Non-steroidal Anti-inflammatory Drug-activated Gene-1 Expression Research Articles

The anti-diabetic effects of NAG-1/GDF15 on HFD/STZ-induced mice

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) plays a role in various diseases. Here, the anti-diabetic effects of NAG-1 were evaluated using a high-fat diet/streptozotocin-induced diabetic mouse model. NAG-1-overexpressing transgenic (NAG-1 Tg) mice exhibited lower body weight, fasting blood glucose levels, and serum insulin levels than wild-type (WT) mice. The homeostatic model assessment of insulin resistance scores of NAG-1 Tg mice were lower than those of WT mice. Hematoxylin and eosin staining revealed a smaller lipid droplet size in the adipose tissues, lower lipid accumulation in the hepatocytes, and larger beta cell area in the pancreas of NAG-1 Tg mice than in those of WT mice. Immunohistochemical analysis revealed downregulated expression of cleaved caspase-3, an apoptosis marker, in the beta cells of NAG-1 Tg mice. Adiponectin and leptin mRNA levels were upregulated and downregulated in NAG-1 Tg mice, respectively. Additionally, the expression of IRS1/PI3K/AKT signaling pathway components, especially Foxo1, which regulates gluconeogenesis in the muscle and white adipose tissue, was downregulated in NAG-1 Tg mice. Furthermore, NAG-1 overexpression promoted the expression of As160 in both muscles and adipocytes, and the mRNA levels of the NLRP3 pathway members were downregulated in NAG-1 Tg mice. Our findings suggest that NAG-1 expression alleviates diabetes in mice.

Oroxylin A inhibits the migration of hepatocellular carcinoma cells by inducing NAG-1 expression.

Hepatocellular carcinoma (HCC), the most prevalent liver cancer, is considered one of the most lethal malignancies with a dismal outcome mainly due to frequent intrahepatic and distant metastasis. In the present study, we demonstrated that oroxylin A, a natural product extracted from Scutellaria radix, significantly inhibits transforming growth factor-beta1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) and metastasis in HCC. Oroxylin A blocked the TGF-β1/Smad signaling via upregulating the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression. Oroxylin A promoted NAG-1 transcription by regulating the acetylation of CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor that binds to the NAG-1 promoter. In terms of the underlying mechanism, oroxylin A may interact with histone deacetylase 1 (HDAC1) by forming hydrogen bonds with GLY149 residue and induce proteasome-mediated degradation of HDAC1 subsequently impairing HDAC1-mediated deacetylation of C/EBPβ and promoting the expression of NAG-1. Taken together, our findings revealed a previously unknown tumor-suppressive mechanism of oroxylin A. Oroxylin A should be further investigated as a potential clinical candidate for inhibiting HCC metastasis.

Grape Seed Proanthocyanidins Induce Apoptosis and Cell Cycle Arrest of HepG2 Cells Accompanied by Induction of the MAPK Pathway and NAG-1.

Hepatocellular carcinoma (HCC) is one of the common malignancies leading to death. Although radiotherapy and chemotherapy have certain effects, their side effects limit their therapeutic effect. Phytochemicals have recently been given more attention as promising resources for cancer chemoprevention or chemotherapy due to their safety. In this study, the effects of grape seed proanthocyanidins (GSPs) on the apoptosis, cell cycle, and mitogen-activated protein kinase (MAPK) pathway-related proteins and non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression of HepG2 cells were investigated. The results showed that GSPs inhibited the viability of HepG2 cells in a time- and dose-dependent manner, induced apoptosis and G2/M phase cell cycle arrest, and regulated cell cycle-related proteins, cyclin B1, cyclin-dependent kinase 1, and p21. GSPs also increased reactive oxygen species production and caspase-3 activity. In addition, GSPs also increased the expression of p-ERK, p-JNK, p-p38 MAPK and NAG-1, and GSPs-induced NAG-1 expression was related to the MAPK pathway-related proteins. These data suggest that GSPs may be promising phytochemicals for HCC chemoprevention or chemotherapy.

