3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate induced Hep G2 cell apoptosis through a ROS-mediated pathway

Article abstract of DOI:10.1016/j.cbi.2009.12.008

3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate (DPB-5) is a synthetic benzothiazole derivative. In the present study, we revealed that DPB-5 had strong cytotoxicity to induce cell apoptosis, which was mediated by ROS. And DPB-5 was more cytotoxic toward hepatoma cells than toward normal hepatic cells, which was resulted from the greater susceptibility of the malignant cells to ROS. DBP-5 caused massive ROS accumulation and GSH decrease, which lead to MMP disruption, caspase activation and finally induced cell apoptosis. Additionally, rotenone, an inhibitor of mitochondria electron transport system, effectively blocked the ROS elevated effect of DPB-5, which suggested that DPB-5-induced ROS generated from the mitochondria. Further studies showed that DPB-5-induced cell apoptosis through caspases-cascade, but failed to activate caspase-9. Hence, we concluded that DPB-5-induced Hep G2 cells apoptosis via a ROS-mediated pathway which was caspase-dependent but did not rely on caspase-9.

Full text of DOI:10.1016/j.cbi.2009.12.008

Chemico-Biological Interactions 183 (2010) 341–348
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Chemico-Biological Interactions
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3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate induced
Hep G2 cell apoptosis through a ROS-mediated pathway
Jianyong Lia, Zhijun Xub, Mengyao Tana, Weike Sub, Xing-guo Gonga,∗
a Institute of Biochemistry, College of Life Sciences, Zhejiang University, 310058, PR China
b Key Laboratory of Pharmaceutical Engineering of Ministry of Education, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, 310014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 October 2009
Received in revised form 4 December 2009
Accepted 8 December 2009
Available online 16 December 2009
3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate (DPB-5) is a synthetic benzoth-
iazole derivative. In the present study, we revealed that DPB-5 had strong cytotoxicity to induce cell
apoptosis, which was mediated by ROS. And DPB-5 was more cytotoxic toward hepatoma cells than
toward normal hepatic cells, which was resulted from the greater susceptibility of the malignant cells
to ROS. DBP-5 caused massive ROS accumulation and GSH decrease, which lead to MMP disruption, cas-
pase activation and finally induced cell apoptosis. Additionally, rotenone, an inhibitor of mitochondria
electron transport system, effectively blocked the ROS elevated effect of DPB-5, which suggested that
DPB-5-induced ROS generated from the mitochondria. Further studies showed that DPB-5-induced cell
apoptosis through caspases-cascade, but failed to activate caspase-9. Hence, we concluded that DPB-5-
induced Hep G2 cells apoptosis via a ROS-mediated pathway which was caspase-dependent but did not
rely on caspase-9.
Keywords:
Benzothiazole
DPB-5
Apoptosis
ROS
Caspase
© 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Thiazole, an important heterocyclic ring, is widely used in anti-
cancer drug development [17]. Many natural chemotherapeutic
Apoptosis is important in chemotherapy-induced tumor-cell
killing, and many anticancer drugs induce restoration of apopto-
sis [1–4]. Many tumors have been associated with the inhibition
of apoptosis, and the disruption of apoptotic function which con-
tribute substantially to the transformation of a normal cell into
a tumor cell [5,6]. Intracellular reactive oxygen species (ROS) is
considered to be a death signal in apoptosis [7–10]. ROS induces
disruption of the mitochondrial membrane potential (MMP) and
release of cytochrome c from mitochondria into the cytosol, where
cytochrome c triggers caspase-9 activation and initiates caspases-
cascade which terminates cell to apoptosis [11,12]. Accumulation
of excessive ROS leads to lipid peroxidation, protein oxidation,
enzyme inactivation, oxidative DNA damage [13,14]. Cancer cells
are more sensitive to ROS than normal cells; therefore, ROS is con-
sidered as an important target for anticancer drug research [15,16].
agents containing thiazole moiety have been discovered and stud-
ied, like tiazofurin [18,19], distamycin [20,21], bleomycin [22]
and netropsin [21]. Among the thiazole derivatives, benzothia-
zole is an important scaffold of drugs, and it has been studied
extensively. Benzothiazole derivatives are known as inhibitors of
topoisomerase II, tyrosine kinase and ubiquitin–proteasome sys-
tem [23–26]. Some studies reported the antioxidant properties
of benzothiazole derivatives [27,28], and their biological activ-
ity was indicated to be strongly related to ROS [29]. In this
respect, we examined the relationship between cytotoxic effect
of 3-(4-(benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl
acetate (DPB-5, Fig. 1B) and ROS. DPB-5 is screened from a
series of compounds derived from 2-(1,3-diphenyl-1H-pyrazol-
4-yl) benzo[d]thiazole (DPB, Fig. 1A), which were designed and
synthesized in earlier work.
In this study, we determined that DPB-5 had a strong specific
cytotoxicity against Hep G2 cells, which was caused by apoptosis
through a ROS-mediated pathway. DPB-5-induced cell death was
caspase-dependent, but independent on caspase-9.
Abbreviations: DPB-5, 3-(4-(benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl)
phenyl acetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; GSH,
reduced glutathione; PI, propidium iodide; ROT, rotenone; DCFH-DA, dichlorodihy-
drofluorescein diacetate; NAC, N-acetyl-l-cysteine; Rh123, rhodamine 123; CAT,
catalase; AMBAN, 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-aminoacrylonitrile.
2. Materials and methods
2.1. Cell culture and reagent
∗
Corresponding author at: Institute of Biochemistry, College of Life Sciences,
Zhejiang University, Hangzhou, Zhejiang Province, 310058, PR China. Tel.: +86 571
88206475; fax: +86 571 88206549.
Human cancer cell lines specific for: breast, MCF-7; hepatoma,
Hep G2; laryngeal epithelium, Hep 2; gastric carcinoma, SGC 7901;
E-mail address: gongxingguo@126.com (X.-g. Gong).
0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2009.12.008

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J. Li et al. / Chemico-Biological Interactions 183 (2010) 341–348
Fig. 1. Chemical structures and reaction. (A) Chemical structures of DPB; (B) chemical structures of DPB-5; (C) reaction for DPB-5 synthesized.
and leukaemia, HL60; were obtained from the Institute of Bio-
chemistry and Cell Biology (Chinese Academy of Sciences). The
human normal embryonic hepatic cell line LO2 was purchased
from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). All cells
used in this study were cultured at 37 ◦C in RPMI-1640 medium
(Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco),
100 U/ml penicillin (Sigma) and 100 mg/ml streptomycin (Sigma),
in a humidified 5% (v/v) CO2 atmosphere. For the drug treatment
experiments, cells were harvested during the exponential growth
phase and seeded for 12–24 h, then the medium was replaced by
RPMI-1640 medium supplemented with 2% FBS and containing var-
ious doses of drug, and kept incubation for further study.
(model ELX800, Bio-Tek Instruments). The IC50, calculated from
50% formazan formation compared with the control without drug
treatment, were determined graphically using the curve-fit algo-
rithm of the software PrismTM 2.0 (Graph-Pad). Each test was
performed in triplicate.
2.3. Trypan blue exclusion
The cell death rate was determined by trypan blue exclusion
method [31]. Cells in the exponential growth phase were plated
at 5 × 104 cells/well in 24-wells culture plates. After 12 h growth,
the medium was replaced by RPMI-1640 medium supplemented
with 2% FBS containing various doses of DPB-5. After incubating for
the indicated times, the viable cells and dead cells were counted
on optical microscope with hemacytometer. Living cells possess
intact cell membranes that exclude trypan blue dyes; however dead
cells take up dyes and turn to blue. (Cell death %) = (total number
of dead cells per ml of aliquot)/(total number of cells per ml of
aliquot) × 100%.
DPB-5 was synthesized as in Fig. 1C, and purified by silica gel
column chromatography. The purity was determined by TLC, and
the structure determined by MS, 1H NMR and 13C NMR.
