NAG-1 Expression Research Articles

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

Simple SummaryThyroid 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.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.

Anti-proliferative activity of A. Oxyphylla and its bioactive constituent nootkatone in colorectal cancer cells

BackgroundA. oxyphylla extract is known to possess a wide range of pharmacological activites. However, the molecular mechanism of A. oxyphylla and its bioactive compound nootkatone in colorectal cancer is unknown.MethodsOur study aims to examine the role of A. oxyphylla and its bioactive compound nootkatone, in tumor suppression using several in vitro assays.ResultsBoth A. oxyphylla extract and nootkatone exhibited antiproliferative activity in colorectal cancer cells. A. oxyphylla displayed antioxidant activity in colorectal cancer cells, likely mediated via induction of HO-1. Furthermore, expression of pro-apoptotic protein NAG-1 and cell proliferative protein cyclin D1 were increased and decreased respectively in the presence of A. oxyphylla. When examined for anticancer activity, nootkatone treatment resulted in the reduction of colony and spheroid formation. Correspondingly, nootkatone also led to increased NAG-1 expression and decreased cyclin D1 expression. The mechanism by which nootkatone suppresses cyclin D1 involves protein level regulation, whereas nootkatone increases NAG-1 expression at the transcriptional level. In addition to having PPARγ binding activity, nootkatone also increases EGR-1 expression which ultimately results in enhanced NAG-1 promoter activity.ConclusionIn summary, our findings suggest that nootkatone is an anti-tumorigenic compound harboring antiproliferative and pro-apoptotic activity.

Expression of lytic enzyme genes involved in antagonistic activity of Trichoderma viridescens during interaction with Sclerotinia sclerotiorum

This study was conducted to evaluate expression of 11 genes, endochitinase (chit33), endochitinase (chit37), endochitinase (chit42), N-acetyl-B-D-glucosaminidase (nag1), α-1,2-mannosidase (gh92), α-1,3-glucanase (a13gluc), β-1,6-glucanase (b16gluc), β-1,3-glucanase (b13gluc), aminopeptidase S9 (s9), trypsin-like (pra1) and serine protease (sprt) which may be involved in biological control of Sclerotinia sclerotiorum by Trichoderma viridescens. Total RNA was extracted from the mycelia where colonies of the pathogen and T. viridescens reached together on potato dextrose agar. As a control, a confrontation assay was conducted where T. viridescens was interacted with itself. The results of reverse transcriptase-PCR detected no band for s9 and sprt. However, expression of a13gluc, b16gluc, chit33 and chit37 was upregulated. No change in expressions was detected in chit42, b13gluc and pra1 compared with the control. The gh92 was upregulated only in one isolate (Tvig2). The expression of nag1 in both isolates of T. viridescens was clearly downregulated compared with the control. This study showed that upregulation of a13gluc, b16gluc, chit33 and chit37 in T. viridescens may play a role in biocontrol of S. sclerotiorum.

Predictive value of microRNA-132 and its target gene NAG-1 in evaluating therapeutic efficacy of non-steroidal anti-inflammatory drugs treatment in patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common chronic rheumatic disorder, accompanied by the differential expression of various microRNAs (miRNAs) in patients suffering from the condition, some of which have the potential to serve as novel complementary AS biomarkers. During this study, AS patients were recruited in connection with our investigation into the correlation of microRNA-132 (miR-132) in peripheral blood and its target gene NAG-1 expressions in relation with the clinical efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) treatment in patients with AS. A total of 218 AS patients who had been previously treated with oral diclofenac sodium and were placed into either the response (n = 175) or non-response groups (n = 43) following a 16-week period of therapeutic evaluation. An additional 113 healthy patients were also recruited for the purposes of the study. AS patient peripheral blood samples were obtained at the 0th, 8th, and 16th week, with the corresponding samples of the healthy patients collected at week 0. The expressions of miR-132 and NAG-1 were detected by RT-qPCR and analyzed using a ROC curve for the elucidation of the diagnostic value of peripheral blood miR-132 expressions as well as their predictive value among AS patients undergoing NSAIDs treatment. The targeting relations of miR-132 and NAG-1 were validated by microRNA.org and luciferase assay. Greater levels of peripheral blood miR-132 expression were observed among AS patients prior to treatment, in comparison to the healthy patients in the study. Prior to treatment, the area under the miR-132 ROC curve (AUC) of AS patients was 0.965, with a critical point of 2.605. The sensitivity and specificity of miR-132 were 91.7 and 97.3%, respectively, in regard to the AS diagnostic clinical efficacy. In comparison with the non-response group, the miR-132 expression of patients in the response group exhibited descended levels while the mRNA expression of NAG-1 increased. The ROC results indicated that the AUC of miR-132 was 0.876 with its sensitivity and specificity observed to be 95.3 and 80.0%, respectively. The AUC of NAG-1 was 0.912 with its sensitivity and specificity observed to be 76.6 and 79.1%, respectively. In comparison with the high miR-132 expression group and the low NAG-1 mRNA expression group, significantly improved blood biochemistry indexes, sign indexes, blood indexes, and adverse reaction rate were observed among the low miR-132 expression group and the high NAG-1 mRNA expression group. The microRNA.org and luciferase assay revealed NAG-1 to be a target of miR-132. Based on the results of this study, it was concluded that the expressions of MiR-132 and NAG-1 could serve as biological markers in the prediction of the therapeutic efficiency of NSAID treatment in AS patients.

