FLOW CYTOMETRIC ANALYSIS AND CONFOCAL IMAGING OF ANTICANCER ALKYLAMINOANTHRAQUINONES AND THEIR N-OXIDES IN INTACT HUMAN-CELLS USING 647-NM KRYPTON LASER EXCITATION

Flow cytometry and laser-scanning confocal fluorescence microscopy hav e been used in the study of the pharmacodynamics, in single intact cel ls, of two novel alkylaminoanthraquinones (AQ4 and AQ6), structurally based upon the mid-red excitable but very weakly fluorescent anticance r agent mitoxantrone, together with their respective N-oxide derivativ es (AQ4NO and AQ6NO). The drug design rationale was that N-oxide modif ications generates prodrug forms suitable for selective bioreductive-a ctivation in hypoxic tumor cells. DNA-binding ranked in the order of m itoxantrone >AQ6 >AQ4 >AQ6NO >>AQ4NO. Using both cytometric methods a similar ranking was found for whole cell and nuclear location in human transformed fibroblasts. However, AQ6 showed enhanced nuclear uptake compared with mitoxantrone, in keeping with its greater capacity to in hibit DNA synthesis. Partial charge neutralisation by N-oxide derivati zation resulted in loss of DNA synthesis inhibition but retention of t he ability to accumulate in the cytosol, an important property for pro drug development. We conclude that both flow cytometry and confocal im aging revealed biologically significant differences between analogues for subcellular distribution and retention properties, The study demon strates the potential for these complementary 647-nm krypton laser lin e-based fluorometric methods to provide relevant structure-activity in formation in anthraquinone drug-design programmes. (C) 1997 Wiley-Liss , Inc.