Skip to main content
PMC home page
Cytotechnology logoLink to Cytotechnology
. 1998 Sep;27(1-3):95–111. doi: 10.1023/A:1008040117882

Intracellular distribution of anthracyclines in drug resistant cells

Giuseppe Arancia 1, Annarica Calcabrini 1, Stefania Meschini 1, Agnese Molinari 1
PMCID: PMC3449566  PMID: 19002786

Abstract

The unresponsiveness of multidrug resistant tumor cells to antineoplastic chemotherapy is often associated with reduced cellular drug accumulation accomplished by overexpressed transport molecules. Moreover, intracellular drug distribution in resistant cells appears to be remarkably different when compared to their wild type counterparts. In the present paper, we report observations on the intracellular accumulation and distribution of doxorubicin, an antitumoral agent widely employed in chemotherapy, in sensitive and resistant cultured tumor cells. The inherent fluorescence of doxorubicin allowed us to follow its fate in living cells by laser scanning confocal microscopy. This study included flow cytometric analysis of drug uptake and efflux and analysis of the presence of the well known drug transporter P-glycoprotein. Morphological, immunocytochemical and functional data evidentiated the Golgi apparatus as the preferential intracytoplasmic site of drug accumulation in resistant cells, capable of sequestering doxorubicin away from the nuclear target. Moreover, P-glycoprotein has been found located in the Golgi apparatus in drug induced resistant cells and in intrinsic resistant cells, such as melanoma cells. Thus, this organelle seems to play a pivotal role in the intracellular distribution of doxorubicin.

Keywords: anthracyclines, doxorubicin, Golgiapparatus, intracellular transport, multidrugresistance, ultrastructure

Full Text

The Full Text of this article is available as a PDF (403.8 KB).

