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Medicarpin and millepurpan, two flavonoids isolated from Medicago sativa, induce apoptosis and overcome multidrug resistance in leukemia P388 cells.

ABSTRACT

Background: High consumption of flavonoids has been associated with a decrease risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in traditional medicine and is currently used as a dietary supplement because of their high nutrient content. We previously reported the cytotoxic activity of alfalfa leaf extracts against several sensitive and multidrug resistant tumor cell lines.

Hypothesis/purpose: We aimed to determine whether medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects against drug-sensitive (P388) and multidrug resistant P388 leukemia cells (P388/DOX).

Study design/methods: Cells were incubated with medicarpin or millepurpan for the appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by flow cytometry technics. Caspases 3 and 9 activities were measured using Promega caspACE assay kits. Proteins and genes expression were visualized respectively by western-blot using specific antibodies and RT-PCR assay.

Results: P-glycoprotein-expressing P388/DOX cells did not show resistance to medicarpin ([IC.sub.50] [approximately equal to] 90 [micro]M for P388 and P388/DOX cells) and millepurpan ([IC.sub.50] = 54 [micro]M and 69 [micro]M for P388 and P388/DOX cells, respectively). Treatment with medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug resistant P388 cells. These effects were mediated through the mitochondrial pathway by modifying the balance pro/anti-apoptotic proteins. While 3 [micro]M doxorubicin alone could not induce cell death in P388/DOX cells, concomitant treatment with doxorubicin and subtoxic concentration of medicarpin or millepurpan restored the pro-apoptotic cascade. Each compound increased sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX cells ([IC.sub.50] = 210 nM to 23 and 25 nM with medicarpin and millepurpan, respectively). This improved sensitivity was mediated by an increased uptake of doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by exposure to medicarpin and millepurpan.

Conclusion: These data indicate that medicarpin and millepurpan possess pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells.

Keywords:

Multidrug resistance

Apoptosis

Medicarpin

Millepurpan

Alfalfa

Flavonoids

Introduction

Resistance to chemotherapy is a major cause of failure in cancer treatment, one of the main mechanisms being the overexpression of drug efflux pumps such as the 170-kDa P-glycoprotein (P-gp). P-glycoprotein is a member of the highly conserved superfamily of ATP-binding cassette (ABC) transporters proteins. It acts as an ATP-dependent drug efflux pump that reduces intracellular accumulation of antineoplastic agents and thereby hampers their effectiveness in clinic (Bates et al. 2001; Borst and Elferink 2002; Gottesman et al. 2002).

A multitude of approaches to attempt reversal of multidrug resistance have been investigated but efforts to reverse drug resistance in tumor cells gave little results due to dose-limiting toxicity in clinical trials (Volm 1998). Therefore, there is a need to develop new compounds capable of inducing apoptosis despite the existence of mechanisms which limit drug accumulation in tumor cells. Apoptosis is a regulated program of cell death that occurs under a variety of physiological and pathological conditions. It is characterized by the activation of a complex intracellular pathway leading to a cascade of biochemical and morphological changes (Hengartner 2000). Induction of apoptosis depends on the balance between pro-apoptotic factors such as Bax or Bak, and anti-apoptotic factors such as Bcl-2 or Bcl-[X.sub.L] (Chao and Korsmeyer 1998; Gross et al. 1999).

In the last twenty years, researches focused on naturally-occurring compounds, especially flavonoids. A lot of evidences have been accumulated, showing that the beneficial effects of plant extracts in cancer chemoprevention may be in part attributed to the presence of these polyphenolic compounds (Khan et al. 2008; Ramos 2008). Flavonoids were shown to possess a wide variety of biological effects, including apoptosis induction and multidrug resistance reversal in tumor cells (Limtrakul et al. 2005; Qian et al. 2005; Ramos 2007).

Alfalfa (Medicago sativa) is a plant from the Fabaceae family whose culture is mainly intended to cattle feeding. However alfalfa leaves are also used as dietary supplements because of their high protein and vitamin contents, and have been widely used for 1500 years to cure various ailments. It displays numerous pharmacological properties, recently reviewed by Bora and Sharma (Bora and Sharma 2011).

