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β-catenin expression in areca quid chewing-associated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells
Journal of the Formosan Medical Association (2012) 111, 194e200
Available online at www.sciencedirect.com
journal homepage: www.jfma-online.com
ORIGINAL ARTICLE
b-catenin expression in areca quid chewingassociated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells Shiuan-Shinn Lee a,y, Chung-Hung Tsai b,c,y, Lo-Lin Tsai d, Ming-Chih Chou b, Ming-Yung Chou e, Yu-Chao Chang d,e,* a
School of Public Health, Chung Shan Medical University, Taichung, Taiwan Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan c Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan d Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan e School of Dentistry, Chung Shan Medical University, Taichung, Taiwan b
Received 25 September 2010; received in revised form 15 November 2010; accepted 18 November 2010
KEYWORDS areca quid; arecoline; b-catenin; oral squamous cell carcinoma; regulatory mechanisms
Background/Purpose: Nuclear localization of b-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare b-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce b-catenin expression. Methods: A total of 40 areca quid chewing-associated OSCCs and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl- L -cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms. Results: b-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p < 0.05). It was demonstrated that normal oral epithelium showed only membranous staining for b-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic/nuclear staining in OSCCs. Arecoline was found to elevate b-catenin
* Corresponding author. School of Dentistry, Chung Shan Medical University, 110, Section 1, Chien-Kuo N. Road, Taichung, Taiwan. E-mail address: [email protected] (Y.-C. Chang). y These authors contributed equally to the results of this study. 0929-6646/$ - see front matter Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved. doi:10.1016/j.jfma.2010.11.002
b-catenin expression in areca quid chewing associated OSCCs
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expression in a dose-dependent manner (p < 0.05). The addition of PD98059, NAC, herbimycinA, SB203580, and LY294002 markedly inhibited the arecoline-induced b-catenin expression (p < 0.05). Conclusion: b-catenin expression is significantly upregulated in areca quid chewing-associated OSCC. The localization of b-catenin expression is correlated with the tumor size and clinical stage. In addition, b-catenin expression induced by arecoline is downregulated by PD98059, NAC, herbimycin-A, SB203580, and LY294002. Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Introduction
Materials and methods
Oral squamous cell carcinoma (OSCC), which accounts for approximately 90% of oral malignancies, is the eighth most common cancer in the world.1 The most common etiologies appear to be related to the oral habit of areca quid chewing, tobacco smoking, and heavy alcohol consumption.2 Areca quid is carcinogenic to humans3 and is an independent risk factor in the development of OSCC among people with this habit in Southeast Asia.4,5 However, the pathogenesis of areca quid chewing-associated OSCC remains to be elucidated. b-catenin, a cytoplasmic protein, is often involved in the regulation of E-cadherin-mediated cell adhesion and in the Wingless/Wnt signaling pathway.6 It plays an important role in the maintenance of epithelial structure with other members of the E-cadherin/catenin complex.7 In normal epithelial cells, in the absence of a Wnt signal, free cytoplasmic b-catenin interacts with the adenomatous polyposis coli (APC) tumor-suppressor protein and is phosphorylated by glycogen synthase kinase-3b at serine/threonine residues, which are encoded in exon 3 of the b-catenin gene.8,9 One of the hallmarks of activated Wnt signaling is the accumulation of nuclear b-catenin,10 a downstream component of the Wnt signaling pathway. Nuclear localization of b-catenin is known to be associated with the malignant transformation of squamous cell carcinoma of the esophagus, adenocarcinoma of the stomach, and adenocarcinoma of the colon.11,12 In addition, reduced cell adhesive properties of this complex have been reported to be associated with tumor development and progression in OSCCs.13e16 Recently, nuclear b-catenin was found to be correlated with the tumor malignancy index in Taiwanese population.17 Areca quid chewing is a major risk factor in the development of OSCCs in Taiwan.18 However, there is limited information on the regulation of b-catenin expression in areca quid chewing-associated OSCCs both in vitro and in vivo. The purpose of this study was to test whether b-catenin expression is regulated in areca quid chewingassociated OSCC specimens and to further explore the possible pathogenic mechanisms that may lead to the induction of b-catenin in vivo. The oral epithelial cell line GNM cells were challenged with arecoline, the major areca nut alkaloid, in vitro. Furthermore, extracellular signalregulated protein kinase (ERK) inhibitor PD98059, glutathione (GSH) precursor N-acetyl-L-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002 were added to investigate the possible mechanisms and their protective effects.
Arecoline, NAC, and herbimycin A were purchased from Sigma (St. Louis, MO, USA). PD098059, SB203580, and LY294002 were obtained from Promega (Madison, WI, USA). b-catenin (sc-59737) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All culture materials were obtained from Gibco (Grand Island, NY, USA). The final concentration of PD098059, NAC, herbimycin-A, SB203580, and LY294002 used in this study were 10 mM, 1 mM, 10 mM, 26 mM, and 163 mM, respectively.
