Cancer Chemotherapy and Pharmacology

, Volume 68, Issue 5, pp 1179–1190 | Cite as

Accumulation and distribution of doxorubicin in tumour spheroids: the influence of acidity and expression of P-glycoprotein

  • Howard R. Mellor
  • Richard CallaghanEmail author
Original Article



The intra-tumour distribution of anticancer drugs remains an important, but often under-estimated, influence on drug efficacy. Tumour acidity and the presence of efflux pumps were examined for their influence on the distribution of doxorubicin in a solid tumour model.


Anticancer drug distribution and overall accumulation was measured in tumour spheroids (TS) of varying sizes. The distribution profiles were examined in normoxic and hypoxic TS, the latter generating metabolic acidosis. Finally, the drug distribution profiles were related to efficacy using radial outgrowth assays.


In large tumour spheroids (TS) (d ~500 μm), intracellular accumulation of doxorubicin was restricted to cells in the outermost layers and failed to accumulate within the viable cells in the ‘intermediate’ hypoxic zone. A similar profile was obtained for another protonatable amine, 7-AAD. In contrast, the distribution of the non-ionisable drug (at physiological pH) BODIPY-Taxol was uniform throughout the TS. In order to independently model the hypoxic and normoxic zones of TS, we compared drug accumulation in small entirely normoxic TS (d ~200 μm) with equivalent sized ones exposed to hypoxia in an anaerobic chamber. Exposure of TS to hypoxia caused a considerable reduction in the pH of the bathing medium and lower tissue accumulation of doxorubicin. Interstitial acidity reduces the proportion of doxorubicin in the non-ionised form.


In TS, the accumulation and distribution of doxorubicin was influenced by both the expression of P-glycoprotein and hypoxia-induced acidity. Therefore, optimisation of doxorubicin chemotherapy for hypoxic tumours will require circumvention of both of these crucial pharmacokinetic determinants.


P-glycoprotein Doxorubicin Solid tumours Bioenergetic metabolism Tumour spheroid Cancer chemotherapy Drug resistance Hypoxia Tumour acidity 



This research was funded by a Cancer Research UK Program grant (SP1861/0401) awarded to RC.


