Arecoline N-Oxide Upregulates Caspase-8 Expression in Oral

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Arecoline N-oxide upregulates caspase-8 expression in oral hyperplastic lesions of mice Ying-Chin Ko, Pei-Ying Chang, Tzer-Min Kuo, Po-Ku Chen, You-Zhe Lin, Chun-Hung Hua, and Yuan-Chien Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03999 • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 6, 2017

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Journal of Agricultural and Food Chemistry 1

1

Arecoline N-oxide upregulates caspase-8 expression in oral hyperplastic lesions

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of mice

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# Pei-Ying Chang§, , Tzer-Min Kuo, Po-Ku Chen, You-Zhe Lin♁, Chun-Hung Hua△,

4

Yuan-Chien Chen

#,

, Ying-Chin Ko* ,, §

♁

5 6 7 8 9

§

Graduate Institute of Clinical Medical Science, China Medical University, Taichung,

Taiwan. #

Department of Oral and Maxillofacial Surgery, China Medical University Hospital,

Taichung, Taiwan

10

11

China Medical University, Taichung 40402, Taiwan.

12 13 14

♁

Environment-Omics-Disease Research Center, China Medical University Hospital,

Graduate Institute of Biomedical Sciences, China Medical University, Taichung,

Taiwan. △

Department of Otorhinolaryngology, China Medical University Hospital, Taichung,

15

Taiwan

16

School of Dentistry, China Medical University, Taichung, Taiwan

17 18

*Correspondence to: Ying-Chin Ko, MD, PhD, Professor, Graduate Institute of

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Clinical Medical Science, China Medical University & Hospital, 2 Yude Road,

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Taichung, 40447, Taiwan. E-mail: [email protected]. TEL 886-4-2205-2121

21

ext.7322

ACS Paragon Plus Environment


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Abstract

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Areca nut is strongly associated with oral squamous cell carcinoma(OSCC)

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occurrence. Arecoline N-oxide (ANO), a metabolite of the areca alkaloid arecoline,

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exhibits an oral fibrotic effect in NOD/SCID mice. Caspase-8, a cysteine protease

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encoded by the CASP8 gene, is a central mediator in extrinsic apoptotic pathway via

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death receptors. Deregulation of caspase-8 in OSCC has been reported. This study

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investigates the regulation of caspase-8 in ANO-induced oral squamous epithelial

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hyperplasia which represents the initial highly-proliferative stage of oral

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carcinogenesis. CASP8 somatic mutations were identified from whole-exome

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sequencing of OSCC samples. Immunohistochemical staining showed up-regulation

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of caspase-8 in ANO-induced hyperplasia of both NOD-SCID and C57BL/6 mice.

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Expressions of CASP8, APAF-1, BAX and BAD increased in ANO-treated DOK cells.

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Co-localization of increased caspase-8 and PCNA was detected in ANO-induced

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hyperplastic lesions, whereas no co-localization between gamma-H2A.X, caspase-3

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and up-regulated caspase-8 was observed. The findings indicate that up-regulation of

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caspase-8 is involved in cell proliferation rather than apoptosis during the initial stage

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of ANO-mediated oral tumorigenesis.

39 40

Keywords: Caspase-8, areca nut, arecoline N-oxide(ANO), oral squamous cell


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carcinoma(OSCC),

42

hyperplasia.

oral

potentially

malignant

disorder(OPMD),

43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59


squamous


60

Introduction

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The areca nuts (betel quid) are the edible seeds of the areca palm (Areca catechu), a

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tropical tree widely cultivated in the tropical Pacific, southeast and south Asia, and

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parts of east Africa. In these areas including Taiwan, chewing betel quid is a prevalent

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habit and highly associated with the occurrence of oral potentially malignant disorders

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(OPMDs)1 and oral squamous cell carcinoma(OSCC).2 The areca nut has been

66

recognized as a group I carcinogen by the International Agency for Research on

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Cancer (IARC) of the World Health Organization.3 Neither oral pathologic lesions nor

68

tumor formation induced by any of the alkaloid components of the areca nut has been

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proven. Arecoline N-oxide(ANO), a metabolite of areca alkaloid, exhibits high

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activities on collagen deposition and severity of OPMD in mouse tongue, suggesting

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that arecoline N-oxide may enhance tumorigenicity.4

72 73

Oral squamous cell carcinoma (OSCC), a major type of oral cancer, accounts for more

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than 90% of all oral malignancies with an estimated 300,000 new cases and 145,000

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deaths in 2012 over the world.5 Etiological factors of OSCC include sunlight exposure,

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HPV infection, smoking, alcohol consumption and use of the areca nuts. The

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formation of OSCC is thought to be the result of multiple stepwise alterations in

