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Cytotoxicity and Apoptosis Induced by Alfalfa (Medicago sativa) Leaf Extracts in Sensitive and Multidrug-Resistant Tumor Cells a

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Grégory Gatouillat , Abdulmagid Alabdul Magid , Eric Bertin , Marie-Genevieve Okiemyd

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Akeli , Hamid Morjani , Catherine Lavaud & Claudie Madoulet

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Laboratoire de Biochimie et Biologie Moléculaire , Faculté de Pharmacie, URCA , Reims , France b

Laboratoire de Pharmacognosie, Faculté de Pharmacie, SFR Cap Santé , ICMR–CNRS UMR 7312, Reims , France c

Service d’Endocrinologie, de Diabétologie et de Nutrition, CHU Robert-Debré , Reims , France d

Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA, Reims, France and Laboratoire de Biochimie et Biologie Moléculaire, ENS, Université Marien Ngouabi , Brazzaville , Congo e

Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA, Reims, France and MEDyC Unité CNRS UMR 6237, SFR Cap Santé, URCA , Reims , France f

Laboratoire de Pharmacognosie, Faculté de Pharmacie, SFR Cap Santé, ICMR–CNRS UMR 7312 , Reims , France g

Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA , Reims , France Published online: 14 Mar 2014.

To cite this article: Grégory Gatouillat , Abdulmagid Alabdul Magid , Eric Bertin , Marie-Genevieve Okiemy-Akeli , Hamid Morjani , Catherine Lavaud & Claudie Madoulet (2014) Cytotoxicity and Apoptosis Induced by Alfalfa (Medicago sativa) Leaf Extracts in Sensitive and Multidrug-Resistant Tumor Cells, Nutrition and Cancer, 66:3, 483-491, DOI: 10.1080/01635581.2014.884228 To link to this article: http://dx.doi.org/10.1080/01635581.2014.884228

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Nutrition and Cancer, 66(3), 483–491 C 2014, Taylor & Francis Group, LLC Copyright  ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2014.884228

Cytotoxicity and Apoptosis Induced by Alfalfa (Medicago sativa) Leaf Extracts in Sensitive and Multidrug-Resistant Tumor Cells Gr´egory Gatouillat Laboratoire de Biochimie et Biologie Mol´eculaire, Facult´e de Pharmacie, URCA, Reims, France

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Abdulmagid Alabdul Magid Laboratoire de Pharmacognosie, Facult´e de Pharmacie, SFR Cap Sant´e, ICMR–CNRS UMR 7312, Reims, France

Eric Bertin Service d’Endocrinologie, de Diab´etologie et de Nutrition, CHU Robert-Debr´e, Reims, France

Marie-Genevieve Okiemy-Akeli Laboratoire de Biochimie et Biologie Mol´eculaire, Facult´e de Pharmacie, URCA, Reims, France and Laboratoire de Biochimie et Biologie Mol´eculaire, ENS, Universit´e Marien Ngouabi, Brazzaville, Congo

Hamid Morjani Laboratoire de Biochimie et Biologie Mol´eculaire, Facult´e de Pharmacie, URCA, Reims, France and MEDyC Unit´e CNRS UMR 6237, SFR Cap Sant´e, URCA, Reims, France

Catherine Lavaud Laboratoire de Pharmacognosie, Facult´e de Pharmacie, SFR Cap Sant´e, ICMR–CNRS UMR 7312, Reims, France

Claudie Madoulet Laboratoire de Biochimie et Biologie Mol´eculaire, Facult´e de Pharmacie, URCA, Reims, France

Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments. However, only a few studies have reported its anticancer effects. In this study, extracts were obtained from alfalfa leaves and their cytotoxic effects were assessed on several sensitive and multidrug-resistant tumor cells lines. Using the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart (P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf extracts was mediated through the induction of apoptosis, as evidenced by DNA fragmentation analysis. The execution of programmed cell death was achieved via the activation of caspase-3, leading to PARP cleavage. Fractionation

