Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status

Accepted Manuscript Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status

Shanmei Du, Kui Liu, Peng Gao, Zhongyou Li, Jie Zheng PII: DOI: Reference:

S0024-3205(18)30585-X doi:10.1016/j.lfs.2018.09.033 LFS 15954

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

30 July 2018 15 September 2018 18 September 2018

Please cite this article as: Shanmei Du, Kui Liu, Peng Gao, Zhongyou Li, Jie Zheng , Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status. Lfs (2018), doi:10.1016/j.lfs.2018.09.033

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Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status Shanmei Dua1, Kui Liua,b1, Peng Gaoc,d, Zhongyou Lie, Jie Zhenga, Department of Pathology, Medical School of Southeast University, Nanjing 210009, China

b

Center of Translational Medicine, Zibo Central Hospital, Zibo 255036, China

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Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of

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a

Philadelphia, Philadelphia, PA19104, USA d

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Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia,

e

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Philadelphia, PA 19104, USA

Department of Molecular Oncology, Jiangsu Institute of Cancer Research, Nanjing, 210009,

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Both authors contributed equally to this work.

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China

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,

Corresponding author:

Dr. Jie Zheng

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Department of Pathology

Medical School of Southeast University

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Nanjing 210009, China

Telephone: (86) 13913820330, Fax: 86-25-3317073 E-mail: [email protected]

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ABSTRACT Aims: Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) are caused by human papillomavirus (HPV) infection. The effect of arsenic trioxide (ATO) on HPV oncogene expression of HNSCC cells remains unknown. In this study, we investigated the anti-cancer activity and possible molecular pathways of ATO on the six HNSCC cell lines (three HPV-positive

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and three HPV-negative). Methods: The effects of ATO on the cell proliferation, apoptosis, cell cycle of HNSCC cells were

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analyzed using CCK-8 assay, colony formation and flow cytometry. Transwell assay was used to examine the effect of ATO on cell migration. The transcriptional and protein expression of key

we assessed the effects of ATO on HNSCC cells in vivo.

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genes were determined by real-time PCR and Western blot, respectively. Using a xenograft model,

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Key findings: HPV-positive and -negative HNSCC cells had different expression of key genes. ATO inhibited HNSCC cell proliferation and migration and induced apoptosis and these effects

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were more significant in HPV-positive HNSCC cells than in HPV-negative HNSCC cells. ATO treatment reduced the expression of HPV16-E6/E7 and cyclin D1 proteins and enhanced the

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expression of p16, pRb, and p53 in HPV-positive HNSCC cells. By contrast, ATO treatment

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reduced the expression of epidermal growth factor receptor, cyclin D1 and mutant p53 and enhanced the expression of pRb in HPV-negative HNSCC cells. Anti-cancer effect of ATO on HNSCCs was confirmed by inhibiting xenograft growth in vivo.

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Significance: Our data suggest that ATO is a potential therapeutic drug for HNSCCs, especially

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HPV-positive HNSCCs.

Keywords:

Apoptosis, Arsenic trioxide, Cell proliferation, Head & neck squamous cell carcinoma, Human papillomavirus

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1. Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer death worldwide [1]. While early-stage HNSCC can often be cured with surgery and adjuvant radiation, the 5-year survival of patients with lymph node metastases is low [2]. In the past decade, the incidence of HNSCC associated with known risk factors, such as tobacco use, alcohol use and

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poor oral hygiene, has been declining [3]. However, human papillomavirus (HPV) infection has been recognized as a new risk factor for HNSCC, particularly in oropharyngeal tumors, and more

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than 20% of HNSCCs contain HPV DNA, especially HPV 16 [4]. HPV-positive and -negative HNSCCs represent different clinicopathological and molecular entities, and HPV-positive HNSCC

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patients have a better prognosis [5,6].

Since its successful use for the treatment of acute promyelocytic leukemia in China [7],

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arsenic trioxide (ATO) has been extensively studied, and its potential application has been extended to solid cancers [8-11]. A study by Zheng et al. indicated that ATO induced apoptosis in

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HPV-positive cervical epithelial cells by down-regulating HPV E7 expression [12]. In a previous study, we systematically investigated the effect of ATO on the proliferation of cervical cancer cells

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with different HPV states and explored the underlying anti-cancer mechanisms by investigating

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the effects of ATO on HPV oncogene expression [13]. However, the effect of ATO on HPV oncogene expression of HNSCC cells remains unknown. In the present study, we aimed to investigate the effects of ATO on cell proliferation, apoptosis, migration and certain key genes

HNSCC cells.

