Dual inhibition of topoisomerase I and tubulin polymerization by BPR0Y007, a novel cytotoxic agent

Article abstract of DOI:10.1016/S0006-2952(03)00197-7

Through the screening of DNA topoisomerase I (Top I) inhibitors, a new cytotoxic agent, BPR0Y007 [2,5-bis(4-hydroxy-3-methoxybenzylidene)cyclopentanone], was identified. BPR0Y007 was less potent than camptothecin (CPT) in the inhibition of Top I in vitro.

Full text of DOI:10.1016/S0006-2952(03)00197-7

Biochemical Pharmacology 65 (2003) 2009–2019
Dual inhibition of topoisomerase I and tubulin polymerization
by BPR0Y007, a novel cytotoxic agent
Jang-Yang Changa,*, Hsing-Pang Hsiehb, Wen-Yu Pana, Jing-Ping Lioub,
Shian-Jy Beyb, Li-Tzong Chena,c, Jin-Fen Liua, Jen-Shin Songb
aDivision of Cancer Research, National Health Research Institutes, 7, Chung-Shan South Road, Taipei, Taiwan, ROC
bDivision of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei, Taiwan, ROC
cDivision of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC
Received 27 August 2002; accepted 25 November 2002
Abstract
Through the screening of DNA topoisomerase I (Top I) inhibitors, a new cytotoxic agent, BPR0Y007 [2,5-bis(4-hydroxy-3-
methoxybenzylidene)cyclopentanone], was identified. BPR0Y007 was less potent than camptothecin (CPT) in the inhibition of Top
I in vitro. Also, in vitro data showed that BPR0Y007 induces DNA cleavage in the presence of Top I at micromolar concentrations, with a
cleavage specificity similar to that of CPT. High concentrations of BPR0Y007 did not produce detectable DNA unwinding, suggesting that
BPR0Y007 is not a DNA intercalator. However, BPR0Y007 displaced Hoechst 33342 dye, suggesting that BPR0Y007 binds to DNA at
the Hoechst 33342 binding site. Furthermore, BPR0Y007 generated protein-linked DNA breaks in a cell-based study. Cell cycle analysis
demonstrated that the cell cycle effect of BPR0Y007 differs from that of CPT. Cells accumulated in the S-phase when treated with high
concentrations of CPT, whereas cells accumulated gradually in the G2/M phase when treated with increasing concentrations of
BPR0Y007. Further studies showed that BPR0Y007 inhibits tubulin polymerization in vivo and in vitro, and induces apoptosis in a
concentration-dependent manner. No cross-resistance with BPR0Y007 was observed in CPT-, VP-16-, or vincristine-resistant cell lines.
The IC50 of BPR0Y007 for various human cancer cell lines ranged from 1 to 8 mM. Taken together, these results suggest that BPR0Y007
acts on both Top I and tubulin. Given its unique biochemical mechanisms of action, BPR0Y007 warrants exploration as an antitumor
compound.
# 2003 Elsevier Science Inc. All rights reserved.
Keywords: Topoisomerase I; Tubulin; Dual inhibitor
1. Introduction
prevents the resealing of the Top I-mediated DNA single-
strand breaks, causing a collision of the replication forks
when replication occurs and, subsequently, cell death [6,7].
CPT is an S-phase-specific DNA-damaging agent [8].
However, high concentrations of CPT can kill both S-phase
and non-S-phase cells [9]. The mechanism of cytotoxicity
of CPT in non-S-phase tumor cells may involve apoptosis
[10]. Although several CPT derivatives, including irinote-
can and topotecan, have been introduced for the clinical
treatment of colon and ovarian cancers, the response rates
remain low, and the overall survival rate has not improved
substantially [11,12]. Thus, interest remains in finding new
Top I inhibitors.
Eukaryotic DNA Top I is an essential enzyme that
regulates the topological changes of DNA that accompany
DNA replication, transcription, recombination, and chro-
mosome segregation during mitosis [1–3]. Top I introduces
transient single-strand DNA breaks in one of the phospho-
diester backbones of the duplex DNA, resulting in a
reversible Top I/DNA covalent complex [4,5]. Top I inter-
active agents, consisting primarily of CPT analogues,
interact with the Top I/DNA complex. This interaction
* Corresponding author. Tel.: þ886-2-2356-2879;
fax: þ886-2-2389-2737.
