Advances in Bioscience and Biotechnology, 2012, 3, 649-656 ABB Published Online October 2012 (
NS-398 induces caspase-dependent,
mitochondria-mediated intrinsic apoptosis of
hepatoma cells
Il Han Song*, Suk Bae Kim, Hyun Duk Shin, Ha Yan Kang, Eun Young Kim
Division of Hepatology and Gastroenterology, Department of Internal Medicine, Dankook University College of Medicine, Dankook
University Hospital Institute of Medical Science, Cheonan, Korea
Email: *
Received 2 July 2012; revised 15 August 2012; accepted 5 September 2012
The present study was conducted to investigate whe-
ther mitochondrial pathway of apoptosis is involved
in cyclooxygenase-2 (COX-2) inhibitor-induced growth
inhibition of hepatoma cells. The g ro wt h ra t e a nd pa t-
tern of NS-398 (selective COX-2 inhibitor)-treated
Hep3B hepatoma cells were analyzed by microscopic
examination, DNA fragmentation gel analysis and
flow cytometry followed by the cleavage of down-
stream caspase 3 and the release of cytosolic fraction
of cytochrome c assessed by Western blot analysis.
NS-398 induced the growth inhibittion of hepatoma
cells depending on the concentration of this COX-2
inhibitor and time sequence. Ladder patterned-DNA
fragmentation and cytometric redistribution to sub-
G1 phase in cell cycle were revealed in NS-398-in-
duced growth inhibition of hepatoma cells. Cyto-
chrome c was translocated from mitochondria to cy-
tosol in time-dependent manner following NS-398
treatment to hepatoma cells. COX-2 inhibitor induces
the growth inhibition of hepatoma cells via caspase-
dependent, mitochondria-mediated intrinsic apop-
tosis pathway. These results strongly suggest the pos-
sibility of therapeutic implication of COX-2 inhibitor
in HCC.
Keywords: Hepatocellular Carcinoma;
Cyclooxygenase-2 (COX-2); COX-2 Inhibitor;
Apoptosis; Western Blotting; Flow Cytometry; DNA
Hepatocellular carcinoma (HCC) is a growing health
problem worldwide, which is the fifth most common
malignancy in incidence and the third leading cause in
cancer-related mortality [1-3]. In advanced stage of HCC
beyond clinical indications for curative treatment mo-
dalities such as surgical resections or percutaneous abla-
tions, no effective systemic therapies but recently intro-
duced molecular targeted agent are present by this time
Cyclooxygenases (COX), the key enzymes involved in
the metabolic conversion of arachidonic acid to pros-
taglandins, consist of at least two isoforms, constitutive
form of COX-1 and inducible form of COX-2. Since the
overexpression of COX-2 was known to be associated
with neoangiogenic, antiapoptotic, and invasive or me-
tastatic property in certain cell types [7-11], COX-2 has
come to the surface as a therapeutic target of several ma-
lignant tumors including HCC. Several growing evi-
dences of preclinical studies have indicated that COX-2
inhibitors exert antineoplastic effects on hepatoma cells
both in vitro and in vivo [12-18]. However, the degree of
impact of COX-2 inhibitor on growth control of heap-
toma cells are controversial and its growth inhibitory
mechanisms remain unclear thus far.
Major signaling pathways of apoptosis, extrinsic death
receptor-mediated and intrinsic mitochondria-mediated,
are usually carried out through the activation of down-
stream effector caspases in cytoplasm, resulting in the
cleavage of cellular substrates relevant to the morpho-
logical and biochemical constellations of apoptotic phe-
notype [19,20]. Among these pathways, mitochondria-
mediated apoptosis progresses to the cascade activation
of initiator caspase 9 and effector caspase 3 via cyto-
plasmic translocation of cytochrome c from mitochon-
dria [21-23].
In the present study, we tried to evaluate whether
COX-2 inhibitor induces the growth inhibition of heap-
toma cells and engages a caspase-dependent, mitochon-
drial-mediated apoptosis signaling pathway in HCC.