Immunohistochemistry Formalin-fixed, paraffin-embedded specimens of 10 normal oral epithelium specimens from non-areca quid chewers, and 40 OSCC specimens from areca quid chewers, were drawn from the files of the Department of Pathology, Chung Shan Medical University Hospital. Diagnosis was based on the histological examination of hematoxylin- and eosinstained sections. Institutional Review Board permission at the Chung Shan Medical University Hospital was obtained for the use of discarded human tissues. Sections (5 mm thick) from formalin-fixed, paraffin-embedded specimens were stained with the monoclonal anti-b-catenin antibody (1:50 dilution) using a standard avidinebiotineperoxidase complex method as described previously.19 Diaminobenzidine (Zymed, South San Francisco, CA, USA) was then used as the substrate for localizing the antibody binding. Negative controls included serial sections from which either the primary or secondary antibodies were excluded. The preparations were counterstained with hematoxylin, mounted with Permount (Merck, Darmstadt, Germany), and examined using light microscopy.
Cell culture The oral epithelial cell line GNM cells,20 derived from a patient with T2N2aM0 (Ref. No. 0-18353) gingival carcinoma and metastasis to the cervical lymph node at the Department of Oral & Maxillofacial Surgery, School of Stomatology, Wuhan University, were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and antibiotics (100 units/mL of penicillin, 100 mg/mL of streptomycin, and 0.25 mg/mL of fungizone). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 and 95% air. Confluent cells were detached with 0.25% trypsin and 0.05% EDTA for 5 minutes, and aliquots of separated cells were subcultured.
196
b-catenin expression analysis Cells arrested in G0 by serum deprivation (0.5% FCS; 48 hours) were used according to our previous experiment.21 Nearly confluent monolayers of GNM cells were washed with serum-free medium and immediately thereafter exposed to 0, 20, 40, 80, and 160 mg/mL arecoline. Cell lysates were collected after 8 hours for Western blot analysis. Cultures without FCS were used as negative control. Subsequently, various pharmacological agents were added to test their regulation effects during the 8-hour coincubation period.
S.-S. Lee et al. 1 hour with biotinylated secondary antibody diluted 1:1000 in the same buffer, washed again as described above, and treated with 1:1000 streptavidineperoxidase solution for 30 minutes. After a series of washing steps, protein expression was detected by chemiluminescence using an ECL detection kit (Amersham Biosciences UK Limited, England), and relative photographic density was quantitated by scanning the photographic negatives on a gel documentation and analysis system (AlphaImager 2000; Alpha Innotech Corp., San Leandro, CA). Each densitometric value was expressed as the mean SD.
Statistical analysis Western blot For Western blot analysis, cell lyates were collected as described previously.22 Briefly, cells were solubilized with sodium dodecyl sulfate-solubilization buffer (5 mM EDTA, 1 mM MgCl2, 50 mM TriseHCl, pH 7.5, and 0.5% Trition X-100, 2 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide) for 30 minutes on ice. Next, cell lysates were centrifuged at 12,000 g at 4 C and the protein concentrations determined with Bradford reagent using bovine serum albumin as standards. Equivalent amounts of total protein per sample of cell extracts were run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immediately transferred to nitrocellulose membranes. The membranes were blocked with phosphate-buffered saline containing 3% bovine serum albumin for 2 hours, rinsed, and then incubated with primary antibodies anti-bcatenin (1:500) in phosphate-buffered saline containing 0.05% Tween 20 for 2 hours. After three washes with Tween 20 for 10 minutes, the membranes were incubated for
The statistical significance of differences between bcatenin localization and various clinico-pathologic features (T-categories, N-categories, age, sex, staging of the patients, and cell differentiation of OSCC) were assessed using Fisher’s exact test for independence. For Western blot assay, triplicate separate experiments were performed throughout this study. The significance of the results obtained from control and treated groups was statistically analyzed using paired Student t-test. A p value <0.05 was considered statistically significant.
Results b-catenin staining was mainly expressed in the OSCC specimens, and the intensity was significantly higher than that in normal epithelial specimens (p < 0.05). As shown in Fig. 1A, cytoplasmic or nuclear staining of b-catenin was not detected in all samples examined in the normal oral epithelium. b-catenin signals were detected in the cell
Figure 1 (A) Strong membranous expression of b-catenin in the normal oral epithelium, with no cytoplasmic/nuclear expression (200). (B) Peripheral loss of membranous of b-catenin in well-differentiated OSCC, keratin pearls or keratinizing cells showed weak or negative b-catenin staining (200). (C) Cytoplasmic and nuclear staining of b-catenin in moderately differentiated OSCC samples (200). (D) Absent membranous expression of b-catenin was recognized in poor differentiated OSCC (200).
