40
Bufalin induces G0/G1 phase arrest through inhibiting
the levels of cyclin D, cyclin E, CDK2 and CDK4, and
triggers apoptosis viamitochondrial signaling pathway in
T24 human bladder cancer cells

Wen-Wen Huanga, 1,

Jai-Sing Yangb, 1,

Shu-Jen Paia,

Ping-Ping Wuc,

Shu-Jen Changc,

Fu-Shin Chuehd,

Ming-Jen Fane,

Shang-Ming Chiouf, g,

Hsiu-Maan Kuoh,

Chin-Chung Yehi,

Po-Yuan Chena,

Minoru Tsuzukij, k,

Jing-Gung Chunga, e,

a
Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan

b
Department of Pharmacology, China Medical University, Taichung 404, Taiwan

c
School of Pharmacy, China Medical University, Taichung 404, Taiwan

d
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan

e
Department of Biotechnology, Asia University, Taichung 413, Taiwan

f
Department of Functional Neurosurgery & Gamma Knife Center, China Medical University Hospital, Taichung 404, Taiwan

g
School of Medicine, China Medical University, Taichung 404, Taiwan

h
Department of Parasitology, China Medical University, Taichung 404, Taiwan

i
Department of Urology, China Medical University Hospital, Taichung 404, Taiwan

j
Department of Biochemistry, Nihon Pharmaceutical University, Saitama 362-0806, Japan

k
,
Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 404, Taiwan
Abstract
Most of the chemotherapy treatments for bladder cancer aim to kill the cancer cells, but a high recurrence rate after
medical treatments is still occurred. Bufalin from the skin and parotid venom glands of toad has been shown to induce
apoptotic cell death in many types of cancer cell lines. However, there is no report addressing that bufalin induced cell
death in human bladder cancer cells. The purpose of this study was investigated the mechanisms of bufalin-induced
apoptosis in a human bladder cancer cell line (T24). We demonstrated the effects of bufalin on the cell growth and
apoptosis in T24 cells by using DAPI/TUNEL double staining, a PI exclusion and flow cytometric analysis. The effects
of bufalin on the production of reactive oxygen species (ROS), the level of mitochondrial membrane potential (ΔΨm),
and DNA content including sub-G1 (apoptosis) in T24 cells were also determined by flow cytometry. Western blot
analysis was used to examine the expression of G0/G1 phase-regulated and apoptosis-associated protein levels in
bufalin-treated T24 cells. The results indicated that bufalin significantly decreased the percentage of viability, induced
the G0/G1 phase arrest and triggered apoptosis in T24 cells. The down-regulation of the protein levels for cyclin D,
CDK4, cyclin E, CDK2, phospho-Rb, phospho-AKT and Bcl-2 with the simultaneous up-regulation of the cytochrome c,
Apaf-1, AIF, caspase-3, -7 and -9 and Bax protein expressions and caspase activities were observed in T24 cells after
bufalin treatment. Based on our results, bufalin induces apoptotic cell death in T24 cells through suppressing AKT
activity and anti-apoptotic Bcl-2 protein as well as inducing pro-apoptotic Bax protein. The levels of caspase-3, -7 and
-9 are also mediated apoptosis in bufalin-treated T24 cells. Therefore, bufalin might be used as a therapeutic agent for
the treatment of human bladder cancer in the future.
Highlights
► Suppression of AKT activity and anti-apoptotic Bcl-2 protein level in bufalin-treated T24 cells. ► Stimulations of Bax
signal and caspases- and mitochondria-dependent pathway in T24 cells after bufalin exposure. ► Inductions of
G0/G1 phase arrest and apoptotic death of T24 cells.
Abbreviations

AIF, apoptosis-inducing factor;

CDK, cyclin-dependent kinase;

CsA, cyclosporine A;

DCFH-DA, 2′-7′-dichlorfluorescein-diacetate;

DiOC6, 3,3′-dihexyloxacarbocyanine iodide;

DMSO, dimethyl sulfoxide;

ECL,enzyme chemiluminescence;

FCS, fetal calf serum;

HRP, horseradish peroxidase;

