Acridine-based agents with topoisomerase II activity inhibit pancreatic cancer cell proliferation and induce apoptosis

Article abstract of DOI:10.1021/jm701228e

A series of substituted 9-aminoacridines is evaluated for antiproliferative activity toward pancreatic cancer cells. The results indicate that the compounds inhibit cell proliferation by inducing a G1-S phase arrest. A model is also developed that explain

Full text of DOI:10.1021/jm701228e

Copyright 2008 by the American Chemical Society
Volume 51, Number 2
January 24, 2008
Letters
studied and act by stabilizing the “cleavable complex”,
resulting in the covalent linkage between the enzyme and
DNA, ultimately leading to irreversible double strand breaks.3–5
Catalytic inhibitors, which have received far less attention,
act at any other stage in the seven step catalytic cycle, such
as enzyme/DNA association occurring in the first step.6
Acridine-Based Agents with Topoisomerase II
Activity Inhibit Pancreatic Cancer Cell
Proliferation and Induce Apoptosis
John R. Goodell,† Andrei V. Ougolkov,‡ Hiroshi Hiasa,§
Harneet Kaur,† Rory Remmel,† Daniel D. Billadeau,‡ and
David M. Ferguson*,†
Recent work in our laboratory investigating compounds
with antiherpes activity resulted in the discovery of a small
library of substituted 9-aminoacridine derivatives that appear
to have novel topo II catalytic inhibitory activity.7 These
compounds demonstrated activity comparable to that of
amsacrine in topo II relaxation assays but did not show
activity in a topo II cleavage assay.7 On the basis of their
potency in preliminary cytotoxicity screens and effectiveness
in inhibiting human topo II catalyzed relaxation reactions,
four representative compounds, shown in Figure 1, were
assayed for antiproliferative activity against three pancreatic
cell lines. To further our understanding of the mechanism of
these compounds at a cellular and molecular level, the
compounds were investigated for their effect on cell prolif-
eration, cell cycle progression, induction of apoptosis and
modeled to investigate their interaction with DNA.
Department of Medicinal Chemistry and Pharmacology and Center for
Drug Design, UniVersity of Minnesota, Minneapolis, Minnesota 55455,
and DiVision of Oncology Research and DeVelopmental
Experimental Therapeutics, Mayo Clinic College of Medicine,
Rochester, Minnesota 55905
ReceiVed September 27, 2007
Abstract: A series of substituted 9-aminoacridines is evaluated for
antiproliferative activity toward pancreatic cancer cells. The results
indicate that the compounds inhibit cell proliferation by inducing a
G1-S phase arrest. A model is also developed that explains the
molecular basis to inhibition through a DNA “threading” mechanism.
We conclude that the drug–DNA complex formed blocks topo-
isomerase II binding and activity leading to catalytic inhibition of
the enzyme and the induction of apoptosis and programmed cell
death.
Three pancreatic cancer cell lines consisting of MiaPaCa-2,
SU86.86, and BXPC-3 were used to allow for the investigation
of growth inhibition based on their different rates of prolifera-
tion. MiaPaCa-2 has the fastest rate of proliferation, whereas
BXPC-3 has the slowest. Percent growth inhibition experiments
using a range of concentrations of 1–4 were conducted over a
72 h period using an MTT assay for cell viability quantification.
The IC50 values for the three cell lines are shown in Table 1,
with all of the compounds showing low micromolar activity in
the three cell lines examined.
Acridine-based compounds have had a long and successful
history as antibacterial, antimalarial, and more recently,
antitumor agents. Most of these antitumor agents act by
inhibiting the essential enzyme, topoisomerase II (topo II).1,2
Topo II plays a critical role in actively replicating cells by
affecting topological changes in DNA, allowing for replica-
tion, transcription, and decatenation.3 Therefore, aggressive
and rapidly replicating cancers, such as small-cell lung
cancer, lymphomas, and a variety of leukemias, appear to
be the most responsive to compounds that inhibit topo II.4,5
Current topo II inhibitors are classified as topo II catalytic
inhibitors or topo II poisons, with most of the current
inhibitors being poisons. Topo II poisons have been well
Previous studies have indicated that topo II poisons
primarily cause irreversible damage during the S phase (when
topo IIR levels are high), ultimately leading to an accumula-
tion of cells in the G2 phase.6,8 Similarly, the topo II catalytic
inhibitor, aclarubicin, also induces DNA damage during the
S phase by preventing relaxation of supercoiled DNA ahead
of the replication fork.6 However, a different morphologic
appearance is observed with an enlarged nucleus (containing
elongated and entangled DNA) compared to the small
apoptotic nuclei (containing fragmented DNA) induced by
topo II poisons.6 Catalytic inhibitors therefore result in
* To whom correspondence should be addressed. Voice mail: (612) 626-
2601. Fax: (612) 626-4429. E-mail: ferguson@umn.edu.
†
Department of Medicinal Chemistry, University of Minnesota.
Mayo Clinic College of Medicine.
Pharmacology and Center for Drug Design, University of Minnesota.
‡
§
10.1021/jm701228e CCC: $40.75
2008 American Chemical Society
Published on Web 12/29/2007

