Rhodamine 123 (R123) is a mitochondria-specific prototype anticancer agent because its target is the energy-producing mechanism of the cell. The goal of this study was to investigate the relationship between intracellular R123 accumulation and cytotoxicity in a R123-sensitive cell line (RS) and a R123-resistant subline (RR) that we developed. Cytotoxicity after exposure to R123 (0-60 μg/ml) was assessed using the clonogenic assay. Intracellular R123 was extracted with acid-alcohol and measured by fluorimetry. The rate of R123 accumulation over 1 hr was significantly higher (P < 0.0001) for RS cells (4.65 ± 0.39 μg/min/106 cells) than for RR cells (1.29 ± 0.24 μg/min/106 cells). R123 accumulation in RS cells was strongly correlated (r = 0.80; P < 0.0001) with cytotoxicity. Treatment of RR cells with verapamil (100 μM) reversed R123 resistance. The resulting dose-survival curve was identical to the dose-response curve of RS cells treated with R123 alone. Cellular content of R123 in RR cells treated with verapamil increased to a level similar to that of RS cells and correlated with cytotoxicity. These data suggest that cytotoxicity of R123 in B16 cells results from increased cellular accumulation of R123.
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