Isochaihulactone-induced DDIT3 causes ER stress-PERK independent apoptosis in glioblastoma multiforme cells.

The endoplasmic reticulum (ER) is a major site of cellular homeostasis regulation. Under the ER stress condition, Glioblastoma multiform (GBM) cells activate the unfolded protein response. In this study, we discovered isochaihulactone, a natural compound extracted from the Chinese traditional herb Nan-Chai-Hu, which can disrupt ER homeostasis in GBM cell lines. It can induce DNA damage inducible transcript 3 (DDIT3) expression which is independent of 78 kDa glucose-regulated protein (GRP78) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) expression. Flow cytometry results revealed that isochaihulactone trigger the cell cycle arrest at G2/M phase and apoptosis in GBM cells. Isochaihulactone induced DDIT3 led to the expression of NAG-1. The in vivo study showed that isochaihulactone suppressed tumor growth, and DDIT3 and Caspase3 overexpressed in the xenograft model, which is consistent with the in vitro study. Overall, the data revealed that isochaihulactone disrupted ER homeostasis in cancer cells by increasing DDIT3 and NAG-1 expression. Our finding also provides a therapeutic strategy by using isochaihulactone for GBM treatment.

Early Epithelial Restitution by Nonsteroidal Anti-Inflammatory Drug–Activated Gene 1 Counteracts Intestinal Ulcerative Injuries

In response to ulcerative mucosal injuries, intestinal epithelial restitution is a critical event in the early defense against harmful attacks by luminal Ags. Based on the assumption that epithelial NAG-1 is an endogenous regulator of ulcerative stress-induced injuries, the expression and functions of NAG-1 were investigated. Genetic ablation of NAG-1 decreased survival of mice with dextran sodium sulfate-induced intestinal ulcer and histologically delayed the epithelial restitution, confirming early protective roles of NAG-1 in ulcerative insults. Moreover, enhanced expression of NAG-1 during the wound-healing process was associated with epithelial cell migration and spreading. In response to ulcerative injury, RhoA GTPase, a cytoskeleton modulator, mediated epithelial restitution via enhanced motility. RhoA expression was prominently elevated in the restituting epithelia cells around the insulted wound bed and was attenuated by NAG-1 deficiency. Pharmacological intervention with RhoA thus attenuated NAG-1-mediated epithelial cell migration during epithelial restitution. Taken together, epithelial restitution was promoted by enhanced NAG-1 expression and subsequent enterocyte locomotion during the early wound-healing process, suggesting clinical usefulness of NAG-1 as a novel endogenous muco-protective factor or an indicator of therapeutic efficacy against the ulcerative gastrointestinal diseases, including inflammatory bowel disease.

한방이화주 주박 추출물에 의한 암세포 항성장 및 세포사멸 기전 연구

본 연구에서는 한국 전통 막걸리 중 하나인 한방 이화주의 주박으로부터 에탄올 추출물과 그것의 순차적인 유기용매 분획물을 획득하고, 이들 시료들이 인간 대장암 세포인 HCT116 세포주의 cell viability와 pro-apoptotic 유전자인 ATF-3와 NAG-1의 발현에 미치는 영향을 연구하였다. 처리된 유기용매 시료 가운데 KSD-E3-2 분획물 (hexane 분획물)과 KSD-E3-3 분획물 (ethyl acetate 분획물)의 처리에 의해 현저하게 세포생존율이 감소하였으며, 두 분획물에 의해 농도의존적으로 세포 생존율이 감소하였다. 또한, 두 분획물의 처리에 의해 pro-apoptotic 유전자인 NAG-1과 ATF-3의 유전자와 단백질의 발현이 증가함을 확인하였다. 특히, KSD-E3-3 분획물 0.1 mg/ml의 처리에 의해 NAG-1 단백질의 발현이 현저하게 증가하였으며, 또한 apoptosis의 지표인 PARP cleavage가 관찰되었다. 이러한 KSD-E3-3 분획물에 의해 유도된 apoptosis는 NAG-1 siRNA transfection에 의해 회복됨을 확인하였다. 따라서, KSD-E3-3 분획물에 의한 NAG-1 단백질의 발현증가와 apoptosis 간의 직접적인 관련성이 있음을 시사한다. 종합적으로 본 연구 결과는 한방이화주 주박에 함유된 생리활성물질의 암세포 항 성장 활성 및 apoptosis기전을 이해하는데 도움을 줄 것으로 생각된다. In the present study, ethanol extracts and their subsequent organic solvent fractions were extracted from the lees of Ehwa Makgeolli containing oriental herbs, a commercialized traditional Korean rice wine, and the prepared lees samples were designated as from KSD-E3-1 to KSD-E3-5. First, their effects on cell viability and on the expression of pro-apoptotic ATF3 and NAG-1 genes in human colorectal HCT116 cells were investigated. Among the treated lees samples, the hexane fraction (KSD-E3-2) and the ethyl acetate fraction (KSD-E3-3) of lees extracts from Ehwa Makgeolli significantly reduced cell viabilities, in a dose dependent manner. The treatment with KSD-E3-2 and KSD-E3-3 also increased the expression of pro-apoptotic NAG-1 and ATF-3 genes and their proteins, which were detected with RT-PCR and Western blot analysis, respectively. In addition, poly-(ADP-ribose) polymerase (PARP) cleavage was detected by treatment with the fraction KSD-E3-3, indicating that KSD-E3-3 could induce apoptosis in HCT116 cells. Interestingly, this PARP cleavage was recovered by transfection of NAG-1 small interfering RNA. The results indicate that NAG-1 is one of the genes responsible for apoptosis induced by the fraction KSD-E3-3 from Ehwa Makgeolli. Overall, the findings may help in understanding the molecular mechanisms of the anti-proliferative and pro-apoptotic activities mediated by the lees of Ehwa Makgeolli.

Abstract 3238: Taiwanin A targets against nonsteroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma

Abstract Taiwanin A (α,β-bis(piperonylidene)-γ-butyrolactone) is isolated from Taiwania cryptomerioides. Similar to paclitaxel, Taiwanin A also extracted from tree barks and have anti-tumor activity in breast, liver, and lung cancer cell lines. The objective of this study is to demonstrate the cytotoxicity of Taiwanin A against tumor cells by increasing the expression of Non-steroidal anti-inflammatory drug-activated gene (NAG-1). NAG-1 has been reported to have antitumor and pro-apoptotic activity, suggesting potential target for cancer therapy. Silencing NAG-1 mRNA expression in A549 reduces the cytotoxicity caused by Taiwanin A. Furthermore, the c-Jun-N-terminal kinase/Ste20-related protein proline/alanine-rich kinase (JNK/SPAK) pathway played a key role in the action of NAG-1 on cell viability, whereas the addition of the JNK pathway inhibitor, SP600125, resulted in the inhibitory effect of NAG-1 and recovery of Taiwanin A treated cells. Xenograft tumor model showed that Taiwanin A dose-dependently significantly decreases tumor mediated growth in nude mice by increasing NAG-1 expression accompanied tumor apoptosis. These data support the conclusion that Taiwanin A inhibits lung carcinoma growth by increasing NAG-1 expression via the JNK pathway both in vivo and in vitro. This result might be contributed to compound design for increasing cytotoxicity activity in the future. Citation Format: Hong-Meng Chuang, Horng-Jyh Harn. Taiwanin A targets against nonsteroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3238. doi:10.1158/1538-7445.AM2015-3238

ATF3 Mediates Anti-Cancer Activity of Trans-10, cis-12-Conjugated Linoleic Acid in Human Colon Cancer Cells.

