α7-Nicotine acetylcholine receptor mediated nicotine induced cell survival and cisplatin resistance in oral cancer

Journal Pre-proof ␣7-nicotine acetylcholine receptor mediated nicotine induced cell survival and cisplatin resistance in oral cancer Chia-Chen Hsu, Kuo-Yang Tsai, Yu-Fu Su, Chu-Yen Chien, Ying-Chen Chen, Yu-Chiao Wu, Shyun-Yeu Liu, Yi- Shing Shieh

PII:

S0003-9969(19)31140-9

DOI:

https://doi.org/10.1016/j.archoralbio.2020.104653

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AOB 104653

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Archives of Oral Biology

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8 November 2019

Revised Date:

7 January 2020

Accepted Date:

7 January 2020

Please cite this article as: Hsu C-Chen, Tsai K-Yang, Su Y-Fu, Chien C-Yen, Chen Y-Chen, Wu Y-Chiao, Liu S-Yeu, Shieh Y-S, ␣7-nicotine acetylcholine receptor mediated nicotine induced cell survival and cisplatin resistance in oral cancer, Archives of Oral Biology (2020), doi: https://doi.org/10.1016/j.archoralbio.2020.104653

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α7-nicotine acetylcholine receptor mediated nicotine induced cell survival and cisplatin resistance in oral cancer

Running title: acetylcholine receptor in oral cancer

Chia-Chen Hsua, Kuo-Yang Tsaib,c, Yu-Fu Sua,d, Chu-Yen Chiena, Ying-Chen Chene,

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Yu-Chiao Wuf, Shyun-Yeu Liug and Yi- Shing Shiehf,h*

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. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan b

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. Department of Oral and Maxillofacial Surgery, Changhua Christian Hospital, Changhua, Taiwan c . College of Nursing and Health Science, Da-Yeh University, Changhua, Taiwan d . Department of Radiation Oncology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan e . Molecular and cell Biology, Taiwan International Graduate Program, Academia

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Sinica and Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan f . Department of Dentistry, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan g . Department of Oral and Maxillofacial Surgery, Chi Mei Medical Center, Tainan, Taiwan h . Department and Graduate Institute of Biochemistry, National Defense Medical Center, Taipei, Taiwan

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*Correspondence: Dr. Yi- Shing Shieh, School of Dentistry, National Defense Medical Center, No.161, Sec.6, Min-Chuan Ease Rd., Nei-Hu, Taipei 114, Taiwan. Tel: +886-2-87923148, Fax: +886-2-87919276, E-mail address: [email protected] Abbreviations: α7-nAChRs, α7-nicotinic acetylcholine receptors; Bcl-2, B-cell lymphoma 2; cleaved-PARP, cleaved-Poly (ADP-ribose) polymerase; EMT, 1

epithelial-to-mesenchymal transition; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MLA, methyllycaconitine; MTT, (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide); OSCC, oral squamous cell carcinoma; sh-RNA, short hairpin RNA;

Highlights

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 Nicotine enhanced cell survival and drug resistance of OSCC cells by α7-nAChRs.  Blocking α7-nAChRs may increase chemosensitivity in patients.  Bcl-2 was involved in nicotine-induced cisplatin resistance.

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ABSTRACT Objective: To investigate the effect of nicotine on cell survival and cisplatin resistance in oral cancer and the possible involvement of α7-nicotinic acetylcholine receptors (α7-nAChRs). Design: The effects of nicotine on cell survival and cisplatin-induced apoptosis were assessed. Knockdown of α7-nAChRs by short hairpin RNA and the specific antagonist methyllycaconitine (MLA) was used to examine the involvement of α7-nAChRs in modulating the effects of nicotine. Apoptosis signal molecules were

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examined in nicotine- and cisplatin-treated cells. Results: Nicotine increased the survival of the oral cancer cells YD8 and OEC-M1 in a dose- and time-dependent manner. Nicotine treatment accelerated cell cycle

progression in the oral cancer cells, and significantly reduced cisplatin-induced cell apoptosis. In the α7-nAChR-silenced cells, the prosurvival effect of nicotine in the

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cisplatin-treated cells was attenuated. Co-treatment of cisplatin and nicotine

attenuated the effect of cisplatin on Bcl-2 expression. In addition, the effect of

the Bcl-2 inhibitor ABT-737.

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nicotine on cell survival under cisplatin treatment was attenuated with the addition of

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Conclusions: Treating oral cancer cells with nicotine increased cell survival and cisplatin resistance, in which α7-nAChRs were involved.

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Key words: Acetylcholine receptors; Apoptosis; Cisplatin resistance; Nicotine

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INTRODUCTION Oral cancer comprises about 4% of all cancers worldwide, and it is the most common head and neck neoplasm with more than 500,000 new cases being diagnosed annually (Siegel, Miller, & Jemal, 2015) . Moreover, more than 90% of oral cavity cancers have been reported to be oral squamous cell carcinoma (OSCC), which in turn has been reported to be an important cause of cancer morbidity and mortality (Siegel et al., 2015). Primary surgery is the most commonly used initial therapy for OSCC, however

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chemotherapy is used in patients with stage III/IV oral cancer, including advanced nodal disease or advanced primary tumors. Despite advances in chemotherapy, the prognosis has not significantly improved over the last 20 years (Forastiere, Koch, Trotti, & Sidransky, 2001), essentially because of drug resistance. Most clinical regimens used to treat OSCC are based on cisplatin combination chemotherapy

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(Dasari & Tchounwou, 2014). Since drug resistance is a major hindrance in treating OSCC, it is important to identify the molecular mechanisms for chemoresistance in

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OSCC.

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Cigarette smoking is known to cause many cancer malignant behaviors, including drug resistance (Gandini et al., 2008). Nicotine is a major component of cigarettes (Y. Y. Wang et al., 2014). Previous studies have shown that nicotine

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promotes the progression of cancer cells in multiple types of cancer (Wu, Lee, & Ho, 2011). For example, Liu et al. reported that cancer cell survival and chemoresistance were induced by nicotine by promoting the phosphorylation of Mcl-1 and via

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interactions with Bak. They concluded that this mechanism could potentially be used to improve the effectiveness of chemotherapy in patients with lung cancer (Liu et al.,

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2019). In bladder cancer, Chen et al showed that an extended duration of treatment with nicotine could induce chemoresistance via the excessive activation of Stat3 and inhibition of the activation of ERK1/2. The authors concluded that chemotherapy may fail due to the nicotine-mediated inhibition of cell death (Chen, Ho, Guo, & Wang, 2010). In oral cancer, nicotine has been reported to participate in carcinogenesis, epithelial-to-mesenchymal transition (EMT), cancer stem cell properties (C. Wang, Niu, et al., 2017; Yu & Chang, 2013), cell proliferation and invasiveness, and the 4

inhibition of apoptosis. However, the mechanisms of the effect of nicotine in drug resistance are unclear and need to be investigated further. Nicotine exerts its effects by binding to and activating cell-surface receptors, and in particular nicotinic acetylcholine receptors (nAChRs) (Dasgupta et al., 2009). nAChRs are composed of five transmembrane subunits which form homo- or heteromeric pentamer channels that are composed of either five identical α subunits (α7, α8, or α9) or combinations of α and β subunits (α2–α6 or α10 subunits combined with β2–β4 subunits (Wu et al., 2011). nAChRs are widely expressed in the peripheral and central nervous systems and at neuromuscular junctions, and also on

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non-neuronal cells such as bronchial epithelium, keratinocytes, and endothelial, immune, vascular smooth muscle and cancer cells (Wu et al., 2011). Given the

somewhat unclear mechanisms of the effect of nicotine in chemoresistance in oral

cancer, the aim of this study was to investigate the potential role and mechanism of

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nicotine and its specific receptors in oral cancer cells.

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MATERIALS AND METHODS Cell culture OSCC cell lines (OEC-M1 and YD8) were kindly provided by Professor Yook (Namseoul University, Korea) and Professor Meng (National Defense Medical Center, Taiwan). The cells were cultured in RPMI1640 medium with 10% fetal bovine serum followed by incubation at 37°C in a 5% CO2 atmosphere incubator. Both cell lines were confirmed to be mycoplasma-free.

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Chemicals Nicotine and propidium iodide were obtained from Sigma-Aldrich (St. Louis, MO, USA), cisplatin was purchased from Selleck chemicals (Houston, TX, USA),

and the α7-nAChR antagonist methyllycaconitine (MLA) was purchased from Tocris Bioscience (Bristol, England, UK). MTT

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(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from

the USB Corporation (Cleveland, OH, USA). All other chemicals were obtained from

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Sigma. ABT-737 was purchased from AdooQ® bioscience (Irvine, CA, USA).

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Protein extraction and Western blot analysis

Cell lysate combinations of RIPA with PIC2 and PPI were used to form the lysis buffer, with a reaction time of 30 min in an ice bottle. A commercial BCA kit

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(Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to measure the protein concentration in each cell lysate. An SDS-polyacrylamide gel electrophoresis system was used for Western blot analysis. Anti-GAPDH (Cell Signaling, Danvers,

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Massachusetts, USA), anti-α7-nAChR (OriGene, Rockville, Maryland, USA), anti-cleaved-Poly (ADP-ribose) polymerase (cleaved-PARP) (Cell Signaling), and

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B-cell lymphoma 2 (Bcl-2) (Santa Cruz Biotechnology, Dallas, Texas, USA) antibodies were used for probing. MTT assay

Cell survival ability was assessed using the MTT assay. The 1×104 cells were seeded in a 96-well plate, and were treated with 1 µM nicotine, 40 µM MLA, 15 and 6

20 µM cisplatin, and 10µM ABT-737 for 24–72 h after they had become attached. MTT reagents were then added to each well, and the cells were incubated at 37°C for 3 h. The supernatant was subsequently removed to preserve the cells, and DMSO was added to each well and shaken. Cell survival ability was measured by reading the color intensity using a plate reader at 570 nm. Short hairpin RNA α7-nAChR Short hairpin RNA (sh-RNA) α7-nAChR was purchased from the RNAi Core Facility of Academia Sinica (Taipei, Taiwan). The sh-α7-nAChR target sequence was

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GCA AAT GTC TTG GAC AGA TCA, and the negative control was Plko-1. Transfection was completed using Polyjet™ In Vitro DNA Transfection Reagent (SignaGen Laboratories, Medical Center Dr, Rockville, Maryland, USA) in accordance with the protocol provided.

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Cell cycle analysis

Cells were stained live with 1 mg/mL propidium iodide in cell culture media for

Cell apoptosis analysis

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analyzed using Cell Quest software.

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40 min at 37°C. Cell-cycle was analyzed using a FACSCalibur flow cytometer and

An APO-DIRECT™ Kit (ThermoFisher Scientific, Waltham, Massachusetts,

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USA) was used to label DNA breaks and total cellular DNA, and flow cytometry was used to detect apoptotic cells (TUNEL assay). In this method, the cells were stained in

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a single step with FITC-labeled dUTP. The cells were seeded in culture plates at a concentration of 1-2 × 106, and

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harvested 24 h after drug treatment. They were then suspended in 1 ml 1% paraformaldehyde, placed on ice for 15 min, washed twice with PBS, and then stored overnight at -20°C after the addition of 5 ml ice-cold 70% ethanol. The ethanol was then removed by aspiration, and the fixed cells were resuspended twice in 1 ml Wash-Buffer. Following resuspension in 50 μl DNA-Labeling-Solution for 60 min at 37°C in a temperature-controlled bath, the cells were washed twice and the cell pellet was resuspended in 500 μl propidium iodide/RNase Staining Buffer. Finally, the cells 7

were incubated in the dark for a minimum of 30 min at room temperature, and then analyzed using flow cytometry ( FACSCalibur, BD Biosciences, Heidelberg, Germany). Statistical analysis All statistical analyses were performed using SPSS 22.0 and GraphPad Prism 5.0 programs (USA), and a P value < 0.05 was considered to be statistically significant. Data were expressed as the mean ± SD. In parametrmic data, statistical significance was calculated by two-tailed Student’s t-test (2 groups) or one way- ANOVA test (>2

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groups) followed by Tukey post-hoc test. In non-parametric data, statistical significance was determined with Kruskal-wallis test (>2 groups) followed by Dunn's

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post-hoc test (Mucchietto et al., 2017).

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RESULTS Nicotine augmented cell survival of OSCC cells The effect of nicotine on the viability of oral cancer cells was evaluated using the MTT assay. YD8 and OEC-M1 cells were treated with various concentrations of nicotine for 24, 48, and 72 hours. After incubation for 24 h, there were significantly more OEC-M1 cells in the 1 μM nicotine group than in the 100 nM and control groups (Figure 1A). Nicotine significantly increased cell survival at the concentration of 1 μM and at 24, 48 and 72 hours (Figure 1B). The results showed that nicotine

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increased YD8 and OEC-M1 survival in a dose- and time-dependent manner. We then investigated the effect of nicotine on the cell cycle. We used flow

cytometry to analyze the proportion of cells in each phase, which showed that nicotine treatment accelerated progression from the S to G2/M phase in the YD8 cells, and

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increased the number of cells entering the S phase in the OEC-M1 cells in a time- and dose-dependent manner (Figure 1C and D). These results demonstrated that nicotine

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treatment increased OSCC cell survival by modulating cell cycle distribution.

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Nicotine reduced cisplatin cytotoxicity in OSCC cells

We then examined the effect of nicotine treatment on cisplatin-induced apoptosis. The viability of YD8 and OEC-M1 cells was assessed by treating them with the

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anti-cancer drug cisplatin in the presence or absence of nicotine. The shape of many of the cisplatin-treated cells became rounded and exhibited fragmented nuclei morphology, characteristic of apoptotic cells. In contrast, the cells treated with both

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cisplatin and nicotine exhibited little nuclear fragmentation or apoptosis. MTT analysis demonstrated that nicotine attenuated the cytotoxic efficacy of cisplatin in the

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YD8 and OEC-M1 cells (Figure 2A and 2B). We then investigated whether nicotine affected cisplatin-induced cell apoptosis. YD8 and OEC-M1 cells were treated with 1µM nicotine and 15 and 20 µM cisplatin, respectively. After 24 h, the cells were subjected to apoptosis assay. The results showed that nicotine significantly reduced cisplatin-induced cell apoptosis from 87% to 30% in the YD8 cells and from 76% to 24% in the OEC-M1 cells (Figure 2C and 2D). 9

Nicotine induced cisplatin resistance through α7-nAChRs in OSCC cells Previous studies have reported that α7-nAChRs are the major receptors in medicating the effect of nicotine in many cancers including oral malignancy (Martinez et al., 2017; Wei et al., 2011; Yoneyama et al., 2016). Therefore, we investigated the potential role of α7-nAChRs in mediating the effect of nicotine-induced cell proliferation and cisplatin resistance in OSCC. We used sh-RNA to knockdown the expression of α7-nAChRs in OEC-M1 cells (Figure 3A). The effects of nicotine on cell survival and cisplatin resistance were attenuated in the α7-nAChR-silenced cells (Figure 3B). In addition, the effect of nicotine on the

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expression of cleaved-PARP, an apoptotic marker, was also suppressed in the α7-nAChR-silenced cells (Figure 3C). Of note, the effects of nicotine on the

pro-survival and anti-apoptosis induced by cisplatin were partially reversed when the OSCC cells were treated with the α7-nAChR antagonist MLA (Figure 3D).

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Changes in the expression of Bcl-2 were involved in nicotine-induced cisplatin

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resistance

We then examined the expression of the anti-apoptotic protein Bcl-2 in nicotine-

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and cisplatin-treated OSCC cells. As shown in Figure 4A, cisplatin treatment decreased the expression of Bcl-2 whereas nicotine treatment increased the expression of Bcl-2. Quantitative results of western blot assay are shown (Figure. 4B). Cisplatin

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and nicotine co-treatment attenuated the effect of cisplatin on Bcl-2 expression. Moreover, the addition of the Bcl-2 inhibitor ABT-737 attenuated the effect of

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nicotine on cell survival under cisplatin treatment (Figure 4C).

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DISCUSSION Tobacco smoking remains the main cause of oral cancer. The molecular mechanisms underlying tobacco smoke-induced chemoresistance in patients with oral cancer have yet to be elucidated. Nicotine is a major constituent of tobacco smoke, and it can be detected in the serum of people who smoke. Therefore, we hypothesized that the main cause of chemoresistance of oral cancer cells may be due to exposure to nicotine. To examine the possible chemoresistant effects of nicotine, we analyzed cell survival and chemoresistance in response to nicotine treatment and the subsequent effects on inhibiting apoptosis and regulating the cell cycle in YD8 and OEC-M1 cells.

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Our results showed that nicotine promoted cell survival and cisplatin resistance

through α7-nAChRs in OSCC cells. To the best of our knowledge, this is the first

study to demonstrate that α7-nAChRs play a crucial role in nicotine-mediated OSCC survival and cisplatin resistance. Our results are consistent with earlier reports that

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nicotine exposure during treatment for oral cancer, such as with cisplatin, may reduce the effects of treatment due to interactions between nicotine and α7-nAChRs.

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In this study, we found that treating tumor cells with 100 nM to 1 μM nicotine, which are concentrations detected in the plasma of smokers (Sastry, Chance, Singh,

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Horn, & Janson, 1995; Wu et al., 2011), significantly increased survival and cisplatin resistance of OSCC cells. In lung, pancreas, and bladder cancer, nicotine has been reported to induce cell proliferation and invasion (Dasgupta et al., 2009). In colon and

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gastric cancer, nicotine treatment has been shown to increase cell migration and EMT change . In oral cancer, nicotine treatment has been shown to decrease E-cadherin expression and alter the morphology of oral cancer cells from an oval shape to a

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slender spindle shape characteristic of EMT (C. Wang, Xu, Jin, & Liu, 2017). EMT is considered to be an important step in the acquisition of malignant phenotypes

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(Dasgupta et al., 2009), as it allows cells to acquire migratory properties, thus enabling detachment of malignant tumor cells from the primary site and metastasis to a new location (Dasgupta et al., 2009). Accumulating evidence has indicated a strong correlation between chemotherapy resistance and EMT; in that tumor cells become more resistant to therapy after undergoing EMT (Theys et al., 2011). Our results are consistent with previous studies (Xu et al., 2007) which indicated that nicotine 11

attenuated the efficacy of cisplatin. Furthermore, relevant studies have demonstrated that nicotine has anti-inflammatory effects and that it can reduce the release of inflammatory cytokines from macrophages, which is crucial in antitumor immunotherapy (Kalra et al., 2004). As nicotine enhances cisplatin resistance of oral cancer cells and suppresses the antitumor function of immune cells, further studies are needed to investigate the effect of nicotine on modulating the efficacy of immunotherapy in patients with smoke-associated cancer. Our findings support the work of other studies which investigated the effects of nicotine in cancer, and further demonstrate that nicotine-mediated OSCC survival and

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cisplatin resistance is α7-nAChR-dependent. Increasing evidence has indicated that

nAChRs participate in cell growth and prosurvival signaling pathways as well as in mediating oncogenic signal transduction during cancer development in a manner

specific to the type of cancer. For example, Zhang et al. demonstrated that in lung

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cancer, α7-nAChRs mediated the effects of nicotine on invasion, migration, and EMT change of cancer cells (Zhang et al., 2016). In addition, Lee et al. demonstrated that

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α9-nAChR carcinogenic signals were related to the development of breast cancer (Lee et al., 2011), and Jia et al. also reported that nicotine-activated α5-nAChR signaling

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was involved in cisplatin resistance in gastric cancer (Jia et al., 2016). These studies indicate that the effects of nicotine on cancer through nAChRs could be specific to the type of cancer. In oral cancer, α3-, α5-, and α7-nAChRs have been identified as major

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receptors that mediate the effect of nicotine (Arredondo, Chernyavsky, & Grando, 2006; Zhao, 2016). We screened the nAChRs expression in a panel of oral cancer cells and found α3-nAChRs were not expressed in these cell lines and cell lines

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expressed similar levels of α5-nAChR. The expression of α7-nAChRs varied in different cells, which is associated with cellular response to nicotine treatment (data

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no shown). Therefore, we investigated the potential role of α7-nAChRs in mediating nicotine-induced cell survival and cisplatin resistance in oral cancer. In the present study, we found that α7-nAChRs mediated nicotine-induced cell survival and anti-apoptosis induced by cisplatin. In oral carcinogenesis, α7-nAChRs have been identified as playing a crucial role in mediating the malignant cell transformation of oral keratinocytes, development of oral precancerous lesions, and oral cancer 12

progression (C. Wang, Niu, et al., 2017) (Carracedo, Rodrigo, Nieto, & Gonzalez, 2007; Scherl et al., 2016). However, the role of α7-nAChRs in cisplatin resistance of OSCC is still not clear. Our results demonstrated that nicotine promoted the protein expression of α7-nAChRs and blocked the activation of α7-nAChRs through shRNA-attenuated nicotine-induced OSCC cell survival under cisplatin treatment. Therefore, α7-nAChRs were not only involved in early carcinogenesis, but also in the progression and chemoresistance of oral cancer. Our results confirm the role of α7-nAChRs in nicotine-related carcinogenesis, and the inhibition of nicotine receptors

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may be a novel and valid therapeutic approach for treating OSCC patients with cisplatin resistance. Clinical trials have assessed the use of nAChR-specific

antagonists in the treatment of various diseases apart from cancer (Wu et al., 2011). However, most of these antagonists have been shown to cause various side effects

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associated with a lack of specificity for the nAChR subtype. Therefore, further studies are needed to identify selective antagonists which specifically target the

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overexpression of α7-nAChRs in oral cancer cells, with the aim of establishing novel therapeutic strategies.

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Cisplatin is currently the standard chemotherapy for patients with oral cancer, however the success rate is limited. The efficacy of cisplatin relies on the induction of DNA damage (Basu & Krishnamurthy, 2010), and so the cisplatin-induced apoptosis

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of cancer cells plays a crucial role in its sensitivity. Nicotine has been shown to inhibit cisplatin-induced apoptosis in many types of cancer (Jia et al., 2016; Nishioka et al., 2014), suggesting that nicotine can both induce the development of cancer through the

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activation of cell growth pathways, and also reduce chemotherapeutic agent efficacy through the stimulation of survival pathways. Consistent with these previous studies,

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we also found that treating the cells with both cisplatin and nicotine resulted in the significant activation of Bcl-2. In addition, nicotine prevented cisplatin-induced apoptosis through the upregulation of the expression of the anti-apoptotic protein Bcl-2. Elevated levels of Bcl-2 have been reported to induce resistance to cisplatin in tongue carcinoma cell lines, and the inhibition of Bcl-2 has been reported to suppress the proliferation of oral cancer cells and to increase their apoptosis, while increasing 13

their sensitivity to cisplatin (Xiong, Tang, Liu, Dai, & Wang, 2016). Previous studies have also reported that nicotine can enhance Bcl-2 phosphorylation in lung cancer cell lines (Mai, May, Gao, Jin, & Deng, 2003), as well as reduce ubiquitin-dependent Bcl-2 degradation (Nishioka et al., 2014), resulting in enhanced chemotherapeutic resistance. Of note, α7-nAChR activation has been reported to mediate Bcl-2 phosphorylation in certain cells (Jin, Gao, Flagg, & Deng, 2004), and nicotine has also been shown to increase the expression of α7-nAChRs in lung cancer cells (Brown et al., 2013), which could then lead to a positive feedback mechanism. The enhanced expression of Bcl-2 could promote the

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assembly and transport of α7-nAChRs, thereby further increasing the nicotine-mediated promotion of Bcl-2 activity and maintaining the cells in an

antiapoptotic state. Further studies are needed to investigate whether there is a direct interaction between Bcl-2 and α7-nAChRs in oral cancer cells.

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Conclusions

In conclusion, our results indicated that nicotine enhanced cell survival and drug

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resistance of OSCC cells through α7-nAChRs, and that these effects were reversed by MLA and sh-α7-nAChR. The clinical implications of these results for oral cancer

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therapy include stopping tobacco smoking and nicotine-based treatment, as this may increase the efficacy of cisplatin therapy. In addition, blocking α7-nAChRs may increase chemosensitivity in patients with oral cancer who develop resistance to

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cisplatin therapy.

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Funding This work was supported by research grants from the Ministry of science and technology (MOST 106-2314-B-016-005-MY3), the National Defense Medical Center (MAB-107-081), the Tri-Service General Hospital (TSGH-C107-003-006-S03), and the Chi Mei Medical Center (CMNDMC10702), Taiwan. Competing interests

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The authors declare no conflict of interest.

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Acknowledgements

The authors acknowledge the technical services (supports) provided by

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Instrument Center of National Defense Medical Center.

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FIGURE LEGENDS Figure 1. Effect of nicotine on oral cancer cell viability. Dose-dependent (A) and time-dependent (B) increased cell viability in nicotine-treated oral cancer cells YD8 and OEC-M1. The analysis was determined with Kruskal-wallis test. Data show the median, 25th and 75th percentiles (box) and the range of the data ('whiskers') by box-and-whisker plots. Cells were treated with nicotine at various concentrations and times. Nicotine induced G2/M progression in YD8 (C) and S phase entry in OEC-M1 (D) cells. Flow cytometry of YD8 and OEC-M1 cells treated with 1 μM nicotine for 24 h was used to examine changes in the cell cycle. The cell cycle data was calculated

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by one way- ANOVA test. Each experiments was repeated three times, and the results were similar. The error bars indicate standard deviation (SD) from three independent

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experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Nicotine attenuated the effect of cisplatin in oral cancer cells. In cisplatin

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and nicotine co-treated cells, nicotine enhanced the viability and reduced the sensitivity to cisplatin in YD8 (A) and OEC-M1 (B) cells. Nicotine decreased

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cisplatin-induced apoptosis in YD8 (C) and OEC-M1 (D) cells. The error bars indicate standard deviation (SD) from three independent experiments. Statistical

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analysis was performed using ANOVA.*P < 0.05, ***P < 0.001.

Figure 3. α7-nAChRs in OSCC cells involved in nicotine-induced cell survival and

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anti-apoptosis. (A). Small interfering (sh) RNA specifically knocked down the expression of α7-nAChRs in OSCC cells. (B). In the α7-nAChR-knockdown cells, the

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pro-survival effect of nicotine in cisplatin-treated OSCC cells was decreased. (C). After knockdown of α7-nAChRs, the anti-apoptosis effect of nicotine in cisplatin-treated OSCC cells was attenuated as shown by the expression of the apoptotic marker cleaved-PARP, comparing lane 3 to lane 4, and lane 7 to lane 8. (D). In OSCC cells treated with the α7-nAChR antagonist MLA, the anti-apoptosis effect of nicotine was suppressed. Cell lysates were subjected to immunoblotting to examine the protein expressions. GAPDH antibody was used to probe the blots to assess the 20

equal loading of total proteins in each lane. The error bars indicate standard deviation (SD) from three independent experiments. Statistical analysis was performed using ANOVA. ***P < 0.001. Figure 4. Bcl-2 was involved in the survival and anti-apoptosis effect of nicotine in cisplatin-treated OSCC cells. (A). YD8 and OEC-M1 cells were treated with nicotine (1 μM) for 24 h, cisplatin (15 and 20 μM) for 24 h, or a combination of both. The cell lysates were then subjected to immunoblotting to examine the expression of Bcl-2. GAPDH antibody was used to probe the blots to assess the equal loading of total proteins in each lane. (B). Quantitative results of Bcl-2 protein level by using Image J

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software. (C) Twenty-four hours after the addition of ABT-737, a specific Bcl-2

inhibitor, the cells were treated with cisplatin, nicotine, or a combination of both for 24 h. The cell lysates were the subjected to MTT assay. The error bars indicate

standard deviation (SD) from three independent experiments. Statistical analysis was

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performed using ANOVA. ***P < 0.001.

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