RETRACTED ARTICLE: RINT1 is a new suppression target to reduce colon cancer cell growth, migration and invasion through regulating ZW10/NAG-1 expression

This study explored the function and mechanism of RAD50 interactor 1 (RINT1), Zeste white 10 (ZW10) or nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) in cell growth of colon cancer. The mRNA expression of RINT1 was suppressed in patients with colon cancer. In addition, disease free survival (DFS) in colon cancer patients with high expression of RINT1, ZW10 and NAG-1 were promoted compared with colon cancer patients with low expression of RINT1, ZW10 and NAG-1. Over-expression of RINT1, ZW10 or NAG-1 reduced colon cancer cell growth, migration and invasion in vitro model. In contrast, down-regulation of RINT1, ZW10 or NAG-1 promoted colon cancer cell growth, migration and invasion in vitro model. Over-expression of RINT1 promoted while down-regulation of RINT1 suppressed ZW10 and NAG-1 expression. Following knockdown of ZW10 and NAG-1 protein expressions, RINT1 induced changes of colon cancer cell growth, migration and invasion were inhibited. Taken together, it is indicated that RINT1 is a new suppression target to reduce cell growth, migration and invasion of colon cancer by targeting ZW10/NAG-1.

Effects of non-steroidal anti-inflammatory drug-activated gene-1 on Ganoderma lucidum polysaccharides-induced apoptosis of human prostate cancer PC-3 cells.

Ganoderma lucidum polysaccharides (GLP) has been demonstrated to elicit antitumorigenic and proapoptotic activities in cancer; however, the molecular mechanisms underlying the anticancer effects of GLP have yet to be elucidated. Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) has been reported to exert proapoptotic effects and therefore, may serve an important role in cancer prevention. The present study aimed to elucidate the molecular mechanism by which GLP stimulates anticancer activity in human prostate cancer (PCa) PC-3 cells. In addition, the role of NAG-1 in GLP-induced cancer inhibition was examined. The results of the present study demonstrated that GLP significantly inhibited cell viability in a time- and dose-dependent manner in PC-3 cells. Flow cytometry indicated that GLP induced late apoptosis, which was accompanied by poly (ADP-ribose) polymerase 1 (PARP) cleavage, and inhibition of pro-caspase-3, -6 and -9 protein expression. Furthermore, it was observed that the expression levels of NAG-1, and its transcriptional factor early growth response-1, were upregulated in a time- and dose-dependent manner upon GLP treatment. The results of a luciferase assay demonstrated that GLP induced the promoter activity of NAG-1, thus indicating that NAG-1 may be transcriptionally regulated by GLP. The secretion of NAG-1 proteins into the cell culture medium was also upregulated upon GLP treatment. Furthermore, inhibition of NAG-1 expression by small interfering RNA significantly, but not completely, prevented GLP-induced apoptosis, and reversed the effects of GLP on PARP and pro-caspase expression. It was further demonstrated that GLP inhibited the phosphorylation of protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in PC-3 cells. The present study is the first, to the best of our knowledge, to report that GLP may induce apoptosis of PCa cells, which is partially mediated through NAG-1 induction. The present findings may be helpful in elucidating the anticancer mechanisms of GLP through NAG-1 induction for its chemopreventive potential in PCa.

Trichostatin A Induces NAG-1 Expression and Apoptosis in Human Endometriotic Stromal Cells.