In a previous study, we described the antiproliferative and apoptosis-inducing effects of alfalfa leaf extracts in several tumor cells lines and the isolation of flavonoids, including medicarpin (MED) and millepurpan (MIL), with potential antitumor activities (Gatouillat et al. 2014).

In this study, we report the in vitro pro-apoptotic effects of medicarpin and millepurpan, which were mediated through the mitochondrial pathway by modifying the balance between pro- and anti-apoptotic proteins. Then, we investigated whether they could sensitize P388 cells overexpressing P-glycoprotein to doxorubicin and vinblastine treatment and, thereby, overcome multidrug resistance.

Materials and methods

Antibodies and reagents

Cell culture reagents and propidium iodide were purchased from InVitrogen (Cergy-Pontoise, France). Doxorubicin (DOX), vinblastine (VBL), verapamil (VPL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (Saint Quentin Fallavier, France). Anti-[beta]-actin, PARP-1, caspase-3 antibodies were from Santa Cruz Biotechnology. Anti-Bcl-2, Bcl-[X.sub.L], Bax antibodies were from Beckman Coulter. Anti-P-gp antibody was purchased from Proteogenix. Medicarpin and millepurpan were extracted and purified from Medicago sativa leaf extracts as previously described (Gatouillat et al. 2014). Briefly, the dried plant was supplied by Prolivim (Reims, FRANCE). After several steps of extraction, fractions eluted with cyclohexane-CH[Cl.sub.3] (9; 1) were purified by column chromatography (CC) over RP-18, eluted with MeOH-[H.sub.2]0 to give millepurpan. Fractions eluted with CHC13 were purified by prep. TLC in C[H.sub.2][Cl.sub.2]-Methanol (98:2) to give medicarpin.

Cell culture

The murine P388 leukemia cell line was supplied by Dr. G. Atassi (Servier Lab, France) and maintained in our laboratory. A doxorubicin-resistant subline (P388/DOX) was established by culturing them with increasing concentrations of the drug. Cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 pig/ml streptomycin at 37 [degrees]C in a humidified atmosphere of 5% C[O.sub.2]. The resistant sublines were cultured in the presence of 1 [micro]M doxorubicin. Before experiments, they were cultured in a drug-free medium for at least 7 days.

Cell viability assay

Cell viability was assessed by the MTT assay. Cells growing in suspension were seeded in a 96-well plate at a density of 5 x [10.sup.3] cells per well and incubated with increasing concentrations of the appropriate compound for 72 h. 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 [micro]l 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 [micro]l DMSO. Absorbance was measured at 540 nm using a microplate reader (Multiskan Ascent, Labsystems). Triplicate experiments were conducted in each test. Percent viability was calculated as (Absorbance of treated sample/Absorbance of non-treated sample) x 100.

RT-PCR assay

Total RNA was extracted from cultured cells using the Qiagen RNeasy Kit procedure (Qiagen; Courtaboeuf, France). One microgram of mRNA was used as a template for each RT-PCR. The primer sets were 5'-TGCTTATGGATCCCAGAGTGAC-3/ and 5'-TTGGTGAGGATCTCTCCGGCT-3' for mdrl; 5'-GAAAGAT GGTGAACTATGCC-3' and S'-TTACCAAAAGTGGCCCACTA-S' for mdr3; 5,-GAAAGATGGTGAACTATGCC-3, and 5'-TTACCAAAAGTGGCCCACTA3' for 18S rRNA.

Western blotting and densitometry

Cells (3 x [10.sup.6]) 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 mixed. After centrifugation (15,000 g, 20 min, 4 [degrees]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 electrotransferred on nitrocellulose membranes. Immunodetection was performed using the Western Breeze chemiluminescence detection system (InVitrogen) according to the manufacturer's instructions with the appropriate monoclonal antibodies. Densitometric measurement of immunoblots was performed using the ImageJ software. Each specific protein expression was normalized compared to [beta]-actin expression.

DNA fragmentation analysis

After treatment, 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 X-100). The lysate was successively treated with RNase A (100 [micro]g/ml) and proteinase K (1 mg/ml) for 1 h at 37 [degrees]C. After centrifugation (15,000 g, 15 min, 4 [degrees]C), DNA was precipitated overnight with absolute ethanol and 3 M sodium acetate at -20 [degrees]C and electrophoresed on 1.2% agarose gel containing ethidium bromide. DNA fragmentation was observed under a UV transilluminator.