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cellular and molecular pathways of the oral squamous epithelium.6 The interaction


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between genetic susceptibility and environmental stimulations like the consumption

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of alcohol, betel-quid, cigarette raises the risk of oral cancer occurrence.7 OSCC may

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be preceded by clinically evident OPMDs,8 which refer to the oral mucosal diseases

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with different rates of malignant transformation. Mild squamous hyperplasia, an

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abnormal proliferation of squamous epithelial cells, may suggest the initiation of

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carcinogenesis.9 Leaving OPMDs unmanaged clinically sometimes leads to eventual

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oral malignancies and even tumor metastases. For high-risk tumors, searching of

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biomarkers in early stages is therefore essential.10

87 88

Caspase-8, a caspase protein encoded by the CASP8 gene, is generally regarded as a

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central mediator of cell apoptosis. Activated caspase-8 regulates death-receptor

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(extrinsic) pathway of apoptosis which involves binding of FADD (Fas-associated

91

death domain-containing protein) to Fas, recruitment of pro-caspase-8, and formation

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of the death-inducing signaling complex (DISC).11 According to recent studies,

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caspase function is not only limited to inducing normal cell death.12 Somatic mutation

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of CASP8 has been reported in hepatocellular carcinomas13 and gastric cancers.14 Loss

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of caspase-8 expression has been shown to enhance metastatic potential of malignant

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neuroblastoma.15 Studies have also suggested the potential correlation between

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caspase-8 and head and neck cancers. Mutations or polymorphisms of CASP8 have



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been reported in head and neck squamous cell carcinoma(HNSCC) and OSCC.16

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Either reduced or strong positive expression of caspase-8 protein has been found in

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OSCC tissues.17 These findings all implicate that caspase-8 may play a key role in

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carcinogenesis of OSCC.

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So far, no available mechanism has been proposed to unveil the link of areca

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nut-induced OPMDs to caspase-8 expression. Considering the potential of malignant

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transformation of OPMDs, preventing them from occurrence or proceeding into

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OSCC is a topic worth investigation. Based on the intriguing findings of caspase-8 in

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HNSCC or OSCC shown in previous studies,16-17 the purpose of this research was to

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investigate the role of caspase-8 in arecoline-N-oxide-induced OPMD and hopefully

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to develop a preventive regimen of OPMD and the subsequent OSCC from

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occurrence.

111 112

Material and Methods

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Patient enrollment

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This study was conducted in Kaohsiung Medical University Hospital and China

115

Medical University Hospital, the medical center in southern and central Taiwan,

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respectively. All male patients who visited the hospital clinic age 18 or older were


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eligible for enrollment in current study. Inclusion criteria were the patients whose

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suspicious oral mucosal lesions have proven OSCC via an incisional biopsy procedure.

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The patients who were reluctant to join this study or not suitable to undergo tumor

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ablative surgery were excluded. All participants were informed of the detailed study

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protocol, required to sign consent forms and instructed to complete a standardized

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questionnaire. Tumor location and staging were recorded. Participants were asked to

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describe their personal habits, including tobacco use, alcohol consumption, and betel

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quid chewing. They agreed the surgeon to collect a small piece of cancer tissue as

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well as a small piece of normal oral mucosa during the operation, without

126

jeopardizing the surgical safe margin, for lab analysis. Each piece of the paired tissue

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samples was around 2x5mm in size.

128 129

Next generation sequencing

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Whole-exome sequencing (WES) screening for candidate somatic mutations (SMs)

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was done for genomic DNA of the paired tissues obtained from our OSCC patients,

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with the approval by the institutional review boards of Kaohsiung Medical University

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Hospital, Kaohsiung, Taiwan(KMUH-IRB-980119) and China Medical University

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Hospital, Taichung, Taiwan(CMUH103-REC2-036(CR-3)). The procedures of NGS

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data processing are modified from the pipelines previously described.18 DNA



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quantitation was determined by using gel-electrophoresis and NanoDrop 2000

137

Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA). The

138

whole-exome regions were captured utilizing SureSelect Target Enrichment System

139

(Agilent Technologies, Santa Clara, CA, USA). The raw sequencing data was

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generated by Solexa Hiseq 2000 sequencing system (Illumina Inc., San Diego, CA,

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USA). Burrows-Wheeler Aligner (BWA, v0.7.8)19 was used for mapping the

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high-quality reads to UCSC human reference genome (hg19). To identify potential

143

variants, the Genome Analysis ToolKit (GATK, v3.4.2)20 was utilized for the local

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realignment and recalibration. And the MuTect (v1.1.7)21 software was used to

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determine the potential somatic mutations. The ANNOVAR was utilized to annotate

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the functions of these variants and to obtain other information about known variants

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that were reported in the dbSNP and 1000 Genome Project databases.22 The novelty of

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SMs was assessed using the Catalogue of Somatic Mutations in Cancers (COSMIC

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v.81).23 The potential variants were validated using Sanger sequencing with designed

150

specific primers.

