title: 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.
  
  
  OBJECTIVE
  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).
  
  
  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 (IC50 ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC50 = 54 µM and 69 µM 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 µM 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 (IC50 = 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA, Reims, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Gatouillat
authors.affiliation: Laboratoire de Pharmacognosie, Faculté de Pharmacie, SFR Cap Santé, ICMR-CNRS UMR 7312, Reims, France.
authors.forename: Abdulmagid Alabdul
authors.initials: AA
authors.lastname: Magid
authors.affiliation: Service d'endocrinologie, de diabétologie et de nutrition, CHU Robert-Debré, Reims, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Bertin
authors.affiliation: MEDyC UMR CNRS/URCA no. 7369, Faculté des Sciences, SFR Cap Santé, URCA, Reims, France.
authors.forename: Hassan
authors.initials: H
authors.lastname: El btaouri
authors.affiliation: MEDyC UMR CNRS/URCA no. 7369, Faculté de Pharmacie, SFR Cap Santé, URCA, Reims, France.
authors.forename: Hamid
authors.initials: H
authors.lastname: Morjani
authors.affiliation: Laboratoire de Pharmacognosie, Faculté de Pharmacie, SFR Cap Santé, ICMR-CNRS UMR 7312, Reims, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Lavaud
authors.affiliation: Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA, Reims, France. Electronic address: claudie.madoulet@univ-reims.fr.
authors.forename: Claudie
authors.initials: C
authors.lastname: Madoulet
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor / Q000187:drug effects; D004317:Doxorubicin; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005419:Flavonoids / Q000494:pharmacology*; D007941:Leukemia P388 / Q000378:metabolism*; D000455:Medicago sativa / Q000737:chemistry*; D051379:Mice; D015394:Molecular Structure; D018515:Plant Leaves / Q000737:chemistry; D036343:Pterocarpans / Q000494:pharmacology*; D000094162:Phytoalexins
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020169:Caspases; D004317:Doxorubicin; D005419:Flavonoids; D036343:Pterocarpans; C000604091:millepurpan; C047353:medicarpin; D000094162:Phytoalexins
keywords: Alfalfa; Apoptosis; Flavonoids; Medicarpin; Millepurpan; Multidrug resistance