(PEITC)-Mediated Generation of Reactive Oxygen Species Causes Cell
Cycle Arrest and Induces Apoptosis via Activation of Caspase-3,
Mitochondria Dysfunction and Nitric Oxide (NO) in Human
Osteogenic Sarcoma U-2 OS Cells
Chang-Lin Wu,
1An-Cheng Huang,
2Jai-Sing Yang,
3Ching-Lung Liao,
4Hsu-Feng Lu,
5,6Su-Tze Chou,
7Chia-Yu Ma,
8Te-Chun Hsia,
4,9Yang-Ching Ko,
10,11Jing-Gung Chung
1,121Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan, 2Department of Nursing, St. Mary’s
Medicine Nursing and Management College, Yilan 266, Taiwan, 3Department of Pharmacology, China Medical University, Taichung 404,
Taiwan,4School of Chinese Medicine, China Medical University, Taichung 404, Taiwan,5Department of Clinical Pathology, Cheng Hsin General
Hospital, Taipei 112, Taiwan,6College of Human Ecology, Fu-Jen Catholic University, Taipei 242, Taiwan,7Department of Food and Nutrition,
Providence University, Taichung 433, Taiwan,8Department of Food and Beverage Management, Technology and Science Institute of Northern
Taiwan, Taipei 112, Taiwan, 9Department of Internal Medicine, China Medical University Hospital, Taichung 404, Taiwan, 10Division of
Pulmonary and Critical Care Medicine, Department of Internal Medicine, St. Martin De Porres Hospital, Chiayi 600, Taiwan,11Department of
Nursing, Chung Jen College of Nursing, Health Sciences and Management, Chiayi 622, Taiwan,12Department of Biotechnology, Asia University,
Taichung 413, Taiwan
Received 14 October 2010; accepted 10 December 2010
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.21350
ABSTRACT: Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, have been shown to exhibit antineoplastic ability against many human cancer cells. In this study, we found that exposure of human osteogenic sarcoma U-2 OS cells to BITC and PEITC led to induce morphological changes and to decrease the percentage of viable cells in a time- and dose-dependent manner. BITC and PEITC induced cell cycle arrest at G2/M phase at 48 h treatment and inhibited the levels of cell cycle regulatory proteins such as cyclin A and B1 in U-2 OS cells but promoted the level of Chk1 and p53 that led to G2/M arrest. BITC and PEITC induced a marked increase in apoptosis (DNA fragmentation) and poly(ADP-ribose)polymerase (PARP) cleavage, which was associated with mitochondrial dysfunction and the activation of caspase-9 and -3. BITC and PEITC also promoted the ROS production in U-2 OS cells and the N-acetylcysteine (NAC, an antoxidant agent) was pretreated and then treated with both compounds which led to decrease the levels of ROS and increase the cell viability. Interestingly, BITC and PEITC promoted the levels of NO production and increased the iNOS enzyme. Confocal laser microscope also demonstrated that BITC and PEITC promoted the release of cytochromec and AIF, suggesting that both compounds induced apoptosis through ROS, caspase-3 and mitochondrial, and NO signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC and PEITC-caused growth inhibition, G2/M arrest, and apoptotic death of U-2 OS cells. ß2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Keywords: BITC; PEITC; apoptosis; NO; human osteogenic sarcoma U-2 OS cells
Bone cancer is one of the major causes of death in the
human population worldwide. In Taiwan, bone cancer
causes about 0.4 persons per 100,000 to die annually
based on the 2008 report from the Department of Health,
ROC (TAIWAN). It also represents about 0.2% of all
malignant tumors with an incidence of 3 cases/million
-population/year.
1Osteosarcoma, a highly malignant
bone tumor, is a primary malignant bone tumor that
usually develops in children and young adults during
periods.
2,3Currently, surgery, radiation, chemotherapy,
or a combination of radiotherapy and chemotherapy
were used in clinical patients for the treatment of
bone cancer but it is still unsatisfying. Numerous studies
have focused on gene and protein levels to investigate
the pathogenesis and development of osteosarcoma, and
the results found many osteosarcoma related genes and
proteins related to familial genetics, cell cycle biology,
DNA damage pathways, and the use of chemotherapy.
4–7Many compounds that have been used for cancer
pre-ventative agents or even as cancer therapy drugs have
been found in natural products.
8Epidemiological studies
suggest that a dietary intake of cruciferous vegetables
may protect against different malignancies.
9–11Benzyl isothiocyanate (BITC) and phenethyl
isothio-cyanate (PEITC) are present in cruciferous plants
and both are a member of the isothiocyanate family,
which have been demonstrated to be protective against
carcinogenesis.
12–14Much evidence has shown that
BITC induced G2/M cell cycle arrest via decreasing
Cdk1, cyclin B1, and Cdc25B protein levels.
15–17It was also reported that BITC produces the formation
of reactive oxygen species (ROS) that induces cell death
through apoptosis.
14,18Other investigators also reported
that BITC treatment effectively inhibits growth of
human breast cancer cells by inducing apoptotic cell
death.