151 152

Chemicals

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The chemical agents, areca nut extract (ANE) and arecoline N-oxide, used in this

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study were prepared as previously described.4, 24


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Animals and treatment

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The tongue tissue samples from NOD.CB17-Prkdcscid/NcrCrl (NOD/SCID Mouse)

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animals of age 10 weeks treated with 500 µg/ml arecoline N-oxide (n=10) by cotton

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swab smearing the oral cavity for 21 weeks were prepared as previously described.4

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Since NOD/SCID mice are genetically immune-deficient and might be more prone to

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uncontrollable gene-regulation, the same procedure was also conducted in

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immune-normal C57BL/6 mice. For treatment of arecoline N-oxide, three

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C57BL/6JNarl mice of age 11 to 12 weeks, obtained from the National Laboratory

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Animal Center, were maintained in a specific pathogen-free environment. All mice

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were treated with 8.77 mM arecoline N-oxide dissolved in 0.1 % DMSO/deionized

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water (1.5mg/ml) by cotton swab smearing the oral cavity for once daily, five days per

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week for 20 weeks. During treatment, a standard laboratory diet was allowed to offer

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to all mice during treatment. In this study, all animal procedures conformed to the

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guidelines published by the National Institutes of Health (NIH Publication No. 85-23)

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and were also approved by the Institutional Animal Care and Use Committee (IACUC)

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of the China Medical University, Taichung, Taiwan.

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Cell culture and cell viability


Journal of Agricultural and Food Chemistry

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To see the transcripts of pro-apoptotic genes in both oral dysplastic and cancer cells,

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DOK (human dysplastic oral keratinocyte cell) and CAL27 (ATCC® CRL2095™) cell

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lines were purchased from Bioresource Collection and Research Center (BCRC,

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Taipei, Taiwan). DOK and CAL27 cells were maintained in Dulbecco’s modified

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Eagle’s medium (DMEM) and DMEM-F12 respectively, supplemented with 10%

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fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. The cell viability was

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assessed using MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation

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Assay, Promega, Madison, WI, USA) according to the manufacturer’s protocol.

182 183

Immunohistochemistry analysis

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For observation of the pathological changes, tongue tissue samples from all treated

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mice were fixed with 4% paraformaldehyde for 3 days, embedded in paraffin. The

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3-µm sections were stained with hematoxylin & eosin (H&E) and specific antibodies

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for histological evaluation. Used primary antibodies for immunohistochemical

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detections

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anti-cleaved-caspase-8

190

anti-cleaved-caspase-3

191

(anti-phospho-histone H2A.X, Ser139; Cell Signaling Technology, Danvers, MA,

192

USA), and anti-Ki67 (SP6, abcam, Cambridge, UK). According to the manufacturer’s

were:

anti-PCNA (Cell

(EPR3821,

Signaling

(Abcam,

abcam,

Technology, Cambridge,


Cambridge,

UK),

Danvers,

MA,

USA),

UK),

anti-γ-H2A.X

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brochure, the anti-cleaved-caspase-8 antibody specifically targets cleaved caspase-8.

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The positive signals of PCNA, cleaved-caspase-8, caspase-3, γ-H2A.X and Ki67 were

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observed in a high power field (200× and 400× magnification). In this study, results of

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all immunohistochemical were interpreted by two pathologists.

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Real-time quantitative reverse transcription PCR (RT-qPCR)

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For determination of genes expressions, cells were seeded and grew confluent for

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chemicals treatment. Cells were treated with arecoline N-oxide as indicated dose for

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48 hours. The concentration of arecoline-N-oxide used for cell treatment is described

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in the legends of Fig. 2 and 3. The cDNA templates production from total RNA of

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treated cells was as previously described.4 The cDNA were then subjected to

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RT-qPCR analysis with the specific primer pairs performed by an ABI StepOnePlus™

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Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA) using Fast

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SYBR® Green Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania). Used

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primer sequences were for human Casp8, APAF-1,BAX, BAD.

208 209

Western blotting

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To detect expression of caspase-8, APAF-1, BAX, BAD, and Actin, the cells were

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lysed in Pierce® RIPA buffer (Thermo Scientific, Rockford, IL, USA) containing a



212

cocktail of protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) on ice for

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10 minutes. The extracts were centrifuged at 15600 ×g for 5 min at 4°C to sediment

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the insoluble fraction. Protein samples were separated by SDS-polyacrylamide gel

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and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica,

216

MA, USA). The membranes were incubated with primary antibodies as indicated and

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peroxidase-conjugated secondary antibodies, protein signals detected by enhanced

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chemiluminescence reagent (Millipore, Billerica, MA, USA). The primary antibodies

219

used were anti-cleaved-caspase-8, anti-APAF-1, anti-BAX and anti-BAD antibodies

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obtained from Cell Signaling Technology (Danvers, MA, USA).