2.1. Cell Line and Culture
The human HCC cell line, Hep3B, was purchased from
*Corresponding author.
I. H. Song et al. / Advances in Bioscience and Biotechnology 3 (2012) 649-656
Korean Cell Line Bank (Seoul, Korea). Hep3B cells
were cultured in Dulbecco’s Modified Eagle Medium
(Gibco BRL, Hyclone laboratories, Lagan, Utah, USA),
supplemented with 10% fetal bovine serum (FBS) and
1% penicillin/streptomycin in a humidified incubator
supplied with 5% CO2 at 37˚C atmosphere.
2.2. Treatment of Selective COX-2 Inhibitor to
Hepatoma Cells
NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methane-
sulfonamide), dissolved in demethyl sulfoxide (DMSO),
was used as a selective COX-2 inhibitor. We prepared the
culture media in concentrations of 0, 10, 100, and 200 μM
NS-398 for concentration-oriented experiments. Hepa-
toma cells were plated at a density of 1 × 105 cells/well
in six-well plastic dishes with 2 mL of 10% FBS-sup-
plemented medium. After 24 h exposure of NS-398, the
media were changed with other new media containing
same concentration of NS-398, and then the cells were
incubated for 72 h.
2.3. Microscopic Examination
After discarding the media with floating cells, we mi-
croscopically observed the cells continuously for three
days for time-oriented experiments under ×20 magnifica-
tion and compared the growth pattern of cell prolifera-
tion between controls (DMSO-treated cells) and NS-398-
treated cells according to sequential time course of 24,
48, and 72 h.
2.4. DNA Fragmentation Gel Analysis
Hepatoma cells were harvested at 24, 48, and 72 h after
treatment of various concentrations of NS-398. The cells
dissolved with lysis buffer were centrifuged at 10,000 g
for 30 min. For DNA extraction, the supernatant was
digested with 50 ng/mL proteinase K at 37˚C for 24 h,
and precipitated with a equal volume of absolute ethanol.
For RNA elimination, the pellet was incubated with a
Tris-EDTA buffer containing 10 μg/mL RNase A at 37˚C
for 1 h. The amount of extracted DNA was measured by
spectrophotometric analysis. Each DNA sample was
electrophoresed on 1.8% agarose gel containing 0.5
mg/L ethidium bromide, and photographed under ultra-
violet (UV) light.
2.5. Flow Cytometric Analysis
Cell cycle distribution was determined by flow cytomet-
ric analysis. After treatment of NS-398, the cells were
collected by trypsinization, washed twice with phos-
phate-buffered saline (PBS), and fixed overnight in 70%
ethanol at 4˚C. The cells were stained with 50 μg/mL
propidium iodide at room temperature for 30 min in the
dark, following to be incubated with 50 μg/mL RNase A
at 37˚C for 1 h. Then cell cycle components were ana-
lyzed by a flow cytometer and CellQuest software.
2.6. Western Blot Analysis
NS-398-treated hepatoma cells were prepared by wash-
ing in PBS and dissolving in lysis buffer (50 mmol/L Tris
pH 7.5, 250 mmol/L NaCl2, 0.5% Triton X-100, 1
mmol/L EDYA, 1 mmol/L PMSF, 1 mmol/L Na3VO4, 1
mmol/L dithiothreitol, 10 μg of leupeptin/mL and 10 μg
of aprotinin/mL). After centrifugation of cell lysates for
10 min at 14,000 g, the protein concentrations of super-
natant in the homogenate were determined by bicin-
choninic acid assay (Pierce Co, Rockford, IL, USA) ac-
cording to the manufacturer’s instructions. 40 μg of pro-
tein in each extract was separated by 15% SDS-poly-
acrylamide gel, and electronically transferred to nitro-
cellulose membrane. The membrane was blocked with
5% fat free dry milk in TBS-T (25 mmol/L Tris-HCl, pH
7.5, 100 mmol/L NaCl, 0.5 Tween-20), incubated with
primary antibody for overnight at 4˚C, washed three
times in TBS-T for 10 min, and then incubated with
horseradish peroxidase-conjugated secondary antibody
for 1 h at room temperature. The immunoblotting signals
were developed with an ECL system (Amersham Life
Sciences, Buckinghamshire, UK). Anti-β-actin (1:1000,
Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA)
was used as a protein loading control.
