Carcinogenesis vol.27 no.6 pp.1273–1284, 2006
doi:10.1093/carcin/bgi357
Advance Access publication February 12, 2006
Ripe areca nut extract induces G1 phase arrests and senescence-associated
phenotypes in normal human oral keratinocyte
Ssu-Yi Lu1,†, Kuo-Wei Chang1,2,†, Chung-Ji Liu1,3,
Yu-Hsin Tseng1, Hsuan-Hsuan Lu1, Suz-Ying Lee1
and Shu-Chun Lin1,
1
Institute of Oral Biology, School of Dentistry, National Yang-Ming
University, Taipei, Taiwan, 2Department of Dentistry, Taipei Veterans
General Hospital, Taipei, Taiwan and 3Oral and Maxillofacial Surgery,
Taipei Mackay Memorial Hospital, Taipei, Taiwan
To whom correspondence should be addressed at: Institute of Oral Biology,
School of Dentistry, National Yang-Ming University, No. 155, Li-Nong Street,
Sector 2, Taipei, Taiwan 112. Fax: +8862 28264053;
E-mail: [email protected]
Around 200–600 million Asians chew areca (also called
betel), which contains a mixture of areca nut and other
ingredients. Epidemiological evidences indicated that
areca use is tightly linked to oral carcinogenesis. This
study investigated the effects of ripe areca nut extract
(ANE) on cultured normal human oral keratinocyte
(NHOK). Acute subtoxic ANE treatment inhibited DNA
synthesis and induced cell cycle arrest at G1 phase in
early passage (<4th passage) cells. This was accompanied
by a slight increase in the sub-G1 cellular fraction. O(6)Methylguanine-DNA methyltransferase (MGMT), Hsp27
and p38MAPK was upregulated. p16 and p21 were
remarkably upregulated early and declined afterwards.
In contrast, the increase of dephosphorylated Rb seemed
to be secondary to the episodes of p16 and p21 upregulation. To simulate the chronic areca exposure in vivo, constant ANE treatment in serial NHOK culture was
performed. It resulted in a significant decrease in the
population doubling, increase in senescence-associated
b-galactosidase (SA-b-Gal) and decrease in cell proliferation in NHOK of late passages (4th passage). Induction of
senescence-associated phenotypes, G2/M accumulation and
genomic instability following long-term ANE treatment
were also observed in a low-grade oral carcinoma cell.
ANE-treated NHOK also had a higher nuclear factor-kB
(NF-kB) fraction and a lower cytosolic IkBa level relative
to the control in late passages. Moreover, electrophoretic
mobility shift assay (EMSA) indicated that ANE treatment
shifted the NF-kB complex from high mobility position to
lower mobility position in late-passaged NHOK. ANE
treatment also upregulated IL-6 and cyclooxygenase-2
(COX-2) mRNA expressions in late-passaged NHOK. In
summary, our findings suggest that ANE induces the cell
Abbreviations: ANE, areca nut extract; BrdU, 5-bromo-20 -deoxyuridine;
COX, cyclooxygenase; EMSA, electrophoretic mobility shift assay; ERK,
extracellular signal-regulated protein kinase; IkBa, inhibitory subunit of
NF-kB a subunits; IL-1a, interleukin-1a; IL-6, interleukin-6; MAPK,
mitogen-activated protein kinase; MGMT, O(6)-methylguanine-DNA methyltransferase; NF-kB, nuclear factor kB; NHOK, normal human oral
keratinocyte; OSCC, oral squamous cell carcinoma; PDL, population
doubling level; SA-b-Gal, senescence-associated b-galactosidase; TPA,
12-O-tetradecanoylphorbol-13-acetate.
†
These authors have contributed equally to this work.
#
cycle arrest at G1/S phase and the occurrence of
senescence-associated phenotypes of NHOK. The upregulation of p38MAPK, p16, p21, NF-kB, IL-6 and COX-2 are
likely to participate in the control of these impacts.
Introduction
Around 200–600 million people in South and Southeastern
Asia are engaged in chewing areca (also called betel),
which is a combination of an areca nut, a nut of tropic
palm, and other ingredients (1,2). Areca chewing is epidemically related to the high incidence of oral squamous cell carcinoma (OSCC) and other lesions (3,4). OSCC has been the
third most common malignancy in developing countries, consisting of 50% of all malignancies in some nations of South
Asia (1,2). Evidences also link areca use to the risk of esophageal (5) and hepatic carcinogenesis (6).
Areca nuts contain polyphenols, arecoline, arecaidine and
other alkaloids (3,7). Previously, Sundqvist et al. (8) have
shown that areca nut extract (ANE) is highly cytotoxic and
genotoxic to cultured human oral epithelial cells. ANE was
found to induce the formation of reactive oxygen species
(ROS) in several types of cells (7,9,10). In addition, ANE
can induce cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in OSCC cell lines or primary
normal human oral keratinocyte (NHOK) (10,11). Recently,
areca ingredients have been shown to induce the production of
cytokine interleukin-6 (IL-6) by the underlying extracellular
signal-regulated protein kinase (ERK) stimulation in a malignant keratinocyte (12). However, the impact of ANE on primary NHOK is still obscure.
When normal human cells gradually lose division and enter
a state of permanent growth arrest, it is termed replicative
senescence. Normal cells also exhibit premature senescence
in cultures when treated with various treatments (13). Except
for the absence of telomere erosion, premature senescence is
similar to replicative senescence on many aspects (14). Premature senescence can be induced by a variety of stresses, such
as DNA damage and aberrant mitogenic signaling (13). A
previous report indicated that high dose of ANE could arrest
malignant keratinocyte (9). Nuclear factor-kB (NF-kB) plays a
central role in regulating genes in response to cellular stresses
(15,16). In the inactive state, NF-kB is present in the cytoplasm
as a heterotrimer consisting of p50, p65 and an inhibitory
subunit of NF-kB (IkB), a subunits. In response to activation
signals, the IkBa is phosphorylated, ubiquitinated and
degraded, thus exposing the nuclear localization signals to
p50 and p65. Activation of NF-kB was found to associate
with senescence (17), growth arrest (18,19) and carcinogenesis
(20,21) of keratinocyte. Our previous studies have shown that
ANE treatment can elicit a rapid activation of ERK and c-Jun
N-terminal kinase 1 (JNK1) mitogen-activated protein kinase
The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1273
S.-Y.Lu et al.
(MAPK) as well as NF-kB in malignant oral keratinocytes
(16,22,23). Besides, ANE-modulated NF-kB activation
could be the basis of COX-2 upregulation associated with
areca exposure (16). In this study, chronic low-dose ANE
treatment was imposed on NHOK to perceive the impacts.
We identified that ANE-arrested NHOK in G1 phase and
induced senescence-associated phenotypes in long-term serial
culture. Moreover, upregulation of p16, degradation of IkBa
and activation of NF-kB may be the likely causes.
Materials and methods
Cell culture and reagents
Sampling of gingival tissue from donors who received flap operation to remove
the impacted third molar was approved by an institute review board. NHOK
was dissociated from the tissue and grown in keratinocyte serum free medium
(KSFM ; Invitrogen, MD) with low Ca++ (0.1 mM) (24). In addition, cells
were passaged when 70% confluence was reached to avoid the confluenceinduced senescence. In KSFM, NHOK retained the feature of parabasal cells,
which could be maintained for 5–7 passages (18–24 PD) before they reached
senescence. OC3 cells, a low-grade areca-associated OSCC cell line established in our laboratory, having wild-type p53 sequence, minor genomic alterations and non-tumorigenic phenotype were cultivated with conditions used
previously (23). The preparation of ANE from ripe areca nut was performed
with the protocols established previously (7,10). In brief, ripe areca nuts were
chopped, ground and immersed to obtain the soluble extract. This soluble
fraction was lyophilized and stored at 20 C until use. NHOK and OC3
cells for serial culture experiments were trypsinized and passaged every
3 days. Cells in serial culture were subjected to ANE treatment for the
whole period of time. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and arecoline were purchased from Sigma (St Louise, MO). TPA was dissolved in
100% dimethyl sulfoxide (DMSO) (Sigma) as a stock. NHOK were treated
with 50 ng/ml TPA for 1 or 6 h after the termination of ANE treatment.
Trypan blue exclusion assay and growth curve
NHOKs were seeded on 6-well plates at a concentration of 5 · 104 cells per
well and cultured for 24 h. The assay was performed with the protocols we
established previously (23). For serial cultures, the population doubling level
(PDL) was calculated at each passage with the following equation: PDL ¼
Ln(number of collected vital cells/number of plated cells)/Ln2.
5-bromo-20 -deoxyuridine (BrdU) incorporation assay
Around 5 · 103 cells were seeded on 8-well chamber slides and grown to 60%
confluence. Following the treatment of ANE for 24 h, BrdU (BD Biosciences,
Franklin Lakes, NJ, USA) was added to KSFM to get a final concentration of
10 mM, and the labeling procedure was performed for 2 h. BrdU incorporation
was measured in control and ANE-treated cells following the protocols established previously (25). In each experiment, at least 1000 cells were counted and
compared.
Senescence-associated b-galactosidase (SA-b-Gal) assays
SA-b-Gal assays were performed as described previously (26). In brief, cells
were fixed in 2% formaldehyde/0.2% glutaraldehyde and incubated at 37 C
in a solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactoside
(X-gal) for 24 h as described previously (26).
Combined SA-b-Gal and BrdU labeling assay
The combined SA-b-Gal and BrdU labeling assay was performed on NHOK
and OC3 cells using a protocol modified from previous publication to determine the proportion of SA-b-Gal labeled and proliferating cells (27). Briefly,
following the treatment of ANE for 24 h, BrdU was added to culture medium to
get a final concentration of 10 mM, and the labeling procedure was performed
for 2 h, then washed and stained for SA-b-Gal activity as described above.
Consequently, slides were washed and stained with anti-BrdU-A using BrdU
in situ detection kit (BD Biosciences). Diaminobenzidine (DAB) was used as
chromogen for detecting BrdU labeling. For each determination, at least
1000 cells were used to count the percentage of positive staining cells.
Flow cytometry
Nuclei of ANE-treated cells were collected and stained with propidium iodate,
using the cycle TEST-PLUS DNA-staining kit, according to the manufacturer’s instructions (BD Biosciences). The DNA content of the stained nuclei
was measured on a FACSCalibur flow cytometer, and results were analyzed
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using the CellQuest and ModFit LT2.0 softwares (BD Biosciences). The
sub-G1 fraction was used as a measure of apoptotic percentage.
Western blot analysis
Cells were harvested and the cytoplasm and the nucleus were fractionated
using NucBuster Protein Extraction Kit (Novagen, Darmstadt, Germany).
Fifty micrograms of proteins from the whole cell lysate, cytosolic fraction or
nuclear extract were resolved by electrophoresis on a 12.5% denaturing polyacrylamide gel. The subsequent procedures were performed following the
standard protocols (23). The primary antibodies used include the following:
anti-actin Ab (no. MAB1501; 1 : 5000, Chemicon, Temecula, CA), antiGAPDH Ab (no. MAB374; 1 : 5000, Chemicon), anti-HO-1 Ab (no.