DPB-5, white solid. 1H NMR (500 MHz, CDCl3) ı: 2.29 (s, 3H),
7.23 (t, 1H, J = 8.0 Hz), 7.32–7.36 (m, 2H), 7.45–7.50 (m, 4H), 7.59
(s, 1H), 7.66 (d, 1H, J = 7.5 Hz), 7.80 (t, 4H, J = 8.0 Hz), 8.01 (d, 1H,
J = 8.0 Hz), 8.60 (s, 1H). 13C NMR (125 MHz, CDCl3) 169.24, 159.77,
153.27, 150.84, 150.72 139.28, 135.03, 133.47, 129.61, 129.39,
128.53, 127.38, 127.07, 126.24, 124.95, 122.80, 122.69, 122.27,
121.45, 119.33, 117.32, and 21.19.
2.4. Evaluation of apoptosis
DPB-5 was dissolved in DMSO (Sigma) and diluted to the desired
concentration before used, with the concentration of DMSO kept
below 0.1% in the treatment groups. 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), rhodamine 123 (Rh123),
dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetylcysteine
(NAC), catalase (CAT), propidium iodide (PI) and rotenone (ROT)
were purchased from Sigma. Naphthalene-2,3-dicarboxyaldehyde
(NDA) was purchased from Molecular Probes. All other chemicals
were analytical grade.
2.4.1. Analysis of DNA fragmentation
Cells were treated with 15 ␮M DPB-5 for different lengths of
time, harvested by centrifugation and washed twice in ice-cold PBS.
An Apoptotic DNA Laddering Kit (Beyotime Institute of Biotechnol-
ogy, China) was used to analyze DNA fragmentation according to
the manufacturer’s protocol, and then the DNA fragments in the
samples were separated by electrophoresis in 1.8% (w/v) agarose
gel.
2.4.2. Hoechst 33258 staining
Cells were seeded onto a glass slide and treated with 15 ␮M DPB-
5 for 24 h, then washed twice with ice-cold PBS and fixed with 4%
(v/v) formaldehyde in PBS for 20 min, washed with PBS, stained
with 1 mg/ml Hoechst 33258 in PBS at 37 ◦C for 15 min, and then
viewed under a fluorescence microscope (Nikon) with an excitation
wavelength of 345 nm through the filter of 420 nm.
2.2. Cell viability assay
Cell viability was measured by the MTT assay method [30].
Briefly, cells in exponential growth phase were trypsinized,
counted, and seeded in 96-well culture plates at a concentration of
5 × 103 cells/well. After cultured for 12 h, the medium was replaced
by RPMI-1640 medium supplemented with 2% FBS containing vari-
ous doses of drug. After incubation for the length of time indicated,
MTT was added (final concentration 0.5 mg/ml) and kept incu-
bating for 4 h. The formazan crystals were dissolved in 150 ␮l of
DMSO and the plates were read at 570 nm with a microplate reader
2.4.3. Annexin V-FITC/PI staining
Apoptosis was determined by translocation of phosphatidylser-
ine to the cell surface using an Annexin V-FITC apoptosis detection
kit (Nanjing KeyGen Biotech. Co. Ltd., China) according to the man-

J. Li et al. / Chemico-Biological Interactions 183 (2010) 341–348
343
Table 1
ufacturer’s protocol. Briefly, after treatment with DPB-5 for 48 h,
Effects of DPB-5 on the growth of human tumor cell
lines.
cells were harvested and washed twice with ice-cold PBS, then
evaluated for apoptosis using a FACSCalibur flow cytometer (BD
Biosciences) with Annexin V–FITC and PI double staining. Fluo-
rescence was measured with an excitation wavelength of 480 nm
through FL-1 filter (530 nm) and FL-2 filter (585 nm).
Cell line
IC50 (␮M)
Hep G2
MCF-7
Hep 2
HL 60
LO2
SCG7901
4.6 1.0
6.4 0.6
11.7 1.3
6.3 0.8
9.6 0.6
8.3 1.2
2.4.4. Cell cycle analysis
Cells were treated with 8 ␮M or 15 ␮M DPB-5 for 48 h. The cells
were harvested and washed twice with PBS, then fixed in ice-cold
70% (v/v) ethanol for 24 h at 4 ◦C. Before analysis, cells were washed
with PBS, suspended in 1 ml of cold PI solution (50 ␮g/ml PI, 1% (v/v)
Triton X-100, 100 ␮g/ml RNase A) and incubated on ice for 30 min in
darkness. Cytometric analysis was performed using flow cytometer
and Cell Quest software. Fluorescence was measured with an exci-
tation wavelength of 480 nm through FL-2 filter. Apoptotic cells
were detected on a PI histogram as a Pro G1 peak.