DNA flow cytometric analysis

After 48 h incubation with each compound, 5 x [10.sup.5] cells were collected and washed twice in PBS. Then, cells were suspended and fixed in ice-cold 70% ethanol overnight at 4[degrees]C. After washing, cells were suspended again in PBS containing 100 [micro]g/ml RNase A and 50 [micro]g/ml propidium iodide (PI) for 30 min in the dark and analyzed in a FACS Calibur flow cytometer (Becton Dickinson). The percentage of cell in sub-G1 phase was analyzed using standard ModiFit and CellQuest softwares.

Caspases activities assay

P388 and P388/DOX cells were treated with 90 [micro]M medicarpin or 70 [micro]M millepurpan for 24 h. Caspase-3 and -9 activities were determined using colorimetric Promega caspACE assay kits according to the manufacturer's instructions. To explore whether only mitochondrial pathway is involved in this apoptosis, a neutralizing anti-FasmAb (clone ZB4; MBL International) was added to target cells, at 0.5 [micro]g/ml concentration. Cells were collected and washed twice in PBS. Then, cells were suspended in lysis buffer at 4[degrees]C for 30 min. The cell lysates were centrifuged at 10000 g at 4[degrees]C for 10 min, and the supernatants were normalized according to their protein content using Bradford method. In 96-well plates, the 50 ml sample was mixed gently with 50 ml of reaction buffer. With the addition of 5 ml of caspase3 colorimetric substrate (DEVD-pNA) or caspase-9 colorimetric substrate (LEHD-pNA), the mixture was incubated for 4 h at 37[degrees]C in a CO2 incubator. The lysis buffer with the reaction buffer served as a blank. The plate was then read with a microplate reader at 405 nm. Activities of caspase-3 and caspase-9 were expressed as the ratio of treated cells to control cells.

Measurement of doxorubicin uptake

For intracellular accumulation of doxorubicin, cells (1 x [10.sup.5]) were grown for 1 h in culture medium containing 10% of each compound. Doxorubicin (1 [micro]M) was added to the cells, gently mixed and incubated at 37[degrees]C. Cells were then centrifuged, suspended in PBS and fluorescence emitted by doxorubicin was analyzed using a FACS Aria flow cytometer (Becton-Dickinson) with excitation and emission wavelengths of 470 and 585 nm.

Statistical analysis

Results are expressed as mean [+ or -] standard deviation (SD) from three independent experiments. Difference in means between two treatment groups was compared by Student's t-test. Significance was considered when p < 0.05.

Results

Medicarpin and millepurpan inhibit the in vitro growth of sensitive and multidrug-resistant P388 cells

A cell viability assay was performed to assess the antiproliferative effect of the two flavonoids medicarpin and millepurpan (Fig. 1A and B) obtained from Medicago sativa leaves on leukemia P388 cells and their resistant counterparts (P388/DOX). These cells express P-gp (Fig. 2A), which was mainly due to the overexpression of mdr3 gene, whereas mdrl gene expression was not up-regulated in P388/DOX cells compared to the sensitive cells (Fig. 2B).

After 72 h-incubation with each compound, cell viability was measured. Medicarpin reduced cell viability in a concentration-dependent manner, giving the same pattern with the two cell lines (Fig. 2C). [IC.sub.50] were respectively 88 [micro]M and 90 [micro]M with P388 and P388/DOX cells. Treatment of cells with millepurpan also showed a dose-dependent cytotoxicity. [IC.sub.50] were respectively 54 [micro]M and 69 [micro]M with P388 and P388/DOX cells (Fig. 2C).

The resistance index was close to 1, which demonstrates that the two compounds have a similar efficiency against P388 cells and their multidrug-resistant counterpart (Fig. 2D). In comparison, resistance index for doxorubicin was approximately 200. Therefore, 90 [micro]M medicarpin and 70 [micro]M millepurpan were chosen for further investigation.