Conjugated linoleic acids (CLA) are a family of isomers of linoleic acid. CLA increases growth arrest and apoptosis of human colorectal cancer cells through an isomer-specific manner. ATF3 belongs to the ATF/CREB family of transcription factors and is associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which t10, c12-CLA stimulates ATF3 expression and apoptosis in human colorectal cancer cells. t10, c12-CLA increased an apoptosis in human colorectal cancer cells in dose dependent manner. t10, c12-CLA induced ATF3 mRNA and luciferase activity of ATF3 promoter in a dose-dependent manner. The responsible region for ATF3 transcriptional activation by t10, c12-CLA is located between −147 and −1850 of ATF3 promoter. mRNA stability of ATF3 was not affected by t10, c12-CLA treatment. t10, c12-CLA increases GSK3β expression and suppresses IGF-1-stimulated phosphorylation of Akt. The knockdown of ATF3 suppressed expression of GSK3β and NAG-1 and PARP cleavage. The results suggest that t10, c12-CLA induces apoptosis through ATF3-mediated pathway in human colorectal cancer cells.

Variations in NAG-1 expression of human gastric carcinoma and normal gastric tissues

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a member of the transforming growth factor β (TGF-β) superfamily, has been demonstrated to possess antitumorigenic and proapoptotic activities in gastric cancer cells. In the present study, the expression of NAG-1 was assessed in human gastric carcinoma, tumor-adjacent normal tissues and normal gastric mucosa, with the aim to investigate the role of NAG-1 in the carcinogenesis and development of gastric carcinoma. NAG-1 protein expression was evaluated using immunohistochemical staining, while the expression of NAG-1 mRNA was evaluated using reverse transcription-polymerase chain reaction. It was observed that adenocarcinoma tissues had a lower expression of NAG-1 than normal gastric tissues. Furthermore, moderately and well-differentiated adenocarcinoma tissues expressed more NAG-1 protein than the poorly differentiated adenocarcinoma tissues. The expression of NAG-1 protein in adenocarcinoma tissues did not correlate with tumor-node-metastasis staging, infiltration degree or tumor size. The NAG-1 mRNA expression in adenocarcinoma tissues was also lower than that in normal gastric tissues. In conclusion, NAG-1 was poorly expressed in adenocarcinoma tissues and inversely correlated with the degree of tumor differentiation. These results indicate that NAG-1 may have an anti-oncogenic function in the carcinogenesis and development of gastric carcinoma, and that its attenuated or absent expression may lead to gastric carcinogenesis.

Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1

A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5–5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

71 Tolfenamic Acid Induces Apoptosis and Growth Inhibition in Head and Neck Cancer: Involvement of NAG-1 Expression

71 Tolfenamic Acid Induces Apoptosis and Growth Inhibition in Head and Neck Cancer: Involvement of NAG-1 Expression

A peroxisome proliferator-activated receptor ligand MCC-555 imparts anti-proliferative response in pancreatic cancer cells by PPARgamma-independent up-regulation of KLF4

MCC-555 is a novel PPARα/γ dual ligand of the thiazolidinedione class and was recently developed as an anti-diabetic drug with unique properties. MCC-555 also has anti-proliferative activity through growth inhibition and apoptosis induction in several cancer cell types. Our group has shown that MCC-555 targets several proteins in colorectal tumorigenesis including nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) which plays an important role in chemoprevention responsible for chemopreventive compounds. NAG-1 is a member of the TGF-β superfamily and is involved in tumor progression and development; however, NAG-1's roles in pancreatic cancer have not been studied. In this report, we found that MCC-555 alters not only NAG-1 expression, but also p21 and cyclin D1 expression. NAG-1 and p21 expression was not blocked by PPARγ-specific antagonist GW9662, suggesting that MCC-555-induced NAG-1 and p21 expression is independent of PPARγ activation. However, decreasing cyclin D1 by MCC-555 seems to be affected by PPARγ activation. Further, we found that the GC box located in the NAG-1 promoter play an important role in NAG-1 transactivation by MCC-555. Subsequently, we screened several transcription factors that may bind to the GC box region in the NAG-1 promoter and found that KLF4 potentially binds to this region. Expression of KLF4 precedes NAG-1 and p21 expression in the presence of MCC-555, whereas blocking KLF4 expression using specific KLF4 siRNA showed that both NAG-1 and p21 expression by MCC-555 was blocked. In conclusion, MCC-555's actions on anti-proliferation involve both PPARγ-dependent and -independent pathways, thereby enhancing anti-tumorigenesis in pancreatic cancer cells.