19–21PEITC has been shown to decrease the
per-centage of viability of cancer cells in culture through
the
induction
of apoptosis
and autophagy.
19,22,23Chang-Lin Wu and An-Cheng Huang contributed equally to this work.
Correspondence to: Jing-Gung Chung (T:þ886-4-2205-3366 ext. 2161; F:þ886-4-2205-3764; E-mail: [email protected])
Furthermore, PEITC has been demonstrated to exert
growth inhibition, the induction of apoptosis and cell
cycle arrest in lung,
24prostate,
25and ovarian cancer.
26Induction of apoptosis from cancer cells is one of the
best strategies in chemotherapy and radiotherapy.
27Apoptosis pathway can be divided into the extrinsic
(death receptor) pathway and the intrinsic
(mitochon-drial) pathway.
28The extrinsic pathway (caspases 8/10
are the initiator caspases)
29begins with external death
receptors on the cell surface, after ligands bind to their
specific receptor resulting in intercellular signaling to
form the cleavage and activation of caspase-8
30,31before
caspase-8 cleave affects caspase-3 to induce apoptosis
directly.
32In some cases, the activated caspase-8 can
also trigger the activation of the intrinsic pathway
involving key mitochondrial events including the
anti-apoptotic protein Bcl-2, Bcl-XL and the pro-anti-apoptotic
protein Bax, Bak, Bik, Bad, and Bid.
33,34This affects
mitochondrial membrane integrity and triggers
cyto-chrome
c release
35to cause the activation of caspase-9
and subsequently leads to the activation of effector
cas-pase-3
36for causing apoptosis. In some cases, the
mito-chondrial dysfunction of cells will release
apoptosis-inducing factor (AIF)
37and endonuclease G (Endo G)
38to induce apoptosis directly.
Although many studies have shown that BITC and
PEITC induced cell cycle arrest and apoptosis in many
human cancer cell lines, there is no available information
to address the effect of BITC and PEITC on human bone
cancer cells. Thus, the present study aims to investigate
the effect of BITC and PEITC on molecular signaling
pathway to cause the cell cycle arrest and induction of
apoptosis in human osteogenic sarcoma U-2 OS cells.
MATERIALS AND METHODS
Reagents
BITC, PEITC, dimethyl sulfoxide (DMSO), propidium iodide (PI), and trypan blue were purchased from Sigma Chemical Co. (St. Louis, MO). McCoy’s 5A medium,L-glutamine, fetal bovine serum (FBS), penicillin–streptomycin, and trypsin–EDTA were obtained from Gibco BRL/Invitrogen (Carlsbad, CA). Primary antibodies (cyclins A and B1, chk1, p53, catalase, Mn–SOD, iNOS, cytochromec, caspase-9 and -3, PARP, and b-actin) and second antibodies for Western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and diluted in PBS Tween-20 before use.
Cell Culture and Treatments
Human osteogenic sarcoma U-2 OS cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). U-2 OS cells were plated onto 75 cm2tissue
culture flasks at 378C under a humidified 5% CO2atmosphere
and grown with 90% McCoy’s 5A medium with 2 mML -gluta-mine adjusted to contain 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.39The cells were maintained in 5% CO2at 378C until reaching approximately 50–70% confluence,
and then treated with different amounts of BITC and PEITC as indicated.
Morphological Changes and Viability
A U-2 OS cell at 2 105cells/well was maintained on 12-well
plates for 24 h before different concentrations of BITC and
PEITC were added to each well at final concentrations of 0, 5, 7.5, and 10 mM or 0, 5, 10, and 15 mM, respectively. They were then incubated for 0, 6, 12, 24, and 48 h. All cells in the well were directly examined and photographed under phase contrast microscope for morphological change examinations. All cells from each treatment were trypsinized and harvested by centrifugation at 1,500 rpm for 5 min, washed twice with PBS before 5 mg/ml PI in PBS was added to the cells and viable cells were determined by using FACSCalibur utilizing Cell-Quest software (Becton-Dickinson, San Jose, CA) for determi-nation of viable cells as previously described.40,41
Cell Cycle Analysis
The U-2 OS cells were maintained on 12-well plates and cul-tured under the conditions described above. After treatment with BITC and PEITC for 48 h, cells were isolated, washed with ice-cold PBS, and then fixed in 70% ethanol overnight. Then cells were re-suspended in PBS containing 40 mg/ml PI and 0.1 mg/ml RNase and 0.1% Triton X-100 in dark room for 30 min at room temperature.42Cell cycle analyses and
sug-G1 (apoptosis) were analyzed with a flow cytometer (Becton-Dickinson) equipped with an argon ion laser at 488 nm wave-length. The analyses were performed in triplicate for statistical evaluation.