  1. 1.
    Baguley BC (2010) Multidrug resistance in cancer. Methods Mol Biol 596:1–14. doi: 10.1007/978-1-60761-416-6_1 Google Scholar
  2. 2.
    Gillet J-P, Gottesman MM (2010) Mechanisms of multidrug resistance in cancer. Methods Mol Biol 596:47–76. doi: 10.1007/978-1-60761-416-6_4 Google Scholar
  3. 3.
    Mellor HR, Callaghan R (2008) Resistance to chemotherapy in cancer: a complex and integrated cellular response. Pharmacology 81(4):275–300PubMedCrossRefGoogle Scholar
  4. 4.
    Tredan O, Galmarini CM, Patel K, Tannock IF (2007) Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst 99(19):1441–1454. doi: djm135[pii]10.1093/jnci/djm135 PubMedCrossRefGoogle Scholar
  5. 5.
    Hietanen P, Blomqvist C, Wasenius VM, Niskanen E, Franssila K, Nordling S (1995) Do DNA ploidy and S-phase fraction in primary tumour predict the response to chemotherapy in metastatic breast cancer? Br J Cancer 71(5):1029–1032PubMedCrossRefGoogle Scholar
  6. 6.
    Jackson RC (1989) The problem of the quiescent cancer cell. Adv Enzyme Regul 29:27–46PubMedCrossRefGoogle Scholar
  7. 7.
    Siu WY, Arooz T, Poon RY (1999) Differential responses of proliferating versus quiescent cells to adriamycin. Exp Cell Res 250(1):131–141. doi: 10.1006/excr.1999.4551S0014-4827(99)94551-2[pii] PubMedCrossRefGoogle Scholar
  8. 8.
    Jiricny J (2006) The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 7(5):335–346PubMedCrossRefGoogle Scholar
  9. 9.
    Rosell R, Taron M, Barnadas A, Scagliotti G, Sarries C, Roig B (2003) Nucleotide excision repair pathways involved in Cisplatin resistance in non-small-cell lung cancer. Cancer Control 10(4):297–305PubMedGoogle Scholar
  10. 10.
    Trivedi RN, Almeida KH, Fornsaglio JL, Schamus S, Sobol RW (2005) The role of base excision repair in the sensitivity and resistance to temozolomide-mediated cell death. Cancer Res 65(14):6394–6400PubMedCrossRefGoogle Scholar
  11. 11.
    Deng X, Kornblau SM, Ruvolo PP, May WS Jr (2001) Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance. J Natl Cancer Inst Monogr 2000(28):30–37Google Scholar
  12. 12.
    Post LE (2002) Selectively replicating adenoviruses for cancer therapy: an update on clinical development. Curr Opin Investig Drugs 3(12):1768–1772PubMedGoogle Scholar
  13. 13.
    Sjostrom J, Blomqvist C, von Boguslawski K, Bengtsson NO, Mjaaland I, Malmstrom P, Ostenstadt B, Wist E, Valvere V, Takayama S, Reed JC, Saksela E (2002) The predictive value of bcl-2, bax, bcl-xL, bag-1, fas, and fasL for chemotherapy response in advanced breast cancer. Clin Cancer Res 8(3):811–816PubMedGoogle Scholar
  14. 14.
    Giaccone G, Gazdar AF, Beck H, Zunino F, Capranico G (1992) Multidrug sensitivity phenotype of human lung cancer cells associated with topoisomerase II expression. Cancer Res 52(7):1666–1674PubMedGoogle Scholar
  15. 15.
    Gokmen-Polar Y, Escuin D, Walls CD, Soule SE, Wang Y, Sanders KL, Lavallee TM, Wang M, Guenther BD, Giannakakou P, Sledge GW Jr (2005) Beta-tubulin mutations are associated with resistance to 2-methoxyestradiol in MDA-MB-435 cancer cells. Cancer Res 65(20):9406–9414PubMedCrossRefGoogle Scholar
  16. 16.
    Hari M, Loganzo F, Annable T, Tan X, Musto S, Morilla DB, Nettles JH, Snyder JP, Greenberger LM (2006) Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules. Mol Cancer Ther 5(2):270–278PubMedCrossRefGoogle Scholar
  17. 17.
    Matsumoto Y, Takano H, Nagao S, Fojo T (2001) Altered topoisomerase IIalpha and multidrug resistance-associated protein levels during drug selection: adaptations to increasing drug pressure. Jpn J Cancer Res 92(9):968–974PubMedGoogle Scholar
  18. 18.
    Desoize B, Jardillier J (2000) Multicellular resistance: a paradigm for clinical resistance? Crit Rev Oncol Hematol 36(2–3):193–207. doi: S104084280000086X[pii] PubMedCrossRefGoogle Scholar
  19. 19.
    Tannock IF, Lee CM, Tunggal JK, Cowan DS, Egorin MJ (2002) Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy. Clin Cancer Res 8(3):878–884PubMedGoogle Scholar
