Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids.
Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas. We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm). Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells. The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr. Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil. Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants. Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS. Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA). Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89. Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity.
Wartenberg
M
M
Department of Neurophysiology, University of Cologne, Cologne, Germany.
Fischer
K
K
Hescheler
J
J
Sauer
H
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D005985:Glyceraldehyde / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010641:Phenotype; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014812:Vitamin K / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D016877:Oxidants; D017382:Reactive Oxygen Species; D025521:Tumor Suppressor Proteins; D014812:Vitamin K; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005985:Glyceraldehyde; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D017868:Cyclic AMP-Dependent Protein Kinases