Rho(0) tumor cells: a model for studying whether mitochondria are targets for rhodamine 123, doxorubicin, and other drugs.
A human osteosarcoma cell line devoid of mitochondrial DNA (rho(0)) and its wild-type parental cell counterpart (wt) are presented as a model to investigate drug targeting. By virtue of the absence of mitochondrial DNA, rho(0) cells cannot perform electron transport or oxidative phosphorylation. Since most of the drugs studied are transported by the efflux pumping systems controlled by the MDR1 and MRP1 genes, both cell lines were examined for the expression of these genes, and it was found that no MDR1 and only low amounts of MRP1 were expressed. Growth inhibition experiments indicated that doxorubicin (Dox), vinblastine, and paclitaxel were equitoxic in these cell lines. On the other hand, the IC(50) for rhodamine 123 (Rho 123) in rho(0) cells was 50 times higher than in wt cells. This result correlates with a lower accumulation of Rho 123 in rho(0) cells as measured by fluorescence microscopy and flow cytometry (3 times less than in wt cells). In contrast, when stained with Dox, both cell types accumulated similar amounts. Surprisingly, in these non-P-glycoprotein expressing cells, verapamil increased both Dox and Rho 123 retention. Overall, these data suggest that: (i) functional mitochondria do not appear to be targets for the growth inhibitory activities of Dox, paclitaxel, or vinblastine; (ii) for lipophilic cations like Rho 123, however, normal functioning mitochondria and maintenance of a normal mitochondrial membrane potential (Deltapsi(mt)) appear to play a critical role in the intracellular accumulation and subsequent cytotoxicities of these compounds; and (iii) verapamil increases drug accumulation in non-P-glycoprotein expressing cell lines, most likely by direct action on Deltapsi(mt) for Rho 123 and safranin O, and on heretofore unidentified plasma membrane transporters, as well as via interaction with low levels of MRP1, for Dox. These results should be considered when Rho 123 and verapamil are used to detect P-glycoprotein.
Hu
Y
Y
Department of Cell Biology and Anatomy and the Sylvester Comprehensive Cancer Center, Miami, FL 33101, USA.
Moraes
C T
CT
Savaraj
N
N
Priebe
W
W
Lampidis
T J
TJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000172:deficiency / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D004347:Drug Interactions; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D020112:Rhodamine 123 / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D002121:Calcium Channel Blockers; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D020112:Rhodamine 123; D004317:Doxorubicin; D014700:Verapamil