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Pharmaceutical Research

, Volume 25, Issue 4, pp 827–835 | Cite as

Constitutive Overexpression of P-glycoprotein, Rather than Breast Cancer Resistance Protein or Organic Cation Transporter 1, Contributes to Acquisition of Imatinib-Resistance in K562 Cells

  • Chie Hirayama
  • Hiroshi Watanabe
  • Reiko Nakashima
  • Takeru Nanbu
  • Akinobu Hamada
  • Akihiko Kuniyasu
  • Hitoshi Nakayama
  • Tatsuya Kawaguchi
  • Hideyuki Saito
Research Paper

Abstract

Purpose

The purpose of this study was to investigate the contribution of drug transporters in acquired imatinib-resistance. Specifically, we focused on the efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and an influx transporter, organic cation transporter 1 (OCT1).

Materials and methods

We established imatinib-resistant K562 cells (K562/IM). Real-time PCR or Western blot analyses were performed to examine the mRNA or protein levels. Alamar blue method was used in the cytotoxicity assay. The transport activities and intracellular imatinib levels were measured by flow cytometry and HPLC, respectively.

Results

K562/IM displayed a 47-fold increase in resistance to imatinib over the parent K562 cells. Both P-gp and BCRP were overexpressed in K562/IM relative to K562. Furthermore, the intracellular imatinib level was markedly reduced in K562/IM. Interestingly, cyclosporin A, a P-gp inhibitor, but not fumitremorgin C, a BCRP inhibitor, restored both imatinib-sensitivity and the intracellular imatinib level. By contrast, no significant difference in the expression and function of OCT1 was observed between K562/IM and K562.

Conclusions

P-gp, rather than BCRP or OCT1, is partially responsible for the development of imatinib-resistance due to constitutive and functional overexpression, leading to reduced intracellular accumulation of imatinib in K562/IM.

Key words

breast cancer resistance protein drug resistance imatinib organic cation transporter 1 P-glycoprotein 

Abbreviations

BCRP

breast cancer resistance protein

IM

imatinib

OCT1

organic cation transporter 1

P-gp

P-glycoprotein

Notes

Acknowledgments

We thank Dr. Robert W. Robey and Dr. Susan E. Bate at National Cancer Institute (Betheda, MD) for providing FTC and PhA. We also acknowledge Novartis Pharma AG (Basel, Switzerland) for kindly providing imatinib and cyclosporin A.