Submitted 27 February 2013; accepted in final form 10 January 2014. Address correspondence to Prof. Claudie Madoulet, Laboratoire de Biochimie et Biologie Mol´eculaire, Facult´e de Pharmacie, Universit´e de Reims Champagne-Ardenne, 51 Rue Cognacq-Jay, 51096, Reims Cedex, France. Phone: +33 3 26 91 37 32. Fax: +33 3 26 91 37 30. E-mail: [email protected]

of toluene extract (To-1), the most active extract obtained from crude extract, led to the identification of 3 terpene derivatives and 5 flavonoids. Among them, (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate that alfalfa leaf extract may have interesting potential in cancer chemoprevention and therapy.

INTRODUCTION Apoptosis plays a crucial role in the control of cell proliferation in physiological and pathological conditions. It is an essential event that is involved in organism development and homeostasis (1–3). Induction of apoptosis depends on the balance between proapoptotic factors such as Bax or Bak, and antiapoptotic factors such as Bcl-2 or Bcl-XL (4,5). Apoptosis can be triggered through the mitochondrial pathway or the death receptor-mediated pathway, both leading to caspase

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activation that ultimately results in nucleus condensation and DNA fragmentation (6,7). However, the deregulation of pathways controlling apoptosis can lead to abnormal cell proliferation and favor development of tumor (8). There are many evidences showing that antineoplastic drugs kill cancer cells by inducing programmed cell death (9). However, the use of chemotherapeutic agents in the treatment of cancer has been hampered by the occurrence of multidrug resistance associated with the overexpression of membrane transporters, such as P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1), which pumps the drugs out of the cells (10,11). Therefore, the search for novel apoptosis-inducing agents remains a major challenge in cancer therapy. Plant extracts have been shown to exert many pharmacological functions, including apoptosis in tumor cells (12–14). These anticancer effects have been attributed to the presence of many compounds, one of the main families including the flavonoid compounds (15–17). Alfalfa (Medicago sativa) is a plant from the Fabaceae family whose culture is mainly intended to cattle feeding. Nevertheless, alfalfa leaves have been widely used in traditional medicine for 1500 years to treat disorders related to the digestive tract or kidneys. Recently, alfalfa leaf extract (ALE) has been approved by the European Food Safety Authority as a dietary supplement because of its high protein and vitamin contents (18). Alfalfa displays a wide variety of biological effects (19), such as estrogenic and antidiabetic activities (20,21). It also lowers cholesterol and triglyceride blood levels (22,23). However, despite the isolation of many compounds with known anticancer effects from Medicago sativa (24,25), little data exists regarding ALE-mediated cytotoxicity against tumor cells. In this study, we showed that the toluene and methyl terbutyl ether (MtBE) extracts from ALE induced cytotoxicity against both sensitive and multidrug-resistant cell lines. Using the P388 cell line and its doxorubicin (DOX)-resistant counterpart, we also showed that ALE triggered caspase-dependent apoptosis. Finally, we initiated a bioguided fractionation of the toluene extract and isolated 8 compounds with potential cancer chemopreventive activities; (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, chrysoeriol, 3-hydroxy-β-ionone, loliolide, and dihydroactinidiolide. MATERIALS AND METHODS Chemicals and Materials Cell culture reagents, propidium iodide (PI) and Hoechst 33342 were purchased from InVitrogen (Cergy-Pontoise, France). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), proteinase K, RNase A, triton X-100 were from Sigma-Aldrich (Saint Quentin Fallavier, France). Antiβ-actin, anti-PARP-1, and anticaspase-3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were obtained using a UV Uvikon 941 spectrophotometer (Kon-