Morphology and Mitotic Phase Assays
After U-2 OS cells were treated with 10 mM BITC and PEITC for 24, 48, and 72 h, cells from each treatment were harvested and smeared on slides. The slides were air dried, fixed in methanol, and stained with Giemsa at room temperature for 15 min. Alterations of nuclei, membrane, and morphological features were observed by light microscopy. Cells in mitotic phase were recognized by the appearance of chromosomes dis-persed in the cytoplasm and by the disappearance of nuclear membranes.43
Comet Assay for DNA Damage
In order to prevent further DNA damage, all steps were per-formed in the dark. The alkaline comet assay was carried out according to the method described previously.41,42U-2 OS cells
(5 104cells/ml) were treated with 7.5 mM of BITC or 10 mM
of PEITC for 24 and 48 h. Cells were harvested by centrifu-gation, isolated and examined for DNA damage by using the Comet assay. We quantified the DNA damage of nuclei with tail randomly selected under microscope at 100 magnification after staining with 2 mg/mL 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 5 min by using a fluorescent micro-scope. The tail moment (TM) was used to evaluate the degree of DNA damage in all samples.
DAPI Staining
U-2 OS cells (5 104cells/ml) were plated onto 6-well plates and were treated with 7.5 mM of BITC or 10 mM of PEITC for 24 and 48 h. Cells were harvested and U-2 OS cells (5 104cells/
ml) were treated with 7.5 mM of BITC or 10 mM of PEITC for 24 and 48 h. Cells were harvested by centrifugation and stained by 10 mg/mL DAPI for apoptotic cells as previously described.40
After staining, the cells were examined and photographed using a fluorescence microscope.
Western Blotting
The U-2 OS cells at density of 1 106cells/well on 12-well plate were treated with 10 mM BITC or PEITC for incubation of 24 h and then the cells were harvested by trypsinized and
were lysed in lysate buffer composed of 50 mM tris (pH 8.0), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, and 0.5% NP-40 with protease inhibitor solution (Roche, Mannheim, Germany). The protein concentration from each treatment was determined using a protein assay (Bio-Rad1
, Hercules, CA). Equal amount of proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide electrophoretic gel (SDS–PAGE) and transferred to nitrocellulose membranes and then were blocked with 5% dry milk in tris buffered saline– Tween-20 and probed with the appropriate primary antibodies and secondary antibodies. Membranes were then developed using enhanced chemiluminescence methods.40,42
Flow Cytometer Assay for the Production of Reactive Oxygen Species and the Levels of Mitochondrial Membrane Potential (DCm)
The U-2 OS cells were plated onto 12-well plates and were treated with 7.5 and 10 mM, respectively, of BITC and PEITC for 0, 12, 18, and 24 h before being harvested, washed twice, and counted from each treatment. 1 105cells were re-suspended
in the 500 ml of ROS indicator 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA), or 500 ml of the DCm indicator 3,30
-dihexyloxacarbocyanine iodide (DiOC6). Then all samples were incubated at 378C for 30 min and the levels of ROS and DCm were measured by using flow cytometric assay as previously described.42,44Cells were pretreated with or without cyclospor-ine A (CsA, an inhibitor of mitochondrial permeability tran-sition pore)45at 0, 2.5, 5, and 10 mM and then were treated with
7.5 and 10 mM, respectively, of BITC and PEITC for 24 h before being harvested to measure the levels of DCm as described elsewhere.41
Confocal Laser Scanning Microscope
The location of cytochromec and AIF was determined by con-focal laser scanning microscopy. The U-2 OS cells at a density (5 104cells/well) were plated on 4-well chamber slides and
were treated individually with 7.5 and 10 mM, respectively, BITC or PEITC for 24 h. Then cells from each treatment were fixed in 4% formaldehyde in PBS for 15 min, permeabilized with 0.3% Triton X-100 in PBS for 1 h with blocking of non-specific binding sites using 2% BSA as described previously.46,47 At the end of fixation, the fixed cells were stained with primary antibodies to cytochrome c and AIF (1:100 dilution) (green fluorescence) for overnight. Then cells were washed and stained by secondary antibody (FITC-conjugated goat antimouse IgG at 1:100 dilution), followed by the staining with PI (red fluor-escence) before being washed twice. Photomicrographs were obtained using a Leica TCS SP2 Confocal Spectral Microscope as described previously.41
Statistical Analysis
Student’st-test was used to analyze the differences between BITC or PEITC-treated and control groups. p < 0.05;
p < 0.01;p < 0.001.
RESULTS
BITC and PEITC Induced Morphological Changes
Decreased the Percentage of Viable U-2 OS Cells
The morphological changes of U-2OS cells were observed
at 24 h after treatment with 5, 10, and 15 mM of BITC
and PEITC as shown in Figure 1A and these effects are
dose-dependent manners. Figure 1A showed that some
cells after being exposed to BITC or PEITC, became
smaller, round, and blunt in size and these observations
in BITC treatment were more obvious than that of
PEITC treatment. After being examined and
photo-graphed, the cells in each well were harvested by
centrifugation to determine the percentage of viable cells
by flow cytometric assay and the results are shown in
Figure 1B. The data indicated that BITC and PEITC
decreased the percentage of viable U-2 OS cells and
the influences are dose- and time-dependent manners.