Cells were cultured for 48 h and the IC50 values
were calculated using non-linear regression anal-
ysis. The data shown represent as means SD of
three-independent experiments.
3. Results
3.1. Cytotoxicity analysis
MTT analysis showed the cell toxicity of DPB-5 against six
human cell lines (Table 1). The hepatoma cell line Hep G2 was
the most sensitive to DPB-5-induced cell toxicity, with an IC50 of
4.6 ␮M, while the laryngeal epithelial cell line Hep 2 and the normal
embryonic hepatic cell line LO2 had a level of resistance more than
twice of that of Hep G2, with IC50 values of 11.7 and 9.6 ␮M, respec-
tively. Because of the particular sensitivity of Hep G2 cell line, we
chose Hep G2 and the corresponding normal hepatic cell LO2 for
further study.
2.5. Measurement of mitochondrial membrane potential (MMP)
MMP was measured by flow cytometer using the cationic
lipophilic green fluorochrome Rh123 [10]. Cells were harvested,
washed twice with PBS, incubated with 1 ␮M Rh123 at 37 ◦C for
30 min, and washed twice with PBS. Fluorescence was determined
by flow cytometer with an excitation wavelength of 480 nm at FL-1
filter.
Next we examined the cytotoxicity of DPB-5 against Hep G2
for various lengths of time and doses. As shown in Fig. 2, DPB-5-
induced Hep G2 cell death in a time- and dose-dependent manner.
2.6. Determination of cellular reactive oxygen species (ROS) and
intracellular glutathione levels (GSH)
3.2. DPB-5-induced apoptosis in Hep G2 cells
Intercellular ROS was determined by flow cytometer and stain-
ing with DCFH-DA. DCFH-DA is deacetylated by intracellular
esterase and converted to nonfluorescent dichlorodihydrofluores-
cein, which is oxidized rapidly to the highly fluorescent compound
dichlorofluorescein (DCF) in the presence of ROS. Cellular ROS con-
tent was measured by incubating the cells with 10 ␮M DCFH-DA at
37 ◦C for 30 min. After incubation with the fluorochrome, cells were
washed with PBS and analyzed immediately by flow cytometer
through FL-1 filter with an excitation wavelength of 480 nm.
The cellular level of reduced glutathione (GSH), another index
reflecting cellular reducing power, was measured by staining cells
with NDA [32]. GSH is oxidized to oxidized glutathione (GSSG)
under the stress of ROS. Cells were stained with 200 ␮M NDA at
37 ◦C for 30 min, the level of GSH was determined by measuring
the fluorescence intensity by flow cytometer with the FL-1 filter
and an excitation wavelength of 480 nm.
In order to identify whether DPB-5-induced cell death in apop-
totic mode, we observed DPB-5-treated Hep G2 cells by staining
with Hoechst 33258 (Fig. 3A), DNA fragment ladder (Fig. 3.B), stain-
ing with Annexin V-FITC (Fig. 3C), and Pro G1 analysis (Fig. 3D).
Cells treated with 15 ␮M DPB-5 for 24 h then stained with Hoechst
33258 showed condensed nuclei and apoptotic bodies. Genomic
DNA of cells treated with 15 ␮M DPB-5 for 24 and 48 h showed
typical DNA fragment ladders. When cells were stained with both
Annexin V-FITC and PI, the cell membrane showed PS external-
ization. Compared to the control, cells treated with 15 ␮M DPB-5
for 48 h had an increase in the percentage of early apoptotic cells
(Annexin V positive but PI negative) from 1.2% to 42.4%, and of late
apoptotic cells (Annexin V and PI double-positive cells) from 1.9% to
35.8%. Analysis of the cell cycle by flow cytometer showed the dose-
2.7. Caspase-9 activity assay
Caspase-9 activity was measured using colorimetric assay kits
(KeyGen Biotech Co., Ltd., Nanjing, China) according to the man-
ufacturer’s instructions. Briefly, cells treated with DPB-5 were
harvested and lysed, then the lysate was incubated at 37 ◦C for 2 h
with 200 ␮M LEHD-pNA (caspase-9 substrate). Samples were read
at 405 nm in a microplate reader (Bio-Tek Instruments). Protein
concentration was determined by Lowry method [33].
