NSAID-activated Gene Research Articles

Abstract 2765: Novel WNT-RIP1-GDF15 pathway promotes migration and invasion of colorectal cancer cells

Abstract Growth-differentiation factor 15 (GDF15) is a member of the transforming growth factor β (TGF-β) family protein, also known as NSAID-activated gene 1 (NAG-1) or macrophage inhibitory cytokine 1 (MIC-1), and is a major cause of cancer cachexia, cardiovascular disease, and various inflammatory diseases. Roles of GDF15 as major inflammatory response factor also have been demonstrated in animal models, and it has also showed various malignant functions such as regulating metabolism, promoting metastasis by inducing EMT in cancer. Here, we revealed a novel potential role for RIP1 in colorectal cancer (CRC) in enhancing metastasis by controlling WNT-RIP1-GDF15 signaling. We showed that WNT3A treatment increased the expression of GDF15 and GFRAL. Immunohistochemical analysis of human CRC tissue arrays consisting of primary cancer and metastatic cancer also confirmed that the expression of GDF15 and GFRAL was increased in metastatic cancer. Human XL cytokine assay, western blot, and ELISA results confirmed that GDF15 expression was reduced in cell lines in which RIP1 was knocked down. Reduction of GDF15 expression and secretion by RIP1 knockdown showed suppression of EMT induction resulting to decrease of migration and invasion of CRC cells. Taken together, these results suggest a novel EMT-enhancing role for RIP1 in the WNT pathway and suggest a novel WNT-RIP1-GDF15 pathway contributing to CRC malignancy by promoting EMT. Citation Format: A-Ram Kang, Do Hoon Kim, Jong Kuk Park. Novel WNT-RIP1-GDF15 pathway promotes migration and invasion of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2765.

Growth differentiation factor 15 (GDF15): A survival protein with therapeutic potential in metabolic diseases.

Growth Differentiation Factor 15 (GDF15), also known as NSAID activated gene-1 (NAG-1), is associated with a large number of biological processes and diseases, including cancer and obesity. GDF15 is synthesized as pro-GDF15, is dimerized, and is cleaved and secreted into the circulation as a mature dimer GDF15. Both the intracellular GDF15 and the circulating mature GDF15 are implicated in biological processes, such as energy homeostasis and body weight regulation. Although there have been many studies on GDF15, GFRAL, a member of the glial-derived neurotropic factor receptor α family, has only been recently identified as a receptor for mature GDF15. In this review, we focused on cancer and energy homeostasis along with obesity and body weight, and the effect of the identification of the GDF15 receptor in these investigations. In addition, the therapeutic potential of GDF15 as a pharmacological agent in obesity and other metabolic diseases was discussed.

Anticarcinogenic effects of water extract of sporoderm-broken spores of Ganoderma lucidum on colorectal cancer in vitro and in vivo.

Ganoderma lucidum (G. lucidum) polysaccharides (GLPs) have been used as traditional Chinese medicine for cancer prevention for many years. However, the mechanism by which GLP exerts its chemopreventive activities remains elusive. In addition, it is unclear whether sporoderm-broken spores of G. lucidum water extract (BSGLWE), which contains mainly GLPs, has anticancer effects on colorectal cancer. The present study investigated the anticancer effects and potential mechanisms of BSGLWE on colorectal cancer in vivo and in vitro. Our results showed that BSGLWE significantly inhibited colorectal cancer HCT116 cell viability in a time- and dose-dependent manner. Flow cytometry analysis indicated that BSGLWE disrupted cell cycle progression at G2/M phase via downregulation of cyclin B1 and cyclin A2, and upregulation of P21 at mRNA levels. Moreover, BSGLWE induced apoptosis by decreasing Bcl-2 and survivin at mRNA levels, and reduced Bcl-2, PARP, pro-caspase-3 and pro-caspase-9 at protein levels. Furthermore, BSGLWE suppressed tumor growth in vivo by regulating the expression of genes and proteins associated with cell cycle and apoptosis, which was further confirmed by a reduction of Ki67, PCNA, and Bcl-2 expression as determined by immunohistochemistry staining. NSAID activated gene-1 (NAG-1), a pro-apoptotic gene, was significantly upregulated in vivo and in vitro upon BSGLWE treatment at both mRNA and protein levels. In addition, the relative amounts of secreted NAG-1 in cell culture medium or serum of nude mice were all upregulated upon BSGLWE treatments, suggesting a role of NAG-1 in BSGLWE-induced anticolorectal cancer activity. This is the first study to show that BSGLWE inhibits colorectal cancer carcinogenesis through regulating genes responsible for cell proliferation, cell cycle and apoptosis cascades. These findings indicate that BSGLWE possesses chemopreventive potential in colorectal cancer which may serve as a promising anticancer agent for clinical applications.

