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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 2002 Sep;93(9):1037–1046. doi: 10.1111/j.1349-7006.2002.tb02481.x

Reversal of P‐Glycoprotein‐mediated Paclitaxel Resistance by New Synthetic Isoprenoids in Human Bladder Cancer Cell Line

Hideki Enokida 1,✉, Takenari Gotanda 1, Shoichi Oku 1, Yoshiharu Imazono 1, Hiroyuki Kubo 1, Toshikatsu Hanada 2, Shigenori Suzuki 3, Kouhei Inomata 3, Takao Kishiye 4, Yoshiyuki Tahara 4, Kenryu Nishiyama 1, Masayuki Nakagawa 1
PMCID: PMC5927134  PMID: 12359058

Abstract

We isolated a paclitaxel‐resistant cell line (KK47/TX30) from a human bladder cancer cell line (KK47/WT) in order to investigate the mechanism of and reversal agents for paclitaxel resistance. KK47/TX30 cells exhibited 700‐fold resistance to paclitaxel and cross‐resistance to vinca alkaloids and topoisomerase II inhibitors. Tubulin polymerization assay showed no significant difference in the ratio of polymerized α‐ and β‐tubulin between KK47/WT and KK47/TX30 cells. Western blot analysis demonstrated overexpression of P‐glycoprotein (P‐gp) and lung resistance‐related protein (LRP) in KK47/TX30 cells. Drug accumulation and efflux studies showed that the decreased paclitaxel accumulation in KK47/TX30 cells was due to enhanced paclitaxel efflux. Cell survival assay revealed that verapamil and cepharanthine, conventional P‐gp modulators, could completely overcome paclitaxel resistance. To investigate whether new synthetic isoprenoids could overcome paclitaxel resistance, we synthesized 31 isoprenoids based on the structure of N‐solanesyl‐N, N′‐bis(3,4‐dimethoxybenzyl)ethylenediamine (SDB), which could reverse multidrug resistance (MDR), as shown previously. Among those examined, trans‐N, N′‐bis(3,4‐dimethoxybenzyl)‐.N‐solanesyl‐l,2‐diaminocyclohexane (N‐5228) could completely reverse paclitaxel resistance in KK47/TX30 cells. N‐5228 inhibited photoaffinity labeling of P‐gp by [3H]azidopine, suggesting that N‐5228 could bind to P‐gp directly and could be a substrate of P‐gp. Next, we investigated structural features of these 31 isoprenoids in order to determine the structural requirements for the reversal of P‐gp‐mediated paclitaxel resistance, suggesting that the following structural features are important for overcoming paclitaxel resistance: (1) a basic structure of 8 to 10 isoprene units, (2) a cyclohexane ring or benzene ring within the framework, (3) two cationic sites in close proximity to each other, and (4) a benzyl group with 3, 4‐dimethoxy functionalities, which have moderate electron‐donating ability. These findings may provide valuable information for the development of P‐gp‐mediated MDR‐reversing agents.

Keywords: Synthetic isoprenoid, Multidrug resistance, Bladder cancer

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REFERENCES

  • 1. ) Bremnes , R. M. , Sundstrom , S. , Vilsvik , J. and Aasebo , U.Multicenter phase II trial of paclitaxel, cisplatin, and etoposide with concurrent radiation for limited‐stage small‐cell lung cancer . J. Clin. Oncol. , 19 , 3532 – 3538 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 2. ) Riccardi , A. , Pugliese , P. , Danova , M. , Brugnatelli , S. , Grasso , D. , Giordano , M. , Bernardo , G. , Giardina , G. , Fava , S. , Montanari , G. , Pedrotti , C. , Trotti , G. , Rinaldi , E. , Poli , M. A. and Tinelli , C.A phase II study of sequential 5‐fluorouracil, epirubicin and cyclophosphamide (FEC) and paclitaxel in advanced breast cancer (Protocol PV BC 97/01) . Br. J. Cancer , 85 , 141 – 146 ( 2001. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. ) Prince , H. M. , Rischin , D. , Quinn , M. , Allen , D. , Planner , R. , Neesham , D. , Gates , P. and Davison , J.Repetitive high‐dose topotecan, carboplatin, and paclitaxel with peripheral blood progenitor cell support in previously untreated ovarian cancer: results of a Phase I study . Gynecol. Oncol. , 81 , 216 – 224 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) Hurwitz , C. A. , Strauss , L. C. , Kepner , J. , Kretschmar , C. , Harris , M. B. , Friedman , H. , Kun , L. and Kadota , R.Paclitaxel for the treatment of progressive or recurrent childhood brain tumors: a pediatric oncology phase II study . J. Pediatr. Hematol. Oncol. , 23 , 277 – 281 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 5. ) Haas , N. , Roth , B. , Garay , C. , Yeslow , G. , Entmacher , M. , Weinstein , A. , Rogatko , A. , Babb , J. , Minnitti , C. , Flinker , D. , Gillon , T. and Hudes , G.Phase I trial of weekly paclitaxel plus oral estramustine phosphate in patients with hormone‐refractory prostate cancer . Urology , 58 , 59 – 64 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 6. ) Vaishampayan , U. , Flaherty , L. , Du , W. and Hussain , M.Phase II evaluation of paclitaxel, alpha‐interferon, and cis‐retinoic acid in advanced renal cell carcinoma . Cancer , 92 , 519 – 523 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 7. ) Meluch , A. A. , Greco , F. A. , Burris , H. A. , 3rd , O'Rourke , T. , Ortega , G. , Steis , R. G. , Morrissey , L. H. , Johnson , V. and Hainsworth , J. D.Paclitaxel and gemcitabine chemotherapy for advanced transitional‐cell carcinoma of the urothelial tract: a phase II trial of the Minnie Peal Cancer Research Network . J. Clin. Oncol. , 19 , 3018 – 3024 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Horwitz , S. B. , Cohen , D. , Rao , S. , Ringel , L , Shen , H. J. and Yang , C. P.Taxol: mechanisms of action and resistance . J. Natl. Cancer Inst. Monogr. , 15 , 55 – 61 ( 1993. ). [PubMed] [Google Scholar]
  • 9. ) Dumontet , C. and Sikic , B. I.Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death . J. Clin. Oncol. , 17 , 1061 – 1070 ( 1999. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Zhou , J. , Cheng , S. C. , Luo , D. and Xie , Y.Study of multidrug resistant mechanism in a taxol‐resistant hepatocellular carcinoma QGY‐TR 50 cell line . Biochem. Biophys. Res. Commun. , 280 , 1237 – 1242 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Bhalla , K. , Huang , Y. , Tang , C. , Self , S. , Ray , S. , Mahoney , M. E. , Ponnathpur , V. , Tourkina , E. , Ibrado , A. M. and Bullock , G.Characterization of a human myeloid leukemia cell line highly resistant to taxol . Leukemia , 8 , 465 – 475 ( 1994. ). [PubMed] [Google Scholar]
  • 12. ) Ohta , S. , Nishio , K. , Kubota , N. , Ohmori , T. , Funayama , Y. , Ohira , T. , Nakajima , H. , Adachi , M. and Saijo , N.Characterization of a taxol‐resistant human small‐cell lung cancer cell line . Jpn. J. Cancer Res. , 85 , 290 – 297 ( 1994. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. ) Fardel , O. , Lecureur , V. and Guillouzo , A.The P‐glycoprotein multidrug transporter . Gen. Pharmacol. , 27 , 1283 – 1291 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Ford , J. M. and Hait , W. N.Pharmacologic circumvention of multidrug resistance . Cytotechnology , 12 , 171 – 212 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Wingo , P. A. , Tong , T. and Bolden , S.Cancer statistics, 1995 . CA Cancer J. Clin. , 45 , 8 – 30 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 16. ) Taya , T. , Kobayashi , T. , Tsukahara , K. , Uchibayashi , T. , Naito , K. , Hisazumi , H. and Kuroda , K.In vitro culture of malignant tumor tissues from the human urinary tract . Jpn. J. Urol. , 68 , 1003 – 1010 ( 1977. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Scheper , R. J. , Broxterman , H. J. , Scheffer , G. L. , Kaaijk , P. , Dalton , W. S. , van Heijningen , T. H. , van Kalken , C. K. , Slovak , M. L. , de Vries , E. G. , van der Valk , P. , Meijer , C. J. and Pinedo , H. M.Overexpression of a M(r) 110,000 vesicular in non‐P‐glycoprotein‐mediated multidrug resistance . Cancer Res. , 53 , 1475 – 1479 ( 1993. ). [PubMed] [Google Scholar]
  • 18. ) Rome , L. H. , Kedersha , N. L. and Chugani , D. C.Unlocking vaults: organelles in search of function . Trends Cell BioL. , 1 , 47 – 50 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 19. ) Chugani , D. C. , Kedersha , N. L. and Rome , L. H.Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex . J. Cell ScL. , 106 , 23 – 29 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Schuurhuis , G. J. , Broxterman , H. J. , de Lange , J. H. , Pinedo , H. M. , van Heijningen , T. H. , Kuiper , C. M. , Scheffer , G. L. , Scheper , R. J. , van Kalken , C. K. , Baak , J. P. and Lankelma , J.Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P‐glycoprotein . Br. J. Cancer , 64 , 857 – 861 ( 1991. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. ) Kitazono , M. , Okumura , H. , Ikeda , R. , Sumizawa , T. , Furukawa , T. , Nagayama , S. , Tani , A. , Takao , S. , Aikou , T. and Akiyama , S.Reversal of LRP‐associated drug resistance in colon carcinoma SW‐620 cells . Int. J. Cancer , 91 , 126 – 131 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Nakagawa , M. , Yamaguchi , T. , Ueda , H. , Shiraishi , N. , Komiyama , S. , Akiyama , S. , Ogata , J. and Kuwano , M.Potentiation by vitamin A of the action of anticancer agents against murine tumors . Jpn. J. Cancer Res. , 76 , 887 – 894 ( 1985. ). [PubMed] [Google Scholar]
  • 23. ) Nakagawa , M. , Akiyama , S. , Yamaguchi , T. , Shiraishi , N. , Ogata , J. and Kuwano , M.Reversal of multidrug resistance by synthetic isoprenoids in the KB human cancer cell line . Cancer Res. , 46 , 4453 – 4457 ( 1986. ). [PubMed] [Google Scholar]
  • 24. ) Yamaguchi , T. , Nakagawa , M. , Shiraishi , N. , Yoshida , T. , Kiyosue , T. , Arita , M. , Akiyama , S. and Kuwano , M.Overcoming drug resistance in cancer cells with synthetic isoprenoids . J. Natl. Cancer Inst. , 76 , 947 – 953 ( 1986. ). [PubMed] [Google Scholar]
  • 25. ) Akiyama , S. , Yoshimura , A. , Kikuchi , H. , Sumizawa , T. , Kuwano , M. and Tahara , Y.Synthetic isoprenoid photoaffinity labeling of P‐glycoprotein specific to multidrug‐resistant cells . Mol. PharmacoL. , 36 , 730 – 735 ( 1989. ). [PubMed] [Google Scholar]
  • 26. ) Suzuki , H. , Tomida , A. and Nishimura , T.Cytocidal activity of a synthetic isoprenoid, N‐solanesyl‐N, N′‐bis(3,4‐dimethoxybenzyl)ethylenediamine, and its potentiation of antitumor drugs against multidrug‐resistant and sensitive cells in vitro . Jpn. J. Cancer Res. , 81 , 298 – 303 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. ) Kimiya , K. , Naito , S. , Soejima , T. , Sakamoto , N. , Kotoh , S. , Kumazawa , J. and Tsuruo , T.Establishment and characterization of doxorubicin‐resistant human bladder cancer cell line, KK47/ADM . J. UroL. , 148 , 441 – 445 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 28. ) Carmichael , J. , DeGraff , W. G. , Gazdar , A. F. , Minna , J. D. and Mitchell , J. B.Evaluation of a tetrazolium‐based semiautomated colorimetric assay: assessment of chemosensitivity testing . Cancer Res. , 47 , 936 – 942 ( 1987. ). [PubMed] [Google Scholar]
  • 29. ) Minotti , A. M. , Barlow , S. B. and Cabral , F.Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin . J. Biol Chem. , 266 , 3987 – 3994 ( 1991. ). [PubMed] [Google Scholar]
  • 30. ) Lever , J. E.Active amino acid transport in plasma membrane vesicles from Simian virus 40‐transformed mouse fibroblasts . J. BioL Chem. , 252 , 1990 – 1997 ( 1977. ). [PubMed] [Google Scholar]
  • 31. ) Bradford , M. A.A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding . Anal. Biochem. , 72 , 248 – 254 ( 1976. ). [DOI] [PubMed] [Google Scholar]
  • 32. ) Laemmli , U. K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature , 227 , 680 – 685 ( 1970. ). [DOI] [PubMed] [Google Scholar]
  • 33. ) Towbin , H. , Staehelin , T. and Gordon , J.Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications . Proc. Natl. Acad. Sci. USA , 76 , 4350 – 4354 ( 1979. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. ) Kyhse , A. J.Electroblotting of multiple gels: simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose . J. Biochem. Biophys. Methods , 10 , 203 – 209 ( 1984. ). [DOI] [PubMed] [Google Scholar]
  • 35. ) Bjerrum , O. J. and Schafer‐Nielsen , C.Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus . In “ Analytical Electrophoresis ,” ed. Dunn M. J. , pp. 315 – 327 ( 1986. ). Verlag Chemie; , Weinheim . [Google Scholar]
  • 36. ) Debenham , P. G. , Kartner , K. , Siminovich , L. , Riordan , J. R. and Ling , V.DNA mediated transfer of multi drug resistance and plasma membrane glycoprotein expression . Mol. Cell. BioL. , 2 , 881 – 884 ( 1982. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. ) Inomata , K. , Takahashi , T. , Inoue , H. , Yanai , M. , Yamazaki , H. , Suzuki , M. , Takasawa , T. , Kawamura , K. , Oshida , N. , Ikemoto , H. and Kishiye , T.Isoprene derivatives . US Patent, No. 5756475 ( 1998. ).
  • 38. ) Inomata , K. , Takahashi , T. , Inoue , H. , Yanai , M. , Yamazaki , H. , Suzuki , M. , Takasawa , T. , Kawamura , K. , Oshida , N. , Ikemoto , H. and Kishiye , T.Isoprene derivatives . US Patent, No. 6011069 ( 2000. ).
  • 39. ) Kartner , N. , Evernden , P. D. , Bradley , G. and Ling , V.Detection of P‐glycoprotein in multidrug‐resistant cell lines by monoclonal antibodies . Nature , 316 , 820 – 823 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 40. ) Horwitz , S. B. , Cohen , D. , Rao , S. , Ringel , I. , Shen , H. J. and Yang , C. P.Taxol: mechanisms of action and resistance . J. Natl. Cancer Inst. Monogr. , 15 , 55 – 61 ( 1993. ). [PubMed] [Google Scholar]
  • 41. ) Giannakakou , P. , Sackett , D. L. , Kang , Y. K. , Zhan , Z. , Buters , J. T. , Fojo , T. and Poruchynsky , M. S.Paclitaxel‐resistant human ovarian cancer cells have mutant (β‐tubulins that exhibit impaired paclitaxel‐driven polymerization . J. BioL Chem. , 272 , 17118 – 17125 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 42. ) Zordan‐Nudo , T. , Ling , V. , Liu , Z. and Georges , E.Effects of nonionic detergents on P‐glycoprotein drug binding and reversal of multidrug resistance . Cancer Res. , 53 , 5994 – 6000 ( 1993. ). [PubMed] [Google Scholar]
  • 43. ) Zhang , L. , Sachs , C. W. , Fine , R. L. and Casey , P. J.Interaction of prenylcysteine methyl esters with the multidrug resistance transporter . J. BioL Chem. , 269 , 15973 – 15976 ( 1994. ). [PubMed] [Google Scholar]
  • 44. ) Wiese , M. and Pajeva , I. K.Structure‐activity relationships of multidrug resistance reversers . Curr. Med. Chem. , 8 , 685 – 713 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 45. ) Seelig , A.How does P‐glycoprotein recognize its substrates ? Int. J. Clin. Pharmacol. Ther. , 36 , 50 – 54 ( 1998. ). [PubMed] [Google Scholar]

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