Modification of uptake and subcellular distribution of doxorubicin by N-acylhydrazone residues as visualised by intrinsic fluorescence.

Purpose: Doxorubicin (1) is commonly used in the treatment of a wide range of cancers. Some N-acylhydrazones of 1 were previously found to have an improved tumour and organ selectivity. In order to clarify the molecular basis for this effect, the cellular uptake into various cancer cells and the localisation in PtK(2) potoroo kidney cells of 1 and its N-acylhydrazones derived from heptadecanoic acid (2) and 11-(menthoxycarbonyl)undecanoic acid (3) were studied drawing on their intrinsic fluorescence.

Methods: The uptake of compounds 1-3 into human cells of HL-60 leukaemia, 518A2 melanoma, HT-29 colon, and resistant KB-V1/Vbl and MCF-7/Topo breast carcinomas was determined fluorometrically from their residual amounts in the supernatant. Their time-dependent accumulation in PtK(2) potoroo kidney cells was visualised by fluorescence microscopy.

Results: The uptake, though not the cytotoxicity, of 2 in multi-drug resistant MCF-7/Topo breast cancer cells was conspicuously greater than that of 1 and 3, probably due to an attractive lipophilic interaction with the lipid-rich membranes of these cells. In non-malignant PtK(2) cells, both 1 and 3 accumulated initially in the nuclei. Upon prolonged incubation, their fluorescent metabolites were visualised in lysosomes neighbouring the nuclei. In contrast, conjugate 2 was not observed in the nuclei at any time. After 2 h, it had accumulated in vesicles scattered all over the cells, and upon prolonged incubation, its fluorescent metabolites were concentrated in the cellular membrane.

Conclusions: Long unbranched fatty acyl residues when attached to doxorubicin via a hydrazone can act as lipophilic membrane anchors. This allows an increased uptake of such derivatives into lipid-rich membranes especially of multi-drug resistant cancer cells, a retarded release from there into the cytosol and the eventual storage of their metabolites again in the cell membrane rather than in lysosomes.