C/EBP homologous protein promotes NSAID-activated gene 1-linked pro-inflammatory signals and enterocyte invasion by enteropathogenic Escherichia coli

NSAID-activated Gene 1 (NAG-1) is a prognostic indicator of chronic inflammatory diseases and aggressive tumors. Among the stress sentinels in response to infection by enteropathogenic Escherichia coli (EPEC) or other pathogenic E. coli, C/EBP homologous protein (CHOP), a representative stress-regulated transcription factor, was prominently increased and assessed for its involvement in NAG-1-mediated pathogenic cellular responses. NAG-1 expression was transcriptionally upregulated by CHOP, which promoted chemokine production through sustained NF-κB activation. Mechanistically, NF-κB activation by NAG-1 was due to TGFβ-activated kinase 1 (TAK-1)-mediated pathway rather than SMAD-associated signals. Moreover, CHOP and subsequent TAK-1-linked signals were also involved in bacterial invasion into human cells. Therefore, CHOP as an infection-induced sentinel played crucial roles in induction of NAG-1 and subsequent prolonged activation of pro-inflammatory responses to EPEC infection or related chronic pathogenic states.

NSAID-activated gene 1 mediates pro-inflammatory signaling activation and paclitaxel chemoresistance in type I human epithelial ovarian cancer stem-like cells.

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in developed countries. Chronic endogenous sterile pro-inflammatory responses are strongly linked to EOC progression and chemoresistance to anti-cancer therapeutics. In the present study, the activity of epithelial NF-κB, a key pro-inflammatory transcription factor, was enhanced with the progress of EOC. This result was mechanistically linked with an increased expression of NSAID-Activated Gene 1 (NAG-1) in MyD88-positive type I EOC stem-like cells, compared with that in MyD88-negative type II EOC cells. Elevated NAG-1 as a potent biomarker of poor prognosis in the ovarian cancer was positively associated with the levels of NF-κB activation, chemokines and stemness markers in type I EOC cells. In terms of signal transduction, NAG-1-activated SMAD-linked and non-canonical TGFβ-activated kinase 1 (TAK-1)-activated pathways contributed to NF-κB activation and the subsequent induction of some chemokines and cancer stemness markers. In addition to effects on NF-κB-dependent gene regulation, NAG-1 was involved in expression of EGF receptor and subsequent activation of EGF receptor-linked signaling. The present study also provided evidences for links between NAG-1-linked signaling and chemoresistance in ovarian cancer cells. NAG-1 and pro-inflammatory NF-κB were positively associated with resistance to paclitaxel in MyD88-positive type I EOC cells. Mechanistically, this chemoresistance occurred due to enhanced activation of the SMAD-4- and non-SMAD-TAK-1-linked pathways. All of the present data suggested NAG-1 protein as a crucial mediator of EOC progression and resistance to the standard first-line chemotherapy against EOC, particularly in MyD88-positive ovarian cancer stem-like cells.

Combined Therapeutic Effects of Vinblastine and Astragalus Saponins in Human Colon Cancer Cells and Tumor Xenograft via Inhibition of Tumor Growth and Proangiogenic Factors