NAC, N-acetyl-cysteine;

PBS, phosphate-buffered saline;

PI, propidium iodide;

SDS, sodium dodecyl sulfate;

TUNEL,terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labeling;

z-VAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone (pan-caspase inhibitor);

z-LEHD-fmk, z-Leu-Glu-His-Asp-fluoromethyl ketone (caspase-9 inhibitor)
Keywords

Bufalin;

T24 human bladder cancer cells;

G0/G1 phase arrest;

Apoptosis;

Mitochondrial signaling pathway
1. Introduction
Bladder cancer is the fifth most common cancer among men and women in the USA in 2008 [1]. In Taiwan, 3.3
persons per 100 thousand die annually from bladder cancer based on the report in 2009 from the Department of
Health, R.O.C. (Taiwan). The major treatments for bladder cancer patients are surgery, radiotherapy and
chemotherapy, or combine with radiotherapy and chemotherapy, but the efficiency of cure rates are not satisfactory.
Therefore, searching for chemoprevention or chemical controls for bladder cancer has become a crucial concern.
Bufalin (Fig. 1) is the major digoxin-like immunoreactive component of Chan-Su extracts from the venom ofBufo bufo
gargarizan[2]. Chan-Su, a traditional Chinese medicine, was obtained from the skin and parotid venom glands of the
toad [3] and its extracts have been applied in the treatment of various cancers in clinical trials in China [4]. Bufalin has
been demonstrated to induce cell cycle arrest and apoptosis in many human cancer cells including
leukemia [5], [6], [7], [8] and [9],
prostatic
cancer [2] and [10],
endometrial
and
ovarian
cancer [11] and
osteosarcoma [12]. Besides, bufalin induced autophagy in human colon cancer cells through promoted the reactive
oxygen species (ROS) generation and the c-Jun NH2-terminal kinase (JNK) signaling[13]. Bufalin has been shown to
inhibit cytochrome P450 3A4 (CYP3A4) in in vitro and in vivo effects and interacted the CYP3A4-metabolized
agent [14]. However, the effects of bufalin on bladder cancer cells have not yet been thoroughly reported and
knowledge of the molecular mechanisms of bufalin-induced apoptosis in bladder cancer cells was rudimentary and
remained to be delineated. Therefore, the purpose of this study was designed to define the biological and therapeutic
effects of bufalin-treated human bladder cancer cells for the first time. We investigated whether or not bufalin was able
to mediate growth inhibition of bladder cells, cell cycle arrest and induction of apoptosis in bladder cancer cells.
Fig. 1. Effects of bufalin on cell viability in human bladder cancer cells. (A) The chemical structure of bufalin. (B)
Bladder cancer cells were treated with 0, 50, 100 and 200 nM of bufalin for 24 h then cell viability was determined
by a PI exclusion method. Data were expressed mean ± SEM of three independent experiments. ***P < 0.001,
significantly different compared with the control (0 nM) group.
Apoptosis, also known as programmed cell death type I, involves a cascade of molecular changes including loss of
organelle trans-membrane potential, swelling of the matrix, and rupture of the outer membrane, DNA fragmentation,
chromatin condensation, apoptotic body, and culminates with the discharge of apoptotic proteins, most notably
cytochrome c in the cytosol [15], [16] and [17]. The anti-apoptotic Bcl-2 proteins regulating outer membrane
integrity [18] of mitochondrial permeability transition [19] have been intensely pursued for novel molecular
therapeutics of some human cancer.
In this study, we demonstrated that bufalin inhibited the growth of T24 human bladder cancer cells through
G0/G1 phase arrest and the inhibitions of cyclin D and E, CDK2 and CDK4, and it induced apoptosis through a
mitochondria-dependent pathway. Our results indicated that a decrease of PKB (protein kinase B)/AKT activity and an
increase in the pro-apoptotic Bax through dissociation from anti-apoptotic Bcl-2, leading to mitochondrial dysfunction,
cytochrome c release, activation of caspase cascades and consequently apoptotic cell death in bufalin-treated T24