References

  1. Arancia G, Bordi F, Calcabrini A, Cametti C, Diociaiuti M, Molinari A. Influence of anthracyclinic antibiotics on membranes of human erythrocytes: a combined radiowave electrical conductivity and electron microscopy study. Bioelectrochem. Bioenerg. 1994;34:45–51. doi: 10.1016/0302-4598(94)80008-1. [DOI] [Google Scholar]
  2. Arancia G, Bordi F, Calcabrini A, Diociaiuti M, Molinari A. Ultrastructural and spectroscopic methods in the study of anthracycline-membrane interaction. Pharmacol Res. 1995;32:255–272. doi: 10.1016/S1043-6618(05)80013-1. [DOI] [PubMed] [Google Scholar]
  3. Arancia G, Molinari A, Crateri P, Calcabrini A, Silvestri L, Isacchi G. Adriamycin-plasma membrane interaction in human erythrocytes. Eur J Cell Biol. 1988;47:379–387. [PubMed] [Google Scholar]
  4. Baldini N, Scotlandi K, Serra M, Shikita T, Zini N, Ognibene A, Santi S, Ferracini R, Maraldi NM. Nuclear immunolocalization of P-glycoprotein in multidrug-resistant cell lines showing similar mechanisms of doxorubicin distribution. Eur J Cell Biol. 1995;68:226–239. [PubMed] [Google Scholar]
  5. Beck WT, Danks MK, Yalowich JC, Zamora JM, Cirtain MC. Mechanisms of Multiple Drug Resistance in Neoplastic Cells. New York: Academic Press; 1989. Different mechanisms of multiple drug resistance in two human leukemic cell lines; pp. 211–220. [Google Scholar]
  6. Boiocchi M, Toffoli G. Mechanism of multidrug resistance in human tumour cell lines and complete reversion of cellular resistance. Eur J Cancer. 1992;28A:1099–1105. doi: 10.1016/0959-8049(92)90465-E. [DOI] [PubMed] [Google Scholar]
  7. Breuninger LM, Saptarshi P, Gaughan K, Miki T, Chan A, Aaronson SA, Kruh GD. Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res. 1995;55:5342–5347. [PubMed] [Google Scholar]
  8. Broxterman HJ, Schurrhuis GJ, Lantelma J, Baak JPA, Pinedo HM. Towards functional screening for multidrug resistant cells in human malignancies. In: Mihich E, editor. Drug Resistance: Mechanism and Reversal. CIC, Rome: John Libbey; 1990. pp. 309–319. [Google Scholar]
  9. Calcabrini A, Villa AM, Molinari A, Doglia SM, Arancia G. Influence of N-methylformamide on the intracellular transport of doxorubicin. Eur J Cell Biol. 1997;72:61–69. [PubMed] [Google Scholar]
  10. Chaires JB, Dattagupta N, Crothers DM. Interaction of anthracycline antibiotics and DNA. Biochemistry. 1982;21:3933–3940. doi: 10.1021/bi00260a005. [DOI] [PubMed] [Google Scholar]
  11. Cianfriglia M, Willingham MC, Tombesi M, Scagliotti V, Frasca Gand Chersi A. P-glycoprotein mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells. Int J Cancer. 1994;56:153–160. doi: 10.1002/ijc.2910560127. [DOI] [PubMed] [Google Scholar]
  12. Coan DE, Wechezack AR, Viggers RF, Sauvage LR. Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells. J Cell Sci. 1993;104:1145–1153. doi: 10.1242/jcs.104.4.1145. [DOI] [PubMed] [Google Scholar]
  13. Coley HM, Amos WB, Twentyman PR, Workman P. Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localization of anthracyclines in parent and multidrug resistant cell lines. Br J Cancer. 1993;67:1316–1323. doi: 10.1038/bjc.1993.244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cornwell MM, Pastan I, Gottesman MM. Certain calcium channel blockers bind specifically to multidrug resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. J Biol Chem. 1987;262:2166–2170. [PubMed] [Google Scholar]
  15. DeLange JHM, Schipper NW, Schurrhuis GJ, TenKate TK, Jan Heijningen T. H., Pinedo HM, Lankelma J, Baak JPA. Quantification of intracellular doxorubicin distribution in multidrug resistant and sensitive cells by laserscan microscopy and digital image processing. Cytometry. 1992;13:572–576. [Google Scholar]
