A novel bis-benzylidenecyclopentanone derivative, BPR0Y007, inducing a rapid caspase activation involving upregulation of Fas (CD95/APO-1) and wild-type p53 in human oral epidermoid carcinoma cells

Article abstract of DOI:10.1016/j.bcp.2004.03.036

BPR0Y007, a bis-benzylidenecyclopentanone derivative (2,5-bis- (4-hydroxy-3-methoxybenzylidene) cyclopentanone), was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells.

Full text of DOI:10.1016/j.bcp.2004.03.036

Biochemical Pharmacology 68 (2004) 293–303
A novel bis-benzylidenecyclopentanone derivative, BPR0Y007, inducing
a rapid caspase activation involving upregulation of Fas (CD95/APO-1)
and wild-type p53 in human oral epidermoid carcinoma cells
a
Shin-Hun Juang , Wen-Yu Pan , Ching-Chuan Kuo , Jing-Ping Liou , Yi-Mei Hung ,
a
a
b
a
a
b
Li-Tzong Chen , Hsing-Pang Hsieh , Jang-Yang Chang
a,*
a
Cancer Cooperative Ward in National Taiwan University Hospital, Division of Cancer Research, National Health Research Institutes,
, Chung-Shan Road, Taipei, Taiwan, ROC
7
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei, Taiwan, ROC
b
Received 8 December 2003; accepted 23 March 2004
Abstract
BPR0Y007, a bis-benzylidenecyclopentanone derivative (2,5-bis- (4-hydroxy-3-methoxybenzylidene) cyclopentanone), was identified
in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. A previous
study showed that BPR0Y007 inhibited DNA topoisomerase I (Top 1) activity and prevented tubulin polymerization. Notably, no cross-
resistance with BPR0Y007 was observed in camptothecin-, VP-16- or vincristine-resistant cell lines. In this study, we further investigated
the cellular and molecular events underlying the antitumoral function of this compound in human oral epidermoid carcinoma KB cells,
focusing on the early cytotoxic effect. Treatment of KB cells with BPR0Y007-induced G /M phase arrest followed by sub-G phase
2
1
accumulation. Annexin-V–propidium iodide (PI) binding assay and DNA fragmentation assay further indicated that BPR0Y007-induced
cell death proceeded through an apoptotic pathway as opposed to via necrosis. This compound produced a time-dependent activation of
caspases-3 and -8, however, another caspase-3 initiator, caspase-9, was only marginally activated at later time point. We further demonstrated
that the activation of the caspases cascade and nuclear fragmentation was not associated with inactivated Bcl-2 and perturbed mitochondrial
membrane potential by BPR0Y007. The finding that BPR0Y007-induced apoptosis through a membrane-mediated mechanism was
supported by up-regulated expression of Fas (CD95/APO-1), but not Fas-L. Furthermore, up-regulation of p53 and its affected gene, MDM2,
in KB cells was found after BPR0Y007 exposure. Overall, our results demonstrated that the BPR0Y007 could induce an early cytotoxic
apoptosis through a caspase-8-dependent but mitochondrial-caspase-9 independent pathway, and involving upregulation of p53.
#
2004 Elsevier Inc. All rights reserved.
Keywords: bis-Benzylidenecyclopentanone derivative; Antitumoral; Apoptosis; Caspase; Fas (CD95/APO-1); p53
1
. Introduction
BPR0Y007 possessed equally effective activity against
parental- and in vitro-derived drug-resistant cells that were
generated by vincristine, VP-16, or camptothecin-driven
selection [1]. Our previous study demonstrated that
BPR0Y007 is a potent inducer of apoptosis in cancer
cells. However, the mode of action of BPR0Y007-induced
apoptosis is not yet fully understood.