b-catenin expression in areca quid chewing associated OSCCs membrane of the basal and spinous layer. The staining is absent in the most superficial layers. In well-differentiated OSCC, cytoplasmic and nuclear staining of b-catenin was detected in all samples (Fig. 1B). In well-differentiated cancer nests, keratin pearls or keratinizing cells showed weak or negative b-catenin staining. In moderately differentiated OSCC, cytoplasmic and nuclear staining of b-catenin was detected in all samples (Fig. 1C). In addition, a significant reduction of membranous staining was observed. The lack of membranous expression of bcatenin was observed in poorly differentiated OSCC (Fig. 1D). A significant increase in cytoplasmic/nuclear b-catenin expression was noted. As shown in Table 1, there was no significant difference in b-catenin expression in the differentiation of OSCC (p Z 0.109) As shown in Table 1, study participants were divided into two age groups: greater than or less than the median age (54 years). The immunostaining localization of b-catenin in OSCC could be classified into two groups: nuclear, 57.5% (23/40), and membrane, 42.5% (17/40). No significant difference in the localization of b-catenin expression was observed with respect to other factors, such as sex, age, differentiation, and lymph nodes metastasis (p > 0.05). The localization of b-catenin expression was significantly associated with tumor size (p Z 0.02) and stage (p Z 0.024). Expression of b-catenin in GNM cells challenged with arecoline was elevated by Western blot. As shown in Fig. 2, arecoline was found to elevate b-catenin expression in a dose-dependent manner (p < 0.05). From the
Table 1 Association of clinical features of patients and immunohistochemical expression of b-catenin. Clinicopathologic features
b-catenin expression
197
Figure 2 Expression of b-catenin in arecoline-treated GNM cells by Western blot. Cells were exposed for 8 hours in serumfree DMEM containing various concentrations of arecoline as indicated. b-actin was performed in order to monitor equal protein loading.
AlphaImager 2000 (Fig. 3), the amount of b-catenin was elevated about 1.1-, 2.2-, 6.0-, and 3.5-fold after exposure to 20, 40, 80, and 160 mg/mL arecoline (p < 0.05), respectively. The peak of b-catenin protein level induced by arecoline was 80 mg/mL. On the basis of these results, experiments described below were performed at a concentration of 80 mg/mL arecoline. PD98059, NAC, herbimycin-A, SB203580, and LY294002 without cytotoxic concentrations were added to search for the possible regulatory mechanisms on arecoline-induced b-catenin expression. These pharmacological agents were found to inhibit the arecoline-induced b-catenin expression (p < 0.05) (Fig. 4). From the AlphaImager 2000 (Fig. 5), PD98059, NAC, herbimycin-A, SB203580, and LY294002 were found to reduce the arecoline-induced b-catenin expression by lowering 2.8-, 2.5-, 4.4-, 4.5-, and 3.6-fold (p < 0.05), respectively.
Discussion
p
Mem (n Z 17)
C/N (n Z 23)
Sex Male Female
13 4
21 2
0.373
Age (y) &54 ＞54
9 8
11 12
1.0
Size &4 cm ＞4 cm
15 2
12 11
0.02
LN involvement No Yes
14 3
12 11
0.092
Differentiation Well Moderate/Poor
11 6
8 15
0.109
Stage I þ II III þ IV
11 6
6 17
0.024
Statistical analysis was performed using Fisher’s exact test. A p value <0.05 was considered statistically significant. LN Z lymph node; Mem Z membranous; C/N Z cytoplasmic/ nuclear.
b-catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant b-catenin signaling has been implicated in promoting many human squamous cell carcinomas.11,12
Figure 3 Levels of b-catenin protein treatment with arecoline were measured by densitometer. The relative level of bcatenin protein expression was normalized against b-actin signal and the control was set as 1.0. Optical density values represent the mean SD. *Significant difference from control values with p < 0.05.
198
Figure 4 The regulatory effects of PD098059, NAC, herbimycin-A, SB203580, and LY294002 on arecoline-induced bcatenin expression in GNM cells. Cells were coincubation with various pharmacological agents in the presence of 80 mg/mL arecoline. b-actin was performed in order to monitor equal protein loading.
The present study marks the first attempt to evaluate the expression of b-catenin in areca quid chewing-associated OSCC both in vitro and in vivo. To the best of our knowledge, b-catenin expression was first found to be highly expressed in areca quid chewing-associated OSCCs as compared with normal epithelium tissues. Normal oral epithelium has shown only membranous staining for b-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic staining in areca quid chewingassociated OSCCs. Similar results were found in previous studies: that b-catenin was highly expressed in oral cancer specimens.13e17 Thus, membranous staining may be lost or markedly reduced according to the cytoplasmic internalization/nuclear staining of b-catenin in the pathogenesis of areca quid chewing-associated OSCCs. The clinical significance of nuclear b-catenin expression in oral cancer is controversial. b-catenin nuclear stain has been shown to associate with proliferation, invasiveness, and worse prognosis of oral cancer.13e17 In the present study, the important finding is that the nuclear b-catenin expression has an inverse correlation with tumor size and
Figure 5 Quantization of arecoline-induced b-catenin expression in GNM cells was achieved by densitometer as described in Fig. 2. *Significant difference from control values with p < 0.05. #Statistically significant between arecoline alone and arecoline with pharmacological agents; p < 0.05.