SC-10789; 1 : 500, Santa Cruz Biotech, Santa Cruz, CA), anti-Hsp27 Ab
(MS-101; 1 : 500, Lab Vision Fremont, CA), anti-Hsp70 Ab (no. 554243;
1 : 1000, Lab Vision), anti-IkBa l Ab (c-21, no. SC-371; 1 : 250, Santa Cruz
Biotech.), anti-p21 Ab (EA10, no. OP64; 1 : 250, Oncogene, Darmstadt,
Germany), anti-p38 Ab (no. SC-7149; 1 : 2000, Santa Cruz Biotech.), antiphospho-p38 Ab (no. SC-7975-R; 1 : 250, Santa Cruz Biotech.), anti-p53 Ab
(DO7; 1 : 1000, Novocastra, Newcastle, UK), anti-phospho-Ser15-p53 Ab
(no. 9284; 1 : 500, Cell Signaling, Beverly, MA), anti-Rad51 (no. 556453;
1 : 1000, BD Biosciences), anti-Rb Ab (1F8 1, no. MS-107; 1 : 250, Neo
Markers, Fremont, CA), anti-MGMT Ab (no. 557045; 1 : 1000, BD Biosciences), anti-MSH2 Ab (no. 556349; 1 : 1000, BD Biosciences), anti-NF-kB
p65 Ab (no. SC-109; 1 : 500, Santa Cruz Biotech.), anti-NFkB p50 Ab (H-119,
no. SC-7178; 1 : 500, Santa Cruz Biotech.) and anti-XPA Ab (no. SC-28353; 1 :
1000, Santa Cruz Biotech.). Horseradish peroxidase (HRP)-conjugated antimouse (Amersham, Piscataway, NJ) or anti-rabbit (Santa Cruz Biotech.)
secondary antibody was used.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were subjected to EMSA analysis. EMSA was performed as
described previously (16,23). Other than anti-p65 and p50, the antibodies used
for the supershift analysis were anti-p52, anti-RelB (p68) and anti-cRel (p75)
(polyclonal antibodies from Santa Cruz Biotech.). The radioactive bands from
the dried gels were quantified by a phosphoimager (Amersham).
RT–PCR
Total RNA purification, cDNA synthesis and RT–PCR reaction followed protocols established previously (24). The primer sequences will be provided upon
request. Amplicons were resolved on a 2% agarose gel, visualized by an
imaging system and quantified (Viber Lourmat, Marne La Valle, France).
The signal of tested genes was normalized with the mRNA expression of
GAPDH.
Statistics
Paired t-test and one-way ANOVA were used for statistical analysis. P < 0.05
was considered to have a statistically significant difference.
Results
ANE inhibits cell viability and proliferation
To investigate the effect of ANE on growth of oral keratinocytes, NHOKs in early passages (3rd passage) were exposed
to 0, 1, 2.5, 5, 7.5 and 10 mg/ml ANE and harvested at 24, 48
and 72 h. There was a dose-dependent reduction in number of
NHOKs following the ANE treatment at different time period
(not shown). Treatment with 5 mg/ml ANE resulted in cytotoxicity. Therefore, treatments with 2.5 and 5 mg/ml ANE,
which caused cytoinhibition and cytostasis, respectively,
were used to evaluate the impacts of ANE on NHOKs. To
test if abnormal proliferation is responsible for the reduced
cell viability, BrdU was added to label cells in S phase. Exposure of NHOK cells to 2.5 and 5 mg/ml ANE for 24 h resulted in
an inhibition of cell proliferation in a dose-dependent manner
with an average of 20 and 43% reductions in BrdU-labeled
cells relative to the control cells, respectively (not shown). The
degrees of reduction in proliferation are similar to those in cell
viability, suggesting that the inhibition of ANE on NHOK
viability is mainly exerted by inhibiting the cell proliferation.
Areca induces cell cycle arrest and senescence
ANE induces G1 arrest and apoptosis
To examine how ANE affects the proliferation of NHOK,
fluorescence-activated cell sorter (FACS) was performed to
analyze the cell cycle status modulated by 0–5 mg/ml ANE
treatment. At 24 h, a progressive decrease in S fraction in
attached NHOK from 20 to 11% was noted upon treatment
with increasing dosage of ANE (Figure 1A). In contrast, the G1
fraction was increased from 66 to 74%. No significant difference in G2/M phase was noted. The concomitant decrease in
S fraction and increase in G1 phase were consistent with G1
arrest. The G1 arrest became more prominent with the ANE
treatment at 48 (Figure 1B) and 72 h (Figure 1C). Trypan blue
dye exclusion assay designated that all floating NHOK following ANE treatment were non-vital. The sub-G1 fraction of total
NHOK was also evaluated by FACS. It showed a dose- and
time-dependent increase in sub-G1 fraction in NHOK following ANE treatment (not shown). There were 1–3% and 5–7%
NHOK that were apoptotic following 2.5 and 5 mg/ml ANE
treatment, respectively. Apoptosis also underlies the reduction
of cell viability following ANE treatment. In this study, there
was 0.25 mM arecoline presenting in 10 mg/ml ANE preparation (16). NHOK treated with 0.25 or 40 mM (160-fold)
arecoline for 24 h did not cause the inhibition of proliferation
or apoptosis.
ANE modulates repair enzyme expression and
p38MAPK activation
ANE is an inducer for DNA damage (7) and cellular signals
(16). The expression of representative repair enzymes including APE1 (for oxidative DNA damage repair), O(6)-methylguanine-DNA methyltransferase (MGMT; for direct reversal
of base damage), MSH2 (for mismatch repair), Rad51 (for
homologous recombination of strand break) and XPA (for
nucleotide excision repair) (28); stress response proteins
including Hsp27 and Hsp70 that are associated with tumorigenesis (29) as well as HO-1 (1); and signal trnasducers
p38MAPK and ERK (16,30–32) was studied. The expression
of MSH2 increased in the first 2–12 h. It was then suppressed in
later period following 5 mg/ml ANE treatment (Figure 2A). A
sustained increase of MGMT and activation of p38MAPK,
A
B
reflected by phosphorylation of p38MAPK (pp38MAPK), in
NHOK was found following 5 mg/ml ANE treatment
(Figure 2B and C). However, ANE-elicited ERK activation
occurred late at 24 h. Hsp27 increased within 24 or 48 h
following treatment, whereas it was not the case for Hsp70
(Figure 2A and B). Slight increase of APE1 for 40% was noted
in the first 2–6 h. With no change in expression level or absence
of expression in control or ANE-treated NHOK being detected,
the involvements of other proteins for pathogenetic changes
have been excluded.
