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«Yi-Chen Li (李宜珍)1, Ann-Joy Cheng (鄭恩加)2 1 Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, 333, Taiwan 2 ...»

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Gene expression profiling of oral cancer cells chronic exposed to

areca nut extract

Yi-Chen Li (李宜珍)1, Ann-Joy Cheng (鄭恩加)2

1

Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, 333, Taiwan

2

Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan 333,

Taiwan

Abstract

Oral cancer is the 6th most frequent cancer in Taiwan. The habit of areca nut

chewing is the main etiological factor of oral cancer. To shed light on molecular basis of areca nut associated oral carcinogenesis, we established two oral cell sublines chronically treated with areca nut extract (ANE) at IC70 dose for 2 months. Affymatrix microarray was used in transcriptome profiling between parental and ANE sublines of oral cancer cells. Algorithmic analysis was applied to analyze the network regulatory pathways. RT-PCR was used to validate the genes altered expressions in ANE-sublines. Total of 35 genes was differentially expressed in both sublines. Several functional pathways were apparently altered, with lipid metabolism (P=1.9510-10), oxidative phosphorylation (P=1.0210-6), and cell adhesion (P=9.6610-6) most significant. Seven genes were confirmed over 2-fold of changes, including HMGCS1, KRT-17 up-regulation and SMC4, CENPF, ID-1, IL1-alpha and Ches1 down-regulation. Further study revealed Ches1 was down regulation upon arecoline treatment. Consistently, this gene was reduced expression in 52% of oral cancer tissues, which was significantly correlated with areca nut chewing habit of patients (p = 0.04). Thus, these results provide information regarding the molecular mechanisms of areca nut-induced oral carcinogenesis.

Background Oral Cancer/ oral cavity cancer Oral cavity includes lips, inside lining of the lips, cheeks (buccal mucosa), teeth, gums, the front two-thirds of the tongue, the floor of the mouth below the tongue, the bony roof of the mouth (hard palate), and the area behind the wisdom teeth.

Oral cancer (ORC) is one of the ten most frequent cancers worldwide.

According to the Oral Cancer Foundation report, an estimated 481,000 new cases were diagnosed annually on 2007. The global estimates from the WHO in the year 2000 have reported an incidence rate for ORC of 14.27 per 100,000. In Taiwan, the mortality rate of ORC is significantly and most increased from 1999 to 2008. The incidence of oral cancer is also sixth leading cancer and still increasing in the recent years. Especially, oral cancer is forth leading cancer in male, appearing that males have high risk suffering oral cancer. Previous study indicates 90% of male oral cancer patient with betel quid chewing and smoking1.

Risk factor of oral cancer- betel quid The habits of alcohol drinking, betel quid chewing, and cigarette smoking have been documented as risk factors for oral precancerous lesions and oral cancer2. Patient combining alcohol drinking, betel quid chewing, and cigarette smoking have 123-fold increase of suffering oral cancer. Moreover, betel quid chewer has 28-fold to suffer oral cancer compared with control, and it is highest risk than other risk factors in Taiwan3.

Betel quid (BQ) is generally known as the complex of areca nut, lime, catechu, piper betel inflorescence, piper betel leaf and so on. It is an old oral habit, and extensively chews in many Asian countries, especially in South and Southeast Asian.

There are about 600 million betel quid chewers, more than 15% of the total population, the 4th most popular oral habit in the world. In the South Asian and

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Southeast Asia which mostly used mature betel fruit, the Taiwan chewer commonly use fresh unripe betel fruit and with slaked lime as an essential ingredient 6-7.

Areca nut extract Among the betel quid, areca nut extracts have been considered to be the major etiologic factors in the pathogenesis of oral cancer and other oral diseases 8. From NHRI report in 2000, areca nut extract is composed of 0.15-0.67% alkaloids, 11.4-26% polyphenols, 1.3-17% fats, 47.2-26% saccharide, unclear percentage of crude fiber and rare tannins. Considerable evidence suggests that alkaloids of areca nut are the major factors for gentoxicity9. Arecoline is the most abundant component of alkaloids. In saliva, arecoline can transfer to arecadine by enzyme of saliva 10. The alkaloids in areca nut extract can undergo nitrosation reactions and the nitroso-derivatives in the oral cavity have been suggested as constituting the primary cause of oral mucosal lesions 11.

Areca nut extract (ANE) is highly cytotoxic and genotoxic to cultured human oral mucosal epithelial cells and fibroblasts. ANE can cause DNA-protein cross-links,

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migration and invasion mainly. There have been some evidences that ANE associate migration and invasion. Matrix metalloproteinases (MMPs) on salivary may participate in tumor invasion and migration. Areca quid consumption stimulates

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areca nut should be highly suspected as a human carcinogen.

CHES1 Using differentiation display technique, we have previously identified several

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Compared to tumor and normal mucosa tissue, CHES1 was found at least 2-fold under-expressed in 46% of the tumor tissue samples. Therefore, CHES1 may lose its function during oral cancer development.





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locates on chromosome 14q24.3-q31, determined by using of the CA repeat in the 3’ untranslated region (UTR) of the mRNA. This repeat was found to be highly polymorphic in human DNA. Initial mapping determined linkage of this marker (named CCC1) with 14q32, which was further refined to the region between 14q24.3 24 and 14q31. CHES1 encodes 2667 nucleotides. The ATG and TAA codons of the open reading frame are 1473 bp. CHES1 encodes a protein of 490-amino acid with predicted molecular mass of 54 kDa. This gene shows a small but significant region of homology to HTLF (human T-cell leukemia enhancer factor), a member of the fork head/Winged Helix family 23,25. There are 51% identical and 69% conserved residues between HTLF and CHES1. In addition to the highly conserved DNA binding domain, there are significant regions of homology between these two proteins in both upstream and downstream of this motif. Therefore, CHES1 is a member of the fork head/Winged Helix subfamily of transcription factor, which include HTLF, rodent WHN, and FKHR 23.

It has been reported that CHES1 is responsible for G2 arrest of the cell cycle after DNA damage and via an MEC1-independent checkpoint pathway. In study of S.

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interacts with Sin3, a member of the S. cerevisiae Sin3/Rad3 histone deacetylase complex (HDAC). Whereas, CHES1 can not suppress the DNA damage response in Sin3 mutant strains, and over-expression of Sin3 blocks CHES1-mediated G2/M arrest after DNA damage 26. Therefore, CHES1 may possess function by inhibition of Sin3 in HDAC effects. However, the function of CHES1 in mammalian cells was not

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Preparations of areca nut extract (ANE) The fresh areca nuts were smashed with sterilized water by juicer, and percolated by strainer. Then, those ANE was filtrated by Grade NO.1 qualitative filter paper (Whatman, UK). The filtrate was lyophilized by FreeZone 2.5 Liter Benchtop Freeze Dry System (Labconco, USA), and re-dissolved in sterilized water. After being filtrated by 0.22 μm filters, ANE became as working solution for training cultured cells and was stored at -20℃.

Liquid chromatography According to previous study, we modified their condition of HPLC to detect our samples 27. Analyses were performed on Waters 2690 Separations Module HPLC system (Waters, USA) with C18 reversed-phase column (Sunfire). Buffer A consisted of 50% acetonitrile plus 50% 0.01M NaH2PO4 with 0.01% TEA. Buffer B consisted of 10% acetonitrile plus 90% 0.01M NaH2PO4 with 0.01% TEA. Mobile phase consisted of 50% buffer A and 50% buffer B (v/v). All chromatographic solvents were degassed with helium before use. Isocratic chromatography was 1 mL/min at 30◦C.

Arecoline (sigma) and arecadine (sigma) were used as the standards for ANE quality control. Using 2.5 μg/ml, 5 μg/ml and 10μg/ml arecoline established standard curve to monitor quantity of arecoline of ANE. Finally, using UV detector detected those compounds on 215 nm.

Establishment of ANE-trained sublines OECM1 and SAS were used and maintained, as previously described.11,12 For each cell line, the IC30 dose of areca nut extract was determined by calculating the concentration at which there was 70% cell viability after 24 hours of treatment. The areca nut extract–trained sublines were established by chronically treating cells with areca nut extract at IC70 doses for 30 passages.

Affymextrix Microarray and Functional Network Analysis The RNA extraction and microarray analyses were similar, as previously described28. Briefly, the Affymetrix U133A microarray and scanning systems were used. The functional network analyses of differentially expressed genes were performed using the MetaCore Analytical suite (GeneGo, St Joseph, MI). MetaCore is a webbased computational platform that provides cluster analysis of gene expression data in the context of regulatory networks and signaling pathways. MetaCore was used to calculate the statistical significance (p-value) based on the probability of assembly from a random set of nodes (genes) of the same size as the input gene list.

Cloning of Ches1 Full-Length Plasmid and Cellular Transfection Full-length open reading frame of Ches1 was produced by RT-PCR.

Polymerase chain reactions (PCRs) were carried out with 35 cycles of denaturation at 94℃for 1 minute, annealing at 58_C for 1 minute, and extension at 68_C for 2

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5’-GGTACCAATGGTCCAGTCATG-3’ and 5’-TCTAGATTAATTTTTTGTGGTTTCCTTTT-3’. The full-length Ches1 gene was cloned in-frame into pGEM-T easy vector (Promega, Fitchburg, WI) to obtain pGEMChes1 plasmid and verified by direct sequencing. The full-length Ches1 fragments were subcloned into pFlag-CMV-2 vector (Sigma, St Louis, MO) using KpnI/XbaI restriction enzyme site to obtain pFlag-Ches1 plasmids. The oral cancer cells, SCC25, were transfected in 100-mm dishes with the mixture 6 lg plasmid DNA and 6 lL Lipofectamin 2000 (Invitrogen, Carlsbad, CA) in 3 mL OPTI-MEM medium (Gibco, Langley, OK) and incubated in 37℃, in a 5% CO2 incubator for 10 hours. After this, the medium was replaced with fresh complete medium and continuously cultured. Cell numbers were determined daily.

Flow Cytometry Analysis Cells transfected with pFlag-Ches1 or the vector plasmids were harvested at a density of 8 *105/mL and washed with phosphate-buffered saline (PBS). The cell pellets were fixed with ice-cold 70% ethyl alcohol in PBS at 20℃ for 1 hour and then centrifuged at 1500 revolutions per minute for 5 minutes. The pellets were suspended and incubated with 0.5% Triton X-100 (Sigma Chemical Co) and 0.05% RNase (Sigma) in 1 mL PBS at 37_C for 30 minutes and then centrifuged at 1500 revolutions per minute for 5 minutes. These cell pellets were resuspended and incubated with 40 mg/mL propidium iodide in 1 mL PBS at room temperature for 30 minutes. Samples were analyzed by FACScan flow cytometry (Becton Dickinson, San Jose, CA). The distribution of cell cycle phases was determined using Cell Quest Pro and ModiFit software.

Patients and clinical tissues Fifty- two consecutive patients seen at the Otorhinolaryngology or Head and Neck Surgery clinics at Chang Gung Memorial Hospital (Taoyuan, Taiwan) were enrolled. Written informed consent was obtained from all subjects prior to this study, and this study was approved by the Institutional Review Board at Chang Gung Memorial Hospital. The characteristics of these head and neck cancer patients were summarized in the Table 1. There were 48 (92%) males and 4 (8%) females. The mean age was 49.8 years, ranging from 30 to 78. A total of 34 (65%) consumed alcohol, 44 (85%) smoked tobacco, and 43 (83%) chewed betel quid. Biopsies of cancer and grossly normal mucosa tissue were obtained from each subject before chemo- or radiotherapy. Part of the diseased tissue was dissected and frozen immediately in liquid nitrogen until used for molecular assays. The remaining sample was fixed in formalin and processed for routine histopathologic examination. All patients had undergone a series of clinical evaluations, including assessment of tumor extension before treatment and the tumor response to therapy. All cancers were histologically graded as well differentiated, moderately differentiated, poorly differentiated, or undifferentiated according to the World Health Organization (WHO) classification. The diagnosis, clinical staging, and identification of the anatomic site of the HNC were based on the American Joint Committee on Cancer (AJCC) TNM Classification of Malignant Tumors.

Reverse transcription and quantitative RT-PCR The RNA extraction and quantitative RT-PCR analysis was performed similarly as previously describe (12, 13). Briefly, Total RNA was extracted by using TRIzol reagent (Gibco BRL, Rockville, MD). Ten micrograms of total RNA was reverse transcribed using poly-T primer. Quantitative PCR reactions were performed using an ABI Prism 5700 Sequence Detection System (Perkin-Elmer Applied Biosystems). The primers used for each specific gene are listed in Table 2. All assays were performed in duplicates with less than 5% of variation. For an internal control, we derived a Ct using Ribosome 18S RNA expression. Ct was the difference in the Ct values derived from the specific gene being assayed and the 18S control, while Ct represented the difference between the paired tissue samples, as calculated by the formula Ct = Ct of normal tissue - Ct of tumor tissue. The value of the expression of a specific gene of a tumor sample compared to the normal counterpart was expressed as 2Ct. A gene differential expression in tumor tissue over 2-fold of the normal counterpart was defined as significance difference.

Statistical Analysis The Pearson chi-square test was used to examine the association of Ches1 expression and clinicopathologic features, including TNM stage and histopathologic characteristics. All P values were two-sided, and the significance level was set at a P

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