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AKT activation
Oral Oncology (2007) 43, 670– 679
available at www.sciencedirect.com
journal homepage: http://intl.elsevierhealth.com/journals/oron/
Areca nut extract treatment down-regulates involucrin in normal human oral keratinocyte through P13K/AKT activation Yu-Hsin Tseng a, Che-Shoa Chang a,b, Tsung-Yun Liu Kuo-Wei Chang a,b,*, Shu-Chun Lin a,* a b c
a,c
, Shou-Yen Kao
a,b
,
Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan Department of Dentistry Taipei Veterans General Hospital, Taipei, Taiwan Department of Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan
Received 14 July 2006; accepted 2 August 2006 Available online 25 October 2006
KEYWORDS
Summary Areca (betel) is an important etiological factor linked to the high prevalence of oral carcinoma and other oral diseases in South Asians. Involucrin is a key component of the cornified envelop and a differentiation marker of keratinocyte. In this study, we found that 5 lg/ml non-toxic areca nut extract (ANE) treatment resulted in the 0.5-fold down-regulation of involucrin and disruption in involucrin distribution in normal human oral keratinocyte (NHOK). Progressive down-regulation of involucrin during oral carcinogenesis was noted. Activation of AKT by 1.7-fold and up-regulation of COX-2 by 2-fold were elicited following ANE treatment in NHOK. Treatment with PI3K/AKT blockers reverted the down-regulation of involucrin. ANE also down-regulated involucrin by 0.6-fold and disturbed both cornified envelope and cell aggregation in calcium-induced differentiated NHOK. However, such phenomena seemed to be independent from the ANE-associated COX-2 activation. The ANE-associated down-regulation of involucrin through AKT pathway could underlie the areca-associated epithelial pathogenesis. c 2006 Elsevier Ltd. All rights reserved.
AKT; Areca; Differentiation; Involucrin; Oral carcinoma
Introduction * Corresponding authors. Address: Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan. Fax: +886 2 28264053 (K.-W. Chang). E-mail addresses: [email protected] (K.-W. Chang), sclin@ym. edu.tw (S.-C. Lin).
Around 200–400 million people in South Asian and Southeast Asian countries are addicted to areca1 Areca-associated oral squamous cell carcinoma (OSCC) is one of the leading cancers in these regions2 Areca nuts contain polyphenols, arecoline, arecaidine and other alkaloids3 Areca nut extract
1368-8375/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2006.08.003
Areca nut extract treatment down-regulates involucrin in normal human (ANE) is highly cytotoxic and genotoxic to cultured human oral epithelial cells4 Studies have identified areca ingredients as the synergistic or promoting agents for multistep chemical carcinogenesis in hamster buccal pouch models.5,6 Recently, we have demonstrated that chronic low dose of ANE treatment induced the senescence-associated phenotype of normal human oral keratinocyte (NHOK) and hyperdiploid chromosomal changes in low grade OSCC cells7,8 However, the pathogenetic roles of areca for NHOK require further elucidation. ANE can induce COX-2 expression and PGE2 production in OSCC or NHOK cells9,10 Our previous studies have shown that ANE elicited a rapid activation of ERK and JNK1 mitogenactivated protein kinase (MAPK) as well as NF-jB in OSCC cells11,12 Besides, ANE-modulated NF-jB activation could be the basis of COX-2 up-regulation associated with areca exposure11 Our study also showed that the activation of Rho and Rac GTPase could underlie the areca-mediated morphological changes of OSCC cells12 In mammals, protein kinase B or AKT (PKB/AKT) belongs to a serine/threonine kinase family consisting of PKBa (AKT1), PKBb (AKT2), and PKBc (AKT3). PKB/AKT is activated in cells exposed to diverse stimuli such as hormones, growth factors, oncogene and extracellular matrix components13 AKT is the primary mediator of PI3K signaling; it has a number of downstream effectors including Bad, procaspase-9, Ij-K, GSK-3, mTOR and so on, that can contribute to the onset of cancer.13–15 Although AKT is highly expressed in OSCC, the activation of AKT following the stimulation of ANE have not been addressed16 The interactive signaling cascades and phenotypic impacts induced by ANE in oral keratinocyte need extensive clarification. The upper epidermis or oral epithelium acts as a barrier, composing mainly of terminally differentiated cells and keratinized layer, which are the endpoint of keratinocyte. During keratinocyte differentiation, cell membrane will be progressively replaced by cornified envelope, which consists of keratins that are enclosed within insoluble protein complexes cross-linked by transglutaminases (TGMs)17 Involucrin is one of the insoluble proteins in cornified envelopes; it is abundant in the upper spinous and granular layers, and is a key marker of keratinocyte differentiation18 A report showed that involucrin expression was reduced in intraepithelial cervical neoplasms as compared with normal epithelia19 Moreover, involucrin expression was significantly downregulated in highly metastatic OSCC cell lines as compared with parental cells with low metastatic potential20 Hypoxia is an adverse status for tumor growth. Interestingly, involucrin seems to be an oxygen-regulated protein, and the majority of hypoxic cells in head and neck cancer express involucrin21 Thereby, dysregulation or loss of involucrin expression is highly associated with neoplastic process of epithelium. When cultured in a low-Ca++ (0.1 mM) medium, NHOK grows as a monolayer polygonal cell, which can be cultivated for 6 or 7 passages (for 20 population doublings)8 In the initial passage, only scarce amount of cytosolic involucrin is present, but it is abundantly dispersed in cytosol during 3–4 passages22 Conversely, when a higher concentration of Ca++ (1 mM) is used, NHOK differentiates with a squamous morphology. The regulation of keratinocyte differentiation by Ca++ is related to phospholipase C (PLC) and protein ki-
671
nase C (PKC) activation, which might activate AKT and MAPK, and subsequently induce proteins required for differentiation23 Activation of p38MAPK is also involved in involucrin up-regulation in keratinocytes24 In addition, NF-jB signaling plays critical roles in regulating cell cycle withdrawal, cell survival, and differentiation in keratinocytes25 In this study, we have identified that ANE is able to downregulate involucrin by means of AKT activation, a signaling element lying downstream of PLC and upstream of NF-jB.
Materials and methods Oral tissues Sampling of tissue pairs from OSCC patients and oral precancerous lesions (OPL) for RT-PCR analysis was approved by an institutional review board. A 0.5 cm3 tissue from OSCC, the matched metastatic OSCC lesions in cervical lymph nodes (abbreviated as LNM), OPL and a non-malignant matched tissue (NMMT) were obtained from patients. Tissue samples were embedded in OCT first, stored immediately in liquid nitrogen until use. Laser capture microdissection (LCM) was performed on frozen-sectioned tissue sections to retrieve pure epithelial components of NMMT, OPL, OSCC and LNM, representing various tumorigenic stages using a PixCell II LCM system and ARC200 v1.0.2 software (Arcturus, Mountain View, CA, USA). The clinicopathological features of the subjects are summarized in Table 1.