  20. 20.
    Konerding MA, Fait E, Gaumann A (2001) 3D microvascular architecture of pre-cancerous lesions and invasive carcinomas of the colon. Br J Cancer 84(10):1354–1362. doi: 10.1054/bjoc.2001.1809S0007092001918099[pii] PubMedCrossRefGoogle Scholar
  21. 21.
    Modok S, Hyde P, Mellor HR, Roose T, Callaghan R (2006) Diffusivity and distribution of vinblastine in three-dimensional tumour tissue: Experimental and mathematical modelling. Eur J Cancer 42:2404–2413PubMedCrossRefGoogle Scholar
  22. 22.
    Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, Jain RK (2004) Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 64(11):3731–3736. doi: 10.1158/0008-5472.CAN-04-007464/11/3731[pii] PubMedCrossRefGoogle Scholar
  23. 23.
    Heldin CH, Rubin K, Pietras K, Ostman A (2004) High interstitial fluid pressure––an obstacle in cancer therapy. Nat Rev Cancer 4(10):806–813. doi: nrc1456[pii]10.1038/nrc1456 PubMedCrossRefGoogle Scholar
  24. 24.
    Patel KJ, Tannock IF (2009) The influence of P-glycoprotein expression and its inhibitors on the distribution of doxorubicin in breast tumors. BMC Cancer 9:356. doi: 1471-2407-9-356[pii]10.1186/1471-2407-9-356 PubMedCrossRefGoogle Scholar
  25. 25.
    Primeau AJ, Rendon A, Hedley D, Lilge L, Tannock IF (2005) The distribution of the anticancer drug doxorubicin in relation to blood vessels in solid tumors. Clin Cancer Res 11(24):8782–8788. doi: 11/24/8782[pii]10.1158/1078-0432.CCR-05-1664 PubMedCrossRefGoogle Scholar
  26. 26.
    Matherly LH (2001) Molecular and cellular biology of the human reduced folate carrier. Prog Nucleic Acid Res Mol Biol 67:131–162PubMedCrossRefGoogle Scholar
  27. 27.
    Gorlick R, Goker E, Trippett T, Steinherz P, Elisseyeff Y, Mazumdar M, Flintoff WF, Bertino JR (1997) Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression. Blood 89(3):1013–1018PubMedGoogle Scholar
  28. 28.
    Ifergan I, Meller I, Issakov J, Assaraf YG (2003) Reduced folate carrier protein expression in osteosarcoma: implications for the prediction of tumor chemosensitivity. Cancer 98(9):1958–1966. doi: 10.1002/cncr.11741 PubMedCrossRefGoogle Scholar
  29. 29.
    Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2(1):48–58PubMedCrossRefGoogle Scholar
  30. 30.
    Modok S, Mellor HR, Callaghan R (2006) Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer. Curr Opin Pharmacol 6(4):350–354PubMedCrossRefGoogle Scholar
  31. 31.
    Keppler D, Konig J (2000) Hepatic secretion of conjugated drugs and endogenous substances. Semin Liver Dis 20(3):265–272PubMedCrossRefGoogle Scholar
  32. 32.
    Loe DW, Almquist KC, Cole SP, Deeley RG (1996) ATP-dependent 17 beta-estradiol 17-(beta-D-glucuronide) transport by multidrug resistance protein (MRP). Inhibition by cholestatic steroids. J Biol Chem 271(16):9683–9689PubMedCrossRefGoogle Scholar
  33. 33.
    Loe DW, Stewart RK, Massey TE, Deeley RG, Cole SP (1997) ATP-dependent transport of aflatoxin B1 and its glutathione conjugates by the product of the multidrug resistance protein (MRP) gene. Mol Pharmacol 51(6):1034–1041PubMedGoogle Scholar
  34. 34.
    Adams DJ (2005) The impact of tumor physiology on camptothecin-based drug development. Curr Med Chem Anticancer Agents 5(1):1–13PubMedCrossRefGoogle Scholar
  35. 35.
    Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324(5930):1029–1033. doi: 324/5930/1029[pii]10.1126/science.1160809 PubMedCrossRefGoogle Scholar
  36. 36.
    Yeluri S, Madhok B, Prasad KR, Quirke P, Jayne DG (2009) Cancer’s craving for sugar: an opportunity for clinical exploitation. J Cancer Res Clin Oncol 135(7):867–877. doi: 10.1007/s00432-009-0590-8 PubMedCrossRefGoogle Scholar
  37. 37.
    Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314PubMedCrossRefGoogle Scholar
  38. 38.
    Shimoda LA, Fallon M, Pisarcik S, Wang J, Semenza GL (2006) HIF-1 regulates hypoxic induction of NHE1 expression and alkalinization of intracellular pH in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 291(5):L941–L949. doi: 00528.2005[pii]10.1152/ajplung.00528.2005 PubMedCrossRefGoogle Scholar