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involved in the molecular biology of HNSCC and to assess the effects of ATO on xenografts of

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2. Materials and methods

2.1. Cell culture and reagents HNSCC cell lines SCC-4 and CAL-27 were obtained from Nanjing Medical University; SCC-25 was obtained from the Tumor Research Institute of the Second Military Medical University; and UM-SCC-47, UPCI-SCC-090, and 93-VU-147T were kindly supplied from Dr. Randall J Kimple at University of Wisconsin - Madison, USA. The human cervical cancer cell line SiHa (HPV16-positive) was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and was used as the HPV-positive control. The basic information regarding the HNSCC cell lines used in this study is listed in Table 1. UM-SCC-47, 3

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UPCI-SCC-090, 93-VU-147T and SiHa cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM) (HyClone). SCC-4, SCC-25 and CAL-27 cells were cultured in DMEM/F-12 (HyClone). All media were supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco-BRL), 100 units/ml penicillin and 100 μg/ml streptomycin (Beyotime, Shanghai, China). Table 1 Basic information on the head and neck cancer cell lines used in this study.

46

Male

57

SCC-4

Male

55

SCC-25

Male

70

CAL-27

Male

56

Ton gue Ton gue Ton gue

+

Chr.9q 31.1; 12p3

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+

Refs .

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Chr.3q 28

TP53/CDNK2 A status

15

Wild type

[1417]

+

739

Wild type

[1417]

Chr.17 q21; 3p21; 5p15.3 3

+

58

Wild type

[1417]

-

-

-

0

Mutated

-

-

-

0

Mutated

-

-

-

0

Mutated

+

[1819] [1819] [1819]

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93-VU-14 7T

Floo r of mou th

+

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Male

53

Episo me

HPV16 copies/β-g lobin copy

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UPCI:SC C-090

Male

Late ral tong ue Base of tong ue

HPV DNA integra tion site

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UM-SCC47

A ge

Gen der

HPV16 E5/E6 /E7

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Cell line

Site of orig in

Abbreviations: HPV, human papillomavirus; Chr, chromosome.

ATO was purchased from Sigma-Aldrich Chemicals and initially dissolved in phosphate-buffered saline (PBS) to a concentration of 10-3 M and stored at 4°C. 2.2. CCK-8 assay The proliferation inhibitory effects of ATO on cancer cells were assessed using the CCK-8 kit

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(Beyotime) according to the manufacturer’s protocol. A total of 2 × 103 cells/well were seeded in 96-well plates, and each group consisted of six parallel wells. The optical density (OD) at 450 nm was measured using a microplate reader (BioTek). Cell viability (%) = ODdosing group mean/ODcontrol group mean

× 100.

2.3. Flow cytometry analysis

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The apoptosis and cell cycle kits (Beyotime) were used according to the manufacturer’s protocol, respectively. In the apoptosis assay, the cells were double-stained with Annexin

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V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) in the dark at room temperature for 10 min. In the cell cycle assay, the cells were harvested, fixed in 70% ethanol, and then stained

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with PI for 30 min at 4°C in the dark. The stained cells were analyzed using a FACSCalibur flow cytometer (BD).

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2.4. Colony formation assay

The cells (1 × 103 cells/well) were plated in 6-well plates and allowed to attach overnight.

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Then the medium was replaced with fresh medium with and without ATO and maintained for 10 days. The medium was changed every three days. At the end of the growth period, the cells were

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fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. The

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cell colonies were imaged, and the number of colonies was counted for statistical analysis. 2.5. Cell migration assay

After treatment with 6 μM ATO for 24 h, the HNSCC cells (5 × 104) were suspended in 0.1 ml

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of medium without FBS and seeded in the upper chamber of a Transwell insert. Then, medium containing 10% FBS was added to the lower chamber as a chemoattractant. Cells that had

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migrated to the lower chamber at 24 h were fixed and stained with 0.1% crystal violet. Three low-magnification areas were randomly selected, and the number of migrated cells was counted. 2.6. Reverse transcription and quantitative PCR (RT-qPCR) Total RNA was extracted from cells using RNAiso Plus (TAKARA, Dalian, China) according to the manufacturer’s instructions. RT-qPCR was performed using a SYBR-Green-based PCR kit (TAKARA) with an Applied Biosystems StepOnePlus Real-Time PCR system (ABI). β-actin was used as an internal control. The comparative Ct method was applied to determine the fold-difference in expression levels relative to a control sample. The primer sequences are listed in Table 2. 5