Table 1
Clinicopathological parameters of OPL and OSCC
Parameters
OPL
OSCC
Age (years) Mean age ± SD Sex (M/F)
38–62 49.6 ± 10.8 5/0
41–79 53.0 ± 9.4 20/1
Site Buccal mucosa Tongue Other sites
0 3 2
5 7 9
Diagnosis Epithelial dysplasia Hyperkeratosis or epithelial hyperplasia
0 5
Differentiation Well Moderate and Poor
12 9
Size T1–T3 T4
4 17
Stage I–III IV
2 19
LNM Presence Absence
7 14
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Cell culture and ANE treatment Sampling of gingival tissue from donors who received flap operation to remove the impacted third molar was approved by an institute review board. NHOK was dissociated from the tissue and grown in keratinocyte serum free medium (KSFMÒ, Invitrogen, Carlsbad, CA, USA) with 0.1 mM Ca++22 In addition, cells were passaged when 70% confluence was reached to avoid the confluence-induced differentiation. By culturing with KSFM, the majority of NHOK, retaining the parabasaloid morphology prior to passage 4, were used
Y.-H. Tseng et al. for experiments. SAS OSCC cells cultivation and the preparation of ANE from ripe areca nut were preformed with protocols previously established11 In brief, ripe areca nuts were chopped, grounded and immersed to obtain the soluble extract. This soluble fraction was lyophilized and stored at 20 °C until use. Dimethyl sulfoxide (DMSO) and wortmannin were purchased from Sigma (St Louis, MO, USA). LY294002 and U0126 were purchased from EMD Biosciences Inc. (San Diego, CA, USA). CAY10404 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). LY294002, wortmannin and U0126 were dissolved in DMSO for experiments.
Figure 1 ANE treatment causes morphological change and disruption of involucrin. Control and ANE-treated NHOK were subjected to inverted microscopy, fluorescence confocal microscopy, RT-PCR and western blotting analysis. (A) NHOK following ANE treatment for 24 h exhibited an increase in cell population with spindle cell morphology (a, b). A transfer of cytosolic involucrin to both cytosol and nucleus was noted (e, f) in comparison with cytokeratin (c, d). A, g and h, sustained ANE treatment for 48 h also exhibited such phenomena, with a cytosolic and nuclear localization of involucrin in a spindle-shaped NHOK. (a and b, x100; c-f, x200; g and h, x1000). (B) A representative RT-PCR analysis displays a decrease in involucrin mRNA expression following ANE treatment for 24 h. (C) Quantitation of four individual experiments indicates a significant decrease of involucrin mRNA expression upon ANE treatment. The expression of TGM1 did not affected by such treatment. (D) A representative western blotting displays a decrease in involucrin protein following 5 lg/ml ANE treatment for 48 h. (E) Quantitation of four individual NHOKs indicates a significant decrease of involucrin protein expression following ANE treatment after 48 h, despite the fact that the involucrin protein was not affected by ANE treatment after 24 h. Values are expressed as means ± SD. *, P < 0.05; **, P < 0.01.
Areca nut extract treatment down-regulates involucrin in normal human
RT-PCR Total RNA purification, cDNA synthesis and RT-PCR reaction were performed following the protocols previously established22 The primer sequences will be provided upon request. Amplicons were resolved on a 2% agarose gel, visualized and quantitated by an imaging system (Viber Lourmat, Marne La Valle, France). The signals of the tested genes, including COX-2, involucrin and TGM1, were normalized with the mRNA expression of GAPDH.
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ing; 1:500), anti-ERK (Cell Signaling; 1:500) and anti-GAPDH (Chemicon, Temecula, CA, USA; 1:5000). Secondary antibodies were horseradish peroxidase-conjugated (Chemicon; 1:1000) antibodies. The signals were detected by the Western Lightening chemiluminescence reagent plus kit (Perkin-Elmer, Wellesley, MA, USA). The densities of the signals were measured by a densitometer (Amersham, Piscataway, NJ, USA). Quantification of signals was achieved by normalization with that of GAPDH.
Statistics Immunofluoresence NHOK were fixed with 4% paraformaldehyde in PBS for 30 min and permeablized with 0.5% Triton X-100/fixative for 5 min at 25 °C. Cells were incubated with anti-involucrin (NovoCastra, Newcastle, UK; 1:500) and anti-pan-cytokeratin (Lab Vision Corp., Fremont, CA, USA; 1: 500) antibodies for 2 h at 25 °C, followed by incubation with Alexa fluorÒ 488-conjugated secondary antibodies (Invitrogen) for 30 min at 37 °C. The immunofluoresence was photographed on a Leica TCS SP2 confocal laser-scanning microscope.
Western blotting Fifty micrograms of protein from the whole cell lysate were resolved by electrophoresis on a 12.5% denaturing polyacrylamide gel. The subsequent procedures were performed following the standard protocols11 The primary antibodies used including anti-COX-2 (BD Biosciences, Franklin Lakes, NJ, USA; 1:500), anti-involucrin (NovoCastra, Newcastle, UK, 1:500), anti-phosphorylated AKT (Cell Signaling, Temecula, CA, USA; 1:1000), anti-AKT (Santa Cruze Biotech., Santa Cruz, CA, USA; 1:500), anti-phosphorylated ERK (Cell Signal-
In this study, t-test or Fisher’s exact test was used for statistical analysis. It was considered to have a statistically significant difference when P < 0.05.
Results Treatment of NHOKs to passages 63 with 5 lg/ml ANE for 24 h caused no cytotoxicity8 It elicited the elongation of NHOK from the original parabasaloid morphology in 30% of cultivated cells12 (Fig. 1A, a, b). We investigated the distribution of cytokeratin using anti-pan-cytokeratin antibody and identified no obvious change following the treatment (Fig. 1A, c, d). To find out the possible influences of ANE on NHOK differentiation, we detected the position of involucrin and TGM1. In control NHOK, involucrin was evenly and intensively localized in cytosol, whereas, it was rather loosely arranged in cytosol and nuclei of ANE treated NHOK (Fig. 1A, e, f). Magnified pictures demonstrate the phenomena more evidently (Fig. 1A, g, h). To further specify the expression of ANE-modulated involucrin, RT-PCR analysis has shown that 24 h ANE treatment significantly down-regulated involucrin mRNA expression (Fig. 1B and C). Involucrin
Figure 2 Involucrin mRNA expression in LCM-retrieved tissues. (A) A representative RT-CR analysis in tissue pairs. (B) Quantitation of involucrin mRNA expression in OPL, OSCC and LNM in comparison to paired NMMT. A horizontal line in each column indicates the median value. #, code number of cases. *, P < 0.05; ***, P < 0.001.
674 protein level in NHOK was also down-regulated significantly to 0.5-fold, when ANE treatment was applied for 48 h (Fig. 1D and E). Since cytokeratin and TGM1 did not appear remarkable changes (Fig. 1B and C) in ANE-treated cells, ANE could be a target on involucrin regulation. To determine the involurcin mRNA expression during oral carcinogenesis, LCM-retrieved pure paired epithelial cells from various oral tissues including that from 26 NMMTs, 5 OPLs, 21 OSCCs and 4 corresponding LNMs were analyzed. The normalized involurcin mRNA expression in OPL, OSCC and LNM relative to NMMT were 0.78 ± 0.20, 0.49 ± 0.09 and 0.41 ± 0.11, respectively (Fig. 2A). A statistically signif-
Y.-H. Tseng et al. icant difference in involucrin mRNA expression was noted between NMMT and OSCC (P < 0.001, paired t-test) as well as NMMT and LNM (P < 0.05, paired t-test), however, such difference was not observed between NMMT and OPL (Fig. 2B). Cases with OSCC/NMMT ratio < median were defined as having low involucrin mRNA expression. Although primary OSCC with low involucrin mRNA expression exhibited a higher propensity for less differentiation (56% vs 42%) and more LNM (57% vs 43%) compared to those had higher involucrin mRNA, involucrin mRNA expression was not associated with tumor site, differentiation, size, LNM and stage (Fisher’s exact test).