Medicarpin and millepurpan promote a mitochondrial-mediated, caspase-dependent-apoptosis in p388 and P388/DOX cells

To determine the mechanisms responsible for growth inhibition, P388 and P388/DOX cells were incubated with each compound and the cells undergoing apoptosis were identified by DNA-PI flow cytometry. Hypodiploid cells corresponding to the apoptotic cells were significantly increased after treatment of the two cell lines for 48 h with 90 [micro]M medicarpin and 70 [micro]M millepurpan (Fig. 3A). The sub-G1 population shifted from 3.4% for untreated cells to 32.4% and 37.6% with medicarpin and millepurpan, respectively. In P388/DOX cells, the sub-G1 fraction increased from 4.2% to 19.1% and 32.7% with medicarpin and millepurpan, respectively. In addition, a typical DNA ladder fragmentation pattern was observed in both P388 and P388/DOX cells incubated for 48 h with the two flavonoids (Fig. 3B), demonstrating that medicarpin and millepurpan cause apoptosis in both sensitive and chemoresistant cells.

To further confirm the ability of the two flavonoids to trigger apoptosis, detection of PARP cleavage, which is a hallmark of apoptosis, was performed by western blotting in sensitive and resistant P388 cells. As shown in Fig. 4, expression of the 85-kDa fragment corresponding to cleaved PARP increased after 24 h and 48 h of incubation with medicarpin and millepurpan. Since PARP is a substrate of executioner caspase-3, expression of procaspase-3, which is cleaved in an active form, was observed. After treatment with medicarpin and millepurpan, expression of the 33-kDa fragment corresponding to uncleaved procaspase-3 decreased in a time-dependent manner (Fig. 4), indicating that caspase-3 was activated upon exposure of cells with each compound.

Flavonoid-mediated apoptosis has frequently been associated with a modulation of mitochondrial apoptotic-related proteins. In order to know whether medicarpin and millepurpan modulate expression of Bcl-2 family members Bax, Bcl-2 and Bcl-[X.sub.L], their expression levels were determined by western blotting (Fig. 4). In P388 as well as P388/DOX cells, Bcl-XL expression, but not Bcl-2 decreased after treatment with medicarpin in a time-dependent manner, whereas medicarpin up-regulated Bax expression. Treatment of P388 cells with millepurpan resulted in a substantial decrease in Bcl-2 and Bd-[X.sub.L] expression levels, but did not alter Bax expression after 48 h of incubation. In P388/DOX cells, Bax expression remained unchanged by millepurpan, whereas Bd-[X.sub.L] was dramatically reduced after 48 h. In contrast, a slight decrease in Bcl-2 was observed after 48 h with millepurpan treatment.

In order to confirm that only the mitochondrial pathway is involved in medicarpin or millepurpan-induced apoptosis, cells were treated with the flavonoids in the presence of FAS/APO-1-induced apoptosis inhibitor (ZB4) and Caspase-3 and 9 activities were analyzed. As show in Fig 5, Caspases-3 and 9 activities were increased in the presence of both flavonoids and were not affected by ZB4 antibody. These data provide an evidence for the involvement of mitochondrial pathway in medicarpin and millepurpan-induced apoptosis.

Medicarpin and millepurpan potentiate doxorubicin-mediated apoptosis in P-gp-expressing P388 cells

To examine the effect of medicarpin and millepurpan on doxorubicin-mediated cytotoxicity in P388 overexpressing P-gp, P388/DOX cells were incubated with doxorubicin and sub-toxic concentrations of each flavonoid, and apoptosis was evidenced by procaspase-3 and PARP cleavages and DNA laddering. While treatment of sensitive P388 cells with 30 nM doxorubicin led to caspase3 activation, PARP cleavage and DNA fragmentation, 3 [micro]M doxorubicin could not trigger apoptosis in P388/DOX cells (Fig. 6A-B). Concomitant treatment with 3 [micro]M doxorubicin and 60 [micro]M medicarpin or 40 [micro]M millepurpan restored the apoptotic cascade in p388/DOX cells. At these concentrations, medicarpin and millepurpan alone did not significantly affect cell viability (Fig. 2C).

