Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung.

Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year round, but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore, in the current study, we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next, we determined the effect of GDF15 on viral replication, antiviral responses, and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally, we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein overexpression in mice led to exaggerated inflammatory responses to HRV, increased infectious particle release, and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover, GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly, Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation, which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e., HRV) infection in cigarette smoke-exposed airways with GDF15 overproduction.

High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin.

In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 +/- 50 pg/ml. In comparison, individuals with beta-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 +/- 9,600 pg/ml; range 4,800-248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.

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 +41 bp region of the promoter. At post-transcriptional levels, MG132 stabilized NAG-1 mRNA by increasing the half-life from 1.5 h to >8 h. 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.

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-ß 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 H6D variant of NAG-1/GDF15 inhibits prostate xenograft growth in vivo.

BACKGROUND: Non-steroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor-beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo. METHODS: Human prostate cancer DU145 cells expressing the H6D NAG-1 or wild-type (WT) NAG-1 were injected subcutaneously into nude mice and tumor growth was monitored. Serum and tumor samples were collected for subsequent analysis. RESULTS: The H6D variant was more potent than the WT NAG-1 and inhibited tumor growth significantly compared to control mice. Mice with tumors expressing the WT NAG-1 have greater reduced both body weight and abdominal fat than mice with H6D variant tumors suggesting different activities of the WT NAG-1 and the H6D NAG-1. A significant reduction in adiponectin, leptin, and IGF-1 serum levels was observed in the tumor-bearing mice with a more profound reduction observed with expression of H6D variant. Cyclin D1 expression was suppressed in the tumors with a dramatic reduction observed in the tumor expressing the H6D variant. CONCLUSION: Our data suggest that the H6D variant of NAG-1 inhibits prostate tumorigenesis by suppressing IGF-1 and cyclin D1 expression but likely additional mechanisms are operative.

Regulation of EP4 expression via the Sp-1 transcription factor: inhibition of expression by anti-cancer agents.

For glioblastomas, COX-2 expression is linked to poor survival. COX-2 effects are mediated by the receptors EP2 and EP4, whose regulation is poorly understood. The expression of EP4, and activation or inhibition of EP4 activity in human glioblastoma T98G cells, was found to correlate with growth on soft agar. Chemoprevention drugs, troglitazone (TGZ) and some COX inhibitors, significantly suppressed EP4 expression in T98G cells in a dose dependant manner. Specificity protein 1 (Sp-1) binding sites, located within region -197 to -160 of the human EP4 promoter, are important for the transcription initiation of the human EP4 gene and are responsible for the EP4 suppression by TGZ. Mutation in the Sp-1 sites altered the promoter activity of luciferase constructs and TGZ effects on the promoter. The inhibitory effect of TGZ on EP4 expression was reversed by PD98059, a MEK-1/Erk inhibitor. Immunoprecipitation-Western blot analysis detected Sp-1 phosphorylation that was dependent on TGZ-induced Erks activation. ChIP assay confirmed that Sp-1 phosphorylation decreases its binding to DNA and as a result, leads to the suppression of EP4 expression. Thus, we propose that the expression of EP4 is regulated by Sp-1, but phosphorylation of Sp-1 induced by TGZ suppresses this expression. This represents a new and unique mechanism for the regulation of the EP4 receptor expression.

A green tea component suppresses posttranslational expression of basic fibroblast growth factor in colorectal cancer.

BACKGROUND & AIMS: Green tea catechins are known to have anticarcinogenic effects. Epigallocatechin-3-gallate (EGCG) accounts for almost 50% of the total catechin content in green tea extract and has very potent antioxidant effects. EGCG also inhibits angiogenesis, possibly through the inhibition of proangiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which in turn, inhibits tumor growth and metastasis. However, the exact molecular mechanism by which EGCG suppresses bFGF expression is not known. Our objective was to elucidate the molecular mechanisms by which EGCG inhibits bFGF expression in colorectal cancer. METHODS: We examined posttranslational regulation of bFGF by EGCG in human colorectal cancer cells. We also examined bFGF in intestinal tumor formation of APC(Min/+) mice with and without catechin treatment. RESULTS: The bFGF protein was quickly degraded in the presence of EGCG, but a proteasome inhibitor suppressed this degradation. EGCG was also found to increase ubiquitination of bFGF and trypsin-like activity of the 20S proteasome, thereby resulting in the degradation of bFGF protein. Furthermore, EGCG suppressed tumor formation in APC(Min/+) mice, compared with vehicle-treated mice, in association with reduced bFGF expression. CONCLUSIONS: The ubiquitin-proteasome degradation pathway contributes significantly to down-regulation of bFGF expression by EGCG. Catechin compounds have fewer adverse effects than chemotherapeutic agents and hence can be used as proof-of-concept in cancer therapeutics to suppress growth and metastasis by targeting proteins such as bFGF.

Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism.

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor-beta superfamily, is involved in many cellular processes, such as inflammation, apoptosis/survival, and tumorigenesis. Vitamin E succinate (VES) is the succinate derivative of alpha-tocopherol and has antitumorigenic activity in a variety of cell culture and animal models. In the current study, the regulation and role of NAG-1 expression in PC-3 human prostate carcinoma cells by VES was examined. VES treatment induced growth arrest and apoptosis as well as an increase in NAG-1 protein and mRNA levels in a time- and concentration-dependent manner. VES treatment induced nuclear translocation and activation of p38 kinase. Pretreatment with p38 kinase inhibitor blocked the VES-induced increase in NAG-1 protein and mRNA levels, whereas an inhibition of protein kinase C, Akt, c-Jun NH(2)-terminal kinase, or MEK activity had no effect on VES-induced NAG-1 levels. Forced expression of constitutively active MKK6, an upstream kinase for p38, induced an increase in NAG-1 promoter activity, whereas p38 kinase inhibitor blocked MKK6-induced increase in NAG-1 promoter activity. VES treatment resulted in >3-fold increase in the half-life of NAG-1 mRNA in a p38 kinase-dependent manner and transient transfection experiment showed that VES stabilizes NAG-1 mRNA through AU-rich elements in 3'-untranslated region of NAG-1 mRNA. The inhibition of NAG-1 expression by small interfering RNA significantly blocked VES-induced poly(ADP-ribose) polymerase cleavage, suggesting that NAG-1 may play an important role in VES-induced apoptosis. These results indicate that VES-induced expression of NAG-1 mRNA/protein is regulated by transcriptional/post-transcriptional mechanism in a p38 kinase-dependent manner and NAG-1 can be chemopreventive/therapeutic target in prostate cancer.

Activation of TRPA1 by volatile organic chemicals leading to sensory irritation.

Many volatile organic chemicals (VOCs) have not been tested for sensory pulmonary irritation. Development of in vitro non-animal sensory irritation assay suitable for a large number of chemicals is needed to replace the mouse assay. An adverse outcome pathway (AOP) is designed to provide a clear description of the biochemical and cellular processes leading to toxicological effects or an adverse outcome. The AOP for chemical sensory pulmonary irritation was developed according to the Organization for Economic Co-operation and Development guidance including the Bradford Hill criteria for a weight of evidence to determine the confidence of the AOP. The proposed AOP is based on an in-depth review of the relevant scientific literature to identify the initial molecular event for respiratory irritation. The activation of TRPA1 receptor (transient receptor potential cation channel, subfamily A, member 1) is the molecular initial event (MIE) leading to sensory irritation. A direct measure of TRPA1 activation in vitro should identify chemical sensory irritants and provide an estimate of potency. Fibroblasts expressing TRPA1 are used to determine TRPA1 activation and irritant potency. We report a linear relationship between the in vivo RD50 and the in vitro pEC50 values (R=0.81) to support this hypothesis. We propose that this in vitro assay after additional analysis and validation could serve as a suitable candidate to replace the mouse sensory irritation assay.

BRCA1 modulates sensitivity to 5F-203 by regulating xenobiotic stress-inducible protein levels and EROD activity.

