Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids.
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.
Wartenberg
M
M
Institute for Neurophysiology, University of Cologne, Germany.
Frey
C
C
Diedershagen
H
H
Ritgen
J
J
Hescheler
J
J
Sauer
H
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D002455:Cell Division; D018118:Chloride Channels / Q000502:physiology; D004317:Doxorubicin; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011471:Prostatic Neoplasms / Q000473:pathology*; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018118:Chloride Channels; D004317:Doxorubicin; D014700:Verapamil; D019833:Genistein