Abstract
Acquired resistance to anthracyclines is characterised by a lower sensitivity to these agents, associated with impaired accumulation of drug. We have examined the ability of aclacinomycin A (ACM) associated with doxorubicin (DOX), to increase intranuclear DOX concentrations and, consequently, to enhance cytotoxic effects against drug resistant cells in vitro. A recently developed microspectrofluorometric technique is used to measure intranuclear DOX concentrations in sensitive and DOX-resistant K562 cells treated with DOX and ACM. Fluorescence emission spectra are collected from a microvolume of single living cell nuclei. From both DOX and ACM model fluorescence spectra (free, DNA-bound and metabolites), the intranuclear spectral profile is analysed according to the amount of each component. This quantitative analysis determines intranuclear DOX concentrations with an error of 10%. Non-cytotoxic doses of ACM, in combination with DOX, increase cytotoxic activity of DOX against K562 resistant cells. When DOX-resistant cells are exposed simultaneously to ACM and DOX, significant increases in intranuclear DOX concentrations are found compared with the case of exposure to DOX alone. The measure of the intranuclear retention of DOX shows that ACM partly blocks the DOX efflux in resistant cell nuclei, resulting in enhanced accumulation of DOX. These data lead us to conclude that ACM-DOX association partly reverses the DOX resistance at clinically achievable concentrations.
Highlights
With the recent development of microspectrofluorometry, we have studied fluorescence signals from microvolumes within a single living cell (Ginot et al, 1984; Manfait et al, 1987)
We have demonstrated the importance of the intranuclear concentration, since the cytotoxic effect induced by DOX was dependent on the amount of drug incorporated into the nucleus (Gigli et al, 1989)
(10pgrml'), in combination with DOX or daunomycin, increased the intracellular amount of the latter compounds with concomitant increased cytotoxicity against resistant cells (Tapiero et al, 1988). We have extended these inhibition growth studies with non-toxic doses of ACM against DOX-resistant K562 cells in order to determine which kind of exposure with ACM and DOX, simultaneously or sequentially, produced the most cytotoxicity
Summary
Stock solutions (10 jAM) of DOX, ACM and ACM metabolites (Laboratoires Roger Bellon, Paris, France) were prepared in Dulbecco's PBS Drug concentrations in PBS solution were determined by their absorbances at 480 nm for DOX and at 430 nm for ACM. Calf thymus DNA (Sigma Chemical Company, type I) was dissolved in Dulbecco's PBS. Concentration of DNA (phosphate) was estimated on the basis of a molar absorption coefficient e = 6600 M-' cm ' at 260 nm. The appropriate amounts of drugs were added to DMEM (Gibco), supplemented with 10% fetal calf serum (Seromed) and 2 mM L-glutamine.
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