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

Oxford University Press 1527

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
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 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
Fig. 2. Effects of AN extract and arecoline on the cell cycle progression of induce apoptosis of KB cells by DNA fragmentation assay. To
KB cells. Changes in the percentage of KB cells residing in G0/G1, S- and investigate whether AN extract or arecoline treatment induced
G2/M-phase can be detected. Results were expressed as percentage of cells apoptosis in oral KB cells, cells grown in monolayer cultures
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
were treated with AN extract or arecoline for 24 h and assayed
KB cells treated with 0.4 and 0.8 mM of arecoline for 24 h (n 4). for the presence of apoptosis by measuring DNA fragmentation.
*Denotes marked difference when compared with control. 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-
cells to AN extract (8001200 g/ml) further induced evidently istic of cells undergoing apoptosis was detected in cells
G2/M-phase cycle arrest (Figure 2a). An average of 59, 20 similarly treated with quercetin (250750 g/ml) (data not
and 21% of untreated KB cells were residing in G0/G1, S- and shown).
G2/M-phase of cell cycle, respectively. Following exposure to
800 g/ml of AN extract for 24 h, marked G2/M cell cycle Effects of AN extract and arecoline on cellular GSH levels
arrest was noted as revealed by increasing the percentage of Cellular GSH has been shown to be crucial for regulation of
cells to about 47%. No evident increasing in sub-G0/G1 peak cell proliferation, cell cycle progression and apoptosis (19
was noted in any assayed AN concentrations (data not shown), 22). We therefore analyzed the changes of GSH levels of KB
indicating no obvious induction of apoptosis. cells by using a flow cytometer to measure the single cell
Effects of AN on the cell cycle control may be related to CMF fluorescence. Untreated KB cells showed two populations
its content of arecoline, the major areca alkaloid. We, therefore, of cells with different GSH content as demonstrated in a flow
tested the effects of arecoline on the cell cycle progression of cytometric histogram (Figure 4a). The M1 population of KB
KB cells. Within 2-h of exposure, no marked changes of cell cells showed higher level of intracellular content, as revealed
cycle was noted at all concentrations (0.11.2 mM) of arecoline by high CMF fluorescence, whereas the M2 population showed
tested (data not shown). Exposure of KB cells to arecoline lower level of GSH content. Twenty-four hour exposure of
(0.4 and 0.8 mM) for 7-h led to slight G2/M cell cycle arrest KB cells to 1.2 mM of arecoline and 1200 g/ml of AN
(data not shown). At concentrations of 0.2 mM, arecoline extract significantly elevated the percentage of cells residing
slightly induced the alterations of DNA contents in KB cells. in the M2 population from 11 (control) to 49 (1.2 mM
At concentration of 0.4 mM or higher, a 24-h exposure to arecoline) and 49% (AN 1200 g/ml), respectively. This
arecoline led to marked G2/M-phase arrest. The percentage of 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 Effects of AN extract and arecoline on cellular H2O2 production
in the M1 population showed higher levels of GSH content Cellular GSH is the principal detoxifying system, capable of
following exposure to AN extract and arecoline. The mean scavenging ROS and maintaining the redox state of cellular
GSH fluorescence in the M1 population of untreated KB cells thiols (36). Depletion of cellular thiol may potentially lead to
was about 118. Following exposure to 0.20.8 mM of arecoline, cellular oxidative stress (37). It is thus interesting to know
the GSH fluorescence of the M1 population increased to an whether AN extract and arecoline may induce oxidative stress
average value of 154178 (Figure 4b). AN extract also on oral epithelial cells. Exposure to 8001200 g/ml of AN
increased the intracellular GSH level of M1 population cells extract for 24 h led to intracellular accumulation of H2O2.
from 121 (in control) to 154 and 548 by 400 and 1200 g/ml Mean DCF fluorescence of KB increased from 114 (control)
of AN extract (data not shown). to 330 and 423, respectively, by 800 and 1200 g/ml of AN
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 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
Fig. 4. Effects of AN extract and arecoline on the single cell fluorescence disrupt mitochondrial functions, both of which may have
detection of cellular GSH. KB cells (5105 cells) in 100 mm culture dishes toxic consequence (39,40). It is critical to evaluate whether
(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
disturbance of mitochondria mediates the toxicity of AN
for 15 min and subjected to flow cytometry immediately. extract and arecoline on oral epithelial cells. The mito-
(a) Histogram of CMF fluorescence of untreated KB cells and KB cells chondrial membrane potential (m) was determined by the
exposure to 1.2 mM arecoline and 1200 g/ml of AN extract. Two m sensitive fluorescent probe rhodomine 123. Exposure
populations of KB cells (M1 and M2) with differential intracellular GSH of KB cells to AN extract (8001200 g/ml) and arecoline
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 (0.21.2 mM) for 24 h profoundly increased the net cellular
mean of GSH fluorescence. *Denotes marked difference when compared fluorescence intensity, as revealed in the flow cytometric
with control (P 0.05). 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.

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
Fig. 6. Effects of AN extract and arecoline on the mitochondrial membrane the induction of cell cycle arrest by AN extract. DNA damage
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. may induce G1/S and G2/M cell cycle checkpoints which
Cells were collected, resuspended in PBS, stained with Rho123 for 15 min provide sufficient time for cells to repair damaged DNA prior
and subjected to flow cytometry immediately. (a) Rhodamine fluorescence to entering the next phase of cell cycle (11,42). Consistently,
of KB cells following exposure to 0.11.2 mM of arecoline for 24 h. (b) AN and arecoline have been shown to induce DNA breaks,
Rhodamine fluorescence of KB cells following exposure to 1001200 g/ml
of AN extract. Results are expressed as mean of rhodamine fluorescence.
chromosomal aberrations and mutagenesis in different assays
*Denote marked difference when compared with control (P 0.05) (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
fluorescence by 20113%, indicating the presence of m GK and buccal keratinocytes (6,9). As arecoline can form
hyperpolarization (Figure 6a). Similar mitochondrial membrane conjugate with GSH and lead to intracellular GSH depletion
hyperpolarization of KB cells was also noted following expo- in fibroblasts and keratinocytes (6,10) that is critical for
sure to 800 and 1200 g/ml of AN extract for 24 h, increasing cell growth and cell cycle progression (1922), induction of
rhodamine fluorescence by 80 and 103%, respectively cell cycle arrest by AN extract is probably mediated by
(Figure 6b). 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
Discussion
cells exhibiting low CMF fluorescence. Interestingly, Agarwal
Using oral KB epithelial cells for study, we found that et al. report the upregulation of p21waf1/cip1, an inhibitor of
AN extract and arecoline inhibited the growth of KB cells. cyclin dependent kinases, in the epithelium of BQ chewers
Concomitantly, AN extract and arecoline also induced the G2/ with proliferating dysplasia and squamous cell carcinoma
M cycle arrest of KB cells. The event of AN extract is preceded (43). The correlation between p21 expression and cell cycle
by the induction of S-phase cycle delay of KB cells early after regulation by AN components is an intriguing question that
7 h of exposure. However, AN extract and arecoline did not should be clarified. Chemical-induced cytotoxicity can be
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 Areca ingredients arrest cell cycle

mediated by either necrosis and/or apoptosis (16,44,45), that accompanied by ROS production. Following administration of
varies with the test chemicals and cell types used. Apoptosis [3H]arecoline into female Chester Beatty rats, five major
is a critical component of the cellular responses to injuries to arecoline metabolites namely arecoline 1-oxide, arecaidine 1-
cell membranes, mitochondria and DNA, or to a dysregulation oxide, arecaidine, N-acetyl-S-(3-carboxyl-1-methylpiperid-4-
of the cell cycle (44,45). Here we noted that the induction of yl)-L-cysteine and an unidentified product were detected (51).
G2/M cycle arrest of KB cells by AN extract and arecoline is Differential reports about the effects of AN and arecoline on
not linked to the induction of apoptosis within 24 h. AN GSH and ROS levels may be because of the difference in the
extract also stimulated the KB cells to synthesize more IL-6 types of cells or organ that exhibit disparities in metabolic
(unpublished observation), a cytokine known to suppress the systems. This point should be further clarified.
p53-induced apoptosis to leukemia cells (46), failure to activate Mitochondria are the major source for endogenous cellular
apoptosis after DNA injury may be one route to carcinogenesis ROS production (26,52). Mitochondria have been shown to
by BQ (45,47). AN ingredients have been shown to exert participate in the process of chemical-induced cell injury that
various types of DNA damage (1,3), lack of apoptotic induction leads to cellular dysfunctions. In addition to ATP synthesis,
by AN may be one of the factors responsible for carcinogenesis. mitochondria are crucial for the modulation of cell redox
Oxidative stress and genotoxic insult may induce cell cycle status, osmotic regulation, pH control and calcium homeostasis
checkpoint functions (13). The GSH redox status is crucial for (26). However, mitochondria are prone to the attack by
a number of biological processes, including transcription of oxidants, electrophiles and lipophilic cations (26). Inducing
specific genes, regulating redox-sensitive signal transduction, the oxidative stress and depletion of mitochondrial GSH by
control of cell proliferation, apoptosis and tissue inflammation toxic chemicals may impair the mitochondrial functions and
(48). In the present study, exposure of KB cells to AN extract bioenergetics, leading to dysregulation of cell cycle control
and arecoline led to depletion of GSH in most of the KB cells. and apoptosis, and necrosis (3840). However, there is scant
Similar reduction of cellular GSH on oral fibroblasts and information available on the role of mitochondrial functions
keratinocytes by AN extract and arecoline is also reported during the toxic processes of AN on the oral tissues.
(6,10). Depletion of GSH by AN and arecoline may explain Rhodamine123 (Rho), as a fluorescent lipophilic cationic dye,
why exposure to AN components leads to cell cycle arrest and has been shown to accumulate in the mitochondria of living
cytotoxicity. This may explain why decreasing of GSH levels cells and has been used for evaluating changes in m of
and concomitant epithelial atrophy in OSF patients with BQ living cells (53). Rho123 incorporation may reveal both
chewing habits is usually observed in vivo (5,49). Interestingly, changes in mitochondrial activity and mitochondrial number
we also found that a fraction of KB cells have higher level of (23). In the present study, AN extract and arecoline induced
GSH content following exposure to AN and arecoline. This hyperpolarization of m. This may be a result of dispersion
reveals the possible presence of cellular heterogeneity. Never- of Rho123 fluorecence from mitochondria as observed by
theless the reasons are not fully clear. Probably, this elevation Hoyt et al. (54). Similarly, proliferation of dysfunctional
of GSH content was an adaptive response of KB cells to AN mitochondria is associated with G2/M arrest in Colo-205 cells
and arecoline via stimulating the GSH synthesis, decreasing treated with herbimycin (55). Membrane hyperpolarization is
GSH degradation, or increasing GSH transport into the cells also shown to inhibit the mitogenesis of lymphocytes and
or decreasing the GSH exit from the cells. spleen cells (56,57). Induction of mitochondrial membrane
Depletion of cellular GSH by AN ingredients may prone hyperpolarization thus may partly be responsible for the growth
the cells to further attack by other oral environmental toxicants, inhibition and cell cycle arrest by AN components. However,
leading to oxidative stress (36,37). In the present study, KB the precise action of AN and arecoline on mitochondrial
cells showed increased ROS production after AN treatment. structure and function of KB cells is not entirely clear.
Accordingly, the induction of ROS production in CHO cells Taken together, the present study reveals that arecoline and
by AN extract was recently reported (35). By feeding the AN ingredients can be crucial in the pathogenesis of oral
lactating mice with 1% AN diet, pronounced increasing in the cancer by inducing the dysregulation of cell cycle control,
hepatic levels of cytochrome b5, cytochrome P450, GSH and GSH homeostasis, mitochondrial function and ROS production.
malondialdehyde is noted, whereas GSH levels are decreased Additional studies on evaluating the source of ROS, the
(50). This indicate that GSH depletion and ROS production metabolism of AN components, and the changes in functional
play crucial roles in the toxicity of AN. As ROS production mitochondrial activity will provide more information for future
by AN components occurs generally at pH value higher than chemoprevention of toxicity of BQ ingredients. As exposure
9.5 (27,28), the ROS was possibly produced intracellularly by to AN components induces marked cellular oxidative stress,
metabolic activation. On the contrary, Sundqvist et al. have this highlights the use of various antioxidants in prevention of
found that inducing the GSH depletion of buccal keratinocytes BQ toxicity in vitro and in vivo in the near future (9,58).
by AN components is not linked to oxidative stress, as
revealed by no concomitant increasing in GSSG formation (6).
Acknowledgements
Interestingly, although exposure of KB cells to arecoline led
to marked GSH depletion, no marked intracellular oxidative The authors thank for Miss H.F.Jeng and Y.S.Chang, and Mr W.Tsai for their
stress was noted. Moreover, intracellular H2O2 production was technical assistance. This study is supported by a grant from National Science
Council (NSC88-2314-B002078-M14).
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 References
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