Carcinogenesis vol.22 no.9 pp.
15271535, 2001
Areca nut extract and arecoline induced the cell cycle arrest but
not apoptosis of cultured oral KB epithelial cells: association of
glutathione, reactive oxygen species and mitochondrial membrane
potential
M.C.Chang1, Y.S.Ho2, P.H.Lee2, C.P.Chan3, J.J.Lee4,
L.J.Hahn4, Y.J.Wang5 and J.H.Jeng4,6
1Team of Biomedical Science, Chang-Gung Institute of Nursing,
2Department of Biomedical Technology, Taipei Medical College,
3Department of Dentistry, Chang-Gung Memorial Hospital, Taipei,
4Laboratory of Dental Pharmacology and Toxicology, Graduate Institute of
Clinical Dental Science, National Taiwan University and Department
of Dentistry, National Taiwan University Hospital and 5Graduate Institute
of Environmental Medicine, National Cheng-Gung University, Taiwan
6To
whom correspondence to be addressed
Email: huei@ha.mc.ntu.edu.tw
There are 600 million betel quid (BQ) chewers in the
world. BQ chewing is a major etiologic factor of oral
cancer. Areca nut (AN) and arecoline may inhibit the
growth of oral mucosal fibroblasts (OMF) and keratino-
cytes. In this study, AN extract (100800 g/ml) and
arecoline (20120 M) inhibited the growth of oral KB
cells by 3690 and 1575%, respectively. Exposure to
arecoline (>0.2 mM) for 24 h induced G2/M cell cycle arrest
of OMF and KB cells. Areca nut extract (>400 g/ml)
also induced G2/M arrest of KB cells, being preceded
by S-phase arrest at 7-h of exposure. No evident sub-
G0/G1 peak was noted. Marked retraction and intracellular
vacuoles formation of OMF and KB cells were observed.
Glutathione (GSH) level, mitochondrial membrane poten-
tial (m) and H2O2 production of KB cells were measured
by flow cytometry. GSH level [indicated by 5-chloromethyl-
fluorescein (CMF) fluorescence] was depleted by 24-h expo-
sure of KB cells to arecoline (0.41.2 mM) and AN extract
(8001200 g/ml), with increasing the percentage of cells
in low CMF fluorescence. By contrast, arecoline (0.11.2
mM) and AN extract (8001200 g/ml) induced decreasing
and increasing H2O2 production (by 2,7-dichloro-
fluorescein fluorescence), respectively. Hyperpolarization
of m (increasing of rhodamine uptake) was noted by
24-h exposure of KB cells to arecoline (0.41.2 mM)
and AN extract (8001200 g/ml). AN extract (100
1200 g/ml) and arecoline (0.11.2 mM) induced little
DNA fragmentation on KB cells within 24 h. These results
indicate that AN ingredients are crucial in the pathogenesis
of oral submucous fibrosis (OSF) and oral cancer by
differentially inducing the dysregulation of cell cycle con-
trol, m, GSH level and intracellular H2O2 production,
these events being not coupled with cellular apoptosis.
Introduction
Betel quid (BQ) chewing in a popular oral habit in India,
South Africa and numerous southeastern Asian countries
(14). It has been estimated that there are ~600 million BQ
Abbreviations: AN, areca nut; BQ, betel quid; DMEM, Dulbeccos modified
Eagles medium; FCS, fetal calf serum; GF, gingival fibroblasts; OMF,
oral mucosal fibroblasts; OSF, oral submucous fibrosis; PBS, phosphate
buffered saline.
Oxford University Press
chewers in the world (4). Chewing BQ shows strong association
to the incidence of oral cancer, leukoplakia and oral submucous
fibrosis (OSF) (1,5). Areca nut (AN) components and arecoline,
a main areca alkaloid, have long been considered to be the
major etiologic factors in the pathogenesis of oral cancer and
OSF (1,35). AN extract induces the DNA breaks, unscheduled
DNA synthesis and differentiation of oral keratinocytes (68).
Arecoline also displays genotoxic effects as assayed by both
the bacterial test systems and cultured mammalian cells (1,3,4).