cells.
2. Materials and methods
2.1. Chemicals and reagents
Bufalin, dimethyl sulfoxide (DMSO), propidium iodide (PI), RNase A, Triton X-100, proteinase K, cyclosporine A (CsA:
a mitochondrial membrane permeability transition inhibitor) and N-acetyl-cysteine (NAC: a ROS scavenger) were
purchased from Sigma – Aldrich Corp. (St. Louis, MO, USA). FCS, L-glutamine, penicillin-streptomycin and
trypsin-EDTA were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). The z-LEHD-fmk (caspase-9
inhibitor), z-VAD-fmk (a pan-caspase inhibitor) were purchased from R&D Systems (Minneapolis, MN, USA). Sources
of antibodies used in this study were as follows: monoclonal antibodies specific for β-actin, cyclin D, CDK4, cyclin E,
CDK2, Rb, phospho-Rb, cytochrome c, Apaf-1, AIF, AKT, Bax, Bcl-2 and all peroxidase-conjugated secondary
antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyclonal antibodies specific
for phospho-AKT, caspase-9, caspase-7 and caspase-3 were obtained from Cell Signaling Technology Inc. (Danvers,
MA, USA). Enhanced chemiluminescence (ECL), a Western blot detection reagent, was purchased from Pierce
Chemical (Rockford, IL, USA).
2.2. Cell culture
The human bladder cancer cell lines (T24, TSGH-8301 and RT4) were purchased from the Food Industry Research
and Development Institute (Hsinchu, Taiwan). The cells were grown in McCoy's 5a medium supplemented with 10%
FCS, 2 mM L-glutamine, and 100 units/ml penicillin/100 μg/ml streptomycin at 37 °C under 5% CO2 in air.
2.3. Cell viability assay
Bladder cancer cell lines (2 × 105 cells/well) were individually maintained in 24-well plates with McCoy's 5a medium for
24 h, and then treated with 0, 50, 100 and 200 nM of bufalin for 24 h at 37 °C, 5% CO2 and 95% air. For incubation
with the specific inhibitors, cells seeded into 24-well plates were pretreated with NAC (10 mM), CsA (5 μM), a
pan-caspase inhibitor (z-VAD-fmk; 10 μM) and a caspase-9 inhibitor (z-LEHD-fmk) for 1 h, followed by treatment with
or without 100 nM bufalin. Cells were harvested from individual well by centrifugation. For viability determination, cells
from each treatment were stained with PI (5 μg/ml) and analyzed by flow cytometry (Becton-Dickinson, FACSCalibur,
San Jose, CA, USA) as previously described[20] and [21].
2.4. Cell morphology were examined by DAPI and TUNEL staining
Aliquots of T24 cells (2 × 105 cells/well) were placed into 24-well plates with McCoy's 5a medium and then were
exposed to 0, 50, 100 and 200 nM of bufalin for 24 h. Cells were examined and photographed under a phase-contrast
microscope. Apoptosis was detected using the DAPI/TUNEL double staining method in situapoptosis detection kit (in
situ cell death detection kit; Roche Diagnostics, Mannheim, Germany). T24 cells were treated with or without 100 nM
bufalin. Cells were fixed in 4% buffered formaldehyde then were mounted on glass slides. After being washed with
PBS, endogenous peroxidase was blocked by the addition of 3% H2O2. Cells were then treated with FITC-label
terminal deoxynucleotidyl transferase and biotinylated dUTP. After stopping the reaction, the samples were stained
with 4′-6-diamidino-2-phenylindole (DAPI, Invitrogen). All cells were stained by DAPI and TUNEL then were examined
and photographed by a fluorescence microscope as described previously [22] and [23].
2.5. DNA content analysis for cell cycle distribution and sub-G1 group
Approximately 2 × 105 cells/well of T24 cells in 24-well plates were treated with 100 nM bufalin for 0, 6, 12 and 24 h.
Cells were harvested and washed twice with cold PBS. Cells were fixed by using 70% ethanol at −20 ̊C overnight and
washed twice with cold PBS, and then cells were re-suspended in PBS containing 40 μg/ml PI and 0.1 mg/ml RNase
and 0.1% triton X-100 in dark room for 30 min at 37 °C. All samples were analyzed by flow cytometry and the cell cycle
and sub-G1 (apoptosis) phase were determined and analyzed as described previously [24] and [25].
2.6. Determination of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm)