2.7. Preparation of Mitochondrial and Cytosolic
Extracts for Localization of Cytochrome c
The cell pellets were obtained from NS-398-treated
hepatoma cells that were washed, centrifugated, and re-
suspended in a buffer containing 25 mM Tris (pH 7.4),
250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM
EDTA, 1 mM EGTA, and 1 mM DTT. The resuspened
cells were homogenized ten times with Dounce homoge-
nizer (Wheaton Scientific Products, Millville, New Jer-
sey, USA) after adding protease inhibitor (10 μg of leu-
peptin/mL, 10 μg of aprotinin/mL, and 1 mM PMSF) and
phosphatase inhibitor (10 μM Na3VO4). Unlysed cells
and nuclei were discarded by centrifugation at 750 g for
10 min. The supernatant was centrifugated at 10,000 g
for 30 min, and the resulting pellet, which indicates the
mitochondrial-enriched fraction, was washed once with
the same buffer. The remnant supernatant was further
centrifugated at 10,000 g for 1 h, representing the cytosol
fraction of final supernatant. Each 40 μg of cytosolic and
mitochondrial fractions were used for cytochome c im-
munoblotting described above.
2.8. Statistical Analysis
Statistical analysis was carried out using SPSS software
Copyright © 2012 SciRes. OPEN ACCESS
I. H. Song et al. / Advances in Bioscience and Biotechnology 3 (2012) 649-656 651
system (SPSS Inc., Chicago, IL, USA). Data were ex-
pressed as the mean ± SD of at least three-times inde-
pendent experiments. Student’s t-test and ANOVA analy-
sis were applied to verify the statistical difference as P <
0.05 between experimental groups.
3.1. COX-2 Inhibitor Induced Growth Inhibition
of Hepatoma Cells
After treatment of Hep3B cells with NS-398, cell number
progressively decreased up to 72 h, while the number of
DMSO-treated cells exponentially increased within the
same time period (Figure 1). This pattern of COX-2 in-
hibitor-induced growth inhibition was more prominent in
cells treated with 200 μM concentration than in cells
treated with 100 μM concentration of NS-398, indicating
the both concentration-dependent and time-dependent
inhibition of hepatoma cells.
3.2. COX-2 Inhibitor Induced DNA
Fragmentation and Cell Cycle
Redistribution of Hepatoma Cells
Regardless of concentrations of this compound applied in
the present study, genomic DNA of Hep3B cells was
fragmented as ladder-pattern at 48 h after the treatment
of NS-398, which represented an apoptosis induced by
this selective COX-2 inhibitor, but no definite DNA lad-
der was found after DMSO treatment (Figure 2). Flow
cytometric shifting to sub-G1 phase, indicating an apop-
totic redistribution of cell cycle, was gradually intensi-
fied from 6 to 48 h after the exposure of NS-398, while it
was not observed in control part of DMSO treatment
(Figure 3(a)). Namely, sub-G1 fraction of NS-398-ex-
posed cells increased from 3.0% ± 2.1% at 6 h to 7.0% ±
3.8% at 48 h (100 μM NS-398) and from 3.0% ± 1.9% at
6 h to 18.0% ± 6.4% at 48 h (200 μM NS-398) (Figure
3(b)). This cytometric redistribution was more conspicu-
ous in 200 μM NS-398-treated cells than in cells treated
with 100 μM concentration.