Percentage of viability decreased by more than 50% in
U-2 OS cells exposed to 10 mM PEITC or 7.5 mM BITC
after 48 h treatment. Thus the concentration of 10 or
7.5 mM, respectively, for both test agents was used in all
further experiments.
BITC and PEITC Induced Cell Cycle Arrest in U-2 OS
Cells
Based on the growth inhibition results, further studies
were conducted to investigate the possible mechanisms
by which PEITC and BITC exhibits the inhibitory effects
in U-2 OS cells in vitro. The results from flow cytometric
assay, as shown in Figure 2A, revealed that BITC
induced dramatic accumulation of U-2 OS cells in
G2/M phase at 5 mM but induced accumulation of cells
G0/G1 phase at 10 mM BITC. However, PEITC induced
dramatic accumulation of U-2 OS cells in G2/M phase at 5
and 7.5 mM but induced dramatic accumulation of U-2
OS cells in S phase at 10 mM. All observations are
obtained from BITC and PEITC treatment for 48 h.
The results also showed that BITC or PEITC caused cell
cycle arrest in different doses.
BITC and PEITC Affected Cell in Mitotic Phase and G2/M
Arrest Associated Protein Levels in U-2 OS Cells
To examine whether the growth inhibition effect of BITC
and PEITC on U-2 OS cells were mediated through
specific inhibition of mitosis, we investigated the mitosis
stain by Gimsa staining and the results are shown in
Figure 2B. The results indicated that both BITC and
PEITC compounds induced the inhibition of mitotic
phase in examined U-2 OS cells. We examined whether
the G2/M arrest in U-2 OS cells by BITC and PEITC were
mediated through the effects on associated protein
levels. The results in Figure 2C show that similar effects
were observed with BITC and PEITC treatment in U-2
OS cells for 48 h, and both compounds decreased the
levels of cyclin A and cyclin B1 but increased the levels
of p53 and Chk1.
BITC and PEITC Induced Apoptosis and DNA Damage in
U-2 OS Cells
To examine the decrease of the percentage of viable U-2
OS cells from treatment of BITC and PEITC through
DNA damage and apoptosis, cells were examined by
DAPI staining and Comet assay. The results shown in
Figure 3 indicated that both examined compounds
induced condensation of nuclei (apoptosis) (Fig. 3A).
The Comet assay showed that both examined compounds
induced DNA damage in U-2 OS cells (Fig. 3B). After the
calculation, both compounds induced apoptosis in U-2
OS cells and these effects are time-dependent (Fig. 3C).
NAC Affected the Effects of BITC and PEITC on the ROS
and NO Production in U-2 OS Cells
To examine whether the induction of apoptosis in U-2 OS
cells came from the treatment of BITC and PEITC
through ROS production, cells also pretreated with or
without
N-acetylcysteine (NAC) were then harvested for
measuring ROS production by flow cytometric assay. The
results indicated that both examined compounds
pro-moted ROS production in U-2 OS cells in a
dose-depend-ent manner (Fig. 4A). Both compounds promoted NO
production and the levels of catalase (Fig. 4C), but BITC
decreased the levels of Mn–SOD and PITC yet did not
significantly affect the levels of Mn–SOD (Fig. 4C). Both
compounds also promoted NO production and the levels
of iNOS (Fig. 4B and D) in a time-dependent manner.
After U-2 OS cells were pretreated with NAC then
treated with BITC or PEITC, cell viability was
deter-mined by flow cytometric assay and the results are shown
in Figure 4E. They indicate that NAC can increase the
percentage of viable cells from both compounds of treated
groups and the percentage over the control group. These
observations indicated that BITC and PEITC induced
growth inhibition through the ROS and NO production
in U-2 OS cells.
CsA Did Not Alter the Effects of BITC and PEITC on the
Level of Mitochondrial Membrane Potential (DCm) and
NAO in U-2 OS Cells
To examine whether the effects of DCm in U-2 OS cells
came from the treatment of BITC and PEITC, cells also
pretreated with or without cyclosporine then were
Figure 1. PEITC and BITC induced morphological changes and decreased the percentage of viable U-2 OS cells. Cells were treated with different concentrations (5–15 mM) of BITC and PEITC for 0, 6, 12, 24, and 48 h and cells morphological changes were examined under phase contrast microscope at 200 (A) and cells were harvested to measure the percentage of viable cells by flow cytometric assay (B). Data are given as relative inhibitory rates compared with untreated control group. The values presented are the mean SD (n ¼ 3) from three independent experiments.p < 0.05. Significantly different from vehicle control treated cells.
harvested for measuring the levels of DCm and the
production of NAO by flow cytometric assay. The
results are shown in Figure 5, indicating that BITC
and PEITC decreased the levels of DCm and these
effects are of a time-dependent manner (Fig. 5A). Both
compounds increased the NAO production after 12 h
treatment (Fig. 5B). However, cells were pretreated
with CsA and did not change the effects of BITC and
PEITC decreasing the levels of DCm in U-2 OS cells
(Fig. 5C).