The growth inhibition of cells was investigated in response to doxorubicin and vinblastine (VBL) with or without medicarpin and millepurpan. Table 1 shows that the two natural compounds increased sensitivity of P388/DOX to doxorubicin and vinblastine. [IC.sub.50] for vinblastine were 9-fold and 8.4-fold higher when P388/DOX cells were treated with medicarpin and millepurpan, respectively, compared to vinblastine alone. For doxorubicin, [IC.sub.50] were 2.1-fold and 2.6-fold higher in the presence of medicarpin and millepurpan, respectively. Verapamil (VPL), known as a reference modulator of multidrug resistance, enhanced doxorubicin cytotoxicity 12.3-fold compared to treatment with doxorubicin alone. In P388 cells, 60 [micro]M medicarpin did not influence doxorubicin cytotoxicity (31 [+ or -] 7.7 nM versus 28 [+ or -] 2.3 nM for doxorubicin alone). Treatment with 40 [micro]M millepurpan slightly increased doxorubicin cytotoxicity (20 [+ or -] 2.1 nM), which may be due to the intrinsic cytotoxicity of millepurpan. This is consistent with the results shown in Fig. 2C. These results demonstrate that medicarpin and millepurpan may potentiate doxorubicin cytotoxicity in P388/DOX cells by overcoming Pgp-mediated multidrug resistance.

Medicarpin and millepurpan increase doxorubicin uptake in P388/DOX cells without competing with doxorubicin and down-regulating P-gp expression

In order to know whether medicarpin and millepurpan could modulate P-gp activity, the intracellular accumulation of doxorubicin was measured by flow cytometry analysis, using the intrinsic fluorescence of doxorubicin. Treatment of P388/DOX cells with either medicarpin or millepurpan at sub-toxic concentrations led to a significant increase in doxorubicin uptake, but this effect remained inferior to verapamil-induced intracellular doxorubicin accumulation (Fig. 7A). Concomitant treatment with verapamil and medicarpin or millepurpan did not affect cell viability in comparison with treatment with the two flavonoids alone (Fig. 7B), suggesting that medicarpin and millepurpan may not be transported by P-gp. This is consistent with our findings showing that the resistance index for medicarpin and millepurpan in P388/DOX cells was close to 1 (Fig. 2D).

In addition, incubation of cells each compound did not alter P-gp expression until 72 h of treatment (Fig. 7C). This demonstrates that the effects of medicarpin and millepurpan could be only due to a modulation of P-gp activity.

Discussion

Flavonoids represent a group of polyphenolic compounds found in fruits, vegetables and beverage such as tea, where they are synthesized in response to bacterial and fungal infections and UV radiations. These bioactive molecules possess anticarcinogenic effects and are considered as potential chemopreventive candidates for cancer treatment (Yang et al. 2001). Their beneficial activities might be attributed to a combination of their cytoprotective effects on normal cells and their cytotoxic effects on tumor cells (Ramos 2007). In this study, we report the effects of two flavonoids from the leaves of alfalfa (Medicago sativa). Consumption of alfalfa has been shown to provide a beneficial impact (Bora and Sharma 2011). Alfalfa leaf extracts have been approved by the European Food Safety Authority as a dietary supplement because of their high protein and vitamin contents (Bresson et al. 2009).

In a previous study, we reported the isolation of flavonoids from alfalfa leaf extracts (Gatouillat et al. 2014). Among them, the two isoflavonoids medicarpin and millepurpan were identified as potential antiproliferative agents on several tumor cell lines. Medicarpin, a pterocarpan, and millepurpan, an isoflavone, were previously isolated from alfalfa (Spencer et al. 1991), in which they are involved in plant defense. Little data exist regarding the biological effects of millepurpan. Ito et al. (Ito et al. 2000) showed that millepurpan inhibited Epstein-Barr virus-induced tumor promotion in Raji cells. Medicarpin exhibited a broad spectrum of biological properties such as osteodastogenesis inhibition in ovariectomized mice (Tyagi et al. 2010) and osteoblast differentiation in rats (Bhargavan et al. 2012), due to its structural similarity to estradiol. It also displayed cytotoxic effects against tumor cells. [IC.sub.50] of medicarpin ranged from 11 [micro]M in colon carcinoma 26-L5 cells (Li et al. 2008) to > 100 [micro]M in KB cells (Ngamrojanavanich et al. 2007). In leukemia HL-60 cells, medicarpin [IC.sub.50] was 83 [micro]M (Militao et al. 2007), which is in agreement with our results ([IC.sub.50] = 88 [micro]M in P388 cells).