tron Instruments, Toulon, France). Infrared spectroscopy (IR) spectra were recorded on a Nicolet Avatar 320 FT-IR spectrometer and 1H and 13C nuclear magnetic resonance (NMR) spectra on a Bruker Avance DRX 500 spectrometer in CD3 OD (Bruker, Wissenbourg, France). 2D NMR experiments were performed using standard Bruker microprograms (XWIN-NMR version 2.6 software). For ESI-MS experiments a Micromass Q-TOF micro instrument (Micromass, Manchester, UK) was used. Column chromatography was carried out on Silica gel 60 (63–200 mesh) (Merck) or LiChroprep RP-18 (40–63 μm) (Merck, Fontenay sous Bois, France). Semipreparative HPLC was performed on a Dionex apparatus equipped with an Interchrom UP5 ODB.25M Uptisphere 5 μm (250 × 10 mm) at 25◦ C and a flow rate at 3 ml/min, an ASI-100 autosampler, a STH 585 column oven, a P580 pump, a diode array detector UVD 340S (λ = 205, 280, and 330 nm), and the Chromeleon software. TLC (Silica gel 60 F254, Merck) spots were detected with a UV lamp and by spraying with 50% H2 SO4 in H2 O, followed by heating. Extraction and Purification of the Active Compounds from ALE The dried ALE was supplied by Prolivim (Reims, France). Powdered ALE (2 kg) was macerated in 60% aqueous acetone (40:l) for 48 h and filtered. The filtrate was concentrated under reduced pressure into 10:l and partitioned successively with 10:l of toluene, MtBE, EtOAc, and n-BuOH, yielding a toluene extract (9.5 g), MtBE extract (4.9 g), EtOAc extract (10 g), n-BuOH extract (26 g), and an aqueous residue (65 g). A part of the toluene extract (8 g) was subjected to vacuum liquid chromatography over RP-18, eluted with MeOH-H2 O (6:4, 8:2, 9:1, and 10:0, each 1: l) to give successively fractions To-1 (1.7 g), To-2 (2.7 g), To-3 (1.3 g), and To-4 (3.4 g). Fraction To-1 was chromatographed over silica gel column (2.7 × 30 cm) using a gradient of cyclohexane-CHCl3 (2:8 to 0:10) and then CHCl3 -MeOH (10:0 to 5:5). Fractions eluted with cyclohexane-CHCl3 (9:1) were purified by CC over RP-18, eluted with MeOH-H2 O (4:6–10:0) to give 15 mg of millepurpan (3); fractions eluted with cyclohexane-CHCl3 (95:5) were purified by semi-preparative HPLC eluted with ACN-H2 O (25:75) to afford 4 mg of (-)-melilotocarpan E (2) and 13 mg of 3-hydroxyβ-ionone (6); fractions eluted with cyclohexane-CHCl3 (99:1) were purified by prep. TLC in CHCl3 to give dihydroactinidiolide (10 mg); fractions eluted with cyclohexane-CHCl3 (97:3) were purified by semi-preparative HPLC with a linear gradient (35 to 50% of ACN in H2 O for 20 min) to give 16 mg of loliolide; fractions eluted with CHCl3 were purified by prep. TLC in CH2 Cl2 -MeOH (98:2) to give (-)-medicarpin, (10 mg); fraction eluted with CHCl3 -MeOH (99:1) afforded by crystallization a mixture (1:1) of tricin and chrysoeriol (10 mg). A part of the MtBE extract (4.5 g) was subjected to vacuum liquid chromatography over reversed phase silica (RP-18), eluted with MeOH-H2 O (6:4, 8:2, and 10:0, each 1L) to give successively fractions MtBE-1 (1.5 g), MtBE-2 (0.8 g), and

