Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311

Abstract

Summary C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.Keywords: C1311; HT29 cells; colorectal cancer; cytotoxicity; DNA intercalation; lysosomes 367British Journal of Cancer (1999) 81(2), 367-375 © 1999 Cancer Research Campaign Article no. bjoc.1999 Received MATERIALS AND METHODS Materials/DrugsThe 5-(ω-aminoalkyl)amino-8-hydroxyimidazoacridinone C1311 (Figure 1) was synthesized at the Technical University of Gdansk, as described elsewhere (Cholody et al, 1990(Cholody et al, , 1992. Stock solutions were prepared in dimethyl sulphoxide (DMSO) or normal saline. Aqueous solutions were prepared in polystyrene tubes to minimize adsorption effects and quantified by spectrophotometry. Hoechst 33258 dye (Sigma-Aldrich Ltd) was used as a control ligand for viscometric studies and quantified using ε 338 = 42 000 M -1 cm -1 (Loontiens et al, 1990). All DNA binding experiments were performed in aqueous phosphate buffer (10 mM NaH 2 PO 4 /Na 2 HPO 4 , 1 mM Na 2 EDTA, pH 7.00 ± 0.01). Doublestranded calf thymus DNA (CT-DNA, type-I [Sigma-Aldrich Ltd.]) was sonicated and purified using a publ...

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