Recently, as part of a program to identify clinically
effective antitumor agents with antiproliferative properties,
we identified a cytotoxic agent, BPR0Y007 [2,5-bis-
(4-hydroxy-3-methoxybenzylidene)cyclopentanone]. The
E-mail address: jychang@nhri.org.tw (J.-Y. Chang).
Abbreviations: Top I, topoisomerase I; CPT, camptothecin; PLDBs,
protein-linked DNA breaks.
0006-2952/03/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0006-2952(03)00197-7

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J.-Y. Chang et al. / Biochemical Pharmacology 65 (2003) 2009–2019
concentration of drug, respectively. KB 7D cells displayed
a decrease in cellular Top II content and overexpression of
multidrug-resistant-associated protein (MRP) [14]. KB
100 cells displayed a decrease in Top I and unidentified
post-protein-linked DNA break (PLDB) resistance to Top I
poisons [15]. KB vin10 cells overexpressed P-glycopro-
tein-170 (P-gp) (unpublished data). The cell doubling
times for KB, KB 7D, KB 100, and KB vin10 cells were
20, 22, 29, and 22 hr, respectively. CPT 30 cells, a CPT-
resistant cell line derived from a human nasopharyngeal
carcinoma cell line, HONE-1, displayed a decrease in Top I
level and a mutation in the Top I gene [16]. The cell
doubling times for HONE-1 and CPT 30 cells were similar,
on the order of 21 hr.
Fig. 1. Chemical structure of BPR0Y007.
structure of BPR0Y007 is shown in Fig. 1. In this study,
we determined the mechanisms of action of BPR0Y007
and found that it could inhibit both Top I and tubulin
polymerization. Furthermore, no cross-resistance with
BPR0Y007 was observed in CPT-, vincristine-, or VP-
16-resistant cells.
2. Materials and methods
2.4. Screening the topoisomerase inhibitor
2.1. Synthesis of BPR0Y007
A two-step combinational screening strategy was used to
identify novel inhibitors for human topoisomerases in the
Division of Biotechnology and Pharmaceutical Research,
National Health Research Institutes. A Beckman Coulter
high-throughput cell screening system was used to screen
the compounds for antiproliferative activity. The growth
inhibition assay using a human gastric cancer cell line,
NUGC3, was performed as described by Goodwin et al.
[17]. If the growth inhibitory activity against NUGC3 cells
was greater than 50% at a concentration of 10 mM, biolo-
gical screening using an in vitro topoisomerase-mediated
DNA relaxation assay [18] was performed.
BPR0Y007 was synthesized according to the procedure
described by Sardjiman et al. [13]. Vanillin and cyclopen-
tanone were heated in a water bath (45–508) until a clear
solution was obtained; then concentrated hydrochloric acid
was added followed by stirring for 2 hr. After standing
overnight, the mixture was treated with cold AcOH/water
(1:1) and filtered. The solid materials were washed first
with cold ethanol, then with hot water, and dried under
vacuum. The yellow substance was recrystallized from
ethanol. The structure of BPR0Y007 was confirmed by
1H-NMR and liquid chromatography-mass spectrometry.
BPR0Y007 was stable for more than 12 months when
stored in the solid phase.
2.5. Inhibition of the catalytic activity of Top I
Top I catalytic activity was determined by the ATP-
independent relaxation of pBR322 supercoiled DNA, as
described previously [18]. Each reaction mixture contained
0.25 mg supercoiled DNA, 50 mM Tris–HCl (pH 7.5),
60 mM KCl, 2.5 mM MgCl2, 0.5 mM EDTA, 50 mg/mL
of BSA, one unit of enzyme, and various concentrations of
drugs. The reaction was terminated by adding 1% SDS, and
separated in a 1% agarose gel. Photographs of the resulting
ethidium bromide-stained agarose gel were taken under
UV light.
2.2. Drugs, enzymes, and chemicals
CPT, adriamycin, etoposide (VP-16), paclitaxel, and
netropsin were purchased from the Sigma Chemical Co.
Stock solutions of these drugs, except for those that were
water-soluble, were prepared in DMSO at 20 mM, stored at
À208, and diluted in water immediately before use. Calf
thymus Top I was purchased from TopGene. Microtubule-
associated protein (MAP)-rich bovine brain tubulin was
purchased from Cytoskeleton, and [a-32P]dATP and
[14C]thymidine (specific activity, 50.5 mCi/mmol) were
obtained from the Amersham Corp. All chemicals were
standard analytical grade or higher.