PURPOSE: We have investigated the effects of BRCA1 over-expression and knockdown on 5F-203-induced gene expression and cytotoxicity in human breast cancer cells. 5F-203 is a chemotherapeutic prodrug that both induces a p450 enzyme, CYP1A1, and is metabolically activated by CYP1A1. METHODS: We used several molecular biological techniques to confirm our findings. BRCA1 regulates sensitivity to 5F-203 by regulating the expression of CYP1A1 mRNA and its EROD activity. XRE-Luc reporter assays, semi-quantitative RT-PCR, Western blot analysis, EROD activity measurements, gene knockdown and MTT cell survival assays were used for this study. RESULTS: Our results show that the ability of 5F-203 treatments to increase CYP1A1 mRNA level and CYP1A1 enzymatic activity (EROD activity) are affected by BRCA1 protein levels. In addition, the ability of 5F-203 treatments to induce proteins, P53 and P53 target genes such as P21, is significantly decreased in BRCA1 knockdown cells, suggesting that BRCA1-related effects could at least partially explain why BRCA1 knockdown increases resistance to 5F-203-mediated cytotoxicity. We also observed altered expression of the two major transcription factors (AhR and ARNT) that affect CYP1A1 expression when BRCA1 protein levels are altered. CONCLUSION: BRCA1 is an important protein, which affects 5F-203-mediated cytotoxicity. Our findings are potentially clinically significant; they suggest that those patients most likely to respond to this new prodrug will have tumors containing normal amounts of BRCA1.

Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways.

Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.

Changes in gene expression contribute to cancer prevention by COX inhibitors.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used primarily for the treatment of inflammatory diseases. However, certain NSAIDs also have a chemopreventive effect on the development of human colorectal and other cancers. NSAIDs inhibit cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2) activity and considerable evidence supports a role for prostaglandins in cancer development. However, the chemopreventive effect of NSAIDs on colorectal and other cancers appears also to be partially independent of COX activity. COX inhibitors also alter the expression of a number of genes that influence cancer development. One such gene is NAG-1 (NSAID-Activated Gene), a critical gene regulated by a number of COX inhibitors and chemopreventive chemicals. Therefore, this article will discuss the evidence supporting the conclusion that the chemo-preventive activity of COX inhibitors is mediated, in part, by altered gene expression with an emphasis on NAG-1 studies. This review may also provide new insights into how chemicals and environmental factors influence cancer development. In view of the cardiovascular and gastrointestinal toxic side effects of COX-2 inhibitors and non-selective COX inhibitors, respectively, the results presented here may provide the basis for the development of a new family of anti-tumorigenic compounds acting independent of COX inhibition.

A novel peroxisome proliferator-activated receptor gamma ligand, MCC-555, induces apoptosis via posttranscriptional regulation of NAG-1 in colorectal cancer cells.

Apoptosis and/or differentiation induction caused by the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand is a promising approach to cancer therapy. The thiazolidinedione derivative MCC-555 has an apoptotic activity in human colorectal cancer cells, accompanied by up-regulation of a proapoptotic nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in a PPARgamma-independent manner. Treatment with MCC-555 resulted in the induction of NAG-1 expression and apoptosis in HCT-116 cells. Down-regulation of NAG-1 by small interfering RNA suppressed MCC-555-induced apoptosis. MCC-555 was found to affect NAG-1 mRNA stability. To further define the underlying mechanism of RNA stability affected by MCC-555, we cloned the 3'-untranslated region (3'UTR) of human NAG-1 mRNA, which contains four copies of an AU-rich element (ARE), downstream from the luciferase gene. The reporter activity was reduced to approximately 70% by inserting the 3'UTR. In addition, deletion of ARE sequences in the 3'UTR or MCC-555 treatment substantially restored activity. This effect of MCC-555 on the ARE-mediated mRNA degradation was inhibited by extracellular signal-regulated kinase (ERK) pathway inhibitors. Subsequently, rapid phosphorylation of ERK1/2 by MCC-555 treatment was detected. Moreover, ERK small interfering RNA suppressed MCC-555-induced NAG-1 expression. These results suggest that ARE sequences in the 3'UTR of the NAG-1 gene contribute to mRNA degradation and ERK1/2 phosphorylation is responsible for the stabilization of NAG-1 mRNA. These findings may provide a novel explanation for the antitumorigenic and/or proapoptotic action of MCC-555 in human colorectal cancer and the ability of pharmacologic approaches to be used against diseases caused by alterations of RNA stability.

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells.

