Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their n‐oxides in intact human cells using 647‐nm krypton laser excitation

Abstract

Flow cytometry and laser-scanning confocal fluorescence microscopy have been used in the study of the pharmacodynamics, in single intact cells, of two novel alkylaminoanthraquinones (AQ4 and AQ6), structurally based upon the mid-red excitable but very weakly fluorescent anticancer agent mitoxantrone, together with their respective N-oxide derivatives (AQ4NO and AQ6NO). The drug design rationale was that N-oxide modifications generates prodrug forms suitable for selective bioreductive-activation in hypoxic tumor cells. DNA-binding ranked in the order of mitoxantrone > 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 inhibit DNA synthesis. Partial charge neutralisation by N-oxide derivatization resulted in loss of DNA synthesis inhibition but retention of the ability to accumulate in the cytosol, an important property for prodrug development. We conclude that both flow cytometry and confocal imaging revealed biologically significant differences between analogues for subcellular distribution and retention properties. The study demonstrates the potential for these complementary 647-nm krypton laser line-based fluorometric methods to provide relevant structure-activity information in anthraquinone drug-design programmes.

True

“…oxic or hypoxic) cell populations. However, with AQ4N the observed enhancement is maintained even when drug is administered up to 16 h before radiation in the SCCVII tumour model (Figure 4); the stability and DNA affinity of AQ4 is likely to explain this phenomenon (Smith et al, 1997a;1997b). Once produced, AQ4 will persist in the hypoxic cell fraction and kill any cell which attempts to re-enter the cell cycle following destruction of oxic cells by irradiation.…”

“…In contrast, we report here preclinical data on a new class of bioreductive agent exemplified by AQ4N (1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}5,8-dihydroxyanthracene-9, 10-dione). This agent is a prodrug designed to be excluded from cell nuclei (Patterson, 1993;Smith et al, 1997b) until metabolized in hypoxic cells to give AQ4, a stable, oxygeninsensitive metabolite (Smith et al, 1997a) (Figure 1). AQ4 is a DNA intercalator and potent inhibitor of DNA type II topoisomerase (Patterson, 1993;Patterson et al, 1994;Smith et al, 1997a).…”

Nuclear cytometry and chromatin organization

“…7 (188)) permits fluorescence microscopy and flow cytometry to determine uptake and nuclear distribution. Subcellular and nuclear distribution patterns reveal drug resistance (189), altered behavior of alkylamino-anthraquinones and the impact of N-oxide derivatization (160). The structurally related far-red fluorescent DNA dyes and the nuclear counterstains DRAQ5™ (128,(190)(191)(192), DRAQ7™ (89,132,(193)(194)(195), and the spectrally shifted CyTRAK Orange™ (196) have applications in nuclear cytometry (197).…”

The unidirectional hypoxia‐activated prodrug OCT1002 inhibits growth and vascular development in castrate‐resistant prostate tumors

“…These unidirectional HAPS (uHAPs) are reduced in hypoxia to the metabolically stable reduction products (AQ4 and OCT1001, respectively). Studies with AQ4N and OCT1002 show that hypoxia‐mediated reduction results in a product with high affinity for DNA and targeting of topoisomerase II,which can effect a long‐term inhibition of both DNA replication and G2/M cell transition …”

“…Studies with AQ4N and OCT1002 show that hypoxiamediated reduction results in a product with high affinity for DNA and targeting of topoisomerase II, 23,24 which can effect a long-term inhibition of both DNA replication and G2/M cell transition. 25,26 Previously we have shown that OCT1002 kills hypoxic prostate cancer cells in vitro and in vivo. 21 We provided the first evidence that OCT1002 has a hypoxia-dependent anti-tumor effect in androgensensitive LNCaP prostate tumor xenografts and the effect can be markedly enhanced when combined with daily bicalutamide administration, a drug which targets the androgen receptor (AR).…”