2.6. Top I-induced DNA cleavage
Evaluation of Top I-induced single-strand DNA breaks
was performed as described previously [7]. Linear pBR322
DNAwas 30-end labeled, as described previously [19]. The
reaction was carried out at 378 for 15 min, stopped by the
addition of 5 mL of 5% SDS and 0.75 mg/mL of proteinase
K, and incubated for 30 min at 508. Prior to loading onto a
1% agarose gel in 1Â TBE (0.09 M Tris-borate and
0.002 M EDTA) with 0.1% SDS, 10-mL samples were
denatured with 0.45 N NaOH, 30 mM EDTA, 15% (v/w)
sucrose, and 0.1% bromophenol blue. After electrophor-
esis, the gel was dried and autoradiographed for 24 hr.
2.3. Cell lines
All cell lines were maintained in RPMI 1640 medium
containing 5% fetal bovine serum. The CPT-resistant cell
lines, KB 100 and CPT 30, were maintained in growth
medium supplemented with 100 or 30 nM CPT, respec-
tively. The VP-16-resistant cell line, KB 7D, and the
vincristine-resistant cell line, KB vin10, were maintained
in the medium supplemented with a 7 mM or a 10 nM

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2.7. Measurement of PLDBs in vivo
2.12. In vivo tubulin polymerization assay
Cells in log phase growth were labeled with [14C]thy-
midine for 24 hr. After labeling, the cells were trypsinized,
resuspended in fresh medium at a density of 5 Â 105/mL,
and shaken gently in a 378 water bath for 30 min in
suspension. Various concentrations of drugs were added,
and incubation was continued for an additional 0.5 hr. The
cells were collected and analyzed for protein-linked DNA
breaks by a potassium-SDS (K-SDS) co-precipitation
method, as described previously [20].
Cells at a density of 1 Â 106/dish were treated with
various concentrations of test agents. Then the cells were
washed with PBS three times before adding a lysis buffer
containing 20 mM Tris–HCl (pH 8.6), 1 mM MgCl2, 2 mM
EGTA, 20 mg/mL of aprotinin, 20 mg/mL of leupeptin,
1 mM phenylmethylsulfonyl fluoride, 1 mM orthovana-
date, and 0.5% Nonidet P-40. Supernatants were collected
after centrifugation at 9168 g for 10 min at 48. The pellets
were dissolved in an SDS–PAGE sampling buffer and
subjected to electrophoresis on a 10% SDS–polyacryla-
mide gel. After electrophoresis, the proteins were trans-
ferred to a nitrocellulose membrane, and the membrane
was incubated with a-tubulin monoclonal antibody
(Sigma). Detection of immunoreactive signal was accom-
plished with Western Blot Chemilluminescent Reagent
Plus (Perkin Elmer Life Sciences).
2.8. DNA unwinding measurements
Drug-induced DNA unwinding was assayed as des-
cribed previously [21]. The DNA circle-ligation assay,
using nicked DNA as a substrate, was performed using
the method of Montecucco et al. [22].
2.9. Measurement of fluorescence of DNA-bound
Hoechst 33342
2.13. Colchicine and tubulin binding assays
Tubulin was incubated with [3H]colchicine in the pre-
sence of different concentrations of either unlabeled col-
chicine or BPR0Y007 in a buffer containing 0.05 M PIPES
(pH 6.9), 1 mM MgCl2, and 1 mM GTP. The reaction
mixtures were incubated at 378 for 1 hr. The samples were
loaded onto a Sephadex G-50 column (Amersham Phar-
macia) previously equilibrated with the buffer solution. The
columns were placed into 1.5-mL tubes, spun at a speed of
750 g for 2 min at room temperature, and radioactivity in
the flow-through was analyzed by a scintillation counter.
The minor groove binding effect of BPR0Y007 was
assayed as described previously [23]. Briefly, salmon
sperm DNA and 0.8 mg Hoechst 33342 were mixed in
200 mL of PBS and preincubated for 10 min at room
temperature in a 96-well plate. Netropsin, a known minor
groove binder, and VP-16 were included as positive
and negative controls, respectively. Then BPR0Y007
was added at various concentrations. After another
10-min incubation, the fluorescence derived from Hoechst
33342 bound to DNA was measured with a fluorometer
(excitation wavelength, 355 nm; and emission wave-
length, 460 nm).