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.

NSAID activated gene (NAG-1), a modulator of tumorigenesis.

The NSAID activated gene (NAG-1), a member of the TGF-beta superfamily, is involved in tumor progression and development. The over-expression of NAG-1 in cancer cells results in growth arrest and increase in apoptosis, suggesting that NAG-1 has anti-tumorigenic activity. This conclusion is further supported by results of experiments with transgenic mice that ubiquitously express human NAG-1. These transgenic mice are resistant to the development of intestinal tumors following treatment with azoxymethane or by introduction of a mutant APC gene. In contrast, other data suggest a pro-tumorigenic role for NAG-1, for example, high expression of NAG-1 is frequently observed in tumors. NAG-1 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression. The expression of NAG-1 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development. Most notable is the increase in NAG-1 expression by the inhibitors of cyclooxygenases that prevent human colorectal cancer development. The regulation of NAG-1 is complex, but these agents act through either p53 or EGR-1 related pathways. In addition, an increase in NAG-1 is observed in inhibition of the AKT/GSK-3beta pathway, suggesting NAG-1 alters cell survival. Thus, NAG-1 expression is regulated by tumor suppressor pathways and appears to modulate tumor progression.

The conventional nonsteroidal anti-inflammatory drug sulindac sulfide arrests ovarian cancer cell growth via the expression of NAG-1/MIC-1/GDF-15.

Although the chemopreventive and antitumorigenic activities of nonsteroidal anti-inflammatory drug (NSAID) against colorectal cancer are well established, the molecular mechanisms responsible for these properties in ovarian cancer have not been elucidated. Therefore, there is an urgent need to develop mechanism-based approaches for the management of ovarian cancer. To this end, the effect of several NSAIDs on ovarian cancer cells was investigated as assessed by the induction of NAG-1/MIC-1/GDF-15, a proapoptotic gene belonging to the transforming growth factor-beta superfamily. Sulindac sulfide was the most significant NSAID activated gene 1 (NAG-1) inducer and its expression was inversely associated with cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. This growth suppression by sulindac sulfide was recovered by transfection of NAG-1 small interfering RNA. These results indicate that NAG-1 is one of the genes responsible for growth suppression by sulindac sulfide. Furthermore, we observed down-regulation of p21 WAF1/CIP1 by introduction of NAG-1 small interfering RNA into sulindac sulfide-treated cells. In addition, to elucidate other potential molecular mechanisms involved in sulindac sulfide treatment of ovarian cancer cells, we did a membrane-based microarray experiment. We found that cyclin D1, MMP-1, PI3KR1, and uPA were down-regulated by sulindac sulfide. In conclusion, a novel molecular mechanism is proposed to explain the experimental results and provide a rationale for the chemopreventive activity of NSAIDs in ovarian cancer.

Suppression of tumor cell invasion by cyclooxygenase inhibitors is mediated by thrombospondin-1 via the early growth response gene Egr-1.

Cyclooxygenase (COX) inhibitors have antitumorigenic activity and increase the expression of the early growth response gene Egr-1, a tumor suppressor gene and transcription factor. In this study, we have investigated the gene regulatory and anti-invasive activity of two traditional nonsteroidal anti-inflammatory drugs (NSAID), sulindac sulfide and indomethacin. These compounds inhibited tumor cell invasion and induced Egr-1 expression in lung adenocarcinoma A549 cells. Overexpression of Egr-1 reduced cellular invasion in the Matrigel system, whereas suppression of Egr-1 by small interference RNA (siRNA) attenuated the inhibition of Matrigel invasion by these compounds, indicating that Egr-1 is responsible for the decrease in invasion reported following treatment with NSAIDs. Egr-1-overexpressing cells were analyzed for genes involved in invasion and metastasis. Thrombospondin-1 (TSP-1) an antiangiogenic and anti-invasion protein was up-regulated by Egr-1 overexpression, which was confirmed following treatment with sulindac sulfide. Furthermore, the induction of TSP-1 by sulindac sulfide was blocked by Egr-1 siRNA. When TSP-1 was sequestered by the addition of anti-TSP-1 antibody, the inhibition of invasion by sulindac sulfide was attenuated, indicating that TSP-1 is involved in the inhibition of invasion by NSAIDs. We used the Min mouse model to determine if sulindac sulfide would increase Egr-1 and TSP-1 in vivo, because this model is widely used to study the effects of NSAIDs on tumor formation. Treatment of Min mice with concentrations of sulindac sulfide that inhibit tumor formation increased the expression of Egr-1 and TSP-1 in colonic tissues and in the polyps of these mice. This is the first report suggesting that COX inhibitors suppress tumor cell invasion via TSP-1, which occurs downstream of Egr-1.