Dietary flavonoids are known to interact with a wide range of molecules involved in both mitochondrial-mediated and death receptor-mediated apoptotic pathways (Ramos 2007). In the intrinsic mitochondrial pathway, increasing Bax/Bcl-2 and Bax/Bcl-[X.sub.L] ratios result in loss of mitochondrial membrane potential and mitochondrial permeabilization. The subsequent release of cytochrome c in the cytosol induces the formation of the apoptosome and the activation of caspase-9. This caspase activates caspase-3 which in turn cleaves and inactivates PARP (Grutter 2000; Hengartner 2000). Our results clearly indicated that medicarpin and millepurpan altered the balance between pro- and anti-apoptotic proteins in favor of cell death at the mitochondrial level, leading to caspase-3 activation, PARP cleavage and DNA fragmentation. In addition, medicarpin and millepurpan also potentiated doxorubicin-mediated apoptosis in P388 cells overexpressing P-gp when subtoxic concentrations of each compound were used, whereas doxorubicin alone at 1 [micro]M had no effect on these cells. At these concentrations, the two flavonoids restored sensitivity of P388/DOX cells to doxorubicin and vinblastine cytotoxicity whereas no effect was observed in the sensitive cells. This suggests that medicarpin and millepurpan may interfere with P-gp activity.

Isoflavonoids were first described as inefficient to inhibit P-gp function (Versantvoort et al. 1993), but this conclusion has been more recently challenged. We and other studies (Castro and Altenberg 1997; Ji and He 2007; Zhang and Morris 2003) showed that isoflavonoids could affect P-gp-mediated drug efflux. In our study, medicarpin and millepurpan caused a marked increase of intracellular doxorubicin accumulation in P388/DOX cells leading to the potentiation of doxorubicin cytotoxicity, without affecting P-gp expression.

Medicarpin and millepurpan themselves had an equal inhibitory effect on the proliferation of both P388 and P388/DOX cells. This indicates that overexpression of P-gp does not confer cell resistance to each compound, and they may not be substrates for P-gp. This was confirmed by the concomitant treatment of each compound and verapamil, a substrate and modulator of P-gp, which did not influence the inhibitory effect of P388/DOX cells. Therefore, medicarpin and millepurpan may inhibit P-gp transport activity without competing with doxorubicin binding site.

The mechanisms underlying flavonoid-P-gp inhibition are uncertain and seem to differ according to the flavonoid used. It has been shown that silymarin, morin and genistein strongly inhibited P-gp labeling with its photoactive substrate 3H-azidopine (Castro and Altenberg 1997; Zhang and Morris 2003), indicating that they may directly interact with the substrate binding site. This has been described for another family of compounds. In fact, Tajima et al. have shown that nitensidine A, a guanidine alkaloid from Pterogyne nitens, was able to inhibit the extrusion of calcein by P-gp by interacting with the substrate binding site (Tajima et al. 2014). Another study has demonstrated that salvianolic acid A, a phenolic active compound extracted from Salvia miltiorrhiza is able to reverse paditaxel resistance through mechanisms involving attenuation of PI3 K/Akt pathway activation and ABC transporter up-regulation (Cai et al. 2014). It was also proposed that flavonoids overlap the ATP-binding site and the vicinal steroid binding site within the nucleotide binding domain 2 (Conseil et al. 1998), but interaction of different flavonoids with the ATP-binding region of P-gp led to opposite results. Silymarin and morin inhibited whereas biochanin A and phloretin stimulated verapamil-induced ATPase activity (Zhang and Morris 2003). Since our results demonstrate for the first time that medicarpin and millepurpan inhibit P-gp function without affecting its expression and that both molecules are not P-gp substrates, further studies are needed to determine the exact mechanisms by which the two flavonoids act.

Flavonoids suffer from very poor oral bioavailability, which makes their utility as such agents very unsubstantiated. This poor bioavailability is highly dependent on their free hydroxyl groups, making them susceptible in particular to glucuronidation and sulfation and oxidation (Otake et al. 2002; Otake and Walle 2002), which commonly, but not always, removes the biological activity of these compounds. This is case of medicarpin and millepurpan. In fact these two molecules harbor one and two hydroxyl groups respectively (Fig. 1).

