Skip to main content

Doxorubicin-transferrin conjugate selectively overcomes multidrug resistance in leukaemia cells

Abstract

Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was determined using the MTT assay. The IC50 for DOX-TRF was lower than the IC50 value for the free drug in both leukaemia cell lines. The IC50 values for the HL60 cells were 0.08 μM for DOX and 0.02 μM for DOX-TRF. The IC50 values for HL60ADR cells were 7 μM for DOX and 0.035 μM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the leukaemia cell lines while having only a very limited effect on normal tissue cells.

Abbreviations

DOX:

doxorubicin

DOX-TRF:

doxorubicin-transferrin conjugate

MDR:

multidrug resistance

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

SD:

standard deviation

SDS:

sodium dodecyl sulfate

References

  1. Gewirtz, D.A. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 57 (1999) 727–741.

    PubMed  Article  CAS  Google Scholar 

  2. Singal, P.K., Iliskovic, N., Li, T. and Kumar, D. Adriamycin cardiomyopathy: pathophysiology and prevention. FASEB J. 11 (1997) 931–936.

    PubMed  CAS  Google Scholar 

  3. Lage, H. ABC-transporters: implications on drug resistance from microorganisms to human cancers. Int. J. Antimicrob. Agents 22 (2003) 188–199.

    PubMed  Article  CAS  Google Scholar 

  4. Li, H. and Qian, Z.M. Transferrin/transferrin receptor-mediated drug delivery. Med. Res. Rev. 22 (2002) 225–250.

    PubMed  Article  CAS  Google Scholar 

  5. Hatano, T., Ohkawa, K. and Matsuda, M. Cytotoxic effect of the proteindoxorubicin conjugates on the multidrug-resistant human myelogenous leukemia cell line, K562, in vitro. Tumour Biol. 14 (1993) 288–294.

    PubMed  CAS  Article  Google Scholar 

  6. Kratz, F., Beyer, U., Roth, T., Tarasova, N., Collery, P., Lechenault, F., Cazabat, A., Schumacher, P., Unger, C. and Falken, U. Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy. J. Pharm. Sci. 87 (1998) 338–346.

    PubMed  Article  CAS  Google Scholar 

  7. Daniels, T.R., Delgado, T., Rodriguez, J.A., Helguera, G. and Penichet, M.L. The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin. Immunol. 121 (2006) 144–158.

    PubMed  Article  CAS  Google Scholar 

  8. Singh, M. Transferrin as a targeting ligand for liposomes and anticancer drugs. Curr. Pharm. Des. 5 (1999) 443–451.

    PubMed  CAS  Google Scholar 

  9. Wang, F., Jiang, X., Yang, D.C., Elliott, R.L. and Head, J.F. Doxorubicingallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells. Anticancer Res. 20 (2000) 799–808.

    PubMed  CAS  Google Scholar 

  10. Singh, M., Atwal, H. and Micetich, R. Transferrin directed delivery of adriamycin to human cells. Anticancer Res. 18 (1998) 1423–1427.

    PubMed  CAS  Google Scholar 

  11. Berczi, A., Barabas, K., Sizensky, J.A. and Faulk, W.P. Adriamycin conjugates of human transferrin bind transferrin receptors and kill K562 and HL60 cells. Arch. Biochem. Biophys. 300 (1993) 356–363.

    PubMed  Article  CAS  Google Scholar 

  12. Sun, I.L., Sun, E.E., Crane, F.L., Morre, D.J. and Faulk, W.P. Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates. Biochim. Biophys. Acta 1105 (1992) 84–88.

    PubMed  Article  CAS  Google Scholar 

  13. Kobayashi, T., Ishida, T., Okada, Y., Ise, S., Harashima, H. and Kiwada, H. Effect of transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells. Int. J. Pharm. 329 (2007) 94–102.

    PubMed  Article  CAS  Google Scholar 

  14. Fritzer, M., Szekeres, T., Szuts, V., Jarayam, H.N. and Goldenberg, H. Cytotoxic effects of a doxorubicin-transferrin conjugate in multidrugresistant KB cells. Biochem. Pharmacol. 51 (1996) 489–493.

    PubMed  Article  CAS  Google Scholar 

  15. Gallagher, R., Collins, S., Trujillo, J., McCredie, K., Ahearn, M., Tsai, S., Metzgar, R., Aulakh, G., Ting, R., Ruscetti, F. and Gallo, R. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54 (1979) 713–733.

    PubMed  CAS  Google Scholar 

  16. Krishnamachary, N. and Center, M.S. The MRP gene associated with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane bound glycoprotein. Cancer. Res. 53 (1993) 3658–3661.