The mechanisms why AN ingredients lead to these toxic
events are also not fully clear. AN extract and arecoline are
cytotoxic and genotoxic to various kinds of cells and inhibits
the growth of oral mucosal fibroblasts (OMF), gingival
fibroblasts (GF) and keratinocytes (1,3,6,810). Growth of
cells is strictly regulated by the cell cycle progression.
Cells also typically exhibit cell cycle arrests in response to
genotoxic stress for allowing more time for DNA repair (11
13). Dysregulation of cell cycle control is one major cause of
cancer induction (14). Impairment of cell cycle by toxic
chemicals usually leads to growth retardation, cytotoxicity and
apoptosis (12,1417). However, little is known about whether
cytotoxicity by AN ingredients is a result of the induction of
cell cycle dysregulation or apoptosis. It is, therefore, interesting
to know whether AN extract or arecoline may induce apoptosis
of KB oral epithelial cells.
Recently, production of reactive oxygen species (ROS)
(12,18), depletion of cellular glutathione (GSH) (1922) and
regulation of mitochondrial functions (2326) have been shown
to influence the cell cycle progression, apoptosis and chemical
toxicity. As AN components have been shown to autooxidize
in the alkaline condition and produce ROS (27,28), it is
interesting to know whether AN component-induced cell cycle
changes are linked to the production of ROS, the changes in
mitochondrial functions and cellular redox. We, therefore,
critically evaluate the effects of AN and arecoline on the cell
cycle kinetics, apoptosis and related changes in the ROS
production, mitochondrial membrane potential (m) and GSH
levels using oral KB epithelial cells.
Materials and methods
Chemicals
AN extract was prepared as described previously (8,9,29). Arecoline hydro-
bromide, propidium iodide, rhodomine 123 and 2,7-dichlorofluorescein
diacetate (DCFH-DA) were from Sigma (Sigma Chemical Company, St Louis,
MO, USA). 5-chloromethylfluorescein diacetate (CMF-DA) was purchased
from Molecular Probes (Eugene, OR, USA). Reagents for flow cytometry
were obtained from Becton Dickinson, Worldwide Inc., San-Jose, California.
Cell culture medium and reagents were from Life Technologies (Gibco, Life
Technologies, NY, USA).
Culture of OMF and oral KB carcinoma cells
OMF were cultured by an explant technique as described previously (9,30).
Briefly, oral buccal mucosa tissues were minced to 111 mm3 small
pieces and cultured in DMEM containing 10% FCS, 100 U/ml penicillin and
100 g/ml streptomycin at 37C in a humidified atmosphere of 95% air/5%
CO2. Cultured OMF in passage numbers between four to eight were used for
these studies. Oral KB carcinoma cells, from American Type Culture Collection
(ATCC), were cultured in DMEM with 10% FCS.
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M.C.Chang et al.

Effects of AN extract and arecoline on the growth of KB cells

Cell growth assay was performed as described previously (8,29). Briefly,
5000 KB cells were inoculated into 24-well culture plate in DMEM supple-
mented with 10% FCS. After 24 h, cells were exposed to fresh medium
containing various concentrations of AN extract (100800 g/ml) and arecoline
(20120 M) for 5 days. Finally, cells were washed with DMEM, three times
and then in DMEM containing 0.5 mg/ml of MTT for 2 h. The formazan
produced was dissolved in DMSO and read against blank reagent at OD540
using a Dynatech Microwell plate reader.
Effects of AN extract and arecoline on the cell cycle kinetics of OMF and
KB cells
For elucidation of whether AN ingredients and arecoline can modulate the
cell cycle progression, 5105 of OMF or KB cells were seeded into 100-mm
culture dishes in DMEM containing 10% FCS. After 24 h, medium was
changed containing different concentrations of AN extract (final concentration
of 100, 200, 400, 800, 1200 g/ml) or arecoline (0.11.2 mM) for further
24 h. Morphological changes were photographed under a phase contrast
microscope.
For measurement of cellular DNA content, flow cytometric analysis was
used as described (31,32). Briefly, floating cells and attached cells were
collected, respectively, and poured together in the centrifuge tube. Attached
OMF and KB cells were washed with phosphate buffered saline (PBS) and
removed from the culture dishes by trypsinEDTA. Cells from two culture
dishes with similar exposure conditions were collected together, resuspended
and fixed in 70% ice-cold ethanol containing 2 mg/ml RNase for 30 min.
They were washed twice with PBS and finally stained with propidium iodide
(PI) (40 g/ml) for 10 min at room temperature. The PI fluorescence of
individual OMF or KB cells was analyzed by FACSCalibur Flow Cytometer
(Becton Dickinson) supplemented with an Argon ion laser. The wavelength
of laser excitation was set at 488 nm and emission collected at longer than
590 nm. FL2 fluorescence was collected in a linear/log scale fashion. Totally
20 000 cells were analyzed for control and each sample. The percentage of
cells in G0/G1, S and G2/M-phase were determined using standard ModiFit
software programs.
DNA fragmentation assay
About confluence KB cells were exposed to AN extract, arecoline and
quercetin (as positive control) for 24 h. Cells in the supernatant were
collected and then attached cells detached with trypsinEDTA. Cells were
then poured together and digested with lysis buffer containing 0.5%
sarkosyl, 0.5 mg/ml proteinase K, 50 mM Tris(hydroxy methyl) aminomethane
(pH 8.0) and 10 mM EDTA at 55C for 3 h (33). RNAase (0.5 g/ml) was
added and further incubated for 24 h. The DNA was extracted with phenol
chloroformisoamyl alcohol, subjected to 1.8% of agarose gel electrophoresis
and photographed under UV light.
Analysis of mitochondrial transmembrane potential, GSH levels and the Fig. 1. Effects of AN extract and arecoline on the growth of KB cells. KB
generation of reactive oxygen species cells (5103 cells) in 24-well culture well were exposed to AN extract or
arecoline for 5 days. Cell number was measured with MTT assay. (a) KB
Briefly 5105 KB cells in DMEM containing 10% FCS were exposed to AN cells treated with AN extract (n 6), (b) KB cells treated with arecoline
extract or arecoline for 24 h. Cells were detached with trypsinEDTA and (n 4). Results were expressed as percentage of control (mean SE).
washed with PBS. Mitochondrial membrane potential (m) was measured *Denotes marked difference when compared with control.
by flow cytometry (34) using the rhodomine 123 (Sigma), a fluorescent dye
being shown to be selectively accumulated in the mitochondria of living cells
by a mechanism which depends on m. For doing this, cells were resuspended
in 0.5 ml containing 10 g/ml of rhodomine for 15 min at 37C and then cells were evaluated. As shown in Figure 1a, AN extract
immediately submitted for flow analysis (Becton Dickinson). To assess the inhibited the growth of oral KB epithelial cells in a dose-
generation of ROS, cells treating with AN extract and arecoline were dependent manner. At concentrations of 100800 g/ml, AN
resuspended in 0.5 ml PBS containing 10 M DCF-DA (Sigma) for 15 min extract inhibited the growth of KB cells by 3690%, as
at 37C (33). For measurement of intracellular GSH content, cells were revealed by MTT assay. Arecoline, the major areca alkaloid,
resuspended in 100 l of PBS with 25 M of CMF-DA for 15 min at 37C.
Cells were then subjected to flow cytometry immediately. also markedly suppressed the growth of KB cells in a dose-
Statistical analysis dependent fashion. As depicted in Figure 1b, a 20120 M of
Three or more separate experiments were performed. Results were expressed arecoline decreased the cell number by 1575%.
as mean SE. Statistical analysis was done by paired Students t test and P Effects of AN extract and arecoline on the cell cycle control
values were determined to evaluate the statistical significance (P 0.05) of of OMF and oral KB carcinoma cells
the changes observed.