3.3. COX-2 Inhibitor Induced
Mitochondria-Mediated Apoptosis
The activation of caspase 3, a down-stream caspase of
apoptosis pathway, was elicited from 24 to 72 h after
treatment of both concentrations 100 and 200 μM NS-
398 to Hep3B cells in time-dependent manner (Figure
4(a)). The relative expression of activated caspase 3 to
β-actin significantly increased up to 72 h following the
exposure of COX-2 inhibitor to hepatoma cells, which
was 0.96 ± 0.18, 1.18 ± 0.21, and 1.50 ± 0.19 (P < 0.05)
at 24, 48, and 72 h in case of 100 μM NS-398, respec-
tively, and 0.96 ± 0.15, 1.19 ± 0.18, and 1.30 ± 0.20 (P <
0.05) at 24, 48, and 72 h in case of 200 μM NS-398, re-
spectively (Figure 4(b)). In company with this observa-
tion, a cytosolic accumulation of cytochrome c, which
means the release of cytochrome c from mitochondria,
gradually increased in sequences of the same time (Fig-
ure 5(a)). The relative expression of cytosolic fraction of
cytochrome c to β-actin significantly increased up to 72 h
following the exposure of COX-2 inhibitor to hepatoma
cells, which was 0.17 ± 0.14, 0.28 ± 0.19, and 0.31 ±
0.21 (P < 0.05) at 24, 48, and 72 h in case of 100 μM NS-
398, respectively, and 0.12 ± 0.11, 0.18 ± 0.15, and 0.31
± 0.16 (P < 0.05) at 24, 48, and 72 h in case of 200 μM
NS-398, respectively (Figure 5(b)). It may be suggested
that NS-398, selective COX-2 inhibitor, engages a cas-
pase-dependent, mitochondria-mediated intrinsic apoptosis
100 μM
200 μM
0 h24 h 48 h 72 h
Figure 1. Microscopic morphology of NS-398-treated cells.
NS-398 induced a progressive decrease in cell number from 24
h to 48 and 72 h after treatment of NS-398, indicating a growth
inhibition of hepatoma cells with the both concentration-de-
pendent and time-dependent manners.
NS-398, μM
Figure 2. DNA fragmentation
gel analysis. Ladderpatterned
DNA fragmentations of Hep3B
cells were noted at 48 h after
treatment of NS-398 in con-
centrations of 10, 100, 200 μM,
while no definite DNA ladder
as found in DMSO control. w
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I. H. Song et al. / Advances in Bioscience and Biotechnology 3 (2012) 649-656
Copyright © 2012 SciRes.
Figure 3. Cell cycle distribution by flow cytometry. Flow cytometric redistribution of cell
cycle was gradually headed toward sub-G1 phase in time sequences from 6 h to 24 h and 48
h after exposure of NS-398, while it was not observed during same periods in control part of
DMSO treatment (a) and (b). This cytometric redistribution to sub-G1 phase was more nota-
ble in 200 μM NS-398-treated cells than in cells treated with 100 μM concentration.
signaling pathway in HCC cells.
The greater parts of HCC are usually beyond the thera-
peutic indications of locoregionally curative measures,
allowing many clinical researchers to keep attempting to
excavate the therapeutic targets with its corresponding
therapeutic agents in clinical studies. Although HCC has
appeared to be chemoresistant in the response rate and to
show no or minimal survival benefit in meta-analysis for
the results of randomized controlled trials of systemic
chemotherapy [24], extensive efforts for further im-
provement of clinical outcome in this liver cancer are
ongoing under intensive investigations.
In malignant tumors, COX-2 is one of the therapeutic
targets, which has been comprehensively studied around
the world. Up to date, there have been several preclinical
studies in vitro that up-regulation of COX-2 was known
to reduce the rate of apoptosis, to promote angiogenesis
and to increase the invasiveness of tumor cells [25-28].