Figure 3 ANE treatment activates AKT and ERK, and up-regulates COX-2. Control and ANE-treated NHOK were subjected to RTPCR and western blotting analysis. (A) A representative western blotting displays an increase of p-AKT following ANE treatment for 0.5 h. Note the slight increase in total AKT. (B) Quantitation of three independent NHOKs shows a significant rapid increase in p-AKT following ANE treatment at 0.5 h and recovered to basal level at 2 h. (C) A representative western blotting displays an increase of pERK following ANE treatment for 24 h. Total ERK here is also increased. (D) Quantitation of two to five independent NHOKs shows a progressive increase in p-ERK following ANE treatment. (E) and (G) Representative RT-PCR and western blotting analysis of COX-2 level, respectively, following ANE treatment for 24 h in a representative NHOK. (F) and (H) Quantitation of five NHOKs indicates significant increases in COX-2 mRNA and protein expression following ANE treatment for 24 h. Values are expressed as means ± SD. *, P < 0.05; **, P < 0.01.
Areca nut extract treatment down-regulates involucrin in normal human
675
Figure 4 PI3K/AKT blockers treatment reverts ANE-associated involucrin down-regulation. NHOKs were pretreated with U0126, wortmannin or LY294002 for 1 h, and then treated with ANE for 24 h. (A) RT-PCR analysis of a representative NHOK exhibits the blockage of ANE-associated down-regulation on involucrin mRNA expression by wortmannin and LY294002. U0126 does not exhibit such blockage effect. (B) Quantitation of (A). Data shown is a representative experiment of two independent experiments.
It was known that ANE can activate MAPKs and COX2,10,11 we further investigated to find out if ANE-mediated signal activation underlies the disruption of involucrin. Western blotting demonstrated a rapid activation of AKT by 1.7-fold increased fraction of phosphorylated (p)-AKT following ANE treatment at 0.5 h (Fig. 3A and B). Whereas ERK was activated slower, which reached the peak of 1.8-fold at 24 h (Fig. 3C and D). Similarly, COX-2 was also up-regulated by ANE through transcriptional activation in NHOK (Fig. 3E–H). Ten nM wortmannin and 10 lM LY294002 treatment blocked AKT activity, and 20 lM U0126 treatment blocked ERK activity. With pretreatment of wortmannin and LY294002 for 1 h, ANE-mediated down-regulation of involucrin was prevented (Fig. 4), but the pretreatment of U0126 did not result in such reversion. However, in a representative NHOK, the nuclear localization of involucrin induced by ANE treatment cannot be reverted by AKT blockers. Other than NHOK, the down-regulation of involucrin and the activation of AKT induced by non-toxic ANE treatment (65 lg/ml) were also observed in SAS oral keratinocyte cell line (Fig. 5). Therefore, SAS cell line could be another cell model for studying the areca-associated pathogenesis. We were interested in knowing if ANE also play similar roles in down-regulating involucrin in differentiated NHOK. Calcium-induced differentiated NHOK was chosen to address this question. NHOKs were treated with 1 mM Ca++ to induce differentiation. Immunofluorescence showed that following Ca++ induction for 24 h, a representative NHOK became aggregated with the formation of cornified envelop,
manifesting as subcortical package of dense involucrin (Fig. 6A, b). In such differentiated NHOK, concomitant ANE treatment reduced the intimate cellular contact, disrupted the continuity of cornified envelop and induced the
Figure 5 ANE treatment causes AKT activation and involucrin down-regulation in SAS OSCC cells. (A) RT-PCR analysis shows the decreased involucrin mRNA expression and increased COX-2 mRNA expression in SAS cells following 5 lg/ml ANE treatment for 24 h. (B) Western blotting analysis reveals the increase in p-AKT expression following ANE treatment. The figure shown is a representative experiment of two independent experiments.
676 cytosolic returning of involucrin (Fig. 6A, c). In a representative NHOK, RT-PCR analysis shows an increase of involucrin mRNA expression for 6-fold following Ca++ induction. Concomitant ANE treatment for 24 h with increasing dosages repressed such up-regulation in a dose-dependent manner (Fig. 6B and C). Concomitant 5 lg/ml ANE treatment significantly repressed the Ca++-induced involucrin by 0.6-fold compared to the untreated cells (Fig. 6D–G). Ca++-induction slightly increased p-AKT, while concomitant ANE treatment further increased both total (t)-AKT and pAKT, but the activation did not reach a statistically significant level (Fig. 7A and B). The treatment also did not significantly trigger the activation of ERK (Fig. 7C and D). Ca++-induction significantly up-regulated COX-2 expression for 1.5-fold, and concomitant ANE treatment further upregulated COX-2 expression for additional 1.6-fold
Y.-H. Tseng et al. (Fig. 7, lower panel of E and F). Transcriptional activation seemed to underlie such ANE effects (Fig. 7E, upper panel). 0.5 lM CAY10404 treatment was sufficient to reduce COX-2 activity.26 However, with a 2 lM CAY10404 treatment, which presumably would repress the COX-2 activity, ANEassociated involucrin down-regulation was not affected in differentiated NHOKs (Fig. 7G and H). Neither the NHOK morphology nor the involucrin localization was affected.
Discussion Areca use tightly linked with the high prevalence of various oral diseases in Asians, including OPL, OSCC and OSF2 Signaling elements, including ERK, JNK and COX-2 and NFj-B, have been shown to be activated by ANE in oral keratinocytes 7,11,12,27 ANE treatment can induce the spindle-shape
Figure 6 ANE treatment disrupts the subcortical localization and involucrin expression in Ca++-induced differentiated NHOKs. (A) Fluorescence microscopy reveals that Ca++ induces NHOK aggregation and subcortical condensation of involucrin in (b) relative to control NHOK (a). (c) Concomitant ANE treatment attenuates the intense NHOK contact and continuous subcortical involucrin condensation. Cytosolic return of involucrin is also seen. (B) RT-PCR analysis reveals that Ca++ treatment induces around 6-fold increase of involucrin mRNA expression in a representative NHOK. Concomitant ANE treatments repress such increase in a dosedependent manner. (C) Quantitation of (B). (D) RT-PCR analysis reveals that 5 lg/ml ANE treatment for 24 h decreases the Ca++induced involucrin mRNA expression in a representative NHOK. (E) Quantitation of six independent NHOKs. (F) Western blotting analysis reveals that ANE treatment down-regulates the Ca++-induced involucrin protein expression in a representative NHOK. (G) Quantitation of seven independent NHOKs. Values are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Areca nut extract treatment down-regulates involucrin in normal human
677
Figure 7 ANE-associated COX-2 activation is not essential for involurcin down-regulation in Ca++-induced differentiated NHOKs. NHOKs were induced by Ca++, and concomitantly treated with 5 lg/ml ANE or not for either 1 h or 24 h. (A) A representative western blotting displays a slight increase of p-AKT following Ca++ treatment. Concomitant ANE treatment for 1 h did not cause further increase in p-AKT, but both t-AKT and p-AKT were increased for 24 h. (B) Quantitation of six NHOKs demonstrates a slight but nonsignificant increase in p-AKT following ANE treatment for 24 h. (C) A representative western blotting displays an increase of p-ERK in a Ca++-induced NHOK following concomitant ANE treatment for 24 h. (D) Quantitation of six NHOKs demonstrates a slight but insignificant increase in p-ERK following ANE treatment for 24 h. (E) Upper panel: a representative RT-PCR analysis displays an increase in COX-2 mRNA expression following concomitant ANE treatment in a Ca++-induced differentiated NHOK. Concomitant ANE treatment results in higher COX-2 mRNA expression. Lower panel, same NHOK displays an increase in COX-2 protein expression following Ca++ induction. Concomitant ANE treatment results in higher COX-2 protein expression. (F) Quantitation of six NHOKs demonstrates a significant increase in COX-2 protein expression in Ca++-induced differentiated NHOK with concomitant ANE treatment for 24 h. (G) RT-PCR analysis of a representative NHOK exhibits no change in ANE-associated involucrin down-regulation when cells are treated with CAY10404. (H) Quantitation of involucrin mRNA expression level in six control NHOKs and three CAY10404-treated NHOKs. Values are expressed as means ± SD. NS, not significant; *, P < 0.05; **, P < 0.01.