2.14. TUNEL assay
To determine whether BPR0Y007 could induce apop-
tosis, a TUNEL assay using an In Situ Cell Death Detection
Kit (Roche Molecular Biochemicals) was performed. Cells
treated with various concentrations of BPR0Y007 for 24 hr
at 378 were subjected to a TUNEL assay according to the
instructions of the manufacturer.
2.10. Cell cycle analysis
Flow cytometric analysis of propidium iodide-stained
cells was performed with a FACS IV flow cytometer
(Becton Dickinson). Cell cycle analysis was performed
according to the mathematical model of Jett [24].
2.15. Caspase-3 activation assay
2.11. In vitro tubulin polymerization assay
After treatment with different BPR0Y007 concentrations
for 24 hr, and 12 mM BPR0Y007 for different durations, in
triplicate at 378 in 96-well microtiter plates, caspase-3
activation by KB ATCC cells was measured by the cleavage
of the fluorometric substrate Ac-DEVD-AMC (Promega
Corp.) according to the procedure of the manufacturer.
Microtubule polymerization was conducted in a 96-well
UV microtiter plate with MAP-rich tubulin and various
concentrations of BPR0Y007 in a buffer containing
100 mM PIPES (pH 6.9), 2 mM MgCl2, 1 mM GTP, and
4% (v/v) DMSO. The increase in absorbance was mea-
sured at 350 nm in a PowerWave X (Biotek) microplate
reader at 378 and recorded every 30 s for 30 min. The
area under the curve (AUC) was used to determine the
concentration that inhibited tubulin polymerization by
2.16. Growth inhibition assay
Cells in logarithmic phase were cultured at a density
of 5000 cells/mL/well in a 24-well plate. The resistant
cells were cultured in drug-free medium for 3 days prior to
use. The cells were exposed to various concentrations of
50% (IC ). The AUC of the untreated control was set to
50
100% polymerization, and the IC was calculated using
50
nonlinear regression.

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the test compounds for 72 hr. The methylene blue dye
assay was used to evaluate the effects of the test com-
pounds on cell growth, as described previously [25], and to
determine the concentration of drug that inhibited 50% of
performed with linearized DNA and T4 ligase. In this assay,
adriamycin, a strong intercalator, and CPT, a non-interca-
lator, were included as positive and negative controls,
respectively. As seen in Fig. 4A, adriamycin produced a
concentration-dependent DNA shift, indicating a change in
the DNA linking number. In the presence of adriamycin at
concentrations greater than 2.5 mM, substrate linear DNA
remained unchanged, indicating that the strong intercalating
activity of adriamycin causes inhibition of T4 ligase, as
reported previously [22]. Similar to the effect of CPT,
BPR0Y007 produced no DNA shift at concentrations up
to 50 mM (Fig. 4A). To determine whether BPR0Y007 binds
to the DNA, a Hoechst 33342 dye displacement assay was
performed. Hoechst 33342 is a fluorochrome that binds to
the DNA minor groove and generates specific fluorescence
[26,27]. In a cell-free system, netropsin, another DNA minor
groove binder [28], and VP-16 were included as positive and
negative controls, respectively. As shown in Fig. 4B, similar
to the effect of netropsin, the fluorescence of DNA-bound
Hoechst 33342 was quenched afterBPR0Y007wasadded in
aconcentration-dependentmanner. Thisresultindicatedthat
BPR0Y007 displaced the Hoechst 33342 bound to the DNA.
cell growth (IC ).
50
3. Results
3.1. Top I-mediated DNA relaxation inhibited by
BPR0Y007
Using a high-throughput cell-based growth inhibition
assay to screen part of a chemical library of 26,000
compounds, we identified 260 compounds with significant
inhibitory activity against human gastric cancer NUGC3
cells. Through the screening of the Top I and II inhibitors
using a DNA relaxation assay, we determined that one
compound, BPR0Y007 (Fig. 1), could inhibit Top I cata-
lytic activity, but not Top II activity (data not shown). The
1
HPLC profile and the H-NMR spectrum of the synthetic
compound were identical with those produced by a refer-
ence sample on the same spectrometer at the same time. As
shown in Fig. 2A, BPR0Y007 inhibited Top I-mediated
DNA relaxation, but was less potent than CPT.