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Table 2. Specific primers for target and control genes. Forward primer (5ʹ–3ʹ)

EGFR

AGAGGATGTTCAATAACTGTGAGGT G

AGGGCAATGAGGACATAACCAG

CDKN2 A

GGCCTCCGACCGTAACTATTC

GAGCAGCATGGAGCCTTCG

CCND1

CTTGAGGGACGCTTTGTCTG

TGGAAACATGCCGGTTACAT

TP53

CAGTCTACCTCCCGCCATAA

CAAGGGTTCAAAGACCCAAA

Rb

ACCCTTACGGATTCCTGGAG

TAGGAGGGTTGCTTCCTTCA

GCATCCACAACATTACTGGCG

GTAGACACAGACAAAAGCAGCG G

AAGGGCGTAACCGAAATCGGT

GTTTGCAGCTCTGTGCATA

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β-actin

GAGGAGGAGGATGAAATA

CTGATCCACATCTGCTGGAA

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CACCCAGCACAATGAAGATC

ACAACCGAAGCGTAGAGT

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HPV-16 E5 HPV-16 E6 HPV-16 E7

Reverse primer (5ʹ–3ʹ)

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Gene

2.7. Western blot analysis

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Proteins extracted from cells were subjected to SDS-PAGE and transferred to polyvinylidene

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fluoride (PVDF) membranes, and the membrane was then blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween -20) for 1 h at room temperature. Then PVDF membranes were incubated with the following primary antibodies at 4 °C overnight: anti-EGFR Biological,

China),

anti-Rb

(Cell

Signaling Technology),

anti-P53

(Cell

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(Ruiying

Signaling Technology), anti-cyclin D1 (Proteintech), anti-HPV-16 E6 (Abcam, Cambridge, UK),

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anti-HPV-16 E7 (Santa Cruz), anti-CDKN2A (R&D Systems) and anti-β-actin (Proteintech) in 5% non-fat milk in TBST. Next, the membranes were washed with TBST and incubated with a horseradish peroxidase-linked secondary antibody (anti-mouse, SA00001-1; anti-rabbit, SA00001-2; anti-goat, SA00001-4, all from Proteintech). Signals were detected by incubating the membranes with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and then exposing and developing the films. 2.8. Mouse xenograft study Six-week-old SCID mice (BALB/c) were handled in accordance with the Guidelines for Animal Experiments of the Southeast University. Each of the nude mice was subcutaneously 6

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injected with 106 cells as indicated in Fig. 7. When the tumor volume reached approximately 80 mm3, the mice were randomized to the different experimental groups (n=4). ATO was intraperitoneally injected at 2 mg/kg/d. Tumor volume was calculated as (L × W2)/2, where L is the length, and W is the width in millimeters (mm). Tumors were excised and weighed at 21 days. 2.9. Statistical analysis

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All the experiments were repeated at least twice. The data were analyzed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) and expressed as the mean values ±

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SEM (standard error of the mean). Statistical analysis was performed using a standard Student’s

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t-test analysis. P<0.05 was considered to indicate significant differences.

3. Results

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3.1. Inhibitory effect of ATO on HPV-positive HNSCC cells differs from that on HPV-negative HNSCC cells

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Our results showed that the response of HPV-positive HNSCC cells to ATO was slightly different from that of HPV-negative HNSCC cells. At a low concentration (2 μM), ATO stimulated

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the proliferation of HPV-negative HNSCC cells; however, at higher concentrations (6-10 μM),

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ATO inhibited the proliferation of these cells (Fig. 1A). For HPV-positive HNSCC cells, ATO obviously inhibited cellular proliferation, with the exception of 2 μM ATO (Fig. 1B). The results

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showed that HPV-positive cells were more sensitive to ATO than HPV-negative cells.

Fig. 1 Inhibitory effects of ATO on the proliferation of HPV-negative (A) and HPV-positive (B) HNSCC cells were determined using the CCK-8 assay. The mean±SEM of three independent experiments are shown.