morphological changes and G1/S arrest in NHOK8,12 This study further elucidated the impacts of ANE on NHOK to address its pathogenetic roles. During the areca-associated oral carcinogenesis process, we identified the significant involucrin down-regulation occurring on OSCC and metastasis stage, although it was not so eminent in OPL. We identified ANE-associated down-regulation of involucrin expression and nuclear localization of involucrin in parabasaloid NHOK. Since involucrin takes part in intensifying cel-
lular structure by organizing the cornified envelop in the subcortical region of mature keratinocytes, our findings may lead to a hypothesis that the chronic ANE exposure in vivo can impair the barrier function oral epithelium and oral mucosa will lost protection for physical or chemical injuries.17 Activation of AKT pathway can affect multiple phenotypes of keratinocytes especially the ones that are crucial for carcinogenesis.16 We have demonstrated for the first
678 time that ANE can activate AKT in NHOK. Notably, this activation is essential for the down-regulation of involucrin, as PI3K/AKT blockers unequivocally reversed the involucrin expression. It is well-known that NF-jB is a key downstream effector of AKT28 and a paradigm for ANE-associated signaling activation.11 Thereby, whether ANE-associated NF-jB activation is involved in the down-regulation of involucrin requires more study. There is no nuclear localization signal existing in involucrin, however, Kawabata et al.29 have shown nuclear localization of involucrin in calcium-induced HaCaT keratinocyte differentiation. It is likely that certain proteins interacting with involucrin can co-migrate into nucleus following ANE treatment. However, using pull-down experiments to identify involucrin-interacting proteins could be difficult due to the low solubility of involucrin.17 Currently, the influences of nuclear involucrin localization on regulating gene expression or cellular proliferation are still unclear. When differentiation of NHOK was induced by calcium, it was noted that involucrin was highly up-regulated, accompanying with slight AKT, ERK and COX-2 activations. The up-regulation of involucrin and COX-2 seemed to occur significantly at transcriptional and post-transcriptional level, respectively. In differentiated NHOK, ANE significantly down-regulated involucrin and up-regulated COX-2. In addition, ANE also disrupted cornified envelop and segregated cells, which might counteract the differentiation process. COX-dependent pathways influencing keratinocyte differentiation have been reported.30,31 Using antagonist against COX-2, we have preliminary identified that COX-2 activity seemed to be unessential for the ANE-associated involucrin down-regulation and disruption in differentiation. Some studies have suggested that transcription factors AP1, C/EBP and Sp1 are required for differentiation-dependent involucrin expression.32,33 Another study also indicated that hypoxia can modulate the involucrin expression. We are testing if ANE can down-regulate involurcin transcription by regulating AP1, C/EBP, HIF or SP1 in SAS cells. More comprehensive characterization on the impacts of ANE-associated PLC and PKC signaling activation will provide better understanding of the adverse effects toward keratinocyte differentiation23 NF-jB signalings play central roles in regulating keratinocyte phenotypes34 NF-jB activation is also essential for ANE-associated COX-2 up-regulation in oral karatinocyte.11 Thereby, further studies are required on the involvement of NF-jB in ANE-associated dysregulation with NHOK cells. Slaked lime (calcium hydroxide) has been used as an areca quid ingredient that resulted in the high content of calcium ion and ANE in areca fluid.35 It was known that ANE can up-regulate COX-2.9 This study further specified that calcium treatment further up-regulate COX-2 to a higher degree. The preliminary findings warrant the additive effects of multiple areca ingredients to oral carcinogenesis through COX-2 activation.10 Use of COX-2 blockers may be beneficial for the abrogation of oral carcinogenesis in areca chewers. Down-regulation of involucrin is frequently accompanied with the loss of keratinocyte differentiation. It was known that tumors having poor epithelial differentiation stage would grow more rapidly, spread more diffusely, be more resistant to chemotherapy, and result in grave prognosis.20 Down-regulation in involucrin has been found in early intra-
Y.-H. Tseng et al. epithelial oncogenic stage in cervical carcinomas.19 This study provides evidences that involucrin was down-regulated in areca-associated oral tumorigenesis and ANE can down-regulate involucrin and change the involucrin localization in both parabasaloid cells and differentiated NHOKs, which might be advantageous for epithelial carcinogenic process. Besides, the AKT activation elicited by ANE being found in this study suggests the potentials of developing anti-AKT regimens for prevention of areca-associated pathogenesis.28 Although more analyses are required for advanced mechanistic understanding, the findings in this study substantiate the impairments of NHOK cells caused by areca.
Acknowledgement This study was supported by Grant VGHUST95-G1-01-2 from Veterans General Hospital-University System in Taiwan Program.
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changes, actin re-organization and signaling activation in oral keratinocytes. J Oral Pathol Med 2003;32:600–5. Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 2002;14:381–95. McCubrey JA, Steelman LS, Abrams SL, Lee JT, Chang F, Bertrand FE, et al. Roles of the RAF/MEK/ERK and PI3K/PTEN/ AKT pathways in malignant transformation and drug resistance. Adv Enzyme Regul, in press (Online available). Chen ML, Xu PZ, Peng XD, Chen WS, Guzman G, Yang X, et al. The deficiency of Akt1 is sufficient to suppress tumor development in Pten+/- mice. Genes Dev 2006;20:1569–74. Nakayama H, Ikebe T, Beppu M, Shirasuna K. High expression levels of nuclear factor kappaB, IkappaB kinase alpha and Akt kinase in squamous cell carcinoma of the oral cavity. Cancer 2001;92:3037–44. Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 2005;6:328–40. Cline PR, Rice RH. Modulation of involucrin and envelope competence in human keratinocytes by hydrocortisone, retinyl acetate, and growth arrest. Cancer Res 1983;43:3203–7. Xu XC, Mitchell MF, Silva E, Jetten A, Lotan R. Decreased expression of retinoic acid receptors, transforming growth factor beta, involucrin, and cornifin in cervical intraepithelial neoplasia. Clin Cancer Res 1999;5:1503–8. Zhang X, Hunt JL, Landsittel DP, Muller S, Adler-Storthz K, Ferris RL, et al. Correlation of protease-activated receptor-1 with differentiation markers in squamous cell carcinoma of the head and neck and its implication in lymph node metastasis. Clin Cancer Res 2004;10:8451–9. Chou SC, Azuma Y, Varia MA, Raleigh JA. Evidence that involucrin, a marker for differentiation, is oxygen regulated in human squamous cell carcinomas. Br J Cancer 2004;90:728–35. Lin SC, Wang CP, Chen YM, Lu SY, Fann MJ, Liu CJ, et al. Regulation of IGFBP-5 expression during tumourigenesis and differentiation of oral keratinocytes. J Pathol 2002;198: 317–25. Bikle DD, Ng D, Tu CL, Oda Y, Xie Z. Calcium- and vitamin Dregulated keratinocyte differentiation. Mol Cell Endocrinol 2001;177:161–71.