S.-S. Lee et al. clinical stage. However, Gasparoni et al23 reported that nuclear b-catenin was rare in oral cancer and found no clear association between intranuclear b-catenin and histopathological and malignancy indexes in vivo. The reason for this contrary result is not clear. This may be due to the different origins of the specimens, different causation of OSCC, or different experimental protocols used in each laboratory. The activation of b-catenin in areca quid chewingassociated OSCCs may be explained as follows. Cyclin D1 have been identified as target genes of the Wnt/b-catenin pathway.24 Cyclin D1 nuclear staining was observed to be highly expressed in areca quid chewing-associated OSCCs in Taiwan.25 In addition, inactivation of the APC gene results in accumulation and translocation of b-catenin, which are important for malignant development.26 APC mutations were found to contribute to the carcinogenesis of at least some OSCCs in Taiwan, especially for users of areca quid.27 It is therefore feasible to suggest that cyclin D1 pathway and/ or APC gene mutation may at least in part be responsible for the upregulation of b-catenin in areca quid chewingassociated OSCCs. However, the interaction among bcatenin, cyclin D1, and APC deserves further investigation. In this study, we first reported the upregulation of bcatenin expression in oral epithelial cell line GNM cells stimulated by arecoline. This suggests that one of the pathogenic mechanisms of OSCC may be the synthesis of b-catenin expression by epithelial cells in response to areca quid challenge. The ERK signaling pathway is one of the mitogenactivated protein kinase cascades and plays important roles in the regulation of cell growth and differentiation.28 Previously, b-catenin was found to be abolished by PD98059 in RK3E cells.29 In this study, PD98059 was found to reduce b-catenin protein expression by arecoline. In accord, Chang et al30 reported that areca nut extract treatment resulted in the significant induction of p-ERK in human oral keratinocytes. These data demonstrate that activation of the ERK pathway may involve in arecoline-induced b-catenin expression in GNM cells. Previously, we demonstrated that arecoline significantly depletes intracellular GSH in human buccal mucosal fibroblasts.31 NAC is easily deacetylated inside the cells and provides cysteine for cellular GSH synthesis and thus stimulates the cellular GSH system.32 In this study, NAC was found to inhibit arecoline-stimulated b-catenin expression. Taken together, GSH may act as an intracellular buffer against arecoline-mediated b-catenin expression. Decreased b-catenin phosphorylation was reported to block cell migration in the tyrosine kinase inhibitor herbimycin-A in the IEC-6 cell line derived from normal rat intestine.33 The cell migration rate was decreased by adding herbimycin A in an oral epithelial cell line derived from buccal mucosa squamous cell carcinoma, HO-1-N-1.34 In this study, herbimycin A was found to inhibit arecoline-stimulated b-catenin expression. Taken together, activation of the tyrosine kinase pathway may involve in arecoline-induced bcatenin expression. Recently, Bikkavilli et al35 reported a novel role for p38 in canonical Wnt-b-catenin signaling. The p38 inhibitor SB203580 strongly attenuates the Wnt3a-induced b-catenin accumulation. The cell migration rate was decreased by
b-catenin expression in areca quid chewing associated OSCCs adding SB203580 in HO-1-N-1 cell line.34 In this study, we also demonstrated that SB203580 inhibited arecolinestimulated b-catenin expression, highlighting a critical role for p38 in b-catenin signaling. The PI3K consists of regulatory regulatory (p85) and catalytic (p110) subunits that participate in multiple cellular processes including cell growth, transformation, differentiation, and survival.36 Inhibition of PI3K signaling with LY294002 was reported to decrease nuclear accumulation of b-catenin in prostate cancer cell line LNCaP.37 In this study, LY294002 was found to reduce b-catenin protein expression by arecoline. These data demonstrate that activation of the PI3K pathway may involve in arecolineinduced b-catenin expression in GNM cells. This study shows that b-catenin is elevated in OSCC specimens from areca quid chewers. Arecoline was capable of stimulating b-catenin expression in GNM cells. This suggests that areca quid chewing may contribute to the pathogenesis of OSCCs via b-catenin expression. b-catenin inhibited by PD98059, herbimycin-A, SB203580, and LY294002 suggest that ERK, tyrosine kinase, p38, and PI3K transduction pathways may be involved in the arecolinestimulated b-catenin expression. b-catenin inhibited by NAC indicates that arecoline-stimulated b-catenin expression may be partially related to the GSH levels. Therefore, studying the regulatory mechanisms involved in b-catenin expression may prove versatile. However, more detailed studies should be undertaken to clarify the areca nut constitutes that can regulate b-catenin in vitro and in vivo. The design of in vitro studies cannot explain the decreased membranous staining and increased nuclear staining of bcatenin in OSCC samples in vivo. Because the proteins were extracted from whole cells in the present study, the protein from nuclear or membrane should be addressed separately in further studies to clarify the actual mechanism of bcatenin nuclear translocation after arecoline treatment.