Furthermore, selective COX-2 inhibition was reported to
elicit an antineoplastic effect on HCC cells, to prevent
the resistance to apoptosis as well as to suppress the
growth of human HCC implants in vivo study using a
nude mice [12,13,18,29]. A series of epidemiologic stud-
ies have revealed that non-steroidal anti-inflammatory
drugs and aspirin could reduce the relative risk of death
by colon cancer [30,31]. A couple of COX-2 selective
drugs is also known to have the therapeutic potential to
decrease the number and size of colonic polyps in pa-
tients with familial adenomatous polyposis [32-34], re-
sulting in the advent of celecoxib approved by US Food
and Drug Administration for chemoadjuvant therapy in
I. H. Song et al. / Advances in Bioscience and Biotechnology 3 (2012) 649-656 653
Caspase 3
Activat ed
caspase 3
24 h 48 h 72 h
100 μM 200 μM
24 h 48 h 72 h
24 h 48 h 72 h
100 μM 200 μM
24 h 48 h 72 h
(Caspase 3/β-actin)
Caspase 3Activated caspase 3
Figure 4. Caspase activity after treatment of NS-398. (a) The
activity of caspase 3, a down-stream caspase of apoptosis, was
evaluated by Western blot analysis; (b) The expression of
cleaved form (19 kDa) of caspase 3 gradually increased on time
sequence from 24 h to 72 h after both concentrations 100 and
200 μM of NS-398 treatment to Hep3B cells in time-dependent
manner. It may be indicated that NS-398 involves a caspase-
dependent apoptosis signaling pathway in HCC cells. *P < 0.05,
compared with the relative expression of activated caspase 3 at
24 h after treatment of NS-398.
these patients. However, the exact mechanisms responsi-
ble for explaining these growth-inhibitory effects of se-
lective COX-2 inhibitor are not clear even to this time.
Based on our knowledges, together with experimental
and clinical evidences mentioned above, that COX-2
might play a pivotal role in tumorigenesis and overex-
pression of COX-2 has been observed in a number of
tumor tissues, including colorectal cancer [9], pancreatic
cancer [35], gastric cancer [27], esophageal cancer [36]
and hepatocellular carcinoma [37], we conducted the
present study that was designed for the clarification of
inhibitory mechanisms and chemotherapeutic impact of
NS-398, a selective COX-2 inhibitor, on the growth of
hepatoma cells.
Our results showed that NS-398 definitely suppressed
the growth of Hep3B HCC cells in both concentration-
dependent and time-dependent manner with a resultant
consequence of decreased tumor cell number. Besides the
decrease of cell number, cell morphology was changed to
be microscopically elongated with its significance being
not defined. This growth-inhibitory effect of NS-398 to
hepatoma cells was verified as a result of the induction
of apoptosis by indicating a distinct ladder patterned-
fragmentation of genomic DNA and significant redistri-
Cytochrome c
24 h 48 h 72 h
100 μM 200 μM NS-398
24 h 48 h 72 h
Cytochrome c
Expresion ratio
(Cytochrome c/β-actin)
100 μM 200 μM
Cytochromec (mitochondria)Cytochromec (cytosol)
24 h 48 h 72 h24 h 48 h 72 h
Figure 5. Localization of cytochrome c after treatment of NS-
398. (a) The activity of cytochrome c was evaluated by Western
blot analysis; (b) The activity of cytosolic fraction of cyto-
chrome c (14 kDa) gradually increased on time sequence from
24 h to 72 h, in opposition to that of mitochondrial fraction,
following the treatment of both concentrations 100 and 200 μM
of NS-398, which means the cytoplasmic release of cytochrome
c from mitochondria. It may be indicated that NS-398 engages
a mitochondria-mediated intrinsic apoptosis signaling pathway
in HCC cells. *P < 0.05, compared with the relative expression
of cytosolic fraction of cytochrome c at 24 h after treatment of
bution of cell cycle shifted to sub-G1 phase following
NS-398 treatment. The strength of apoptosis-mediated
growth inhibition of hepatoma cells was proportionally
intensified according to the concentration of COX-2 in-
hibitor, which is allowed to be able to predict the dose-
dependent growth suppression of tumor cell.