3.5. Effect of BPR0Y007 on the cell cycle
The impact of different concentrations of BPR0Y007
and CPTon the cell cycle progression of HONE-1 cells was
studied after one generation of drug exposure (Fig. 5).
Consistent with previous reports [15], cells treated with
CPTaccumulated in S-phase with concomitant losses from
the G0/G1 and G2/M phases, especially at higher drug
concentrations. However, with increasing BPR0Y007 con-
centrations, cells accumulated gradually in the G2/M phase
with concomitant losses from the G0/G1 phase. No change
of S-phase was observed.
3.2. Top I-mediated DNA cleavage induced by BPR0Y007
Induction of DNA cleavage in the presence of Top I was
tested in a linearized pBR322 DNA. As shown in Fig. 2B,
Top I-mediated cleavage in the presence of BPR0Y007 was
detectable at 5 mM and reached a plateau at 15 mM. A
number of cleavage sites detected in the presence of
BPR0Y007 were also induced by CPT. The relative inten-
sity of similar sites varied between BPR0Y007 and CPT.
3.3. Induction of protein-linked DNA breaks
3.6. Microtubule assembly studies
Drug-induced PLDBs were evaluated in HONE-1 cells
and their CPT-resistant variant (CPT 30) cells by K-SDS
assay. BPR0Y007 induced PLDBs in HONE-1 cells in a
concentration-dependent manner, which reached a plateau
at a concentration of 10 mM. The maximal level of PLDBs
generated by BPR0Y007 was one-fifth of that of CPT
(Fig. 3). Moreover, in CPT 30 cells, which displayed a
reduced level of Top I and a mutation in the Top I gene [16],
the number of PLDBs induced by CPT was much lower
than that in its parental cells, HONE-1. There were almost
no detectable PLDBs in CPT 30 cells in the presence of 1–
25 mM BPR0Y007.
The effect of BPR0Y007 on microtubule polymerization
was examined in vitro. Figure 6A shows the result of
microtubule assembly in vitro using MAP-rich tubulin.
In control samples (without the addition of any test agents),
absorbance at 350 nm (A350 nm) increased with time. The
initial rate of increase was slow but accelerated within
ꢀ3 min after initiation of the reaction. The increase in
A350 nm reached a plateau in ꢀ12 min. In the presence of
10 mM colchicine, tubulin polymerization was inhibited
completely throughout the reaction (data not shown).
Furthermore, in the presence of different concentrations
of BPR0Y007, tubulin polymerization was inhibited in a
concentration-dependent manner. The inhibitory concen-
tration that reduced the amount of polymerized tubulin by
50% was 1:5 Æ 0:2 mM (N ¼ 2).
3.4. Interaction of BPR0Y007 with DNA
Most of the non-CPT Top I inhibitors are DNA inter-
calators and/orminorgroove binders. Toinvestigatewhether
BPR0Y007 intercalates into DNA, an unwinding assay was
In Fig. 6B, the effect of BPR0Y007 on microtubule
assembly was compared with those of paclitaxel, vincris-
tine, and colchicine during an in vivo study. In the presence

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Fig. 2. (A) Effect of BPR0Y007 on Top I catalytic activity. Supercoiled pBR322 DNA was incubated with one unit of Top I in the presence of CPT and
BPR0Y007 in a reaction mixture for 30 min at 378. The DNA was analyzed by electrophoresis in a 1% agarose gel. Lane 1, control DNA; lane 2, no drug;
lanes 3–6, CPT 5, 10, 25, and 50 mM, respectively; lanes 7–10, BPR0Y007 5, 10, 25, and 50 mM, respectively. (B) DNA cleavage pattern induced by CPT and
BPR0Y007 in the presence of Top I. 30-Ended-32P-labeled pPR322 DNA was incubated with 20 units of purified Top I in the presence of CPT and BPR0Y007
in a reaction mixture for 15 min at 378. The reactions were terminated by SDS-proteinase K treatment. Samples were analyzed by alkali gel electrophoresis.