221 222

Statistical analysis

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Fisher’s exact test was used to examine the difference of tumor location and adverse

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oral habits between grouped OSCC participants. For cell culture experiments, the

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statistical differences were evaluated using the Student’s t-test and unpaired Student’s

226

t-test accordingly.

227 228

Results

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Human samples: OSCC patient characteristics and WES analysis

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The paired tissues from 14 OSCC patients were obtained to conduct WES analysis.


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The information of 1 patient’s (ID 0063) lesion location, tumor staging and adverse

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oral habits was lost whereas complete NGS results were still well kept (Fig. S1). WES

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revealed 4 somatic mutations in CASP8 across 14 (28.6%) OSCC samples, including

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1 (25%) synonymous and 3 (75%) missense mutations. The distribution of mutations

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in the coding region was depicted as Table 1, with one missense mutation (p.P31L)

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occurring in the first death effector domain (DED1), one synonymous mutation

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(p.S226S) occurring in the linker between DED2 and the large subunit of peptidase

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(p18), and two missense mutations (p.L365F and p.P369S) occurring in the p18

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subunit. The characteristics of OSCC patients were shown in Table S1. The mean age

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of OSCC patients with somatic mutations(SM group) and OSCC patients with wild

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type (WT group) of CASP8 were 64.7±5.0 and 51.8±10.9 years old, respectively. 4

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samples out of the 10 OSCC patients in WT group were derived from buccal mucosa,

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3 from the tongue, and 3 from other sites. Meanwhile, 2 samples of the 3 OSCC

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patients in SM group were from buccal mucosa while the other, tongue. Between the

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two grouped participants, no significant difference was observed regarding cancer

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location, tumor staging and adverse oral habits. These results confirmed that CASP8

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somatic mutations can present in OSCC.

248 249

Animal study: Caspase-8 upregulation in the sublingual squamous hyperplastic



250

lesion of immune-deficient NOD/SCID mice

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Since OSCC may originate in high-risk OPMDs, this finding evoked our curiosity to

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see caspase-8 expression in OPMDs. To assess the level of caspase-8 protein in

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OPMD, immunohistochemical staining with anti-caspase-8 antibody for sublingual

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squamous hyperplastic lesions of arecoline N-oxide-treated NOD/SCID mice were

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performed. A higher expression of cleaved-caspase-8 protein in the pathologically

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hyperplastic part than in the adjacent normal part was measured (Fig. 1A, 1B). The

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results indicate that the expression of activated caspase-8 protein was up-regulated in

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the arecoline N-oxide-induced sublingual squamous hyperplasia of NOD/SCID mice.

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Animal study: Caspase-8 upregulation in the sublingual squamous hyperplastic

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lesion of immune-normal C57BL/6 mice

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NOD/SCID mice are genetically immune-deficient and might be more prone to

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uncontrollable gene-regulation. Therefore, we made an attempt to induce oral

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squamous hyperplastic lesions in immune-normal C57BL/6 mice. 2 of the 3 mice

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treated with 1.5mg/ml (8.77mM) arecoline N-oxide for 20 weeks developed squamous

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hyperplasia at sublingual tongue. The immunohistochemical staining showed

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substantial upregulation of cleaved-caspase-8 protein in the pathologically

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hyperplastic part of sublingual tongue, similar to the findings in NOD/SCID mice


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(Fig. 1C, 1D).

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Cell study: arecoline N-oxide increases the transcripts of pro-apoptotic genes

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As mentioned above, caspase-8 protein upregulation was noticed under microscopic

273

examination. We wondered the mRNA expression of CASP-8 and associated

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pro-apoptotic genes. RT-qPCR of arecoline N-oxide-treated DOK(human dysplastic

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oral keratinocyte cell) and CAL27 cells was conducted to examine the expression of

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pro-apoptotic genes including CASP-8, APAF-1, BAX and BAD which are all

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important genes regulating intrinsic apoptosis pathway. After 48 hours of treatment

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with either 0.1% DMSO/deionized water, arecoline N-oxide 200μM or 400μM, the

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total RNA of the abovementioned genes was estimated by RT-qPCR. For DOK cells,

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arecoline N-oxide stimulation significantly increased the mRNA expression of

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CASP-8, APAF-1, BAX and BAD at 400 μM( p < 0.05, vs. DMSO)(Fig. 2). Viability

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of the DOK cells treated with arecoline N-oxide 400 µM for 48 hours did not show

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significant difference compared to DMSO group(Fig. 3A). Arecoline N-oxide also

284

enhanced CASP-8 expression at 400 μM( p < 0.05, vs. DMSO) in CAL27 cells(Fig.

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3B).

286 287

Cell study: arecoline N-oxide increases the expression of pro-apoptotic proteins



288

After checking the mRNA level, the protein expression of caspase-8, APAF-1, BAX

289

and BAD were further examined. DOK cells treated for 48 hours. Areca nut here was

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used as a positive control. Arecoline N-oxide significantly increased levels of APAF-1,

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cleaved-caspase-8, and BAX proteins at concentration of 400 μM, whereas BAD

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protein was also trend to increase in arecoline N-oxide–treated cells. Arecoline

293

N-oxide exhibited higher activity on APAF-1 and cleaved-caspase-8 up-regulation

294

than areca nut. (Fig. 4).

295

APAF-1, BAX and BAD transcripts in DOK cells.

The results confirm arecoline N-oxide effects on caspase-8,

296 297

Animal samples: up-regulation of PCNA and Ki67 proteins in the sublingual

298

squamous hyperplastic lesion

299

Hyperplasia is excessive cell proliferation. Proliferating cell nuclear antigen (PCNA)

300

and Ki67 proteins are well-known cell proliferation markers, and are up-regulated in

301

the tissues that have progressed from normal oral epithelium to hyperplastic and

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premalignant and malignant lesions of the oral cavity.25 Here, sectioned tongue tissue

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samples from arecoline N-oxide-treated NOD-SCID mice were immunostained with

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anti-PCNA and anti-Ki67 antibodies. PCNA and Ki67 protein expression was

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enhanced in the cell nuclei of squamous hyperplastic lesions (Fig. 5). The results

306

confirm the proliferative condition in the sublingual hyperplastic tissues in response


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to arecoline N-oxide treatment.

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Animal samples: co-localization of PCNA and caspase-8 proteins in the

310

squamous hyperplasia

311

To see the distribution of PCNA and caspase-8 in cells, the sublingual tissue samples

312

from NOD-SCID mice were co-stained immunochemically with anti-PCNA and

313

anti-caspase-8 antibodies. Co-localization of PCNA and caspase-8 proteins was seen

314

(Fig. 6A). The result suggests that the fate of the cells presenting with caspase-8

315

upregulation in the sublingual squamous hyperplastic tissues may go toward

316

proliferation rather than apoptosis.

317 318

Animal samples: negative expression of caspase-3 in caspase-8-up-regulated cells

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Caspase-8 is generally regarded as an important molecule involved in cell apoptosis.

320

In some cell types, the autocatalytic activation of caspase-8 at the DISC turns

321

pro-caspase-3 into active caspase-3 via proteolysis, resulting in mitochondria-

322

independent apoptosis.11a To further observe the status of caspase-3 in caspase-8-

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up-regulated cells, co-immunohistochemical staining of anti-caspase-8 and anti-

324

caspase-3 antibodies for the sublingual tissue samples of NOD/SCID mice was

325

performed. The result showed negative expression of caspase-3 protein in the main



326

hyperplastic lesions where caspase-8 protein expressed robustly (Fig.6B). It indicates

327

that the up-regulation of caspase-8 had no effect on triggering the apoptosis cascade.

328 329

Animal samples: no co-localization of γ-H2A.X and caspase-8 proteins in the

330

squamous hyperplasia

331

Phosphorylation of histone H2A.X at serine 139 (γ-H2A.X) is a sensitive marker of

332

DNA double-strand breaks (DSBs). Co-staining with anti-γ-H2A.X and anti-caspase-8

333

antibodies for the sublingual samples from NOD-SCID mice showed a small amount

334

of scattered cells positively expressing γ-H2A.X. No co-localization of these two

335

proteins was seen. The result implicates that the cells positively expressing caspase-8

336

might not meanwhile be under stress leading to DSBs(Fig. 6C).

337 338

Discussion

339

Mutations or polymorphisms of CASP8 have been reported in head and neck

340

squamous cell carcinoma(HNSCC) and OSCC.16 CASP8 polymorphism was observed

341

in Han Chinese OSCC patients16c whose genetic profile may be close to Taiwanese

342

population. Since the interaction between genetic susceptibility and environmental

343

stimulations can raise the risk of oral cancer occurrence,7 different carcinogens may

344

be the reason for different findings between the studies while Taiwanese OSCC


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patients mostly chew betel quid as an adverse oral habit in addition to smoking. A

346

recent study by Su et al. using whole-exome sequencing (WES) to examine

347

Taiwanese male OSCC samples reported a result of 23.3%(28/120) of CASP8

348

mutation, including 12 missense, 8 nonsense, 6 indel and 1 multiple mutations.26

349

Comparably, the current research showed somatic mutation of CASP8 in 28.6% (4/14)

350

of the studied individuals with 1 synonymous and 3 missense mutations (Fig. 1). It is

351

no surprise that the two studies carried out in the same ethnic group of similar adverse

352

oral habits have yielded comparable results with respect to mutation rate of CASP8, as

353

well as implicated missense mutation as the most common type of genetic alteration

354

in Taiwanese OSCC victims. To date, exome sequencing technique has become a

355

commonly-used, practical tool for genomic analysis, which is key to precision

356

medicine. Via genomic sequencing, a handful of genetic mutations have been linked

357

to OSCC of Taiwanese.5, 26-27 However, few studies have focused on the relationship

358

between CASP-8 and OPMDs which thus still serves as an enigmatic area to explore.

359 360

No oral cancerous lesion to date has been experimentally induced by any alkaloid

361

components of areca nut. Concerning the potential tumorigenicity of this edible seed,

362

areca nut extract (ANE) has been shown to inhibit repair of DNA double-strand

363

breaks.2b Arecoline N-oxide(ANO), a metabolite of areca nut alkaloids, also has been



364

reported to induce OPMDs.4 The chemical composition of areca nut is complicated

365

and the components may enhance the formation of OPMDs or oral malignancies via

366

different genetic impacts than other carcinogens. The correlation between betel-quid

367

chewing and the prevalence of OPMDs has been studied in Taiwan,1 but the

368

mechanism of them proceeding into oral cancer is still unclear. Leukoplakia,

369

erythroplakia and oral submucous fibrosis(OSF) are the most common types of

370

OPMDs with different rates of malignant transformation.28 Pathological diagnosis of

371

an OPMD ranges from squamous cell hyperplasia to squamous cell carcinoma

372

depending on the cellular characteristics under microscopic examination. Positive

373

caspase-8 expression was reported by Shi et al. in oral leukoplakia of 85.7% (12/14)

374

of the studied subjects, the potential role of caspase-8 in the occurrence and

375

progression of oral precancerous lesions was suggested.17a Due to the small number of

376

patients willing to join the current research program, we turned to animal models to

377

see caspase-8 expression. Although no cancerous lesion was developed, sublingual

378

squamous hyperplasia as an OMPD in both immune-deficient and -normal mice was

379

induced by arecoline N-oxide(ANO). To our surprise, up-regulation of activated

380

caspase-8, a molecule mainly thought to regulate cell apoptosis, was noticed.

381

Meanwhile, negative expression of caspase-3, a downstream molecule of caspase-8 in

382

the apoptosis pathway, was identified in the lesions. Cell proliferation in hyperplastic


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tissues was confirmed by up-regulation of PCNA and Ki67 proteins in this study. In

384

addition to an increase in the transcripts of CASP-8, APAF-1, BAX and BAD in

385

ANO-treated-precancerous cells, the data of corresponding proteins also showed a

386

trend of upregulation. Putting together, the cells expressing high level of caspase-8

387

were undergoing proliferation rather than apoptosis. The increased mRNA of

388

pro-apoptotic genes and corresponding proteins may not be ready to drive

389

precancerous DOK cells to death. In a study of T-cell saw that the caspase-8 inhibitor,

390

c-FLIPL, can modulate T-cell proliferation in vivo by decreasing the T-cell receptor

391

signaling threshold.29 FLIP may play a crucial role in turning signals for T-cell death

392

into those for cell survival via activation of NF-κB and Erk.30 With respect to oral

393

squamous cells, nevertheless, it takes more research to verify the exact functional role

394

of caspase-8 and the associated pro-apoptotic genes in OPMDs. The animal model

395

used in present study could probably be further modified to induce other OPMDs for

396

more research.

397 398

DNA can be damaged by environmental factors such as ionizing radiations and

399

chemicals which induce the formation of DNA double-stranded breaks (DSBs). DSBs

400

should be repaired in a timely fashion to prevent the disruption of genome integrity

401

which contributes to cancer development.31 γ-H2A.X is formed by phosphorylation of



402

histone H2A.X at serine 139 and appears to be a sensitive marker of DSBs. In the

403

present study, cells positively expressing γ-H2A.X were identified in mice OPMD,

404

indicating that these cells may be under stress from arecoline N-oxide stimulation and

405

undergoing genome damage. On the contrary, the cells positively expressing

406

caspase-8 did not show the signs of DSBs. It is possible that caspase-8 not only

407

involves in triggering apoptosis cascade just as its most well-known molecular

408

function, but also plays a protective role in maintaining DNA integrity for

409

ANO-stimulated oral squamous cells.

410 411

In conclusion, the finding of this research in regards to caspase-8 expression in

412

squamous hyperplasia is interesting. Previous studies have suggested caspase-8

413

involves in suppressing cellular necrosis, promoting differentiation, regulating

414

autophagy, and promoting cellular migration32, it is possible that the multifunctional

415

caspase-8 might promote the pathogenesis of some cancer-related or precancerous

416

lesions. Further studies should be conducted to find the mechanism how an OPMD

417

proceeds into an OSCC.

418 419

Abbreviations Used: ANO, Arecoline N-oxide; OPMD, oral potentially malignant

420

disorders; OSCC, oral squamous cell carcinoma; DOK, human dysplastic oral


Page 22 of 45

Page 23 of 45


421

keratinocyte cell; WES, whole-exome sequencing.

422 423

Acknowledgment

424

This study is supported by Taiwan Ministry of Health and Welfare surcharge of

425

tobacco products MOHW106-TDU-B-212-144003, Taiwan, and Ministry of Science

426

and Technology MOST105-2314-B-039-019,

427

China medical university & Hospital DMR-107-032, Experiments and data analysis

428

were performed in part through the use of the Medical Research Core Facilities Center,

429

Office of Research & Development at China Medical University Hospital, Taichung,

430

Taiwan.

MOST106-2314-B-039-016-MY3,

431 432

Competing financial interests

433

The authors declare that they have no competing interests.

434 435

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Figure legends

577

Figure 1. Arecoline N-oxide increased caspase-8 expression in the squamous

578

hyperplasia lesions of NOD/SCID and C57BL/6 mice. NOD/SCID and C57BL/6

579

mice were consecutively treated with arecoline N-oxide (500 µg/ml for NOD/SCID

580

and 1.5 mg/ml for C57BL/6) for 21 and 20 weeks, respectively. Sectioned tongue

581

tissues were stained with H&E staining and anti-caspase 8 monoclonal antibody.

582

Over expression of caspase-8 (brown) was observed in the sublingual hyperplastic

583

part of NOD/SCID(B) and C57BL/6 mice(D) but not seen in the adjacent normal

584

part(A, C). (Magnification, 200×; Scale bar, 50 µm.).

585 586

Figure 2. RT-qPCR for DOK cells: after 48 hours of treatment with either 0.1%

587

DMSO/deionized water, arecoline N-oxide 200µM or 400µM, total RNA of four

588

apoptotic genes was estimated. Arecoline N-oxide stimulation increased the

589

expression of CASP-8, APAF-1, BAX and BAD at 200 and 400 µM(*p < 0.05, vs.

590

DMSO).

591



592

Figure 3. (A) MTS assay: viability of the DOK cells treated with arecoline N-oxide

593

400 µM for 48 hours did not show significant difference compared to DMSO group.

594

(B) Arecoline N-oxide enhanced CASP-8 expression in CAL27 cells at 200 and 400

595

µM(*p < 0.05, vs. DMSO).

596 597

Figure 4. Western blotting procedures were conducted to identify the protein

598

expression of caspase-8, APAF-1, BAX and BAD in DOK cells treated for 48 hours

599

with either 0.1% DMSO/deionized water, areca nut extract(ANE) 200 ug/ml,

600

arecoline N-oxide(ANO) 200µM or 400µM. (A) The result showed a significant

601

increase of cleaved-caspase-8 at ANO 400 µM, and a slight increase of

602

cleaved-caspase-8 at ANO 200 µM and APAF-1 at ANO 400 µM. (B) Signals were

603

quantified by densitometry analysis. The graphs represent mean (±SD) values, and

604

each experiment was performed more than three times in triplicate. (∗) p < 0.05, vs.

605

DMSO group; (#) p < 0.05, vs. ANE; ($) p < 0.05, vs. ANO 200 µM.

606 607

Figure 5. Comparison of Ki67 and PCNA expression in ANO-induced squamous

608

hyperplasia. Significantly higher expression of both proteins in the hyperplastic areas

609

than the normal part.

610

(C), (D) Normal and hyperplastic lesion with PCNA staining. (magnification, 400×;

(A), (B) Normal and hyperplastic lesion with Ki67 staining.


Page 32 of 45

Page 33 of 45


611

Scale bar, 50 µm)

612 (A) ANO induced upregulation and co-localization of caspase-8(green)

613

Figure 6.

614

and PCNA(brown) in mice sublingual squamous hyperplasia.

615

caspase-3(brown) expression in ANO-induced lesion where caspase-8(green)

616

expressed robustly. (C) Both γ-H2A.X(brown) and caspase-8(green) were upregulated

617

but no co-localization was observed. (Magnification, 400×; Scale bar, 20 µm).

618 619 620 621 622 623 624 625 626 627 628 629


(B) Absence of


Page 34 of 45

34

Tables Table 1.

WES analysis : CASP8 somatic mutations in 4 out of 14 OSCC samples,

including 1 synonymous and 3 missense mutations.

Patient

Gene

140092

Chr

Position

Ref Alt

Variant type

mRNA change

AA change

COSMIC

CASP8 2

202139643

G

A

synonymous

c.G678A

p.S226S

COSM3713585

140093

CASP8 2

202149778

C

T

missense

c.C1093T

p.L365F

COSM3713588

0063

CASP8 2

202149790

C

T

missense

c.C1105T

c.P369S

COSM4308566

0069

CASP8 2

202131301

C

T

missense

c.C92T

p.P31L

a. All genetic coordinates were mapped to GRCh 37.p13 (NC_000009.11) annotation release 105 and used human transcript annotation imported from Ensemble database release 75, and the somatic mutations were compared with COSMIC database v81. b. Nucleotide and amino acid numbers grounded on GenBank accession numbers NM_001228 and NP_001219, respectively


Page 35 of 45


Figures Figure 1.

Figure 2.



Figure 3.

Figure 4.


Page 36 of 45

Page 37 of 45


Figure 5.

Figure 6.



TABLE OF CONTENTS GRAPHICS


Page 38 of 45

Page 39 of 45


Figure 1. Arecoline N-oxide increased caspase-8 expression in the squamous hyperplasia lesions of NOD/SCID and C57BL/6 mice. NOD/SCID and C57BL/6 mice were consecutively treated with arecoline Noxide (500 µg/ml) for 21 and 20 weeks, respectively. Sectioned tongue tissues were stained with H&E staining and anti-caspase 8 monoclonal antibody. Over expression of caspase-8 (brown) was observed in the sublingual hyperplastic part of NOD/SCID(B) and C57BL/6 mice(D) but not seen in the adjacent normal part(A, C). (Magnification, 200×; Scale bar, 50 µm.) 264x198mm (150 x 150 DPI)



Figure 2. RT-qPCR for DOK cells: after 48 hours of treatment with either 0.1% DMSO/deionized water, arecoline Noxide 200µM or 400µM, total RNA of four apoptotic genes was estimated. Arecoline N-oxide stimulation increased the expression of CASP-8, APAF-1, BAX and BAD at 200 and 400 µM(*p < 0.05, vs. DMSO). 142x86mm (144 x 144 DPI)


Page 40 of 45

Page 41 of 45


Figure 3. (A) MTS assay: viability of the DOK cells treated with arecoline N-oxide 400 µM for 48 hours did not show significant difference compared to DMSO group. (B) Arecoline N-oxide enhanced CASP-8 expression in CAL27 cells at 200 and 400 µM(*p < 0.05, vs. DMSO). 160x67mm (144 x 144 DPI)



Figure 4. Western blotting procedures were conducted to identify the protein expression of caspase-8, APAF-1, BAX and BAD in DOK cells treated for 48 hours with either 0.1% DMSO/deionized water, areca nut extract(ANE) 200 ug/ml, arecoline N-oxide(ANO) 200µM or 400µM. (A) The result showed a significant increase of cleaved-caspase-8 at ANO 400 µM, and a slight increase of cleaved-caspase-8 at ANO 200 µM and APAF-1 at ANO 400 µM. (B) Signals were quantified by densitometry analysis. The graphs represent mean (±SD) values, and each experiment was performed more than three times in triplicate. (∗) p < 0.05, vs. DMSO group; (#) p < 0.05, vs. ANE; ($) p < 0.05, vs. ANO 200 µM. 251x129mm (144 x 144 DPI)


Page 42 of 45

Page 43 of 45


Figure 5. Comparison of Ki67 and PCNA expression in ANO-induced squamous hyperplasia. Significantly higher expression of both proteins in the hyperplastic areas than the normal part. (A), (B) Normal and hyperplastic lesion with Ki67 staining. (C), (D) Normal and hyperplastic lesion with PCNA staining. (magnification, 400×; Scale bar, 50 µm)

174x123mm (144 x 144 DPI)



Figure 6. (A) ANO induced upregulation and co-localization of caspase-8(green) and PCNA(brown) in mice sublingual squamous hyperplasia. (B) Absence of caspase-3(brown) expression in ANO-induced lesion where caspase-8(green) expressed robustly. (C) Both γ-H2A.X(brown) and caspase-8(green) were upregulated but no co-localization was observed. (Magnification, 400×; Scale bar, 20 µm).

238x181mm (150 x 150 DPI)


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Table of Contents 121x44mm (144 x 144 DPI)


Arecoline N-Oxide Upregulates Caspase-8 Expression in Oral

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