ANTICANCER EFFECTS OF ALFALFA LEAF EXTRACTS

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MtBE-3 (0.9 g). The description and spectral data of those compounds are available in the Supplementary Material. Cell Lines and Cultures The human erythroleukemia K562 and mammary adenocarcinoma MCF-7 were supplied by the laboratory of Biochemistry in the University of Pharmacy of Reims. The mouse melanoma B16 cells were from the National Tumor Institute of Milan. The murine leukaemia cell lines L1210 and P388 were supplied by the Jean Godinot Institute, Reims, and Dr. G. Atassi (Servier Laboratories, France), respectively. For each cell line, a DOXresistant subline was established by culturing them with increasing concentrations of the drug. All the resistant cell sublines were shown to overexpress P-gp (26,27). Cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37◦ C in a humidified atmosphere of 5% CO2 . The resistant sublines were cultured in the presence of 1 μM DOX. Before experiments, they were cultured in a drug-free medium for at least 7 days. Cell Viability Assay (MTT) Cell viability was assessed by the MTT assay. Cells growing in suspension were seeded in a 96-well plate at a density of 5 × 103 cells per well and incubated with increasing concentrations of extracts or compounds for the indicated periods. Adherent cells were seeded at the same density 24 h before treatment. In every control experiment, an equal volume of DMSO which is used to dissolve extracts or compounds was added in each well. After the period of incubation, 20 μl MTT (2.5 mg/ml) were added to each well for 3 h. Then, the medium was removed and formazan crystals were dissolved in 200 μl DMSO. Absorbance was measured at 540 nm using a microplate reader (Multiskan Ascent, Labsystems, Colombes, France). Triplicate experiments were conducted in each test. The relative percent viability was calculated as (Absorbance of treated sample/Absorbance of nontreated sample) × 100. Hoechst 33342 Staining After treatment with alfalfa extracts for 24 h, cells were washed in PBS and centrifuged (1000 g, 5 min, 4◦ C). The cell pellet was suspended in 4% paraformaldehyde containing 1% Triton X-100 (Sigma). After fixation for 10 min, the cells were applied to cytospin chambers and centrifuged at 700 rpm for 2 min. After air-drying, Hoechst 33342 (0.5 μg/ml diluted in PBS) was added for 15 min in the dark. The slides were rinsed in water and mounting medium was added before visualization with a fluorescence microscope under UV excitation. DNA Fragmentation Assay Cells were washed in PBS and lysed on ice for 15 min in a lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5% Triton X100). Then, the lysate was successively treated with RNase A (100 μg/ml) and proteinase K (1 mg/ml) for 1 h at 37◦ C. After centrifugation (15000 g, 15 min, 4◦ C), DNA was precipitated overnight with ethanol and 3 M sodium acetate at −20◦ C, and

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an electrophoresis on 1.2% agarose gel containing ethidium bromide was performed. DNA fragmentation was observed under a UV transilluminator. Western Blot Analysis PARP cleavage and caspase-3 activation were analyzed by Western blot. Cells (3 × 106) were lysed on ice for 15 min in RIPA lysis buffer (50 mM Tris-HCl, 150 mM EDTA, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 0.5 mM PMSF and 0.1% protease inhibitor. After centrifugation (15,000 g, 20 min, 4◦ C), the supernatant was collected and total protein concentration was determined using the Bradford method. Equal amounts of proteins were separated on a 4–12% polyacrylamide gel and transferred on nitrocellulose membranes. Immunodetection was performed using the Western Breeze chemiluminescence detection system (InVitrogen, Cergy-Pontoise, France) according to the manufacturer’s instructions with the appropriate monoclonal antibodies. Statistics Data are given as the means ± SD. For each assay, Student’s t-test was used for statistical comparison with the untreated control cells or between sensitive and resistant cells. A limit of P ≤ 0.05 was accepted for significant differences. All data represent at least 3 independent experiments. RESULTS Cytotoxicity Effects of ALE in Sensitive and Multidrug-Resistant Tumor Cell Lines Five extracts were obtained from ALE, and the growth of several murine and human, sensitive and P-gp-expressing cell lines was examined in the presence of each extract. IC50 are shown in Table 1. Toluene extracts (TE) and MtBE extracts (ME) exhibited the highest cytotoxicity effect against all cell lines while EtOAc, butanol, and aqueous extracts were poorly cytotoxic. IC50 ranged between 17 and 105 μg/ml for the TE and between 38 and 145 μg/ml for the ME. The lowest IC50 were obtained on the P388, L1210, and K562 leukemia cell lines. Surprisingly, some differences of sensitivity were observed between the sensitive and resistant cells for a given cell line. For the K562 cell line, the IC50 of TE was higher with the sensitive compared to the resistant cells whereas an inverse result was obtained with the other cell lines. We then focused on the effect of TE) and ME extracts on the growth of P388 cell line and its DOX-resistant counterpart (P388/DOX). Cells were treated with each extract for 24, 48, and 72 h and their viability was measured by the MTT assay. Both TE and ME inhibited cell growth in a dose-dependent manner (Fig. 1.). TE also induced a time-dependent cytotoxicity in P388 cells, as IC50 was reduced from 82 μg/ml for a 24-h exposure to 48.5 μg/ml for 72 h, whereas this effect was not observed in P388/DOX. However, a time-dependent cytotoxicity of ME was observed in P338/DOX although it was less marked in the