Lane 1, control DNA; lane 2, no drug; lane 3, CPT 10 mM; lanes 4–6, BPR0Y007 5, 15, and 50 mM, respectively.
of 100 nM colchicine and 10 nM vincristine, inhibition of
microtubule assembly was observed, whereas 100 nM
paclitaxel promoted tubulin polymerization. Similar to
the effect of colchicine and vincristine, BPR0Y007 pre-
vented tubulin polymerization and reached a plateau at a
concentration of 5 mM. We further determined the nature of
BPR0Y007 interactions with tubulin by binding colchi-
cine-binding domains on tubulin using a spin column
assay. As shown in Fig. 6C, tubulin incubation with
[3H]colchicine in the presence of different concentrations
of unlabeled colchicine reduced the amount of [3H]colchi-
cine found in the flow-through in a concentration-depen-
dent manner. At concentrations up to 10 mM, BPR0Y007
reduced the amount of [3H]colchicine in the flow-through.
No further reduction in radioactivity was observed at
BPR0Y007 concentrations greater than 10 mM.
3.7. Apoptosis induction by BPR0Y007
The effect of BPR0Y007 on the induction of apoptosis
was also examined. Using the TUNEL assay (Fig. 7A),
green fluorescein was detectable after treating cells
with 10 mM BPR0Y007 and was increased with concen-
trations up to 50 mM. We also observed an increase in the
ability of cell lysates to clear the caspase-3 substrate, Ac-
DEVD-AMC, in a concentration-dependent (Fig. 7B) and

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Fig. 3. Measurement of PLDBs in vivo by a K-SDS co-precipitation assay. HONE-1 and CPT 30 cells were treated with 1, 5, 10, and 25 mM CPT and
BPR0Y007 for 30 min at 378, respectively. The experimental procedures were described in Section 2. Data represent means Æ SD from three independent
experiments performed in duplicate.
Fig. 4. (A) DNA unwinding assay. Linear DNA and an excessive amount of T4 ligase were incubated with different concentrations of adriamycin, CPT, and
BPR0Y007, as indicated, at 158 for 60 min. Unwinding measurements were performed as described in Section 2. (B) Quenching of the fluorescence of DNA-
bound Hoechst 33342 in a cell-free system by BPR0Y007. Netropsin and VP-16 were used as a positive and negative control, respectively. The
concentrations were 1, 5, 10, 25, and 50 mM for each compound.

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Table 2
Growth inhibition of BPR0Y007 against human tumor cell lines
Cell lines
Origin
IC (mM)
50
KB
Epidermoid carcinoma
Nasopharyngeal carcinoma
Breast carcinoma
3.5 Æ 0.3
1.9 Æ 0.1
7.7 Æ 1.0
4 Æ 0.5
HONE-1
MCF-7
TSGH
HT-29
H460
Stomach carcinoma
Colon carcinoma
7.2 Æ 0.4
1.8 Æ 0.3
4.6 Æ 1.0
Non-small cell lung cancer
Glioblastoma multiformi
DBTRG
Values are averages Æ SD of at least three independent experiments.
more, two CPT-resistant cell lines, KB 100 and CPT 30,
which exhibited a decrease in Top I content and a mutation
in the Top I gene, were as sensitive to BPR0Y007 as their
parental cells. The IC of BPR0Y007 to various human
50
cancer cell lines tested ranged from 1 to 8 mM (Table 2).
4. Discussion
By screening potential antitumor agents using a high-
throughput cytotoxicity assay, we identified BPR0Y007 as
an agent possessing antiproliferative properties. We further
determined that BPR0Y007 inhibits Top I-mediated DNA
relaxation with less potency than does CPT (Fig. 2A).
Furthermore, BPR0Y007 induced Top I-mediated single-
strand DNA breaks. The cleavage specificity was similar to
that of CPT, whereas the relative cleavage intensity
induced by BPR0Y007 was less than that of CPT. In a
cellular-based study, BPR0Y007 generated protein-linked
DNA breaks in a concentration-dependent manner. More-
over, the number of PLDBs generated by BPR0Y007 was
lower than that generated by CPT, consistent with the in
vitro findings that BPR0Y007 possessed lower inhibitory
potency against Top I than did CPT. Similar to CPT, the
number of PLDBs induced by BPR0Y007 in a CPT-
resistant cell line was lower than that in parental
HONE-1 cells, further indicating that BPR0Y007 targets
Top I. Several lines of evidence have shown that non-CPT
Top I inhibitors are DNA intercalators and/or minor groove
binders (for review, see Ref. [29]). Our unwinding assay
data suggest that BPR0Y007 does not intercalate into DNA
(Fig. 4A). However, similar to the effect of netropsin,
Fig. 5. Effects of CPT and BPR0Y007 on cell cycle distribution of KB
cells. The cell cycle analysis was done as described in Section 2. Cells
were treated with various concentrations of CPT (top panel) and
BPR0Y007 (bottom panel) expressed as multiples of IC values [(CPT
50
or BPR0Y007): IC50] for 24 hr. The IC50 was based on the growth inhibition
assay. This result is representative of three independent experiments.
time-dependent (Fig. 7C) manner, which was independent
of caspase-1 (data not shown).