To investigate the effects of trichostatin A (TSA) on nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression and apoptosis in human endometriotic stromal cells (HESCs), ectopic endometrial tissues were obtained from 15 patients with endometriotic cysts who underwent cystectomy. Human endometriotic stromal cells were isolated and cultured with different concentrations of TSA. Nonsteroidal anti-inflammatory drug-activated gene-1 messenger RNA (mRNA) and protein levels were evaluated by real-time polymerase chain reaction and Western blotting, respectively, and apoptosis was assessed by flow cytometry. Viability of HESCs was reduced in a dose-dependent manner by treatment with TSA. The percentage of early and late apoptotic HESCs was increased upon treatment with TSA. Nonsteroidal anti-inflammatory drug-activated gene-1 mRNA and protein expression was induced in a dose-dependent manner by TSA treatment. Gene knockdown experiments using small-interfering RNA confirmed an association between NAG-1 expression and TSA-induced apoptosis. Whether effects of TSA on NAG-1 gene expression are enhanced in the presence of 5-aza-2'-deoxycytidine (5-aza-dC) are also investigated; however, TSA-induced apoptosis was unaffected by 5-aza-dC. In conclusion, TSA induced apoptosis in HESCs via induction of NAG-1 expression. These results suggest that upregulation of NAG-1 contributes to TSA-induced apoptosis in HESCs.

Abstract 3510: Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) are usually used for the treatment of inflammatory diseases. However, certain NSAIDs also have antitumor activities in various cancers, including head and neck cancer, through cyclooxygenase-dependent or independent pathways. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a TGF-β superfamily protein, is induced by NSAIDs and has been shown to be induced by several antitumorigenic compounds and to exhibit proapoptotic and antitumorigenic activities. In this report, we demonstrate for the first time that tolfenamic acid (TA) transcriptionally induced the expression of NAG-1 during TA-induced apoptosis of anaplastic thyroid cancer (ATC) cells. TA reduced the viability of ATC cells in a dose-dependent manner and induced apoptosis, findings that were coincident with NAG-1 expression. Overexpression of the NAG-1 gene using cDNA enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. Subsequently, we found that intracellular ROS generation plays an important role in activating the proapoptotic protein NAG-1. Then, we confirmed antitumorigenic effects of TA in a nude mouse orthotopic ATC model, and this result accompanied the augmentation of NAG-1 expression and ROS generation in tumor tissue. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression and ROS generation in in vitro and in vivo ATC models, providing a novel mechanistic explanation and indicating a potential chemotherapeutic approach for treatment of ATC. Citation Format: Yeon Soo Kim, Jae Won Chang, SungUn Kang, JaeWon Choi, YooSeob Shin, Seung Joon Baek, Seong-Ho Lee, Kyoung Ho Oh, Chul-Ho Kim. Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3510.

Taiwanin A targets non-steroidal anti-inflammatory drug-activated gene-1 in human lung carcinoma

Taiwanin A (α,β-bis(piperonylidene)-γ-butyrolactone) is extracted from Taiwania cryptomerioides. Taiwanin A is extracted from tree bark and exhibits antitumor activity in breast, liver, and lung cancer cell lines. The objective of this study was to demonstrate the cytotoxicity of Taiwanin A against tumor cells by increasing the expression of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). NAG-1 has been reported to exhibit antitumor and proapoptotic activities, suggesting potential use in cancer therapy. Inhibiting NAG-1 mRNA expression in A549 reduced 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 influence of NAG-1 on cell viability, whereas the addition of the JNK pathway inhibitor SP600125 resulted in an inhibitory effect on NAG-1 and recovery of Taiwanin-A-treated cells. A xenograft tumor model demonstrated that Taiwanin A dose-dependently significantly decreases tumor-mediated growth in nude mice by increasing the NAG-1 expression accompanying tumor apoptosis. These data supported the hypothesis that Taiwanin A inhibits lung carcinoma growth by increasing NAG-1 expression through the JNK pathway both in vivo and in vitro. This result can contribute to a compound design for increasing cytotoxicity activity in the future.

Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells.

Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: Involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) are usually used for the treatment of inflammatory diseases. However, certain NSAIDs also have antitumor activities in various cancers, including head and neck cancer, through cyclooxygenase-dependent or independent pathways. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a TGF-β superfamily protein, is induced by NSAIDs and has been shown to be induced by several antitumorigenic compounds and to exhibit proapoptotic and antitumorigenic activities. In this report, we demonstrate for the first time that tolfenamic acid (TA) transcriptionally induced the expression of NAG-1 during TA-induced apoptosis of anaplastic thyroid cancer (ATC) cells. TA reduced the viability of ATC cells in a dose-dependent manner and induced apoptosis, findings that were coincident with NAG-1 expression. Overexpression of the NAG-1 gene using cDNA enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. Subsequently, we found that intracellular ROS generation plays an important role in activating the proapoptotic protein NAG-1. Then, we confirmed antitumorigenic effects of TA in a nude mouse orthotopic ATC model, and this result accompanied the augmentation of NAG-1 expression and ROS generation in tumor tissue. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression and ROS generation in in vitro and in vivo ATC models, providing a novel mechanistic explanation and indicating a potential chemotherapeutic approach for treatment of ATC.

Proteasome inhibitor MG132 induces NAG-1/GDF15 expression through the p38 MAPK pathway in glioblastoma cells

The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is regulated by the p53 and Egr-1 tumor suppressor pathways. Many anti-cancer drugs and chemicals induce NAG-1 expression, but the mechanisms are not fully understood. Transgenic mice expressing human NAG-1 are resistant to intestinal and prostate cancer, suggesting that NAG-1 is a tumor suppressor. Proteasome inhibitors exhibit anti-glioblastoma activities in preclinical studies. Here, we show that the proteasome inhibitors MG132 and bortezomib induced NAG-1 expression and secretion in glioblastoma cells. MG132 increased NAG-1 expression through transcriptional and post-transcriptional mechanisms. At the transcriptional level, the induction of NAG-1 required the −133 to +41bp region of the promoter. At post-transcriptional levels, MG132 stabilized NAG-1 mRNA by increasing the half-life from 1.5h to >8h. Because of the dramatic increase in mRNA stability, this is likely the major contributor to MG132-mediated NAG-1 induction. Further probing into the mechanism revealed that MG132 increased phosphorylation of the p38 MAPK pathway. Consequently, inhibiting p38 phosphorylation blocked activation of the NAG-1 promoter and decreased mRNA stability, indicating that p38 MAPK activation mediates both MG132-dependent promoter activation and mRNA stabilization of NAG-1. We propose that the induction of NAG-1 by p38 MAPK is a potential contributor to the anti-glioblastoma activity of proteasome inhibitors.

Expression of NSAID-activated gene-1 by EGCG in head and neck cancer: involvement of ATM-dependent p53 expression

Epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, possesses remarkable chemopreventive and therapeutic potential against various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanism involved is not completely understood. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a transforming growth factor β superfamily protein, is shown to be induced by several antitumorigenic compounds and to exhibit proapoptotic and antitumorigenic activities. In this report, we demonstrate that EGCG transcriptionally induced the expression of NAG-1 during EGCG-induced apoptosis of HNSCC cells. Reporter assays, using the luciferase constructs containing the NAG-1 promoter, demonstrate that p53 is required for EGCG-mediated activation of NAG-1. Overexpression of NAG-1 enhanced the apoptotic effect of EGCG, whereas suppression of NAG-1 expression by small interfering RNA attenuated EGCG-induced apoptosis in HNSCC cells. Subsequently, we found that ataxia-telangiectasia mutated (ATM) plays an important role in activating these proapoptotic proteins (NAG-1 and p53) and cell cycle inhibitor (p21). Furthermore, EGCG significantly inhibited tumor formation as assessed by xenograft models, and this result is accompanied with induction of apoptotic cells and NAG-1 expression in tumor tissue samples. Taken together, these results demonstrate for the first time that EGCG induces apoptosis via ATM/p53-dependent NAG-1 expression in HNSCC, providing an additional mechanistic explanation for the apoptotic activity of EGCG.