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24. Jans R, Atanasova G, Jadot M, Poumay Y. Cholesterol depletion upregulates involucrin expression in epidermal keratinocytes through activation of p38. J Invest Dermatol 2004;123:564–73. 25. Kaufman CK, Fuchs E. It’s got you covered. NF-kappaB in the epidermis. J Cell Biol 2000;149:999–1004. 26. Koenigsknecht-Talboo J, Landreth GE. Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines. J Neurosci 2005;25: 8240–9. 27. Jeng JH, Chang MC, Hahn LJ. Role of areca nut in betel quidassociated chemical carcinogenesis: current awareness and future perspectives. Oral Oncol 2001;37:477–92. 28. Greten FR, Karin M. The IKK/NF-kappaB activation pathway-a target for prevention and treatment of cancer. Cancer Lett 2004;206:193–9. 29. Kawabata H, Kawahara K, Kanekura T, Araya N, Daitoku H, Hatta M, et al. Possible role of transcriptional coactivator P/ CAF and nuclear acetylation in calcium-induced keratinocyte differentiation. J Biol Chem 2002;277:8099–105. 30. Tiano HF, Loftin CD, Akunda J, Lee CA, Spalding J, Sessoms A, et al. Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis. Cancer Res 2002;62:3395–401. 31. Akunda JK, Lao HC, Lee CA, Sessoms AR, Slade RM, Langenbach R. Genetic deficiency or pharmacological inhibition of cyclooxygenase-1 or -2 induces mouse keratinocyte differentiation in vitro and in vivo. Faseb J 2004;18:185–7. 32. Crish JF, Gopalakrishnan R, Bone F, Gilliam AC, Eckert RL. The distal and proximal regulatory regions of the involucrin gene promoter have distinct functions and are required for in vivo involucrin expression. J Invest Dermatol 2006;126:305–14. 33. Eckert RL, Crish JF, Efimova T, Dashti SR, Deucher A, Bone F, et al. Regulation of involucrin gene expression. J Invest Dermatol 2004;123:13–22. 34. Kaufman CK, Sinha S, Bolotin D, Fan J, Fuchs E. Dissection of a complex enhancer element: maintenance of keratinocyte specificity but loss of differentiation specificity. Mol Cell Biol 2002;22:4293–308. 35. Nair UJ, Obe G, Friesen M, Goldberg MT, Bartsch H. Role of lime in the generation of reactive oxygen species from betel-quid ingredients. Environ Health Perspect 1992;98:203–5.
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Areca nut extract treatment down-regulates involucrin in normal human oral keratinocyte through P13K/AKT activation Yu-Hsin Tseng a, Che-Shoa Chang a,b, Tsung-Yun Liu Kuo-Wei Chang a,b,*, Shu-Chun Lin a,* a b c
a,c
, Shou-Yen Kao
a,b
,
Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan Department of Dentistry Taipei Veterans General Hospital, Taipei, Taiwan Department of Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan
Received 14 July 2006; accepted 2 August 2006 Available online 25 October 2006
KEYWORDS
Summary Areca (betel) is an important etiological factor linked to the high prevalence of oral carcinoma and other oral diseases in South Asians. Involucrin is a key component of the cornified envelop and a differentiation marker of keratinocyte. In this study, we found that 5 lg/ml non-toxic areca nut extract (ANE) treatment resulted in the 0.5-fold down-regulation of involucrin and disruption in involucrin distribution in normal human oral keratinocyte (NHOK). Progressive down-regulation of involucrin during oral carcinogenesis was noted. Activation of AKT by 1.7-fold and up-regulation of COX-2 by 2-fold were elicited following ANE treatment in NHOK. Treatment with PI3K/AKT blockers reverted the down-regulation of involucrin. ANE also down-regulated involucrin by 0.6-fold and disturbed both cornified envelope and cell aggregation in calcium-induced differentiated NHOK. However, such phenomena seemed to be independent from the ANE-associated COX-2 activation. The ANE-associated down-regulation of involucrin through AKT pathway could underlie the areca-associated epithelial pathogenesis. c 2006 Elsevier Ltd. All rights reserved.
AKT; Areca; Differentiation; Involucrin; Oral carcinoma
Introduction * Corresponding authors. Address: Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan. Fax: +886 2 28264053 (K.-W. Chang). E-mail addresses: [email protected] (K.-W. Chang), sclin@ym. edu.tw (S.-C. Lin).
Around 200–400 million people in South Asian and Southeast Asian countries are addicted to areca1 Areca-associated oral squamous cell carcinoma (OSCC) is one of the leading cancers in these regions2 Areca nuts contain polyphenols, arecoline, arecaidine and other alkaloids3 Areca nut extract
1368-8375/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2006.08.003
Areca nut extract treatment down-regulates involucrin in normal human (ANE) is highly cytotoxic and genotoxic to cultured human oral epithelial cells4 Studies have identified areca ingredients as the synergistic or promoting agents for multistep chemical carcinogenesis in hamster buccal pouch models.5,6 Recently, we have demonstrated that chronic low dose of ANE treatment induced the senescence-associated phenotype of normal human oral keratinocyte (NHOK) and hyperdiploid chromosomal changes in low grade OSCC cells7,8 However, the pathogenetic roles of areca for NHOK require further elucidation. ANE can induce COX-2 expression and PGE2 production in OSCC or NHOK cells9,10 Our previous studies have shown that ANE elicited a rapid activation of ERK and JNK1 mitogenactivated protein kinase (MAPK) as well as NF-jB in OSCC cells11,12 Besides, ANE-modulated NF-jB activation could be the basis of COX-2 up-regulation associated with areca exposure11 Our study also showed that the activation of Rho and Rac GTPase could underlie the areca-mediated morphological changes of OSCC cells12 In mammals, protein kinase B or AKT (PKB/AKT) belongs to a serine/threonine kinase family consisting of PKBa (AKT1), PKBb (AKT2), and PKBc (AKT3). PKB/AKT is activated in cells exposed to diverse stimuli such as hormones, growth factors, oncogene and extracellular matrix components13 AKT is the primary mediator of PI3K signaling; it has a number of downstream effectors including Bad, procaspase-9, Ij-K, GSK-3, mTOR and so on, that can contribute to the onset of cancer.13–15 Although AKT is highly expressed in OSCC, the activation of AKT following the stimulation of ANE have not been addressed16 The interactive signaling cascades and phenotypic impacts induced by ANE in oral keratinocyte need extensive clarification. The upper epidermis or oral epithelium acts as a barrier, composing mainly of terminally differentiated cells and keratinized layer, which are the endpoint of keratinocyte. During keratinocyte differentiation, cell membrane will be progressively replaced by cornified envelope, which consists of keratins that are enclosed within insoluble protein complexes cross-linked by transglutaminases (TGMs)17 Involucrin is one of the insoluble proteins in cornified envelopes; it is abundant in the upper spinous and granular layers, and is a key marker of keratinocyte differentiation18 A report showed that involucrin expression was reduced in intraepithelial cervical neoplasms as compared with normal epithelia19 Moreover, involucrin expression was significantly downregulated in highly metastatic OSCC cell lines as compared with parental cells with low metastatic potential20 Hypoxia is an adverse status for tumor growth. Interestingly, involucrin seems to be an oxygen-regulated protein, and the majority of hypoxic cells in head and neck cancer express involucrin21 Thereby, dysregulation or loss of involucrin expression is highly associated with neoplastic process of epithelium. When cultured in a low-Ca++ (0.1 mM) medium, NHOK grows as a monolayer polygonal cell, which can be cultivated for 6 or 7 passages (for 20 population doublings)8 In the initial passage, only scarce amount of cytosolic involucrin is present, but it is abundantly dispersed in cytosol during 3–4 passages22 Conversely, when a higher concentration of Ca++ (1 mM) is used, NHOK differentiates with a squamous morphology. The regulation of keratinocyte differentiation by Ca++ is related to phospholipase C (PLC) and protein ki-
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nase C (PKC) activation, which might activate AKT and MAPK, and subsequently induce proteins required for differentiation23 Activation of p38MAPK is also involved in involucrin up-regulation in keratinocytes24 In addition, NF-jB signaling plays critical roles in regulating cell cycle withdrawal, cell survival, and differentiation in keratinocytes25 In this study, we have identified that ANE is able to downregulate involucrin by means of AKT activation, a signaling element lying downstream of PLC and upstream of NF-jB.
