Novel acridine-based compounds that exhibit an anti-pancreatic cancer activity are catalytic inhibitors of human topoisomerase II

Abstract

We have identified a small library of novel substituted 9-aminoacridine derivatives that inhibit cell proliferation of pancreatic cancer cell lines by inducing apoptosis [Goodell, J.R. et al., 2008. J. Med. Chem. 51, 179-182.]. To further investigate their antiproliferative activities, we have assessed the antiproliferative activity of these acridine-based compounds against several pancreatic cancer cell lines. All four compounds used in this study inhibited the proliferation of pancreatic cancer cell lines in vitro. In addition, we have employed a xenograft tumor model and found that these compounds also inhibit the proliferation of pancreatic cancer in vivo. In light of the potential importance of the anticancer activity of these acridine-based compounds, we have conducted a series of biochemical assays to determine the effect of these compounds on human topoisomerase II. Unlike amsacrine, these compounds do not poison topoisomerase II. Similar to amsacrine, however, these compounds intercalate into DNA in a way that they would alter the apparent topology of the DNA substrate. Thus, inhibition of the relaxation activity of topoisomerase II by these compounds has been reexamined using a DNA strand passage assay. We have found that these compounds, indeed, inhibit the catalytic activity of topoisomerase II. Thus, these novel acridine-based compounds with anti-pancreatic cancer activity are catalytic inhibitors, not poisons, of human topoisomerase II.

Fig. 1. Structures of novel acridine-acridine based compounds with anti-pancreatic cancer activity

Structures of the four acridine-based compounds used in this study are shown along with amsacrine.

Fig. 2. In vivo inhibition of pancreatic cancer cell proliferation

Representative sections from SU86.86 xenografts treated with diluent (DMSO), compound 2, or compound 3 and stained for either BrdU (Panel A) or Ki-67 (Panel B). One hundred tumor cells were counted per microscopic field. The percentage of positively stained cells from 5 randomly selected fields is graphically depicted. 2, compound 2; 3, compound 3.

Fig. 3. The acridine-based compounds intercalate into DNA

The DNA unwinding reaction mixtures contained 0.3 μg of relaxed plasmid DNA, 2 units of human topoisomerase I, and 100 μM of the various drugs. Two independent experiments for each assay exhibited essentially identical results; representative results are shown. Drugs are denoted as follows: 1, compound 1; 2, compound 2; 3, compound 3; 4, compound 4; A, amsacrine; and E, etoposide.

Fig. 4. Determination of the effect of amsacrine on human topoisomerase II catalytic activity using the strand passage assay

Both the DNA unwinding assay for human topoisomerase I (Panel A) and the strand passage assay for human topoisomerase II (Panel B) were performed in the presence of indicated concentrations of amsacrine.

Fig. 4. Determination of the effect of amsacrine on human topoisomerase II catalytic activity using the strand passage assay

Both the DNA unwinding assay for human topoisomerase I (Panel A) and the strand passage assay for human topoisomerase II (Panel B) were performed in the presence of indicated concentrations of amsacrine.

Fig. 5. The acridine-based compounds inhibit the catalytic activity of human topoisomerase II

The DNA unwinding assay for human topoisomerase I (Panel A) and the strand passage assay for human topoisomerase II (Panel B) were performed in the presence of indicated concentrations of the acridine-based compounds. Three independent experiments for each assay exhibited essentially identical results; representative results are shown. Abbreviations are as indicated in the legend to Fig. 3.

Fig. 5. The acridine-based compounds inhibit the catalytic activity of human topoisomerase II

The DNA unwinding assay for human topoisomerase I (Panel A) and the strand passage assay for human topoisomerase II (Panel B) were performed in the presence of indicated concentrations of the acridine-based compounds. Three independent experiments for each assay exhibited essentially identical results; representative results are shown. Abbreviations are as indicated in the legend to Fig. 3.

Fig. 6. The acridine-based compounds do not poison human topoisomerase II

DNA cleavage activity of human topoisomerase II (6 units per reaction) was measured in the presence of the indicated concentrations of the various drugs using relaxed plasmid DNA (0.3 μg) as the substrate. Two independent experiments for each assay exhibited essentially identical results; representative results are shown. Lane 1 in all panels shows the DNA substrate.

Fig. 6. The acridine-based compounds do not poison human topoisomerase II

DNA cleavage activity of human topoisomerase II (6 units per reaction) was measured in the presence of the indicated concentrations of the various drugs using relaxed plasmid DNA (0.3 μg) as the substrate. Two independent experiments for each assay exhibited essentially identical results; representative results are shown. Lane 1 in all panels shows the DNA substrate.

Fig. 6. The acridine-based compounds do not poison human topoisomerase II

DNA cleavage activity of human topoisomerase II (6 units per reaction) was measured in the presence of the indicated concentrations of the various drugs using relaxed plasmid DNA (0.3 μg) as the substrate. Two independent experiments for each assay exhibited essentially identical results; representative results are shown. Lane 1 in all panels shows the DNA substrate.