Cells (2 × 105 cells/well) in 24-well plates were exposed to 100 nM bufalin and incubated for 0, 2, 4, 6 and 12 h. At the
end of incubation, cells from each treatment were harvested by centrifugation and were washed twice by PBS, then
were re-suspended in 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; 10 μM, Invitrogen) for ROS determination
and were re-suspended in DiOC6 (1 μM, Invitrogen) for measurement of ΔΨm. Then cells were incubated at 37 °C
under dark room for 30 min and were analyzed immediately by flow cytometry as described previously [26] and [27].
2.7. Assays of caspase-3, caspase-7 and caspase-9 activity
Approximately 2 × 105 cells/well of T24 cells in 10 cm culture dish were treated with 100 nM bufalin and incubated for
0 and 24 h, and then the activities of caspase-3, -7 and -9 were assessed according to manufacturer's instruction of
Caspase colorimetric kit (R&D system Inc.). Cells were harvested and lysed in 50 μl lysis buffer which containing
2 mM DTT for 10 min. After centrifugation, the supernatant containing 200 μg protein were incubated with caspase-3,
caspase-7 and caspase-9 substrate in reaction buffer. Then all samples were incubated in 96-well flat bottom
microplate at 37 °C for 1 h. Levels of released pNA were measured with ELISA reader (Anthos Reader 2001, Anthos
Labtec) at 405 nm wavelength [23] and [28].
2.8. Determinations of G0/G1 phase arrest and apoptosis-associated protein levels by Western blotting
T24 cells at a density of 1 × 106 cells in 75 T flasks were exposed to 100 nM bufalin and then incubated for 0, 1, 2, 6,
12, 18 and 24 h for examining the protein levels correlated with cell cycle arrest and apoptosis. Cell were harvested
from each treatment, washed with cold PBS, and lysed in the PRO-PREP™ protein extraction solution (iNtRON
Biotechnology, Seongnam, Gyeonggi-Do, Korea). The total proteins were collected before the levels of cyclin D and E,
CDK4, CDK2, Rb, p-Rb, p-AKT, AKT, Bax and Bcl-2 were detected using immunoblotting. The total protein was
collected before the cytochrome c, Apaf-1, AIF, caspase-9, caspase-3 and caspase-7 was detected by Western
blotting. In brief, about 30 μg protein from each sample was resolved over 10% sodium dodecylsulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The blot was soaked in blocking buffer
(5% non-fat dry mik/0.05% Tween 20 in 20 mM Tris buffered saline (TBS) at pH 7.6) at room temperature for 1 h then
incubated with individual monoclonal antibodies in blocking buffer at 4 °C for overnight. Then followed by secondary
antibody-conjugated horseradish peroxidase (HRP) and detected by chemiluminescence kit (Millipore, Bedford, MA,
USA) and autoradiography using X-ray film as described elsewhere [20],[22] and [29]. To ensure equal protein loading,
each membrane was stripped and reprobed with anti-β-actin antibody.
2.9. In vitro AKT kinase assay
This assay was followed as the protocol of the manufacturer's instructions from an AKT kinase assay kit (Cell
Signaling Technology, Beverly, MA, USA). Briefly, about 2 × 106 cells/well of T24 cells in 10 cm dish were treated with
100 nM bufalin for 0, 1, 2 and 6 h. At the end of incubation, cells were harvested and lyzed using the AKT kinase
assay kit and then 200 mg of protein from each time point treatment was immunoprecipitated with 2 mg of anti-AKT
antibody overnight. Then all samples were extensive washing, the immunoprecipitates were incubated with 1 mg of
glycogen synthase kinase-3 α/β (GSK-3 α/β) fusion protein substrate in 50 ml of kinase buffer for 30 min at 30 °C.
Reactions were stop by SDS loading buffer. The samples were separated on 12% SDS-PAGE, and the
phospho-GSK-3 α/β (Ser219) was detected by immunoblotting [30], [31] and [32].
2.10. Statistical analyses
Data are presented as the mean ± SEM for the indicated number of separate experiment. Statistical analyses of data
were done by Student's t-test, and *P < 0.05, ***P < 0.001 were considered significant.
3. Results