MCC‐555 induces apoptosis through the combination of PPARγ‐dependent and ‐independent pathways in pancreatic cancer cells

MCC‐555 is a novel PPARα/γ dual ligand of the thiazolidinedione class that was recently developed as an anti‐diabetic drug with anti‐cancer activity. Nonsteroidal anti‐inflammatory drug (NSAID)‐activated gene (NAG‐1) is involved in pro‐apoptotic and anti‐tumorigenic properties in colorectal and lung cancer, as assessed by NAG‐1 transgenic mice; however, its roles in pancreatic cancer have not been studied. We found that MCC‐555 elicits changes of various genes such as NAG‐1, p21, and cyclin D1, which are involved in cell proliferation and apoptosis. NAG‐1 and p21 expression was not blocked by PPARγ‐specific antagonist GW9662, suggesting that MCC‐555‐induced NAG‐1 and p21 expression is independent on PPARγ activation. However, decreasing cyclin D1 by MCC‐555 seems to be affected by PPARγ activation. For NAG‐1 transcriptional regulation by MCC‐555, the GC box located in the ‐133/+41 NAG‐1 promoter plays an important for NAG‐1 transactivation by MCC‐555 and KLF4 binds to this region. Chip assay confirmed that KLF4 binds to the GC box region in the NAG‐1 promoter and regulates NAG‐1 expression in response to MCC‐555. Further, expression of KLF4 precedes NAG‐1 and p21 expression in the presence of MCC‐555. Therefore, MCC‐555′s actions on anti‐tumorigenesis involve in both PPARγ‐dependent and ‐independent pathways, thereby enhancing anti‐tumorigenesis in pancreatic cancer cells.

브로콜리 유래 sulforaphane에 의한 NAG-1과 p21 유전자의 발현 조절

본 연구에서는 대장암 세포주 모델에서 브로콜리 유래 sulforaphane에 의한 항 성장 활성과 항암 단백질 NAG-1과 p21의 발현 및 발현 조절에 대해서 연구하였다. 그 결과, 처리한 sulforaphane의 농도가 증가됨에 따라 세포사멸이 증가되었고, 세포 생존율은 감소하였다. 또한, sulforaphane에 의한 항암 단백질 NAG-1과 p21의 발현증가를 농도별, 시간대별로 확인한 결과, 두 단백질 모두 sulforaphane 농도와 처리시간 의존적으로 발현이 증가됨을 확인하였다. Sulforaphane에 의한 NAG-1과 p21의 발현의 p53 의존성을 연구한 결과, p21의 발현은 p53에 의존적 이지만 NAG-1의 발현은 p53에 비 의존적인 것으로 생각된다. 또한, sulforaphane이 dietary histone deacetylase inhibitor로서 NAG-1의 발현을 증가시킬 가능성은 매우 미미한 것으로 생각된다. 전사조절인자인 ATF3의 발현을 sulforaphane을 시간대 별로 처리한 후 확인한 결과, 2시간째부터 발현이 증가되어 NAG-1의 발현 보다 먼저 증가됨을 확인하였다. 이러한 연구 결과는 sulforaphane의 항암 기능을 이해하는 새로운 기전을 제시해 줄 것으로 기대된다. We investigated the anti-proliferative activity of sulforaphane and expression changes of NAG-1 and p21 genes in response to sulforaphane treatment in human colorectal HCT116 cells. The results showed that sulforaphane decreased cell viabilities in a dose-dependent manner and induced expression of NAG-1 and p21 proteins in a dose-dependent and time-dependent manner. In addition, we found that NAG-1 expression by sulforaphane was not dependent on the presence of p53, whereas p21 expression was dependent on p53 presence. The results indicated that up-regulation of NAG-1 was not related with the activity of a dietary histone deacetylase inhibitor of sulforaphane. ATF3 induction was detected from 2 hr after sulforaphane treatment, indicating that ATF3 could be a transcription factor to up-regulate NAG-1 expression. The results of this study may help to increase our understanding of the molecular mechanism of anti-cancer activity mediated by sulforaphane in human colorectal cancer cells.