The novel synthetic bis-benzylidenecyclopentanone
derivative, 2,5-bis-(4-hydroxy-3-methoxybenzylidene)-
cyclopentanone (BPR0Y007) (Fig. 1), was identified as
a dual inhibitor of DNA topoisomerase I (Top 1)s and
microtubule in our laboratory [1]. Notably, we showed that
Apoptosis is an active cell suicide mechanism consisting
of an evolutionarily conserved cascade that includes char-
acteristic features such as cell shrinkage, condensation
of chromatin, and formation of specific oligonucleotide
fragments. All apoptotic pathways converge on a family
of cysteine-aspartases, named caspases [2]. To date, two
major caspases pathways, led by caspases-8 and -9 have
Abbreviations: BPR0Y007, 2,5-bis-(4-hydroxy-3-methoxybenzylidene)
0
cyclopentanone; DiOC (3), 3,3 -Dihexyloxacarbocyanine iodide; MPT,
6
mitochondrial potential transition; PBS, phosphate buffered saline; PI,
propidium iodide; Top 1, DNA topoisomerase I
*
Corresponding author. Tel.: þ886-2-23562879;
fax: þ886-2-23892737.
E-mail address: jychang@nhri.org.tw (J.-Y. Chang).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.03.036

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S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
O
2.2. Reagents
O
O
Monoclonal antibodies to Bcl-2, Fas (CD95/APO-1),
MDM2, p53, PARP, as well as horseradish peroxidase-
conjugated secondary antibody were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-
body to Fas-L was purchased from Oncogene Research
Products (Darmstadt, Germany). Monoclonal antibody for
a-tubulin was purchased from Sigma Chemical (St. Louis,
MO). Caspase inhibitor Z-DEVE-fmk, Z-IETD-fmk,
and Z-LEHD-fmk were purchased from Calbiochem-
Novabiochem Co (La Jolla, CA). Cell culture reagents
were obtained from Gibco-BRL Life Technologies
HO
OH
Fig. 1. Chemical structure of BPR0Y007.
been described and shown to mediate distinct sets of
signals [3–5]. The death receptor-mediated pathway is
triggered by rapid formation of death-inducing signaling
complex (DISC) [6] by Fas, tumor necrosis factor (TNF),
or TNF-a-related apoptosis-inducing ligand (TRAIL).
On activation, these receptors recruit Fas (CD95/APO-1)-
associated death domain (FADD), which in turn binds to
procaspase-8 and cleaves into its active form. The stress-
induced mitochondrial-dependent pathway is triggered
by the cytochrome c release from mitochondria, leading
to the formation of a complex with Apaf-1 that, via its
caspase-recruitment domain, binds to procaspase-9. This
in turn activates effector caspases [7].
It is well known that antitumor agents induce apoptosis
through various signaling pathways in tumor cells [8]. In
the present study, we investigated the contribution of the
major apoptotic pathways to the cytotoxic effect of
BPR0Y007 in human oral epidermoid carcinoma KB cells.
We showed that BPR0Y007-induced apoptosis involves
the induction of Fas (CD95/APO-1), and activation of
p53, caspase-8, and downstream caspases. We further
demonstrated that the activation of the caspase cascade
was not associated with perturbed mitochondrial mem-
brane potential by BPR0Y007. Triggering of this signaling
route can explain the rapid and potent proapoptotic activity
of BPR0Y007. These data suggest that BPR0Y007 may
be a promising candidate in the treatment of neoplastic
disease.
0
(Gaitherburg, MD). 3,3 -Dihexyloxacarbocyanine iodide
[(DiOC )(3)] was purchased from Molecular Probes Inc
(Eugene, OR). Acrylamide, bisacryamide, ammonium per-
6
0
0
sulfate, and N,N,N ,N -tetramethylethylene diamine were
from Bio-Rad (Richmond, CA). Western blot chemillumi-
nescent reagent was purchased from Perkin-Elmer Life
Sciences (Boston, MA). All of the other chemicals were
from E. Merck Co. (Darmstadt, Germany) or Sigma
Chemical (St. Louis, MO), and were standard analytic
grade or higher.
2.3. Cell line and growth inhibitory assay
The human oral epidermoid carcinoma KB cells were
obtained from American Type Culture Collection (Rock-
ville, MD) and maintained in RPMI-1640 medium sup-
plied with 5% fetal bovine serum, 2 mM L-glutamine,
100 units/mL penicillin, and 100 mg/mL streptomycin.
For cytotoxic assay, cells in logarithmic phase were cul-
tured at a density of 5000 cells/well in a 24-well plate. The
cells were exposed to various concentrations of BPR0Y007
for indicated times. The methylene blue dye assay was
used to evaluate the effects of the test compound on cell
growth, as described previously [10], to determine the
concentration of BPR0Y007 that inhibited 50% of cell
growth (IC ).
2
. Materials and methods
5
0
2
.1. Synthesis of BPR0Y007
2.4. Flow cytometric analysis
BPR0Y007 was synthesized at the Division of Biotech-
Flow cytometry was performed on a single-cell suspen-
sion on adherent as well as floating cells after pooling
them. For cell cycle analysis, the exponentially growing
cells were incubated with BPR0Y007-containing medium
for the selected treatment duration; then cells were fixed
and stained with 50 mg/mL of propidium iodide (PI). DNA
content was evaluated using FACSVantage (Becton Dick-
inson Labware, Franklin Lakes, NJ) and the percentage of
cells in various cell cycle phases were determined using the
ModFit LT software (Verity Software House, Topsham,
ME). For each analysis, 10,000 events were recorded. For
Annexin-V–PI binding assay, Annexin-V-FLUOS Staining
Kit (Roche Applied Science, Mannheim, Germany) was
used according the manufacturer’s instruction. Briefly, the
nology and Pharmaceutical Research, National Health
Research Institutes, Taipei, Taiwan, Republic of China,
according to the procedure described by Sardjiman et al.
[
9]. Briefly, vanillin and cyclopentanone were heated in a
water bath (45–50 8C) until a clear solution was obtained;
concentrated hydrochloric acid was then added followed
by 2 h of stirring. After standing overnight, the mixture
was treated with cold acetic acid/water (1:1) and filtered.
The solid material was washed first with cold ethanol,
then with hot water, and dried under a vacuum. The
yellow substance was recrystallized from ethanol. The
1
structure of BPR0Y007 was confirmed by H NMR and
EI–MS.

S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
295
BPR0Y007-treated cells were trypsinized and collected
2.6. Caspases inhibitory assay
with in phosphate buffered saline (PBS) by centrifugation
and then resuspended in 100 ml of Annexin-V-FLUOS
labeling solution. After incubation at room temperature
for 15 min in dark, the cells were analyzed by flow cyto-
metry (FACSVantage, Becton Dickinson Labware, Franklin
Lakes, NJ). Control cells stained with Annexin-V or PI
alone was used to compensate for the flow cytometric
analysis. Annexin-V and PI double-negative cells are
defined as live cells; Annexin-V-positive, PI-negative cells
are defined as early apoptotic cells; and annexin-V and PI
double-positive cells are defined as late apoptotic and
necrotic cells. For mitochondrial membrane potential assay,
In the caspase inhibitory experiments, the cell-perme-
able and specific irreversible inhibitors of caspases-3, -8, or
-9 were added to the medium 1 h prior to BPR0Y007
administration. Cell survival was determined by the methy-
lene blue dye assay to evaluate the effects of the test
compound on cell growth as described previously [10].
The stock solution of caspase inhibitors was prepared in
DMSO and all additions were 0.5% (v/v) or less.
2.7. Preparation of cell lysates and western
blot analysis
mitochondrial potential transition (MPT; Dc ) was deter-
m
6
2
mined as proportional to the retention of the dye DiOC (3).
Cells were initially seeded at 1 ꢀ 10 cells in 100 mm
6
Cells were treated with 5 mM BPR0Y007 for the selected
treatment duration. Thirty minutes prior to the conclusion of
each treatment duration, in vitro labeling of cells was
performed with 40 nM DiOC (3) and cells were incubated
dishes. After treatment for the indicated time with various
concentrations of BPR0Y007, adherent cells were washed
twice with PBS, gently scraped from the dishes, then
centrifuged, lysed in ice-cold lysis buffer (50 mM Tris
6
,
at 37 8C in a humidified atmosphere at 95% O and 5%
[pH 7.4], 0.8 M NaCl, 5 mM MgCl , 0.5% NP40, 1 mM
2
2
CO following the instructions of the manufacturer. After
PMSF, 1 mg/mL pepstatin, and 50 mg/mL leupeptin), and
cleared by microcentrifugation. Protein concentrations of
lysates were determined using the BCA Protein Assay
Reagent (PIERCE Biotechnology, Rockford, IL). Equiva-
lent protein amounts of each sample were separated on
SDS-PAGE gels and blotted to nitrocellulose membrane.