Abstract B53: Proteasome inhibitor MG132 induces NAG-1 expression through activation of p38 MAPK pathway

Abstract Glioblastoma multiforme (GBM) is the highly invasive primary tumors with a poor prognosis despite current surgical, radiation and chemotherapy approaches. Proteasome inhibitor, an approved drug for treatment of hematological cancer is currently being tested in clinical trials against various malignancies, including GBM. The pro-apoptotic and antitumorigenic protein NAG-1(nonsteroidal anti-inflammatory drug-activated gene-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily. We showed that the proteasome inhibitor, MG132 induces NAG-1 protein and mRNA expression and apoptosis in a concentration-dependent manner in U87 cells. Studies with NAG-1 luciferase reporter constructs show that the −133 to +41 region of the NAG-1 promoter is activated by MG132 however activation does not require the Sp-/Egr-1 binding site. Using the selective pharmacologic p38 mitogen -activated protein kinase (MAPK) inhibitor, we showed that NAG-1 induction following MG132 is mediated by the p38 MAPK pathway. Pretreatment with MAPK inhibitor, SB203580 and SB202190, blocked the MG132-induced NAG-1 expression, whereas an inhibition of p53, JUNK, MEK, PKC, PKA activity had no effect on NAG-1 levels. In summary, our data indicate linkage between p38MAPK, NAG-1 and the proteasome inhibitor, MG132 and provides a new clue to the molecular mechanism of the antitumorigenic activity of NAG-1.

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

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is induced by nonsteroidal anti-inflammatory drugs and possesses proapoptotic and antitumorigenic activities. Although tolfenamic acid (TA) induces apoptosis in head and neck cancer cells, the relationship between NAG-1 and TA has not been determined. This study investigated the induction of apoptosis in head and neck cancer cells treated by TA and the role of NAG-1 expression in this induction. TA reduced head and neck cancer cell viability in a dose-dependent manner and induced apoptosis. The induced apoptosis was coincident with the expression of NAG-1. Overexpression of NAG-1 enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. TA significantly inhibited tumor formation as assessed by xenograft models, and this result accompanied the induction of apoptotic cells and NAG-1 expression in tumor tissue samples. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression in head and neck squamous cell carcinoma, providing an additional mechanistic explanation for the apoptotic activity of TA.

Rottlerin induces apoptosis of HT29 colon carcinoma cells through NAG-1 upregulation via an ERK and p38 MAPK-dependent and PKC δ-independent mechanism

Rottlerin, a selective inhibitor of novel isoforms of protein kinase C δ (PKC δ), has been shown to exert multiple effects on cancer cells, including inhibition of cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We found that rottlerin dramatically induced non-steroidal anti-inflammatory drug activated gene-1 (NAG-1) expression in both p53 wild-type and p53-null cancer cell lines, suggesting that NAG-1 upregulation is a common response to rottlerin that occurs independently of p53 in multiple cell lines. Although rottlerin is known to inhibit PKC δ, PKC δ siRNA and overexpression of dominant-negative (DN)-PKC δ did not affect rottlerin-mediated induction of NAG-1. These results suggest that rottlerin induces NAG-1 upregulation via a PKC δ-independent pathway. We also observed that CHOP protein levels were significantly increased by rottlerin, but CHOP siRNA did not affect rottlerin-induced NAG-1 expression. In addition, we demonstrated the involvement of the mitogen-activated protein kinase (MAP kinase) signal transduction pathway in rottlerin-induced NAG-1 expression. Inhibitors of MEK (PD98059) and p38 MAP kinase (SB203580) prevented rottlerin-induced NAG-1 expression. Furthermore, we found that down-regulation of NAG-1 attenuated rottlerin-induced apoptosis. Collectively, the results of this study demonstrate, for the first time, that upregulation of NAG-1 contributes to rottlerin-induced apoptosis in cancer cells.

