Determination and reversal of resistance to epirubicin intravesical chemotherapy. A confocal imaging study

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Abstract

Objective

To evaluate the use of confocal microscopy in the study of resistance to epirubicin and to determine the effect of temperature, viability and a resistance-reversing agent on the intracellular distribution of this drug in sensitive and resistant derivatives of a superficial bladder cancer cell line.

Materials and methods

Viable and non-viable adherent cells were incubated in epirubicin solutions under various conditions. After incubation, the distribution of intracellular epirubicin fluorescence was visualized using confocal microscopy and a × 50 water-immersion lens.

Results

There was a striking and consistent difference between resistant and sensitive cells in the intracellular distribution of the drug. In addition to having greater overall levels of epirubicin fluorescence, sensitive cells accumulated epirubicin predominantly in the nucleus. Epirubicin fluorescence in resistant cells was cytoplasmic and granular in appearance. When incubated at 0°C, both cell lines showed no nuclear uptake and thus resembled resistant cells at 37°C. However, dead cells rapidly acquired brightly fluorescent nuclei. The resistance-reversing agent verapamil appeared to cause reversion of the resistant to the sensitive phenotype.

Conclusion

Confocal microscopy allows epirubicin-sensitive and resistant cultured tumour cells to be differentiated reliably and provides information about the mechanisms of action of, and resistance to, epirubicin. Applying this technique to clinical specimens should enable patients who have the resistant phenotype to be detected and the efficacy of intravesical resistance-reversing agents to be evaluated in such cases.

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