Acknowledgments This study was supported by research grants from National Science Council, Taiwan (NSC 96-2321-B-040-006).
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ORIGINAL ARTICLE
b-catenin expression in areca quid chewingassociated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells Shiuan-Shinn Lee a,y, Chung-Hung Tsai b,c,y, Lo-Lin Tsai d, Ming-Chih Chou b, Ming-Yung Chou e, Yu-Chao Chang d,e,* a
School of Public Health, Chung Shan Medical University, Taichung, Taiwan Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan c Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan d Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan e School of Dentistry, Chung Shan Medical University, Taichung, Taiwan b
Received 25 September 2010; received in revised form 15 November 2010; accepted 18 November 2010
KEYWORDS areca quid; arecoline; b-catenin; oral squamous cell carcinoma; regulatory mechanisms
Background/Purpose: Nuclear localization of b-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare b-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce b-catenin expression. Methods: A total of 40 areca quid chewing-associated OSCCs and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl- L -cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms. Results: b-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p < 0.05). It was demonstrated that normal oral epithelium showed only membranous staining for b-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic/nuclear staining in OSCCs. Arecoline was found to elevate b-catenin
* Corresponding author. School of Dentistry, Chung Shan Medical University, 110, Section 1, Chien-Kuo N. Road, Taichung, Taiwan. E-mail address: [email protected] (Y.-C. Chang). y These authors contributed equally to the results of this study. 0929-6646/$ - see front matter Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved. doi:10.1016/j.jfma.2010.11.002
b-catenin expression in areca quid chewing associated OSCCs
195
expression in a dose-dependent manner (p < 0.05). The addition of PD98059, NAC, herbimycinA, SB203580, and LY294002 markedly inhibited the arecoline-induced b-catenin expression (p < 0.05). Conclusion: b-catenin expression is significantly upregulated in areca quid chewing-associated OSCC. The localization of b-catenin expression is correlated with the tumor size and clinical stage. In addition, b-catenin expression induced by arecoline is downregulated by PD98059, NAC, herbimycin-A, SB203580, and LY294002. Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Introduction
Materials and methods
Oral squamous cell carcinoma (OSCC), which accounts for approximately 90% of oral malignancies, is the eighth most common cancer in the world.1 The most common etiologies appear to be related to the oral habit of areca quid chewing, tobacco smoking, and heavy alcohol consumption.2 Areca quid is carcinogenic to humans3 and is an independent risk factor in the development of OSCC among people with this habit in Southeast Asia.4,5 However, the pathogenesis of areca quid chewing-associated OSCC remains to be elucidated. b-catenin, a cytoplasmic protein, is often involved in the regulation of E-cadherin-mediated cell adhesion and in the Wingless/Wnt signaling pathway.6 It plays an important role in the maintenance of epithelial structure with other members of the E-cadherin/catenin complex.7 In normal epithelial cells, in the absence of a Wnt signal, free cytoplasmic b-catenin interacts with the adenomatous polyposis coli (APC) tumor-suppressor protein and is phosphorylated by glycogen synthase kinase-3b at serine/threonine residues, which are encoded in exon 3 of the b-catenin gene.8,9 One of the hallmarks of activated Wnt signaling is the accumulation of nuclear b-catenin,10 a downstream component of the Wnt signaling pathway. Nuclear localization of b-catenin is known to be associated with the malignant transformation of squamous cell carcinoma of the esophagus, adenocarcinoma of the stomach, and adenocarcinoma of the colon.11,12 In addition, reduced cell adhesive properties of this complex have been reported to be associated with tumor development and progression in OSCCs.13e16 Recently, nuclear b-catenin was found to be correlated with the tumor malignancy index in Taiwanese population.17 Areca quid chewing is a major risk factor in the development of OSCCs in Taiwan.18 However, there is limited information on the regulation of b-catenin expression in areca quid chewing-associated OSCCs both in vitro and in vivo. The purpose of this study was to test whether b-catenin expression is regulated in areca quid chewingassociated OSCC specimens and to further explore the possible pathogenic mechanisms that may lead to the induction of b-catenin in vivo. The oral epithelial cell line GNM cells were challenged with arecoline, the major areca nut alkaloid, in vitro. Furthermore, extracellular signalregulated protein kinase (ERK) inhibitor PD98059, glutathione (GSH) precursor N-acetyl-L-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002 were added to investigate the possible mechanisms and their protective effects.
Arecoline, NAC, and herbimycin A were purchased from Sigma (St. Louis, MO, USA). PD098059, SB203580, and LY294002 were obtained from Promega (Madison, WI, USA). b-catenin (sc-59737) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All culture materials were obtained from Gibco (Grand Island, NY, USA). The final concentration of PD098059, NAC, herbimycin-A, SB203580, and LY294002 used in this study were 10 mM, 1 mM, 10 mM, 26 mM, and 163 mM, respectively.
