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
1 Team of Biomedical Science, Chang-Gung Institute of Nursing,
2 Department of Biomedical Technology, Taipei Medical College,
3 Department of Dentistry, Chang-Gung Memorial Hospital, Taipei,
4 Laboratory of Dental Pharmacology and Toxicology, Graduate Institute of Clinical Dental Science, National Taiwan University and Department of Dentistry, National Taiwan University Hospital and
5 Graduate Institute of Environmental Medicine, National Cheng-Gung University, Taiwan
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Abstract
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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 keratinocytes. In this study, AN extract (100800 µg/ml) and arecoline (20120 µM) 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 µg/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 potential (
ßm) 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 exposure of KB cells to arecoline (0.41.2 mM) and AN extract (8001200 µg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.11.2 mM) and AN extract (8001200 µg/ml) induced decreasing and increasing H2O2 production (by 2',7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of
ßm (increasing of rhodamine uptake) was noted by 24-h exposure of KB cells to arecoline (0.41.2 mM) and AN extract (8001200 µg/ml). AN extract (100 1200 µg/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 control,
ßm, GSH level and intracellular H2O2 production, these events being not coupled with cellular apoptosis.
Abbreviations: AN, areca nut; BQ, betel quid; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GF, gingival fibroblasts; OMF, oral mucosal fibroblasts; OSF, oral submucous fibrosis; PBS, phosphate buffered saline
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Introduction
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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 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 (1113). 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 (
ßm) and GSH levels using oral KB epithelial cells.
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Materials and methods
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Chemicals
AN extract was prepared as described previously (8,9,29). Arecoline hydrobromide, 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 1x1x1 mm3 small pieces and cultured in DMEM containing 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C 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.
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 supplemented with 10% FCS. After 24 h, cells were exposed to fresh medium containing various concentrations of AN extract (100800 µg/ml) and arecoline (20120 µM) 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, 5x105 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 µg/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 µg/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 55°C for 3 h (33). RNAase (0.5 µg/ml) was added and further incubated for 24 h. The DNA was extracted with phenolchloroformisoamyl alcohol, subjected to 1.8% of agarose gel electrophoresis and photographed under UV light.
Analysis of mitochondrial transmembrane potential, GSH levels and the generation of reactive oxygen species
Briefly 5x105 KB cells in DMEM containing 10% FCS were exposed to AN extract or arecoline for 24 h. Cells were detached with trypsinEDTA and washed with PBS. Mitochondrial membrane potential (
ßm) was measured 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
ßm. For doing this, cells were resuspended in 0.5 ml containing 10 µg/ml of rhodomine for 15 min at 37°C and then immediately submitted for flow analysis (Becton Dickinson). To assess the generation of ROS, cells treating with AN extract and arecoline were resuspended in 0.5 ml PBS containing 10 µM DCF-DA (Sigma) for 15 min at 37°C (33). For measurement of intracellular GSH content, cells were resuspended in 100 µl of PBS with 25 µM of CMF-DA for 15 min at 37°C. Cells were then subjected to flow cytometry immediately.
Statistical analysis
Three or more separate experiments were performed. Results were expressed as mean ± SE. Statistical analysis was done by paired Student's t test and P values were determined to evaluate the statistical significance (P < 0.05) of the changes observed.
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Results
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Effects of AN extract and arecoline on the growth of KB epithelial cells
As primary oral keratinocytes are more difficult for culture, we have tried to study the effects of AN ingredients on the KB epithelial cells and related mechanisms. At the beginning, the effects of AN extract and arecoline on the growth of KB cells were evaluated. As shown in Figure 1a
, AN extract inhibited the growth of oral KB epithelial cells in a dose-dependent manner. At concentrations of 100800 µg/ml, AN extract inhibited the growth of KB cells by 3690%, as revealed by MTT assay. Arecoline, the major areca alkaloid, also markedly suppressed the growth of KB cells in a dose-dependent fashion. As depicted in Figure 1b
, a 20120 µM of arecoline decreased the cell number by 1575%.


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Fig. 1. Effects of AN extract and arecoline on the growth of KB cells. KB cells (5x103 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 cells treated with AN extract (n = 6), (b) KB cells treated with arecoline (n = 4). Results were expressed as percentage of control (mean ± SE).
*Denotes marked difference when compared with control.
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Effects of AN extract and arecoline on the cell cycle control of OMF and oral KB carcinoma cells
AN extracts and arecoline have been shown to suppress the growth of several kind of cells (1,6,8,29). However, the precise reasons are not well understood. Growth of mammalian cells has been reported to be tightly regulated by cell cycle control (12,1417). OMF and KB cells treated with AN and arecoline demonstrated growth arrest, being more evident after 24 h of treatment. Exposure of KB cells to AN extract (400 and 800 µg/ml) for 4 and 7 h led to transient S-phase cycle arrest (data not shown). Twenty-four hour exposure of KB cells to AN extract (8001200 µg/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 µg/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.


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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.
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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 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
). Incubation of OMF with 800 µg/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 µg/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 µg/ml) and arecoline (0.11.2 mM) led to no pronounced DNA ladder formation with 24 h. On the contrary, DNA laddering characteristic of cells undergoing apoptosis was detected in cells similarly treated with quercetin (250750 µg/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 (1922). 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 µg/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 µg/ml), respectively. This indicated the depletion of intracellular GSH content of KB 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 µg/ml of AN extract (data not shown).


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Fig. 4. Effects of AN extract and arecoline on the single cell fluorescence detection of cellular GSH. KB cells (5x105 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 µg/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).
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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 µg/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 µg/ml of AN 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
).


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Fig. 5. Effects of AN extract and arecoline on the cellular production of H2O2. KB cells (5x105 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).
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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 mitochondrial membrane potential (
ßm) was determined by the
ßm sensitive fluorescent probe rhodomine 123. Exposure of KB cells to AN extract (8001200 µg/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 fluorescence by 20113%, indicating the presence of
ßm hyperpolarization (Figure 6a
). Similar mitochondrial membrane hyperpolarization of KB cells was also noted following exposure to 800 and 1200 µg/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 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 µM 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 carboxylesterase and GSH levels have been shown to be the major metabolic pathway of arecoline (9,30,41), perhaps the differential 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 formation 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. Nevertheless 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 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
ßm 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
ßm. 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).
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Notes
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6 To whom correspondence to be addressed
Email: huei{at}ha.mc.ntu.edu.tw 
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Acknowledgments
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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.