AN extracts and arecoline have been shown to suppress the
Results growth of several kind of cells (1,6,8,29). However, the
precise reasons are not well understood. Growth of mammalian
Effects of AN extract and arecoline on the growth of KB cells has been reported to be tightly regulated by cell cycle
epithelial cells control (12,1417). OMF and KB cells treated with AN and
As primary oral keratinocytes are more difficult for culture, arecoline demonstrated growth arrest, being more evident after
we have tried to study the effects of AN ingredients on the 24 h of treatment. Exposure of KB cells to AN extract (400
KB epithelial cells and related mechanisms. At the beginning, and 800 g/ml) for 4 and 7 h led to transient S-phase cycle
the effects of AN extract and arecoline on the growth of KB arrest (data not shown). Twenty-four hour exposure of KB
1528

Fig. 2. Effects of AN extract and arecoline on the cell cycle progression of
KB cells. Changes in the percentage of KB cells residing in G0/G1, S- and
G2/M-phase can be detected. Results were expressed as percentage of cells
in G0/G1, S- and G2/M-phase (mean SE). (a) Untreated KB cells and KB
cells treated with AN extract for 24 h (n 6). (b) Untreated KB cells and
KB cells treated with 0.4 and 0.8 mM of arecoline for 24 h (n 4).
*Denotes marked difference when compared with control.
cells to AN extract (8001200 g/ml) further induced evidently
G2/M-phase cycle arrest (Figure 2a). An average of 59, 20
and 21% of untreated KB cells were residing in G0/G1, S- and
G2/M-phase of cell cycle, respectively. Following exposure to
800 g/ml of AN extract for 24 h, marked G2/M cell cycle
arrest was noted as revealed by increasing the percentage of
cells to about 47%. No evident increasing in sub-G0/G1 peak
was noted in any assayed AN concentrations (data not shown),
indicating no obvious induction of apoptosis.
Effects of AN on the cell cycle control may be related to
its content of arecoline, the major areca alkaloid. We, therefore,
tested the effects of arecoline on the cell cycle progression of
KB cells. Within 2-h of exposure, no marked changes of cell
cycle was noted at all concentrations (0.11.2 mM) of arecoline
tested (data not shown). Exposure of KB cells to arecoline
(0.4 and 0.8 mM) for 7-h led to slight G2/M cell cycle arrest
(data not shown). At concentrations of 0.2 mM, arecoline
slightly induced the alterations of DNA contents in KB cells.
At concentration of 0.4 mM or higher, a 24-h exposure to
arecoline led to marked G2/M-phase arrest. The percentage of
Areca ingredients arrest cell cycle
KB cells in G2/M increased from an average of 13% (control)
to 43% (Figure 2b). No evident sub-G0/G1 peak was noted
following exposure of KB cells to arecoline. However, a minor
proportion of cells showed aneuploidy. Similarly, 24-h exposure
of OMF to arecoline and AN extract also induced G2/M-phase
cell-cycle arrest (data not shown).
Morphological alterations of OMF and KB cells following
exposure to AN extract and arecoline
OMF were spindle-shaped in appearance with extended cellular
processes (filopodi and lamellipodia) (Figure 3a). Following
exposure to 0.2 mM of arecoline for 24 h, some cells became
retracted and rounded in appearance. Further retraction and loss
of functional organization of OMF were observed following
exposure to 0.8 mM of arecoline for 24 h (Figure 3b). Incuba-
tion of OMF with 800 g/ml of AN extract for 24 h led to
intracellular vacuoles formation in most cells (Figure 3c,
arrow head).
Untreated oral KB carcinoma cells are cuboid and polygonal
in appearance with clear intercellular space (Figure 3d).
Exposure of KB cells to 0.4 mM of arecoline for 24 h led to
retraction and rounding in some sensitive cells. On exposure
to 0.8 mM of arecoline, KB cells seemed larger and lost sharp
intercellular space. Intracellular vacoules are present in some
cells (Figure 3e). Similarly, exposure of KB cells to AN
extract (400 g/ml) also induced evident intracellular vacuole
formation (data not shown).
Lack of apoptotic effects of AN extract and arecoline on
KB cells
AN components have been shown to produce ROS in the
alkaline condition (27,28) and also induce oxidative stress in
culture Chinese hamster ovary (CHO) cells (35). As production
of oxidative stress and dysregulation of cell cycle by toxic
chemicals has been closely linked to the induction of apoptosis
(15,18), we further evaluated whether AN ingredients can
induce apoptosis of KB cells by DNA fragmentation assay. To
investigate whether AN extract or arecoline treatment induced
apoptosis in oral KB cells, cells grown in monolayer cultures
were treated with AN extract or arecoline for 24 h and assayed
for the presence of apoptosis by measuring DNA fragmentation.