ANE affects expression of cell cycle regulators
To investigate the mechanism of G1 arrest induced by ANE, we
analyzed changes in p53, p21(Waf1/Cip1/Sdi1/CAP20),
p16(INK4a) and pRb, which regulate the process of G1-S
transition (33). A pilot study showed no remarkable change
in the expression level of aforementioned regulators in control
NHOK at different time points. Figure 3A and C show the
results of a representative western blot with 2.5 and 5 mg/ml
ANE treatment, respectively. The expressions of p53, p21 and
p16 were quantitatively normalized with actin. The dephosphorylated Rb fraction is indicated as the ratio of pRb versus
ppRb (Figure 3B and D). The most prominent change among
all the tested proteins was observed in p16, which was upregulated by 7–30-fold between 2 and 48 h. It was nearly
decreased to the baseline level at 72 h. The 2.5 mg/ml ANE
treatment only slightly upregulated p21 for <2-fold between
2–48 h, whereas 5 mg/ml ANE persistently upregulated p21 by
3–6 fold. There was a slight increase of p53 following the
ANE treatment, not exceeding 2-fold. No apparent change in
phosphorylation of p53 at Ser-15 was noted in the first 24 h.
However, phosphorylated p53 (pp53) level decreased below
the baseline at 72 h. We have observed the maintenance of
dephosphorylated Rb fraction between 2 and 24 h. It was
followed by a reduction of ppRb and an increase of dephosphorylated Rb fraction by 2–5-fold at 48 and 72 h. This
increase appeared secondary to the increase of p16 or p21
between 2 and 24 h. The reduction of pp53 and ppRb at 48
and 72 h could be very likely caused by the upregulation of
unknown phosphatases during this period.
C
Fig. 1. Effect of ANE on arresting the cell cycle of NHOK at G1 phase. (A) 24 h, (B) 48 h and (C) 72 h. Cell cycle data in graphs represent the relative
percentage of cells in G1 and G2/M (left axis) or S (right axis) phases in the cell cycle. Values are expressed as means ± SE from triplicate experiments.
The figure shown is a representative experiment of three independent experiments.
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S.-Y.Lu et al.
ANE induced senescence-associated phenotypes
A previous study has shown the fibroblastoid morphological
changes of NHOK induced by ANE (22). This was observed in
our experiments, despite the fact that such cellular fraction
is much lower since the treatment dosage of ANE was
lower in this study. The serial culture of NHOK simulates
the in vivo lifespan of oral keratinocytes (24). To examine
whether ANE could reproduce a proliferation blockage similar
to that of senescent cells, serial NHOK cultures were grown.
A
Phase-contrast microscopy was used to evaluate the changes
associated with senescence, including enlargement, elongation
and flattening in cell morphology, with cytoplasmic vacuoles
and occasional multinuclei. The late-passaged control cells
exhibited more senescence-associated morphologies than
the early passaged control cells (not shown). In the same passage, ANE-treated NHOKs also exhibited more senescenceassociated morphologies than the controls. We measured the
growth curves of NHOK in the serial culture. NHOK reached
B
C
Fig. 2. Western blot analysis of representative repair enzymes, stress response proteins and signal transducers in NHOK following ANE treatment. (A) and
(B) Protein expression of MSH2, MGMT, Hsp27 and Hsp70 following 2.5 and 5 mg/ml ANE treatment for 0–72 h, respectively. (C) p38MAPK expression
following 5 mg/ml ANE treatment for 0–72 h. Protein level was normalized with GAPDH or actin. Quantification values are shown below the illustration.
A
C
B
D
Fig. 3. Western blot analysis of cell cycle regulatory proteins p53, pp53, p16, p21 and Rb in NHOK, following ANE treatment. (A) and (C) Representative
blots of NHOKs treated with 2.5 mg/ml and 5 mg/ml ANE, respectively, for 0–72 h. (B) and (D) Quantified protein level changes following 2.5 and 5 mg/ml
ANE treatment, respectively. Bands for Rb are designated as ppRb, (hyperphosphorylated protein, top band) and pRb (hypophosphorylated protein, bottom
band). Protein level was normalized with actin. Values are expressed as means ± SE from two experiments.
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Areca induces cell cycle arrest and senescence
replicative senescence at PDL > 22, as indicated by the appearance of growth plateau. In comparison to the controls, ANEtreated NHOKs exhibited 7 or 12 reduction in PDL to reach
replicative senescence, which was at Days 35 or 20, respectively (Figure 4A). The results suggested that ANE could
modulate the senescence-associated phenotypes of NHOK.
In addition, there was a significant increase in the SA-b-Gal
staining cells in late-passaged NHOKs, but not the earlier ones.
Treatment of ANE further increased the percentage of NHOK
exhibiting SA-b-Gal staining (Figure 4B). The progressive
increase of SA-b-Gal staining cells and decrease of BrdU
labeling cells in control NHOK during serial passage and in
NHOK following ANE treatment indicating the induction of
senescence-associated phenotypes was confirmed by combined SA-b-Gal staining and decrease of BrdU labeling
assay (Figure 5). In a representative NHOK with 2.5 mg/ml
ANE treatment for five continuous passages, flow cytometry
showed that the fraction of sub-G1, G1, S and G2/M phase was
2, 73, 12 and 12% in control cells, respectively, whereas it was
6, 76, 3 and 12%, respectively, in treated cells (not shown). In
addition, morphological changes of cells also substantiated the
occurrence of senescence-associated phenotypes. There was
no NHOK exhibiting both SA-b-Gal staining and BrdU
labeling indicating that cells positive for SA-b-Gal staining
were not proliferating (Figure 5A).