3.8. No cross-resistance to CPT-, vincristine- and VP-16-
resistant cells
The cytotoxicity of BPR0Y007 toward various human
cancer and drug-resistant cell lines was evaluated. As
shown in Table 1, KB vin10 cells, which displayed over-
expressed P-gp and down-regulated Top II, and KB 7D
cells, which overexpressed MRP, were 3-fold more sensi-
tive to BPR0Y007 than their parental KB cells. Further-
Table 1
Growth inhibition of various compounds against different drug-resistant cell lines
IC
50
KB
KB vin10
KB 100
KB 7D
HONE-1
CPT 30
BPR0Y007 (mM)
Vincristine (nM)
CPT (nM)
3.5 Æ 0.3
1.2 Æ 0.2
3 Æ 0.5
1 Æ 0.2
1.9 Æ 0.2
1.4 Æ 0.3
27 Æ 5
1.3 Æ 0.1
1.6 Æ 0.2
355 Æ 30
0.4 Æ 0.1
0.8 Æ 0.1
30 Æ 1.2
0.6 Æ 0.2
50 Æ 10
0.9 Æ 0.2
4 Æ 1
65 Æ 5
420 Æ 15
25 Æ 2.3
0.7 Æ 0.3
1286 Æ 33
3.1 Æ 0.5
7.9 Æ 0.3
33 Æ 2.5
1.2 Æ 0.4
15 Æ 1.5
44 Æ 3.5
7.9 Æ 0.5
55 Æ 7
VP-16 (mM)
Taxol (nM)
0.5 Æ 0.1
1.2 Æ 0.3
31 Æ 0.8
16500 Æ 707
115 Æ 7
Colchicine (nM)
10.5 Æ 1
Values are averages Æ SD of at least three independent experiments.

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Fig. 6. (A) Effect of BPR0Y007 on microtubule assembly in vitro. MAP-rich tubulin in a reaction buffer was incubated at 378 in the presence of 4% (v/v)
DMSO (^), and 1 mM (^), 2.5 mM (&), 5 mM (~) and 10 mM (*) BPR0Y007. This result is representative of two independent experiments. (B) Effect of
tubulin polymerization in vivo. Cells were treated with colchicine (100 nM), paclitaxel (100 nM), vincristine (10 nM), and 1, 5, 10, and 25 mM BPR0Y007 for
24 hr at 378. The in vivo tubulin polymerization assay was done as described in Section 2. (C) Binding of BPR0Y007 to the colchicine site of tubulin, as
determined by a spin column assay, as described in Section 2. The concentrations for colchicine and BPR0Y007 were 1, 5, 10, 25, and 50 mM. Data represent
means Æ SD from three independent experiments performed in duplicate.
BPR0Y007 displaced Hoechst 33342 dye in a cell-free
system (Fig. 4B). Both Hoechst 33342 and netropsin bind
to AT-rich sites in the minor groove of DNA and cause
widening of the minor grooves [28]. Therefore, these
results suggest that BPR0Y007 binds to DNA, probably
in a way similar to that of Hoechst 33342. Previous studies
of non-CPT Top I inhibitors indicate that Top I-mediated
DNA cleavage by these strong intercalators is suppressed
at higher drug concentrations because the change of DNA
conformation blocks Top I access to DNA [30,31]. In the
case of BPR0Y007, however, Top I-mediated DNA clea-
vage reached a plateau at 15 mM, and no suppression of
DNA cleavage occurred at concentrations up to 50 mM.
Thus, BPR0Y007 can be categorized as a drug with low
DNA binding affinity, which induces DNA cleavage with-
out suppression at high concentrations.

J.-Y. Chang et al. / Biochemical Pharmacology 65 (2003) 2009–2019
2017
Fig. 7. (A) Effect of BPR0Y007 concentration on the induction of apoptosis. This effect was determined by a TUNEL test as described in Section 2.