Exposure of KB cells to AN extract (1001200 g/ml) and
arecoline (0.11.2 mM) led to no pronounced DNA ladder
formation with 24 h. On the contrary, DNA laddering character-
istic of cells undergoing apoptosis was detected in cells
similarly treated with quercetin (250750 g/ml) (data not
shown).
Effects of AN extract and arecoline on cellular GSH levels
Cellular GSH has been shown to be crucial for regulation of
cell proliferation, cell cycle progression and apoptosis (19
22). We therefore analyzed the changes of GSH levels of KB
cells by using a flow cytometer to measure the single cell
CMF fluorescence. Untreated KB cells showed two populations
of cells with different GSH content as demonstrated in a flow
cytometric histogram (Figure 4a). The M1 population of KB
cells showed higher level of intracellular content, as revealed
by high CMF fluorescence, whereas the M2 population showed
lower level of GSH content. Twenty-four hour exposure of
KB cells to 1.2 mM of arecoline and 1200 g/ml of AN
extract significantly elevated the percentage of cells residing
in the M2 population from 11 (control) to 49 (1.2 mM
arecoline) and 49% (AN 1200 g/ml), respectively. This
indicated the depletion of intracellular GSH content of KB
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M.C.Chang et al.

Fig. 3. Morphological alterations of OMF and KB cells following exposure to AN extract and arecoline. (a) Control OMF, (b) OMF exposed to 0.8 mM
arecoline for 24 h, (c) OMF exposed to 800 g/ml of AN extract for 24 h. Intracellular vacuoles can be noted (arrow head). (d) Untreated oral KB carcinoma
cells. (e) Exposure of KB cells to 0.8 mM of arecoline for 24 h (100, original magnification).

cells by AN extract and arecoline. Interestingly, some KB cells
in the M1 population showed higher levels of GSH content
following exposure to AN extract and arecoline. The mean
GSH fluorescence in the M1 population of untreated KB cells
was about 118. Following exposure to 0.20.8 mM of arecoline,
the GSH fluorescence of the M1 population increased to an
average value of 154178 (Figure 4b). AN extract also
increased the intracellular GSH level of M1 population cells
from 121 (in control) to 154 and 548 by 400 and 1200 g/ml
of AN extract (data not shown).
1530
Effects of AN extract and arecoline on cellular H2O2 production
Cellular GSH is the principal detoxifying system, capable of
scavenging ROS and maintaining the redox state of cellular
thiols (36). Depletion of cellular thiol may potentially lead to
cellular oxidative stress (37). It is thus interesting to know
whether AN extract and arecoline may induce oxidative stress
on oral epithelial cells. Exposure to 8001200 g/ml of AN
extract for 24 h led to intracellular accumulation of H2O2.
Mean DCF fluorescence of KB increased from 114 (control)
to 330 and 423, respectively, by 800 and 1200 g/ml of AN

Fig. 4. Effects of AN extract and arecoline on the single cell fluorescence
detection of cellular GSH. KB cells (5105 cells) in 100 mm culture dishes
(10 ml, DMEM with 10% FCS) were exposed to AN extract or arecoline
for 24 h. Cells were collected, resuspended in PBS, stained with CMF
for 15 min and subjected to flow cytometry immediately.
(a) Histogram of CMF fluorescence of untreated KB cells and KB cells
exposure to 1.2 mM arecoline and 1200 g/ml of AN extract. Two
populations of KB cells (M1 and M2) with differential intracellular GSH
content were noted. (b) CMF fluorescence of M1 population of KB cells
following exposure to 0.11.2 mM of arecoline. Results are expressed as
mean of GSH fluorescence. *Denotes marked difference when compared
with control (P 0.05).
Areca ingredients arrest cell cycle
Fig. 5. Effects of AN extract and arecoline on the cellular production of
H2O2. KB cells (5105 cells) in 100 mm culture dishes (10 ml, DMEM
with 10% FCS) were exposed to (a) AN extract and (b) arecoline for 24 h.