    PubMed  CAS  Google Scholar 

  17. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680–685.

    PubMed  Article  CAS  Google Scholar 

  18. Wistop, A., Keller, U., Sprung, C.N., Grabenbauer, G.G., Sauer, R. and Distel, L.V. Individual radiosensitivity does not correlate with radiationinduced apoptosis in lymphoblastoid cell lines or CD3+ lymphocytes. Strahlenther. Onkol. 181 (2005) 326–335.

    PubMed  Article  Google Scholar 

  19. Lubgan, D., Distel, L., Grabenbauer, G., Jozwiak, Z. and Sauer, R. Kopplung von Doxorubicin an Transferrin ermöglicht erhöhte Wirkspiegel in multidrug-resistenten Zellen zu erreichen. Strahlenther. Onkol. 181 (2005) 95.

    Google Scholar 

  20. Lubgan, D., Distel, L., Grabenbauer, G.G. and Sauer, R. Selektive Therapie von Tumorzellen durch Kopplung von Doxorubicin an Transferrin. Strahlenther. Onkol. 180 (2004) 7.

    Google Scholar 

  21. Bryszewska, M., Piasecka, A., Zavodnik, L.B., Distel, L. and Schussler, H. Oxidative damage of Chinese hamster fibroblasts induced by t-butyl hydroperoxide and by X-rays. Biochim. Biophys. Acta 1621 (2003) 285–291.

    PubMed  CAS  Google Scholar 

  22. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1983) 55–63.

    PubMed  Article  CAS  Google Scholar 

  23. Binaschi, M., Bigioni, M., Cipollone, A., Rossi, C., Goso, C., Maggi, C.A., Capranico, G. and Animati, F. Anthracyclines: selected new developments. Curr. Med. Chem. Anti-Canc. Agents 1 (2001) 113–130.

    Article  CAS  Google Scholar 

  24. Ward, J.H., Kushner, J.P. and Kaplan, J. Transferrin receptors of human fibroblasts. Analysis of receptor properties and regulation. Biochem. J. 208 (1982) 19–26.

    PubMed  CAS  Google Scholar 

  25. Gomme, P.T., McCann, K.B. and Bertolini, J. Transferrin: structure, function and potential therapeutic actions. Drug. Discov. Today 10 (2005) 267–273.

    PubMed  Article  CAS  Google Scholar 

  26. Berczi, A., Ruthner, M., Szuts, V., Fritzer, M., Schweinzer, E. and Goldenberg, H. Influence of conjugation of doxorubicin to transferrin on the iron uptake by K562 cells via receptor-mediated endocytosis. Eur. J. Biochem. 213 (1993) 427–436.

    PubMed  Article  CAS  Google Scholar 

  27. Daniels, T.R., Delgado, T., Helguera, G. and Penichet, M.L. The transferrin receptor part II: targeted delivery of therapeutic agents into cancer cells. Clin. Immunol. 121 (2006) 159–176.

    PubMed  Article  CAS  Google Scholar 

  28. Barabas, K., Sizensky, J.A. and Faulk, W.P. Transferrin conjugates of adriamycin are cytotoxic without intercalating nuclear DNA. J. Biol. Chem. 267 (1992) 9437–9442.

    PubMed  CAS  Google Scholar 

  29. Inoue, T., Cavanaugh, P.G., Steck, P.A., Brunner, N. and Nicolson, G.L. Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials. J. Cell. Physiol. 156 (1993) 212–217.

    PubMed  Article  CAS  Google Scholar 

  30. Buchholz, T.A., Davis, D.W., McConkey, D.J., Symmans, W.F., Valero, V., Jhingran, A., Tucker, S.L., Pusztai, L., Cristofanilli, M., Esteva, F.J., Hortobagyi, G.N. and Sahin, A.A. Chemotherapy-induced apoptosis and Bcl-2 levels correlate with breast cancer response to chemotherapy. Cancer. J. 9 (2003) 33–41.

    PubMed  Article  CAS  Google Scholar 

  31. Sizensky, J.A., Barabas, K. and Faulk, W.P. Characterization of the anticancer activity of transferrin-adriamycin conjugates. Am. J. Reprod. Immunol. 27 (1992) 163–166.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dorota Łubgan.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Łubgan, D., Jóźwiak, Z., Grabenbauer, G.G. et al. Doxorubicin-transferrin conjugate selectively overcomes multidrug resistance in leukaemia cells. Cell Mol Biol Lett 14, 113–127 (2009). https://doi.org/10.2478/s11658-008-0037-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-008-0037-2

Key words

  • Doxorubicin-transferrin
  • Promyelocytic leukaemia
  • Apoptosis
  • DNA double-strand breaks
  • Multidrug resistance