  16. Diociaiuti M, Molinari A, Calcabrini A, Arancia G. Electron energy-loss spectroscopy analysis of adriamycinplasma membrane interaction. J Microsc. 1991;164:95–106. doi: 10.1111/j.1365-2818.1991.tb03196.x. [DOI] [PubMed] [Google Scholar]
  17. Diociaiuti M, Molinari A, Calcabrini A, Arancia G, Isacchi G, Bordi F, Cametti C. Alteration of the passive-electrical properties of adriamycin-treated red cell membrane deduced from dielectric spectroscopy. Bioelectrochem Bioenerg. 1991;26:177–192. doi: 10.1016/0302-4598(91)80022-U. [DOI] [Google Scholar]
  18. Diociaiuti M, Calcabrini A, Meschini S, Arancia G. Intracellular mapping of 4′-deoxy-4′-iododoxorubicin in sensitive and multidrug resistant cells by electron spectroscopic imaging. Micron. 1997;28:389–395. doi: 10.1016/S0968-4328(97)00047-4. [DOI] [PubMed] [Google Scholar]
  19. Duarte-Karim M, Ruysschaert JM, Hildebrand J. Affinity of adriamycin to phospholipids: a possible explanation for cardiac mitochondrial lesions. Biochim Biophys Acta. 1976;71:658–663. doi: 10.1016/0006-291x(76)90838-x. [DOI] [PubMed] [Google Scholar]
  20. Duffy PM, Hayes MC, Cooper A, Smart CJ. Confocal microscopy of idarubicin localization in sensitive and multidrug-resistant bladder cancer cell lines. Br J Cancer. 1996;74:906–909. doi: 10.1038/bjc.1996.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fojo A, Akiyama S, Gottesman MM, Pastan I. Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines. Cancer Res. 1985;45:3002–3007. [PubMed] [Google Scholar]
  22. Fragu P, Klijanienko J, Gandia D, Halpern S, Armand JP. Quantitative mapping of 4′-iododeoxyrubicin in metastatic squamous cell carcinoma by secondary ion mass spectroscopy (SIMS) microscopy. Cancer Res. 1992;52:974–977. [PubMed] [Google Scholar]
  23. Frezard F, Garnier-Suillerot A. DNA-containing liposomes as a model for the study of cell membrane permeation by anthracyclines derivatives. Biochemistry. 1991;30:5038–5043. doi: 10.1021/bi00234a028. [DOI] [PubMed] [Google Scholar]
  24. Fritzsche H, Triebel H, Chaires JB, Dattagupta W, Crothers DM. Studies on interaction of anthracyclines antibiotics and DNA. Biochemistry. 1982;21:3940–3946. doi: 10.1021/bi00260a006. [DOI] [PubMed] [Google Scholar]
  25. Gervasoni JE, Jr, Fields SZ, Krishna S, Baker MA, Rosado M, Thuraisamy K, Hindenburg AA, Taub RN. Subcellular distribution of daunorubicin in P-glycoprotein-positive and-negative drug-resistant cell lines using laser-assisted confocal microscopy. Cancer Res. 1991;51:4955–4963. [PubMed] [Google Scholar]
  26. Goormaghtigh E, Ruysschaert JM. Anthracycline glycoside-membrane interactions. Biochim Biophys Acta. 1984;779:271–288. doi: 10.1016/0304-4157(84)90013-3. [DOI] [PubMed] [Google Scholar]
  27. Guffy MM, North JA, Burns CP. Effects of cellular fatty acids alteration on adriamycin sensitivity in cultured L1210 murine leukemia cells. Cancer Res. 1984;44:1863–1866. [PubMed] [Google Scholar]
  28. Henry N, Fantine EO, Bolard J, Garnier-Suillerot A. Interaction of adriamycin with negatively charged model membranes: evidence of two types of binding sites. Biochemistry. 1985;24:7085–7092. doi: 10.1021/bi00346a010. [DOI] [PubMed] [Google Scholar]
  29. Hindenburg AA, Gervasoni JE, Jr, Krishna S, Stewart VJ, Rosado M, Lutzky J, Bhala K, Baker MA, Taub RN. Intracellular distribution and pharmacokinetics of daunorubicin in anthracycline-sensitive and-resistant HL60 cells. Cancer Res. 1989;49:4607–4614. [PubMed] [Google Scholar]
  30. Huxham HG, Pinedo HM, Schuurhuis GJ, Joenje H. The use of parallel EELS spectral imaging and elemental mapping in the rapid assessment of anti-cancer drug localization. J Microsc. 1992;166:367–380. doi: 10.1111/j.1365-2818.1992.tb01535.x. [DOI] [PubMed] [Google Scholar]
  31. Keizer HG, Schurrhuis GJ, Broxtermann HJ, Landelma J, Schoonen WGEJ, van Rijn T, Pinedo HJ, Joenje H. Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expressed in cultured SW-1573 human lung tumor cells. Cancer Res. 1989;49:2988–2993. [PubMed] [Google Scholar]