Cells were collected, resuspended in PBS, stained with DCF-DA for 15 min
and subjected to flow cytometry immediately. Results are expressed as mean
of DCF fluorescence. *Denotes marked difference when compared with
control (P 0.05).
extract (Figure 5a). Unexpectedly, arecoline (0.11.2 mM)
markedly suppressed the cellular H2O2 production of KB cells
by 1855%, following 24 h of exposure (Figure 5b).
Alterations of mitochondrial membrane potential
GSH may also function in mitochondria to provide protection
against ROS (38) and exposure to oxidative stress may also
disrupt mitochondrial functions, both of which may have
toxic consequence (39,40). It is critical to evaluate whether
disturbance of mitochondria mediates the toxicity of AN
extract and arecoline on oral epithelial cells. The mito-
chondrial membrane potential (m) was determined by the
m sensitive fluorescent probe rhodomine 123. Exposure
of KB cells to AN extract (8001200 g/ml) and arecoline
(0.21.2 mM) for 24 h profoundly increased the net cellular
fluorescence intensity, as revealed in the flow cytometric
histogram (data not shown). At concentrations ranging from
0.2 to 1.2 mM, arecoline markedly increased the rhodamine
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M.C.Chang et al.
Fig. 6. Effects of AN extract and arecoline on the mitochondrial membrane
potential (m). KB cells (5105 cells) in 100 mm culture dishes (10 ml,
DMEM with 10% FCS) were exposed to AN extract and arecoline for 24 h.
Cells were collected, resuspended in PBS, stained with Rho123 for 15 min
and subjected to flow cytometry immediately. (a) Rhodamine fluorescence
of KB cells following exposure to 0.11.2 mM of arecoline for 24 h. (b)
Rhodamine fluorescence of KB cells following exposure to 1001200 g/ml
of AN extract. Results are expressed as mean of rhodamine fluorescence.
*Denote marked difference when compared with control (P 0.05)
fluorescence by 20113%, indicating the presence of m
hyperpolarization (Figure 6a). Similar mitochondrial membrane
hyperpolarization of KB cells was also noted following expo-
sure to 800 and 1200 g/ml of AN extract for 24 h, increasing
rhodamine fluorescence by 80 and 103%, respectively
(Figure 6b).
Discussion
Using oral KB epithelial cells for study, we found that
AN extract and arecoline inhibited the growth of KB cells.
Concomitantly, AN extract and arecoline also induced the G2/
M cycle arrest of KB cells. The event of AN extract is preceded
by the induction of S-phase cycle delay of KB cells early after
7 h of exposure. However, AN extract and arecoline did not
1532
elicit the apoptosis of KB cells at all test concentrations. The
effects of AN extract on the KB cells occurred simultaneously
with the induction of cellular GSH deprivation, ROS production
and mitochondrial hyperpolarization. On the other hand,
although arecoline induced GSH depletion and mitochondrial
hyperpolarization, this event is not coupled with cellular
oxidative stress.
Arecoline and AN extract exhibit cytotoxicity and inhibit
the growth of a number of cells (1,8,29). In the current
work, AN extract and arecoline also markedly suppressed the
proliferation of KB cells. As a concentration of 4080 M of
arecoline is sufficient to effectively inhibit the cell growth,
KB cells seem to be more susceptible to the toxic effect of
arecoline when compared with primary oral keratinocytes and
GF (6,8,29). Difference in experimental condition may partially
explain the differential results. Recently, cellular carboxyl-
esterase and GSH levels have been shown to be the major
metabolic pathway of arecoline (9,30,41), perhaps the differen-
tial cytotoxic effects of arecoline on different kinds of cells
may also be a result of variation in the activity of cellular
carboxylesterase or intracellular GSH homeostasis.
Impeding the growth of KB epithelial cells by AN extract
and arecoline could be partially explained by their induction
of cell cycle arrest. AN extract induced G2/M cell cycle arrest
that was preceded by S-phase cycle arrest at 7 h of exposure.
