Acquired Resistance to Peloruside A and Laulimalide is Associated with Downregulation of Vimentin in Human Ovarian Carcinoma Cells



Acquired β-tubulin alterations in human ovarian carcinoma 1A9 cells were previously shown to confer resistance to the microtubule stabilizing agents peloruside A (PLA) and laulimalide (LAU). We examined the proteome of resistant cells to see what other protein changes occurred as a result of the acquired drug resistance.


Two-dimensional differential in-gel electrophoresis was performed to explore differentially expressed proteins in the resistant 1A9-R1 (R1) and 1A9-L4 (L4) cells. The proteins on the gels were identified by MALDI-TOF MS, and altered protein abundance was confirmed by Western blotting and immunocytochemistry. Vimentin expression was restored in vimentin-deficient L4 cells by transfecting a full-length human vimentin cDNA, and sensitivity to PLA and LAU were tested using an MTT cell proliferation assay.


Proteomic analysis identified several proteins that were significantly altered in the resistant cells relative to the parental 1A9 cells. Using Western blotting and immunocytochemistry, a decreased vimentin abundance in the L4 cells was validated. Vimentin levels were unchanged in PLA-resistant R1 cells and paclitaxel/epothilone-resistant derivatives of 1A9 cells. Vimentin cDNA transfection into L4 cells partially restored PLA and LAU sensitivity.


Downregulation of vimentin contributes to the resistance of 1A9 cells to the microtubule stabilizing agents, PLA and LAU.

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two-dimensional differential in-gel electrophoresis


biological variation analysis










matrix assisted laser desorption/ionization time of flight




peloruside A




vimentin degradation products


  1. 1.

    Steinert PM, Roop DR. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Franke WW, Schmid E, Winter S, Osborn M, Weber K. Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp Cell Res. 1979;123(1):25–46.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Dráberová E, Dráber P. A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules. J Cell Sci. 1993;106(4):1263–73.

    PubMed  Google Scholar 

  4. 4.

    Esue O, Carson AA, Tseng Y, Wirtz D. A direct interaction between actin and vimentin filaments mediated by the tail domain of vimentin. J Biol Chem. 2006;281(41):30393–9.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 2011;68(18):3033–46.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Zhu QS, Rosenblatt K, Huang KL, Lahat G, Brobey R, Bolshakov S, et al. Vimentin is a novel AKT1 target mediating motility and invasion. Oncogene. 2011;30(4):457–70.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Nijkamp MM, Span PN, Hoogsteen IJ, van der Kogel AJ, Kaanders JH, Bussink J. Expression of E-cadherin and vimentin correlates with metastasis formation in head and neck squamous cell carcinoma patients. Radiother Oncol. 2011;99(3):344–8.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Liang Y, McDonnell S, Clynes M. Examining the relationship between cancer invasion/metastasis and drug resistance. Curr Cancer Drug Targets. 2002;2(3):257–77.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov. 2010;9(10):790–803.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP -dependent transporters. Nat Rev Cancer. 2002;2(1):48–58.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer. 2010;10(3):194–204.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Verrills NM, Po’uha ST, Liu ML, Liaw TY, Larsen MR, Ivery MT, et al. Alterations in gamma-actin and tubulin-targeted drug resistance in childhood leukemia. J Natl Cancer Inst. 2006;98(19):1363–74.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Zhou M, Liu Z, Zhao Y, Ding Y, Liu H, Xi Y, et al. MicroRNA-125b confers the resistance of breast cancer cells to paclitaxel through suppression of pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) expression. J Biol Chem. 2010;285(28):21496–507.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Grzanka A, Grzanka D, Orlikowska M. Cytoskeletal reorganization during process of apoptosis induced by cytostatic drugs in K-562 and HL-60 leukemia cell lines. Biochem Pharmacol. 2003;66(8):1611–7.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Grzanka A, Grzanka D, Orlikowska M, Zuryn A, Grzanka A. Estimation of taxol influence on changes in tubulin and vimentin systems in K-562 and HL-60 cell lines by immunofluorescence microscopy. Neoplasma. 2005;52(3):193–8.

    PubMed  Google Scholar 

  16. 16.

    Sun QL, Sha HF, Yang XH, Bao GL, Lu J, Xie YY. Comparative proteomic analysis of paclitaxel sensitive A549 lung adenocarcinoma cell line and its resistant counterpart A549-Taxol. J Cancer Res Clin Oncol. 2011;137(3):521–32.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Kabos P, Haughian JM, Wang X, Dye WW, Finlayson C, Elias A, et al. Cytokeratin 5 positive cells represent a steroid receptor negative and therapy resistant subpopulation in luminal breast cancers. Breast Cancer Res Treat. 2011;128(1):45–55.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Bauman PA, Dalton WS, Anderson JM, Cress AE. Expression of cytokeratin confers multiple drug resistance. Proc Natl Acad Sci U S A. 1994;91(12):5311–4.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Mooberry SL, Tien G, Hernandez AH, Plubrukarn A, Davidson BS. Laulimalide and isolaulimalide, new paclitaxel-like microtubule stabilizing agents. Cancer Res. 1999;59(3):653–60.

    PubMed  CAS  Google Scholar 

  20. 20.

    Hood KA, West LM, Rouwé B, Northcote PT, Berridge MV, Wakefield SJ, et al. Peloruside A, a novel antimitotic agent with paclitaxel-like microtubule stabilizing activity. Cancer Res. 2002;62(12):3356–60.

    PubMed  CAS  Google Scholar 

  21. 21.

    Kanakkanthara A, Wilmes A, O’Brate A, Escuin D, Chan A, Gjyrezi A, et al. Peloruside- and laulimalide-resistant human ovarian carcinoma cells have βI-tubulin mutations and altered expression of βII- and βIII-tubulin isotypes. Mol Cancer Ther. 2011;10(8):1419–29.

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Gaitanos TN, Buey RM, Díaz F, Northcote PT, Spittle PT, Andreu JM, et al. Peloruside A does not bind to the taxoid site on β-tubulin and retains its activity in multidrug-resistant cell lines. Cancer Res. 2004;64(15):5063–7.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Pryor DE, O’Brate A, Bilcer G, Díaz JF, Wang Y, Wang Y, et al. The microtubule stabilizing agent laulimalide does not bind in the taxoid site, kills cells resistant to paclitaxel and epothilones, and may not require its epoxide moiety for activity. Biochemistry. 2002;41(29):9109–15.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Kanakkanthara A, Northcote PT, Miller JH. βII-Tubulin and βIII-tubulin mediate sensitivity to peloruside A and laulimalide, but not paclitaxel or vinblastine, in human ovarian carcinoma cells. Mol Cancer Ther. 2012;11(2):393–404.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Giannakakou P, Sackett DL, Kang YK, Zhan Z, Buters JTM, Fojo T, et al. Paclitaxel-resistant human ovarian cancer cells have mutant β-tubulins that exhibit impaired paclitaxel-driven polymerization. J Biol Chem. 1997;272(27):17118–25.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Giannakakou P, Gussio R, Nogales E, Downing KH, Zaharevitz D, Bollbuck B, et al. A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc Natl Acad Sci U S A. 2000;97(6):2904–9.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Kajiyama H, Shibata K, Terauchi M, Yamashita M, Ino K, Nawa A, et al. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. Int J Oncol. 2007;31(2):277–83.

    PubMed  CAS  Google Scholar 

  28. 28.

    Işeri OD, Kars MD, Arpaci F, Atalay C, Pak I, Gündüz U. Drug resistant MCF-7 cells exhibit epithelial-mesenchymal transition gene expression pattern. Biomed Pharmacother. 2011;65(1):40–5.

    PubMed  Article  Google Scholar 

  29. 29.

    Goldman RD, Khuon S, Chou YH, Opal P, Steinert PM. The function of intermediate filaments in cell shape and cytoskeletal integrity. J Cell Biol. 1996;134(4):971–83.

