Quercetin Induces Anticancer Activity by Upregulating Pro-NAG-1/GDF15 in Differentiated Thyroid Cancer Cells

Simple Summary Thyroid cancer is one of the most common cancers worldwide, and its incidence has increased over the last few decades. It is difficult to diagnose different types of thyroid cancer. Tumor tissues from papillary thyroid cancer patient showed higher expression of mature NAG-1, whereas adjacent normal tissues showed higher expression of pro-NAG-1. Several anti-cancer compounds increased pro-NAG-1 expression in thyroid cancer cell line. Quercetin (3,3’,4’,5,7-pentahydroxyflavone) is a flavonoid that is a major component of various plants, including raspberries, grapes, and onions. Quercetin induced apoptosis by inducing only pro-NAG-1 expression, but not mature NAG-1, mediated by the transcription factor C/EBP. This study indicates that pro-NAG-1 could be used as a useful biomarker for thyroid cancer and also provides a potential therapeutic target for the treatment of thyroid cancer with quercetin. Abstract Although the treatment of thyroid cancer has improved, unnecessary surgeries are performed due to a lack of specific diagnostic and prognostic markers. Therefore, the identification of novel biomarkers should be considered in the diagnosis and treatment of thyroid cancer. In this study, antibody arrays were performed using tumor and adjacent normal tissues of patients with papillary thyroid cancer, and several potential biomarkers were identified. Among the candidate proteins chosen based on the antibody array data, mature NAG-1 exhibited increased expression in tumor tissues compared to adjacent normal tissues. In contrast, pro-NAG-1 expression increased in normal tissues, as assessed by western blot analysis. Furthermore, pro-NAG-1 expression was increased when the thyroid cancer cells were treated with phytochemicals and nonsteroidal anti-inflammatory drugs in a dose-dependent manner. In particular, quercetin highly induced the expression of pro-NAG-1 but not that of mature NAG-1, with enhanced anticancer activity, including apoptosis induction and cell cycle arrest. Examination of the NAG-1 promoter activity showed that p53, C/EBPα, or C/EBPδ played a role in quercetin-induced NAG-1 expression. Overall, our study indicated that NAG-1 may serve as a novel biomarker for thyroid cancer prognosis and may be used as a therapeutic target for thyroid cancers.


Introduction
The incidence of thyroid cancer has increased in recent years, being the most commonly detected cancer in the USA (52,070 new cases in 2019) [1]. It is also the second most frequently diagnosed cancer in Korea [2]. Cancer biomarkers can be classified into three broad categories: DNA, RNA, and protein markers. New biomarkers for cancer research are highly desirable, as early detection and correct diagnosis are essential for

Antibody Array
Whole proteins were extracted from thyroid tissues by sonication in radioimmunoprecipitation assay (RIPA) buffer (GenDEPOT, Katy, TX, USA) supplemented with proteinase and phosphatase inhibitors. The antibody array was performed using a RayBio ® C-Series Human Cancer Discovery Antibody Array 3 (RayBiotech, Peachtree Corners, GA, USA) according to the manufacturer's protocol.

Protien Isolation and Western Blot Analysis
Cells were grown to 80% confluence and then treated with the indicated compounds. After 24 h of incubation with serum-free media, protein lysates were obtained using RIPA buffer supplemented with proteinase and phosphatase inhibitors and separated on sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels (10,12, and 15% gels for tissue samples and 12% gels for BCPAP cells). To obtain conditional media, cells were grown in a 10 cm culture dish with 10 mL of serum-free media and incubated for 24 h. The media were harvested, and the cell debris was removed and concentrated using an Amicon Ultra-15 (UFC901024; Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). The separated protein bands were transferred onto nitrocellulose membranes (GVS filter technology, Zola Predosa BO, Italy) and blocked with TBS buffer containing 0.05% Tween 20 (TBS-T) with 5% non-fat milk at room temperature for 1 h, followed by overnight incubation with an appropriate primary antibody in TBS-T containing 5% non-fat milk at 4 • C. The primary antibodies used were anti-NAG-1 (specific to both pro-NAG-1 and mature NAG-1) [8], anti-galectin-3 (sc-32790; Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiosteoprotegerin (OPG; sc-390518; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TIMP-1(sc-6832; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (sc-47724; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were washed three times with TBS-T buffer for 10 min and incubated with secondary antibodies dissolved in TBS-T buffer containing 5% non-fat milk at room temperature for 2 h. The membranes were washed again, and protein expression was detected by chemiluminescence using an enhanced chemiluminescence (ECL) western blotting detection reagent (Thermo Fisher Scientific, Waltham, MA, USA) on a chemiluminescence analyzer, Alliance Q9 Advanced (UVTEC CAMBRIDGE, Cambridge, England, UK) according to the manufacturer's instructions.

Dual-Luciferase Assay
BCPAP cells were seeded in a 24-well plate and incubated for 24 h at 37 • C. Four luciferase constructs were used (pNAG-1 -133/+70/LUC, pNAG-1 -133/+41/LUC, pNAG-1 -474/+41/LUC, and pNAG-1 -1086/+41/LUC) [27]. For the co-transfection experiments, each luciferase construct with the empty vector, p53, ATF3, CREB, RARα, C/EBPα, C/EBPδ, or EGR-1 expression vector was transfected into BCPAP cells, and either DMSO or quercetin (10 and 50 µM) was added to the transfected cells in serum-free media. After 24 h, the media were removed, and the cells were washed twice with 1x PBS. Then, 200 µL of 1x passive lysis buffer was added to each well, and the plate was shaken until the cells were detached completely on ice. The cell lysate was transferred to a new tube and centrifuged at 12,000× g for 15 s. Luciferase activity was measured using a Dual-Luciferase kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.

Apoptosis Analysis by Flow Cytometry
BCPAP cells were cultured in a 6-well plate until they reached 60-80% confluence. The cells were treated with the compound in serum-free media and incubated for 24 h. After washing and trypsinization, cells were stained with FITC Annexin V Apoptosis Detection Kit with propidium iodide (PI; BioLegend, San Diego, CA, USA) according to the manufacturer's protocol. The cells were then analyzed using Sony SH800 Cell Sorter (Sony Biotechnology Inc., Tokyo, Japan). The data is analyzed by FlowJo software (BD Life Sciences, Franklin Lakes, NJ, USA).2.9. Cell Cycle Analysis Cells grown on a 6-well plate to 100% confluence were treated with the compounds in serum-free media for 24 h. Then, the cells were harvested in a microcentrifuge tube and fixed with 0.5 mL of cold 70% EtOH (Merck, Billerica, MA, USA) for 1 h. Cells were collected by centrifugation and resuspended in 0.5 mL of phosphate-buffered saline (PBS) with 0.25% Triton-X 100 (Glentham Life Sciences Ltd., Corsham, Wiltshire, UK) for 15 min on ice. After centrifugation, cell pellets were resuspended in PBS (0.5 mL) containing 10 µg/mL RNase A (iNtRON Biotechnology, Seongnam, Gyeonggi Province, Korea) and 20 µg/mL PI (Invitrogen, Carlsbad, CA, USA) and incubated for 30 min in the dark. Cells were analyzed by Sony SH800 Cell Sorter (Sony Biotechnology Inc., Tokyo, Japan). The data BCPAP cells were seeded in 60 mm dishes and transiently transfected with PolyJet Transfection Reagent (SignaGen, Gaithersburg, MD, USA) according to the manufacturer's protocol. After 24 h post-transfection, serum-free media containing DMSO and quercetin (1, 10, and 50 µM) was added to the dishes. After 24 h of treatment with the compounds, the cells were harvested and analyzed by western blotting.

Enzyme-Linked Immunosorbent Assay (ELISA)
Plasma NAG-1 levels were measured using the Human GDF15 Quantikine ELISA Kit (DGD150; R&D Systems, Minneapolis, MN, USA). Samples, reagents, and buffers were prepared according to the manufacturer's instructions. The detection sensitivity of NAG-1 was 4.39 pg/mL, and the assay range was 23.4-1500 pg/mL. To determine the optical density, a microplate reader was used to measure the intensity of the wells. The microplate reader was set to 450 nm and corrected by subtracting the intensity at 570 nm. The concentration of each sample was calculated using a standard curve.

Statistical Analysis
Statistical analysis was conducted using Microsoft Office Excel, SPSS, and GraphPad Prism 8. Unpaired Student's t-test and one-way analysis of variance were used to analyze the data. For all analyses, results were considered significant at p < 0.05 (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Identification of Differentially Expressed Proteins in Thyroid Normal and Tumor Tissues
To identify novel biomarkers in thyroid cancer, we performed an antibody array using normal human thyroid and tumor tissues. Tumor and adjacent normal tissues were obtained from patients with papillary thyroid cancer, and an antibody array was performed. Four candidate proteins, galectin-3, NAG-1, TIMP-1, and osteoprotegerin (OPG), were identified as potential biomarkers of thyroid cancer ( Figure 1A). The protein expression of galectin-3, TIMP-1, and NAG-1 was higher in tumor tissues than in normal tissues. In contrast, OPG expression was higher in normal tissues than in tumor tissues. To confirm the results of the antibody array, western blot analysis was performed using the proteins extracted from three patients with papillary cancer ( Table 1). The expression of OPG was higher in normal tissues, whereas that of galectin-3 and TIMP-1 was higher in tumor tissues ( Figure 1B). Interestingly, two forms of NAG-1/GDF15 were detected in the tissues. Mature NAG-1 was expressed more in tumor tissues, whereas pro-NAG-1 exhibited higher expression in normal tissues. Since NAG-1 is expressed as a pro-(~35 kDa) and a mature form (~12 kDa), the antibody array data were consistent with the mature form of NAG-1. Pro-NAG-1 and mature-NAG-1 have been reported to exhibit different biological activities in tumorigenesis [7,14]. Since mature NAG-1 has been detected in the antibody array and mature serum NAG-1 is linked to thyroid pro-tumorigenesis [28], we measured serum NAG-1 levels to identify the linkage in different types of thyroid cancer: non-aggressive benign thyroid nodules (BTN: FA and NH) and aggressive differentiated thyroid cancer (DTC: FVPTC, PTC, and FTC). Serums from forty-nine patients ( Figure S1E) were obtained, and NAG-1 expression was measured by ELISA. The data were analyzed for NAG-1 concentration by tumor type, sex, BMI, and age. NAG-1 concentration was lower in the plasma samples of patients with benign tumors, such as follicular adenoma and nodular hyperplasia than in those with malignant thyroid cancer, such as PTC, FVPTC, and FTC, but the difference was not statistically significant ( Figure S1A). In addition, no difference was noted in NAG-1 concentrations according to sex and BMI ( Figure S1B,C). Interestingly, the concentration of NAG-1 in the plasma increased with patient age [29] ( Figure S1D). According to these data, the levels of mature serum NAG-1 change with age regardless of the patient's sex, BMI, or cancer type. Together, these results indicate that pro-and mature NAG-1 not only exhibit great potential as a biomarker for diagnosis but also as a therapeutic target for thyroid cancer.
in the plasma samples of patients with benign tumors, such as follicular adenoma and nodular hyperplasia than in those with malignant thyroid cancer, such as PTC, FVPTC, and FTC, but the difference was not statistically significant ( Figure S1A). In addition, no difference was noted in NAG-1 concentrations according to sex and BMI ( Figure S1B and S1C). Interestingly, the concentration of NAG-1 in the plasma increased with patient age [29] ( Figure S1D). According to these data, the levels of mature serum NAG-1 change with age regardless of the patient's sex, BMI, or cancer type. Together, these results indicate that pro-and mature NAG-1 not only exhibit great potential as a biomarker for diagnosis but also as a therapeutic target for thyroid cancer. Figure 1. Antibody array using human thyroid tissues. (A) Antibody array (RayBio ® C-Series Human Cancer Discovery Antibody Array 3, RayBiotech) showed that 30 cytokines related to cancer biology were differentially expressed between thyroid normal and tumor tissues. One pair of PTC samples was used to analyze the expression of these cytokines. Four different proteins (Galectin-3, NAG-1/GDF15, TIMP-1, and osteoprotegerin) were selected as biomarker candidates for the diagnosis of thyroid cancer (rectangle). The right graph represents the intensity of each protein. (B) Western blot was performed using three pairs of PTC samples to confirm the data of antibody array. The protein expression of galectin-3, mature NAG-1, pro-NAG-1, TIMP-1, and OPG was examined using thyroid tissue samples. The right graph is from the average quantification of protein expression from three patients. N, thyroid normal tissue; T, thyroid tumor tissue. The number of patients is indicated (see Table 1 for details). Uncropped versions of blots presented in Figure S2 and Figure S3.