(B) Concentration-dependent increase in caspase-3 activation (Ac-DEVD-AMC) in KB cells after treatment for 24 hr. (C) Time-dependent increase in
caspase-3 activation in KB cells after treatment with 12 mM BPR0Y007 for the indicated times. Results are representative of two independent experiments.
We were surprised to observe no cross-resistance with
BPR0Y007 in two CPT-resistant cell lines (Table 1), CPT
30 and KB 100, despite the fact that BPR0Y007 generated
fewer PLDBs in CPT 30 cells than in HONE-1 cells.
Furthermore, it has been demonstrated that CPT is an
S-phase-specific DNA-damaging agent [8]. In our cell
cycle study, cells treated with CPT accumulated in
S-phase with concomitant losses from the G0/G1 and
G2/M phases, especially at higher drug concentrations,
whereas BPR0Y007 arrested cells in the G2/M phase
(Fig. 5). These results suggest that BPR0Y007 might act
on other nuclear targets. Using in vitro and in vivo tubulin
polymerization assays (Fig. 6A and B), we found that
BPR0Y007 inhibits tubulin polymerization in a concentra-
tion-dependent manner. The mechanism of tubulin-inter-
acting agents includes disruption of microtubule assembly
(vinca alkaloids and colchicine) and stabilization of micro-
tubules (paclitaxel and docetaxel). In contrast to paclitaxel-
promoted tubulin polymerization, the pattern of inhibition
of tubulin polymerization of BPR0Y007 was similar to
those of colchicine and vincristine (Fig. 6B), which pre-
vented tubulin polymerization. BPR0Y007 is thus classi-
fied as a ‘‘microtubule depolymerizing agent.’’
BPR0Y007 cytotoxicity exhibits features that are some-
what unusual, compared with those of other agents that
inhibit Top I and tubulin. For example, a decrease in a Top
I intracellular level and/or a mutation in the Top I gene,
which are the common mechanisms of resistance to Top I
inhibitors, does not inhibit cell sensitivity to BPR0Y007,
as observed in two CPT-resistant cell lines, KB 100 and
CPT 30. This indicates that BPR0Y007 cytotoxicity likely
correlates with parameters other than anti-Top I activity,
such as anti-tubulin activity. Furthermore, as previously
reported, overexpression of P-gp or MRP best described
the mechanism of resistance to tubulin-binding agents,
including vincristine, colchicine, and paclitaxel [32,33].
In our study, different levels of resistance to vincristine,
colchicine, and paclitaxel in a vincristine-resistant cell
line (KB vin10) that overexpressed P-gp were observed,
yet BPR0Y007 exhibited greater cytotoxicity toward KB
vin10 cells than toward KB cells (Table 1). Similar find-
ings were obtained from a VP-16-resistant cell line
(KB 7D), which decreased cellular Top II content and
overexpressed MRP. Therefore, BPR0Y007 exhibits a
broad spectrum of activity and is cytotoxic to cells that
are resistant to either Top I or Top II or tubulin inhibitors. It

2018
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will be interesting to determine whether Top I or tubulin is
the principal target of BPR0Y007 in tumor cells. Inves-
tigating the cytotoxic effects on mammalian cells, which
are resistant to BPR0Y007, may be able to supply the
answer.
Bis-benzylidenecyclopentanone derivatives have been
shown to possess antioxidative properties [13]. We demon-
strated for the first time that this class of compounds
possesses cytotoxicity, which acts through inhibition of
Top I and tubulin. In addition, BPR0Y007 also induces
apoptosis in solid tumor cells (Fig. 7).
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Diferuloylmethane (curcumin), an antioxidant, induced
apoptosis in a human leukemia cell line [34]. BCL-2 may
play a role in the early stage of curcumin-triggered apop-
totic cell death. Whether the BPR0Y007-induced apoptotic
pathway is similar to curcumin is under investigation.
In summary, BPR0Y007 is a dual inhibitor of Top I and
tubulin. The structure of this compound resembles neither
the structure of CPTs nor the structure of known tubulin
inhibitors. Moreover, no cross-resistance with BPR0Y007
in CPT-, vincristine-, and VP-16-resistant cells was
observed. Given its unique biochemical properties,
BPR0Y007 warrants further investigation as an antitumor
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