PEITC and BITC Affected Apoptosis Associated Protein
Levels and Translocation in U-2 OS Cells
Based on the results from apoptotic cell death, further
studies were conducted to investigate the possible
mechanisms by which BITC and PEITC induced
apoptosis in U-2 OS cells in vitro. The results from
Western blotting revealed that PEITC and BITC
increased the protein levels of AIF, cytochrome
c,
cas-pase-9, caspase-3, and PARP (Fig. 6A) in U-2 OS cells
and these effects are time-dependent manner. The
Figure 2. BITC and PEITC induced cell cycle arrest, affected cell in mitotic index, and G2/M arrest associated protein levels in U-2 OS cells. Cells were treated with different concentrations (5–10 mM) of BITC and PEITC for 24 and 48 h and then were harvested for measuring the cell cycle distribution as described in the Materials and Methods Section. (A) Percentage of cells in G0/G1, S, and G2/M phase of cell cycle. The values presented are the mean SD (n ¼ 3) from three independent experiments.p < 0.05. Significantly different from vehicle control treated cells. Cells were treated with 10 mM PEITC and 7.5 mM BITC for different time periods and then were harvested for measuring the mitotic index by Gimsa staining (B) and also for measuring the changes of G2/M arrest associated protein levels by Western blotting (C). Results were obtained from three independent experiments.
confocal
laser
microscope
examination
indicated
that PEITC and BITC promoted the release of
cyto-chrome
c and AIF, but only AIF moved into the nuclei
(Fig. 6B,C).
DISCUSSION
It is well known that some anticancer and DNA damage
agents work are via the cell cycle arrest at different
phases and then induce apoptosis in cancer cells.
48–50Figure 3. BITC and PEITC induced apoptosis and DNA damage in U-2 OS cells. Cells were treated with different concentrations of BITC and PEITC for different time periods and then were harvested for measuring the apoptosis by DAPI staining (A) and for DNA damage examination by Comet assay (B) then to calculate the percentage of apoptosis based on sub-G1 from flow cytometric assay (C). The values presented are the mean SD (n ¼ 3) from three independent experiments.p < 0.05. Significantly different from vehicle control treated cells.
It was also well documented that cell cycle have
check-points for ensuring cells have time to repair the damaged
DNA, whereas apoptotic cell death can eliminate
irrep-arable or unrepaired damaged cells. Although numerous
studies have reported that BITC and PEITC can induce
cell cycle arrest and apoptosis in many human cancer cell
lines, the molecular mechanism is still unclear.
Herein, the aim of the present study was to elucidate
the molecular mechanism of action by which BITC and
PEITC induced cytotoxic effects on human osteogenic
sarcoma U-2 OS cells in vitro. The results can be
summarized as (1) BITC and PEITC induce
morphologi-cal changes and decreased the percentage of viable cells;
(2) BITC and PEITC induced dramatic accumulation of
U-2 OS cells in G2/M phase at 5 mM, however, PEITC
induced dramatic accumulation of U-2 OS cells in S
phase at 10 mM; (3) BITC and PEITC both induced
apop-tosis in time-dependent manners; (4) BITC and PEITC
both promoted the production of ROS and Ca
2þbut
decreased the levels of MMP; (5) BITC and PEITC both
promoted the production of NO but decreased the levels
of NAO.
Figure 4. NAC affected the effects of BITC and PEITC on the ROS and NO production in U-2 OS cells. Cells were pre-treated with or without NAC and then were treated with 10 mM PEITC and 7.5 mM BITC for different time periods before being harvested for measuring the ROS production by H2DCF-DA (A) and NO production by DAF/FM (B) were analyzed by flow cytometric assay. For determination of ROS (C)
and NO (D) production associated proteins by Western blotting and then to calculate the percentage of ROS production and whether or not it was affected by NAC from flow cytometric assay (E). The values presented are the mean SD (n ¼ 3) from three independent experiments.
Figure 2 indicates that BITC and PEITC induced an
accumulation of U-2 OS cells in the G2/M phase of the cell
cycle. It was reported that the microtubule-stabilizing
agents
51induced G2/M phase arrest in cancer cells. Our
results showed that the novel finding of BITC and PEITC
induced G2/M phase arrest in U-2 OS cells. In
mamma-lian cells, a number of Cdks have shown to regulate the
cell cycle event,
52,53and Cdk1 and Cdk2 kinases are
activated primarily in association with cyclin A and
B1 in the G2/M phase progression. Results also showed
that BITC and PEITC both decreased the protein levels
of cyclin A and B1 (Fig. 2C). It was reported that the
cyclin B1/Cdk1 complex is the primary regulator of
tran-sition from G2 to M phase.
54BITC and PEITC both
decreased the percentage of viable cells via the apoptotic
cell death with cell cycle arrest.