Activation of NAG-1 via JNK signaling revealed an isochaihulactone-triggered cell death in human LNCaP prostate cancer cells

BackgroundWe explored the mechanisms of cell death induced by isochaihulactone treatment in LNCaP cells.MethodsLNCaP cells were treated with isochaihulactone and growth inhibition was assessed. Cell cycle profiles after isochaihulactone treatment were determined by flow cytometry. Expression levels of cell cycle regulatory proteins, caspase 9, caspase 3, and PARP were determined after isochaihulactone treatment. Signaling pathway was verified by inhibitors pre-treatment. Expression levels of early growth response gene 1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) were determined to investigate their role in LNCaP cell death. NAG-1 expression was knocked down by si-NAG-1 siRNA transfection. Rate of cell death and proliferation were obtained by MTT assay.ResultsIsochaihulactone caused cell cycle arrest at G2/M phase in LNCaP cells, which was correlated with an increase of p53 and p21 levels and downregulation of the checkpoint proteins cdc25c, cyclin B1, and cdc2. Bcl-2 phosphorylation and caspase activation were also observed. Isochaihulactone induced phosphorylation of c-Jun-N-terminal kinase (JNK), and JNK inhibitor partially reduced isochaihulactone-induced cell death. Isochaihulactone also induced the expressions of EGR-1 and NAG-1. Expression of NAG-1 was reduced by JNK inhibitor, and knocking down of NAG-1 inhibited isochaihulactone-induced cell death.ConclusionsIsochaihulactone apparently induces G2/M cell cycle arrest via downregulation of cyclin B1 and cdc2, and induces cellular death by upregulation of NAG-1 via JNK activation in LNCaP cells.

Role of nonsteroidal anti-inflammatory drug-activated gene-1 in docetaxel-induced cell death of human colorectal cancer cells with different p53 status

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression is upregulated not only by NSAIDs such as sulindac sulfide, but also by several antitumorigenic dietary compounds, suggesting that NAG-1 is a specific target for the development of effective anticancer agents. Despite being a downstream target of p53, NAG-1 induction is both p53-dependent and p53-independent. It is not clear whether NAG-1 induction is the responsible factor in cancer cell apoptosis with mutated p53. In this study, we report that NAG-1 induction alone cannot determine apoptotic cell fate in colon cancer cells. Although docetaxel induced an increase in NAG-1 and apoptosis in both HCT-116 (wild-type p53) and HT-29 (mutant p53) colon cancer cells, NAG-1 knockdown with siRNA prevented docetaxel-induced cell death in only HCT-116 cells. Docetaxel decreased Bcl-2 in HCT-116 cells, which have functionally active p53, according to luciferase reporter gene analyses, and docetaxel-induced cell death and changes in Bcl-2 and NAG-1 expression were blocked by PFT-α, a p53 inhibitor. In HT-29 cells with functionally inactive p53, the docetaxel-induced Bcl-xL decrease, NAG-1 increase, and cell death were not blocked by PFT-α. On the other hand, sulindac sulfide at concentrations that significantly induced NAG-1 did not decrease cell viability comparable to docetaxel, and it did not affect the level of p53, Bax, Bcl-2, and Bcl-xL in either cell line. The present study demonstrates that p53-dependent NAG-1 induction is linked to cell death and that NAG-1 induction without accompanying alteration of antiapoptosis protein Bcl-2 family members may not lead to cancer cell death.

Expression and possible role of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in the human endometrium and endometriosis

Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is involved in cellular processes such as inflammation, apoptosis and tumorigenesis. However, little is known about the expression and function of NAG-1 in the endometrium. This study aimed to evaluate the expression of NAG-1 in the endometrium and in the absence or presence of endometriosis and to investigate the effect of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on NAG-1 mRNA levels and apoptosis in human endometrial stromal cells (HESCs). Eutopic endometrial samples were obtained during surgery from 40 patients with, and 40 patients without, endometriosis. Real-time PCR was used to quantify NAG-1 mRNA levels and immunohistochemistry was used to localize NAG-1 protein in the endometrium. To investigate the effects of celecoxib, HESCs were isolated and cultured with different concentrations of celecoxib or with 100 µM celecoxib at different times. Apoptosis was assessed by flow cytometry. NAG-1 mRNA levels and immunoreactivity showed cyclical changes through the menstrual cycle, increasing during the late secretory and menstrual phases. NAG-1 mRNA and protein levels were significantly lower in patients with endometriosis, compared with the control group. Celecoxib induced NAG-1 mRNA levels and apoptosis in cultured HESCs, with the effects dependent on drug concentrations and duration of treatment. Celecoxib treatment had no effect on prostaglandin E(2) levels in the culture supernatants. NAG-1 may be important in maintaining homeostasis in the normal endometrium and alterations in NAG-1 expression may be associated with the establishment of endometriosis. NAG-1 might be a therapeutic target for endometriosis.

MCC-555-induced NAG-1 expression is mediated in part by KLF4

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in cell differentiation, metabolism and tumorigenesis. We have investigated the therapeutic properties of 5-[[6-[(2-fluorophenyl)-methoxy]-2-napthalenyl]-methyl]-2,4-thiazolidinedione (MCC-555) a PPARgamma agonist in human colorectal cancer cells. To elucidate the molecular mechanism(s), by which MCC-555 exerts its effects on the human colorectal cancer cells, we have analyzed the expression of two pro-apoptotic proteins, Krüppel-like factor 4 (KLF4) and nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1). MCC-555-induced expression of the transcription factor, KLF-4 was blocked by a PPARgamma specific antagonist GW9662 in PPARgamma-dependent manner in HCT-116 cells. We further identified a new KLF4 target gene NAG-1, which shows a pro-apoptotic activity. We confirmed that PPARgamma agonists-induced NAG-1 expression was abolished using KLF4 siRNA in HCT-116 cells. Subsequently, KLF4 expression enhances the NAG-1 promoter activity in HCT-116 cells, and functional KLF4 binding sites in the NAG-1 promoter were also identified. MCC-555, a PPARgamma agonist induced the expression of Klf4 mRNA and protein in murine intestinal tumors from MCC-555-treated mice, as assessed by RT-PCR and immunohistochemistry. This study shows that PPARgamma agonists up-regulate KLF4 expression in receptor-dependent manner, and KLF4 was identified as a novel transcription factor that controls NAG-1 promoter activity in human and mouse colorectal cancers.

Involvement of NSAID‐activated gene‐1 in a novel synthetic hexahydrocannabinol analogue‐induced growth inhibition and apoptosis of colon cancer cells

Nonsteroidal anti‐inflammatory drug (NSAID)‐activated gene‐1 (NAG‐1) has received greater attention as a novel molecular target for various cancers including colorectal cancer. In this study, we identified a novel synthetic hexahydrocannabinol analogue LYR‐8 as a potent NAG‐1 inducer which inhibited growth and induced apoptosis in a panel of human cancer cells irrespective of p53‐status. In colon cancer cells, LYR‐8 dramatically induced NAG‐1expression in a concentration‐ and time‐dependent manner. The induction of NAG‐1 protein expression was independent of p53‐activation even in wild‐type HCT116 cells which had increased nuclear p53 after LYR‐8 treatment. The induction of NAG‐1 promoter activity by the LYR‐8 was strongly associated with increased Sp1 activation as noted in various luc‐promoters activities. Furthermore, pre‐treatment of specific Sp1 inhibitor Mithramycin A completely reversed the LYR‐8‐induced NAG‐1 expression. Genetically knockdown of NAG‐1 using siRNA significantly reversed cell death induced by LYR‐8 in both wild‐type as well as mutant p53‐expressing colon cancer cells, thus suggesting the role of p53‐independent NAG‐1 activation. These results suggest that p53‐indepenent induction of NAG‐1 via Sp1 activation is a promising therapeutic approach and novel compound like LYR‐8 could be a potent chemotherapeutic agent targeting colon cancers irrespective of p53‐mutation.