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TABLE 1 Effect of alfalfa leaf extract on the growth of sensitive and multidrug-resistant cell lines IC50 (μg/ml)

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Cell line B16 B16/DOX MCF7 MCF7/DOX P388 P388/DOX K562 K562/DOX L1210 L1210/DOX

Toluene extract

MtBE extract

EtOAc extract

Butanol extract

Aqueous extract

70 ± 3.1 90 ± 5.9 81 ± 7.8 105 ± 9.5 48.5 ± 4.2 71 ± 5.1∗ 45 ± 2.4 17 ± 1.9∗ 29.5 ± 3.2 62 ± 4.1∗

85 ± 6.5 145 ± 9.5 108 ± 4.6 100 ± 6.7 79.5 ± 4.8 63 ± 3 38 ± 2.1 130 ± 10.3∗ 48.5 ± 6.1 71 ± 3.2∗

195 ± 13.9 171 ± 11.1 203 ± 15.6 163 ± 12.6 182 ± 21.5 >250 220 ± 13.3 287 ± 28 152 ± 9.7 235 ± 12.6

>250 ND ND 180 ± 12.3 >250 >300 >300 >350 ND ND

>400 ND ND ND >400 >400 >350 >350 ND ND

IC50 were determined from dose-response curves. Each value represents means ± SD of 3 or 4 experiments. MtBE = methyl ter-butyl ether; ND = not determined; DOX = doxorubicin. ∗ P < 0.05 significantly different between the sensitive cells and the resistant counterpart cell line.

sensitive cell line. In P388, ME IC50 were 119 to 79.5 μg/ml at 24 and 72 h, respectively. TE and ME Induce Morphological Changes and DNA Fragmentation in Mouse P388 Cells and Their Multidrug-Resistant Counterparts To determine the mechanism by which TE and ME exert their cytotoxic effects, we studied the morphological changes of nuclei using Hoechst 33342 staining. TE- and ME-mediated cytotoxicity was accompanied by nuclear shrinkage, chromatin

FIG. 1. Effect of toluene (TE) and MtBE (ME) extracts on the viability of P388 and its doxorubicin (DOX)-resistant cell lines. Cells were treated with increased concentrations of each extract for 24, 48, and 72h. Control cells were cultured in the presence of 0.2% DMSO for the same period. Relative cell viability was determined by the MTT assay and expressed as mean ± SD of 3 independent experiments. ∗ P < 0.05 compared to untreated cells.

condensation, and fragmentation (Fig. 2), which is consistent with the morphological hallmarks of an apoptotic nucleus. In contrast, untreated cells exhibited round and homogenous nuclei. These observations suggest that TE and ME may induce apoptosis in both P388 and P388/DOX cells. To further confirm the apoptosis-inducing effect of TE and ME in both sensitive and resistant P388 cells, a DNA fragmentation assay was performed. P388 and P388/DOX cells were exposed to 100 μg/ml TE and ME for 24 or 48 h, and DNA was extracted. Agarose gel electrophoresis showed that TE and

FIG. 2. Hoechst staining of toluene (TE)- and MtBE (ME)-treated or untreated cells. After exposure to each extract, cells were fixed and stained with Hoechst 33342. Observations by fluorescence microscope revealed condensed and fragmented nuclei (indicated by arrows) in cells treated with TE and ME.

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TABLE 2 Cytotoxicity induced by the 4 fractions obtained from the toluene extract

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IC50 (μg/ml)

FIG. 3. DNA fragmentation induced by toluene (TE) and MtBE (ME). After treatment with 100 μg/ml of each extract for 24 or 48 h, genomic DNA was extracted. DNA ladders were observed using agarose gel electrophoresis containing ethidium bromide. Typical results from 3 repeated experiments are shown. M = molecular weight markers (100 bp DNA ladder).

ME induced fragmentation of genomic DNA in sensitive and resistant cell lines (Fig. 3). These DNA fragments were multiples of 180–200 bp, whereas untreated cells did not show any evident DNA ladder. DNA laddering was time-dependent, as the intensity of the low molecular weight fragments was enhanced after 48 h. TE- and ME-Induced PARP Cleavage and Caspase-3 Activation To determine whether TE and ME from ALE of Medicago sativa activate the caspase-dependent pathway of programmed cell death, the activation of caspase-3 was analyzed by Western blot. Treatments of sensitive and resistant P388 cells with both extracts led to the dose- and time-dependent activation of caspase-3, as seen by the progressive disappearance of the inactive 33 kDa enzyme (Fig. 4). TE- and ME-induced activation of caspase-3 was followed by an increased level of the 86 kDafragment of PARP (Fig. 4), which suggests that the 2 extracts induce PARP cleavage in a dose- and time-dependent manner. Overall, these results demonstrate that the cytotoxic effects of TE and ME from alfalfa leaves are mediated through the induction of caspase-dependent apoptosis in P388 cells, as well as in their multidrug-resistant counterpart. Fractionation of TE and Isolation of Compounds with Cytotoxic Effect from the To-1 Subfraction A bioguided fractionation of ALE was initiated to identify the compounds responsible for their anticancer activities. We first studied the TE. Vacuum liquid chromatography was performed to yield 4 subfractions (To-1/4) from TE. Using the MTT assay, the To-1 and To-2 subfractions were shown to be the most cytotoxic against P388 and P388/DOX cells (Table 2). There-

Cell line

ALE-To-1

ALE-To-2

ALE-To-3

P388 P388/DOX K562 K562/DOX L1210 L1210/DOX

63 ± 1.4 51 ± 4.9 >100 100 ± 2.4∗ 69 ± 1.4∗ >200 75.2 ± 5 75.5 ± 4.3 >150 84 ± 6.8 66 ± 6.1 >200 66.7 ± 6.7 47.5 ± 3.1 92, 5 ± 6.9 84 ± 5.3∗ 91.5 ± 1.7∗ 176, 5 ± 9.8∗

ALE-To-4 >200 >200 ND ND >200 >200

IC50 were determined from dose-response curves. Each value represents means ± SD of 3 or 4 experiments. ND = not determined; DOX = doxorubicin; ALE = alfalfa leaf extract. ∗ P < 0.05 compared to the sensitive cell line counterpart.