Materials and methods Oral tissues Sampling of tissue pairs from OSCC patients and oral precancerous lesions (OPL) for RT-PCR analysis was approved by an institutional review board. A 0.5 cm3 tissue from OSCC, the matched metastatic OSCC lesions in cervical lymph nodes (abbreviated as LNM), OPL and a non-malignant matched tissue (NMMT) were obtained from patients. Tissue samples were embedded in OCT first, stored immediately in liquid nitrogen until use. Laser capture microdissection (LCM) was performed on frozen-sectioned tissue sections to retrieve pure epithelial components of NMMT, OPL, OSCC and LNM, representing various tumorigenic stages using a PixCell II LCM system and ARC200 v1.0.2 software (Arcturus, Mountain View, CA, USA). The clinicopathological features of the subjects are summarized in Table 1.
Table 1
Clinicopathological parameters of OPL and OSCC
Parameters
OPL
OSCC
Age (years) Mean age ± SD Sex (M/F)
38–62 49.6 ± 10.8 5/0
41–79 53.0 ± 9.4 20/1
Site Buccal mucosa Tongue Other sites
0 3 2
5 7 9
Diagnosis Epithelial dysplasia Hyperkeratosis or epithelial hyperplasia
0 5
Differentiation Well Moderate and Poor
12 9
Size T1–T3 T4
4 17
Stage I–III IV
2 19
LNM Presence Absence
7 14
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Cell culture and ANE treatment Sampling of gingival tissue from donors who received flap operation to remove the impacted third molar was approved by an institute review board. NHOK was dissociated from the tissue and grown in keratinocyte serum free medium (KSFMÒ, Invitrogen, Carlsbad, CA, USA) with 0.1 mM Ca++22 In addition, cells were passaged when 70% confluence was reached to avoid the confluence-induced differentiation. By culturing with KSFM, the majority of NHOK, retaining the parabasaloid morphology prior to passage 4, were used
Y.-H. Tseng et al. for experiments. SAS OSCC cells cultivation and the preparation of ANE from ripe areca nut were preformed with protocols previously established11 In brief, ripe areca nuts were chopped, grounded and immersed to obtain the soluble extract. This soluble fraction was lyophilized and stored at 20 °C until use. Dimethyl sulfoxide (DMSO) and wortmannin were purchased from Sigma (St Louis, MO, USA). LY294002 and U0126 were purchased from EMD Biosciences Inc. (San Diego, CA, USA). CAY10404 was purchased from Cayman Chemicals (Ann Arbor, MI, USA). LY294002, wortmannin and U0126 were dissolved in DMSO for experiments.
Figure 1 ANE treatment causes morphological change and disruption of involucrin. Control and ANE-treated NHOK were subjected to inverted microscopy, fluorescence confocal microscopy, RT-PCR and western blotting analysis. (A) NHOK following ANE treatment for 24 h exhibited an increase in cell population with spindle cell morphology (a, b). A transfer of cytosolic involucrin to both cytosol and nucleus was noted (e, f) in comparison with cytokeratin (c, d). A, g and h, sustained ANE treatment for 48 h also exhibited such phenomena, with a cytosolic and nuclear localization of involucrin in a spindle-shaped NHOK. (a and b, x100; c-f, x200; g and h, x1000). (B) A representative RT-PCR analysis displays a decrease in involucrin mRNA expression following ANE treatment for 24 h. (C) Quantitation of four individual experiments indicates a significant decrease of involucrin mRNA expression upon ANE treatment. The expression of TGM1 did not affected by such treatment. (D) A representative western blotting displays a decrease in involucrin protein following 5 lg/ml ANE treatment for 48 h. (E) Quantitation of four individual NHOKs indicates a significant decrease of involucrin protein expression following ANE treatment after 48 h, despite the fact that the involucrin protein was not affected by ANE treatment after 24 h. Values are expressed as means ± SD. *, P < 0.05; **, P < 0.01.
Areca nut extract treatment down-regulates involucrin in normal human
RT-PCR Total RNA purification, cDNA synthesis and RT-PCR reaction were performed following the protocols previously established22 The primer sequences will be provided upon request. Amplicons were resolved on a 2% agarose gel, visualized and quantitated by an imaging system (Viber Lourmat, Marne La Valle, France). The signals of the tested genes, including COX-2, involucrin and TGM1, were normalized with the mRNA expression of GAPDH.
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ing; 1:500), anti-ERK (Cell Signaling; 1:500) and anti-GAPDH (Chemicon, Temecula, CA, USA; 1:5000). Secondary antibodies were horseradish peroxidase-conjugated (Chemicon; 1:1000) antibodies. The signals were detected by the Western Lightening chemiluminescence reagent plus kit (Perkin-Elmer, Wellesley, MA, USA). The densities of the signals were measured by a densitometer (Amersham, Piscataway, NJ, USA). Quantification of signals was achieved by normalization with that of GAPDH.
Statistics Immunofluoresence NHOK were fixed with 4% paraformaldehyde in PBS for 30 min and permeablized with 0.5% Triton X-100/fixative for 5 min at 25 °C. Cells were incubated with anti-involucrin (NovoCastra, Newcastle, UK; 1:500) and anti-pan-cytokeratin (Lab Vision Corp., Fremont, CA, USA; 1: 500) antibodies for 2 h at 25 °C, followed by incubation with Alexa fluorÒ 488-conjugated secondary antibodies (Invitrogen) for 30 min at 37 °C. The immunofluoresence was photographed on a Leica TCS SP2 confocal laser-scanning microscope.
Western blotting Fifty micrograms of protein from the whole cell lysate were resolved by electrophoresis on a 12.5% denaturing polyacrylamide gel. The subsequent procedures were performed following the standard protocols11 The primary antibodies used including anti-COX-2 (BD Biosciences, Franklin Lakes, NJ, USA; 1:500), anti-involucrin (NovoCastra, Newcastle, UK, 1:500), anti-phosphorylated AKT (Cell Signaling, Temecula, CA, USA; 1:1000), anti-AKT (Santa Cruze Biotech., Santa Cruz, CA, USA; 1:500), anti-phosphorylated ERK (Cell Signal-
In this study, t-test or Fisher’s exact test was used for statistical analysis. It was considered to have a statistically significant difference when P < 0.05.
Results Treatment of NHOKs to passages 63 with 5 lg/ml ANE for 24 h caused no cytotoxicity8 It elicited the elongation of NHOK from the original parabasaloid morphology in 30% of cultivated cells12 (Fig. 1A, a, b). We investigated the distribution of cytokeratin using anti-pan-cytokeratin antibody and identified no obvious change following the treatment (Fig. 1A, c, d). To find out the possible influences of ANE on NHOK differentiation, we detected the position of involucrin and TGM1. In control NHOK, involucrin was evenly and intensively localized in cytosol, whereas, it was rather loosely arranged in cytosol and nuclei of ANE treated NHOK (Fig. 1A, e, f). Magnified pictures demonstrate the phenomena more evidently (Fig. 1A, g, h). To further specify the expression of ANE-modulated involucrin, RT-PCR analysis has shown that 24 h ANE treatment significantly down-regulated involucrin mRNA expression (Fig. 1B and C). Involucrin
Figure 2 Involucrin mRNA expression in LCM-retrieved tissues. (A) A representative RT-CR analysis in tissue pairs. (B) Quantitation of involucrin mRNA expression in OPL, OSCC and LNM in comparison to paired NMMT. A horizontal line in each column indicates the median value. #, code number of cases. *, P < 0.05; ***, P < 0.001.
674 protein level in NHOK was also down-regulated significantly to 0.5-fold, when ANE treatment was applied for 48 h (Fig. 1D and E). Since cytokeratin and TGM1 did not appear remarkable changes (Fig. 1B and C) in ANE-treated cells, ANE could be a target on involucrin regulation. To determine the involurcin mRNA expression during oral carcinogenesis, LCM-retrieved pure paired epithelial cells from various oral tissues including that from 26 NMMTs, 5 OPLs, 21 OSCCs and 4 corresponding LNMs were analyzed. The normalized involurcin mRNA expression in OPL, OSCC and LNM relative to NMMT were 0.78 ± 0.20, 0.49 ± 0.09 and 0.41 ± 0.11, respectively (Fig. 2A). A statistically signif-
Y.-H. Tseng et al. icant difference in involucrin mRNA expression was noted between NMMT and OSCC (P < 0.001, paired t-test) as well as NMMT and LNM (P < 0.05, paired t-test), however, such difference was not observed between NMMT and OPL (Fig. 2B). Cases with OSCC/NMMT ratio < median were defined as having low involucrin mRNA expression. Although primary OSCC with low involucrin mRNA expression exhibited a higher propensity for less differentiation (56% vs 42%) and more LNM (57% vs 43%) compared to those had higher involucrin mRNA, involucrin mRNA expression was not associated with tumor site, differentiation, size, LNM and stage (Fisher’s exact test).
Figure 3 ANE treatment activates AKT and ERK, and up-regulates COX-2. Control and ANE-treated NHOK were subjected to RTPCR and western blotting analysis. (A) A representative western blotting displays an increase of p-AKT following ANE treatment for 0.5 h. Note the slight increase in total AKT. (B) Quantitation of three independent NHOKs shows a significant rapid increase in p-AKT following ANE treatment at 0.5 h and recovered to basal level at 2 h. (C) A representative western blotting displays an increase of pERK following ANE treatment for 24 h. Total ERK here is also increased. (D) Quantitation of two to five independent NHOKs shows a progressive increase in p-ERK following ANE treatment. (E) and (G) Representative RT-PCR and western blotting analysis of COX-2 level, respectively, following ANE treatment for 24 h in a representative NHOK. (F) and (H) Quantitation of five NHOKs indicates significant increases in COX-2 mRNA and protein expression following ANE treatment for 24 h. Values are expressed as means ± SD. *, P < 0.05; **, P < 0.01.
Areca nut extract treatment down-regulates involucrin in normal human
675
Figure 4 PI3K/AKT blockers treatment reverts ANE-associated involucrin down-regulation. NHOKs were pretreated with U0126, wortmannin or LY294002 for 1 h, and then treated with ANE for 24 h. (A) RT-PCR analysis of a representative NHOK exhibits the blockage of ANE-associated down-regulation on involucrin mRNA expression by wortmannin and LY294002. U0126 does not exhibit such blockage effect. (B) Quantitation of (A). Data shown is a representative experiment of two independent experiments.
It was known that ANE can activate MAPKs and COX2,10,11 we further investigated to find out if ANE-mediated signal activation underlies the disruption of involucrin. Western blotting demonstrated a rapid activation of AKT by 1.7-fold increased fraction of phosphorylated (p)-AKT following ANE treatment at 0.5 h (Fig. 3A and B). Whereas ERK was activated slower, which reached the peak of 1.8-fold at 24 h (Fig. 3C and D). Similarly, COX-2 was also up-regulated by ANE through transcriptional activation in NHOK (Fig. 3E–H). Ten nM wortmannin and 10 lM LY294002 treatment blocked AKT activity, and 20 lM U0126 treatment blocked ERK activity. With pretreatment of wortmannin and LY294002 for 1 h, ANE-mediated down-regulation of involucrin was prevented (Fig. 4), but the pretreatment of U0126 did not result in such reversion. However, in a representative NHOK, the nuclear localization of involucrin induced by ANE treatment cannot be reverted by AKT blockers. Other than NHOK, the down-regulation of involucrin and the activation of AKT induced by non-toxic ANE treatment (65 lg/ml) were also observed in SAS oral keratinocyte cell line (Fig. 5). Therefore, SAS cell line could be another cell model for studying the areca-associated pathogenesis. We were interested in knowing if ANE also play similar roles in down-regulating involucrin in differentiated NHOK. Calcium-induced differentiated NHOK was chosen to address this question. NHOKs were treated with 1 mM Ca++ to induce differentiation. Immunofluorescence showed that following Ca++ induction for 24 h, a representative NHOK became aggregated with the formation of cornified envelop,
manifesting as subcortical package of dense involucrin (Fig. 6A, b). In such differentiated NHOK, concomitant ANE treatment reduced the intimate cellular contact, disrupted the continuity of cornified envelop and induced the
Figure 5 ANE treatment causes AKT activation and involucrin down-regulation in SAS OSCC cells. (A) RT-PCR analysis shows the decreased involucrin mRNA expression and increased COX-2 mRNA expression in SAS cells following 5 lg/ml ANE treatment for 24 h. (B) Western blotting analysis reveals the increase in p-AKT expression following ANE treatment. The figure shown is a representative experiment of two independent experiments.
676 cytosolic returning of involucrin (Fig. 6A, c). In a representative NHOK, RT-PCR analysis shows an increase of involucrin mRNA expression for 6-fold following Ca++ induction. Concomitant ANE treatment for 24 h with increasing dosages repressed such up-regulation in a dose-dependent manner (Fig. 6B and C). Concomitant 5 lg/ml ANE treatment significantly repressed the Ca++-induced involucrin by 0.6-fold compared to the untreated cells (Fig. 6D–G). Ca++-induction slightly increased p-AKT, while concomitant ANE treatment further increased both total (t)-AKT and pAKT, but the activation did not reach a statistically significant level (Fig. 7A and B). The treatment also did not significantly trigger the activation of ERK (Fig. 7C and D). Ca++-induction significantly up-regulated COX-2 expression for 1.5-fold, and concomitant ANE treatment further upregulated COX-2 expression for additional 1.6-fold
Y.-H. Tseng et al. (Fig. 7, lower panel of E and F). Transcriptional activation seemed to underlie such ANE effects (Fig. 7E, upper panel). 0.5 lM CAY10404 treatment was sufficient to reduce COX-2 activity.26 However, with a 2 lM CAY10404 treatment, which presumably would repress the COX-2 activity, ANEassociated involucrin down-regulation was not affected in differentiated NHOKs (Fig. 7G and H). Neither the NHOK morphology nor the involucrin localization was affected.
Discussion Areca use tightly linked with the high prevalence of various oral diseases in Asians, including OPL, OSCC and OSF2 Signaling elements, including ERK, JNK and COX-2 and NFj-B, have been shown to be activated by ANE in oral keratinocytes 7,11,12,27 ANE treatment can induce the spindle-shape
Figure 6 ANE treatment disrupts the subcortical localization and involucrin expression in Ca++-induced differentiated NHOKs. (A) Fluorescence microscopy reveals that Ca++ induces NHOK aggregation and subcortical condensation of involucrin in (b) relative to control NHOK (a). (c) Concomitant ANE treatment attenuates the intense NHOK contact and continuous subcortical involucrin condensation. Cytosolic return of involucrin is also seen. (B) RT-PCR analysis reveals that Ca++ treatment induces around 6-fold increase of involucrin mRNA expression in a representative NHOK. Concomitant ANE treatments repress such increase in a dosedependent manner. (C) Quantitation of (B). (D) RT-PCR analysis reveals that 5 lg/ml ANE treatment for 24 h decreases the Ca++induced involucrin mRNA expression in a representative NHOK. (E) Quantitation of six independent NHOKs. (F) Western blotting analysis reveals that ANE treatment down-regulates the Ca++-induced involucrin protein expression in a representative NHOK. (G) Quantitation of seven independent NHOKs. Values are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Areca nut extract treatment down-regulates involucrin in normal human
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Figure 7 ANE-associated COX-2 activation is not essential for involurcin down-regulation in Ca++-induced differentiated NHOKs. NHOKs were induced by Ca++, and concomitantly treated with 5 lg/ml ANE or not for either 1 h or 24 h. (A) A representative western blotting displays a slight increase of p-AKT following Ca++ treatment. Concomitant ANE treatment for 1 h did not cause further increase in p-AKT, but both t-AKT and p-AKT were increased for 24 h. (B) Quantitation of six NHOKs demonstrates a slight but nonsignificant increase in p-AKT following ANE treatment for 24 h. (C) A representative western blotting displays an increase of p-ERK in a Ca++-induced NHOK following concomitant ANE treatment for 24 h. (D) Quantitation of six NHOKs demonstrates a slight but insignificant increase in p-ERK following ANE treatment for 24 h. (E) Upper panel: a representative RT-PCR analysis displays an increase in COX-2 mRNA expression following concomitant ANE treatment in a Ca++-induced differentiated NHOK. Concomitant ANE treatment results in higher COX-2 mRNA expression. Lower panel, same NHOK displays an increase in COX-2 protein expression following Ca++ induction. Concomitant ANE treatment results in higher COX-2 protein expression. (F) Quantitation of six NHOKs demonstrates a significant increase in COX-2 protein expression in Ca++-induced differentiated NHOK with concomitant ANE treatment for 24 h. (G) RT-PCR analysis of a representative NHOK exhibits no change in ANE-associated involucrin down-regulation when cells are treated with CAY10404. (H) Quantitation of involucrin mRNA expression level in six control NHOKs and three CAY10404-treated NHOKs. Values are expressed as means ± SD. NS, not significant; *, P < 0.05; **, P < 0.01.
morphological changes and G1/S arrest in NHOK8,12 This study further elucidated the impacts of ANE on NHOK to address its pathogenetic roles. During the areca-associated oral carcinogenesis process, we identified the significant involucrin down-regulation occurring on OSCC and metastasis stage, although it was not so eminent in OPL. We identified ANE-associated down-regulation of involucrin expression and nuclear localization of involucrin in parabasaloid NHOK. Since involucrin takes part in intensifying cel-
lular structure by organizing the cornified envelop in the subcortical region of mature keratinocytes, our findings may lead to a hypothesis that the chronic ANE exposure in vivo can impair the barrier function oral epithelium and oral mucosa will lost protection for physical or chemical injuries.17 Activation of AKT pathway can affect multiple phenotypes of keratinocytes especially the ones that are crucial for carcinogenesis.16 We have demonstrated for the first
678 time that ANE can activate AKT in NHOK. Notably, this activation is essential for the down-regulation of involucrin, as PI3K/AKT blockers unequivocally reversed the involucrin expression. It is well-known that NF-jB is a key downstream effector of AKT28 and a paradigm for ANE-associated signaling activation.11 Thereby, whether ANE-associated NF-jB activation is involved in the down-regulation of involucrin requires more study. There is no nuclear localization signal existing in involucrin, however, Kawabata et al.29 have shown nuclear localization of involucrin in calcium-induced HaCaT keratinocyte differentiation. It is likely that certain proteins interacting with involucrin can co-migrate into nucleus following ANE treatment. However, using pull-down experiments to identify involucrin-interacting proteins could be difficult due to the low solubility of involucrin.17 Currently, the influences of nuclear involucrin localization on regulating gene expression or cellular proliferation are still unclear. When differentiation of NHOK was induced by calcium, it was noted that involucrin was highly up-regulated, accompanying with slight AKT, ERK and COX-2 activations. The up-regulation of involucrin and COX-2 seemed to occur significantly at transcriptional and post-transcriptional level, respectively. In differentiated NHOK, ANE significantly down-regulated involucrin and up-regulated COX-2. In addition, ANE also disrupted cornified envelop and segregated cells, which might counteract the differentiation process. COX-dependent pathways influencing keratinocyte differentiation have been reported.30,31 Using antagonist against COX-2, we have preliminary identified that COX-2 activity seemed to be unessential for the ANE-associated involucrin down-regulation and disruption in differentiation. Some studies have suggested that transcription factors AP1, C/EBP and Sp1 are required for differentiation-dependent involucrin expression.32,33 Another study also indicated that hypoxia can modulate the involucrin expression. We are testing if ANE can down-regulate involurcin transcription by regulating AP1, C/EBP, HIF or SP1 in SAS cells. More comprehensive characterization on the impacts of ANE-associated PLC and PKC signaling activation will provide better understanding of the adverse effects toward keratinocyte differentiation23 NF-jB signalings play central roles in regulating keratinocyte phenotypes34 NF-jB activation is also essential for ANE-associated COX-2 up-regulation in oral karatinocyte.11 Thereby, further studies are required on the involvement of NF-jB in ANE-associated dysregulation with NHOK cells. Slaked lime (calcium hydroxide) has been used as an areca quid ingredient that resulted in the high content of calcium ion and ANE in areca fluid.35 It was known that ANE can up-regulate COX-2.9 This study further specified that calcium treatment further up-regulate COX-2 to a higher degree. The preliminary findings warrant the additive effects of multiple areca ingredients to oral carcinogenesis through COX-2 activation.10 Use of COX-2 blockers may be beneficial for the abrogation of oral carcinogenesis in areca chewers. Down-regulation of involucrin is frequently accompanied with the loss of keratinocyte differentiation. It was known that tumors having poor epithelial differentiation stage would grow more rapidly, spread more diffusely, be more resistant to chemotherapy, and result in grave prognosis.20 Down-regulation in involucrin has been found in early intra-
Y.-H. Tseng et al. epithelial oncogenic stage in cervical carcinomas.19 This study provides evidences that involucrin was down-regulated in areca-associated oral tumorigenesis and ANE can down-regulate involucrin and change the involucrin localization in both parabasaloid cells and differentiated NHOKs, which might be advantageous for epithelial carcinogenic process. Besides, the AKT activation elicited by ANE being found in this study suggests the potentials of developing anti-AKT regimens for prevention of areca-associated pathogenesis.28 Although more analyses are required for advanced mechanistic understanding, the findings in this study substantiate the impairments of NHOK cells caused by areca.
Acknowledgement This study was supported by Grant VGHUST95-G1-01-2 from Veterans General Hospital-University System in Taiwan Program.
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