Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids

Int J Cancer. 1998 Mar 16;75(6):855-63. doi: 10.1002/(sici)1097-0215(19980316)75:6<855::aid-ijc7>;2-u.


Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400+/-70 microm), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100+/-50 microm), which consist entirely of proliferating cells. After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity. At a depth 80 microm from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids. The P-glycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect. Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 microm distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence. This was corroborated by whole-cell patch-clamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Cell Division
  • Chloride Channels / physiology
  • Doxorubicin
  • Drug Resistance, Multiple*
  • Drug Resistance, Neoplasm*
  • Genistein / pharmacology
  • Humans
  • Male
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Prostatic Neoplasms / pathology*
  • Spheroids, Cellular
  • Tumor Cells, Cultured
  • Verapamil / pharmacology


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Chloride Channels
  • Doxorubicin
  • Verapamil
  • Genistein