Immunohistochemistry Formalin-fixed, paraffin-embedded specimens of 10 normal oral epithelium specimens from non-areca quid chewers, and 40 OSCC specimens from areca quid chewers, were drawn from the files of the Department of Pathology, Chung Shan Medical University Hospital. Diagnosis was based on the histological examination of hematoxylin- and eosinstained sections. Institutional Review Board permission at the Chung Shan Medical University Hospital was obtained for the use of discarded human tissues. Sections (5 mm thick) from formalin-fixed, paraffin-embedded specimens were stained with the monoclonal anti-b-catenin antibody (1:50 dilution) using a standard avidinebiotineperoxidase complex method as described previously.19 Diaminobenzidine (Zymed, South San Francisco, CA, USA) was then used as the substrate for localizing the antibody binding. Negative controls included serial sections from which either the primary or secondary antibodies were excluded. The preparations were counterstained with hematoxylin, mounted with Permount (Merck, Darmstadt, Germany), and examined using light microscopy.
Cell culture The oral epithelial cell line GNM cells,20 derived from a patient with T2N2aM0 (Ref. No. 0-18353) gingival carcinoma and metastasis to the cervical lymph node at the Department of Oral & Maxillofacial Surgery, School of Stomatology, Wuhan University, were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and antibiotics (100 units/mL of penicillin, 100 mg/mL of streptomycin, and 0.25 mg/mL of fungizone). Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2 and 95% air. Confluent cells were detached with 0.25% trypsin and 0.05% EDTA for 5 minutes, and aliquots of separated cells were subcultured.
196
b-catenin expression analysis Cells arrested in G0 by serum deprivation (0.5% FCS; 48 hours) were used according to our previous experiment.21 Nearly confluent monolayers of GNM cells were washed with serum-free medium and immediately thereafter exposed to 0, 20, 40, 80, and 160 mg/mL arecoline. Cell lysates were collected after 8 hours for Western blot analysis. Cultures without FCS were used as negative control. Subsequently, various pharmacological agents were added to test their regulation effects during the 8-hour coincubation period.
S.-S. Lee et al. 1 hour with biotinylated secondary antibody diluted 1:1000 in the same buffer, washed again as described above, and treated with 1:1000 streptavidineperoxidase solution for 30 minutes. After a series of washing steps, protein expression was detected by chemiluminescence using an ECL detection kit (Amersham Biosciences UK Limited, England), and relative photographic density was quantitated by scanning the photographic negatives on a gel documentation and analysis system (AlphaImager 2000; Alpha Innotech Corp., San Leandro, CA). Each densitometric value was expressed as the mean SD.
Statistical analysis Western blot For Western blot analysis, cell lyates were collected as described previously.22 Briefly, cells were solubilized with sodium dodecyl sulfate-solubilization buffer (5 mM EDTA, 1 mM MgCl2, 50 mM TriseHCl, pH 7.5, and 0.5% Trition X-100, 2 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide) for 30 minutes on ice. Next, cell lysates were centrifuged at 12,000 g at 4 C and the protein concentrations determined with Bradford reagent using bovine serum albumin as standards. Equivalent amounts of total protein per sample of cell extracts were run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immediately transferred to nitrocellulose membranes. The membranes were blocked with phosphate-buffered saline containing 3% bovine serum albumin for 2 hours, rinsed, and then incubated with primary antibodies anti-bcatenin (1:500) in phosphate-buffered saline containing 0.05% Tween 20 for 2 hours. After three washes with Tween 20 for 10 minutes, the membranes were incubated for
The statistical significance of differences between bcatenin localization and various clinico-pathologic features (T-categories, N-categories, age, sex, staging of the patients, and cell differentiation of OSCC) were assessed using Fisher’s exact test for independence. For Western blot assay, triplicate separate experiments were performed throughout this study. The significance of the results obtained from control and treated groups was statistically analyzed using paired Student t-test. A p value <0.05 was considered statistically significant.
Results b-catenin staining was mainly expressed in the OSCC specimens, and the intensity was significantly higher than that in normal epithelial specimens (p < 0.05). As shown in Fig. 1A, cytoplasmic or nuclear staining of b-catenin was not detected in all samples examined in the normal oral epithelium. b-catenin signals were detected in the cell
Figure 1 (A) Strong membranous expression of b-catenin in the normal oral epithelium, with no cytoplasmic/nuclear expression (200). (B) Peripheral loss of membranous of b-catenin in well-differentiated OSCC, keratin pearls or keratinizing cells showed weak or negative b-catenin staining (200). (C) Cytoplasmic and nuclear staining of b-catenin in moderately differentiated OSCC samples (200). (D) Absent membranous expression of b-catenin was recognized in poor differentiated OSCC (200).
b-catenin expression in areca quid chewing associated OSCCs membrane of the basal and spinous layer. The staining is absent in the most superficial layers. In well-differentiated OSCC, cytoplasmic and nuclear staining of b-catenin was detected in all samples (Fig. 1B). In well-differentiated cancer nests, keratin pearls or keratinizing cells showed weak or negative b-catenin staining. In moderately differentiated OSCC, cytoplasmic and nuclear staining of b-catenin was detected in all samples (Fig. 1C). In addition, a significant reduction of membranous staining was observed. The lack of membranous expression of bcatenin was observed in poorly differentiated OSCC (Fig. 1D). A significant increase in cytoplasmic/nuclear b-catenin expression was noted. As shown in Table 1, there was no significant difference in b-catenin expression in the differentiation of OSCC (p Z 0.109) As shown in Table 1, study participants were divided into two age groups: greater than or less than the median age (54 years). The immunostaining localization of b-catenin in OSCC could be classified into two groups: nuclear, 57.5% (23/40), and membrane, 42.5% (17/40). No significant difference in the localization of b-catenin expression was observed with respect to other factors, such as sex, age, differentiation, and lymph nodes metastasis (p > 0.05). The localization of b-catenin expression was significantly associated with tumor size (p Z 0.02) and stage (p Z 0.024). Expression of b-catenin in GNM cells challenged with arecoline was elevated by Western blot. As shown in Fig. 2, arecoline was found to elevate b-catenin expression in a dose-dependent manner (p < 0.05). From the
Table 1 Association of clinical features of patients and immunohistochemical expression of b-catenin. Clinicopathologic features
b-catenin expression
197
Figure 2 Expression of b-catenin in arecoline-treated GNM cells by Western blot. Cells were exposed for 8 hours in serumfree DMEM containing various concentrations of arecoline as indicated. b-actin was performed in order to monitor equal protein loading.
AlphaImager 2000 (Fig. 3), the amount of b-catenin was elevated about 1.1-, 2.2-, 6.0-, and 3.5-fold after exposure to 20, 40, 80, and 160 mg/mL arecoline (p < 0.05), respectively. The peak of b-catenin protein level induced by arecoline was 80 mg/mL. On the basis of these results, experiments described below were performed at a concentration of 80 mg/mL arecoline. PD98059, NAC, herbimycin-A, SB203580, and LY294002 without cytotoxic concentrations were added to search for the possible regulatory mechanisms on arecoline-induced b-catenin expression. These pharmacological agents were found to inhibit the arecoline-induced b-catenin expression (p < 0.05) (Fig. 4). From the AlphaImager 2000 (Fig. 5), PD98059, NAC, herbimycin-A, SB203580, and LY294002 were found to reduce the arecoline-induced b-catenin expression by lowering 2.8-, 2.5-, 4.4-, 4.5-, and 3.6-fold (p < 0.05), respectively.
Discussion
p
Mem (n Z 17)
C/N (n Z 23)
Sex Male Female
13 4
21 2
0.373
Age (y) &54 ＞54
9 8
11 12
1.0
Size &4 cm ＞4 cm
15 2
12 11
0.02
LN involvement No Yes
14 3
12 11
0.092
Differentiation Well Moderate/Poor
11 6
8 15
0.109
Stage I þ II III þ IV
11 6
6 17
0.024
Statistical analysis was performed using Fisher’s exact test. A p value <0.05 was considered statistically significant. LN Z lymph node; Mem Z membranous; C/N Z cytoplasmic/ nuclear.
b-catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant b-catenin signaling has been implicated in promoting many human squamous cell carcinomas.11,12
Figure 3 Levels of b-catenin protein treatment with arecoline were measured by densitometer. The relative level of bcatenin protein expression was normalized against b-actin signal and the control was set as 1.0. Optical density values represent the mean SD. *Significant difference from control values with p < 0.05.
198
Figure 4 The regulatory effects of PD098059, NAC, herbimycin-A, SB203580, and LY294002 on arecoline-induced bcatenin expression in GNM cells. Cells were coincubation with various pharmacological agents in the presence of 80 mg/mL arecoline. b-actin was performed in order to monitor equal protein loading.
The present study marks the first attempt to evaluate the expression of b-catenin in areca quid chewing-associated OSCC both in vitro and in vivo. To the best of our knowledge, b-catenin expression was first found to be highly expressed in areca quid chewing-associated OSCCs as compared with normal epithelium tissues. Normal oral epithelium has shown only membranous staining for b-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic staining in areca quid chewingassociated OSCCs. Similar results were found in previous studies: that b-catenin was highly expressed in oral cancer specimens.13e17 Thus, membranous staining may be lost or markedly reduced according to the cytoplasmic internalization/nuclear staining of b-catenin in the pathogenesis of areca quid chewing-associated OSCCs. The clinical significance of nuclear b-catenin expression in oral cancer is controversial. b-catenin nuclear stain has been shown to associate with proliferation, invasiveness, and worse prognosis of oral cancer.13e17 In the present study, the important finding is that the nuclear b-catenin expression has an inverse correlation with tumor size and
Figure 5 Quantization of arecoline-induced b-catenin expression in GNM cells was achieved by densitometer as described in Fig. 2. *Significant difference from control values with p < 0.05. #Statistically significant between arecoline alone and arecoline with pharmacological agents; p < 0.05.