Our previous study had demonstrated that Astragalus saponins (AST) could reduce the side effects of orthodox chemotherapeutic drugs, while concurrently promote antitumor activity. In the present study, we attempted to investigate the potential synergistic anticarcinogenic effects of AST and a vinca alkaloid vinblastine (VBL). Reduced expression of key proangiogenic and metastatic factors including VEGF, bFGF, metalloproteinase (MMP)-2, and MMP-9 was detected in VBL-treated colon cancer cells, with further downregulation by combined VBL/AST treatment. Subsequently, VBL or AST decreased LoVo cell invasiveness, with further reduction when the drugs were cotreated. Significant growth inhibition and cell cycle arrest at G2/M phase were achieved by either drug treatment with apparent synergistic effects. VBL-induced apoptosis was confirmed but found to be unrelated to induction of the novel apoptotic protein NSAID-activated gene 1. In vivo study in tumor xenograft indicates that combined VBL/AST treatment resulted in sustained regression of tumor growth, with attenuation of the neutropenic and anemic effects of VBL. In addition, downregulation of proangiogenic and proliferative factors was also visualized, with boosting effect by combined drug treatment. These findings have provided evidence that AST combined with adjuvant chemotherapeutics like VBL could alleviate cancer development through diversified modes of action, including the regulation of angiogenesis.

NAG-1/GDF-15 prevents obesity by increasing thermogenesis, lipolysis and oxidative metabolism

SummaryObjectiveObesity is a major health problem associated with high morbidity and mortality. NSAID activated gene, (NAG-1) is a TGF-β superfamily member reported to alter adipose tissue levels in mice. We investigated whether hNAG-1 acts as a regulator of adiposity and energy metabolism.Design/SubjectshNAG-1 mice, ubiquitously expressing hNAG-1, were placed on a control or high fat diet (HFD) for 12 weeks. hNAG-1 expressing B16/F10 melanoma cells were used in a xenograft model to deliver hNAG-1 to obese C57BL/6 mice.ResultsAs compared to wild-type littermates, transgenic hNAG-1 mice have less white fat and brown fat despite equivalent food intake, improved glucose tolerance, lower insulin levels and are resistant to dietary- and genetic-induced obesity. hNAG-1 mice are more metabolically active with higher energy expenditure. Obese C57BL/6 mice treated with hNAG-1 expressing xenografts show decreases in adipose tissue and serum insulin levels. hNAG-1 mice and obese mice treated with hNAG-1 expressing xenografts show increased thermogenic gene expression (UCP1, PGC1α, ECH1, Cox8b, Dio2, Cyc1, PGC1β, PPARα, Elvol3) in brown adipose tissue (BAT) and increased expression of lipolytic genes (Adrb3, ATGL, HSL) in both white adipose tissue (WAT) and BAT, consistent with higher energy metabolismConclusionhNAG-1 modulates metabolic activity by increasing the expression of key thermogenic and lipolytic genes in BAT and WAT. hNAG-1 appears to be a novel therapeutic target in preventing and treating obesity and insulin resistance.

Lower NLRP3 inflammasome activity in NAG‐1 transgenic mice is linked to a resistance to obesity and increased insulin sensitivity

OBJECTIVEThe NLRP3 inflammasome plays an important regulatory role in obesity-induced insulin resistance. NSAID activated gene-1 (NAG-1) is a divergent member of the TGF-β superfamily. NAG-1 Tg mice are resistant to dietary- and genetic-induced obesity and have improved insulin sensitivity. Our objective was to examine whether NLRP3 inflammasome activity is associated with this observed phenotype in NAG-1 Tg mice.DESIGN AND METHODSKey components of the NLRP3 inflammasome were examined in NAG-1 Tg mice on both regular and high fat diet (HFD) conditions.RESULTSThe expression of caspase-1 and ASC, key components of the NLRP3 inflammasome, is significantly reduced at mRNA and protein levels in white adipose tissue (WAT) of NAG-1 Tg mice. HFD increases the expression of caspase-1 and ASC in WT mice, but their expression is reduced in NAG-1 Tg mice. Furthermore, there is reduced IL-18, IL-1β, and TNF-α expression in the WAT of NAG-1 Tg mice. NAG-1 Tg mice have significantly lower serum leptin and insulin levels and reduced expression of macrophage infiltration markers (F4/80, CD11b, and CD11c) in WAT.CONCLUSIONSOur study suggests the lower NLRP3 inflammasome activity may play a role in the resistance of NAG-1 Tg mice to diet-induced obesity and improved insulin sensitivity.