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3.2. ATO induces apoptosis of HNSCC cells Apoptotic assays showed that under normal culture conditions apoptotic rates of SCC-4, SCC-25, CAL-27, UM-SCC-47, UPCI-SCC-090 and 93-VU-147T cells was 1.8%, 2.4%, 5.5%, 15.1%, 4.6% and 7.4%, respectively, while it reached 7.5%, 8.1%, 10.3%, 32.2%, 9.3% and 84%,

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respectively, after treatment with 4 μM ATO for 72 h (Fig. 2). Histogram analysis displays that

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these changes are significant for all cell lines, particularly 93-VU-147T cells (Fig. 2).

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Fig. 2 ATO induces apoptosis in HNSCC cells. HNSCC cells were treated with ATO for 48 h, and the apoptosis rates were determined by flow cytometry. The experiment was performed in triplicate, and the data are presented

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as the mean±SEM. ***P<0.001.

3.3. ATO induces G2 phase arrest of HNSCC cells Fig. 3 showed that in the normal conditions, the cell cycle distribution was different in these cell lines. In general, the percentage of cells in the G1 phase is higher in HPV-negative HNSCC cells than in HPV-positive HNSCC cells. For example, the percentage of cells in the G1, S and G2 phases was 76.7, 4.7 and 18.5 in SCC-25 cells, and 48.5, 9.3 and 42.2 in UPCI-SCC-090 cells, and 32.8, 23.7and 43.5 in 93-VU-147T cells, respectively. When DNA is damaged, the cell cycle is arrested at the G1/S checkpoint or G2/M checkpoint for DNA repair or cells will undergo

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apoptosis if repair efforts fail. Fig. 3 showed the cell-cycle distribution of HNSCC cells after treatment with 4 μM ATO for 72 h. The S/G2 phase arrest was seen in most cell lines but only G2-arrest in UPCI-SCC090. The marked sub-G1 population (apoptotic cells) was seen in

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93-VU-147T cells, consistent with the results of apoptotic assay (Fig. 2).

Fig. 3 ATO induces G2 arrest in HNSCC cells. Flow cytometry showed that HNSCC cell growth was inhibited at

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the G2 phase following exposure to 4 µM ATO for 48 h. The experiment was performed in triplicate, and the data

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are presented as the means±SEM.

3.4. ATO inhibits the colony formation and migration of HNSCC cells

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For SCC-4, SCC-25, CAL-27, UM-SCC-47, UPCI-SCC-090 and 93-VU-147T cells, the

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colony formation assay showed that the average number of colonies was 251, 320, 143, 163, 183 and 78 in the control groups, and these numbers were decreased to 60, 76, 40, 31, 63 and 28, respectively, when the same cell lines were treated with 0.5 μM ATO (Fig. 4A). HNSCC colony

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formation was inhibited in the presence of 2 μM ATO.

Fig. 4 Inhibitory effects of ATO on the colony formation and migration of HNSCC cells. (A) HNSCC cells were treated with ATO for 10 days. ATO inhibited the colony numbers of these cells. (B) HNSCC cells were treated with 9

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ATO for 24 h. ATO inhibited these cell migration as determined using a Transwell assay (100×). The experiments were performed in triplicate, and the data are presented as the mean±SEM. **P<0.01, ***P<0.001.

Transwell assays showed that the number of migrating SCC-4, SCC-25, CAL-27, UM-SCC-47, UPCI-SCC-090 and 93-VU-147T cells was 258, 205, 131, 188, 245 and 205, respectively, in the control and these numbers decreased to 113, 106, 58, 21, 58 and 21,

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respectively, after treatment with 6 μM ATO for 24 h (Fig. 4B).

3.5. Differential expression of key genes in HPV-positive and -negative HNSCC cells

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We evaluated the expression of HPV16 oncogenes in three HPV-positive HNSCC cell lines and found that the levels of E6 and E7 were higher in UPCI-SCC-090 and 93-VU-147T cells than

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in UM-SCC-47 cells (Fig. 5), consistent with the fact that UPCI-SCC-090 and 93-VU-147T cells contain more copies of HPV DNA (Table 1). We also found transcription of the HPV 16 E5 gene

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in these three cell lines, but not in SiHa cells (Fig. 5A), suggesting the existence of viral episomes in these cells. As shown in Fig. 5, HPV-negative HNSCC cells had relatively higher levels of

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EGFR, TP53 (p53 gene) and CCND1 (cyclin D1 gene) expression than HPV-positive HNSCC

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cells. There was a relatively high expression level of CDKN2A (p16 gene) in HPV-positive HNSCC cells. However, its expression was very low in HPV-negative HNSCC cells and not

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detectable in SCC-25 cells (Fig. 5).

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Fig. 5 Different expression of key genes in HNSCC cells in steady-state growth. (A) RT-qPCR revealed high levels of CDKN2A and low levels of EGFR, TP53 and CCND1 in HPV-positive HNSCC cells, whereas high levels of EGFR, TP53 and CCND1 and low levels of CDKN2A were observed in HPV-negative HNSCC cells. The relative

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mRNA levels of key genes normalized to β-actin. (B) Basal expression levels of key proteins in HNSCC cells

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measured by Western blot were consistent with the qPCR results.

3.6. ATO alters the expression of key proteins in HNSCC cells Western blot assays showed that ATO altered the expression of key proteins in a time-dependent manner (Fig. 6A). The expression levels of HPV16-E6 and -E7 proteins were significantly down-regulated in HPV-positive HNSCC cells compared with the control after treatment with 6 μM ATO. In addition, the expression levels of cyclin D1 were reduced, and the expression levels of Rb, p53 and p16 were up-regulated in these cells. In HPV-negative HNSCC cells, the expression levels of EGFR, mutant p53 and cyclin D1 were reduced, whereas the expression levels of Rb were up-regulated following treatment with ATO. 11

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Fig. 6 Time- and dose-dependent effects of ATO on protein expression in HNSCC cells. (A) Time-course. HNSCC

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cells were treated with 6 µM ATO for the indicated times, and then, the cells were harvested for Western blot analysis. (B) Dose-course. HNSCC cells were treated with the indicated concentrations of ATO for 72 h, and then,

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the cells were harvested for Western blot analysis.

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In addition, Western blot revealed that ATO altered the expression of key proteins in a dose-dependent manner (Fig. 6B). For example, E6 and E7 protein expression in UM-SCC-47 cells was inhibited by approximately 36% and 34%, respectively, after treatment with 4 μM ATO

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and by up to 75% and 87%, respectively, after treatment with 6 μM ATO compared with the

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control at the end of a 72-h period. The expression levels of cyclin D1 were reduced, and the expression levels of Rb, p53 and p16 were up-regulated to different levels in HPV-positive

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HNSCC cells after treatment with ATO. In HPV-negative HNSCC cells, the expression levels of EGFR, mutant p53 and cyclin D1 were reduced, and the expression levels of Rb were

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up-regulated following treatment with ATO. 3.7. ATO suppresses xenograft growth of HNSCC cells After treatment with ATO for 21 days, ATO inhibited tumor growth compared with the control, and HPV-positive xenograft tumors showed a greater response to ATO than did HPV-negative xenograft tumors (Fig. 7).

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Fig. 7 Inhibitory effects of ATO on xenograft growth of HNSCC cells. When the xenograft tumors reached ~80 mm3, the mice were treated daily with ATO or vehicle by intraperitoneal injection for 21 days. The tumor volumes

***P<0.001.

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4. Discussion

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and weights were determined after sacrifice. The data are presented as the mean±SEM. *P<0.05, **P<0.01,

Our data showed that HPV-positive HNSCC cells exhibited a slightly different response pattern to ATO than HPV-negative HNSCC cells. In HPV-negative HNSCC cells, ATO at less than 4 μM mildly stimulated cellular proliferation; however, ATO at 6-10 μM inhibited cellular proliferation (Fig. 1A). For HPV-positive HNSCC cells, ATO obviously inhibited cellular proliferation, with the exception of 2 μM ATO (Fig. 1B). In addition, HPV-positive HNSCC cells exhibited greater levels of apoptosis than did HPV-negative HNSCC cells upon treatment with 4 μM ATO for 72 h (data not shown). In general, the response of HPV-positive HNSCC cells to ATO was superior to that of HPV-negative HNSCC cells. These results are consistent with those of a 13

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study by Wen et al., who showed that HPV-positive cells (HeLa and CaSki) were more sensitive to ATO than HPV-negative cells (C33A) [11]. Why did a low concentration of ATO stimulate cellular proliferation? The reason may be related to the properties of arsenic and the tissue type because many properties of arsenic have both anti- and pro-cancer effects. Liu et al. reported that a low concentration of ATO (0.1-0.2 μM) increased the proliferation, migration and invasion of the human epidermoid carcinoma A431 cells, while a high concentration of ATO (5-20 μM) induced

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cell apoptosis [20]. Burnichon et al. also reported that 5-20 μM ATO increased the viability of

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human fibroblasts; however, cell survival decreased after 25 μM ATO [21]. Our data suggest that optimal concentrations should be chosen to avoid negative effects if ATO is used with

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HPV-negative HNSCC cells.