DNA methylation‐mediated silencing of nonsteroidal anti‐inflammatory drug‐activated gene (NAG‐1/GDF15) in glioma cell lines

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

The Role of NAG-1/GDF15 in the Inhibition of Intestinal Polyps in APC/Min Mice by Sulindac

The antitumor effects of nonsteroidal anti-inflammatory drugs (NSAID) are assumed to be due to the inhibition of COX activity, but COX-independent mechanisms may also play an important role. NSAID-activated gene (NAG-1/GDF15) is induced by NSAIDs and has antitumorigenic activities. To determine the contribution of COX-2 inhibition and NAG-1/GDF15 expression to the prevention of colon carcinogenesis by NSAIDs, we evaluated several sulindac derivatives [des-methyl (DM)-sulindac sulfide and its prodrug DM-sulindac] that do not inhibit COX-2 activity. Sulindac sulfide and DM-sulindac induced the expression of NAG-1/GDF15 in HCT116 cells as determined by quantitative real-time PCR and Western blot. We fed APC/Min mice with 320 ppm of sulindac and doses of DM-sulindac. Only sulindac significantly inhibited tumor formation inAPC/Min mice. To determine the pharmacokinetic properties of sulindac and DM-sulindac in vivo, wild-type C57/B6 mice were fed with sulindac and DM-sulindac at 80, 160, and 320 ppm. High-performance liquid chromatography analysis revealed that the conversion of DM-sulindac to DM-sulindac sulfide (active form) was less efficient than the conversion of sulindac to sulindac sulfide (active form) in the mice. Lower levels of DM-sulindac sulfide accumulated in intestinal and colon tissues in comparison with sulindac sulfide. In addition, NAG-1/GDF15 was induced in the liver of sulindac-fed mice but not in the DM-sulindac-fed mice. Collectively, our results suggest that the tumor-inhibitory effects of sulindac in APC/Min mice may be due to, in part, NAG-1/GDF15 induction in the liver. Our study also suggests that pharmacologic properties should be carefully evaluated when developing drug candidates.

Troglitazone induces apoptosis in gastric cancer cells through the NAG-1 pathway

The non-steroidal anti-inflammatory drug-activated gene (NAG-1) is a newly identified member of the transforming growth factor (TGF)-β superfamily and plays significant roles in regulating proliferation and pro-apoptotic activities. In the present study, we studied the regulation of NAG-1 by troglitazone in the cultured gastric cancer cell line BGC-823. MTT and TUNEL assays demonstrated that troglitazone potentially inhibits the proliferation of the gastric cancer cell line and induces apoptosis in vitro in a dose- and time-dependent manner. Troglitazone induced concentration-dependent NAG-1 expression in the BGC-823 cells, as assessed using immunocytochemistry. Furthermore, troglitazone increased Egr-1 protein levels in a concentration-dependent manner. In conclusion, the present study suggests that troglitazone markedly impedes proliferation and pro-apoptotic activities in BGC-823 cells, and the mechanism may partly be through the Egr-1 pathway.

Pleurospermum kamtschaticumExtract Induces ApoptosisviaMitochondrial Pathway and NAG-1 Expression in Colon Cancer Cells

To evaluate the anticarcinogenic activity of methanol extract of Pleurospermum kamtschaticum (PKE), we assessed its apoptosis-inducing capability in HT-29 colon carcinoma cells. PKE treatment for 2 h reduced cell viability in a dose-related manner, and induced apoptotic morphological changes. Flow cytometric analysis indicated that PKE treatment at 0.05 mg/ml induced early apoptosis in 66.2% of HT-29 cells. Additionally, Bcl-2 expression was substantially reduced in PKE-treated HT-29 cells, increasing the Bax/Bcl-2 ratio. The protein levels of procaspase-9 and procaspase-3 were decreased markedly, reflecting caspase-9 and caspase-3 activation, and resulting PARP cleavage was noted in the PKE-treated HT-29 cells. Furthermore, we detected increased NAG-1 expression in the PKE-treated HT-29 cells. In an in vivo study, intraperitoneal PKE administration suppressed the formation of tumor nodules in the lungs of mice. These results indicate that PKE can serve as a beneficial supplement in the treatment and the prevention of colon cancer.