2.8. Statistical analysis
Unless otherwise stated, data are reported as mean SD. Com-
parisons between multiple treatments were made with analysis of
variance (ANOVA) whereas comparisons between two treatments
were made using a Student’s t-test with P ≤ 0.05 considered statis-
tically significant.
Fig. 2. Time- and dose-dependent effects of DPB-5 on Hep G2 cells. Cell viability
was determined by a MTT assay as described in the text. Results are expressed as
means SD of data obtained in three independent experiments.

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J. Li et al. / Chemico-Biological Interactions 183 (2010) 341–348
Fig. 3. Apoptotic effects of DPB-5 on Hep G2 cells. (A) Fluorescent microscopic analysis of apoptotic cells. Cells stained with Hoechst 33258 after treated with 15 ␮M DPB-5
for 24 h. (a) Control, (b) 15 ␮M DPB-5 for 24 h. (B) DNA fragmentation analysis for apoptosis. Hep G2 cells were pretreated with 15 ␮M DPB-5 for 24 and 48 h. M: marker, 1:
50 ␮M H2O2 for 6 h as positive control, 2: 15 ␮M DPB-5 for 48 h, 3: 15 ␮M DPB-5 for 24 h, C: control. (C) Annexin V-FITC and PI staining for apoptosis. Data were obtained
in three separate experiments. (a) Control; (b) 15 ␮M DPB-5 for 48 h. (*P ꢀ 0.05 versus control) (D) Pro G1 analysis for apoptotic cells. Hypodiploid cells (apoptotic cells) are
shown in region with green colour by ModFit LT software and marked as Pro G1 Peak. (a) Control; (b) 8 ␮M DPB-5 for 48 h; (c) 15 ␮M DPB-5 for 48 h. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of the article.)
dependent increased Pro G1 peak. These results are all hallmarks
of apoptotic cell death, and demonstrated the ability of DPB-5 to
induce apoptosis in Hep G2 cells.
cence intensity was increased 10-fold after 12 h of drug treatment.
However, DPB-5-induced less ROS change on LO2 cells with 1.5-
fold after 12 h treatment compared to Hep G2 cells. We analyzed
changes of the GSH level in Hep G2 cells and LO2 cells using NDA
fluorescence. DPB-5 caused a significant decrease of the intracellu-
lar GSH content after 12 h of drug treatment compared to the less
change on LO2 cells (Fig. 4B).
3.3. DPB-5-induced MMP disruption
It is generally agreed that MMP disruption irreversibly leads to
cell death, resulting in mitochondrial release of apoptogenic factors
and a decrease of ATP generation [4,34,35]. To gain a better under-
standing of the mechanism underlying DPB-5-induced apoptosis,
we examined the effect of DPB-5 on MMP by examining the reten-
tion of Rh123. As shown in Fig. 4C, there was a time-dependent
decrease of Rh123 fluorescence after treatment with DPB-5 com-
pared with the untreated control, indicating that DPB-5-induced
MMP disruption in Hep G2 cells. In contrast, normal cell LO2 was
less sensitive to DPB-5.
3.5. Antioxidants protected Hep G2 cell from DPB-5 cytotoxicity
To verify the relationship between ROS generation and cell
toxicity, we examined the effects of ROS scavengers, NAC (a
well-known antioxidant and GSH precursor) and catalase (CAT, a
H2O2-scavenging enzyme), on DPB-5-induced cell death. Before
treated with drugs, the cells were treated with 1 mM NAC or
2000 U/ml catalase for 30 min, and then the change of MMP and cell
toxicity were determined. As shown in Fig. 5, both NAC and catalase
inhibited the loss of MMP and protected cells from DPB-5 cytotox-
icity. Further they both significantly reduced DPB-5-induced cell
apoptosis as indicated by Hoechst staining and trypan blue exclu-
sion. It is suggested that ROS accumulation caused MMP disruption
and mediated cell apoptosis.
3.4. DPB-5-induced ROS burst and GSH depletion
ROS and GSH are indices of cell redox status. The generation
of intracellular ROS and depletion of GSH are always associated
with MMP disruption and cell apoptosis [36,37]. Therefore, we
examined the levels of ROS and GSH in Hep G2 cells and LO2
cells treated with DPB-5. ROS was monitored by the oxidation-
sensitive fluorescent dye DCFH-DA. A time-dependent increase in
DCF fluorescence was detected in DPB-5-treated cells (Fig. 4A).
Rapid generation of ROS, up to 3.56-fold faster than the control,
was detected only 2 h after drug treatment, while the fluores-
3.6. ROS generation that triggered by DPB-5 against Hep G2
originated from mitochondria
We identified and clarified the source of DPB-5-induced ROS
burst in Hep G2 cells (Fig. 6). Mitochondria are considered to be

J. Li et al. / Chemico-Biological Interactions 183 (2010) 341–348
345
nificantly increased its activity. However as the cells pretreated
with zVADfmk (a synthetic pan-caspase inhibitor, purchased from
Promega) to inhibit the caspase activity, the cell death rate was
massively reduced and cell apoptosis was significantly inhibited
(Fig. 7). It is suggested that DPB-5-induced cell apoptosis in a
caspase-dependent pathway but not depend on caspase-9.
4. Discussion
In this study, Hep G2 cells were shown to be much more sen-
sitive to DPB-5-induced cytotoxicity than four other tumor cells
and LO2 normal cells; and the DPB-5-induced Hep G2 cell death
was both dose- and time-dependent. The cytotoxic effect of DPB-5
caused apoptotic cell death, as determined by DNA fragmentation,
morphological change of the nucleus, increase of pro-G1 DNA con-
tent, and externalization of phosphatidylserine.
ROS plays an important role in apoptosis. The apoptotic effect
of DPB-5 on Hep G2 cells was associated with an early elevated
level of intracellular ROS. After treatment with DPB-5 for 1 h, ROS
was increased to 2.56-fold, and continued to increase with time,
reaching almost 10-fold after 12 h treatment. However, the ROS
change to normal cells LO2 was not so significant with only 0.5-
fold increase after 12 h. In conjunction, the ROS generation was
supported by the significant decrease of intercellular GSH, a main
non-protein antioxidant in the cell, which is able to clear intra-
cellular ROS [41] and inversely proportional to ROS level [42].
Consistent with continuous ROS generation, our result indicated
that DPB-5-induced significant reduction of intracellular GSH on
Hep G2 cells, while less change on LO2 cells. The higher vulner-
ability to ROS of Hep G2 cells than LO2 cells lead to the higher
cytotoxicity of Hep G2 cells on DPB-5 than that of LO2 cells. Can-
cer cells, function with a heightened basal level of ROS-mediated
signal, which is required for the increased rate of growth, may
be more vulnerable to oxidant stress. A further oxidative stress
could exhaust the cellular antioxidant capacity and push the ROS
stress level beyond a “threshold”, which would cause cell apoptosis
[16].
Moreover, in order to confirm that the apoptotic effect of DPB-5
was mediated by ROS, antioxidants NAC and catalase were used.
Both NAC and GSH inhibited the cytotoxicity of DPB-5 associated
with suppressed ROS generation and GSH depletion. And they both
can ensure cell survival as shown by trypan blue exclusion and
significantly reduced cell apoptosis as demonstrated by Hoechst
staining. Our results also indicated that DPB-5 induced a decrease
of MMP in Hep G2 cells. In addition, concerning the time course of
ROS burst and MMP depolarization, the ROS burst occurred before
intracellular MMP depolarization. This opinion was supported by
the results obtained from Hep G2 co-incubated with DPB-5 and
antioxidant NAC or catalase. Both NAC and catalase blocked the
MMP depolarization completely. These results demonstrated that
the ROS burst was a prerequisite for MMP collapse and cell death
induced by DPB-5.
Fig. 4. DPB-5-induced ROS generation, GSH depletion and mitochondria membrane
potential (MMP) disruption. (A) Effect of DPB-5 on cellular ROS production. After
treatment with 10 ␮M DPB-5 for 0–12 h, cells were incubated with 10 ␮M DCFH-DA
for 30 min and then immediately subjected to flow-cytometric analysis. Results are
expressed as a ratio of relative fluorescent intensity compared to the control group.
(B) Effect of DPB-5 on cellular GSH level. Cellular GSH level was examined by flow-
cytometric analysis as describe in text after treated with 10 ␮M DPB-5 for 0–12 h.
Results are expressed as a percentage of relative fluorescent intensity compared
to the control group. (C) Effect of DPB-5 on MMP. After treated with 10 ␮M DPB-
5 for 0–12 h, cells determined by flow-cytometric analysis stained with Rho 123
for 30 min. Results are expressed as a percentage of relative fluorescent intensity
compared to the control group. The data represent means SD of three independent
experiments; *P ꢀ 0.05 versus control.
the main source of ROS in most tumor cells [38]. It is estimated
that about 2% of the oxygen in a cell leaks from the mitochondrial
electron transport chain and becomes ROS under physiological con-
dition [39]. We investigated the effect of rotenone (ROT, an inhibitor
of mitochondrial electron transport chain complex I) on DPB-5-
induced ROS generation. Hep G2 cells were treated with 5 ␮M
rotenone for 30 min, and then treated with or without 10 ␮M DPB-5
for 2 h. Compared with the 2.56-fold increase without any pretreat-
ment, the treatment with rotenone blocked the DPB-5-induced ROS
generation significantly (Fig. 6). This result suggested that DPB-5
triggered the generation of ROS by the mitochondria.
In addition, we also determined that DPB-5-induced ROS pro-
duction by the mitochondria. When inhibiting the cell respiration
by rotenone (an inhibitor of the mitochondrial electron transport
chain complex I), the DPB-5-induced generation of ROS in Hep G2
cells was blocked completely. It is well accepted that ROS is gener-
ated from ETC primarily in the form of the superoxide anion (O2−),
which is quickly transformed to hydrogen peroxide (H2O2), a sta-
ble form of ROS. In our study, however, O2− in Hep G2 cells treated
with DPB-5 showed no significant change when stained with dihy-
droethidium (data not shown). Meanwhile, there was a dramatic
increase of intracellular ROS (examined by DCFH-DA), which was
generated by the mitochondria. We postulate that DPB-5-induced
3.7. DPB-5-induced cell apoptosis in a caspase-dependent
pathway but not caspase-9 dependent
Caspases play a critical role in the process of cell apoptosis [40].
Caspase-9 is a major mediator of apoptosis. Surprisingly, DPB-5
failed to activate caspase-9 while H2O2, as a positive control, sig-
ROS generation is related to the mitochondrial protein p66shc
,

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J. Li et al. / Chemico-Biological Interactions 183 (2010) 341–348
Fig. 5. Effects of exogenous application of NAC and catalase (CAT) on the DPB-5-induced ROS generation, GSH depletion MMP disruption, and cell viability of Hep G2 cells.
(A) The ROS, GSH and MMP changes were examined by flow-cytometric analysis. (B) Cell viability was analyzed using MTT assay. Cells treated with DPB-5 for 48 h with or
without pretreated with 1 mM NAC or 2000 U/ml catalase. (C) Cell death rate was determined by trypan blue exclusion. Control, cells treated with solvent as control; DPB-5,
cells treated with 6 ␮M DPB-5 for 48 h; CAT+ DPB-5, cells treated with 6 ␮M DPB-5 for 48 h after incubated with 2000 U/ml catalase for 30 min; NAC + DPB-5, cells treated
with 6 ␮M DPB-5 for 48 h after incubated with 1 mM NAC for 30 min. (D) catalase and NAC protected cells from apoptotic effect of DPB-5 determined by Hoechst staining.
DPB-5: cells treated with 6 ␮M DPB-5 for 36 h; CAT+ DPB-5: cells treated with 6 ␮M DPB-5 for 36 h after pretreated with 2000 U/ml catalase for 30 min; NAC + DPB-5: cells
treated with 6 ␮M DPB-5 for 36 h after pretreated with 1 mM NAC for 30 min. (The data represent means SD of three independent experiments; *P ꢀ 0.05, cells treated with
DPB-5 after incubated with NAC or catalase were compared with cells treated with DPB-5 alone.)
which acts downstream of ETC to oxidize reduced cytochrome c,
The caspases-cascade system plays crucial roles in cell
apoptosis. It is reported that caspases are the executors of
apoptosis. Benzothiazole containing phthalimide induced human
hepatoma cell SKHep1 and Burkitt’s lymphoma cell CA46
apoptosis in both caspase-dependent and caspase-independent
pathways [45], while 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-
aminoacrylonitrile (AMBAN) induced human leukemia cells HL60
and U937 apoptosis through a caspase-dependent pathway [29].
In the present paper, caspases played a critical role in DPB-
5-induced cell death. When preincubated with pan-caspase
inhibitor zVADfmk, the apoptotic effect of DPB-5 was signifi-
cantly inhibited and the death rate was obviously reduced. AMBAN
induced ROS increase as a death signal, which is similar to
DPB-5. AMBAN decreased the mitochondrial membrane potential,
released cytochrome c, and then activated caspase-9 to initiate
caspases-cascade, which finally lead to cell apoptosis. Surprisingly,
different from AMBAN, DPB-5 neither increased nor reduced the
activity of caspase-9. It is supposed that other initiator caspases
could be recruited and activated caspases-cascade.
generating H2O2 directly without O2 [43]. Further, p66shc acts as
−
a regulator of mitochondrial metabolism, so the small increase of
MTT reduction induced in a short time by a low concentration of
DPB-5 (Fig. 2) may be related to the increase of cell mitochondrial
metabolism induced by the activation of p66shc [44].
In conclusion, the apoptotic effect of DPB-5 is mediated by ROS
and relied on caspases-cascade. DPB-5 exerts higher cytotoxicity to
Hep G2 cells than normal liver cells LO2, which is resulted from the
vulnerability of cancer cells to ROS. Moreover this selective cyto-
toxic effect to Hep G2 suggested the anticancer potential of DPB-5.
Fig. 6. Effect of rotenone (ROT) on the ROS induced by DPB-5. ROS level was analyzed
using flow-cytometric analysis stained with DCFH-DA. Cells co-treated with DPB-5
for 2 h after pretreated with 5 ␮M rotenone for 30 min. (The data represents the
means SD of three independent experiments with triplicates. *P < 0.05 compared
to DPB-5 alone group.)

J. Li et al. / Chemico-Biological Interactions 183 (2010) 341–348
347
Fig. 7. DPB-5-induced Hep G2 cells apoptosis in a caspase-dependent pathway but independent on caspase-9. (A) Change of caspase-9 activity after treated with 6 ␮M or
12 ␮M of DPB-5 12 h. NC: negative control; PC: positive control, 50 ␮M H2O2 treated for 6 h. (*P ꢀ 0.05 compared with control.) (B) Pan-caspase inhibitor zVADfmk protected
cells death induced by DPB-5. Cell death rate after 48 h DPB-5 treatment with or without zVADfmk pretreated, which was determined by trypan blue exclusion as described
in text. Control, cells treated with DPB-5 as control; zVADfmk, cells treated with DPB-5 after incubated 20 ␮M zVADfmk for 30 min. (The data represent means SD of three
independent experiments; *P ꢀ 0.05 compared with DPB-5 alone group.) (C) zVADfmk inhibits DPB-5-induced cell apoptosis showed by DNA fragment analysis. c: control,
cells treated with solvent as control; 1: cells treated with 6 ␮M DPB-5 for 36 h; 2: cells which pretreated with 20 ␮M zVADfmk for 30 min treated with 6 ␮M DPB-5 for 36 h.
Conflict of interest statement
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