The different responses of HNSCC cells with and without HPV to ATO reflect their different

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genetic backgrounds. In general, HPV-positive HNSCC cells harbour less genetic changes, and the HPV oncogenes play important roles in tumorigenesis. In contrast, HPV-negative HNSCC cells

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have more obvious genomic changes and highly express EGFR, cyclin D1 and mutant p53 and lack p16 expression (Fig. 5B). It is known that TP53 and CDKN2A genes are wild-type in

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HPV-positive HNSCC cells but are mutated in HPV-negative HNSCC cells (Table 1). Functional

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p53 could be recovered due to E6 inhibition by ATO, which may play a positive role in the response of HPV-positive cells to ATO. The up-regulation of p16 and Rb by ATO treatment may also contribute to the better response of HPV-positive cells to ATO.

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Since there were differences in the cell cycle distribution in HPV-negative and -positive HNSCC cells (Fig. 3), further work may need to elucidate whether they influence the differential

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treatment response in these cells. The observed inhibitory effects of ATO on HNSCCs are attributed to G2 phase arrest and apoptosis, which were also reported by Seol et al. [22]. It is considered that the up-regulation of cyclin-dependent kinase inhibitor p21 or p53 following ATO treatment is responsible for G2/M arrest [22,23]. The carcinogenic risk associated with HPV is primarily due to the activity of three viral oncoproteins: E5, E6 and E7 [24]. Both E6 and E7 play vital roles in viral replication and host cell immortalization and transformation. Therefore, the E6 and E7 oncogenes are ideal targets for HPV-associated cancers, including HPV-positive HNSCC [25]. Of the three oncoproteins expressed by HPV, E5 protein has weak oncogenic properties that occur through increasing the 14

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expression of EGFR and inhibiting the expression of major histocompatibility (MHC-I and II) on the plasma membrane [26]. E5 is generally considered to play a role in the early stage of HPV tumorigenesis because E5 is no longer expressed after the viral DNA integration into the host genome [27]. Fig. 5A shows E5 expression in the three HPV-positive HNSCC cell lines, suggesting that HPV episomes exist in these cells, and E5 may still modulate EGFR turnover and

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other biological activities in these cells. A previous report has indicated that the UPCI-SCC-090 and 93-VU-147T cell lines express HPV episomes [17]. Um et al. reported that variable HPV16

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E5 transcripts were detected from 59 HPV16-positive oropharyngeal tumor samples [28], suggesting that episomal HPV may contribute to the development of HNSCC.

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In this study, the expression levels of E6 and E7 were reduced by ATO in HPV-positive HNSCC cells. However, the effects of ATO on p53 and pRb were variable. ATO up-regulated p53

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in HPV-positive HNSCC cells and down-regulated p53 in HPV-negative cells, suggesting that mutant p53 was targeted by ATO [29]. Mutant p53 was highly expressed in HPV-negative

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HNSCC cells (Fig. 5B), suggesting that its protein stability was increased and had gained oncogenic function. ATO promotes mutant p53 degradation, suggesting the value of ATO in

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therapy for p53-mutant tumors. By contrast, ATO significantly increased pRb expression in both

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HPV-positive and -negative HNSCC cells (Fig. 6), suggesting that ATO could restore pRb expression via E7 inhibition. Our data also suggest that the Rb gene might be wildtype in these HNSCC cells.

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The p16 is a major negative regulator of cell cycle and is inactive in many epithelial malignancies [30]. Inactivation of CDKN2A by aberrant hypermethylation plays a role in the

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process of carcinogenesis in various cancers [31,32]. However, it is well known that p16 is over-expressed in HPV-positive cancer cells and sometime used as a surrogate for HPV infection [33-35]. The increased expression of p16 is considered to be due to the lost function of pRb, which is degraded by the HPV E7 oncoprotein [4]. Our results confirmed this expression pattern of p16 in HPV-positive and -negative HNSCCs. Previous studies have shown that CDKN2A is mutated in SCC-4 and CAL-27 cells and deleted in SCC-25 cells [19,36]. Similar to p53, functional p16 in HPV-positive HNSCC cells may be associated with a positive response to ATO treatment.

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We found that ATO treatment can increase the expression of p16 in HPV-positive HNSCCs. Several studies have shown that ATO restored the expression of CDKN2A via demethylation of CDKN2A promoter in non-HPV cancer cells [37]. However, the effect of ATO on the expression of CDKN2A in HPV-positive cancer cells has not been determined. Generally, the expression of p16 parallels the expression of HPV E7 in HNSCC cells [38], and the transcriptional silencing of

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HPV E7 results in the up-regulation of p53 and the down-regulation of p16 in cervical cancer cells [39,40]. Our data showed that ATO down-regulated the expression of HPV16 E7 and up-regulated

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the expression of p16. The different results may be related to the different experimental systems. In Zhou and Sato’s experimental systems, the authors specifically blocked E6/E7 expression using

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short interfering RNA (siRNA) or an adeno-associated virus vector encoding short hairpin RNA (shRNA) [39,40]. However, as a drug the effect of ATO on cellular molecules is more complex,

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our data suggest that ATO may regulate the expression of HPV16 E7 and CDKN2A via different mechanisms in this study.

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EGFR appears to play a critical role in HNSCC as it is over-expressed in almost all HNSCCs (>90%), which is associated with poor prognoses and reduced survival of patients [41,42].

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Therefore, a large focus of current research is on the development of inhibitors that block EGFR

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function. Cetuximab, a human/mouse chimeric monoclonal antibody against EGFR, has been approved by the FDA for patients with HNSCC [43]. Our results showed that EGFR expression was high in HPV-negative HNSCC cells, whereas its expression was very low or absent in

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HPV-positive cancer cells, which is consistent with other studies [44]. In fact, approximately 15% of HPV-negative HNSCCs exhibit EGFR gene amplification; however, this situation has not been

may

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found in HPV-positive HNSCCs [15,45,46]. The methylation of the EGFR gene by HPV infection explain

the

low

expression

in

HPV-positive

tumors

[47,48].

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‘HPV-positive/EGFR-negative cancers’ hypothesis reflects the inverse relationship between EGFR expression and HPV status. Our results showed that ATO inhibited EGFR expression in a time- and dose-dependent manner, and this inhibitory effect of ATO was associated with its anticancer effects in HPV-negative HNSCC cells. Previous reports have indicated that ATO inhibits EGFR expression in human epidermoid carcinoma A431 cells in vitro and in vivo [49,50]. This inhibitory effect was considered to occur through p21 activation, leading to cell death via the EGFR-Ras-Raf-ERK1/2 16

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pathway [49]. These findings suggest that ATO might be used as a chemotherapy drug to treat tumors with high EGFR expression. Cyclin D1 is a key cell cycle regulatory protein for the mammalian G1-S phase transition and is involved in the regulation of cell proliferation and differentiation. Similar to EGFR, cyclin D1 over-expression is widely observed in HNSCCs, especially in HPV-negative cases [15]. Both

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cyclin D1 and EGFR may be the major targets of ATO in HPV-negative HNSCCs. Ai et al. reported that ATO inhibited gallbladder carcinoma cell growth via the down-regulation of cyclin

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D1 transcription through the transcription factor Sp1 [51]. Lo et al. also reported that ATO down-regulated the expression of cyclin D1 in mantle cell lymphoma cells in a dose- and

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time-dependent manner [52]. 5. Conclusions

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HPV-positive and -negative HNSCC cells have different expression of key genes. ATO inhibits the proliferation of HPV-positive and -negative HNSCC cells in vitro and in vivo and the

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inhibitory effect is more significant in HPV-positive HNSCC cells than in HPV-negative HNSCC cells. The status of TP53 and CDKN2A is a key factor determining the response of HPV-positive

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and -negative cells to ATO. HPV16 E6 and E7 are major targets of ATO in HPV-positive HNSCC

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cells, while EGFR, cyclin D1 and mutant p53 are major targets of ATO in HPV-negative HNSCC cells. These data suggest that ATO is a potential therapeutic drug for HNSCCs, especially

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HPV-positive HNSCCs.

Conflict of interest statement

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The authors declare that there are no conflicts of interest.

Acknowledgements

We thank Dr. Randall J Kimple for the three HPV-positive cell lines. This work was supported by grants from the National Natural Science Foundation of China (No. 81602330) and the Natural Scientific Foundation of Shandong Province (No. ZR2015PH056).

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