fore, the isolation of metabolites from the To-1 subfraction was carried out and gave 8 compounds, 5 flavonoids, and 3 terpene derivatives (Fig. 5). The structure of each compound was identified by comparison of physical and spectral data with those reported in the literature (NMR, mass spectrometry, IR, UV absorption spectroscopy, and optical rotation [α]D ). These compounds were identified as: 3-hydroxy-β-ionone (28), loliolide, and dihydroactinidiolide (29), and among flavonoids, 2 pterocarpans (-)-medicarpin (30) and (-)-melilotocarpan E (31), the isoflavane millepurpan (31), and 2 flavones, tricin, and chrysoeriol (25) (isolated together). The cytotoxic activity of each compound was measured by the MTT assay. Although the 3 terpene derivatives did not show any cytotoxicity (IC50 > 200 μM, data not shown), all the flavonoids exhibited a dose-dependent cytotoxic effect against P388 and P388/DOX cells, with IC50 included between 20 and 100 μM (Table 3). Resistance index for each compound was close to 1 (1.02–4.7), suggesting that the flavonoids may act on P388 and P388/DOX cells to a similar extent. As expected, doxorubicin, a P-gp substrate, was >200 times more efficient in P388 than in P388/DOX cells, which overexpress the drug transporter. DISCUSSION Alfalfa (Medicago sativa) has been used to cure various diseases, and its pharmacological effects were recently reviewed (19). In this study, we show that extracts obtained from alfalfa leaves possess antitumor activities. Other few studies reported antitumor activities of alfalfa. This includes in vivo anticarcinogenic effect (32), estrogenic activity of alfalfa sprouts (33), and antineoplastic activity. The latter has been described for purified compounds from alfalfa seeds, such as biochanin A (34) or the arginine antimetabolite L-canavanine (35). Our data show that other compounds contained in alfalfa leaves, such as (-)medicarpin, (-)-millepurpan, tricin, and chrysoeriol are able to induce a cytotoxic effect in cancer cells. Besides, it should be noted that in the crude leaf extract used in our study, which are

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FIG. 4. Western blot analysis of caspase-3 activation and PARP cleavage in P388 and P388/DOX cells treated with toluene (TE) (a) and MtBE (ME) (b). Cells were treated with 25, 50, 75, or 100 μg/ml of each extract or with 75 μg/ml for different periods of incubation. Protein extracts were analyzed by Western blot using specific monoclonal antibodies. β-actin was used as a loading control. Results are representative of 3 repeated experiments.

used as a dietary supplement, the concentration of L-canavanine, considered as an “antinutrient,” is very low compared to the concentration found in alfalfa seeds. Fractionation of crude ALE gave 5 extracts whose cytotoxicity was assessed on several sensitive and resistant cell lines. Extracts obtained with low polarity solvents (toluene and MtBE) induced the highest cytotoxicity as it has previously been described (36,37), in particular with the leukaemia cell lines. When comparing their efficacy in sensitive and DOX-resistant cell lines, TE and ME were not equally active. As the IC50 were not higher with the DOX-resistant sublines in all cell lines, these differences in sensitivity to TE and ME may not be due only to the expression of P-gp since all the resistant sublines tested overexpress the transporter. In some cell lines, such as K562/DOX cells, the IC50 of TE was approximately threefold lower when compared to that observed in the parental cell line. This phenomenon, known as collateral sensitivity, confers hypersensitivity of multidrug-resistant cells expressing P-gp to other drugs

(38,39). Further investigations to identify other mechanisms of resistance in DOX-resistant cell sublines, as well as compounds contained in TE and ME are needed to clarify these discrepancies. In P388 and P388/DOX cells, TE and ME induced a timeand dose-dependent cytotoxicity. This reduced cell growth was achieved by the induction of apoptosis after treatment of cells with 100 μg/ml TE and ME. Caspase-3 was involved in TEand ME-induced programmed cell death. A time- and dosedependent cleavage of the inactive procaspase-3, suggesting the subsequent activation of the enzyme, was observed in both cell lines treated with TE and ME. PARP is a 116-kDa nuclear protein involved in the reparation of DNA damages. During apoptosis, activated caspase-3 cleaves various proteins, PARP being one of them. This generates 85- and 24-kDa fragments, leading to its inactivation (40). After treatment with TE and ME, an increased level of the 85-kDA fragment was observed in both cell lines. Taken together, these results suggest that apoptosis

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ANTICANCER EFFECTS OF ALFALFA LEAF EXTRACTS

FIG. 5.