  32. Koh HK. Cutaneous melanoma. New Engl J Med. 1991;325:171–182. doi: 10.1056/NEJM199107183250306. [DOI] [PubMed] [Google Scholar]
  33. Lewis W, Gonzales B. Anthracycline effects on actin and actin containing thin filaments in cultured neonatal rat myocardial cells. Lab Invest. 1986;54:416–423. [PubMed] [Google Scholar]
  34. Lothstein L, Wright HM, Sweatman TW, Israel M. N-benzyladriamycin-14-valerate and drug resistance: correlation of anthracycline structural modification with intracellular accumulation and distribution in multidrug resistant cells. Oncol Res. 1992;4:341–347. [PubMed] [Google Scholar]
  35. Meschini S, Molinari A, Calcabrini A, Citro G, Arancia G. Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study. J Microsc. 1994;176:204–210. doi: 10.1111/j.1365-2818.1994.tb03516.x. [DOI] [PubMed] [Google Scholar]
  36. Mizuno NS, Zakis B, Decker RW. Binding of daunomycin to DNA and the inhibition of RNA and DNA synthesis. Cancer Res. 1975;35:1542–1546. [PubMed] [Google Scholar]
  37. Molinari A, Calcabrini A, Crateri P, Arancia G. Interaction of anthracyclinic antibiotics with cytoskeletal components of cultured carcinoma cells. Exp Mol Pathol. 1990;53:11–33. doi: 10.1016/0014-4800(90)90021-5. [DOI] [PubMed] [Google Scholar]
  38. Molinari A, Calcabrini A, Crateri P, Arancia G. Effects of daunomycin on the microtubular network: a cytochemical study on a human melanoma cell line. Eur J Cell Biol. 1991;54:291–298. [PubMed] [Google Scholar]
  39. Molinari A, Cianfriglia M, Meschini S, Calcabrini A, Arancia G. P-glycoprotein expression in the Golgi apparatus of multidrug-resistant cells. Int J Cancer. 1994;59:789–795. doi: 10.1002/ijc.2910590614. [DOI] [PubMed] [Google Scholar]
  40. Molinari A, Calcabrini A, Meschini S, Stringaro A, DelBufalo D, Cianfriglia M, Arancia G. Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells. Int J Cancer. 1998;75:885–893. doi: 10.1002/(SICI)1097-0215(19980316)75:6<885::AID-IJC11>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  41. Monparler RL, Karon M, Siegel SE, Avila E. Effects of adriamycin on DNA, RNA and protein synthesis in cell free systems and intact cells. Cancer Res. 1976;36:2891–2895. [PubMed] [Google Scholar]
  42. Oth D, Begin M, Bischoff P, Leroux JY, Mercier G, Bruneau C. Induction by adriamycin and mitomycin C of modifications in lipid composition, size distribution, membrane fluidity and permeability of cultured RDM4 lymphoma cells. Biochim Biophys Acta. 1987;900:198–208. doi: 10.1016/0005-2736(87)90334-8. [DOI] [PubMed] [Google Scholar]
  43. Peterson C, Trouet A. Transport and storage of daunorubicin and doxorubicin in cultured fibroblasts. Cancer Res. 1978;38:4645–4649. [PubMed] [Google Scholar]
  44. Rabkin SW, Sunga P. The effects of doxorubicin (adriamycin) on cytoplasmic microtubule system in cardiac cells. J Mol Cell Cardiol. 1987;19:1073–1083. doi: 10.1016/s0022-2828(87)80352-8. [DOI] [PubMed] [Google Scholar]
  45. Ralph WE, Marshall B, Darkin S. Anticancer drugs which intercalate DNA: how do they act? TIBS. 1983;8:212–214. [Google Scholar]
  46. Rutherford AV, Willingham MC. Ultrastructural localization of daunomycin in multidrug resistant cells with modulation of the multidrug transporter. J Histochem Cytochem. 1993;41:1573–1577. doi: 10.1177/41.10.7902372. [DOI] [PubMed] [Google Scholar]
  47. Saeki T, Ueda K, Tanigawara Y, Hori R, Komano T. Human P-glycoprotein transports cyclosporin A and FK506. J Biol Chem. 1993;268:6077–6080. [PubMed] [Google Scholar]
  48. Safa AR, Glover CJ, Sewell JL, Meyers MB, Biedler JL, Felsted RL. Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blocker. J Biol Chem. 1987;262:7884–7888. [PubMed] [Google Scholar]