ANE induced senescence-associated phenotypes and
aneuploidity in OC3 cells
The progressive induction of SA-b-Gal activity was also
observed in OC3 OSCC cell line treated with non-toxic
dosages of ANE for 20 continuous passages (Figure 6A).
The induction was particularly eminent following 20 mg/ml
treatment for >10 passages. It was observed that the percentage
of BrdU labeling proliferating cells decreased gradually in
OC3 cells following 20 mg/ml ANE treatment (Figure 6B).
Furthermore, OC3 cells treated with 2.5 and 5 mg/ml for 32
A
or 60 continuous passages exhibited a time- and dosedependent increase in G2/M and hyperdiploid (>4N) cellular
fractions (Figure 7). The induction of G2/M and aneuploidity
was particularly eminent with 5 mg/ml ANE treatment for
60 passages (Figure 7F). In a representative NHOK with
2.5 mg/ml treatment for five continuous passages, 1% control
cells and 2% treated cells exhibited hyperdiploidity. It is
unlikely that ANE can induce aneuploidity in NHOK by
such treatment owing to NHOK’s limited life span, which
might render the insufficiency in treatment duration for
induction of abnormalities.
ANE affects cellular localization of NF-kB and
expression of IkBa
NF-kB has been shown as an important signaling element
involved in the senescence of keratinocyte (17). To evaluate
if the ANE-induced senescence-associated phenotypes are
related to changes in p65, p50 and IkBa, western blot was
performed on cytosolic fraction and nuclear extract of a serially cultivated NHOK following the 2.5 mg/ml ANE treatment
(Figure 8A). Since p65 and p50 were the major NF-kB subunits in NHOK (see Figure 9B for details) and their expression
levels were fairly consistent, the summation of normalized p65
and p50 proteins may represent the protein expression of
NF-kB. The protein changes in different passages were summarized in Figure 8B. The total expression of NF-kB protein,
including that in nucleus and cytosol, was progressively but
slightly increased in ANE-treated cells compared with the
controls. However, there was a rapid increase in the level of
nuclear NF-kB at Passage 2, followed by a reduction at Passage 3. The cytosolic IkBa was exactly opposite to the status
of NF-kB in the early passaged NHOK. This is interpreted
as the degradation of cytosolic IkBa, conferring the nuclear
NF-kB translocation at Passage 2 following the ANE treatment; and an auto-regulatory feedback of IkBa upregulation
at Passage 3. In late passages, nuclear NF-kB was kept
B
Fig. 4. Occurrence of senescence-associated phenotypes in NHOK following ANE treatment. (A) PDL. Control and ANE-treated NHOK were passaged at
Days 10, 14, 18, 20, 29, 36 and 46. The vital cells were counted. Note the significant reduction in PDL in treated NHOK, other than controls, to reach a
plateau growth phase. (B) Quantification of SA-b-Gal activity. It showed the dose-dependent increase in cells exhibiting SA-b-Gal activity following ANE
treatment, particularly in late-passaged NHOK. Values are expressed as means ± SE from triplicate experiments. , P < 0.05; , P < 0.01; , P < 0.001.
P, passage.
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S.-Y.Lu et al.
B
A
C
Fig. 5. Concomitant increase of SA-b-Gal activity and decrease of proliferation in NHOK cells following ANE treatment. (A) Representative results of
NHOK in Passage 1, 3 and 5. The brown color in nuclei indicates the BrdU labeling. The perinuclear blue precipitate designates SA-b-Gal staining.
Senescence-associated phenotypes including SA-b-Gal activity, elongated and enlargement of cells and appearance of cytoplasmic vacuoles were evidently
seen in late-passaged NHOK (P5) and ANE-treated NHOK, particularly for those treated with 5 mg/ml ANE. For late-passaged NHOK, slight trapping of
chromogen in the periphery of cytoplasm was also present most likely because of the surface irregularity, since it also presents in negative control settings
(not shown). However, this did not affect the quantification since only a nuclear labeling was counted (·200). (B) and (C) Quantification of SA-b-Gal
staining and BrdU labeling, respectively. Increase of SA-b-Gal staining and decrease of BrdU labeling proliferating cells was seen in serially passaged
NHOK, and NHOK treated with ANE. Values are expressed as means ± SE from duplicate or triplicate experiments. , P < 0.05; , P < 0.01;
, P < 0.001. P, passage.
A
B
Fig. 6. Concomitant increase of SA-b-Gal activity and decrease of
proliferation in OC3 cells following ANE treatment. OC3 cells were treated
with 10 or 20 mg/ml ANE for 20 consecutive passages. (A) Quantification
of SA-b-Gal staining exhibits a progressive increase in SA-b-Gal activity
in serially passaged cells following treatment. (B) Quantification of BrdU
labeling cells exhibits a progressive decrease in proliferation in serially
passaged cells following 20 mg/ml ANE treatment. Values are expressed
as means ± SE from duplicate or triplicate experiments. , P < 0.05;
, P < 0.01; , P < 0.001. P, passage.
1278
upregulated following the ANE treatment, whereas the cytosolic IkBa was downregulated and completely diminished at the
terminal passage (Figure 8B).
ANE induces NF-kB activation
To test if the changes described above reflect activation of the
NF-kB signaling pathway, activation of NF-kB by 2.5 mg/ml
ANE treatment in serial passages was assayed by EMSA.