Mature and Pro-NAG-1 Expression in Various Cancer Cells
To identify whether various cancer cells express NAG-1 at the transcriptional level, we first measured the mRNA levels of NAG-1 in thyroid and other cancer cells. As shown in Figure 2A, BCPAP cells exhibited higher expression of NAG-1 mRNA among thyroid cancer cell lines, whereas U2OS osteosarcoma cells showed the highest NAG-1 mRNA expression. Since NAG-1 is synthesized as pro-NAG-1 and cleaved into mature NAG-1, Figure 1. Antibody array using human thyroid tissues. (A) Antibody array (RayBio ® C-Series Human Cancer Discovery Antibody Array 3, RayBiotech) showed that 30 cytokines related to cancer biology were differentially expressed between thyroid normal and tumor tissues. One pair of PTC samples was used to analyze the expression of these cytokines. Four different proteins (Galectin-3, NAG-1/GDF15, TIMP-1, and osteoprotegerin) were selected as biomarker candidates for the diagnosis of thyroid cancer (rectangle). The right graph represents the intensity of each protein. (B) Western blot was performed using three pairs of PTC samples to confirm the data of antibody array. The protein expression of galectin-3, mature NAG-1, pro-NAG-1, TIMP-1, and OPG was examined using thyroid tissue samples. The right graph is from the average quantification of protein expression from three patients. N, thyroid normal tissue; T, thyroid tumor tissue. The number of patients is indicated (see Table 1 for details). Uncropped versions of blots presented in Figures S2 and S3.

Mature and Pro-NAG-1 Expression in Various Cancer Cells
To identify whether various cancer cells express NAG-1 at the transcriptional level, we first measured the mRNA levels of NAG-1 in thyroid and other cancer cells. As shown in Figure 2A, BCPAP cells exhibited higher expression of NAG-1 mRNA among thyroid cancer cell lines, whereas U2OS osteosarcoma cells showed the highest NAG-1 mRNA expression. Since NAG-1 is synthesized as pro-NAG-1 and cleaved into mature NAG-1, western blot analysis was conducted to determine the mature and pro-NAG-1 expression in various cancer cell lines. Western blot analysis revealed that BCPAP cells showed higher NAG-1 expression in the cell lysates and conditioned medium among the thyroid cancer cells ( Figure 2B), which was consistent with the RT-qPCR data. Among the non-thyroid cancer cells, BT-20 breast cancer cells and U2OS osteosarcoma cells expressed significant amounts of NAG-1 in cell lysates and conditioned media ( Figure 2C).

Quercetin Increases Pro-NAG-1 Levels but Not Mature NAG-1 Levels
Since two forms of NAG-1 (mature and pro-) are differentially expressed in the cell lysates with opposing activities in cancer cells, we examined several compounds that increase pro-NAG-1 expression in thyroid cancer cells. BCPAP cells were treated with various anticancer compounds, such as phytochemicals and nonsteroidal anti-inflammatory drugs (NSAIDs). Among these, sulindac sulfide and quercetin dramatically increased pro-NAG-1 expression compared to DMSO treatment ( Figure 3A). Treatment with sulindac sulfide (SS), trans-chalcone (TC), and quercetin (QUE) also increased pro-NAG-1 expression in a dosedependent manner ( Figure 3B). Since quercetin treatment increased pro-NAG-1 expression among the tested compounds, cells were treated with different doses of quercetin, and proand mature NAG-1 expressions were measured. Interestingly, quercetin only increased pro-NAG-1 but did not alter the expression of mature NAG-1 in a dose-dependent manner ( Figure 3C). This result indicates that pro-NAG-1 expression is preferentially increased by quercetin, and pro-NAG-1 is a chemotherapeutic target for thyroid cancer. western blot analysis was conducted to determine the mature and pro-NAG-1 expression in various cancer cell lines. Western blot analysis revealed that BCPAP cells showed higher NAG-1 expression in the cell lysates and conditioned medium among the thyroid cancer cells ( Figure 2B), which was consistent with the RT-qPCR data. Among the nonthyroid cancer cells, BT-20 breast cancer cells and U2OS osteosarcoma cells expressed significant amounts of NAG-1 in cell lysates and conditioned media ( Figure 2C). Conditioned medium and total cell lysates were isolated as described in the Methods section, and NAG-1 antibodies against pro-and mature NAG-1 were hybridized into the membrane. Uncropped versions of blots presented in the Figure S4 and Figure S5.

Quercetin Increases Pro-NAG-1 Levels but Not Mature NAG-1 Levels
Since two forms of NAG-1 (mature and pro-) are differentially expressed in the cell lysates with opposing activities in cancer cells, we examined several compounds that increase pro-NAG-1 expression in thyroid cancer cells. BCPAP cells were treated with various anticancer compounds, such as phytochemicals and nonsteroidal anti-inflammatory drugs (NSAIDs). Among these, sulindac sulfide and quercetin dramatically increased pro-NAG-1 expression compared to DMSO treatment ( Figure 3A). Treatment with sulindac sulfide (SS), trans-chalcone (TC), and quercetin (QUE) also increased pro-NAG-1 expression in a dose-dependent manner ( Figure 3B). Since quercetin treatment increased pro-NAG-1 expression among the tested compounds, cells were treated with different doses of quercetin, and pro-and mature NAG-1 expressions were measured. Interestingly, quercetin only increased pro-NAG-1 but did not alter the expression of mature NAG-1 in a dose-dependent manner ( Figure 3C). This result indicates that pro-NAG-1 expression is Conditioned medium and total cell lysates were isolated as described in the Methods section, and NAG-1 antibodies against pro-and mature NAG-1 were hybridized into the membrane. Uncropped versions of blots presented in the Figures S4 and S5.

Quercetin Induces Apoptosis and Cell Cycle Arrest
To confirm the anticancer activity of quercetin, a high-throughput platform was used. Representative images showed reduced fluorescence intensity of SYTOX and MitoTracker in quercetin-treated cells ( Figure 4A). Cell permeability was increased ( Figure 4B), and mitochondrial membrane potential was decreased by quercetin treatment in a dose-dependent manner ( Figure 4C), indicating apoptosis induction. Annexin V assay was performed to measure the percentage of apoptotic cells in quercetin-treated cells. The data showed that quercetin increased the percentage of apoptotic cells ( Figure 4D). Additionally, PI staining data suggested that quercetin treatment affected cell cycle arrest at the S phase ( Figure 4E). Taken together, quercetin induced cell apoptosis and cell cycle arrest. preferentially increased by quercetin, and pro-NAG-1 is a chemotherapeutic target for thyroid cancer.

Quercetin Induces Apoptosis and Cell Cycle Arrest
To confirm the anticancer activity of quercetin, a high-throughput platform was used. Representative images showed reduced fluorescence intensity of SYTOX and Mito-Tracker in quercetin-treated cells ( Figure 4A). Cell permeability was increased ( Figure 4B), and mitochondrial membrane potential was decreased by quercetin treatment in a dosedependent manner ( Figure 4C), indicating apoptosis induction. Annexin V assay was performed to measure the percentage of apoptotic cells in quercetin-treated cells. The data showed that quercetin increased the percentage of apoptotic cells ( Figure 4D). Additionally, PI staining data suggested that quercetin treatment affected cell cycle arrest at the S phase ( Figure 4E). Taken together, quercetin induced cell apoptosis and cell cycle arrest.

Quercetin Increases NAG-1 Promoter Activity through p53, C/EBPα, and C/EBPδ
To determine the mechanism by which quercetin affects NAG-1 expression at the transcriptional level, we examined NAG-1 promoter activity in the presence of quercetin. First, quercetin induced NAG-1 mRNA expression in a dose-dependent manner ( Figure 5A). To elucidate the molecular mechanism, we conducted a dual-luciferase assay using several NAG-1 promoters linked to the luciferase gene. Several luciferase constructs, including pNAG-1-133/+70/LUC, pNAG-1-133/+41/LUC, pNAG-1-474/+41/LUC, pNAG-1-1086/+41/LUC, and pRL null construct, were co-transfected into BCPAP, and transfected cells were treated with DMSO or 50 µM quercetin. After 24 h of incubation, luciferase activity was measured, and NAG-1 promoter activity was marginally increased by quercetin in all constructs ( Figure 5B), indicating that the quercetin response element may be located within the −133 to +70 region. Furthermore, this promoter region was examined in the presence of quercetin, revealing that quercetin increased NAG-1 promoter activity in a dose-dependent manner ( Figure 5C). Quercetin increases the level of p53 tumor suppressor protein in human colorectal cancer cells [30]. To investigate whether the increase in NAG-1 promoter activity depends on p53, we transfected an empty vector or p53 expression vector with pNAG-1-133/+70/LUC and pRL null and treated them with DMSO or quercetin. The luciferase activity was higher in the p53-transfected group ( Figure 5D), indicating that quercetin increased NAG-1 promoter activity via p53 expression. However, since BCPAP is a p53 mutant cell line, we hypothesized that there would be another pathway. To identify additional factors that cause NAG-1 induction, we used the pNAG-1-133/+41/LUC construct to perform the dual-luciferase assay. ATF3, CREB, RAR, C/EBPα, C/EBPδ, or EGR-1 can bind to the NAG-1 promoter within the -133 bp region [15,31,32]. Thus, several expression vector plasmids were co-transfected with the NAG-1 promoter in BCPAP cells. As a result, C/EBPα and C/EBPδ levels were significantly increased compared to the empty vector (EV)-transfected group (Figure 5E), in a dose-dependent manner ( Figure 5F). Taken together, quercetin may affect C/EBPα and C/EBPδ expression, followed by the induction of NAG-1 expression in BCPAP cells.

Quercetin Increases NAG-1 Promoter Activity through p53, C/EBPα, and C/EBPδ
To determine the mechanism by which quercetin affects NAG-1 expression at the transcriptional level, we examined NAG-1 promoter activity in the presence of quercetin. First, quercetin induced NAG-1 mRNA expression in a dose-dependent manner ( Figure  5A). To elucidate the molecular mechanism, we conducted a dual-luciferase assay using 1-133/+41/LUC construct to perform the dual-luciferase assay. ATF3, CREB, RAR, C/EBPα, C/EBPδ, or EGR-1 can bind to the NAG-1 promoter within the -133 bp region [15,31,32]. Thus, several expression vector plasmids were co-transfected with the NAG-1 promoter in BCPAP cells. As a result, C/EBPα and C/EBPδ levels were significantly increased compared to the empty vector (EV)-transfected group (Figure 5E), in a dose-dependent manner ( Figure 5F). Taken together, quercetin may affect C/EBPα and C/EBPδ expression, followed by the induction of NAG-1 expression in BCPAP cells.

Discussion
Although thyroid cancer is a common and relatively indolent cancer with a low mortality rate, some types of thyroid cancer show aggressive clinical features, such as rapid progression, lymph node, distant metastases, and even death from persistent and recurrent disease. Therefore, it is important to identify reliable and clinically applicable novel biomarkers for thyroid cancer diagnosis and prediction.
To elucidate this problem, we conducted an antibody array using papillary thyroid cancer and normal tissues. Four proteins were identified that showed increasing or decreasing expression in thyroid tumorigenesis ( Figure 1A,B). Galectin-3 controls cellular proliferation and apoptosis in normal cells as well as malignant transformation and metastasis in cancer cells [33]. Galectin-3 is a notable protein marker for thyroid tumors, and we confirmed the induction of galectin-3 in an antibody array and western blot analysis ( Figure 1A,B). The expression of TIMP-1 in the plasma and tissues of patients with cancer is highly increased, with more significant levels related to worse clinical results in various cancers, including prostate and colon cancers [34]. However, it is not clear whether TIMP-1 serves only as a biomarker of cancer progression or functions to promote cancer progression; thus, it could serve as an important cancer therapeutic target in thyroid cancer. Osteoprotegerin, which is engaged in many biological systems, plays a key role in the regulation of bone resorption [35]. The use of serum OPG as a prognostic marker has also been investigated in breast cancer and was found to be a potential diagnostic marker [36]. However, OPG was increased in normal thyroid tissues according to antibody array and western blot data, highlighting the differences between tissue and serum OPG levels. The exact biological activity of OPG in thyroid cancer remains to be elucidated.
NAG-1/GDF15 has been identified as an NSAID-induced gene [8]. It is by several anti-cancer agents, including phytochemicals [37], NSAIDs [27], and PPARγ ligands [38]. Although the biological activity of NAG-1 in obesity has been well established [39], the role of NAG-1 in tumorigenesis is contradictory in several cancers [7]. In general, an antitumorigenic effect during tumor development was observed in transgenic mice expressing NAG-1 [40,41]. In contrast, most results showing the pro-cancer activity of NAG-1 were obtained from in vitro experiments using cultured cells [42]. Recently, Kang et al. reported that NAG-1/GDF15 is a mitokine that increases the invasiveness of thyroid cancer [43]. This discrepancy may result from the different activities of pro-NAG-1 and mature NAG-1, the different expression of mature and pro-NAG-1, or multiple activities of NAG-1 depending on the cell context. In this study, antibody array data indicated that NAG-1 expression was increased in tumor tissues due to the abundant expression of mature NAG-1. However, size differentiation by western blot analysis clearly indicated that pro-NAG-1 was more highly expressed in normal tissues, whereas mature NAG-1 was more highly expressed in tumor tissues. This is consistent with our previous report that NAG-1 may function as a moonlighting protein in tumorigenesis [12,14].
Many pharmacological approaches have been proposed for conventional drug therapies. In the present study, we screened dietary compounds, phytochemicals, and NSAIDs to examine their effects on pro-NAG-1 induction in thyroid cancer cells. The levels of pro-NAG-1 were increased by sulindac sulfide or quercetin, a conventional NSAID and a phytochemical, respectively; these have been reported to increase pro-NAG-1 levels and exert anticancer activity in several cancers [44,45]. Interestingly, in the presence of quercetin, only pro-NAG-1 expression was increased in BCPAP cells ( Figure 3C). This result supports the fact that many phytochemicals induce anticancer activities via NAG-1 expression, even though mature NAG-1 is linked to pro-tumorigenic activity. Although detailed mechanisms need to be elucidated, this is the first report to suggest that a phytochemical preferentially increases the expression of pro-NAG-1 but not that of mature NAG-1 in BCPAP cells.
Several transcriptional factors have been shown to increase NAG-1 expression at the transcriptional level. Among these, C/EBPα and C/EBPδ were identified as NAG-1 inducers at the transcriptional level. The overexpression of CCAAT/enhancer-binding protein (C/EBP) α, β, and δ caused a significant increase in basal and capsaicin-induced NAG-1 promoter activity [46], and quercetin increased C/EBPβ mRNA and protein expression [47]. Thus, it is likely that quercetin increases the expression of C/EBP isotypes, followed by increased NAG-1 expression. However, the detailed molecular mechanism by which quercetin affects NAG-1 expression at the transcriptional level remains to be elucidated.
Taken together, this study highlights a potential biomarker for the diagnosis of thyroid cancer, especially in differentiating between pro-and mature NAG-1. Further investigation may be required to elucidate the molecular mechanism of quercetin-induced NAG-1 expression; however, our data indicate that C/EBP proteins contribute at least, in part, to the quercetin-induced NAG-1 expression (Figure 6). by increased NAG-1 expression. However, the detailed molecular mechanism by which quercetin affects NAG-1 expression at the transcriptional level remains to be elucidated.
Taken together, this study highlights a potential biomarker for the diagnosis of thyroid cancer, especially in differentiating between pro-and mature NAG-1. Further investigation may be required to elucidate the molecular mechanism of quercetin-induced NAG-1 expression; however, our data indicate that C/EBP proteins contribute at least, in part, to the quercetin-induced NAG-1 expression ( Figure 6).  Table S1 in human thyroid cancer, leading to an increase in pro-NAG-1 only, and not mature NAG-1.

Conclusions
The expression of mature NAG-1 in BCPAP cells was not significantly altered in the presence of quercetin, but pro-NAG-1 expression was significantly higher. This report suggests that pro-NAG-1 may be used as a therapeutic target in thyroid cancer.

Conclusions
The expression of mature NAG-1 in BCPAP cells was not significantly altered in the presence of quercetin, but pro-NAG-1 expression was significantly higher. This report suggests that pro-NAG-1 may be used as a therapeutic target in thyroid cancer.