3.1. Bufalin decreased the viability of human bladder cancer cells
We determined the growth inhibition effects of bufalin on the cell viability by using a PI exclusion and flow cytometric
assay, and the results are shown in Fig. 1B. Increasing the dose of bufalin led to the decrease of the percentage of
viability in examined 3 different bladder cancer cell lines (Fig. 1B). Bufalin at 100 nM decreased by almost 45, 26 and
22% the viable cells of T24, TSGH-8301 and RT4, respectively, at 24 h treatment. The highest sensitive cell is T24
cells; therefore, we selected T24 cells for further experiments.
3.2. Bufalin induced morphological changes and apoptosis in T24 cells
T24 cells were treated with 0, 50, 100 and 200 nM bufalin for 24 h. As shown in Fig. 2A and B, bufalin induced cell
morphological changes and decreased the cells number, and cells became smaller, round and blunt in size when
compared with control in T24 cells. These effects are dose-dependent (Fig. 2A; arrow). The assay demonstrated that
bufalin induced DNA condensation and apoptosis which were examined by DAPI/TUNEL double staining (Fig. 2B).
Taken together, we concluded that 100 nM bufalin decreased the percentage of viable T24 cells through the apoptotic
cell death.
3.3. Bufalin induced cell cycle arrest and affected associated protein levels in T24 cells
Based on the results from growth inhibition, further studies were conducted to investigate the possible mechanisms
which are involved in bufalin-induced cell cycle arrest and associated protein levels in T24 cellsin vitro. The results
from flow cytometric assay revealed that 100 nM bufalin induced accumulation of G0/G1phase in T24 cells and this
effect is a time-dependent manner (Fig. 3A). Western blotting also showed that bufalin down-regulated the
expressions of cyclin D and E, CDK2 and CDK4 and p-Rb and up-regulated the expressions of Rb, leading to
G0/G1 phase arrest in T24 cells and this effect is a time-dependent response (Fig. 3B).
3.4. Bufalin induced reactive oxygen species (ROS) production and pre-treated with N-acetyl-cysteine
(NAC) and a pan-caspase inhibitor (z-VAD-fmk) to increase the viability in treated T24 cells
To verify that ROS and caspase cascade are involved in bufalin-induced cell death of T24 cells. Cells were pretreated
with 10 mM ROS scavenger (NAC) or 10 μM z-VAD-fmk and then exposed to 100 nM bufalin before being harvested
for measuring the levels of ROS and cell viability by flow cytometric assay. The results in Fig. 4A indicated that bufalin
promoted the ROS production and this effect is a time-dependent response. T24 cells were also measured the
percentage of viability in T24 cells and results are shown in Fig. 4B, which indicated that NAC and z-VAD-fmk can
increased the percentage of viable T24 cells after bufalin exposure. Based on these observations, bufalin-induced
cytotoxic effects were mediated through ROS production and increased caspase activity in T24 cells.
3.5. Bufalin decreased the level of mitochondria membrane potential (ΔΨm) and pre-incubated with
cyclosporine A (CsA) and caspase-9 inhibitor to protect against the viability in T24 cells after treatment
To investigate whether mitochondria are involved in bufalin-triggered cell death, T24 cells were pretreated with 5 μM
CsA (a mitochondrial membrane permeability transition inhibitor) or 10 μM z-LEHD-fmk (a caspase-9 inhibitor). Cells
then were treated with 100 nM bufalin. Cells were harvested for examining of ΔΨmand viability. The results shown
in Fig. 5A and B indicated that 100 nM bufalin decreased the level of ΔΨm. Furthermore, cells were pretreated with
CsA or z-LEHD-fmk and then treated with bufalin, leading to increase the percentage of viable cells, respectively,
when compared to the bufalin-treated only cells (Fig. 5B). These results indicated that bufalin-induced cytotoxic effects
were mediated through mitochondria-dependent apoptotic signaling pathways.
3.6. Bufalin increased the activities of caspase-3, -7 and -9 and affected the apoptosis-associated protein
levels in T24 cells
To determine whether apoptosis is mediated via the activation of caspase-3, -7 and -9 in bufalin-treated T24 cells.
Cells were harvested after exposure to 100 nM bufalin and then determined the activities of caspase-3, -7 and -9 by
colorimetric assays. The changes of apoptosis-associated protein levels were determined by Western blotting. The
results shown in Fig. 6A indicated that bufalin promoted the activation of caspase-3, -7 and -9 for a 24-h treatment.
Results in Fig. 6B indicated 100 nM bufalin increased the levels of cytosolic cytochrome c, AIF, Apaf-1 and active form
of caspase-3, -7 and -9. Our results suggest that bufalin-induced apoptosis is
done through the
mitochondria-dependent signaling pathway in T24 cells.
3.7. Bufalin inhibited the activity of AKT and affected the Bcl-2 family protein levels in T24 cells
To examine whether bufalin-induced apoptosis is through the inhibition of AKT and involved in Bcl-2 family protein
levels in T24 cells. Cells were harvested after treatment with 100 nM bufalin, and then determined the AKT activity and
Bcl-2 family-related protein levels by Western blotting. Our results in Fig. 7A revealed that bufalin decreased the AKT
activity after bufalin for 2 and 6 h-treatment and this effect is time-dependently. Results from Western blotting also
showed that bufalin decreased the levels of p-AKT, AKT and Bcl-2, but it increased the level of Bax (Fig. 7B) in T24
cells. Based on these observations, it is suggested that bufalin-induced apoptosis in T24 cells is mediated through the
changes of ratio of Bax/Bcl-2 and a decrease in the activities of AKT.
4. Discussion
It was reported that bufalin induced cytotoxic effects in many human cancer cell lines through cell cycle arrest and
induction of apoptosis [2], [5], [6], [7], [8], [9], [10], [11] and [12]. In this study, we first demonstrated that bufalin
induced cytotoxic effects through G0/G1 arrest (Fig. 3A) and inducing apoptosis in T24 cells. This is in agreement with
the reports from Nasu et al. indicated that bufalin inhibited the cell proliferation through induction of apoptosis and the
G0/G1 phase arrest of the cell cycle of endometriotic stromal cells in vitro[33]. We also used DAPI/TUNEL double
staining to confirm that bufalin induced apoptosis in T24 cells (Fig. 2A and B). Western blotting analysis indicated that
bufalin induced the down-regulation of cyclin D and cyclin E, CDK2 and CDK4 and p-Rb, but it increased the level of
Rb in T24 cells. These regulations of cell cycle associated proteins indicated bufalin induced G0/G1 phase arrest in T24
cells.
Our results showed that bufalin promoted the production of ROS in T24 cells and this effect is time dependently (Fig.
4A). T24 cells were pretreated with ROS scavenger (NAC) and then led to increase the viable T24 cells when
compared to the bufalin-treated only cells (Fig. 4B). This observation indicated that ROS was involved in
bufalin-induced cell death. This is in agreement with other report demonstrated that bufalin induced
apoptosis via ROS-dependent mitochondrial death pathway in human lung adenocarcinoma ASTC-α-1 cells [34]. Our
results also showed in Fig. 5A indicated that bufalin decreased the level of ΔΨmfrom T24 cells and this is also
agreement with Sun et al. reported that bufalin decreased the level of ΔΨmand mitochondria play an important role in
bufalin-induced apoptotic death in ASTC-α-1 cells [34].
It is well known that caspases can be activated in two major apoptotic pathways, the death-receptor and
mitochondria-mediated signaling pathways. Bufalin promoted caspase-3, -7 and -9 in T24 cells (Fig. 6A). As shown
in Fig. 6B, bufalin increased cytosolic protein levels of cytochrome c, Apaf-1, Pro-caspase-9 and AIF in T24 cells. This
is also in agreement with other report showed that the activation of caspase-9, an initiator caspase closely coupled to
pro-apoptotic
signals,
was
observed
after
bufalin
treatment,
suggesting
that
caspase-9-mediated
mitochondria-mediated signaling pathway is involved in the mechanism of bufalin-induced apoptosis [11]. However,
their report did not show that caspase-3 and -7 also involved in bufalin induced apoptosis. This is our novel finding
mechanism of bufalin-induced apoptosis, which is involved in activations of caspase-9, -3 and -7 in T24 cells. Our
result does not rule out the involvement of the death receptor apoptotic signaling pathway. The levels of Fas, FasL and
FADD protein levels and caspase-8 activity have no significant influence on bufalin-treated T24 cells (data not shown).
Our results suggest that the mitochondrial signaling pathway is mediated bufalin-induced apoptotic response in T24
cells.
Our results showed that bufalin induced the down-regulation of the expression of Bcl-2 (Fig. 7B), and the simultaneous
up-regulation of the Bax (Fig. 7B) and activated caspase-3, -7 and -9 expressions (Fig. 6B) in T24 cells and this is in
agreement with reports from Sun et al. in ASTC-α-1 cells after bufalin exposure (27). We determined caspase activity
assay to confirm that bufalin promoted the activities of caspase-3, -7 and -9 in T24 cells (Fig. 6A). This is also
agreement with other report indicated that activation of caspase-9 as observed after bufalin treatment, suggesting that
caspase-9-mediated cascade is involved in the mechanism of bufalin-induced apoptosis [35]. However, another report
showed that bufalin did not affect caspase-3 activity in ASTC-α-1 cells [34]. Therefore, it is suggested that the
cell-specific effects of bufalin on cancer cells such as the mechanisms of bufalin-induced apoptosis of human
leukemia cells by the activation of AP-1 and the c-Jun N-terminal protein kinase (JNK) [8], cdc2 kinase and casein
kinase II [5] and [6], the induction of Tiam1 expression [9] and Bcl-2 and c-myc expression [7] and the inhibition of
protein kinase A and C [5] and [6]. The interesting point is that other report indicated bufalin induced G2/M phase
arrest in leukemia cells [5] and [6]. Therefore, we suggest that the effects of bufalin may be cell-type specific.
It was reported that phosphorylated Bax on Ser184 by AKT and then inhibition of conformational change and inability of
Bax to translocate to the mitochondrial membrane [36] and [37]. This action, then, blocks the pore formation and
inhibits the release of cytochrome c, Apaf-1, pro-caspase-9 and AIF proteins from mitochondrial into cytosol. It also
reported that the phosphorylated Bax heterodimerizes with Bcl-xl, the binding of Bcl-2 family members may prevent
the translocation of Bax to the mitochondrial membrane, and then inhibit apoptosis [38]. In this study, bufalin inhibited
the activity of AKT in T24 cells. It is well known that AKT is involved in cell survival or death dependent the associated
signal pathway. Oka et al. investigated that the high expression of activated AKT was observed in T24 cells, whereas
low expression of that was shown in RT4 cells [39]. We also demonstrated that treatments of T24 cells with 100 nM
bufalin reversed the high constitutive activity of AKT in comparison to those from TSGH-8301 and RT4 human bladder
cancer cells (data not shown). It is suggested that more sensitivity in the PI3K inhibitors and AKT protein expression
exhibited in T24 cells, a highly malignant grade III human urinary bladder carcinoma [39]. In the present study, the
AKT activity may play an important role in regulating the Bcl-2 family protein levels to the induction of apoptosis in
bufalin-treated T24 cells.
Overall, the outline of molecular signaling pathways is summarized in Fig. 8. These results indicated that bufalin could
be used as a novel therapeutic agent for the medical treatment and/or prevention of bladder cancer.
Conflict of interest statement
None.
Acknowledgments
This study was supported by research grant CMU99-TC-05 from China Medical University, Taichung, Taiwan. We
also thank the National Science Council of the Republic of China for financial support (NSC 97-2320-B-039-004-MY3).
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