Cells failing to progress to mitosis may be destined to
apoptosis by BITC and PEITC because our results from
Gimsa staining also showed that both compounds
inhib-ited the mitotosis (Fig. 2B). We also saw that BITC and
Figure 5. CsA affected the effects of PEITC and BITC on the level of mitochondrial membrane potential (DCm) and NAO production in U-2 OS cells. Cells were pre-treated with or without CsA and then were treated with 10 mM PEITC and 7.5 mM BITC for different time periods and then were harvested for measuring the level of DCm (A) and for determination of NAO production (B) then finally to calculate the level of DCm and whether or not they were affected by CsA from flow cytometric assay (C). The values presented are the mean SD (n ¼ 3) from three independent experiments.p < 0.05. Significantly different from vehicle control treated cells.
PEITC induced apoptosis in U-2 O cells (Fig. 3C) and this
is also confirmed through the morphological changes,
DNA fragmentation, sub-G1 increase, and PARP
cleav-age (Fig. 6A). It is well documented that some of the
anticancer agents induced apoptosis via caspases.
55In
particular, the caspase-3 is an executioner caspase,
which can be activated directly from caspase-8 or
-9
56,57before leading to apoptosis. Based on the results
(Fig. 6A) from Western blotting, it indicated that BITC
and PEITC increased the active form of caspase-9 and -3.
Our results also showed that BITC and PEITC
decreased the levels of DCm which may be through
the release of caspase-9 and then activated the
caspase-3 for apoptosis to occur. However, the cells were
Figure 6. PEITC and BITC affected apoptosis associated protein levels and translocation in U-2 OS cells. Cells were treated with 10 mM PEITC and 7.5 mM BITC for 0, 12, 18, and 24 h and then were harvested for measuring the apoptotic associated proteins by Western blotting (A). Cells were also measured for the translocation of cytochromec and AIF by confocal laser microscope at 200 (B). Results were obtained from three independent experiments. The proposed signaling pathway for BITC and PEITC affecting the induction of cell cycle arrest and apoptosis in U-2 OS cells (C).
pretreated with CsA, and then cells were exposed to
BITC and PEITC for examining the levels of DCm.
Results indicated that there was no significant difference
in BITC or PEITC exposure (Fig. 5C). Therefore, BITC
and PEITC induced apoptosis in U-2 OS cells may be
through other different signaling pathways. We also
used confocal laser microscope to examine the
translo-cation of AIF which indicated that BITC and PEITC
both promoted the release of AIF from mitochondria to
cytoplasm, and this observation indicated that both
com-pounds may promote the AIF release to induce apoptosis
(Fig. 6C). Our results also showed that BITC and PEITC
both promoted the productions of ROS and Ca
2þin U-2
OS cells. However, cells were pretreated with NAC, then
exposed to BITC and PEITC then led to decrease the
production of ROS and Ca
2þbut increased the
percent-age of viable cells. This observation also showed that
BITC and PEITC induced cytotoxic effects was through
the induction of ROS (Fig. 4). However, the NAC
pre-treatment (Fig. 4D) did not completely rule out the dead
cells which means that BITC and PEITC compounds
induced cell death through other signal pathways. Thus,
we investigated the levels of NO and the results indicated
that both compounds promoted the NO production which
then also led to apoptosis in U-2 OS cells. These results
suggested involvement of ROS, NO, and mitochondrial
pathways in BITC and PEITC-induced apoptosis.
In conclusion, BITC and PEITC arrested G2/M phase
in the cell cycle distribution and induced apoptosis of U-2
OS cells and the possible signal pathways are
summar-ized in Figure 6D. BITC and PEITC-induced G2/M phase
arrest was associated with reduction of cyclins A and B1.
BITC and PEITC-induced may have gone through the
ROS production, dysfunction of mitochondria, caspase-3
activation, AIF release from mitochondrial, and
pro-motion of NO for causing apoptosis in U-2 OS cells. Taken
together, these findings provide more information
regarding the possible molecular mechanisms and
possible signal pathways of the anticancer activity of
BITC and PEITC.
ACKNOWLEDGMENTS
This work was supported by the grant CMU99-S-20 from China Medical University, Taichung, Taiwan.
REFERENCES
1. Picci P. 2007. Osteosarcoma (osteogenic sarcoma). Orphanet J Rare Dis 2:6.
2. Thompson RC Jr, Cheng EY, Clohisy DR, et al. 2002. Results of treatment for metastatic osteosarcoma with neoadjuvant che-motherapy and surgery. Clin Orthop Relat Res 397:240–247. 3. Arndt CA, Crist WM. 1999. Common musculoskeletal tumors
of childhood and adolescence. N Engl J Med 341:342–352. 4. Lopez-Guerrero JA, Lopez-Gines C, Pellin A, et al. 2004.
Dereg-ulation of the G1 to S-phase cell cycle checkpoint is involved in the pathogenesis of human osteosarcoma. Diagn Mol Pathol 13:81–91.
5. Toguchida J, Ishizaki K, Sasaki MS, et al. 1989. Preferential mutation of paternally derived RB gene as the initial event in sporadic osteosarcoma. Nature 338:156–158.