Figure 9A shows the presence of two major EMSA complexes,
which bound to the NF-kB probe in nuclear extracts of the
control (lanes 2, 4, 6 and 8), but these complexes were not seen
in the negative control (lane 1). Besides, the expression of the
lower (L) complex was generally much stronger than the upper
(U) complex. The NF-kB binding activity was slightly upregulated in Passages 2–5, following the ANE treatment as reflected by the summation of U complex and L complex
(Figure 9C). However, the U complex was remarkably upregulated following the ANE treatment (Figure 9A, lanes 2–9;
Figure 9C). In the late-passaged NHOK, the ratio of U : L
complex was increased by 4–5-fold (Figure 9C). The specificity of NF-kB binding activities was confirmed by the competitive inhibition with an excessive unlabeled probe
(Figure 9A, lanes 10 and 11) and the supershift of NF-kB
binding complex with anti-p65 and anti-p50 antibodies
(Figure 9A, lanes 12 and 13). To identify the subunits responsible for ANE-modulated NF-kB activation, EMSA was performed after preincubating another treated NHOK (at Passage
4) with the whole panel of antibodies against NF-kB subunits
(Figure 9B). Since nearly all NF-kB signals were supershifted
by antibodies of p65 or p50, but not p52, p68 and p75, p65
and p50 were considered as the major NF-kB subunits of
ANE-treated NHOK. The data supported the detection of
p65 and p50 subunits by western blotting in the previous
experiment (Figure 8). Although both U and L complexes
Areca induces cell cycle arrest and senescence
A
B
C
D
Fig. 7. Long-term effects of ANE on OC3 cells. Early passage OC3 cells were continuously treated with 2.5 or 5 mg/ml ANE for 32 or 60 passages.
(A and B) Controls, no treatment; (C and D) 2.5 mg/ml ANE treatment; (E and F) 5 mg/ml ANE treatment. (A, C and E) 32 passages; (B, D and F) 60
passages. Progressive increase of G2/M and hyperdiploid (>4N) cellular fractions was noted following the increase of duration and ANE dosage for ANE
treatment. Values are expressed as means ± SE from triplicate experiments. The figure shown is a representative experiment of three independent
experiments.
A
B
Fig. 8. Western blotting of NF-kB and IkBa in serial NHOK culture. (A) NF-kB p65 and p50 and I-kBa protein expression in the cytosolic fraction (CF)
and nuclear extract (NE) treated with 2.5 mg/ml ANE for serial passages as indicated. Protein loading was normalized with GAPDH. (B) Quantification of
ANE-mediated NF-kB activation. Total NF-kB represented the summation of cytosolic and nuclear NF-kB. Nuclear NF-kB represented nuclear p65 plus
nuclear p50. There were two episodes of NF-kB activation, early passage and late passage, and there was refractory occurrence of IkBa upregulation during
early passage. P, passage.
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S.-Y.Lu et al.
A
B
C
Fig. 9. ANE-mediated NF-kB activation. Each nuclear extract was incubated with 32P-labeled probe containing NF-kB in the absence or presence of an
unlabeled competitor DNA. (A) A serial passage of NHOK from P2 to P5. Lane 1, free probe control. Lanes 2–9, controls and ANE-treated cells from P2 to
P5. Lanes 10 and 11 contained 100-fold excess of unlabeled probe as competitor DNA to compete the reaction in lanes 1 and 9. Lane 12 and 13, nuclear
extract of lane 9 was preincubated with the antibodies against either p65 or p50, followed by standard EMSA. Com., competition. Supershifts are indicated
by SS. There were two NF-kB complexes at different mobility positions. ANE mediated the shift of L complex to U complex in late-passaged cells.
(B) Another NHOK at P4 was used to confirm the ANE-mediated shift in complex and the subunits of activated NF-kB. Lanes 1 and 2, NF-kB basal activity
and that following ANE treatment, respectively. Lanes 3 and 4 contained 100-fold excess of unlabeled probe as competitor DNA to compete the reaction in
lanes 1 and 2. Lanes 5–9, nuclear extract of lane 2 was preincubated with the antibodies against NF-kB p65, p50, p52, p68 and p75, respectively, followed
by standard EMSA. p65 and p50 account for the major NF-kB subunits. There was no conspicuous supershift of p52, p68 or p75. NS, non-specific band.
(C) Quantification of NF-kB activity with (A). Treatment of ANE slightly activates NF-kB, whereas it profoundly upregulated NF-kB U complex in
late-passaged NHOK. P, passage.
were composed of p65 and p50 subunits, U complex might be
defective in transcriptional activation because IkBa was not
induced in these cells (Figure 8).
Regulation of IL-6, COX-2 and c-fos in ANE-treated NHOK
IL-6 and COX-2 are known to be induced by ANE. Interleukin1a (IL-1a) is an important cytokine secreted by keratinocytes
(34,35). Proto-oncogene c-fos expression is frequently upregulated in response to various stimuli, and c-myc expression is
high in proliferating cells. In addition, IL-6, COX-2 and IL-la
were important for tumorigenesis and were NF-kB-inducible
in keratinocyte (16,36,37). Therefore, we studied the potential
modulation of IL-6, COX-2, IL-1a, c-myc and c-fos in serial
passage of NHOK. Figure 10A shows a representative mRNA
expression in a serially cultured NHOK following constant
2.5 mg/ml ANE treatment. Figure 10B illustrates the mRNA
expression profile of theses genes in two serial cultures of
NHOK. There was a gradual decrease in the IL-6 and
COX-2 mRNA expressions in serial cultures of NHOK.
However, ANE mediated the upregulation in their mRNA
expressions in comparison with controls. The upregulation
became rather prominent in the late passages. There was no
change in IL-1a mRNA expression level. A progressive
increase in c-fos mRNA expression was observed in controls;
1280
it was downregulated by ANE treatment in late-passaged
NHOK. A remarkable downregulation in c-myc mRNA
expression was noted in ANE-treated NHOK at the terminal
passage.
TPA is a well-known tumor promoter; it evokes higher
increases in IL-6 and c-fos expressions of photoaged normal
human epidermal keratinocyte than those that are younger
(35). We observed that, under the process of senescence,
the late-passaged control or ANE-treated NHOK still maintained the ability for TPA induction, as indicated by the regulation of mRNA expressions of IL-6, COX-2 and c-fos
(Figure 11). The data suggested that the occurrence of
ANE-mediated senescence-associated process on NHOK
could maintain the ability on promoter induction.