Identification of TNM stage-specific genes in lung adenocarcinoma by genome-wide expression profiling

The present study aimed to investigate the molecular basis of lung cancer development using a microarray to identify the differentially-expressed genes associated with the various tumor-node-metastasis (TNM) stages of lung adenocarcinoma. This subtype of lung cancer has increased in incidence within recent years in China. A 35K oligo gene array covering ~25,100 genes was used to screen the differentially-expressed genes among 90 lung adenocarcinoma samples of various TNM stages. To verify the data from the gene arrays, three genes [human zinc finger-containing, Miz1, PIAS-like protein on chromosome 7 (Zimp7), GINS complex subunit 2 (GINS2) and NSAID activated gene 1 (NAG-1)] were validated using quantitative (q)PCR in an alternative set of samples to the gene array. A total of 640 genes were identified that were differentially-expressed in lung adenocarcinoma compared with the surrounding normal lung tissues. From these 640 candidate genes, 10 were observed to be differentially-expressed among TNM stages I, II and IIIA, of which, the Zimp7, GINS2 and NAG-1 genes were reported for the first time to be expressed at high levels in lung adenocarcinoma. The results of the qPCR for the three genes were consistent with those from the gene array. In total, 10 candidate genes were identified to be associated with the various TNM stages of lung adenocarcinoma in the population studied, which may provide new insights into the molecular basis underlying the development of lung adenocarcinoma and offer new targets for the diagnosis, therapy and prognosis.

NSAIDs may regulate EGR-1-mediated induction of reactive oxygen species and non-steroidal anti-inflammatory drug-induced gene (NAG)-1 to initiate intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer

This study aims to investigate the unclear molecular relationship involved in the activation of intrinsic pathway of apoptosis and NSAID-activated gene-1 (NAG-1) induction as a putative target in NSAIDs-mediated chemoprevention of colorectal cancer. Male Sprague-Dawley rats were administered with a colon-specific pro-carcinogen, 1,2-dimethylhydrazine dihydrochloride to achieve the early stages of colorectal cancer. Histopathological examination was performed for the analysis of neoplastic lesions while flow cytometry was performed for the relative quantification of intracellular reactive oxygen species (ROS), differential mitochondrial membrane potential (MMP or ΔΨ(M)), and apoptotic events. Various target biomolecules were analyzed either for their mRNA or protein expression profiles via RT-PCR and quantitative Real-Time PCR, or Western blotting and immunofluorescence, respectively. Enhanced gene as well as protein expression of pro-apoptotic agents was observed with the daily oral administration of two NSAIDs viz. Sulindac (cyclooxygenase (COX)-non-specific) and Celecoxib (a selective COX-2 inhibitor). A significant increase in early growth response-1 (EGR-1) protein expression and nuclear localization in NSAIDs co-administered animals may have positively regulated the expression of NAG-1 with a significant enhancement of intracellular ROS in turn decreasing the ΔΨ(M) to initiate apoptosis. In silico molecular docking analysis also showed that Sulindac and Celecoxib can block the active site pocket of B-cell lymphoma-extra large (Bcl-xL, anti-apoptotic transmembrane mitochondrial protein) which could be a putative mechanism followed by these NSAIDs to overcome anti-apoptotic properties of the molecule. NSAIDs-mediated up-regulation of EGR-1 and thereby NAG-1 along with implication of higher ROS load may positively regulate the intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer.

Early growth response protein 1 upregulation and nuclear translocation by 2′-benzoyloxycinnamaldehyde induces prostate cancer cell death

2′-Benzoyloxycinnamaldehyde (BCA) induces apoptosis in human cancer cells through ROS generation. BCA upregulates proapoptotic genes such as activating transcription factor 3 (ATF3), NSAID-activated gene 1 protein (NAG-1), and growth arrest and DNA-damage-inducible protein alpha (GADD45A) in prostate cancer cells. These genes are known to be induced by transcription factor early growth response protein 1 (EGR1). BCA induces significant EGR1 upregulation, while EGR1 knockdown decreases the induction of these genes with concurrent alleviation of cell death by BCA. Antioxidant glutathione pretreatment with BCA removes EGR1 expression increase, suggesting that EGR1 upregulation is dependent on oxidative stress generated by BCA. In prostate cancer cells, EGR1 localizes in the cytoplasm; however, BCA remarkably upregulates EGR1 nuclear translocalization, suggesting its possible effect as a transcriptional activator. BCA induces transient upregulation of importin-7 (IPO7) which is critical for EGR1 nuclear translocation, and IPO7 knockdown led to a significant decrease in chemosensitivity to BCA. Taken together, our findings suggest that BCA induces prostate cancer cell death via EGR1 upregulation and nuclear translocalization, followed by activation of proapoptotic target genes.