After soaking in a blocking solution containing PBS,
0.05% Tween 20, and 5% skim milk, the blot was incu-
bated with the primary antibody at 1–2 mg/mL, and anti-
body binding was detected using the appropriate secondary
antibody coupled with horseradish peroxidase according to
the instructions of manufacturer. Enhanced chemilumines-
cence was used to detect the relevant proteins following
protocols suggested by the manufacturer.
2
removal of the medium supernatant and rinsing of the cell
dish with PBS, cells were harvested and suspended in PBS.
Measurement of the retained DiOC (3) in 10,000 cells of
6
each sample was performed in a flow cytometer (FACS-
Vantage, Becton Dickinson Labware, Franklin Lakes, NJ).
2
.5. Nuclear fragmentation assay
6
2
Cells were initially seeded at 1 ꢀ 10 cells in 100 mm
dishes overnight, and BPR0Y007 was added to the final
concentration of 5 mM. At predetermined time intervals,
the non-adherent cells were collected by aspiration, and
adherent cells were harvested using 0.25% trypsin/0.05%
EDTA. DNA was extracted from adherent and non-adher-
ent cells using Wizard Genomic DNA Purification Kit
2.8. p53 transactivation assay
(Promega, Madison, WI), according to the instructions
of the manufacturer. The isolated DNA samples were
fractionated by electrophoresis in 1.5% agarose gels and
visualized by ethidium bromide staining (0.5 mg/mL)
under ultraviolet transillumination.
Determination of Caspase Activity. The activity of
caspases-3, -8 and -9 on BPR0Y007-treated cells were
determined by using Caspase Fluorometric Assay kits
p53 status was studied by transactivation assay. KB cells
were seeded 24 h before transiently transfected with 2 mg
of p53-Luc reporter plasmid (Stratagene, La Jolla, CA)
using the electropration method following the manufac-
turer’s instructions (Cellject Pro, Thermo Hybaid, MA).
Sixteen hours after transfection, cells were cultured in the
presence of 5 mM BPR0Y007 for the selected treatment
duration. After incubation, cells were collected and lysed
with the reported lysis buffer (Promega, Madison, WI).
Luciferase activity was then determined on a Victor 1420
Multilable Counter (Wallac, Turku, Finland) following the
manufacturer’s instructions.
(R&D System Inc., Minneapolis, MN) or CaspACE Assay
System-Fluorometric (Promega Corporation, Madison,
WI) for the cleavage of respectively specific fluorogenic
peptide substrate according to the instruction of the man-
6
ufacturer. Briefly, 3 ꢀ 10 cells were collected, lysed in a
lysis buffer, and clarified by centrifugation. Equal amounts
of the extract supernatants were incubated with 50 mM of
fluorescent substrate. After 1 h of incubation at 37 8C,
fluorescence of the cleaved substrate was determined using
the Victor 1420 Multilable Counter (Wallac, Turku, Fin-
land), and caspase activity was calculated by subtracting
the value obtained in the untreated sample.
2.9. Statistical analysis
All assays were carried out in triplicate. Data were
expressed as means with standard deviations. Student’s
t-test (P < 0:05) was calculated to compare the mean of
each group with that of the control group.

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3
. Results
Annexin-V–PI binding assay. A significant increase from
.9 to 5 and 12.8% of apoptotic cells (Annexin-V positive/
2
3
.1. Treatment with BPR0Y007 caused concentration-
PI-negative) were found in the 12 and 18 h-BPR0Y007
treated KB cells, respectively (Fig. 3B). Furthermore, the
DNA fragmentation assay revealed a typical laddering of
the DNA in KB cells exposed to BPR0Y007 as early as
12 h, and a strong laddering signal was found after 24 h
(Fig. 3C). These results demonstrated that BPR0Y007
and time-dependent growth inhibition in human oral
epidermoid carcinoma KB cells
The growth inhibitory effect of BPR0Y007 was exam-
ined at doses between 0.5 and 10 mM for the selected
treatment duration. Cell growth was markedly inhibited
by BPR0Y007 treatment in a concentration- and time-
dependent manner (Fig. 2). Approximately 60, 76, and
inhibited KB cells by arresting the cells in the G /M
2
phase, and this, in turn, activated the apoptotic signaling
pathway.
9
3% growth inhibition were detected after 24, 48, and 72 h
BPR0Y007 treatment, respectively. Thus, we chose 5 mM
BPR0Y007 to detect changes in molecular events in the
following experiments.
3.3. Activation of caspase-3 and cleavage of
PARP by BPR0Y007
To gain insights into the mechanism by which
BPR0Y007 induces apoptosis, we investigated the effects
of BPR0Y007 on caspase. Of the many caspases involved
in apoptosis, we first examined the in vivo effect of
BPR0Y007 on caspase-3, which is activated by a number
of apoptotic signals [11]. As shown in Fig. 4A, incubation
of KB cells with BPR0Y007 resulted in the activation of
caspase-3 in a time-dependent manner; this effect started
from 4 h of exposure (1.56-fold increase compared with
control cells), with a maximum of 3.64-fold induction
being observed by 12 h of treatment. Activation of cas-
pase-3 during BPR0Y007-induced apoptosis was also
confirmed by examining PARP, a known endogenous
substrate for caspase-3 and an early marker of apoptosis
[12,13]. As shown in Fig. 4B, caspase-3 activation was
accompanied by the cleavage of PARP (116 kDa) into an
85-kDa C-terminal fragment that became evident 12 h after
BPR0Y007 treatment.
3
BPR0Y007
.2. Induction of G /M phase arrest and apoptosis by
2
To examine the effect of BPR0Y007 on cell cycle
progression, KB cells were treated with 5 mM BPR0Y007
for the selected treatment duration and the cell cycle
distribution was analyzed by flow cytometry. As shown
in Fig. 3A, treatment with BPR0Y007 resulted in accu-
mulation of cells in the G /M phase starting from 2 h of
2
exposure, with a maximum accumulation being observed
at 12 h of treatment (70% in the drug-treated cells versus
3
G /G phase. The hypodiploid population of KB cells
2% in the control cells) with a concomitant loss of the
0
1
increased from approximately 2% in untreated control
cells to approximately 18% in 24 h in BPR0Y007-treated
cells. To further assess the hypodiploid population in
the BPR0Y007-treated KB cells, we performed the
100
0
1
.5 µM
.0 µM
80
60
40
20
0
2.5 µM
5.0 µM
µM
10
0
1
2
3
4
Incubation Period (day)
Fig. 2. BPR0Y007-induced growth inhibition in human epidermoid carcinoma KB cells. Cell were treated with various concentrations of BPR0Y007 at
7 8C for the indicated times. Growth inhibition of cells was determined by methylene blue assay as described under Section 2.
3

S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
297
Fig. 3. Effect of BPR0Y007 on cell cycle progression and apoptosis in oral epidermoid carcinoma KB cells. (A) Time effect of BPR0Y007 on cell cycle
profile in KB cells. Cells were treated with 5 mM BPR0Y007 for the indicated time and analyzed for PI-stained DNA content by flow cytometry.
(B) BPR0Y007-induced cells underwent apoptosis as demonstrated by Annexin-V positivity. KB cells were treated with 5 mM BPR0Y007 for the indicated
times then incubated with Annexin-V and PI and subjected to flow cytometric analysis. (C) BPR0Y007 induces DNA laddering fragmentation. KB cells
were treated with indicated concentrations of BPR0Y007 for the selected treatment duration. After treatment, cells were lysed with lysis buffer, and
DNA was extracted, then dissolved in TE buffer, and subjected to electrophoresis in a 1% agarose gel. DNA laddering was visualized by ultraviolet
transillumination.

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S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
Fig. 4. Activation of caspase-3 and proteolytic cleavage of PARP induced by BPR0Y007 in oral epidermoid carcinoma KB cells. KB cells were culture in
control medium only, or 5 mM BPR0Y007 for different time intervals as indicated. (A) Caspase-3 activity was determined as described in Section 2 using
caspase-3 fluorogenic peptide substrate DEVD-AMC. Values are the means of triplicate analysis. Error bars show the standard deviations. (ꢁ), (ꢁꢁ), (ꢁꢁꢁ),
significantly different form the control at P < 0:05, <0.01, <0.001, respectively, with Student’s t-test. (B) The cleavage of PARP was examined by Western
blot analysis as described in Section 2.
3.4. Apoptosis in BPR0Y007-treated cells is associated
with activation of caspase-8, but not caspase-9
KB cells with membrane-permeable irreversible inhibitor
of caspases-3, -8, or -9 and measured the cell growth under
the influence of BPR0Y007. Results demonstrated that in
the presence of caspase-8-specific inhibitor Z-IETD-fmk
and the caspase-3 inhibitor Z-DEVD-fmk, BPR0Y007-
induced apoptosis is significantly reduced from 50 to
27 and 22%, respectively at 24 h (Fig. 5B). In contrast
to caspases-3 and -8, cells pretreated with the caspase-
9-specific inhibitor, Z-LEHD-fmk, did not prevent
BPR0Y007-induced cell death (Fig. 5B), indicating that
the activation of caspase-9 might not involve BPR0Y007-
induced apoptosis.
To understand the mechanism by which caspase-3 is
activated by BPR0Y007, we further investigated the effect
of BPR0Y007 on the activity of caspases-8 and -9,
upstream activators of caspase-3. Upon treatment of KB
cells with BPR0Y007, a strong activity toward the caspase-
8
specific IETD-AFC fluorogenic tetrapeptide substrate
were detected as early as 4 h after treatment with a further
increase with time (Fig. 5A). In contrast to the high level of
caspase-8-specific activity, weak caspase-9-specific activ-
ity was detected (Fig. 5A), with only 1.37-fold of caspase-9
activity being observed in cells treated with BPR0Y007 for
3.6. Apoptosis in BPR0Y007-treated cells is not related
to mitochondria-mediated caspase-9 pathway
2
4 h. In addition, BPR0Y007-induced caspase-8 activation
showed similar time courses compared with caspase-3
activation. Thus, we propose that BPR0Y007 induces
apoptosis through a caspase-8 mediated pathway.
Because weak caspase-9-specific activity was detected
until treatment with 5 mM BPR0Y007 for 24 h, and
because a previous report by Ferreira et al. [14] showed
that caspase-8 can be activated in a mitochondria-con-
trolled, caspase-9 independent manner. Therefore, we
examined whether mitochondria were involved in initiat-
ing BPR0Y007-induced apoptosis in KB cells. We com-
pared BPR0Y007-treated and -untreated KB cells by
3.5. Inhibition of caspase-3 or caspases-8 activity
suppresses BPR0Y007-induced apoptosis
To further examine the relationship between caspase acti-
vation and BPR0Y007-induced cytotoxicity, we pretreated

S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
.5
299
2
2
1
1
0
0
Caspase-8
Caspase-9
.0
.5
.0
.5
.0
0
4
8
12
24
(
A)
Time (hours)
70
60
50
40
30
20
10
**
**
(
B)
0
.5% DMSO
-
-
-
-
-
+
-
+
+
-
+
+
+
-
+
-
+
+
-
5
µM BPR0Y007
Z-DEVD-fmk
Z-IETD-fmk
+
+
-
-
-
-
-
-
Z-LEHD-fmk
-
-
+
Fig. 5. (A) Effect of BPR0Y007 on caspase-8 and caspase-9 activity in oral epidermoid carcinoma KB cells. Cells were cultured in control medium only, or
mM BPR0Y007 for different time intervals as indicated. Caspases-8 and -9 activities were determined as described in Section 2 using fluorogenic peptide
5
substrates IETD-AFC and LEHD-AFC, respectively. (B) BPR0Y007-induced cell death in KB cells is reduced with caspase inhibitors. KB cells were
pretreated with 100 mM of the caspase inhibitors (Z-DEVD-fmk, caspase-3 inhibitor; Z-IETD-fmk, caspase-8 inhibitor; Z-LEHD-fmk, caspase-9 inhibitor)
for 1 h, then treated with 5 mM BPR0Y007 for 24 h. The cell survival was determined by the methylene blue dye assay. Values are the means of triplicate
analysis. Error bars show the standard deviations. (ꢁ), (ꢁꢁ), (ꢁꢁ), significantly different form the control at P < 0:05, <0.01, <0.001, respectively, with
Student’s t-test.
staining mitochondria with fluorochrome DiOC (3) and
6
BPR0Y007 treatment. Furthermore, we then examined
the effect of BPR0Y007 in antiapoptotic protein, Bcl-2
[15,16]. The immunoblot analysis presented in Fig. 6B
indicates that KB cells treated with 5 mM BPR0Y007
remained unchanged on Bcl-2 expression within the first
20 h of treatment. However, very faint slower migration
form of Bcl-2 band was observed at 24 h of treatment.
In comparison with caspase-3 activation, PARP cleavage
and DNA ladder were observed in the first 12 h of
determined MPT by measuring the retained DiOC (3)
6
using a flow cytometer. Treatment with carbamyl cyanide
m-chlorophenyl hydrazine (mCCCP), a direct mitochon-
drial activator that can completely abrogate the retention of
DiOC (3) in the cell, was used as a positive control. The
6
results are presented in Fig. 6A, MPT remains unchanged
after KB cells are treated for 24 h with BPR0Y007. How-
ever, MPTonly showed a slight increase until 48 h of 5 mM

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S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
Fig. 6. Effect of BPR0Y007 on mitochondrial potential transition and Bcl-2 expression in oral epidermoid carcinoma KB cells. KB cells were treated with
mM BPR0Y007 for the selected treatment duration. (A) Mitochondrial potential transition was detected by used DiOC assay as described in Section 2. (B)
Bcl-2 expression was examined by Western blot analysis as described in Section 2.
5
6
treatment. These results suggest that mitochondria-medi-
cated caspase-9 activation and Bcl-2 hyperphosphorylation
are not the major and critical steps in the early induction of
apoptosis in KB cells.
classical initiator caspase of the Fas (CD95/APO-1) path-
way [17,18]. The Fas (CD95/APO-1) pathway has also been
shown to contribute to chemotherapeutic agents-induced
apoptosis in various cellular systems [19–21], therefore, the
expression of Fas (CD95/APO-1) was assessed by immu-
noblotting. As shown in Fig. 7, BPR0Y007 significantly
induced the level of Fas (CD95/APO-1) after 4 h treatment.
In contrast to Fas upregulation, no appreciable change in the
expression of Fas-L in KB cells treated with the same
concentration of BPR0Y007 from 4 to 24 h was seen
(Fig. 7). This finding suggests that BPR0Y007-induced
Fas (CD95/APO-1) expression is independent of its ligand.
3
.7. Induction of apoptosis by BPR0Y007 is mediated
by an increase in expression of Fas (CD95/APO-1)
but Not Fas-L in KB cells
The results obtained thus far suggest that caspase-8 may
function as an apical caspase in the pathway that triggers
apoptosis in KB cells. Caspase-8 has been shown to be the
Fig. 7. Effect of BPR0Y007 on Fas (CD95/APO-1) and Fas-L expression on oral epidermoid carcinoma KB cells. KB cells were treated with 5 mM
BPR0Y007 for the selected treatment duration. Cellular extracts were prepared and analyzed by SDS-PAGE and Western blot analysis as described in
Section 2.

S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
301
Fig. 8. BPR0Y007-induced apoptosis involving upregulation of p53 in KB cells. (A) BPR0Y007 increased protein levels of p53 and MDM2. KB cells were
treated with 5 mM BPR0Y007 for the selected treatment duration. Cellular extracts were prepared and analyzed by SDS-PAGE and Western blot analysis as
described in Section 2. (B) Enhanced p53 transcriptional transactivating activity after BPR0Y007 treatment. KB cells were transiently transfected with a
p53-responsive (p53-Luc) luciferase indicator construct. Luciferase activities in cell lysates were determined on a Victor 1420 Multiable Counter. Values are
the means of triplicate analysis. Error bars show the standard deviations. (ꢁ), (ꢁꢁ), (ꢁꢁꢁ), significantly different form the control at P < 0:05, <0.01, <0.001,
respectively, with Student’s t-test.
3
.8. BPR0Y007 up-regulates wild-type p53
dual inhibitor of Top 1 and microtubule [1]. In the present
report, we describe the cellular and molecular events
underlying the growth inhibitory effect of this compound
in human oral epidermoid carcinoma KB cells, focusing on
early cytotoxic characteristics.
Because Fas (CD95/APO-1) is a p53-regulated gene, we
further determined the expression and activity of p53 in KB
cells in response to BPR0Y007 exposure. As shown in
Fig. 8A, BPR0Y007 significantly increased the protein
levels of p53 and its affected gene, MDM2, in a time-
dependent manner. The 1.8-fold of p53 expression has
been increased after 4 h treatment, with a maximum of
Our data showed that treatment with BPR0Y007 results
in a rapid accumulation of the G /M phase starting from
2
2 h of exposure, with a maximum accumulation being
observed at 12 h of treatment. The typical apoptotic char-
acteristics, the hypodiploid DNA content peak (sub-G1),
Annexin-V-positive/PI -negative pattern and DNA ladder-
ing can be detected with treatment duration over 12 h
(Fig. 3). These results demonstrate that BPR0Y007-
4
.8-fold induction being observed by 30 h of treatment. In
addition, by transiently transfecting a reporter gene under
the control of a p53 response element, increases in the level
of p53 transactivation were observed in a time-dependent
manner after BPR0Y007 treatment (Fig. 8B).
induced cells cause an arrest in the G /M phase before
2
apoptotic cell death. The percentage of G /M cells
2
decreases and the percentage of cells in G /G goes
0
1
4
. Discussion
back up to a level similar the one observed at 0 h
after BPR0Y007-treated for 12 h which indicated that
KB cell might escape the BPR0Y007-induced G /M
arrest, however, the detail mechanism is under active
investigation.
The novel synthetic BPR0Y007 is efficacious in sup-
pressing cell growth in a variety of human cancer cells
despite their P-gp170/MDR or MRP status, and acts as a
2

302
S.-H. Juang et al. / Biochemical Pharmacology 68 (2004) 293–303
Caspases are at the heart of the apoptotic machinery
22]. Several caspases have been shown to be key execu-
and activity of p53 in KB cells. Results, illustrated in Fig. 8,
show that p53 was activated in response to BPR0Y007. To
the best of our knowledge, this is the first report that a bis-
benzylidenecyclopentanone compound could up-regulate
both Fas (CD95/APO-1) and p53 expression in human
cancer cells.
In conclusion, our results provide evidence that
BPR0Y007 induces early apoptotic cell death via a Fas/
caspase-8 dependent but mitochondria/caspase-9 indepen-
dent pathway. Our data suggest that upregulation of Fas
(CD95/APO-1) and p53 may account for the early cyto-
toxicity of BPR0Y007 in the oral epidermoid carcinoma
cell line. Because p53 has been reported to up-regulate
transcription of Fas (CD95/APO-1) [35], the detailed
molecular mechanism between BPR0Y007-induced acti-
vation of p53 and the Fas (CD95/APO-1)/Fas-L system
observed in KB cells is currently being investigated.
[
tioners of apoptosis, mediated by various inducers includ-
ing antitumor agents [23]. Recently data have suggested
that caspase-3 is involved in Top 1 and microtubule
inhibitors-induced apoptosis in tumor cells [24,25]. As
shown in Fig. 4, caspase-3 activation was accompanied
by the cleavage of PARP (116 kDa) into an 85-kDa C-
terminal fragment in the BPR0Y007-treated KB cells.
Questions remain as to how caspase-3 is activated by
BPR0Y007. Previous reports have demonstrated that cas-
pases-8 and -9 are upstream initiator caspases that can
directly activate caspase-3 [3].
The Bcl-2 located on the outer mitochondrial membrane
is important for the suppression of apoptosis and mito-
chondrial manifestations of apoptosis [15]. Bcl-2 prevents
the initiation of the cellular program by stabilizing the
mitochondrial permeability and avoiding the subsequent
release of cytochrome c to prevent caspase activation [16].
Several microtubule-disrupting drugs that induce phos-
phorylation and inactivate Bcl-2 lead to accumulation in
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