Not only NS-398 but other COX-2 inhibitors such as
nimesulide [13], CAY 10404 [13,17,38], celecoxib [16,
18], 2,5-dimethyl-celecoxib [39], meloxicam [12], JTE-
522 [40], sulindac [12] and indomethacin [13], have been
introduced to the preclinical and clinical investigations
designed for the identification of its growth-inhibitory
mechanisms in tumorous conditions. Until now, the regu-
latory mechanisms of COX-2 inhibitors on the growth of
tumor cells have appeared to depend on the various con-
ditions, namely, a kind of COX-2 inhibitor compounds, a
selectivity of COX-2 inhibition, a type of tumor cells,
whether or not COX-2 expression in tumor cells, and an
inhibitory dominancy of COX-2 inhibitor to COX-2 ac-
tivity. Especially, The pattern of NS-398-induced growth
suppression is thought to be variable according to the
type of tumor cells: COX-2 expressing tumor cell lines
such as GKCI-4 as well as Hep3B and COX-2 nonex-
pressing cell line such as HepG2 and PLC/PRE/5 [41].
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I. H. Song et al. / Advances in Bioscience and Biotechnology 3 (2012) 649-656
The antitumor effects of NS-398 COX-2 inhibitor in
HCC cells have been reported to be performed through
the inhibitory signals via apoptosis, necrosis, or cell cy-
cle arrest [19,40-43], however, most COX-2 inhibitors
are generally accepted to be involved in the activation of
apoptosis pathways which are advanced through the
death receptor-mediated, mitochondria-mediated, or both
signaling. There are several death receptors such as CD
95 (Fas receptor), tumor necrosis factor (TNF)-R, TNF-
related apoptosis-inducing ligand (TRAIL)-R1, and
TRAIL-R2, that are known to be triggered by COX-2
inhibition [17,18].
Besides apoptotic activity via death receptors, several
different mechanisms of COX-2 inhibitor relevant to
apoptosis signal were investigated as follows: 1) down-
regulation of myeloid cell leukemia-1 (Mcl-1); 2) de-
creased expression of Bcl-2 antiapoptotic family; 3)
down-regulation of surviving; 4) inhibition of mitogen-
antivated protein (MAP)/extracellular signal-regulated
kinase (ERK) kinase (MEK)/ERK (MEK/ERK) signaling
pathway; 5) reduction of serine/threonine protein kinase
B (Akt) phosphorylation; 6) up-regulation of peroxisome
proliferator-activated receptor (PPAR)-γ protein; 7) in-
creased expression of Fas ligand [18,26,38,40-42,44].
Caspase 3, one of down-stream proteases for the exe-
cution of apoptotic programmed death in any type of
insulted cells, is a final common pathway for the propa-
gation of both extrinsic/death receptor-mediated or in-
trinsic/mitochondria-mediated apoptosis signals [19].
Our study revealed that the enzymatic activation of cas-
pase 3 increased up to 72 h following treatment of
Hep3B cells with NS-398. This finding was accompanied
by a gradual accumulation of cytoplasmic fraction of
cytochrome c up to 72 h after NS-398 treatment in both
concentrations of 100 and 200 μM, suggesting a gradual
release of cytochrome c from mitochondria. Both active-
tion of cleaved form of down-stream caspase and cyto-
plasmic translocation of cytochrome c are a hallmark of
intrinsic aspoptosis signaling pathway.
NS-398, a selective COX-2 inhibitor, induced the growth
inhibition of hepatoma cells through the caspase-de-
pendent, mitochondria-mediated intrinsic apoptosis, pro-
viding a strong insight into the anti-neoplastic effects of
selective COX-2 inhibitors as novel one of therapeutic
agents for hepatocellular carcinoma. In the near future
selective COX-2 inhibitors would be expected to try for
the application to managements of HCC in clinical fields.
The financial support provided by the Institute of Medical Science
Research of Dankook University Medical Center in 2008 for the pre-
sent work is thankfully acknowledged.
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