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Rho(0) tumor cells: a model for studying whether mitochondria are targets for rhodamine 123, doxorubicin, and other
1Department of Cell Biology and Anatomy and the Sylvester Comprehensive Cancer Center, Miami, FL 33101, USA.
Abstract:
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.
Insights
Mitochondria are not targets for common chemotherapy drugs like doxorubicin. However, functional mitochondria are crucial for the accumulation and toxicity of rhodamine 123, impacting drug targeting strategies.
Area of Science:
- Cell Biology
- Pharmacology
- Cancer Research
Background:
- Mitochondrial DNA (mtDNA) absence in rho(0) cells prevents electron transport and oxidative phosphorylation.
- Osteosarcoma cell lines (rho(0) and wt) were used to model drug targeting.
- MDR1 and MRP1 gene expression was low in both cell lines, indicating minimal efflux pump activity.
Purpose of the Study:
- To investigate the role of functional mitochondria in cellular drug accumulation and cytotoxicity.
- To determine if mitochondria are targets for common chemotherapy agents.
- To understand the mechanism of verapamil's effect on drug retention in non-P-glycoprotein expressing cells.
Main Methods:
- Comparison of drug-induced growth inhibition and intracellular accumulation in rho(0) and wt osteosarcoma cells.
- Fluorescence microscopy and flow cytometry were used to measure drug accumulation.
- Assessment of gene expression for MDR1 and MRP1 efflux pumps.
Main Results:
- Doxorubicin, vinblastine, and paclitaxel showed similar toxicity in both cell lines, suggesting mitochondria are not their primary target.
- Rhodamine 123 accumulation and toxicity were significantly higher in wild-type cells compared to rho(0) cells.
- Verapamil enhanced doxorubicin and rhodamine 123 retention in both cell types, independent of P-glycoprotein.
Conclusions:
- Functional mitochondria are essential for the accumulation and cytotoxicity of lipophilic cations like rhodamine 123.
- Mitochondria are not targets for the growth inhibitory effects of doxorubicin, vinblastine, or paclitaxel.
- Verapamil's effect on drug accumulation may involve direct interaction with mitochondrial membrane potential or unidentified plasma membrane transporters.