6. Ochi K, Daigo Y, Katagiri T, et al. 2004. Prediction of response to neoadjuvant chemotherapy for osteosarcoma by gene-expression profiles. Int J Oncol 24:647–655.
7. Abbott DW, Holt JT. 1997. Finkel-Biskis-Reilly mouse osteo-sarcoma virus v-fos inhibits the cellular response to ionizing radiation in a myristoylation-dependent manner. J Biol Chem 272:14005–14008.
8. AbouEl Hassan MA, Braam SR, Kruyt FA. 2006. Paclitaxel and vincristine potentiate adenoviral oncolysis that is associated with cell cycle and apoptosis modulation, whereas they differ-entially affect the viral life cycle in non-small-cell lung cancer cells. Cancer Gene Ther 13:1105–1114.
9. Verhoeven DT, Goldbohm RA, van Poppel G, et al. 1996. Epi-demiological studies on brassica vegetables and cancer risk. Cancer Epidemiol Biomarkers Prev 5:733–748.
10. Kolonel LN, Hankin JH, Whittemore AS, et al. 2000. Vegetables, fruits, legumes and prostate cancer: a multiethnic case–control study. Cancer Epidemiol Biomarkers Prev 9:795– 804.
11. Ambrosone CB, McCann SE, Freudenheim JL, et al. 2004. Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocya-nates, but is not modified by GST genotype. J Nutr 134:1134– 1138.
12. Nakamura Y, Miyoshi N. 2006. Cell death induction by iso-thiocyanates and their underlying molecular mechanisms. Bio-factors 26:123–134.
13. Srivastava SK, Xiao D, Lew KL, et al. 2003. Allyl isothiocya-nate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo. Carcinogen-esis 24:1665–1670.
14. Miyoshi N, Uchida K, Osawa T, et al. 2004. Benzyl isothiocya-nate modifies expression of the G2/M arrest-related genes. Biofactors 21:23–26.
15. Zhang R, Loganathan S, Humphreys I, et al. 2006. Benzyl isothiocyanate-induced DNA damage causes G2/M cell cycle arrest and apoptosis in human pancreatic cancer cells. J Nutr 136:2728–2734.
16. Nakamura Y, Kawakami M, Yoshihiro A, et al. 2002. Involve-ment of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis. J Biol Chem 277: 8492–8499.
17. Srivastava SK, Singh SV. 2004. Cell cycle arrest, apoptosis induction and inhibition of nuclear factor kappa B activation in anti-proliferative activity of benzyl isothiocyanate against human pancreatic cancer cells. Carcinogenesis 25:1701–1709. 18. Singh SV, Srivastava SK, Choi S, et al. 2005. Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species. J Biol Chem 280:19911–19924. 19. Xiao D, Powolny AA, Singh SV. 2008. Benzyl isothiocyanate
targets mitochondrial respiratory chain to trigger reactive oxy-gen species-dependent apoptosis in human breast cancer cells. J Biol Chem 283:30151–30163.
20. Xiao D, Vogel V, Singh SV. 2006. Benzyl isothiocyanate-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak. Mol Cancer Ther 5:2931–2945.
21. Tseng E, Scott-Ramsay EA, Morris ME. 2004. Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines. Exp Biol Med (Maywood) 229:835–842.
22. Xiao D, Powolny AA, Moura MB, et al. 2010. Phenethyl iso-thiocyanate inhibits oxidative phosphorylation to trigger reac-tive oxygen species-mediated death of human prostate cancer cells. J Biol Chem 285:26558–26569.
23. Bommareddy A, Hahm ER, Xiao D, et al. 2009. Atg5 regulates phenethyl isothiocyanate-induced autophagic and apoptotic
cell death in human prostate cancer cells. Cancer Res 69:3704– 3712.
24. Kuang YF, Chen YH. 2004. Induction of apoptosis in a non-small cell human lung cancer cell line by isothiocyanates is associated with P53 and P21. Food Chem Toxicol 42:1711– 1718.
25. Xiao D, Zeng Y, Choi S, et al. 2005. Caspase-dependent apop-tosis induction by phenethyl isothiocyanate, a cruciferous veg-etable-derived cancer chemopreventive agent, is mediated by Bak and Bax. Clin Cancer Res 11:2670–2679.
26. Satyan KS, Swamy N, Dizon DS, et al. 2006. Phenethyl iso-thiocyanate (PEITC) inhibits growth of ovarian cancer cells by inducing apoptosis: role of caspase and MAPK activation. Gynecol Oncol 103:261–270.
27. Bold RJ, Termuhlen PM, McConkey DJ. 1997. Apoptosis, cancer and cancer therapy. Surg Oncol 6:133–142.
28. Sheridan JP, Marsters SA, Pitti RM, et al. 1997. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277:818–821.
29. Vincenz C, Dixit VM. 1997. Fas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95-and p55-mediated death signaling. J Biol Chem 272:6578– 6583.