  49. Schadendorf D, Makki A, Stahr C, van Dick A, Wanner R, Scheffer GL, Flens MJ, Scheper R, Henz BM. Membrane transport proteins associated with drug resistance expressed in human melanoma. Am J Pathol. 1995;147:1545–1552. [PMC free article] [PubMed] [Google Scholar]
  50. Scheper RJ, Broxterman HJ, Scheffer JL, Kaaijk P, Dalton WS, van Heiningen THM, van Kalken CK, Slovack ML, DeVries EGE, vander Valk P, Meijer CJLM, Pinedo HM. Overexpression of a 110 kD vesicular protein in non-P-glycoprotein mediated multidrug resistance. Cancer Res. 1993;53:1475–1479. [PubMed] [Google Scholar]
  51. Schurrhuis GJ, Broxteman HJ, Cervantes A, van Heijningen THM, de Lange JHM, Baak JPA, Pinedo HM, Lankelma J. Quantitative determination of factors contributing to doxorubicin resistance in multidrug-resistant cells. J Natl Cancer Inst. 1989;81:1887–1892. doi: 10.1093/jnci/81.24.1887. [DOI] [PubMed] [Google Scholar]
  52. Schurrhuis GJ, Broxteman HJ, de Lange JHM, Pinedo HM, van Heijningen THM, Kuiper CK, Baak JPA, Lankelma J. Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein. Br J Cancer. 1991;64:857–861. doi: 10.1038/bjc.1991.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schurrhuis GJ, van Heijningen THM, Cervantes A, Pinedo HM, de Lange JHM, Keizer HG, Broxteman HJ, Baak JPA, Lankelma J. Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumor cells. Br J Cancer. 1993;68:898–908. doi: 10.1038/bjc.1993.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sehested M, Skovsgaard T, van Deurs B, Winther-Nielsen H. Increase in nonspecific adsorptive endocytosis in anthracycline-and vinca alkaloid-resistant Ehrlich ascites tumor cell lines. J Natl Cancer Inst. 1987;78:171–179. doi: 10.1093/jnci/78.1.171. [DOI] [PubMed] [Google Scholar]
  55. Seidel A, Hasmann M, Loser R, Bunge A, Schaefer B, Herzig I, Steidtmann K, Dietel M. Intracellular localization, vesicular accumulation and kinetics of daunorubicin in sensitive and multidrug-resistant gastric carcinoma EPG85-257 cells. Wirchows Archiv. 1995;426:249–256. doi: 10.1007/BF00191362. [DOI] [PubMed] [Google Scholar]
  56. Siegfried J, Kennedy AK, Sartorelli AC, Tritton TR. The role of membranes in the mechanism of action of the antineoplastic agent adriamycin. J Biochem Chem. 1983;258:339–343. [PubMed] [Google Scholar]
  57. Toffoli G, Corona G, Simone F, Gigante M, De Angeli S, Boiocchi M. Cellular pharmacology of idarubicinol in multidrug-resistant LoVo cell lines. Int J Cancer. 1996;67:129–137. doi: 10.1002/(SICI)1097-0215(19960703)67:1<129::AID-IJC21>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  58. Tritton TR, Yee G. The anticancer agent adriamycin can be actively cytotoxic without entering the cells. Science. 1982;217:248–250. doi: 10.1126/science.7089561. [DOI] [PubMed] [Google Scholar]
  59. White JG, Amos WB, Fordham M. An evaluation of confocal vs conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol. 1987;105:41–48. doi: 10.1083/jcb.105.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Willingham MC, Cornwell MM, Cardarelli CO, Gottesman MM, Pastan I. Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and-sensitive KB cells: effect of verapamil and other drugs. Cancer Res. 1986;46:5941–5946. [PubMed] [Google Scholar]
  61. Willingham MC, Richert ND, Cornwell MM, Tsuruo T, Hamada H, Gottesman MM, Pastan I. Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells. J Histochem Cytochem. 1987;35:1451–1456. doi: 10.1177/35.12.2890686. [DOI] [PubMed] [Google Scholar]
  62. Wilson T. Confocal microscopy. San Diego: Academic Press Inc; 1990. [Google Scholar]
  63. Zaman GRL, Versantvoort OHM, Smit JJM, Eijdems EWHM, De Haas M, Smith AJ, Broxterman HJ, Mulder NH, De Vries EGE, Baas F, Borst P. Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines. Cancer Res. 1993;53:1747–1750. [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES