On the role of topoisomerase I in mediating the cytotoxicity of 9-aminoacridine-based anticancer agents

Abstract

The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In previous work, compounds of this type were shown to catalytically inhibit topoisomerase II, leading to a G1-S phase arrest of the cell cycle and apoptosis in pancreatic cancer cells in vitro and in vivo. The present study expands the potential utility of these compounds in the development of cancer therapeutics by showing that these compounds inhibit proliferation of cell lines derived from the nine most common human cancers. Further results show that at least one of the compounds effectively stabilizes topoisomerase I-DNA adduct formation in intact cells. RNA interference experiments, however, indicate that this interaction does not contribute to the drug-induced killing of cancer cells indicating the compounds may be non-lethal poisons of topoisomerase I.

Figure 1

Substituted 9-aminoacridines.

Figure 2. Effects of compounds 1, 2, 3, and 4 on topoisomerase I catalytic activity (A), formation of covalent topoisomerase I-DNA complexes (B) and ICE assay (C)

A. Effect on topoisomerase I activity. Supercoiled plasmid was incubated with 2 units of topoisomerase I in the absence (lane 2) or presence of the positive control pyrazoloacridine (8 μM) or compounds 1, 2, 3, and 4 (lanes 4-11) at a final concentration of 50 or 100 μM as indicated. Lane 1, DNA incubated without enzyme. SC, supercoiled DNA; R, relaxed DNA. Results are representative of 4 experiments. B. Stabilization of topo I-DNA complexes. Aliquots of supercoiled plasmid were incubated for 30 min at 37 °C with nuclear extract containing 50 units of topoisomerase I in the presence of TPT (1.6 μM), PA (200 or 100 μM) or compounds 1, 2, 3, and 4 (100 μM), as indicated. After treatment with SDS and proteinase K, samples were separated on 1% agarose gels in the presence of 0.5 μg/ml ethidium bromide to better separate nicked (N) from relaxed (R) DNA. Compare the increase amount of nicked DNA (lanes 3-9) with the nuclear extract only (lane 2). (L), linear DNA. Results were representative of 3 independent experiments. C. Stabilization of DNA-topoisomerase I complexes in vivo. MiaPACA cells treated with 0.1 % DMSO, 20 μM TPT or 100 μM Compounds 1, 2, 3, and 4 for 1 h at 37 °C were lysed with sarkosyl and subjected to the ICE bioassay as described in the Supporting Materials. Immunoblot of gradient fractions that were positive for DNA is shown (starting from the bottom of the tube). Because of the limited slots available, two membranes (blotted simultaneously) were used to test all the compounds at once. Results are representative of 4 independent experiments.

Figure 3. Effect of topoisomerase I siRNA on drug sensitivity

After transfection with luciferase siRNA (open circles) or topoisomerase I siRNA (filled circles), HeLa cells were treated for 24 h with diluent (0.1% DMSO) or the indicated concentration of camptothecin (A) or compound 2 (B) for 24 h, washed and incubated in drug-free medium for 8 d to allow colonies to form. The Y axis is graphed on a logarithmic scale as is common practice with colony forming assays to emphasize the log-linear effects of these agents. Error bars, ± S.D. are from triplicate plates. Inset in A, whole cell lysates were prepared from additional transfected cells at the time of drug treatment, subjected to SDS-PAGE and probed for topoisomerase I. Lamins A and C served as loading controls.