References

  1. 1.
    M. Kalidas, H. Kantarjian, and M. Talpaz. Chronic myelogenous leukemia. JAMA 286:895–898 (2001).PubMedCrossRefGoogle Scholar
  2. 2.
    R. Capdeville, E. Buchdunger, J. Zimmermann, and A. Matter. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat. Rev. Drug Discov. 1:493–502 (2002).PubMedCrossRefGoogle Scholar
  3. 3.
    B. J. Druker, C. L. Sawyers, H. Kantarjian, D. J. Resta, S. F. Reese, J. M. Ford, R. Capdeville, and M. Talpaz. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344:1038–1042 (2001).PubMedCrossRefGoogle Scholar
  4. 4.
    C. L. Sawyers, A. Hochhaus, E. Feldman, J. M. Goldman, C. B. Miller, O. G. Ottmann, C. A. Schiffer, M. Talpaz, F. Guilhot, M. W. N. Deininger, T. Fischer, S. G. O’Brien, R. M. Stone, C. B. Gambacorti-Passerini, N. H. Russell, J. J. Reiffers, T. C. Shea, B. Chapuis, S. Coutre, S. Tura, E. Morra, R. A. Larson, A. Saven, C. Peschel, A. Gratwohl, F. Mandelli, M. Ben-Am, I. Gathmann, R. Capdeville, R. L. Paquette, and B. J. Druker. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 99:3530–3539 (2002).PubMedCrossRefGoogle Scholar
  5. 5.
    O. G. Ottmann, B. J. Druker, C. L. Sawyers, J. M. Goldman, J. Reiffers, R. T. Silver, S. Tura, T. Fischer, M. W. Deininger, C. A. Schiffer, M. Baccarani, A. Gratwohl, A. Hochhaus, D. Hoelzer, S. Fernandes-Reese, I. Gathmann, R. Capdeville, and S. G. O’Brien. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 100:1965–1971 (2002).PubMedCrossRefGoogle Scholar
  6. 6.
    M. E. Gorre, M. Mohammed, K. Ellwood, N. Hsu, R. Paquette, P. N. Rao, and C. L. Sawyers. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293:876–880 (2001).PubMedCrossRefGoogle Scholar
  7. 7.
    M. E. O’Dwyer, M. J. Mauro, G. Kurilik, M. Mori, S. Balleisen, S. Olson, E. Magenis, R. Capdeville, and B. J. Druker. The impact of clonal evolution on response to imatinib mesylate (STI571) in accelerated phase CML. Blood 100:1628–1633 (2002).PubMedCrossRefGoogle Scholar
  8. 8.
    J. E. Cortes, M. Talpaz, F. Giles, S. O’Brien, M. B. Rios, J. Shan, G. Garcia-Manero, S. Faderl, D. A. Thomas, W. Wierda, A. Ferrajoli, S. Jeha, and H. M. Kantarjian. Prognostic significance of cytogenetic clonal evolution in patients with chronic myelogenous leukemia on imatinib mesylate therapy. Blood 101:3794–3800 (2003).PubMedCrossRefGoogle Scholar
  9. 9.
    S. Branford, Z. Rudzki, S. Walsh, A. Grigg, C. Arthur, K. Taylor, R. Herrmann, K. P. Lynch, and T. P. Hughes. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 99:3472–3475 (2002).PubMedCrossRefGoogle Scholar
  10. 10.
    C. Roche-Lestienne, V. Soenen-Cornu, N. Grardel-Duflos, J.-L. Lai, N. Philippe, T. Facon, P. Fenaux, and C. Preudhomme. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 100:1014–1018 (2002).PubMedCrossRefGoogle Scholar
  11. 11.
    N. C. Wolff, D. R. Veach, W. P. Tong, W. G. Bornmann, B. Clarkson, and R. L. Ilaria, Jr. PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia. Blood 105:3995–4003 (2005).PubMedCrossRefGoogle Scholar
  12. 12.
    H. Kantarjian, F. Giles, L. Wunderle, K. Bhalla, S. O’Brien, B. Wassmann, C. Tanaka, P. Manley, P. Rae, W. Mietlowski, K. Bochinski, A. Hochhaus, J. D. Griffin, D. Hoelzer, M. Albitar, M. Dugan, J. Cortes, L. Alland, and O. G. Ottmann. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N. Engl. J. Med. 354:2542–2551 (2006).PubMedCrossRefGoogle Scholar
  13. 13.
    M. Talpaz, N. P. Shah, H. Kantarjian, N. Donato, J. Nicoll, R. Paquette, J. Cortes, S. O’Brien, C. Nicaise, E. Bleickardt, M. A. Blackwood-Chirchir, V. Iyer, T.-T. Chen, F. Huang, A. P. Decillis, and C. L. Sawyers. Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N. Engl. J. Med. 354:2531–2541 (2006).PubMedCrossRefGoogle Scholar
  14. 14.
    F. X. Mahon, M. W. Deininger, B. Schultheis, J. Chabrol, J. Reiffers, J. M. Goldman, and J. V. Melo. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 96:1070–1079 (2000).PubMedGoogle Scholar
  15. 15.
    N. J. Donato, J. Y. Wu, J. Stapley, G. Gallick, H. Lin, R. Arlinghaus, and M. Talpaz. BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571. Blood 101:690–698 (2003).PubMedCrossRefGoogle Scholar
  16. 16.
    Y. Dai, M. Rahmani, S. J. Corey, P. Dent, and S. Grant. A Bcr/Abl-independent, Lyn-dependent form of imatinib mesylate (STI-571) resistance is associated with altered expression of Bcl-2. J. Biol. Chem. 279:34227–34239 (2004).PubMedCrossRefGoogle Scholar
  17. 17.
    F.-X. Mahon, F. Belloc, V. Lagarde, C. Chollet, F. Moreau-Gaudry, J. Reiffers, J. M. Goldman, and J. V. Melo. MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models. Blood 101:2368–2373 (2003).PubMedCrossRefGoogle Scholar
  18. 18.
    A. Hamada, H. Miyano, H. Watanabe, and H. Saito. Interaction of imatinib mesilate with human P-glycoprotein. J. Pharmacol. Exp. Ther. 307:824–828 (2003).PubMedCrossRefGoogle Scholar
  19. 19.
    N. Widmer, S. Colombo, T. Buclin, and L. A. Decosterd. Functional consequence of MDR1 expression on imatinib intracellular concentrations. Blood 102:1142PubMedCrossRefGoogle Scholar
  20. 20.
    T. Illmer, M. Schaich, U. Platzbecker, J. Freiberg-Richter, U. Oelschlagel, M. von Bonin, S. Pursche, T. Bergemann, G. Ehninger, and E. Schleyer. P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate. Leukemia 18:401–408 (2004).PubMedCrossRefGoogle Scholar
  21. 21.
    A. Radujkovic, M. Schad, J. Topaly, M. R. Veldwijk, S. Laufs, B. S. Schultheis, A. Jauch, J. V. Melo, S. Fruehauf, and W. J. Zeller. Synergistic activity of imatinib and 17-AAG in imatinib-resistant CML cells overexpressing BCR-ABL—inhibition of P-glycoprotein function by 17-AAG. Leukemia 19:1198–1206 (2005).PubMedCrossRefGoogle Scholar
  22. 22.
    M. Mukai, X.-F. Che, T. Furukawa, T. Sumizawa, S. Aoki, X.-Q. Ren, M. Haraguchi, Y. Sugimoto, M. Kobayashi, H. Takamatsu, and S.-i. Akiyama. Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells. Cancer Sci. 94:557–563 (2003).PubMedCrossRefGoogle Scholar
  23. 23.
    F. J. Giles, H. Kantarjian, J. Cortes, D. Thomas, M. Talpaz, T. Manshouri, and M. Albitar. Multidrug resistance protein expression in chronic myeloid leukemia: Associations and significance. Cancer 86:805–813 (1999).PubMedCrossRefGoogle Scholar
  24. 24.
    H. Burger, H. van Tol, A. W. Boersma, M. Brok, E. A. Wiemer, G. Stoter, and K. Nooter. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood 104:2940–2942 (2004).PubMedCrossRefGoogle Scholar
  25. 25.
    P. Breedveld, D. Pluim, G. Cipriani, P. Wielinga, O. van Tellingen, A. H. Schinkel, and J. H. M. Schellens. The Effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics and brain penetration of imatinib mesylate (Gleevec): implications for the use of breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain penetration of imatinib in patients. Cancer Res. 65:2577–2582 (2005).PubMedCrossRefGoogle Scholar
  26. 26.
    P. J. Houghton, G. S. Germain, F. C. Harwood, J. D. Schuetz, C. F. Stewart, E. Buchdunger, and P. Traxler. Imatinib mesylate is a potent inhibitor of the ABCG2 (BCRP) transporter and reverses resistance to topotecan and SN-38 in vitro. Cancer Res. 64:2333–2337 (2004).PubMedCrossRefGoogle Scholar
  27. 27.
    N. E. Jordanides, H. G. Jorgensen, T. L. Holyoake, and J. C. Mountford. Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate. Blood 108:1370–1373 (2006).PubMedCrossRefGoogle Scholar
  28. 28.
    J. Thomas, L. Wang, R. E. Clark, and M. Pirmohamed. Active transport of imatinib into and out of cells: implications for drug resistance. Blood 104:3739–3745 (2004).PubMedCrossRefGoogle Scholar
  29. 29.
    L. C. Crossman, B. J. Druker, M. W. N. Deininger, M. Pirmohamed, L. Wang, and R. E. Clark. hOCT 1 and resistance to imatinib. Blood 106:1133–1134 (2005).PubMedCrossRefGoogle Scholar
  30. 30.
    D. L. White, V. A. Saunders, P. Dang, J. Engler, A. C. Zannettino, A. C. Cambareri, S. R. Quinn, P. W. Manley, and T. P. Hughes. OCT-1-mediated influx is a key determinant of the intracellular uptake of imatinib but not nilotinib (AMN107): reduced OCT-1 activity is the cause of low in vitro sensitivity to imatinib. Blood 108:697–704 (2006).PubMedCrossRefGoogle Scholar
  31. 31.
    H. M. Kantarjian, M. Talpaz, S. O’Brien, F. Giles, G. Garcia-Manero, S. Faderl, D. Thomas, J. Shan, M. B. Rios, and J. Cortes. Dose escalation of imatinib mesylate can overcome resistance to standard-dose therapy in patients with chronic myelogenous leukemia. Blood 101:473–475 (2003).PubMedCrossRefGoogle Scholar
  32. 32.
    J. O’Brien, I. Wilson, T. Orton, and F. Pognan. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267:5421–5426 (2000).PubMedCrossRefGoogle Scholar
  33. 33.
    G. W. Dewald, W. A. Wyatt, A. L. Juneau, R. O. Carlson, A. R. Zinsmeister, S. M. Jalal, J. L. Spurbeck, and R. T. Silver. Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood 91:3357–3365 (1998).PubMedGoogle Scholar
  34. 34.
    R. W. Robey, K. Steadman, O. Polgar, K. Morisaki, M. Blayney, P. Mistry, and S. E. Bates. Pheophorbide a is a specific probe for ABCG2 function and inhibition. Cancer Res. 64:1242–1246 (2004).PubMedCrossRefGoogle Scholar
  35. 35.
    C. Özvegy-Laczka, T. Hegedüs, G. Varady, O. Ujhelly, J. D. Schuetz, A. Varadi, G. Keri, L. Örfi, K. Nemet, and B. Sarkadi. High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter. Mol. Pharmacol. 65:1485–1495 (2004).PubMedCrossRefGoogle Scholar
  36. 36.
    H. Burger, H. van Tol, M. Brok, E. A. Wiemer, E. A. de Bruijn, G. Guetens, G. de Boeck, A. Sparreboom, J. Verweij, and K. Nooter. Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps. Cancer Biol. Ther. 4:747–752 (2005).PubMedCrossRefGoogle Scholar
  37. 37.
    T. Nakanishi, K. Shiozawa, B. A. Hassel, and D. D. Ross. Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression. Blood 108:678–684 (2006).PubMedCrossRefGoogle Scholar
  38. 38.
    E. R. Gardner, H. Burger, R. H. van Schaik, A. T. van Oosterom, E. A. de Bruijn, G. Guetens, H. Prenen, F. A. de Jong, S. D. Baker, S. E. Bates, W. D. Figg, J. Verweij, A. Sparreboom, and K. Nooter. Association of enzyme and transporter genotypes with the pharmacokinetics of imatinib. Clin. Pharmacol. Ther. 80:192–201 (2006).PubMedCrossRefGoogle Scholar
  39. 39.
    M. Warmuth, N. Simon, O. Mitina, R. Mathes, D. Fabbro, P. W. Manley, E. Buchdunger, K. Forster, I. Moarefi, and M. Hallek. Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate-resistant Bcr-Abl kinases. Blood 101:664–672 (2003).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Chie Hirayama
    • 1
  • Hiroshi Watanabe
    • 1
  • Reiko Nakashima
    • 1
  • Takeru Nanbu
    • 1
  • Akinobu Hamada
    • 1
  • Akihiko Kuniyasu
    • 2
  • Hitoshi Nakayama
    • 2
  • Tatsuya Kawaguchi
    • 3
  • Hideyuki Saito
    • 1
  1. 1.Department of PharmacyKumamoto University HospitalKumamotoJapan
  2. 2.Department of Molecular Cell Function, Graduate School of Medical and Pharmaceutical SciencesKumamoto UniversityKumamotoJapan
  3. 3.Department of Hematology and Infectious DiseasesKumamoto University HospitalKumamotoJapan

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