Effects of AN extract cannot be fully explained by its content
of arecoline. Although exposure of KB epithelial cells to
arecoline also led to G2/M cell cycle arrest; however, no
induction of S-phase arrest was noted. Consistently, it
is also similarly noted that the cytotoxicity and genotoxicity
of AN extract on GK was not directly a result of arecoline
content (8,29). Arecoline is not able to induce DNA breaks,
unscheduled DNA synthesis and intracellular vacuole forma-
tion in GF, GK and even KB cells in the current study
(6,8,10,29). The arecoline content of AN is usually ~0.151%
in different preparations (1). Whereas we did not directly
measure arecoline content in our AN extract, it was reasonable
to estimate that arecoline content was not fully responsible for
the induction of cell cycle arrest by AN extract. DNA damage
may induce G1/S and G2/M cell cycle checkpoints which
provide sufficient time for cells to repair damaged DNA prior
to entering the next phase of cell cycle (11,42). Consistently,
AN and arecoline have been shown to induce DNA breaks,
chromosomal aberrations and mutagenesis in different assays
(1,3). It is thus possible that inducing the cell cycle arrest of
KB cells by AN extract and arecoline may be a result of their
genotoxicity. However, this point should be further addressed,
because arecoline is unable to evoke DNA strand breaks on
GK and buccal keratinocytes (6,9). As arecoline can form
conjugate with GSH and lead to intracellular GSH depletion
in fibroblasts and keratinocytes (6,10) that is critical for
cell growth and cell cycle progression (1922), induction of
cell cycle arrest by AN extract is probably mediated by
interaction of arecoline with other AN ingredients. Consistently,
exposure of KB cells to AN extract and arecoline led to
depletion of GSH, as revealed by increasing the number of
cells exhibiting low CMF fluorescence. Interestingly, Agarwal
et al. report the upregulation of p21waf1/cip1, an inhibitor of
cyclin dependent kinases, in the epithelium of BQ chewers
with proliferating dysplasia and squamous cell carcinoma
(43). The correlation between p21 expression and cell cycle
regulation by AN components is an intriguing question that
should be clarified. Chemical-induced cytotoxicity can be

mediated by either necrosis and/or apoptosis (16,44,45), that
varies with the test chemicals and cell types used. Apoptosis
is a critical component of the cellular responses to injuries to
cell membranes, mitochondria and DNA, or to a dysregulation
of the cell cycle (44,45). Here we noted that the induction of
G2/M cycle arrest of KB cells by AN extract and arecoline is
not linked to the induction of apoptosis within 24 h. AN
extract also stimulated the KB cells to synthesize more IL-6
(unpublished observation), a cytokine known to suppress the
p53-induced apoptosis to leukemia cells (46), failure to activate
apoptosis after DNA injury may be one route to carcinogenesis
by BQ (45,47). AN ingredients have been shown to exert
various types of DNA damage (1,3), lack of apoptotic induction
by AN may be one of the factors responsible for carcinogenesis.
Oxidative stress and genotoxic insult may induce cell cycle
checkpoint functions (13). The GSH redox status is crucial for
a number of biological processes, including transcription of
specific genes, regulating redox-sensitive signal transduction,
control of cell proliferation, apoptosis and tissue inflammation
(48). In the present study, exposure of KB cells to AN extract
and arecoline led to depletion of GSH in most of the KB cells.
Similar reduction of cellular GSH on oral fibroblasts and
keratinocytes by AN extract and arecoline is also reported
(6,10). Depletion of GSH by AN and arecoline may explain
why exposure to AN components leads to cell cycle arrest and
cytotoxicity. This may explain why decreasing of GSH levels
and concomitant epithelial atrophy in OSF patients with BQ
chewing habits is usually observed in vivo (5,49). Interestingly,
we also found that a fraction of KB cells have higher level of
GSH content following exposure to AN and arecoline. This
reveals the possible presence of cellular heterogeneity. Never-
theless the reasons are not fully clear. Probably, this elevation
of GSH content was an adaptive response of KB cells to AN
and arecoline via stimulating the GSH synthesis, decreasing
GSH degradation, or increasing GSH transport into the cells
or decreasing the GSH exit from the cells.
Depletion of cellular GSH by AN ingredients may prone
the cells to further attack by other oral environmental toxicants,
leading to oxidative stress (36,37). In the present study, KB
cells showed increased ROS production after AN treatment.
