Effect of Multidrug Resistance-Reversing Agents on Transporting Activity of Human Canalicular Multispecific Organic Anion Transporter

  1. Zhe-Sheng Chen1,
  2. Takeshi Kawabe2,
  3. Mayumi Ono2,
  4. Shunji Aoki1,3,
  5. Tomoyuki Sumizawa1,
  6. Tatsuhiko Furukawa1,
  7. Takeshi Uchiumi2,
  8. Morimasa Wada2,
  9. Michihiko Kuwano2 and
  10. Shin-Ichi Akiyama
  1. 1Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka Kagoshima (Z-S.C., T.S., T.F., S.A.); 2Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka (T.K., M.O., T.U., M.W., M.K.); and 3Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (S.A.)

    Abstract

    The canalicular multispecific organic anion transporter (cMOAT), also termed MRP2, is a recently identified ATP-binding cassette transporter. We previously established stable human cMOAT cDNA-transfected cells, LLC/cMOAT-1 from LLC-PK1 cells, and LLC/CMV cells that were transfected with an empty vector. We found that LLC/cMOAT-1 cells have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxy-camptothecin, and cisplatin but not to etoposide. The multidrug resistance-reversing agents cyclosporin A (CsA) and 2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) almost completely reversed the resistance to VCR, 7-ethyl-10-hydroxy-camptothecin, and cisplatin of LLC/cMOAT-1 cells; anddl-buthionine-(S,R)-sulfoximine, (3′-oxo-4-butenyl-4-methyl-threonine1, (valine2) cyclosporin (PSC833), and 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid (MK571) partially reversed the resistance to these drugs. CsA and PAK-104P at 10 μM enhanced the accumulation of VCR in LLC/cMOAT-1 cells almost to the level in LLC/CMV cells without the agents. The efflux of VCR from LLC/cMOAT-1 cells was enhanced compared with LLC/CMV cells and inhibited by CsA and PAK-104P. Transport of leukotriene C4 (LTC4) and S-(2, 4-dinitrophenyl)glutathione also was studied with membrane vesicles prepared from these cells. LTC4 and S-(2, 4-dinitrophenyl)glutathione were actively transported into membrane vesicles prepared from LLC/cMOAT-1 cells. TheKm and Vmaxvalues for the uptake of LTC4 by the LLC/cMOAT-1 membrane vesicles were 0.26 ± 0.05 μM and 7.48 ± 0.67 pmol/min/mg protein, respectively. LTC4 transport was competitively inhibited by PAK-104P, CsA, MK571, and PSC833, withKi values of 3.7, 4.7, 13.1, and 28.9 μM, respectively. These findings demonstrate that cMOAT confers a novel drug-resistance phenotype. CsA and PAK-104P may be useful for reversing cMOAT-mediated drug resistance in tumors.

    Footnotes

    • Send reprint requests to: Dr. Shin-ichi Akiyama, Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, 8–35-1 Sakuragaoka Kagoshima 890-8520, Japan. E-mail:akiyamas{at}khosp2.kufm.kagoshima-u.ac.jp

    • This work was supported by grants from the Ministry of Education, Science, and Culture; the Ministry of Health and Welfare, Japan; and Japan Society for the Promotion of Science.

    • Abbreviations:
      MDR
      multidrug resistance
      Pgp
      P-glycoprotein
      MRP
      multidrug resistance protein
      GSH
      reduced glutathione
      GS-X pump
      ATP-dependent glutathione-S conjugate export pump
      cMOAT
      canalicular multispecific organic anion transporter
      VCR
      vincristine
      ADM
      doxorubicin (Adriamycin)
      CPT-11
      7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin
      SN-38
      7-ethyl-10-hydroxy-camptothecin
      MX
      mitoxantrone
      CsA
      cyclosporin A
      PSC833
      (3′-oxo-4-butenyl-4-methyl-threonine1,(valine2)cyclosporin
      PAK-104P
      2-[4-(diphenylmethyl)-1-piperazinyl]-5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide
      MK571
      3-([{3(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid
      BSO
      dl-buthionine-(S,R)-sulfoximine
      LTC4
      leukotriene C4
      DNP-SG
      S-(2, 4-dinitrophenyl)glutathione
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      • Received May 17, 1999.
      • Accepted August 24, 1999.
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    1. Molecular Pharmacology December 1, 1999 vol. 56 no. 6 1219-1228
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