Discussion
Chronic areca chewing has been strongly linked to oral pathogenesis. Evidences indicated that ANE was a genotoxic substance (7–9,24). A low concentration of ANE was used in this
study to dissect the phenotypic impacts on target NHOKs. We
identified that ANE could mediate the inhibition of cell growth.
Furthermore, G1 arrest seems to be underlying such cytoinhibition. In cells, G1 phase of the cell cycle is responsible for
Areca induces cell cycle arrest and senescence
A
B
Fig. 10. mRNA expressions of IL-6, COX-2, IL-1a, c-fos and c-myc genes in serial NHOK culture. (A) A representative NHOK showing the downregulation
of IL-6 and COX-2, as well as the upregulation of c-fos during the replicative senescence process, whereas, in ANE-mediated premature senescence, mRNA
expression profile of these genes was reverted. No significant change was observed in IL-1a and c-myc genes. (B) Quantification of mRNA expression in
ANE-treated NHOKs relative to control. Remarkable upregulation of IL-6 and COX-2 as well as downregulation in c-fos were noted in ANE-treated NHOKs,
especially in late-passaged cells. Values are expressed as means ± SE from two distinctive cells.
growth factor-dependent signals (33). Dysregulation of G1
phase is advantageous to tumorigenic cell growth; as such,
G1 regulation is frequently disrupted in cancer through the
impairments of regulators. Cultivated NHOK maintained proliferative characteristics of parabasal cells and had a minimal
p16 expression. Kang et al. (38) have demonstrated that the
onset of limited NHOK growth was accompanied with a
remarkable induction of p16. p16 is an inhibitor of Cdk4/6,
the cyclin D kinases that initiate the phosphorylation of the Rb,
and arrests cells in G1 phase. We identified the rapid and
drastic p16 upregulation following the ANE treatment.
Since this upregulation occurred early and sustained through
48 h, it is probably a major reason for the G1 arrest. The
mechanisms by which the cellular responses are triggered
are not understood. It is postulated that ROS elicited by
ANE could be an important trigger (7).
p21 is involved in the assembly of cyclin D–Cdk4/6 and
plays a key role in cell cycle arrest at the G1 checkpoint in
response to DNA damages. Following an ANE treatment for
cytostasis, p21 is rapidly and sustainedly upregulated other
than p16. Thereby, the upregulation of p21 could also be an
important element underlying the G1 arrest. DNA damages can
activate p53 to protect cells by inducing target genes. The
molecules activated by p53 induce apoptosis, cell arrest and
DNA repair to conserve the genome (33). However, the activation of p53, reflected by the amount of protein or the phosphorylation level of Ser-15, was not a case of ANE treatment.
Therefore, ANE damage may bypass the surveillances of p53
and undergo genetic pathogenesis predisposing tumorigenesis.
Our data also suggested that the activation of p21 by ANE is
p53-independent. Cyclin D–Cdk4/6 phosphorylates Rb, which
drives the cell cycle through late G1 phase. The late increase of
dephosphorylated Rb at 48 h could be secondary to the p16 or
p21 upregulation prior to 48 h. Our study suggested that p16
and p21 are important in modulating cell arrest for the protection of areca-mediated cell damage in NHOK. The frequent
impairments of such genes in areca-associated OSCC might
substantiate the fact that keratinocyte lacking such genomic
protection may confer tumorigenic propensity (39,40). For
cytostatic ANE treatment (5 mg/ml at 24 h), >40% NHOK
was arrested in the proliferation stage, whereas only 5%
NHOK cells underwent apoptosis. This could be derived
from the consequent effects of extensive cell arrest or mild
cytotoxicity exerted by ANE (7,41).
In response to DNA damages, activation of repair or stress
response systems might trigger cell cycle arrest or apoptosis
(28). Previous study has specified that the oxidative DNA
damage resulted from ANE (7). In NHOK, a slight increase
in APE1 expression for a short period that might respond to
oxidative DNA damage was found following ANE treatment.
Moreover, more sustained increase of MGMT, a repair enzyme
for removal of alkylating DNA adduct, in response to higher
dose ANE was noted. Whether this implicates that ANE
ingredients might also induce alkylation lesion deserves further characterization. Interestingly, MSH2 expression
increased during the first 12 h of treatment, then remarkably
decreased at 48 h and 72 h. Deficiency in MSH2 is prone to
mutator phenotypes and genesis of colorectal carcinoma (42).
It is possible that a higher dose and longer duration of ANE
exposure could trigger more prominent MSH2 suppression that
warrants a susceptibility of oral carcinogenesis. Implication of
Hsp27 in tumor progression and resistance to chemotherapy
1281
S.-Y.Lu et al.
A
B
C
Fig. 11. IL-6, COX-2 and c-fos mRNA expressions in ANE-mediated premature senescence in response to TPA stimulation. (A) NHOK at P2–P7 treated
with TPA for 1 or 6 h resulted in increases in IL-6, COX-2 and c-fos mRNA expressions. NHOK with 2.5 mg/ml ANE treatment resulted in upregulation of
IL-6 and COX-2 and downregulation of c-fos. ANE-treated NHOK followed by TPA treatment enhanced ANE-mediated IL-6 and COX-2 upregulation, as
well as the reversal of c-fos downregulation. (B) and (C) Quantification of the TPA-induced mRNA expression in ANE-treated NHOK relative to controls.
(B) 1 h TPA treatment. (C) 6 h TPA treatment. P, passage.
further substantiates the fact that the upregulation of Hsp27
evoked by ANE could be beneficial for neoplastic progression
(29). Unlike the vast majority of MAPKs that elicit cell survival, p38MAPK has been identified for negative regulation of
cell survival and growth (43). Such process implies the activation of p53, and stabilizes of p21 or p16 that leads to cell cycle
arrest or premature senescence (30–32). We identified a rather
high and sustained activation of p38MAPK in NHOK following ANE treatment, which could be a potential element underlying ANE-induced cell cycle arrest and activation of negative
regulators.
NHOK enters the terminal proliferation arrest state of senescence following 20–24 PDL in the defined medium (38).