  39. 39.
    Ullah MS, Davies AJ, Halestrap AP (2006) The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. J Biol Chem 281(14):9030–9037. doi: M511397200[pii]10.1074/jbc.M511397200 PubMedCrossRefGoogle Scholar
  40. 40.
    Wykoff CC, Beasley NJ, Watson PH, Turner KJ, Pastorek J, Sibtain A, Wilson GD, Turley H, Talks KL, Maxwell PH, Pugh CW, Ratcliffe PJ, Harris AL (2000) Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res 60(24):7075–7083PubMedGoogle Scholar
  41. 41.
    Comerford KM, Wallace TJ, Karhausen J, Louis NA, Montalto MC, Colgan SP (2002) Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 62(12):3387–3394PubMedGoogle Scholar
  42. 42.
    Wartenberg M, Ling FC, Muschen M, Klein F, Acker H, Gassmann M, Petrat K, Putz V, Hescheler J, Sauer H (2003) Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species. FASEB J 17(3):503–505. doi: 10.1096/fj.02-0358fje02-0358fje[pii] PubMedGoogle Scholar
  43. 43.
    Lee CM, Tannock IF (2006) Inhibition of endosomal sequestration of basic anticancer drugs: influence on cytotoxicity and tissue penetration. Br J Cancer 94(6):863–869. doi: 6603010[pii]10.1038/sj.bjc.6603010 PubMedCrossRefGoogle Scholar
  44. 44.
    Mellor HR, Ferguson DJ, Callaghan R (2005) A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs. Br J Cancer 93(3):302–309PubMedCrossRefGoogle Scholar
  45. 45.
    Mellor HR, Snelling S, Hall MD, Modok S, Jaffar M, Hambley TW, Callaghan R (2005) The influence of tumour microenvironmental factors on the efficacy of cisplatin and novel platinum (IV) complexes. Biochem Pharmacol 70(8):1137–1146PubMedCrossRefGoogle Scholar
  46. 46.
    Martin C, Walker J, Rothnie A, Callaghan R (2003) The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models. Br J Cancer 89:1581–1589PubMedCrossRefGoogle Scholar
  47. 47.
    Walker J, Martin C, Callaghan R (2004) Inhibition of P-glycoprotein function by XR9576 in a solid tumour model can restore anticancer drug efficacy. Eur J Cancer 40:594–605PubMedCrossRefGoogle Scholar
  48. 48.
    De Milito A, Fais S (2005) Tumor acidity, chemoresistance and proton pump inhibitors. Future Oncol 1(6):779–786. doi: 10.2217/14796694.1.6.779 PubMedCrossRefGoogle Scholar
  49. 49.
    Henning Ta, Kraus Mb, Brischwein Ma, AMa Otto, Ba Wolf (2004) Relevance of tumor microenvironment for progression, therapy and drug development. Anti-Cancer Drugs 15(1):7–14PubMedCrossRefGoogle Scholar
  50. 50.
    Marie JP, Zhou DC, Gurbuxani S, Legrand O, Zittoun R (1996) MDR1/P-glycoprotein in haematological neoplasms. Eur J Cancer 32A(6):1034–1038PubMedCrossRefGoogle Scholar
  51. 51.
    Trock BJ, Leonessa F, Clarke R (1997) Multidrug resistance in breast cancer: a meta-analysis of MDR1/gp170 expression and its possible functional significance. J Natl Cancer Inst 89(13):917–931PubMedCrossRefGoogle Scholar
  52. 52.
    Liu L, Ning X, Sun L, Zhang H, Shi Y, Guo C, Han S, Liu J, Sun S, Han Z, Wu K, Fan D (2008) Hypoxia-inducible factor-1 alpha contributes to hypoxia-induced chemoresistance in gastric cancer. Cancer Sci 99(1):121–128. doi: CAS643[pii]10.1111/j.1349-7006.2007.00643.x PubMedCrossRefGoogle Scholar
  53. 53.
    Fradette C, Batonga J, Teng S, Piquette-Miller M, du Souich P (2007) Animal models of acute moderate hypoxia are associated with a down-regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and up-regulation of CYP3A6 and P-glycoprotein in liver. Drug Metab Dispos 35(5):765–771. doi: dmd.106.013508[pii]10.1124/dmd.106.013508 PubMedCrossRefGoogle Scholar
  54. 54.
    Xiao-Dong L, Zhi-Hong Y, Hui-Wen Y (2008) Repetitive/temporal hypoxia increased P-glycoprotein expression in cultured rat brain microvascular endothelial cells in vitro. Neurosci Lett 432(3):184–187. doi: S0304-3940(07)01279-7[pii]10.1016/j.neulet.2007.12.017 PubMedCrossRefGoogle Scholar
