NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells

Journal Pre-proof NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells Yih Ho, Chien-Yi Wu, Yu-Tang Chin, Zi-Lin Li, Yi-shin Pan, Tung-Yung Huang, Po-Yu Su, Sheng-Yang Lee, Dana R. Crawford, Kuan-Wei Su, Hsien-Chung Chiu, Ya-Jung Shih, Chun A. Changou, Yu-Chen S.H. Yang, Jaqulene Whang-Peng, Yi-Ru Chen, Hung-Yun Lin, Shaker A. Mousa, Paul J. Davis, Kuan Wang PII:

S0278-6915(19)30882-8

DOI:

https://doi.org/10.1016/j.fct.2019.111092

Reference:

FCT 111092

To appear in:

Food and Chemical Toxicology

Received Date: 21 June 2019 Revised Date:

25 October 2019

Accepted Date: 24 December 2019

Please cite this article as: Ho, Y., Wu, C.-Y., Chin, Y.-T., Li, Z.-L., Pan, Y.-s., Huang, T.-Y., Su, P.Y., Lee, S.-Y., Crawford, D.R., Su, K.-W., Chiu, H.-C., Shih, Y.-J., Changou, C.A., Yang, Y.-C.S.H., Whang-Peng, J., Chen, Y.-R., Lin, H.-Y., Mousa, S.A., Davis, P.J., Wang, K., NDAT suppresses proinflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2019.111092. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

NDAT Suppresses Pro-inflammatory Gene Expression to Enhance Resveratrol-induced Anti-proliferation in Oral Cancer Cells

Yih Ho1*, Chien-Yi Wu2, 3*, Yu-Tang Chin4, Zi-Lin Li5,6, Yi-shin Pan5,6, Tung-Yung Huang6,7, Po-Yu Su5, Sheng-Yang Lee4,8, Dana R. Crawford9, Kuan-Wei Su10, Hsien-Chung Chiu11, Ya-Jung Shih5,6, Chun A. Changou12, 13, 14, Yu-Chen S.H. Yang15, Jaqulene Whang-Peng6, 8, 16, Yi-Ru Chen5,6, Hung-Yun Lin6, 8, 16, 17, 18,19, Shaker A. Mousa19, Paul J. Davis19, 20, Kuan Wang5, 6 1

School of Pharmacy, Taipei Medical University, Taipei Medical University, Taipei 11031 Department of Pediatrics, E-DA Hospital, Kaohsiung 82445, Taiwan 3 School of Medicine, I-Shou University, Kaohsiung 84001, Taiwan 4 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei 11031, Taiwan 5 Graduate Institute of Nanomedicine and Medical Engineering, College of Medical Engineering, Taipei Medical University, Taipei 11031, Taiwan 6 Taipei Cancer Center, Taipei Medical University, Taipei 11031, Taiwan 7 Graduate Institute for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan 8 Department of Dentistry, Wan-Fang Medical Center, Taipei Medical University, Taipei 11031, Taiwan 9 Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208, USA. 10 Department of Dentistry, Hsinchu MacKay Memorial Hospital, Hsinchu City 30071, Taiwan 11 Department of Periodontology, School of Dentistry, National Defense Medical, Center and Tri-Service General Hospital, Taipei 11490, Taiwan 12 Core Facility Center, Office of Research and Development, Taipei Medical University, Taipei 11031, Taiwan 13 PhD Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan 14 Integrated Laboratory, Center of Translational Medicine, Taipei Medical University, Taipei 11031, Taiwan 15 Joint Biobank, Office of Human Research, Taipei Medical University, Taipei, 11031, Taiwan 16 Cancer Center, Wan Fang Hospital, Taipei Medical University, Taipei, 11031, Taiwan 17 Traditional Herbal Medicine Research Center of Taipei Medical University Hospital, Taipei Medical University, Taipei 11031, Taiwan 18 TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, 11031, Taiwan 2

1

19

Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY 12208, USA 20 Albany Medical College, Albany, NY 12208, USA

Keywords: NDAT, Resveratrol, L-thyroxine, Programmed death ligand 1, Oral cancer *These authors contributed equally to this article Address correspondence to: Hung-Yun Lin Graduate Institute for Cancer Molecular Biology and Drug Discovery College of Medical Science and Technology Taipei Medical University 250 Wu-Hsing Street Taipei, Taiwan E-Mail: [email protected]

2

ABSTRACT

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1β and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced antiproliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

3

INTRODUCTION

Resveratrol induces anti-proliferative activities in different cancer cells to inhibit cancer growth in vivo. By binding to its receptor on integrin αvβ3, resveratrol activates extracellular signal-regulated kinase-1 and -2 (ERK1/2). Activated ERK1/2 is essential for resveratrolinduced expression of cyclooxygenase-2 (COX-2) and its nuclear accumulation to promote antiproliferation in cancer cells. The nuclear translocated pERK1/2-COX-2 complex binds with p53 to trigger phosphorylation of p53 at Ser-15 and anti-proliferation sequentially (Cheng et al., 2018; Chin et al., 2018; Lin et al., 2017; Nana et al., 2018a; Nana et al., 2018b). Thyroid hormone (thyroxine, T4) induces expression of pro-inflammatory genes, interleukin-1 beta (IL-1β), transforming growth factor beta-1 (TGF-β1), and PD-L1 expression. T4 stimulated both pro-inflammatory and proliferative genes expression and oral cancer cell proliferation. In contrast, resveratrol inhibited those genes and activated anti-proliferative genes. Although resveratrol suppresses thyroid hormone-induced anti-apoptotic activity by inhibiting PD-L1 expression (Chen et al., 2019), resveratrol-induced nuclear COX-2 accumulation is trapped by thyroxine-induced PD-L1 in the cytosol and causes attenuation of anti-proliferative activity in cancer cells when physiological concentration of thyroxine exists (Chin et al., 2018). Tetraidothyronine, a deaminated analog of thyroxine (T4) and its nano-derivative (NDAT) are able to block thyroid hormone-induced cell proliferation in cancers via competing for receptor on cell surface integrin αvβ3. NDAT reduces expression of pro-inflammatory genes, IL1α, IL-1β and IL-6 (Davis et al., 2013). However, it increases expression of IL-11, a desirable stimulator of hematopoietic stem cell proliferation (Davis et al., 2013). In addition, expression of apoptosis XIAP (Inhibitors X-linked inhibitor of apoptosis) and MCL1 (Induced myeloid

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leukemia cell differentiation protein Mcl-1) are downregulated by NDAT whereas apoptosispromoting CASP2 (Caspase-2) and BCL2L14 (Apoptosis facilitator Bcl-2-like protein 14) are upregulated by NDAT. NDAT increases expression of angiogenesis inhibitor THBS1 (Thrombospondin-1) gene, as is the expression of CBY1 (Protein chibby homolog 1), a nuclear inhibitor of catenin activity (Lin et al., 2016b; Lin et al., 2009). NDAT also down-regulates expression of KRAS and epidermal growth factor receptor (EGFR). Additionally, it potentiates other anti-cancer agents such as cetuximab- (Lee et al., 2016) or gefitinib- (Chang et al., 2018) induced anti-cancer activities. Tetrac and NDAT are also demonstrated to enhance resveratrolinduced anti-proliferation in colorectal cancers in vitro and in vivo xenograft model (Nana et al., 2018a; Nana et al., 2018b). Paradoxically, when colorectal cancer cells treated with resveratrol activates ribonucleotide reductase regulatory subunit M2 (RRM2) gene expression which inhibits resveratrol-induced COX-2 nuclear accumulation, NDAT reduces RRM2 expression and thus enhances resveratrol-induced COX-2 gene expression and apoptosis (Nana et al., 2018b). Programmed death-ligand 1 (PD-L1) plays an important role in evading immune surveillance (Guan et al., 2017). Therefore, overexpression of PD-L1 affects cell cycle, cell proliferation, apoptosis, and carcinogenesis (Song et al., 2014). Expression of PD-L1 can be induced by a variety of pro-inflammatory cytokines such as IL-1 (Guo et al., 2017), TGF-β (Park et al., 2016; Sun et al., 2018) and IFN-γ (Li et al., 2018). In addition to the expressions of proinflammatory genes (Davis et al., 2016), thyroid hormone (thyroxine, T4) induces PD-L1 expression to modulate inflammatory activities, which may be linked to cancer progression. Activation of different signal transduction pathways, such as activated extracellular signalregulated kinase 1/2 (ERK1/2), phosphatidylinositol-3-kinase (PI3K), and signal transducer and activator of transcription 3 (STAT3), plays important roles in PD-L1 expression (Li et al., 2019;

5

Liu et al., 2017; Wang et al., 2017). Activated ERK1/2 (Chin et al., 2018; Lin et al., 2016a; Lin et al., 2018), PI3K (Lin et al., 2018) and STAT3 (Chen et al., 2019) are involved in thyroxineinduced PD-L1 expression in different cancer cells. In current studies, we investigated the enhancing effect of NDAT on resveratrol-induced anti-proliferation in two different oral cancer cell lines. Both resveratrol and NDAT suppressed expression of IL-1β and TNF-α. Additionally, they inhibited expression of proliferative genes and PD-L1. Combined treatment of resveratrol and NDAT was significantly enhanced in reducing PD-L1 expression and anti-proliferation as compared with resveratrol treatment alone in both oral cancer cell lines. Combined treatment of resveratrol and NDAT was significantly more effective in reducing PD-L1 expression and anti-proliferation as compared with resveratrol treatment alone in both oral cancer cell lines. A specific STAT3 inhibitor, S31-201 and NDAT showed similar effect to enhance resveratrol-reduced PD-L1 accumulation. Although combined treatment of NDAT and resveratrol may not enhance NDAT-induced gene expression and antiproliferation in two oral cancer cell lines, evidence indicated that it enhanced resveratrolregulated gene expression and anti-proliferation significantly. Blocking PI3K-STAT3 signaling led to the blocking of the inhibitory effect of pro-inflammatory genes induced by thyroxine thus resumed resveratrol-induced anti-proliferation.

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EXPERIMENTAL METHODS

Cell cultures Human oral epidermoid carcinoma cell line, OEC-M1 cells (an indigenous oral cancer cell line in Taiwan), was established from the gingival epidermal carcinoma of a patient in Taiwan by Dr. Ching-Liang Meng (Yang and Meng, 1994). This cell line was contributed by Dr. Hsien-Chung Chiu. Human squamous carcinoma of the tongue, SCC-25 cells (ATCC® CRL-1628™) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). SCC-25 cells had been tested and authenticated with BCRC (isoenzyme analysis, Mycoplasma, cytogenetics, tumorigenesis, receptor expression testing). Cells were maintained in RPMI-1640 supplemented with 10% FBS in the incubator with 5% CO2 at 37ºC, and then used for experiments until passage 15. Before the study, cells were placed in 0.25% hormone-depleted serum-supplemented medium for 2 days. Then the drug treatment was in 5% hormone-depleted serum-supplemented medium.

Cell viability assay OEC-M1 and SCC-25 cells were plated at a density of 4×103 cells/well in 96-well plates. Cell viability was determined by using the alamarBlue Assay Kit (Thermo Fisher Scientific Inc.) at 72 h after treatment. Medium containing different drugs was replaced daily. At the time of detection, medium was removed, and cells were incubated with alamarBlue reagent for 2 h at 37°C according to the manufacturer's instruction. Plates were then analyzed by using a microplate reader (VERSAmaxTM Tunable Microplate Reader, Molecular Devices) at wavelength of 570 nm and 600 nm as reference.

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Quantitative real-time PCR (qPCR) Total RNA was extracted with genomic DNA removed by Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare Life Sciences, Buckinghamshire, UK). One µg of DNase I-treated total RNA was reverse-transcribed using RevertAid H Minus First Strand cDNA Synthesis Kit (Life Technologies Corp.) into cDNA. cDNAs were used as the template for real-time PCR reaction and analyse. The real-time PCR reactions were conducted using QuantiNovaTM SYBR® Green PCR Kit (QIAGEN, Hilden, Germany) on CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). Reaction procedure involved an initial denaturation at 95ºC for 5 min, followed by 40 cycles of denaturing at 95ºC for 5 sec and combined annealing/extension at 60ºC for 10 sec, as shown in detail in the manufacturer’s instructions. The primer sequences were shown as follows: Homo sapiens cyclin D1

(CCND1),

forward

5’-CAAGGCCTGAACCTGAGGAG-3’

GATCACTCTGGAGAGGAAGCG-3’

(Accession

No.:

and

NM_053056);

reverse Homo

5’-

sapiens

programmed death-ligand 1 (PD-L1) (CD274), forward 5’-GTTGAAGGACCAGCTCTCCC-3’ and reverse 5’-ACCCCTGCATCCTGCAATTT-3’ (Accession No. AY254342.1); Homo sapiens cyclooxygenase 2 (COX-2), forward 5’-GCCAAGCACTTTTGGTGGAG-3’ and reverse 5’GGGACAGCCCTTCACGTTAT-3 (Accession No. AY462100.1); Homo sapiens BCL2associated agonist of cell death (BAD), forward 5’-CTTTAAGAAGGGACTTCCTCGCC-3’ and reverse 5’-AAGTTCCGATCCCACCAGGA-3’ (Accession No.: NM_032989.2); Homo sapiens interleukin-1 β (IL-1β), forward 5’-CTTCGAGGCACAAGGCACA-3’ and reverse 5’GCTTCAGACACTTGAGCAATGA-3’ (Accession number NM_000576.2); Homo sapiens tumor necrosis factor-α (TNF-α), forward 5’-GCCCATGTTGTAGCAAACCC-3’ and reverse

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5’-TATCTCTCAGCTCCACGCCA-3’ (Accession number NM_000594.3); and Homo sapiens 18S ribosomal RNA (18S), forward 5’-GTAACCCGTTGAACCCCATT-3’ and reverse 5’CCATCCAATCGGTAGTAGCG-3’ (accession No. NR_003286). Relative gene expression (normalized to 18s reference gene) was calculated according to the ∆∆CT method. Fidelity of the PCR reaction was determined by melting temperature analysis.

Confocal Microscopy This technique was used in our studies previously (Chin et al., 2018; Chin et al., 2015). In brief, exponentially growing oral cancer OEC-M1 and SCC-25 cells were seeded on sterilized cover glasses (Paul Marienfeld, Lauda-Königshofen, Germany). Cells were treated with resveratrol, NDAT or the combination for 24 h. Cells treated with S31-201 and resveratrol were used as positive control. Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes and then permeabilized in 0.1% Triton X-100 in PBS for 20 minutes. Samples were incubated with a rabbit monoclonal antibody to PD-L1 followed by an Alexa-647labeled goat anti-rabbit antibody (Abcam, Cat. No.: GTX104763) and mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Fremont, CA). The fluorescent signals from PD-L1 were recorded and analyzed with the TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems, Wetzlar, Germany). The figures shown are representative of at least four fields for each experimental condition.

Statistical Analysis All of the collected data for immunoblot and nucleotide densities were analyzed by IBM®SPSS® Statistics software version 19.0 (SPSS Inc., Chicago, IL, USA). Student’s t-test was

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conducted and p-values < 0.05 (*, # or $), 0.01 (**, ## or $$) and 0.001 (***, ### or $$$) as the threshold for significance, was used to evaluate the significance of effects of hormone, resveratrol, T4, or combination treatment.

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RESULTS

Resveratrol and NDAT attenuate the expression of pro-inflammatory cytokines in OECM1 cells. Oral cancer OEC-M1 cells were treated with either 10 µM resveratrol, 10-7 M NDAT or the combination for 24 h to investigate effect of resveratrol, NDAT and their combination on pro-inflammatory gene expression. Total RNA was extracted and qPCR was conducted for expression of IL-1β and TNF-α. Expression of IL-1β and TNF-α were not only significantly attenuated by resveratrol but also significantly attenuated by NDAT in OEC-M1 cells (Fig. 1A and Fig 1B). The combination of resveratrol and NDAT further enhanced inhibitory effect on expression of TNF-α significantly as compared with treatment of resveratrol alone (Fig 1B).

NDAT enhances effect of resveratrol on gene expression in OEC-M1 cells. In order to evaluate the antiproliferative effect of resveratrol and NDAT in OEC-M1 cells, cells were treated with either 10 µM resveratrol, 10-7 M NDAT or the combination for 24 h. Total RNA was extracted and qPCR was conducted. Expression of genes involved in proliferation, and immunomodulation and pro-apoptosis was examined. Resveratrol and NDAT significantly inhibited proliferative gene, CCND1, and immunomodulating gene, PD-L1 in OEC-M1 cells (Fig. 1C and 1D). Suppression of CCND1 by resveratrol was enhanced by NDAT significantly (p<0.01) (Fig. 1C). Expression of COX-2 was significantly induced by resveratrol (Fig. 1E), but NDAT attenuated resveratrol-induced COX-2 significantly (p<0.05) (Fig. 1E). Expression of pro-apoptotic gene, BAD, was significantly induced by both resveratrol and NDAT in OEC-M1 cells (Fig. 1F). However, there was no enhanced effect in their combination.

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Thyroxine restored inhibitory effect of resveratrol on PD-L1 accumulation in OEC-M1 cells. We have shown that thyroid hormone (thyroxine, T4) interferes with resveratrol-induced anti-proliferation by trapping resveratrol-induced COX-2 with T4-induced PD-L1 in cytosol (Chen et al., 2019; Chin et al., 2018). We investigated if resveratrol-induced inhibitory effect of PD-L1 expression and accumulation can be restored by thyroxine treatment in OEC-M1 cells by confocal microscopy. OEC-M1 cells were starved for 48 h and re-fed with hormone-stripped serum-containing medium and then treated with either 10 µM resveratrol, T4 (10-7 M), or the combination for another 24 h. Resveratrol not only attenuated the expression of PD-L1 but also inhibit the accumulation of nuclear PD-L1 (Fig. 2). On the other hand, T4 induced PD-L1 expression in both cytoplasm and nuclei in OEC-M1 cells (Fig. 2). When T4 was present, resveratrol diminished T4-induced PD-L1 accumulation (Fig. 2). We then examined if the enhanced effect of T4 on PD-L1 accumulation can be depleted by an inhibitor of STAT3. OECM1 cells were pre-treated with STAT3 inhibitor, S31-201 (40 µM) for 30 min and were treated with resveratrol, thyroxine or the combination for another 24 h. The constitutive PD-L1 in control or resveratrol-treated cells was reduced when cells were pre-treated with STAT3 inhibitor, S31-201 (Fig. 2). T4-induced PD-L1 accumulation was inhibited by pre-treatment of STAT3 inhibitor, S31-201 in OEC-M1 cells (Fig. 2). These results suggest that inactivation of STAT3 may be part of mechanisms in PD-L1 inhibition. NDAT enhances resveratrol-attenuated PD-L1 expression in OEC-M1 cells. In our previous study (Lin et al., 2018), we revealed that NDAT attenuated PD-L1 expression in oral cancer cells. In order to explore if the inhibitory effect of resveratrol on PD-L1 expression would be further enhanced by NDAT, OEC-M1 cells were treated with 10 µM resveratrol, 10-7 M NDAT or the combination for 24 h. Studies by confocal microscopy indicate that NDAT (10-7 M) alone not 12

only reduced PD-L1 accumulation but also enhanced inhibition of resveratrol on PD-L1 accumulation (Fig. 3). STAT3 inhibitor, S31-201 (40 µM) showed similar results to enhance resveratrol’s inhibition on PD-L1 accumulation (Fig. 3). These results suggest that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. NDAT plays a crucial role in potentiating resveratrol attenuated T4-induced cancer progression in OEC-M1 cells. Both resveratrol and NDAT inhibited OEC-M1 cell growth and their combination showed growth inhibition significantly as compared with resveratrol treatment alone (p<0.05) (Fig. 4A). Parallel studies were conducted to investigate STAT3 status and IL-1β accumulation for OEC-M1 cells responded to treatment of resveratrol and NDAT. Both resveratrol and NDAT inhibited IL-1β accumulation (Fig. 4B). Resveratrol reduced not only STAT3 phosphorylation but also STAT3 accumulation. Interestingly, NDAT only inhibited STAT3 phosphorylation but increased STAT3 accumulation (Fig. 4B). The combination treatment reduced IL-1β, STAT3 phosphorylation and STAT3 accumulation. These results suggest that inhibition of STAT3 may play roles in resveratrol- and NDAT-induced antiproliferation. To determine the effect of NDAT inhibitory effect on T4-induced cancer progression by resveratrol, OEC-M1 cells were treated with T4 (10-7 M), 10 µM resveratrol, NDAT (10-7 M) or the combination for 24 h. Total RNA was extracted and qPCR was performed. T4-induced cancer progressive genes such as proliferative gene (CCND1) (Fig. 4C) and immunomodulating gene (PD-L1) (Fig. 4D) were attenuated by NDAT, resveratrol and their combination. In addition, T4inhibited anti-proliferative genes, COX-2 (Fig. 4E) and BAD (Fig. 4F), were restored by resveratrol. Interestingly, although expression of COX-2 was slightly attenuated by NDAT and

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the combined treatments with resveratrol (Fig. 4E), T4-inhibited BAD expression was restored by the combined treatment of NDAT and resveratrol (Fig. 4F).

Resveratrol and NDAT attenuated pro-inflammatory cytokines and enhanced antiproliferative effect in SCC-25 cells. Another oral cancer SCC-25 cell line which shows different characteristics compared to OEC-M1 (Chen et al., 2019) was used to explore enhancing effect of NDAT on resveratrol-induced anti-inflammatory and anti-proliferative activities. SCC25 cells were treated with either 10 µM resveratrol, 10-7 M NDAT or the combination for 24 h to investigate anti-inflammatory and anti-proliferative effects of resveratrol and NDAT. Total RNA was extracted and qPCR was performed. Expression of IL-1β and TNF-α were significantly attenuated by resveratrol, NDAT and their combination in SCC-25 cells (Fig 5A and 5B). Furthermore, NDAT significantly potentiated resveratrol-attenuated expression of IL-1β and TNF-α in SCC-25 cells (p<0.001) (Fig 5A and 5B). In addition, resveratrol, and NDAT significantly inhibited expression of CCND1 and PD-L1 in SCC-25 cells (Fig. 5C and 5D). The combination significantly inhibited expression of CCND1 (p<0.01) and PD-L1 (p<0.05) even more than resveratrol treatment alone. While expression of COX-2 was significantly induced by resveratrol, COX-2 expression was inhibited by NDAT (Fig. 5E). Expression of pro-apoptotic gene, BAD, was significantly enhanced by both resveratrol and NDAT in SCC-25 cells (Fig. 5F). We performed confocal microscopy to evaluate the inhibitory effect of resveratrol on hormone-induced PD-L1 accumulation and the role of STAT3 signal pathway in SCC-25 cells, confocal microscopy was performed. SCC-25 cells were starved for 48 h and re-fed with hormone-stripped serum-containing medium. After pretreatment with STAT3 inhibitor, S31-201 (40 µM), for 30 min, SCC-25 cells were treated either 10 µM resveratrol, T4 (10-7 M), or the

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combination for another 24 h. Resveratrol diminished T4-induced PD-L1 accumulation in cytoplasm and nuclei (Fig. 6) in SCC-25 cells. Furthermore, T4-induced PD-L1 accumulation was also diminished by pre-treatment of S31-201 (Fig. 6).

NDAT potentiates resveratrol attenuated T4-induced cancer progressive effects in SCC-25 cells. Cell proliferation of SCC-25 cells was inhibited by resveratrol and NDAT (Fig. 7A). Combined treatment of resveratrol and NDAT showed significant growth inhibition as compared with resveratrol treatment alone (p<0.01) (Fig. 7A). Parallel studies were conducted to study if resveratrol and NDAT suppressed IL-1β accumulation and STAT3 status in SCC-25 cells. Both resveratrol and NDAT inhibited IL-1β accumulation (Fig. 7B). Resveratrol reduced not only STAT3 phosphorylation but also STAT3 accumulation. Interestingly, NDAT only inhibited STAT3 phosphorylation but increased STAT3 accumulation (Fig. 7B). The combination treatment reduced IL-1β, STAT3 phosphorylation and STAT3 accumulation. These results suggest that inhibition of STAT3 may play roles in resveratrol- and NDAT-induced antiproliferation. In order to determine NDAT inhibitory effect on T4-induced cancer progression by resveratrol, SCC-25 cells were treated with T4 (10-7 M), 10 µM resveratrol, NDAT (10-7 M) or the combination for 24 h. Total RNA was extracted and qPCR was performed for proliferative gene (CCND1), immune-modulating gene (PD-L1), COX-2 and BAD. Expression of T4-induced CCND1 (Fig. 7C) and PD-L1 (Fig. 7D) was inhibited by NDAT, resveratrol and their combination. In addition, expression of genes inhibited by T4, COX-2 (Fig. 7E) and BAD (Fig. 7F), was restored by resveratrol. Interestingly, although expression of COX-2 was slightly

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attenuated by NDAT and the combined treatments with resveratrol (Fig. 7E), T4-inhibited BAD expression was restored by the combined treatment of NDAT and resveratrol (Fig. 7F). In summary, although resveratrol and NDAT both induced anti-proliferation in two oral cancer cell lines by inhibiting different gene expression, their combination treatment only showed enhancing effects on resveratrol-induced activities based on our current results.

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DISCUSSION

Combination treatment of thyroid hormone deaminated analogues with other anti-cancer agents has been studies recently (Chang et al., 2018; Lee et al., 2016; Nana et al., 2018a; Nana et al., 2018b). Although resveratrol has been shown to induce anti-proliferation in various types of cancer cell lines and in vivo xenograft animal models, its low bioavailability is always a big concern. In addition, we have recently shown that thyroid hormone interferes with resveratrolinduced anti-cancer growth via inducible PD-L1 trapping COX-2 in cytosol (Chin et al., 2018). Thyroxine also induces RRM2 (Lee et al., 2016) or HMGA2 (Nana et al., 2018a) to reduce resveratrol-induced anti-proliferative activities in cancer cells. Even more, resveratrol, itself would induce RRM2 (Nana et al., 2018b) to reduce anti-proliferative activities. NDAT not only blocks expression of pro-inflammatory genes but also blocks PD-L1 component of the PD-1/PD-L1 checkpoint in oral cancer cells (Fig.1 and 5). In current studies, IL-1, TNF-α, and PD-L1 might be induced in vitro by free thyroxine which had already existed in the serum. The inhibitory effect of NDAT on the expression of PD-L1 by thyroxine in tumor cells suggests that the hormone receptor on αvβ3 mediated the event and the inhibitory effects of NDAT on tumor cells and dividing endothelial cells are limited to the thyroxine receptor on the extracellular domain of integrin (Lin et al., 2016a). NDAT down-regulates expression of nuclear factor (NF)-κB via the integrin (Davis et al., 2015) and NF-κB de-activation is an antiangiogenic target. Receptor-mediated signaling pathways such as NF-κB (Peng et al., 2015), ERK1/2 (An et al., 2016), phosphoinositide 3-kinase (PI3K) (Lastwika et al., 2016; Zhao et al., 2019), mammalian target of rapamycin (mTOR) (Lastwika et al., 2016; Zhao et al., 2019), and Janus kinase/signal transducers and activators of transcription (JAK/STAT) (Doi et al., 2017) are linked to tumor cells’ PD-L1 expression. PD-L1 gene expression is able to be activated by 17

thyroxine via ERK1/2 activation and PD-L1 protein abundance consequently in different types of cancers such as colorectal, breast, ovarian and oral cancer (Chin et al., 2018; Lin et al., 2016a; Lin et al., 2018). Thyroid-stimulated

expression of pro-inflammatory cytokines has been

well-

demonstrated. Thyroid hormone (triiodothyronine, T3) significantly induces interleukin-6 (IL-6) and IL-8 in human osteoblast-like (MG63) cells and human bone marrow stromal cells (Siddiqi et al., 1998). In contrast, effects of IL-1β and IL-3, IL-4, and IL-11 are unaffected in the same studies (Siddiqi et al., 1998). Therefore, hormone’s actions are selective and substantial. Siddiqi et al (Siddiqi et al., 1998) and Mascanfroni et al. (Mascanfroni et al., 2008) suggest that IL responses to T3 may have depended upon integrin-mediated, nongenomic actions of the hormone. If integrin αvβ3 receptor for thyroid hormone is involved in this interleukin response, it is important to consider physiological concentration of thyroxine, since the affinity of integrin αvβ3 receptor is higher for T4 than T3 (Bergh et al., 2005), thus thyroxine may be a more potent stimulator of IL responses. Additionally, thyroid hormone may regulate transcription of monomeric αv gene, but it is unknown whether this action is initiated at the αvβ3 protein or requires nuclear TR. Modulation of pro-inflammatory gene products and inducible PD-L1 gene expression can occur via integrin αvβ3 by thyroid hormone and its antagonists via STAT3 signal transduction pathway (Fig. 2, 3 and 6). Thyroid hormone-induced PD-L1 expression is activated ERK1/2 (Lin et al., 2016a), PI3K (Lin et al., 2018) or STAT3 (Chen et al., 2019) -dependent in different cancer cells. NDAT inhibits activation of ERK1/2 (Lin et al., 2016a), PI3K (Lin et al., 2018) and STAT3 (Fig. 2, 3, and 6). Thyroid hormone has been shown to potentiate epidermal growth factor (EGF)-induced c-Fos expression by STAT3-dependent signal pathway (Lin et al., 1999).

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Reduced thyroxine decreases not only oncogenic protein SAT3 expression but also cell proliferation in renal cancer cells (Calvino et al., 2016; Poplawski et al., 2017). Those studies indicate that thyroid hormone activates STAT3 signal transduction pathway. Resveratrol can suppress STAT3 activation and inhibit thyroxine-induced PD-L1 expression (Chen et al., 2019). Resveratrol suppressed PD-L1 expression which was enhanced by STAT3 inhibitor and NDAT (Fig. 2, 3 and 6). Expression of pro-inflammatory genes and PD-L1 is involved in cancer proliferation in oral cancer cells. Resveratrol in combination with NDAT moderates ligands of the integrin which inhibit the expression of pro-inflammatory genes and the activity of the PD-1/PD-L1 checkpoint. NDAT inhibited expression of CCND1 and PD-L1 in oral cancer cells (Fig. 1 and 5) and colorectal cancer cells (Lin et al., 2016a). Meanwhile, NDAT potentiated resveratrolsuppressed expression of inflammatory genes and PD-L1. Integrin αvβ3 receptor might regulate the inhibitory effects of NDAT on thyroxine-induced cell growth in tumor cells. However, mechanisms involved in NDAT potentiated resveratrol-regulated gene expression should happen on downstream signal transduction pathway, STAT3 which is suppressed by NDAT and resveratrol. Other actions of NDAT on cancer cells such as inhibitory HMGA2 and RRM2 may also play roles in potentiating effect of NDAT on resveratrol-induced anti-proliferation in oral cancer cells. Basically, nonmalignant cells and normal immune system surveillance will escape from this novel effect of NDAT (Lin et al., 2016a; Lin et al., 2018). NDAT targets integrin αvβ3 receptor which is expressed primarily on cancer cells and only expressed in promptly growing endothelial cells and osteoclasts but not in normal cells, thus restricting the disruption of the PD1/PD-L1 checkpoint by the agent against nonmalignant cells. In addition, integrin αvβ3 may also

19

involve in overexpression of PD-L1 checkpoint in cancer cells as our recent studies have suggested (Lin et al., 2016a). In conclusion, we verified that inflammatory genes and PD-L1 elaborated in the antagonistic effect of thyroxine on resveratrol-induced anti-proliferation in oral cancer cells in normal physiological condition. Inhibiting SATA3 signal transduction pathway by NDAT blocks expression of inflammatory genes and PD-L1 in oral cancer cells to enhance resveratrol-induced anti-proliferation in euthyroid condition. Clinical PD-L1-directed immunotherapy is possibly suppressed by endogenous thyroxine and such therapy might be improved by lowering circulating thyroxine by pharmacologic intervening agents such as resveratrol, NDAT, or their combination.

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ACKNOWLEDGMENTS

This work was supported in part by an intra-institutional grant from E-Da Medical Center (EDAHP108043 to Dr. CY Wu), by a collaborative grant of Taipei Medical University (TMU) and National Taipei University of Technology (NTUT-TMU-101-10 to Dr. Y. Ho), by Research Award from Dr. Ta-Cheng Tung Foundation, by Chair Professor Research Fund to Dr. Kuan Wang, and to Dr. Jaqulene Whang-Peng, by the “TMU Research Center of Cancer Translational Medicine” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan, general grants from the Ministry of Science and Technology Taiwan (MOST108-2119-M-038-001 to J. WhangPeng and MOST108-2314-B-038-050 to H.Y. Lin) and a gift from Dr. Paul J. Davis to Albany College of Pharmacy and Health Sciences. Authors would like to send their most sincere appreciation to Dr. Yi-shin Pan, Miss Zi-Lin Li, Miss Ya-Jung Shih, and Miss Yi-Ru Chen for their extraordinary contribution to this work. Authors express sincere appreciation to Gloria for her stunning proofreading.

AUTHOR CONTRIBUTIONS CY Wu, Y Ho, ZL Li, YC Yang, YR Chen and K Wang conceived the experiments. CY Wu, ZL Li, PJ Davis, and K Wang designed the experiments. YT Chin, ZL Li, YS Pan, TY Huang, PY Su, CA Changou, YJ Shih, and YR Chen performed the experiments. YT Chin, CY Wu, ZL Li, HC Chiu, SY Lee, KW Su, SA Mousa and HY Lin analyzed the data. ZL Li, YS Pan, YJ Shih, and YR Chen prepared figures.

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HY Lin, DR Crawford, J Whang-Peng, PJ Davis and K Wang prepared the manuscript. REFERENCES

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FIGURE LEGENDS

Figure 1. NDAT potentiates resveratrol-affected expression of inflammatory proliferative and anti-proliferative genes in oral cancer OEC-M1 cells. OEC-M1 cells were treated with either 10 µM resveratrol (RV), 10-7 M NDAT or the combination for 24 h. Total RNA was extracted and qPCR was performed for IL-1β (A), TNF-α (B), CCND1 (C), PD-L1 (D), COX-2 (E), and BAD (F). Numbers of Independent studies (N) = 5, **p < 0.01, ***p < 0.001, compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with resveratrol.

Figure 2. Thyroxine but not resveratrol induces PD-L1 in human oral cancer OEC-M1 cells. OEC-M1 cells were seeded on cover glass. Cells were starved for 48 h and re-fed with hormone-stripped serum containing medium, and then treated with either 10 µM resveratrol, 10-7 M T4, or their combination in the presence or absence of 40 µM S31-201 for another 24 h. Cells were fixed for confocal microscopic analysis of PD-L1 expression (red color). Nuclei were stained by DAPI as counter staining (blue color).

Figure 3. NDAT enhances resveratrol-attenuated PD-L1 in human oral cancer OEC-M1 cells. OEC-M1 cells were seeded on cover glass and starved for 48 h with hormone-stripped serum containing medium. Then cells were treated with either 10 µM resveratrol, 10-7 M NDAT, or their combination in the presence or absence of 40 µM S31-201 for another 24 h. Cells were fixed for confocal microscopic analysis of PD-L1 expression (red color). Nuclei were stained by DAPI as counter staining (blue color).

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Figure 4. NDAT inhibits PD-L-1 expression to reverse the inhibitory effect of thyroxine on resveratrol-induced anti-proliferation in oral cancer OEC-M1 cells. (A). OEC-M1 cells were plated at a density of 4x103 cells/well in 96-well plates. Cell viability was determined by using the alamarBlue Assay Kit at 72 h after treatment. (B). OEC-M1 cells were treated with 10 µM resveratrol, 10-7 M NDAT or their combination for 24 h. Cells were harvested and total proteins were extracted. Western blots were conducted for IL-1β, total STAT3 and phosphorylated STAT3. N = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control. OEC-M1 cells were treated for 24 h with 10 µM resveratrol or 10-7 M T4 in the presence or absence of 10-7 M NDAT. Total RNA was extracted and qPCR was performed for CCND1 (C), PD-L1 (D), COX-2 (E), and BAD (F). N = 5, *p < 0.05, **p < 0.01, ***p < 0.001, compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with T4; $ p < 0.05,

$$

p < 0.01, $$$ p < 0.001, compared with the

combined treatment of T4 and resveratrol.

Figure 5. NDAT potentiates resveratrol-affected expression of inflammatory proliferative and anti-proliferative genes in oral cancer SCC-25 cells. SCC-25 cells were treated with either 10 µM resveratrol, 10-7 M NDAT or the combination for 24 h. Total RNA was extracted and qPCR was performed for IL-1β (A), TNF-α (B), CCND1 (C), PD-L1 (D), COX-2 (E), and BAD (F). N = 5, *p < 0.05, ***p < 0.001, compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with resveratrol.

Figure 6. Thyroxine-induced PD-L1 was attenuated by resveratrol in oral cancer SCC-25 cells. SCC-25 cells were seeded on cover glass. Cells were starved for 48 h and re-fed with hormone-stripped serum containing medium, and then treated with either 10 µM resveratrol, 10-7

29

M T4, or their combination in the presence or absence of 40 µM S31-201 for another 24 h. Cells were fixed for confocal microscopic analysis of PD-L1 expression (red color). Nuclei were stained by DAPI as counter staining (blue color).

Figure 7. NDAT inhibits PD-L-1 expression to restore the inhibitory effect of thyroxine on resveratrol-induced anti-proliferation in oral cancer SCC-25 cells. (A). Cells were plated at a density of 4x103 cells/well in 96-well plates. Cell viability was determined by using the alamarBlue Assay Kit at 72 h after treatment. (B). SCC-25 cells were treated with 10 µM resveratrol, 10-7 M NDAT or their combination for 24 h. Cells were harvested and total proteins were extracted. Western blots were conducted for IL-1β, total STAT3 and phosphorylated STAT3. N = 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control. SCC-25 cells were treated for 24 h with 10 µM resveratrol or 10-7 M T4 in the presence or absence of 10-7 M NDAT. Total RNA was extracted and qPCR was performed for CCND1 (C), PD-L1 (D), COX-2 (E), and BAD (F). N = 5, *p < 0.05, **p < 0.01, ***p < 0.001, compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with T4; $$ p < 0.01, $$$ p < 0.001, compared with the combined treatment of T4 and resveratrol.

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OEC-M1 cells

A

B

IL-1β

TNF-α

1.0 0.8

0.6

***

*** ***

0.4 0.2 0.0

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- + -+ - -

+ +

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**

0.6

*** ***

- + -+ - -

+ +

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0.6

#

0.6

RV (10 μM)

0.8

NDAT (10-7 M)

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0.0

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Relative mRNA expression

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0.0 RV (10 μM)

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0.4

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1.0

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RV (10 μM)

##

1.2

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1.0

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C

E

Relatived mRNA expression

1.2

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Relatived mRNA expression

1.2

+ +

1.4 1.2 0.8 0.6 0.4 0.2

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Fig. 1

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1.0

- + -+ - -

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T4+RV

RV (10 μM)

T4 (10-7 M)

Control

PD-L1 (Red) PD-L1 (Red) Magnification Magnification Nuclei (Blue) Nuclei (Blue)

Fig. 2

OEC-M1 cells RV (10 µM)

S31-201 (40 µM)

NDAT (10-7 M)

Control

PD-L1 (Red) PD-L1 (Red) Magnification Magnification Nuclei (Blue) Nuclei (Blue)

Fig. 3

OEC-M1 cells A

100 kD

B 120

*

*

80

***

STAT3

60

-- -+ -+

CTL

NDAT (10-7 M)

RV

NDAT

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RV+NDAT

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0.0

C

CCND1

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##

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0.5

RV (10 μM) NDAT (10-7M)

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###

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E

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NDAT

NDATT4

NDATRV

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$

##

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0

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#

$$$

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Relative mRNA expression

0.0

-

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RV (10 μM) NDAT (10-7M)

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T4 (10-7M)

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0.0

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Relative mRNA expression

*

(10-7 M)

D

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*

0.5

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*

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*** ** ***

40

**

Relative I.O.D.

pSTAT3-S727

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0.0

GAPDH

35 kD

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20

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35 kD

*** **

Cell Viability (%)

pSTAT3-S727 75 kD

100

1.0

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75 kD

#

1.5

1.0

#

**

$

0.5

0.0

T4 (10-7M) RV (10 μM) NDAT (10-7M)

Fig. 4

**

**

-- -+ -+ ++ -- -+ -+ - - - - + + +

+ + +

SCC-25 cells

A

B

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TNF-α

0.6

*** ***

0.2

- + -+ - -

+ +

Relative mRNA expression

1.0 0.8

***

***

***

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(10-7 M)

0.2

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+ +

# *

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Fig. 5

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0.6 0.4 0.2

(10-7 M)

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NDAT NDATRV

BAD 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

RV (10 μM) NDAT

*

0.8

F

#

- + -+ - -

1.0

0.0 RV (10 μM) NDAT

+ +

PD-L1

Relatived mRNA expression

Relative mRNA expression

RV (10 μM)

*** ***

1.2

0.4

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

0.4

D

COX-2

E

***

0.6

NDAT (10-7 M)

0.6

(10-7 M)

###

0.8

RV (10 μM)

##

1.2

1.0

0.0

CCND 1

0.0 RV (10 μM)

NDAT

***

0.4

(10-7 M)

C

Relative mRNA expression

0.8

RV (10 μM)

NDAT

###

1.0

0.0 NDAT

1.2

Relative mRNA expression

Relative mRNA expression

1.2

(10-7 M)

*** ***

- + -+ - -

+ +

SCC-25 cells S31-201 (STAT3 inhibitor)

T4+RV

RV (10 μM)

T4 (10-7 M)

Control

PD-L1 (Red) PD-L1 (Red) Magnification Magnification Nuclei (Blue) Nuclei (Blue)

Fig. 6

SCC-25 cells A

100 kD

B

STAT3

75 kD

##

pSTAT3-S727 75 kD

***

1.5

STAT3 pSTAT3-S727 IL-1β

60

Relative I.O.D.

0.0

--

NDAT

***

*

$

##

0.5

0.0

T4 (10-7M) RV (10 μM) NDAT (10-7M)

-- - -+ ++ -- -+ -+ - - - - + + + 0

+0

0

0

T4 (10-7M)

RV (10 μM) NDAT (10-7M)

-- -+ -+ ++ -- -+ -+ - - - - + + + 0

0

0

0

0

0

0

0

0

+0 + +

BAD 1.5

*

1.0

**

*

$$

0.5

0.0

+0 T (10 M) + RV (10 μM) + NDAT (10 M) Fig. 7 4

0

-7

-- -+ -+ ++ -- -+ -+ - - - - + + +

-7

4

##

$

+ + +

AT RV N D AT T4 R

** 1.0

0.0

F

COX-2

1.5

##

*

D

E

+ + +

+ +

RV+NDAT

0.5

R V

RV (10 μM) NDAT (10-7M)

-- +- -+ ++ -- -+ -+ - - - - + + +

*

T4

0.0

##

N

0.5

(10-7M)

Relative mRNA expression

*

$

** 1.0

ol

**

###

-

AT T

###

NDAT

PD-L1

on tr

1.0

Relative mRNA expression

**

RV

1.5

C

T4

D

CCND1

1.5

Relative mRNA expression

Relative mRNA expression

C

(10-7 M)

-+

+

RV (10 μM) CTL

AT

-

0.5

+ +

RV+NDAT

D

NDAT

D

NDAT (10-7 M)

-+

+

RV

N

--

CTL

*** ***

0 RV (10 μM)

***

20

**

1.0

*** **

40

N

80

GAPDH

35 kD

R V

*

T4

*

100

IL-1β

35 kD

** ***

Cell Viability (%)

120

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: