., 2006). Isoneorautenol showed antimicrobiological activity against Staphylococcus aureus (Mitscher et al., 1988), phaseollin as well as erycristagallin and erythrabyssin II showed antimicrobial activities against Staphylococcus aureus and Mycobacterium smegmatis (Telikepalli et al., 1990). To our best knowledge, no data is available in the literature concerning the toxicity of the isolated six pterocarpans in cellular systems. Here we showed that between these isolated pterocarpans great differences in cytotoxicity exists: While calopocarpin, cristacarpin, orientanol c, and isoneorautenol showed weak or moderate toxicity against H4IIE cells (EC50 -value > 25 ␮M), the toxicity of neorautenol and phaseollin was in the low micromolar range (EC50 -value: 1 and 1.5 ␮M). In the literature, only weak toxicity was reported for pterocarpans. For example, Ngamrojanavanich et al. (2007) reported that medicarpin showed no cytotoxic activity in KB and BC cell lines. In KB cells also no toxic effects were found for 3,4-dimethoxy8,9-(methylenedioxy)pterocarpan (Alvarez et al., 1998). Sakurai et al. (2006) demonstrated that rautandiol A, rautandiol B, 2-hydroxypterocarpin, and neodulin showed little or no toxic activity against MCF-7 and A549 cells. The pterocarpan maackiain was reported to be weakly cytotoxic against the KB cell line (Chaudhuri et al., 1995). A similar result was found by Blatt et al. (2002) analysing the cytotoxicity of this substance in 10 different cell lines. Medicarpin but not maackiain showed moderate cytotoxic activity in the KB cell line with an EC50 value of 2.4 ␮g/mL (Seo et al., 2001). We further analysed the mode of cell death caused by neorautenol and phaseollin: Both compounds increased caspase 3/7 activity and nuclear fragmentation indicating an apoptotic mode of cell death. In the literature, only

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few reports about proapoptotic effects of pterocarpans are available: recently, Militao et al. (2006) reported that (+)-2,3,9-trimethoxy-pterocarpan induced apoptotic cell death in HL60 cells. Maurich et al. (2006) reported that bitucarpin A induces apoptosis in LoVo and HT29 cells and erybraedin C caused a sub-G1 peak characteristic for apoptosis. Here we report that both neorautenol and phaseollin cause a disruption of cell membrane as a marker of necrotic cell death. Induction of necrosis by other pterocarpans was also recently reported in the literature: Militao et al. (2006) reported that medicarpin and vesticarpan induced necrotic cell death in HL60 cells, Cottiglia et al. (2005) reported that erybraedin C induced necrosis in leukaemia Jurkat T cells. In case of neorautenol, but not phaseollin we found an increase in DNA strand breaks in H4IIE cells. For different flavonoids the formation of DNA strand breaks is reported (W¨atjen et al., 2005), maybe due to topoisomerase poison activity or generation of reactive oxygen species. Recently, Chaudhuri et al. (1995) reported that (+)-3,4-dihydroxy-8,9-methylenedioxypterocarpan demonstrated activity in an in vitro DNA strand-scission assay, but two other related pterocarpans were found to be inactive in this assay. Maurich et al. (2006) also discussed that the induction of cell death by the pterocarpan erybraedin C may be mediated via topoisomerase II poison activity since this compound contains regio-specific hydroxyl and prenyl groups. As a further mechanism of apoptosis-induction by pterocarpans we investigated the effects of neorautenol and phaseollin on intracellular signalling pathways. We found no interference of these pterocarpans with NF-␬B, a transcription factor generally associated with cellular survival: Neither basal NF-␬B activity nor increase of NF-␬B activity after stimulation with TNF-␣ was affected by these substances (SEAP reporter gene assay). We further analysed interference of the pterocarpans with mitogen activated protein kinases (MAPK). The ERK pathway, leading to proliferation of the cells, is constitutively active in our cellular model system since cells are cultivated with 10% FCS consisting of a variety of growth factors which are strong activators of this pathway. A disruption of this pathway results in the induction of apoptosis. Both pterocarpans decreased the phosphorylation of ERK, this may be responsible for the induction of apoptosis. Ohkawara et al. (2005) reported that astrapterocarpan inhibited platelet-derived growth factor-induced cell proliferation and DNA synthesis in rat vascular smooth muscle cells. This pterocarpan inhibited PDGF-BB-induced phosphorylation of extracellular signal-regulated kinase

1/2 (ERIC1/2) mitogen-activated protein (MAP) kinase. Additionally, this inhibition was not attributed to toxicity of astrapterocarpan. In general, the inhibition of intracellular signalling pathways which are responsible for proliferation is an important mechanism for various anti-cancer pharmacons, e.g. the class of tyrosine kinase inhibitors (e.g. dasatinib), therefore the effects reported for phaseollin and neorautenol are mechanistically interesting. We conclude that the bark of Erythrina addisoniae contains several pharmacologically interesting compounds. Prenylated pterocarpans (phaseollin and neorautenol) exhibited a prominent toxicity in H4IIE cells inducing apoptotic cell death. This apoptotic cell death was accompanied by a disruption of the ERK signalling pathway. This effect may be responsible for the anticarcinogenic actions of the plant extract. Further analysis of these substances may lead to new pharmacons to be used in cancer therapy. Acknowledgements We thank the Deutsche Forschungsgemeinschaft (DFG graduate collegue 1427: “Food constituents as triggers of nuclear receptor-mediated intestinal signaling”) and the “Forschungs- und Innovationsfonds of the Heinrich-Heine University” for financial support, Mrs. E. M¨uller and Mrs. S. Ohler for excellent technical assistance. References Ahmad, V.U., Iqbal, S., Nawaz, S.A., Choudhary, M.I., Farooq, U., Ali, S.T., Ahmad, A., Bader, S., Kousar, F., Arshad, S., Tareen, R.B., 2006. Isolation of four new pterocarpans from Zygophyllum eurypterum (Syn. Z. atriplicoides) with enzyme-inhibition properties. Chem. Biodivers. 3, 996–1003. Alvarez, L., Rios, M.Y., Esquivel, C., Chavez, M.I., Delgado, G., Aguilar, M.I., Villarreal, M.L., Navarro, V., 1998. Cytotoxic isoflavans from Eysenhardtia polystachya. J. Nat. Prod. 61, 767–770. Bae, E.Y., Na, M., Njamen, D., Mbafor, J.T., Fomum, Z.T., Cui, L., Choung, D.H., Kim, B.Y., Oh, W.K., Ahn, J.S., 2006. Inhibition of protein tyrosine phosphatase 1B by prenylated isoflavonoids isolated from the stem bark of Erythrina addisoniae. Planta Medica 72, 945–948. Blatt, C.T., Chavez, D., Chai, H., Graham, J.G., Cabieses, F., Farnsworth, N.R., Cordell, G.A., Pezzuto, J.M., Kinghorn, A.D., 2002. Cytotoxic flavonoids from the stem bark of Lonchocarpus aff. fluvialis. Phytother. Res. 16, 320–325. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burkill, M., 1995. The Useful Plants of West Tropical Africa, vol. 3., second ed. Royal Botanical Gardens Kew, London, pp. 350. Chaudhuri, S.K., Huang, L., Fullas, F., Brown, D.M., Wani, M.C., Wall, M.E., 1995. Isolation and structure identification of

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