180 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Letters
Table 2. Cell Cycle Arrest by Acridine Compounds 1–4a
G1/S/G2M
compd
MiaPaCa-2
SU86.86
BXPC-3
DMSO
46/36/17
32/47/21
28/52/20
29/51/20
32/50/18
39/42/19
56/27/17
53/29/18
50/26/24
60/24/16
50/37/13
83/10/7
77/16/7
81/12/7
76/20/4
1
2
3
4
a Results were tabulated after 36 h of treatment. The pancreatic cancer
cells were incubated for 36 h with diluent or the indicated compounds 1-4
(15 µM final concentration). Samples were then fixed in 50% ethanol,
rehydrated, treated with RNase, stained with 50 µg/mL propidium iodide
in 0.1% sodium citrate, and analyzed (at least 20 000 events per sample)
using a Becton Dickinson FACScan flow cytometer. Very small debris
(forward scatter less than 10% of intact cells) was excluded from the analysis
by gating. Cell-cycle profiles were determined using Modfit software (Verity,
Topsham, ME).
Figure 1. Structures of the compounds investigated in the current study.
Syntheses of the compounds were previously reported.7
Table 1. Inhibition of Pancreatic Cell Lines by Acridine Compounds
1–4a
IC50 (µM)
compd
MiaPaCa-2
SU86.86
BXPC-3
1
2
3
4
4 ( 0.7
1 ( 0.25
6 ( 1.3
10 ( 0.8
6 ( 0.4
8 ( 1.2
10 ( 1.5
18 ( 2.8
7 ( 0.35
12 ( 1.5
14 ( 1.8
11 ( 1.6
a IC50 estimated from MTT assay. The pancreatic cancer cell lines
MiaPaCa-2, SU86.86, and BXPC-3 were obtained from ATCC (Rockville,
MD) and were grown in DMEM medium containing 10% fetal calf serum
and L-glutamine.
Figure 2. Induction of apoptosis following treatment with 9-amino-
acridine derivatives. BXPC-3 cells were treated with diluent or the
indicated concentrations of 3 and 4. (Results for 1 and 2 are similar.)
Forty-eight hours after exposure, cells were collected and stained
with Hoechst. Three-hundred nuclei were counted per field and
scored for apoptotic changes (fragmentation of the nucleus into
multiple discrete fragments) and are graphically represented (lower
panel). Cell lysates from the same treated cells were probed for
PARP cleavage (upper panel), a hallmark of apoptosis. ꢀ-Actin was
used as a loading control.
cellular arrest at the G2 phase by stopping the cell cycle at
the catenation checkpoint. However, it is unclear at which
stage in the cell cycle this novel set of acridine-based
compounds is affecting tumor cell proliferation. To examine
this, we treated the indicated pancreatic tumor cell lines with
diluent or 1–4 and determined cell cycle accumulation at 36 h
using propidium iodide staining and flow cytometry quan-
tification. Two of the cell lines, SU86.86 and BXPC-3,
demonstrate a loss of cells from the S and G2/M phases of
the cell cycle with a corresponding accumulation of cells in
G1 (Table 2). The MiaPaCa-2 cell line, on the other hand,
shows an accumulation of cells in the S phase following
treatment with 1–4 (Table 2). These data are consistent with
the notion that these novel compounds are topo II inhibitors,
as they would be predicted to impede the G1 f S transition,
since topo II is involved in unwinding DNA during the
initiation of replication. The reason that the MiaPaCa-2 cells
demonstrate more of an S-phase arrest in response to
treatment with the compounds is unclear but may be due to
its relatively fast proliferation rate compared to the other two
cell lines.
result in crisis and initiation of apoptosis. To examine this, we
treated BXPC-3 cells with diluent or the indicated concentrations
of 1–4, measured apoptosis using Hoechst staining of nuclei
from the treated cells, and scored them for the presence of
apoptosis (fragmentation of the nucleus into multiple discrete
fragments). The results, shown in Figure 2, demonstrate that
the BXPC-3 cell line undergoes apoptosis in a concentration-
and time-dependent manner. Cell lysates were also prepared
and analyzed for PARP cleavage, a hallmark of cells undergoing
apoptosis. Consistent with the Hoechst staining results, the
BXPC-3 cells treated with the compound show PARP cleavage.
Similar induction of apoptosis was seen in the SU86.86 and
MiaPaCa-2 cell lines (see Table 3). Together, these results
indicate that the acridine-based compounds not only inhibit
pancreatic cancer cell proliferation but also induce apoptosis,
which is a characteristic of catalytic topo II inhibitors. This is
in contrast to topo II poisons, which can also induce cellular
necrosis.9
Since the compounds result in a replication block, we next
investigated whether the novel compounds lead to programmed
cell death, since stalling the proliferation of cancer cells can