Structure of compounds isolated from the To-1 fraction.

mediated by TE and ME may be triggered via the mitochondrial pathway leading to caspase-3 activation and PARP cleavage. To identify the compounds responsible for the cytotoxic and apoptosis-inducing effects of ALE, the TE was fractionated using vacuum liquid chromatography to give 4 fractions To1/4. Eight compounds, already known from Medicago sativa (25,31,41–44), were isolated within the To-1 fraction, which was the most bioactive fraction. Among these compounds, 5 flavonoids and 3 terpene derivatives were identified as the main

constituents of To-1. Three-hydroxy-β-ionone, which was previously isolated from Phaseolus vulgaris, is involved in the light-induced growth inhibition (45). Loliolide and dihydroactinidiolide are generated from β-carotene degradation (46,47). The former was found to be a prostaglandin inhibitor (48). In contrast to these 3 compounds, which were poorly cytotoxic, the isolated flavonoids exhibited interesting cytotoxic effects on P388 and P388/DOX cells. These five compounds, except for melilotocarpan E, have been shown to display

TABLE 3 Cytotoxic activity of flavonoids isolated from the To-1 fraction (IC50 in μM) Compound

1

2

3

4/5 a

DOX

P388 P388/DOX RIb

88.3 ± 4.8 90.5 ± 4.1 1.02

22.5 ± 4.3 47.8 ± 4.2∗ 2.1

53.7 ± 3.5 68.9 ± 4.3 1.3

6.6 ± 1.9 31.2 ± 7.2∗ 4.7

0.03 ± 0.0021 6.1 ± 0.94∗∗ 203.3

Each value represents means ± SD of 3 experiments. DOX = doxorubicin. a IC50 in μg/ml. bResistance index (RI) was expressed as IC50 (P388/DOX)/IC50 (P388). ∗ P < 0.05. ∗∗ P < 0.01 compared to sensitive P388 cells.

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anticarcinogenic properties. Medicarpin was found to be cytotoxic against a wide variety of tumor cell lines, including 26-L5 colon (49), KB carcinoma (50), or HL-60 leukemia cell lines (51). Isoflavan millepurpan showed inhibitory effects on Epstein-Barr virus early antigen activation induced by 12O-tetradecanoylphorbol-13-acetate (52). Chrysoeriol inhibited benzo[a]pyrene metabolism (53) whereas tricin inhibited the growth of MDA-MB-468 breast tumor cells (54) as well as cyclooxygenase-mediated carcinogenesis (55) and modulated resistance to doxorubicin in P-gp-expressing MCF-7 cells (56). In our study, all these compounds induced cytotoxicity with different efficiencies according to their structure. Although medicarpin and melilotocarpan E have a similar structure, the cytotoxicity induced by the 2 compounds was quite different. In a structure/activity relationship study, Li et al. (49) established that the cytotoxic activity of pterocarpans was correlated to the number of methoxy substituents, which is in agreement with our results. The IC50 of each compound was almost equal in sensitive and DOX-resistant P388 cells. This suggests that these compounds may not be transported by P-gp, in contrast to DOX, a substrate of the efflux pump overexpressed in our P388/DOX cell line (57). Thus, these naturally occurring compounds may be useful for the eradication of chemoresistant tumor cells, which remains a major concern in cancer therapy. Additional studies may be useful to determine whether a continuous exposure to the extracts and purified compounds could induce resistance in the cell lines used. Overall, our results showed the cytotoxic and apoptosisinducing effects of ALE of Medicago Sativa in both sensitive and chemoresistant tumor cells. This might be attributed in part to the presence of flavonoids. Crude ALE may have, in addition to its high nutritional value, a clinical significance in cancer chemoprevention and therapy. ACKNOWLEDGMENTS The authors thank Miss Rawan Zeitoun for her technical assistance in the purification of compounds and Prolivim company for given us crude alfalfa leaf extracts. FUNDING This work was supported by the University of Reims and the French National Center for Scientific Research. SUPPLEMENTAL DATA Supplemental data for this article can be accessed on the publisher’s website at http://dx.doi.org/10.1080/01635581.2014. 884228. REFERENCES 1. Henson PM and Hume DA: Apoptotic cell removal in development and tissue homeostasis. Trends Immunol 27, 244–250, 2006.

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