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Inhibition of P-Glycoprotein Transport Function and Reversion of MDR1 Multidrug Resistance by Cnidiadin

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Inhibition of P-Glycoprotein Transport Function and Reversion of MDR1 Multidrug Resistance by Cnidiadin Cancer Chemother Pharmacol (2005) 56: 173–181 DOI 10.1007/s00280-004-0914-y ORIGINAL ARTICLE Chantal Barthomeuf Æ Je´roˆme Grassi Michel Demeule Æ Chantal Fournier Dominique Boivin Æ Richard Be´liveau Inhibition of P-glycoprotein transport function and reversion of MDR1 multidrug resistance by cnidiadin Received: 13 September 2004 / Accepted: 15 December 2004 / Published online: 12 April 2005 Ó Springer-Verlag 2005 Abstract Purpose: Overexpression of P-glycoprotein cells. The dose-dependent increase in [3H]-VBL uptake (Pgp) encoded by the MDR1 gene is one of the major (IC50 26.5 lM) induced by cnidiadin in the dose range obstacles to successful cancer chemotherapy. The goal of 1–100 lM correlated with inhibition of Pgp photola- this study was to evaluate if, among other natural cou- beling. At 10 lM cnidiadin inhibited photolabeling by marins, cnidiadin, a furanocoumarin present in tradi- 59% and sensitized both MDCK-MDR1 and KB/VCR tional Chinese medications and in a spice commonly cells to vinca alkaloids. Conclusion: Cnidiadin is a used in Greek food, inhibits Pgp transport activity and cytotoxic agent capable in vitro of competitively inhib- has the potential to reverse MDR1 multidrug resistance. iting the binding and efflux of drug by Pgp and of Methods: Using MDR1-transfected Madin-Darby ca- enhancing the cell toxicity of vinca alkaloids in two cell nine kidney (MDCK-MDR1) cells as a model of cells lines (MDCK-MDR1 and mutant human carcinoma expressing the human MDR1 phenotype, and verapamil KB/VCR) overexpressing Pgp. This suggests that diet or or CsA or both as positive control, we tested the traditional preparation containing cnidiadin may con- capacity of six natural coumarins (umbelliferone, escu- tribute to the reversal of MDR1 multidrug resistance lin, esculetin, cnidiadin, angelicin and psoralen) to in- and may affect the bioavailability of Pgp substrates or- duce the accumulation of rhodamine-123 (R-123) and ally administered. However, due to its cell toxicity, [3H]-vinblastine ([3H]-VBL) and to modulate the phot- clinical interest in cnidiadin as a chemosensitizer appears olabeling of Pgp by SDZ 212-122, a diazirin cyclosporin to be limited. A. The growth-inhibitory effect of cnidiadin and its capacity to enhance the cell toxicity of vinblastine Keywords Coumarin Æ P-glycoprotein (Pgp) Æ (VBL) or vincristine (VCR) was then evaluated by the Multidrug resistance (MDR) Æ Cancer Æ Reversal WST-1 assay in two cell lines overexpressing Pgp activity Æ Drug interaction (MDCK-MDR1 and vincristine-resistant KB/VCR). Results: Cnidiadin was the only tested coumarin capa- Abbreviations CsA: Cyclosporin A Æ dz-CsA: The ble of significantly accumulating R-123 and [3H]-VBL diazerine-cyclosporin A derivative SDZ 212-122 Æ KB/ and inhibiting Pgp photolabeling in MDCK-MDR1 VCR cells: KB cell line resistant to vincristine Æ MDR: Multidrug resistance Æ MDR1: Multidrug resistance C. Barthomeuf (&) encoded by the MDR1 gene Æ MDCK-MDR1 cells: UMR-INSERM U-484, Laboratoire de Pharmacognosie MDR1-transfected Madin-Darby canine kidney cells Æ et Biotechnologies, Faculte´de Pharmacie, MRP: Multidrug resistance-associated protein Æ Pgp: Universite´d’Auvergne, Place H. Dunant, P-glycoprotein Æ R-123: Rhodamine-123 Æ TLC: 63001 Clermont-Fd Cedex, France E-mail: [email protected] Thin-layer chromatography Æ VBL: Vinblastine Æ VCR: Tel.: +33-4-73178026 Vincristine Fax: +33-4-73150801 J. Grassi Æ M. Demeule Æ C. Fournier Æ R. Be´liveau Laboratoire d’Oncologie mole´culaire, Groupe de Recherche en Transport Membranaire, Universite´du Que´bec a` Montre´al, Introduction Succursale Centre-Ville, C.P. 8888, Montre´al, QC, Canada D. Boivin Æ R. Be´liveau P-glycoprotein (Pgp) is an inducible protein transporter Laboratoire de Me´decine Mole´culaire Hoˆ pital Ste-Justine, Centre de Cance´rologie Charles-Bruneau, belonging to the ATP-binding cassette (ABC) protein Centre de Recherche de l’Hoˆ pital Ste-Justine, family that mediates the energy-dependent efflux of Montre´al, QC, Canada many drugs, including chemotherapeutics, out of cells. 174 In cancer cells, the overexpression of Pgp encoded by the that have both demonstrated clinical activity as reversal MDR1 gene contributes to multidrug resistance (MDR) agents [4]. To provide information on structure-activity [1], and is already considered as one of the major relationships, cnidiadin was compared to angelicin obstacles to successful cancer chemotherapy [2, 3]. The (7,8-furano-1,2-benzopyrone), psoralen (6,7-furano-1,2- MDR1 phenotype is commonly observed as the escape benzopyrone), umbelliferone (7-hydroxycoumarin), from chemotherapy of leukemic and breast cancer, and esculetin (6,7-dihydroxycoumarin) and esculin (the 6-b- in kidney and colon cancer cells that overexpress Pgp at D-glucoside of esculetin). Esculin is present in large baseline, and are therefore intrinsically resistant to che- amounts in Aesculum hippocastenum seeds whose ex- motherapy [4]. The discovery two decades ago that tracts are traditionally used in the EU to treat venous verapamil, trifluoroperazide, cyclosporin A (CsA) and insufficiency. The results demonstrated that cnidiadin certain CsA analogues can contribute to the sensitiza- was the only tested coumarin that competitively inhib- tion of resistant cancer cells to chemotherapy by com- ited Pgp transport activity in a significant manner. At petitively inhibiting Pgp transport triggered research 10 lM, cnidiadin increased both the uptake of 3H-VBL into chemosensitizers (also termed MDR-reversal and the cell toxicity of VBL on MDCK-MDR1 cells and agents) [4–7]. Although disappointing results have been decreased the IC50 value of vincristine (VCR) in a hu- obtained with the first generation of reversal agents due man mutant carcinoma cell line overexpressing resis- to dose-limiting toxicity and other side effects, three re- tance to this vinca alkaloid. These results suggest that cent randomized trials have confirmed the clinical po- cnidiadin has potential as a chemosensitizer. tential of this approach. Indeed, significant benefits have been observed in patients treated with combinations of Pgp inhibitors and chemotherapy (see reference 4 for a Material and methods review). However, effective and clinically applicable reversal agents remain to be discovered. The observation Cells and materials that numerous plant-derived dietary compounds mod- ulate Pgp transport has led to interest in the possible use MDR1-transfected Madin-Darby canine kidney of natural compounds, or related chemicals, in combi- (MDCK-MDR1) cells were a gift from Amanda Yancy nation with chemotherapy [8–14]. (AstraZeneca Pharmaceuticals). They were cultured in The goal of the present study was to assess whether DMEM (Gibco) supplemented with 10% heat-inacti- cnidiadin, a furanocoumarin present in certain tradi- vated fetal bovine serum. Stock cell cultures were main- tional Chinese medications and in Tordylium apulum tained as monolayers in 75-cm2 culture flasks in (Apiaceae), a plant commonly used as a spice in Greek Glutamax Eagle’s minimum essential medium (MEM) cooking with the name ‘‘kafkalithra’’, can affect Pgp with Earle’s salts supplemented with 10% fetal calf serum, transport activity, and whether it can increase the up- 5 ml of 100 mM solution of vitamins, 5 ml 100 mM so- take and cell toxicity of vinblastine (VBL) in a stable dium pyruvate, 5 ml ·100 non-essential amino acids and clone of MDR1-transfected Madin-Darby canine kidney 2 mg gentamicin base. Cells were grown at 37°C under a (MDR1-MDCK) cells. In all experiments, cnidiadin humidified atmosphere containing 5% CO2. (2¢S,3¢R-2¢ hydroxyisopropyl-3¢ hydroxyisopropyl-dihy- Resistant KB/VCR cells resulted from long-term droangelicin, Fig. 1) was tested versus verapamil or CsA exposure of the parental sensitive KB human oral epi- dermoid carcinoma cell line to VCR. Wild-type KB (ECACC, Salisbury, UK) and mutant KB/VCR cells were grown on RPMI-1640 medium (GibcoBRL) sup- plemented with 10% fetal calf serum, 100 U/ml penicillin and 100 g/ml streptomycin. All cells were grown at 37°C under a humidified atmosphere containing 5% CO2. 3[H]-VBL (11.3 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Oakville, Ontario). CsA, SDZ 212-122, a diazirine-CsA (dz-CsA) derivative and the monoclonal antibody (mAb) directed against CsA were gifts from Novartis Pharma Canada. The mAb Ab- 2 directed against human MDR1 was purchased from NeoMarkers (Fremont, Calif.). The antimouse IgG horseradish peroxidase-linked whole antibody was pur- chased from Jackson Immunoresearch Laboratories (West Grove, Pa.). All components of the medium were from Gibco (Gaithersburg, Md.) or Gibco-BRL (Pais- ley, UK). Umbelliferone, esculetin, esculin, angelicin and psoralen were purchased from Extrasynthe` se (St Genay, France). R-123 and all other chemicals were Fig. 1 Structures of the coumarins tested from Sigma-Aldrich Canada (Oakville, Ontario). 175 Isolation of cnidiadin [6]. MDCK-MDR1 membranes (50 lg) isolated as pre- viously described [6] were incubated for 1 h in the dark Cnidiadin, was isolated with a yield of 0.035% from at 25°Cin10mM Tris-HCl pH 7.5, containing SDZ dried leaves of Tordylium apulum collected during June 212-122 (0.1 lM) and verapamil (10 lM) or coumarins 2002 near Delphes (Greece). A voucher specimen of the at the indicated concentrations, or DMSO at 0.1% collected leaves has been deposited in the Laboratory of (controls). The membranes were then crosslinked at Pharmacognosie et Biotechnologies (Universite´d’Au- 254 nm for 5 min at 4°C with a Stratalinker UV 2400 vergne) under the reference CBGTA-03. Cnidiadin was lamp (Stratagene, La Jolla, Calif.). Laemmli electro- isolated, as described previously [15], by ether extraction phoresis buffer (62.5 mM Tris-HCl, pH 6.8, 10% glyc- (n=3) of powdered leaves, followed by TLC of the dried erol, 2% SDS, 5% b-mercaptoethanol, 0.01% residue on silica gel 60 with n-hexane-ethyl acetate (5:1, bromophenol blue) was added to the membrane sam- v/v), and preparative TLC on silica gel with n-hexane- ples, and the proteins were resolved by SDS-PAGE on ethyl acetate [3:1, v/v].
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