Accordingly, the induction of ROS production in CHO cells
by AN extract was recently reported (35). By feeding the
lactating mice with 1% AN diet, pronounced increasing in the
hepatic levels of cytochrome b5, cytochrome P450, GSH and
malondialdehyde is noted, whereas GSH levels are decreased
(50). This indicate that GSH depletion and ROS production
play crucial roles in the toxicity of AN. As ROS production
by AN components occurs generally at pH value higher than
9.5 (27,28), the ROS was possibly produced intracellularly by
metabolic activation. On the contrary, Sundqvist et al. have
found that inducing the GSH depletion of buccal keratinocytes
by AN components is not linked to oxidative stress, as
revealed by no concomitant increasing in GSSG formation (6).
Interestingly, although exposure of KB cells to arecoline led
to marked GSH depletion, no marked intracellular oxidative
stress was noted. Moreover, intracellular H2O2 production was
decreased. The reasons for this event were not known until
now. This result further supports that effects of AN are not
merely because of arecoline. This is generally consistent with
our previous reports that arecoline-induced cytotoxicity on
OMF can be prevented by thiols, but not catalase, superoxide
dismutase and mannitol (9,30), three specific extracellular
ROS scavengers. GSH depletion is, therefore, not definitely
Areca ingredients arrest cell cycle
accompanied by ROS production. Following administration of
[3H]arecoline into female Chester Beatty rats, five major
arecoline metabolites namely arecoline 1-oxide, arecaidine 1-
oxide, arecaidine, N-acetyl-S-(3-carboxyl-1-methylpiperid-4-
yl)-L-cysteine and an unidentified product were detected (51).
Differential reports about the effects of AN and arecoline on
GSH and ROS levels may be because of the difference in the
types of cells or organ that exhibit disparities in metabolic
systems. This point should be further clarified.
Mitochondria are the major source for endogenous cellular
ROS production (26,52). Mitochondria have been shown to
participate in the process of chemical-induced cell injury that
leads to cellular dysfunctions. In addition to ATP synthesis,
mitochondria are crucial for the modulation of cell redox
status, osmotic regulation, pH control and calcium homeostasis
(26). However, mitochondria are prone to the attack by
oxidants, electrophiles and lipophilic cations (26). Inducing
the oxidative stress and depletion of mitochondrial GSH by
toxic chemicals may impair the mitochondrial functions and
bioenergetics, leading to dysregulation of cell cycle control
and apoptosis, and necrosis (3840). However, there is scant
information available on the role of mitochondrial functions
during the toxic processes of AN on the oral tissues.
Rhodamine123 (Rho), as a fluorescent lipophilic cationic dye,
has been shown to accumulate in the mitochondria of living
cells and has been used for evaluating changes in m of
living cells (53). Rho123 incorporation may reveal both
changes in mitochondrial activity and mitochondrial number
(23). In the present study, AN extract and arecoline induced
hyperpolarization of m. This may be a result of dispersion
of Rho123 fluorecence from mitochondria as observed by
Hoyt et al. (54). Similarly, proliferation of dysfunctional
mitochondria is associated with G2/M arrest in Colo-205 cells
treated with herbimycin (55). Membrane hyperpolarization is
also shown to inhibit the mitogenesis of lymphocytes and
spleen cells (56,57). Induction of mitochondrial membrane
hyperpolarization thus may partly be responsible for the growth
inhibition and cell cycle arrest by AN components. However,
the precise action of AN and arecoline on mitochondrial
structure and function of KB cells is not entirely clear.
Taken together, the present study reveals that arecoline and
AN ingredients can be crucial in the pathogenesis of oral
cancer by inducing the dysregulation of cell cycle control,
GSH homeostasis, mitochondrial function and ROS production.
Additional studies on evaluating the source of ROS, the
metabolism of AN components, and the changes in functional
mitochondrial activity will provide more information for future
chemoprevention of toxicity of BQ ingredients. As exposure
to AN components induces marked cellular oxidative stress,
this highlights the use of various antioxidants in prevention of
BQ toxicity in vitro and in vivo in the near future (9,58).
Acknowledgements
The authors thank for Miss H.F.Jeng and Y.S.Chang, and Mr W.Tsai for their
technical assistance. This study is supported by a grant from National Science
Council (NSC88-2314-B002078-M14).
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Received March 8, 2001; revised June 4, 2001; accepted June 5, 2001
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