Several studies have shown that the methyl capping of hydroxyl groups of flavonoids results in an increase in metabolic stability (Walle 2007; Wen and Walle 2006). Thus this methylation appears to be a simple and effective way of increasing both metabolic resistance of the flavonoids and their biological activities. This strategy could be used also for medicarpin and millepurpan to have a high oral bioavailability compared with their non-methylated forms, which could make them more likely to be useful as anti-cancer drug and chemosensitizers.

In conclusion, we show that medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, trigger apoptosis through the mitochondrial pathway and overcome multidrug resistance, leading to the potentiation of doxorubicin cytotoxicity in murine leukemia cells overexpressing P-gp. Hemi-synthesis of methylated medicarpin and millepurpan derivatives may improve their chemosensitizing effect and strengthen their role in cancer therapy, especially in multidrug resistant tumors.

ARTICLE INFO

Article history:

Received 29 June 2015

Revised 15 September 2015

Accepted 24 September 2015

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

The authors thank Miss Rawan Zeitoun for her technical assistance in the purification of compounds and the Prolivim Company for providing us crude alfalfa leaf extracts. This work was supported by the University of Reims and the French National Center for Scientific Research.

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Gregory Gatouillat (a), Abdulmagid Alabdul Magid (b), Eric Bertin (c), Hassan El btaouri (d), Hamid Morjani (e), Catherine Lavaud (b), Claudie Madoulet (a),*

(a) Laboratoire de Biochimie et Biologie Moleculaire, Faculte de Pharmacie, URCA, Reims, France

(b) Laboratoire de Pharmacognosie, Faculte de Pharmacie, SFR Cap Sante, ICMR-CNRS UMR 7312, Reims, France

(c) Service d'endocrinologie, de diabetologie et de nutrition, CHU Robert-Debre, Reims, France

(d) MEDyC UMR CNRS/URCA no. 7369, Faculte des Sciences, SFR Cap Sante, URCA, Reims, France

(e) MEDyC UMR CNRS/URCA no. 7369, Faculte de Pharmacie, SFR Cap Sante, URCA, Reims, France

Abbreviations: DOX, doxorubicin; MED, medicarpin; MIL, millepurpan; P-gp, P-glycoprotein; VBL, vinblastine; VPL, verapamil.

* Corresponding author at: Laboratoire de Biochimie et Biologie Moleculaire, Faculte de Pharmacie, Universite de Reims Champagne-Ardenne, 51 me Cognacq-Jay, 51096 Reims cedex, France. Tel.: +33 3 26 91 37 32; fax: +33 3 26 91 37 30.

E-mail address: claudie.madoulet@univ-reims.fr (C. Madoulet).

http://dx.doi.org/10.1016/j.phymed.2015.09.005

Table 1
Reversal effects of MED and MIL on multidrug/
resistance against DOX and VLB in P388/DOX cells.

                                                   Relative
DOX treatment                ICso (nM) (a)         resistance (b)

P388                           31 [+ or -] 7.7
P388 + 60 [micro]M MED         28 [+ or -] 2.3
P388 + 40 [micro]M MIL         20 [+ or -] 2.1
P388/DOX                     6100 [+ or -] 90      197
P388/DOX + 60 [micro]M MED   2800 [+ or -] 23 *     90
P388/DOX + 40 [micro]M MIL   2400 [+ or -] 45 *     77
P388/DOX + 3 [micro]M VPL     510 [+ or -] 31 *     16
VBL treatment
P388                         0.51 [+ or -] 0.37
P388/DOX                      210 [+ or -] 8.7     412
P388/DOX + 60 [micro]M MED     23 [+ or -] 5.3 *    45
P388/DOX + 40 [micro]M MIL     25 [+ or -] 4.7 *    49

(a) Determined by the MTT assay. Values are means
[+ or -] SD of three independent experiments.

(b) Expressed as [IC.sub.50] (P388/DOX) / [IC.sub.50]
(P388).

* p < 0.05 compared to DOX treatment alone in P388/DOX
cells.
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Author:Gatouillat, Gregory; Magid, Abdulmagid Alabdul; Bertin, Eric; btaouri, Hassan El; Morjani, Hamid; La
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Dec 1, 2015
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