We pursued the pathological role of ANE in the irreversible
growth of NHOK in serial culture and identified the increase in
SA-b-Gal level and decrease in BrdU labeling of ANE-treated
cells. Besides, ANE treatment reduced the PDL of NHOK in a
dose-dependent manner. The effects of ANE for chronic
exposure in inducing senescence-associated phenotypes
were also observed in OC3 cells, a cell with unlimited lifespan.
Intriguingly, with low-dose ANE treatment for long duration,
OC3 cells gradually exhibited accumulation of G2/M fraction
and prominent genetic instability reflected by the remarkable genesis of hyperdiploid cells. The process simulates the
conversion from a low-grade malignancy to a high-grade
malignancy. It is not known whether aneuploid cells arose
1282
from cells resistant to ANE exposure or emerged from
senescent population. For the increase in G2/M fraction,
further dissection to elucidate a G2 arrest or increase of
mitosis is necessary. Since the increase of G2/M and generation
of aneuploidity occurred concordantly, we postulate that
long-term ANE treatment may impair G2/M check, leaving
unrepaired cells for mitosis that finally resulted in mitotic
abnormalities or aneuploidity (44). For this instance, our
findings might propose important evidences supporting the
roles of ANE in oral carcinogenesis. Alternatively, evidences
should be provided to highlight the fact that the hyperdiploidity
being detected represents the genesis of binuclear OC3 cells
during the senescence induced by ANE.
Recent evidences have indicated that NF-kB was involved
in senescence of keratinocyte (17). We found that activation of
NF-kB followed the occurrence of senescence-associated process induced by ANE. NF-kB activation can induce the reciprocal upregulation of IkBa, and the subsequent reduction in
the NF-kB activity (36). Such regulation is similar to findings
in SAS OSCC cells treated with 5 mg/ml ANE (16). The
molecular mechanisms of such regulation deserve further
study. We observed that early passaged NHOK maintained
such homeostatsis even following the ANE treatment. However, late-passaged NHOK seemed to have poor maintenance
of such homeostasis. The findings might suggest that, following ANE treatment, late-passaged NHOK may enter a state
Areca induces cell cycle arrest and senescence
of worse homeostasis that is prone to regulatory imbalance
and disease susceptibility. EMSA analysis revealed that
late-passaged NHOK had a higher proportion of NF-kB complex in the upper mobility position following the ANE treatment. Our studies showed that NF-kB in nuclear extract was
not degraded and therefore excluded the possibility of forming
degraded NF-kB complex in lower mobility position (detailed
western blot analysis not shown). Some nuclear proteins that
bind to NF-kB can alter the activity of NF-kB and affect its
EMSA pattern (45). It is also possible that the protein bindings
mediated by ANE disrupted the transcription activity of
NF-kB, which might inhibit the reciprocal upregulation of
IkBa. The significance of complex shifts in premature senescence needs to be clarified. NF-kB is involved in multiple
phenotypes in addition to the senescence of keratinocyte
(17–21). Thereby, whether it plays an oncogenic or antioncogenic role in keratinocyte is still controversial. Our
study provided scenarios that ANE, a carcinogenic substance
that activates NF-kB through the degradation of IkBa, could
be associated with the premature senescence of target NHOK.
Activation of NF-kB has been implicated in the development
and the progression of OSCC (20,21). Strategies that mediate
NF-kB abrogation may reveal its potential against arecaassociated tumorigenesis.
NF-kB is involved in the regulation of multiple target genes,
which include IL-6, COX-2, IL-1a, MMP9, cyclin D1, and so
on (36,37). COX-2 is frequently overexpressed in arecaassociated OSCC (10). Its overexpression in neoplastic cells
has been shown to enhance malignant phenotypes. IL-6 has
been linked with the migration of keratinocyte (46) and the
increase of growth and invasion in OSCC (47). Suppression of
IL-6 renders the chemosensitivity and radiosensitivity of
OSCC cells (48). COX-2 and IL-6 can be upregulated by
areca ingredients (10–12,49). However, the induction of cfos in oral keratinocyte following the treatment of areca
ingredients has been controversial (12,50). We identified the
downregulation of COX-2 and IL-6 and upregulation of c-fos in
the replicative senescence process of NHOK. In contrast, the
reversal regulation of these genes was observed in ANEtreated NHOK. Such discrepancies were particularly prominent in late-passaged NHOK. Our data confirmed that ANE can
mediate the upregulation of COX-2 and IL-6, and implicated
that oral keratinocytes following chronic exposure of ANE is
advantageous for neoplastic phenotypes possibly by the
upregulation of COX-2 and IL-6. The finding of c-fos downregulation is consistent with the remarkable decrease in cell
proliferation following ANE treatment. Moreover, tumor promoter TPA can increase the mRNA expressions of IL-6,
COX-2 and c-fos in NHOK underlying ANE treatment. It further suggests that ANE-treated NHOK may retain the
responses to certain carcinogenic insults (35).
ANE has pleiotropic effects on many biological systems.
Our findings implied for the first time that p38MAPK, p16, p21
and NF-kB were activated by areca in NHOK, which could be
linked to senescence-associated phenotypes induced by ANE.
The mechanisms directing the activation need to be elucidated.
The findings in ANE-mediated COX-2 and IL-6 upregulation
in serial NHOK culture could substantiate the potential of
areca-exposed oral mucosa on neoplastic transformation.
Anti-NF-kB strategy might be advantageous in the prevention
or treatment of areca-associated neoplastic or senescent
processes (15).
Acknowledgements
We thank Dr Tsang-Yun Liu and Dr Meei-Hua Lin for critical comments on
this manuscript. This study was supported by National Research Program for
Genomic Medicine Project 91GMP004-3 and National Science Council grant
NSC 93-3112-B010-001, Taiwan.
Conflict of Interest Statement: None declared.
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Received March 25, 2005; revised January 19, 2006;
accepted January 27, 2006