S.-S. Lee et al. clinical stage. However, Gasparoni et al23 reported that nuclear b-catenin was rare in oral cancer and found no clear association between intranuclear b-catenin and histopathological and malignancy indexes in vivo. The reason for this contrary result is not clear. This may be due to the different origins of the specimens, different causation of OSCC, or different experimental protocols used in each laboratory. The activation of b-catenin in areca quid chewingassociated OSCCs may be explained as follows. Cyclin D1 have been identified as target genes of the Wnt/b-catenin pathway.24 Cyclin D1 nuclear staining was observed to be highly expressed in areca quid chewing-associated OSCCs in Taiwan.25 In addition, inactivation of the APC gene results in accumulation and translocation of b-catenin, which are important for malignant development.26 APC mutations were found to contribute to the carcinogenesis of at least some OSCCs in Taiwan, especially for users of areca quid.27 It is therefore feasible to suggest that cyclin D1 pathway and/ or APC gene mutation may at least in part be responsible for the upregulation of b-catenin in areca quid chewingassociated OSCCs. However, the interaction among bcatenin, cyclin D1, and APC deserves further investigation. In this study, we first reported the upregulation of bcatenin expression in oral epithelial cell line GNM cells stimulated by arecoline. This suggests that one of the pathogenic mechanisms of OSCC may be the synthesis of b-catenin expression by epithelial cells in response to areca quid challenge. The ERK signaling pathway is one of the mitogenactivated protein kinase cascades and plays important roles in the regulation of cell growth and differentiation.28 Previously, b-catenin was found to be abolished by PD98059 in RK3E cells.29 In this study, PD98059 was found to reduce b-catenin protein expression by arecoline. In accord, Chang et al30 reported that areca nut extract treatment resulted in the significant induction of p-ERK in human oral keratinocytes. These data demonstrate that activation of the ERK pathway may involve in arecoline-induced b-catenin expression in GNM cells. Previously, we demonstrated that arecoline significantly depletes intracellular GSH in human buccal mucosal fibroblasts.31 NAC is easily deacetylated inside the cells and provides cysteine for cellular GSH synthesis and thus stimulates the cellular GSH system.32 In this study, NAC was found to inhibit arecoline-stimulated b-catenin expression. Taken together, GSH may act as an intracellular buffer against arecoline-mediated b-catenin expression. Decreased b-catenin phosphorylation was reported to block cell migration in the tyrosine kinase inhibitor herbimycin-A in the IEC-6 cell line derived from normal rat intestine.33 The cell migration rate was decreased by adding herbimycin A in an oral epithelial cell line derived from buccal mucosa squamous cell carcinoma, HO-1-N-1.34 In this study, herbimycin A was found to inhibit arecoline-stimulated b-catenin expression. Taken together, activation of the tyrosine kinase pathway may involve in arecoline-induced bcatenin expression. Recently, Bikkavilli et al35 reported a novel role for p38 in canonical Wnt-b-catenin signaling. The p38 inhibitor SB203580 strongly attenuates the Wnt3a-induced b-catenin accumulation. The cell migration rate was decreased by
b-catenin expression in areca quid chewing associated OSCCs adding SB203580 in HO-1-N-1 cell line.34 In this study, we also demonstrated that SB203580 inhibited arecolinestimulated b-catenin expression, highlighting a critical role for p38 in b-catenin signaling. The PI3K consists of regulatory regulatory (p85) and catalytic (p110) subunits that participate in multiple cellular processes including cell growth, transformation, differentiation, and survival.36 Inhibition of PI3K signaling with LY294002 was reported to decrease nuclear accumulation of b-catenin in prostate cancer cell line LNCaP.37 In this study, LY294002 was found to reduce b-catenin protein expression by arecoline. These data demonstrate that activation of the PI3K pathway may involve in arecolineinduced b-catenin expression in GNM cells. This study shows that b-catenin is elevated in OSCC specimens from areca quid chewers. Arecoline was capable of stimulating b-catenin expression in GNM cells. This suggests that areca quid chewing may contribute to the pathogenesis of OSCCs via b-catenin expression. b-catenin inhibited by PD98059, herbimycin-A, SB203580, and LY294002 suggest that ERK, tyrosine kinase, p38, and PI3K transduction pathways may be involved in the arecolinestimulated b-catenin expression. b-catenin inhibited by NAC indicates that arecoline-stimulated b-catenin expression may be partially related to the GSH levels. Therefore, studying the regulatory mechanisms involved in b-catenin expression may prove versatile. However, more detailed studies should be undertaken to clarify the areca nut constitutes that can regulate b-catenin in vitro and in vivo. The design of in vitro studies cannot explain the decreased membranous staining and increased nuclear staining of bcatenin in OSCC samples in vivo. Because the proteins were extracted from whole cells in the present study, the protein from nuclear or membrane should be addressed separately in further studies to clarify the actual mechanism of bcatenin nuclear translocation after arecoline treatment.
Acknowledgments This study was supported by research grants from National Science Council, Taiwan (NSC 96-2321-B-040-006).
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