Target-oriented Mechanisms of Novel Herbal Therapeutics in the Chemotherapy of Gastrointestinal Cancer and Inflammation

A prominent group of effective cancer chemopreventive drugs has been derived from natural products having low toxicity while possessing apparent benefit in the disease process. It is plausible that there are multiple target molecules critical to cancer cell survival. Herbal terpenoids have demonstrated excellent target-specific anti-neoplastic functions by suppression of cell proliferation and induction of apoptosis. Transcriptional molecules in the NF-κB, MEK/ERK and PI3K/Akt/mTOR pathways are important molecular targets of chemotherapy that play distinctive roles in modulating the apoptosis cascades. It is recently suggested that NSAID-activated gene (NAG-1), a novel proapoptotic protein, is the upstream anti-carcinogenic target of NSAIDs, PPAR ligands and herbal chemotherapeutic agents that triggers some of the events mentioned above. Besides, angiogenesis, oxidative stress as well as inflammation are important factors that contribute to the development and metastasis of cancer, which could be actively modulated by novel agents of plant origin. The aim of the present review is to discuss and summarize the contemporary use of herbal therapeutics and phytochemicals in the treatment of human cancers, in particular that of the colon. The major events and signaling pathways in the carcinogenesis process being potentially modulated by natural products and novel herbal compounds will be evaluated, with emphasis on some terpenoids. Advances in eliciting the precise cellular and molecular mechanisms during the anti-tumorigenic process of novel herbal therapeutics will be of imperative clinical significance to increase the efficacy and reduce prominent adverse drug effects in cancer patients through target-specific therapy.

Target-oriented Mechanisms of Novel Herbal Therapeutics in the Chemotherapy of Gastrointestinal Cancer and Inflammation

A prominent group of effective cancer chemopreventive drugs has been derived from natural products having low toxicity while possessing apparent benefit in the disease process. It is plausible that there are multiple target molecules critical to cancer cell survival. Herbal terpenoids have demonstrated excellent target-specific anti-neoplastic functions by suppression of cell proliferation and induction of apoptosis. Transcriptional molecules in the NF-κB, MEK/ERK and PI3K/Akt/mTOR pathways are important molecular targets of chemotherapy that play distinctive roles in modulating the apoptosis cascades. It is recently suggested that NSAID-activated gene (NAG-1), a novel proapoptotic protein, is the upstream anti-carcinogenic target of NSAIDs, PPAR ligands and herbal chemotherapeutic agents that triggers some of the events mentioned above. Besides, angiogenesis, oxidative stress as well as inflammation are important factors that contribute to the development and metastasis of cancer, which could be actively modulated by novel agents of plant origin. The aim of the present review is to discuss and summarize the contemporary use of herbal therapeutics and phytochemicals in the treatment of human cancers, in particular that of the colon. The major events and signaling pathways in the carcinogenesis process being potentially modulated by natural products and novel herbal compounds will be evaluated, with emphasis on some terpenoids. Advances in eliciting the precise cellular and molecular mechanisms during the anti-tumorigenic process of novel herbal therapeutics will be of imperative clinical significance to increase the efficacy and reduce prominent adverse drug effects in cancer patients through target-specific therapy. Keywords: Herbal therapeutics, phytochemicals, chemotherapy, anti-inflammation, colon cancer, molecular targets, natural products, apoptosis, angiogenesis, oxidative stress

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.

Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 Plays a Role in the Impairing Effects of Cyclooxygenase Inhibitors on Gastric Ulcer Healing

Nonsteroidal anti-inflammatory drugs (NSAIDs) can impair gastric ulcer healing. This study investigates the involvement of NSAID-activated gene-1 (NAG-1) in ulcer repair impairment by cyclooxygenase (COX) inhibitors. Gastric ulcers were induced in rats by acetic acid. Four days later, animals received daily intragastric indomethacin (nonselective COX-1/COX-2 inhibitor; 1 mg/kg), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (selective COX-1 inhibitor; 2.5 mg/kg), (5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl) phenyl-2(5H)-furanone (DFU) (selective COX-2 inhibitor; 5 mg/kg), celecoxib (selective COX-2 inhibitor; 1 mg/kg), and valdecoxib (selective COX-2 inhibitor; 1 mg/kg), for 1, 3, or 7 days. Ulcerated tissues were processed to assess: 1) COX-1, COX-2, NAG-1, proliferating cell nuclear antigen (PCNA), and activated caspase-3 expression; 2) ulcer area; and 3) prostaglandin E(2) (PGE(2)) levels. COX-1 expression in ulcerated tissues was decreased, whereas COX-2 expression was enhanced. Ulcer healing was delayed by indomethacin, DFU, and SC-560, but not by celecoxib and valdecoxib. Ulcer PGE(2) levels were decreased by SC-560, DFU, celecoxib, valdecoxib, and indomethacin. NAG-1 was overexpressed in ulcerated tissues and further enhanced by indomethacin, DFU, and SC-560, but not by celecoxib or valdecoxib. PCNA expression in ulcerated areas was reduced by indomethacin, but not by the other test drugs. The expression of activated caspase-3 in ulcers was increased and enhanced further by indomethacin, DFU, and SC-560, but not by celecoxib and valdecoxib. These findings indicate that: 1) COX inhibitors exert differential impairing effects on gastric ulcer healing, through mechanisms unrelated to the inhibition of COX isoforms and prostaglandin production; and 2) NAG-1 induction, followed by activation of proapoptotic pathways, can contribute to the impairing effects of COX inhibitors on ulcer healing.

Synergistic anti‐tumor activity of isochaihulactone and paclitaxel on human lung cancer cells

Drug resistance frequently develops in tumors during chemotherapy. Therefore, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Here, we show that isochaihulactone (K8) enhanced paclitaxel-induced apoptotic death in human lung cancer cells, and the enhancing effect was related to increased NSAID-activated gene-1 (NAG-1) expression. CalcuSyn software was used to evaluate the synergistic interaction of K8 and paclitaxel on human lung cancer cells; the synergistic effect of K8 in combination with paclitaxel was increased more than either of these drugs alone. Furthermore, the activity of ERK1/2 was enhanced by the combination of K8 and paclitaxel, and an ERK1/2 inhibitor dramatically inhibited NAG-1 expression in human lung cancer cells. Therefore, this synergistic apoptotic effect in human lung cancer cells may be directly associated with K8-induced NAG-1 expression through ERK1/2 activation. Moreover, over-expression of NAG-1 enhanced K8/paclitaxel-induced apoptosis in human lung cancer cells. In addition, treatment of nude mice with K8 combined with paclitaxel induced phospho-ERK1/2 and NAG-1 expression in vivo. Targeting of NAG-1 signaling could enhance therapeutic efficacy in lung cancer. Our results reveal that activation of NAG-1 by K8 enhanced the therapeutic efficacy of paclitaxel in human lung cancer cells via the ERK1/2 signaling pathway.

COX inhibitors directly alter gene expression: role in cancer prevention?

Inflammation is an important contributor to the development and progression of human cancers. Inflammatory lipid metabolites, prostaglandins, formed from arachidonic acid by prostaglandin H synthases commonly called cyclooxygenases (COXs) bind to specific receptors that activate signaling pathways driving the development and progression of tumors. Inhibitors of prostaglandin formation, COX inhibitors, or nonsteroidal anti-inflammatory drugs (NSAIDs) are well documented as agents that inhibit tumor growth and with long-term use prevent tumor development. NSAIDs also alter gene expression independent of COX inhibition and these changes in gene expression also appear to contribute to the anti-tumorigenic activity of these drugs. Many NSAIDs, as illustrated by sulindac sulfide, alter gene expressions by altering the expression or phosphorylation status of the transcription factors specificity protein 1 and early growth response-1 with the balance between these two events resulting in increases or decreases in specific target genes. In this review, we have summarized and discussed the various genes altered by this mechanism after NSAID treatment and how these changes in expression relate to the anti-tumorigenic activity. A major focus of the review is on NSAID-activated gene (NAG-1) or growth differentiation factor 15. This unique member of the TGF-β superfamily is highly induced by NSAIDs and numerous drugs and chemicals with anti-tumorigenic activities. Investigations with a transgenic mouse expressing the human NAG-1 suggest it acts to suppress tumor development in several mouse models of cancer. The biochemistry and biology of NAG-1 were discussed as potential contributor to cancer prevention by COX inhibitors.

Abstract 982: Transgenic expression of NSAID-activated gene-1 (NAG-1) protects mice from high fat diet-induced obesity

Abstract Previously, we generated transgenic mice which over-express human NAG-1/GDF15 gene. These mice are resistant to the development of colorectal cancer and have reduced responses to inflammatory stimuli. In the present study, we observed that NAG-1 transgenic mice (NAG-1 Tg) are smaller and leaner than their wild-type counterparts and have less abdominal fat despite comparable food intake. Interestingly, the transgenic mice have lower circulating levels of the inflammatory adipokine, leptin, and lower expression in abdominal fat, the major source of leptin. Placed on a high fat diet (60% fat), body weight gain in NAG-1 Tg mice is very small or negligible compared to wild-type mice. Analysis of the metabolic activity showed that NAG-1 Tg mice on the high fat diet have higher energy expenditure than WT mice. In addition, global gene expression profiling analysis showed that expression of several genes involved in lipid metabolism were significantly down-regulated in the liver of NAG-1 Tg mice. Real-time PCR analysis confirmed decreased expression of genes such as Cidea, Cidec, Mogat1, CD36, and Apoa4, while the expression of SIRT-1 and phosphorylated SIRT-1 was not altered in the NAG-1 Tg mice. Taken together, our data indicate increased energy expenditure and decreased expression of lipid metabolism genes have pivotal roles for the lower observed abdominal fat levels. Furthermore, lower leptin levels, a critical mediator of intestinal inflammation, found in NAG-1 transgenic mice, may contribute to lower intestinal tumor growth and reduced inflammatory responses reported for the NAG-1 Tg mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 982. doi:10.1158/1538-7445.AM2011-982

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.

NSAID Inhibition of Prostate Cancer Cell Migration Is Mediated by Nag-1 Induction via the p38 MAPK-p75NTR Pathway

The nonsteroidal anti-inflammatory drugs (NSAID) R-flurbiprofen and ibuprofen have been shown to induce expression of p75(NTR) (neurotrophin receptor) in prostate cancer cell lines. p75(NTR), a tumor necrosis factor receptor superfamily member, is a proapoptotic protein that functions as a tumor suppressor in the human prostate. Expression of p75(NTR) is lost as prostate cancer progresses and is minimal in several metastatic prostate cancer cell lines. NSAIDs induce p75(NTR) through activation of the p38 mitogen-activated protein kinase (MAPK) pathway, with a concomitant decrease in cell survival. Here, we show that treatment with R-flurbiprofen and ibuprofen induces expression of the NSAID-activated gene-1 (Nag-1) protein, a divergent member of the TGF beta (TGF-β) family, in PC-3 cells. Using the selective pharmacologic inhibitor of p38 MAPK, SB202190, and p38 MAPK-specific siRNA (small interfering RNA), we show that Nag-1 induction following NSAID treatment is mediated by the p38 MAPK pathway. p75(NTR)-specific siRNA pretreatment shows that Nag-1 induction by NSAIDs is downstream of p75(NTR) induction. Decreased survival of NSAID-treated cells is rescued by p75(NTR)-specific siRNA but not by Nag-1 siRNA. Transwell chamber and in vitro wound healing assays demonstrate decreased cell migration upon NSAID treatment. Pretreatment of PC-3 cells with p75(NTR) and Nag-1-specific siRNA shows that NSAID inhibition of cell migration is mediated by Nag-1 and p75(NTR). These results demonstrate a role for Nag-1 in NSAID inhibition of cell migration, but not survival.