Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells
© Giri et al; licensee BioMed Central Ltd. 2010
Received: 15 December 2009
Accepted: 6 July 2010
Published: 6 July 2010
Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells.
Using the human Ishikawa endometrial cancer cell line, we investigated the effects of arecoline on expression, localization and functional connections between the ZO-1 tight junction protein and the HER2 EGF receptor family member. Treatment of Ishikawa cells with arecoline coordinately down-regulated expression of both ZO-1 and HER2 protein and transcripts in a dose dependent manner. Biochemical fractionation of cells as well as indirect immunofluorescence revealed that arecoline disrupted the localization of ZO-1 to the junctional complex at the cell periphery. Compared to control transfected cells, ectopic expression of exogenous HER2 prevented the arecoline mediated down-regulation of ZO-1 expression and restored the localization of ZO-1 to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid reported to up-regulate expression of HER2 in Ishikawa cells, precluded arecoline from down-regulating ZO-1 expression and disrupting ZO-1 localization.
Arecoline is known to induce precancerous lesions and cancer in the oral cavity of betel nut users. The arecoline down-regulation of ZO-1 expression and subcellular distribution suggests that arecoline potentially disrupts cell-cell interactions mediated by ZO-1, which may play a role in arecoline-mediated carcinogenesis. Furthermore, our study has uncovered the dependency of ZO-1 localization and expression on HER2 expression, which has therefore established a new cellular link between HER2 mediated signaling and apical junction formation involving ZO-1.
Areca nut (Areca catechu Linn) chewing in the form of betel quid is popular in southeast Asian countries and plays a major role in the pathogenesis of precancerous lesions and several cancer of the oral cavity, including precancerous lesions such as leukoplakia and oral submucous fibrosis [1, 2]. Epidemiological studies also indicate adverse birth outcome including spontaneous abortion, still birth, low birth weight and birth length reduction among pregnant women who consumed betel quid during pregnancy [3, 4]. The meconium, urine and cord serum of newborns whose mother chewed betelquid during pregnancy was found to contain arecoline as detected by mass spectrometric assays. Arecoline and its derivatives are being used clinically to treat Alzheimer's disease based on their use as centrally active muscarinic agents .
The mechanism of arecoline mediated carcinogenesis in the oral cavity is not fully understood. However, there are reports which indicate that arecoline induces immunodepression, hepatotoxicity and depression of natural antioxidants such as superoxide dismutase, catalase, reduced glutathione and glutathione-s-transferase that are known to neutralize reactive oxygen species in mice . Arecoline has also been found to elicit mutagenicity, genotoxicity, cytotoxicity and chromosomal aberration in different biological systems , and has been shown to mediate the cell cycle arrest, ROS generation, change in the mitochondrial membrane potentials in oral mucosal fibroblasts and oral KB epithelial cells . Furthermore, arecoline was recently reported to alter metallothionein-1  and Heme Oxygenase-1 expression [11, 12] in clinicopathological profile of oral submucous fibrosis samples. Our earlier study shows that arecoline is metabolized to N-oxide of arecoline in mouse in vivo and human in vitro, which is Flavin monooxygenase-1 dependent [13, 14]. Thus, exposure to arecoline has pleiotropic responses in a variety of tissue types that together account for its carcinogenic properties.
Relatively little is known about the potential cellular effects of arecoline on plasma membrane associated signaling components in human cancers. Two types of plasma membrane signaling components that can be altered in transformed cells are apical junction proteins involved in regulating cell-cell interactions and members of specific tyrosine kinase receptors. Tight junctions comprise the more apical structure of junctional complexes that restrict solute diffusion along the paracellular space conferring barrier properties to epithelial and endothelial sheets. Loss of normal junctional formation and cell-cell interactions is thought to play an important role in cancer progression due to significant changes in epithelial compartmentalization and the tissue microenvironment. A key component of junctional complexes that regulates tight junction formation is zonula occludens-1 (ZO-1) . Z0-1 is a 220 kDa protein member of the MAGUK (membrane-associated guanylate kinase homologs) gene family that interacts directly with the transmembrane protein occludin, with ZO-2 and with AF- 6, a target of the ras oncogene, which is involved in acute myeloid leukemia . ZO-1 is an important marker for tight junction integrity, which is disrupted in many intestinal diseases and highly invasive cancer types, and has been shown to be down regulated in poorly differentiated, highly invasive breast cancer cell lines . Immunohistochemical analysis revealed a gradual decrease of ZO-1 protein from normal breast tissue to well differentiate to moderately differentiate to poorly differentiate human breast cancer tissue samples .
HER2 is a transmembrane tyrosine kinase receptor that is a member of the epidermal growth factor (EGF) receptor gene family [19, 20] that is expressed at high levels in several human cancers including in late stage endometrial carcinomas and other reproductive cancers [20–22]. Expression of the HER2 gene has been extensively studied in a variety of ovarian and breast adenocarcinomas, with most studies correlating HER2 overexpression with a poor prognosis. Steroid hormones can alter the expression of HER2 in these two types of tumors. For example, in human neoplastic mammary cells estrogens inhibit HER2 expression , whereas, in ovarian adenocarcinoma cells glucocorticoids exert a stabilizing effect on existing HER2 transcripts .
In the present study, we have established in human Ishikawa endometrial cancer cells that arecoline downregulates expression and disrupts the junctional localization of ZO-1 in a process that requires the downregulation of HER2. Our findings implicate a role for HER2 signaling in the arecoline disruption of apical junction organization in human cancer cells, and have uncovered a new cellular link between HER2 and the control of ZO-1 expression and localization.
Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), calcium- and magnesium-free phosphate-buffered saline, L-glutamine and trypsin-versene mixtures were purchased from Biowhittaker (Walkersville, MD). Insulin (bovine) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St Louis, MO). Arecoline hydrobromide was purchased from Aldrich (Milwaukee, WI). The sources of other reagents are either listed below were of the highest purity available. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Invitrogen.MG132 and Dexamethasone were purchased from Sigma Chemical Co.
Ishikawa human endometrial adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). Ishikawa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% Fetal bovine Serum, 10 μg/ml bovine insulin and 50 U/ml penicillin, 50 U/ml streptomycin and 2 mM Lglutamine. The cells were grown to subconfluency in a humidified air chamber at 37°C containing 5% CO2. Arecoline (99.9% high-performance liquid chromatography grade) was dissolved in appropriate concentrations in DMSO. DMSO was used as vehicle control for all experiments. All the experiments utilized cultured Ishikawa cells in passage 25 to passage 28.
Western Blot Analysis
After the indicated treatments, cells were harvested in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% deoxycholate, 0.1% NoNidet-p40 (Nonidet P-40, Flulta Biochemitra, Switzerland), 0.1% SDS, 50 mM Tris) containing protease and phosphatase inhibitors (50 g/ml phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 5 g/ml leupeptin,0.1 g/ml NaF, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 0.1 mM_-glycerol phosphate). These extracts were then quantified using the Lowry Method (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total cellular protein were mixed with loading buffer (25% glycerol, 0.075% SDS, 1.25 ml β-mercaptoethanol,10% bromphenol blue, 3.13% 0.5 M Tris-HCl, and 0.4% SDS (pH 6.8) and fractionated on 10% polyacrylamide/0.1% SDS resolving gels by electrophoresis. Spectra Multicolor Broad range Protein Ladder from Fermentas life sciences was used as the molecular weight standard. Proteins were electrically transferred to nitrocellulose membranes (Micron Separations, Inc., Westboro, MA). Equal protein loading was confirmed by Ponceau S staining of blotted membranes. Proteins were blocked for one and half hour at room temperature with Western wash buffer-5% NFDM (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20, 5% nonfat dry milk). Protein blots were subsequently incubated for overnight at 4 degree temperature with antibody in western buffer. The antibodies used were rabbit anti-ZO-1 (Invitrogen); rabbit anti-Claudin-1 (Santa Cruz Biotechnology); rabbit anti-E-cadherin (Santa Cruz Biotechnology); rabbit anti-beta-catenin (Santa Cruz Biotechnology); and rabbit anti-HER2/neu (Santa cruz Biotechnology). The working concentration for all antibodies was 1 μl/ml in Western wash buffer. Immunoreactive proteins were detected after incubation with horseradish peroxidase conjugated secondary antibody diluted to 0.25 μl/ml in Western wash buffer (goat anti-rabbit IgG and rabbit anti-mouse IgG (Bio-Rad). Blots were treated with ECL western blotting detection reagent (GE healthcare) and detected on the high performance chemiluminescence film (GE healthcare, UK).
Reverse Transcription PCR
Ishikawa cells were harvested in PBS and total RNA was isolated. RNA was quantified. 5 μg of total RNA was subjected to reverse transcription using murine myelogenous leukemia reverse transcriptase with First strand Buffer, random Primer (hexamers), dNTPs. 2 μl of cDNA was then subjected to PCR using Platinum Taq, 10 × PCR buffer, and 200 μM each dNTP (Invitrogen) along with the following primer sets and conditions: HER2 Forward 5'-CCAGCTCTTTGAGGACAACT - 3' and Reverse 5'-ATGTCCTTCCACAAAATCGT- 3', and the cycling conditions were 30 seconds at 95°C followed by 30 seconds at 52°C for annealing and finally 30 seconds at 72°C for extension for 26 cycles. ZO-1 Forward 5'-CGAGTTGCAATGGTTAACGGA-3' and Reverse 5' -TCAGGATCAGGACGACTTACTGG- 3', and the cycling conditions were 30 seconds at 95°C followed by 30 seconds at 55°C for annealing and finally 30 seconds at 72°C for extension for 26 cycles. GAPDH primers 5'-TGAAGGTCGGAGTCAACGGATTTG-3', GAPDH Reverse: 5'-CATGTGGGCCATGAGGTCCACCAC-3' (Ambion, Austin TX) served as a control, and PCR was performed according to the manufacturer's instructions. The PCR products were run on 1.1% agarose gels with Ethidium bromide along with a 1-kb plus DNA ladder (Invitrogen).
Indirect Immunofluorescence Assay
For indirect immunofluorescence assays, cells were grown on two well chamber slides from Nunc (Fisher scientific, Rochester, NY). The cells were fixed with 3.75% formaldehyde in PBS for 20 min on ice. After three additional washes with PBS, the plasma membrane was permeabilized with 0.1% Triton X-100; 10 mM Tris HCl at PH 7.5, 120 mM NaCl; 25 mM KCl; 2 mM EGTA; and 2 mM EDTA for 10 min at room temperature. Cells were incubated with 3% Bovine serum albumin (Sigma) in PBS before incubation with primary antibodies. Rabbit anti-ZO-1 antibody (61-7300 from Invitrogen) and rabbit anti-E-Cadherin (C212 from Santa Cruz Biotechnology) were used at a 1:400 dilution. Secondary Alexa 488 anti-rabbit (Molecular Probes, Inc., Eugene, OR) were used at a 1:400 dilution. Stained cells were mounted with Vectashield Mounting media containing DAPI (Vector Laboratories, Inc., Burlingame, CA). Stained and mounted cells were then processed with a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Thornwood, NY).
Transfection of Ishikawa cells
To generate stably transfected cells, Ishikawa cells at passage number 25, were transfected with either 0.2 μg of CMV-neo empty vector or CMV-HER2 (CMV empty vector and CMV-HER2 were generously provided by the laboratory of Dr. Bjeldanes, UC Berkeley, CA, USA), using polyfact (Qiagen, CA) and following the manufacturer's suggested protocol. Cells were fed 24 h after transfection with DMEM, supplemented with 10% fetal calf serum, penicillin/streptomycin. The media was replaced with same media containing 0.7 mg/ml G418 (neomycin analog, Mediatech, Herndon, VA) to select for transfected cells. Selection media was replaced every 24 hours for a month and surviving cell populations were propagated in selection media. Experimental treatments were not performed in selection media.
The nuclear and nonnuclear subcellular fractions were harvested from cell extracts using the NE-PER Nuclear Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer's instruction. The total protein was quantified using Bradford reagents (BioRad). Cell fractions were examined by Western blots as described above. Anti-lamin was used as a marker for nuclear fraction.
Effects of Arecoline on expression of the ZO-1 tight junction protein and the HER2 tyrosine kinase receptor
To determine if the arecoline-induced loss of HER2 and ZO-1 protein was due to ubiquitin-26 S proteasome mediated degradation, Ishikawa cells were treated with or without 0.3 mM arecoline for 24 hr and 48 hr in the presence or absence of MG132, an inhibitor of proteasome peptidase enzymatic activity. As shown in Figure 1B, western blot analysis indicated that the downregulation of both ZO-1 and HER2 protein strongly occurs in the presence of MG132, suggesting that the loss of both proteins are not due to proteasomal degradation.
Arecoline Downregulates ZO-1 and HER 2 Transcript Levels in Ishikawa endometrial cancer cells
Effects of Arecoline on expression of Tight Junction and Adherens Junction proteins
Arecoline disruption of the localization of ZO-1 protein
Expression of exogenous HER2 prevents the arecoline down-regulation of ZO-1 and overrides the disruption of ZO-1 localizalization in Ishikawa cells
Dexamethasone treatment overrides the arecoline disruption of ZO-1 localization in Ishikawa cells
We have established that arecoline has profound effects on plasma membrane associated signaling proteins in the human endometrial Ishikawa cell line. Arecoline was shown to coordinately down regulate the expression and disrupt localization of the ZO-1 tight junction component of the apical junction complex as well as decrease expression of the HER2 member of the epidermal growth factor receptor gene family. Our studies have uncovered a functional link between the arecoline down regulation of ZO-1 and HER2 because expression of exogenous HER2 completely prevents the ability of arecoline to disrupt ZO-1 expression and localization to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid that has been shown to upregulate HER2 expression in Ishikawa endometrial cancer cells , also overrides the disruptive effects of arecoline on ZO-1 localization. A functional connection between HER2 levels and the control of ZO-1 localization or expression has not been previously observed in human cancer cells.
HER2 plays an important role in the regulation of cell growth, differentiation and survival through its heterodimerization with other members of the EGF receptor gene family . A variety of cell and tissue types expresses HER2 , and a number of human cancers frequently over-express HER2 due to gene amplification including many reproductive cancers [21, 30–33] as well as lung, gastric and oral cancers [34–39]. Patients with HER2-overexpressing breast or ovarian cancer have significantly shorter overall survival rate and time of relapse relative to patients with tumors without HER2 overexpression [21, 30, 31]. Because of HER2 overexpression in many cancers, its accessible location on the cell surface and its role in carcinogenesis HER2 has been under intensive scrutiny as a therapeutic target. HER2 is expressed at low levels in normal tissue compared to cancer cells , which suggests the existence of a suitable therapeutic window to minimize damage to normal cells but still be able to target HER2-positive cancers by inhibiting either HER2 protein function or expression .
Studies examining ZO-1 protein stability have uncovered a range of ZO-1 protein half lives (ranging between 5 and 20 hours) that can differ depending on the cell type and cell cultured conditions such as cell confluency [42, 43]. Although in many systems, regulated changes in the stability of ZO-1 protein can potentially play a role in its cellular regulation, we have shown that in Ishikawa endometrial cancer cells, the loss of ZO-1 protein is accounted for an a corresponding loss in ZO-1 transcript levels. We have also determined that arecoline concurrently reduces HER2 protein and transcript expression along with that of ZO-1 expression, and that ectopic expression of HER2 reverses the arecoline down regulation of ZO-1. We are currently attempting to establish the precise mechanism by which the arecoline-mediated loss of HER2 levels leads to these effects on ZO-1 utilization. In this regard, is thought that over-expression of HER2 in human cancer cells due to amplification enhances the preferential binding of the low-affinity arm of ligands to HER2 resulting in increased intracellular signaling  that could ultimately lead to the control of ZO-1 and potential regulation of ZO-1 mediated cell-cell interactions. Interestingly, a transcriptional factor that binds to the SH3 domain of ZO-1 (ZONAB, ZO-1-associated nucleic acid binding protein) was shown in MDCK cells to functionally interact with the nuclear form of ZO-1 to modulate expression of HER2 in a cell density dependent manner . This study, in combination with our results, suggests that the expression and cellular use of ZO-1 and HER2 may be linked thorough a mutual feedback system in certain human cancer cells. Because dexamethasone, a synthetic glucocorticoid, regulates the transcription of glucocorticoid receptor target genes and overrides the effects of arecoline on ZO-1 localization, it is tempting to speculate that this steroid hormone alters the transcriptional dynamics of HER2 in this system and thereby stabilizes ZO-1 expression and localization.
Arecoline induced cellular changes in the oral cavity in areca nut chewers leading to oral precancerous lesions may be due to disrupted expression and junctional localization of the ZO-1 tight junctional protein. Furthermore, we have established that the ability of arecoline to control ZO-1 in human Ishikawa cancer cells requires the coordinate down regulation of the HER2 member of the EGF receptor gene family. This observation represents a previously unknown functional connection between HER2 expression and the cellular accessibility of ZO-1. Thus, the physiological control of HER2 expression in human tissues may play a direct role in the susceptibility of humans to the carcinogenic effects of arecoline.
The research described in this paper was supported by National Institute of Health Grant DK-42799 (to G.L.F.) and by Indo-US Science and Technology Forum Fellowship by Department of Science and Technology, Government of India, awarded to S. G. (Ref:IUSSTF/Fellowship/2007-08/8-2008). We are thankful to Dr. Crystal Marconett and Dr. Ida Aronchik for helpful discussion. Technical support of Bhumika Kapadia and Kelvin Tran in the Firestone Lab during the course of study is thankfully acknowledged.
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