  55. 55.
    Lotz C, Kelleher DK, Gassner B, Gekle M, Vaupel P, Thews O (2007) Role of the tumor microenvironment in the activity and expression of the p-glycoprotein in human colon carcinoma cells. Oncol Rep 17(1):239–244PubMedGoogle Scholar
  56. 56.
    Sauvant C, Nowak M, Wirth C, Schneider B, Riemann A, Gekle M, Thews O (2008) Acidosis induces multi-drug resistance in rat prostate cancer cells (AT1) in vitro and in vivo by increasing the activity of the p-glycoprotein via activation of p38. Int J Cancer 123(11):2532–2542. doi: 10.1002/ijc.23818 PubMedCrossRefGoogle Scholar
  57. 57.
    Thews O, Gassner B, Kelleher DK, Gekle M (2008) Activity of drug efflux transporters in tumor cells under hypoxic conditions. Adv Exp Med Biol 614:157–164. doi: 10.1007/978-0-387-74911-2_19 PubMedCrossRefGoogle Scholar
  58. 58.
    Netti PA, Hamberg LM, Babich JW, Kierstead D, Graham W, Hunter GJ, Wolf GL, Fischman A, Boucher Y, Jain RK (1999) Enhancement of fluid filtration across tumor vessels: implication for delivery of macromolecules. Proc Natl Acad Sci USA 96(6):3137–3142PubMedCrossRefGoogle Scholar
  59. 59.
    Stohrer M, Boucher Y, Stangassinger M, Jain RK (2000) Oncotic pressure in solid tumors is elevated. Cancer Res 60(15):4251–4255PubMedGoogle Scholar
  60. 60.
    Swietach P, Patiar S, Supuran CT, Harris AL, Vaughan-Jones RD (2009) The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths. J Biol Chem 284(30):20299–20310. doi: M109.006478[pii]10.1074/jbc.M109.006478 PubMedCrossRefGoogle Scholar
  61. 61.
    Glunde K, Düßmann H, Juretschke H-P, Leibfritz D (2002) Na < sup >+</sup >/H < sup >+</sup > exchange subtype 1 inhibition during extracellular acidification and hypoxia in glioma cells. J Neurochem 80(1):36–44PubMedCrossRefGoogle Scholar
  62. 62.
    Whittingtons DA, Grubb JH, Waheed A, Shah GN, Sly WS, Christianson DW (2004) Expression, assay, and structure of the extracellular domain of murine carbonic anhydrase XIV. J Biol Chem 279(8):7223–7228. doi: 10.1074/jbc.M310809200 CrossRefGoogle Scholar
  63. 63.
    Winum J-Y, Rami M, Scozzafava A, Montero J-L, Supuran C (2008) Carbonic anhydrase IX: a new druggable target for the design of antitumor agents. Med Res Rev 28(3):445–463PubMedCrossRefGoogle Scholar
  64. 64.
    Kaluz S, Kaluzová M, Liao S-Y, Lerman M, Stanbridge EJ (2009) Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: a one transcription factor (HIF-1) show? Biochim Biophys Acta (BBA)––Rev Cancer 1795(2):162–172CrossRefGoogle Scholar
  65. 65.
    Rodriguez-Enriquez S, Gallardo-Perez JC, Aviles-Salas A, Marin-Hernandez A, Carreno-Fuentes L, Maldonado-Lagunas V, Moreno-Sanchez R (2008) Energy metabolism transition in multi-cellular human tumor spheroids. J Cell Physiol 216(1):189–197. doi: 10.1002/jcp.21392 PubMedCrossRefGoogle Scholar
  66. 66.
    Enerson BE, Drewes LR (2003) Molecular features, regulation, and function of monocarboxylate transporters: Implications for drug delivery. J Pharm Sci 92(8):1531–1544PubMedCrossRefGoogle Scholar
  67. 67.
    Sonveaux P, Vegran F, Schroeder T, Wergin MC, Verrax J, Rabbani ZN, De Saedeleer CJ, Kennedy KM, Diepart C, Jordan BF, Kelley MJ, Gallez B, Wahl ML, Feron O, Dewhirst MW (2008) Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J Clin Invest 118(12):3930–3942. doi: 36843[pii]10.1172/JCI36843 PubMedGoogle Scholar
  68. 68.
    Semenza GL (2008) Tumor metabolism: cancer cells give and take lactate. J Clin Invest 118(12):3835–3837PubMedGoogle Scholar
  69. 69.
    Miraglia E, Viarisio D, Riganti C, Costamagna C, Ghigo D, Bosia A (2005) Na +/H + exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin. Int J Cancer 115(6):924–929PubMedCrossRefGoogle Scholar

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© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Nuffield Department of Clinical Laboratory Sciences, John Radcliffe HospitalUniversity of OxfordOxfordUK

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