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Vilalta PM, Zhang L, Hamm-Alvarez SF. A novel taxol-induced vimentin phosphorylation and stabilization revealed by studies on stable microtubules and vimentin intermediate filaments. J Cell Sci. 1998;111(13):1841–52.

    PubMed  CAS  Google Scholar 

  31. 31.

    Janosch P, Kieser A, Eulitz M, Lovric J, Sauer G, Reichert M, et al. The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments. FASEB J. 2000;14(13):2008–21.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Goto H, Tanabe K, Manser E, Lim L, Yasui Y, Inagaki M. Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK). Genes Cells. 2002;7(2):91–7.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Goto H, Yasui Y, Kawajiri A, Nigg EA, Terada Y, Tatsuka M, et al. Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process. J Biol Chem. 2003;278(10):8526–30.

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Nitta T, Kim JS, Mohuczy D, Behrns KE. Murine cirrhosis induces hepatocyte epithelial-mesenchymal transition and alterations in survival signaling pathways. Hepatology. 2008;48(3):909–19.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Tzivion G, Luo ZJ, Avruch J. Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo. J Biol Chem. 2000;275(38):29772–8.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Peñuelas S, Noé V, Ciudad CJ. Modulation of IMPDH2, survivin, topoisomerase I and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells. FEBS J. 2005;272(3):696–710.

    PubMed  Article  Google Scholar 

  37. 37.

    Lopes EC, García MG, Vellón L, Alvarez E, Hajos SE. Correlation between decreased apoptosis and multidrug resistance (MDR) in murine leukemic T cell lines. Leuk Lymphoma. 2001;42(4):775–87.

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Wilson KS, Roberts H, Leek R, Harris AL, Geradts J. Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues. Am J Pathol. 2002;161(4):1171–85.

    PubMed  Article  CAS  Google Scholar 

  39. 39.

    Miller JH, Rouwé B, Gaitanos TN, Hood KA, Crume KP, Bäckström BT, et al. Peloruside A enhances apoptosis in H-ras-transformed cells and is cytotoxic to proliferating T cells. Apoptosis. 2004;9(6):785–96.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Prasad SC, Thraves PJ, Kuettel MR, Srinivasarao GY, Dritschilo A, Soldatenkov VA. Apoptosis-associated proteolysis of vimentin in human prostate epithelial tumor cells. Biochem Biophys Res Commun. 1998;249(2):332–8.

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Morishima N. Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action. Genes Cells. 1999;4(7):401–14.

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Byun Y, Chen F, Chang R, Trivedi M, Green KJ, Cryns VL. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ. 2001;8(5):443–50.

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    Lahat G, Zhu QS, Huang KL, Wang S, Bolshakov S, Liu J, et al. Vimentin is a novel anti-cancer therapeutic target; insights from in vitro and in vivo mice xenograft studies. PLoS One. 2010;5(4):e10105.

    PubMed  Article  Google Scholar 

  44. 44.

    Wilmes A, Chan A, Rawson P, William Jordan T, Miller JH. Paclitaxel effects on the proteome of HL-60 promyelocytic leukemic cells: comparison to peloruside A. Invest New Drugs. 2012;30(1):121–9.

    PubMed  Article  CAS  Google Scholar 

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The authors thank Dr. Paraskevi Giannakakou for kindly providing the 1A9 and the resistant L4, PTX-10, and A8 cell lines. This research was supported by grants to J.H.M from the Cancer Society of New Zealand, the Wellington Medical Research Foundation, and Victoria University of Wellington.

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Correspondence to John H. Miller.

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Kanakkanthara, A., Rawson, P., Northcote, P.T. et al. Acquired Resistance to Peloruside A and Laulimalide is Associated with Downregulation of Vimentin in Human Ovarian Carcinoma Cells. Pharm Res 29, 3022–3032 (2012).

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  • cancer resistance
  • laulimalide
  • ovarian carcinoma cells
  • peloruside A
  • vimentin