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 181
Table 3. Percentages of Cells in Apoptosis after 48 h of Exposure to
Compounds 1–4
cells in apoptosis (%)
compd
MiaPaCa-2
SU86.86
BXPC-3
1 (50 µM)
2 (25 µM)
3 (15 µM)
4 (15 µM)
29 ( 4
36 ( 9
20 ( 5
42 ( 7
23 ( 6
30 ( 5
34 ( 7
17 ( 3
32 ( 8
52 ( 11
44 ( 6
40 ( 5
The results are consistent with our previous work that linked
the antiherpes virus activity of these compounds with catalytic
inhibition of topo II.7 As anticipated, the acridine-based
compounds in the current investigation exhibited notable activity
against all three pancreatic cell lines. Here, we have further
shown that these compounds not only inhibit cancer cell
proliferation but also induce a buildup of cells at the G1 f S
transition of the cell cycle. Since topo II plays a critical role in
the unwinding of DNA in front of the replication machinery,
such a buildup is easily explained and is a well-known hallmark
of topo II inhibition. Disruption or prevention of topo II activity
results in a replication block that in turn leads to the initiation
of programmed cell death, as indicated by the Hoechst staining
and PARP cleavage results. This is also expected, since the
stalling of cancer cell proliferation results in a cellular crisis
and subsequent induction of apoptotic pathways.
Figure 3. Drug–DNA complex of 2 threaded into a representative
DNA fragment. The 9-amino substitution is in the minor grove, and
the 3-amidesubstitution is in the major groove with maximal π-π
overlap between the acridine core tricycle and adjacent DNA base
pairs.
While the results presented here support our proposed
mechanism of action at a cellular level, there is still little known
about how substituted tricycles of this type function at the
molecular level. It has long been postulated that acridine- and
anthracycline-based topo II inhibitors (e.g., doxorubicin) func-
tion via intercalation with DNA, resulting in the disruption of
topo II activity. Most of these intercalating inhibitors, however,
are classified as poisons because of their ability to produce
irreversible double strand breaks. The important exception is
aclarubicin, a catalytic inhibitor and intercalator that functions
by preventing the association of DNA and topo II by blocking
the minor groove.6,10 This is, of course, the major difference in
catalytic inhibitors and poisons; the former prevents topo II from
binding, while the latter traps the enzyme-DNA complex in
the cleavable form.
To begin elucidation of the structure-based mechanism to
activity of the 9-aminoacridines, preliminary molecular modeling
studies were conducted. Ethidium bromide displacement studies
performed in our laboratory have shown that these compounds
intercalate DNA with high affinity.7 By utilization of a reported
X-ray crystal structure of a related acridine derivative complexed
with DNA as a template (9-amino-DACA with the hexanucleo-
side d(CGTACG)2),11,12 the compounds were modeled into the
DNA followed by side chain relaxation/optimization using the
Schrödinger suite of software.13 The resulting complexes, shown
in Figure 3, indicate that the acridine core intercalates DNA
base pairs through π-stacking interactions, while the side
chain substitutions extend into the major and minor grooves.
This type of interaction is referred to as a “threaded” binding
mode and has been proposed for other disubstituted acridines
but never subjected to X-ray crystallography or molecular
modeling.14–16
Figure 4. Structures of known topo II inhibitors.
amsacrine (a related acridine-based topo poison) and 1–4. While
both acridine cores are capable of strong intercalation, amsacrine
lacks the steric bulk required to block minor or major groove
binding of topo II to DNA. This not only explains the differences
in the mechanism of action of these related compounds but also
offers new insight to the structure-based design of catalytic
inhibitors and poisons of topo II activity. A complete mecha-
nistic evaluation of 1–4 and comparisons with related topo drugs
are forthcoming.17
Since catalytic inhibitors may hold some inherent advantages
over poisons in blocking topo II activity (stemming from the
inability to induce DNA cleavage), the ability to rationally
design catalytic inhibitors may prove to be extremely useful in
the development of improved anticancer agents. The observation
that 1–4 are active in all three cells lines, including the slowest
growing cells (BXPC-3) is encouraging because a number of
problematic solid tumors, prostate cancer in particular, have
relatively slower rates of proliferation compared to small cell
carcinomas and leukemias. Although not tested here, we fully
expect the catalytic inhibitors reported here will in fact be active
against a wide range of cancers, offering new strategies for the
design of anticancer agents with lower toxicities.
Given the prior work on anthracyclines, it is fair to conclude
that the substituted acridines function by sterically blocking topo
II recognition of the minor and/or major groove of DNA. A
comparison of the structures of doxorubicin with aclarubicin
shows, the latter contains two additional sugar moieties (see
Figure 4). These sugars block topo association with the minor
groove of DNA. A similar analogy can be drawn between
Acknowledgment. This research has been supported in part
by the University of Minnesota Academic Health Center, the

182 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2
Letters
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Mayo Foundation, and a specialized program of research
excellence Grant P50 CA102701 from the National Cancer
Institute.
Supporting Information Available: Experimental procedures
and descriptions of biology experiments. This material is available
free of charge via the Internet at http://pubs.acs.org.
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JM701228E