30. Kamachi M, Le TM, Kim SJ, et al. 2002. Human autoimmune sera as molecular probes for the identification of an autoan-tigen kinase signaling pathway. J Exp Med 196:1213–1225. 31. Ashkenazi A, Dixit VM. 1999. Apoptosis control by death and
decoy receptors. Curr Opin Cell Biol 11:255–260.
32. Porter AG, Janicke RU. 1999. Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6:99–104.
33. Gross A, McDonnell JM, Korsmeyer SJ. 1999. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911.
34. Green DR, Reed JC. 1998. Mitochondria and apoptosis. Science 281:1309–1312.
35. Kulkarni K, Selesniemi K, Brown TL. 2006. Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways. Apoptosis 11:2205–2215.
36. Caserta TM, Smith AN, Gultice AD, et al. 2003. Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis 8:345–352.
37. Yang L, Liu X, Lu Z, et al. 2010. Ursolic acid induces doxor-ubicin-resistant HepG2 cell death via the release of apoptosis-inducing factor. Cancer Lett 298:128–138.
38. Diener T, Neuhaus M, Koziel R, et al. 2010. Role of endonu-clease G in senescence-associated cell death of human endo-thelial cells. Exp Gerontol 45:638–644.
39. Hsu SC, Yang JS, Kuo CL, et al. 2009. Novel quinolone CHM-1 induces apoptosis and inhibits metastasis in a human ostero-genic sarcoma cell line. J Orthop Res 27:1637–1644. 40. Lu CC, Yang JS, Huang AC, et al. 2010. Chrysophanol induces
necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cells. Mol Nutr Food Res 54:967–976.
41. Yang JS, Hour MJ, Huang WW, et al. 2010. MJ-29 inhibits tubulin polymerization, induces mitotic arrest, and triggers apoptosis via cyclin-dependent kinase 1-mediated Bcl-2 phos-phorylation in human leukemia U937 cells. J Pharmacol Exp Ther 334:477–488.
42. Wu SH, Hang LW, Yang JS, et al. 2010. Curcumin induces apoptosis in human non-small cell lung cancer NCI-H460 cells through ER stress and caspase cascade- and mitochondria-dependent pathways. Anticancer Res 30:2125–2133. 43. Yang H, Pang JY, Cai YC, et al. 2006. Cytotoxic activity and
cytostatic mechanism of novel 2-arylbenzo[b]furans. J Pharm Pharmacol 58:1281–1287.
44. Ji BC, Hsu WH, Yang JS, et al. 2009. Gallic acid induces apoptosis via caspase-3 and mitochondrion-dependent path-ways in vitro and suppresses lung xenograft tumor growth in vivo. J Agric Food Chem 57:7596–7604.
45. Zhang SS, Zhang HQ, Li D, et al. 2008. A novel benzotriazole derivative inhibits proliferation of human hepatocarcinoma cells by increasing oxidative stress concomitant mitochondrial damage. Eur J Pharmacol 584:144–152.
46. Chiang JH, Yang JS, Ma CY, et al. 2011. Danthron, an anthra-quinone derivative, Induces DNA damage and caspase cas-cades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways. Chem Res Toxicol 24:20–29. 47. Chiu TH, Lai WW, Hsia TC, et al. 2009. Aloe-emodin induces
cell death through S-phase arrest and caspase-dependent pathways in human tongue squamous cancer SCC-4 cells. Anticancer Res 29:4503–4511.
48. Orren DK, Petersen LN, Bohr VA. 1997. Persistent DNA dam-age inhibits S-phase and G2 progression, and results in apop-tosis. Mol Biol Cell 8:1129–1142.
49. Fujimoto K, Hosotani R, Doi R, et al. 1999. Induction of cell-cycle arrest and apoptosis by a novel retinobenzoic-acid deriva-tive, TAC-101, in human pancreatic-cancer cells. Int J Cancer 81:637–644.
50. Kessel D, Luo Y. 2000. Cells in cryptophycin-induced cell-cycle arrest are susceptible to apoptosis. Cancer Lett 151:25–29. 51. Ling YH, Consoli U, Tornos C, et al. 1998. Accumulation of
cyclin B1, activation of cyclin B1-dependent kinase and induc-tion of programmed cell death in human epidermoid carcinoma KB cells treated with taxol. Int J Cancer 75:925–932. 52. Molinari M. 2000. Cell cycle checkpoints and their inactivation
in human cancer. Cell Prolif 33:261–274.
53. Hartwell LH, Weinert TA. 1989. Checkpoints: controls that ensure the order of cell cycle events. Science 246:629–634. 54. Doree M, Galas S. 1994. The cyclin-dependent protein kinases
and the control of cell division. FASEB J 8:1114–1121. 55. Shi Y. 2002. Mechanisms of caspase activation and inhibition
during apoptosis. Mol Cell 9:459–470.
56. Budihardjo I, Oliver H, Lutter M, et al. 1999. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15:269–290.
57. Wolf BB, Green DR. 1999. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274:20049– 20052.