The anti-invasive activity of cyclooxygenase inhibitors is regulated by the transcription factor ATF3 (activating transcription factor 3).

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.

Reinterpreting the best biomarker of oxidative stress: The 8-iso-prostaglandin F2α/prostaglandin F2α ratio shows complex origins of lipid peroxidation biomarkers in animal models.

Oxidative stress is elevated in numerous environmental exposures and diseases. Millions of dollars have been spent to try to ameliorate this damaging process using anti-oxidant therapies. Currently, the best accepted biomarker of oxidative stress is the lipid oxidation product 8-iso-prostaglandin F2α (8-iso-PGF2α), which has been measured in over a thousand human and animal studies. 8-iso-PGF2α generation has been exclusively attributed to nonenzymatic chemical lipid peroxidation (CLP). However, 8-iso-PGF2α can also be produced enzymatically by prostaglandin-endoperoxide synthases (PGHS) in vivo. When failing to account for PGHS-dependent generation, 8-iso-PGF2α cannot be interpreted as a selective biomarker of oxidative stress. We investigated the formation of 8-iso-PGF2α in rats exposed to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) using the 8-iso-PGF2α/PGF2α ratio to quantitatively determine the source(s) of 8-iso-PGF2α. Upon exposure to a 120mg/kg dose of CCl4, the contribution of CLP accounted for only 55.6±19.4% of measured 8-iso-PGF2α, whereas in the 1200mg/kg dose, CLP was the predominant source of 8-iso-PGF2α (86.6±8.0% of total). In contrast to CCl4, exposure to 0.5mg/kg LPS was characterized by a significant increase in both the contribution of PGHS (59.5±7.0) and CLP (40.5±14.0%). In conclusion, significant generation of 8-iso-PGF2α occurs through enzymatic as well as chemical lipid peroxidation. The distribution of the contribution is dependent on the exposure agent as well as the dose. The 8-iso-PGF2α/PGF2α ratio accurately determines the source of 8-iso-PGF2α and provides an absolute measure of oxidative stress in vivo.

Expression of Human NSAID Activated Gene 1 in Mice Leads to Altered Mammary Gland Differentiation and Impaired Lactation.

Transgenic mice expressing human non-steroidal anti-inflammatory drug activated gene 1 (NAG-1) have less adipose tissue, improved insulin sensitivity, lower insulin levels and are resistant to dietary induced obesity. The hNAG-1 expressing mice are more metabolically active with a higher energy expenditure. This study investigates female reproduction in the hNAG-1 transgenic mice and finds the female mice are fertile but have reduced pup survival after birth. Examination of the mammary glands in these mice suggests that hNAG-1 expressing mice have altered mammary epithelial development during pregnancy, including reduced occupancy of the fat pad and increased apoptosis via TUNEL positive cells on lactation day 2. Pups nursing from hNAG-1 expressing dams have reduced milk spots compared to pups nursing from WT dams. When CD-1 pups were cross-fostered with hNAG-1 or WT dams; reduced milk volume was observed in pups nursing from hNAG-1 dams compared to pups nursing from WT dams in a lactation challenge study. Milk was isolated from WT and hNAG-1 dams, and the milk was found to have secreted NAG-1 protein (approximately 25 ng/mL) from hNAG-1 dams. The WT dams had no detectable hNAG-1 in the milk. A decrease in non-esterified free fatty acids in the milk of hNAG-1 dams was observed. Altered milk composition suggests that the pups were receiving inadequate nutrients during perinatal development. To examine this hypothesis serum was isolated from pups and clinical chemistry points were measured. Male and female pups nursing from hNAG-1 dams had reduced serum triglyceride concentrations. Microarray analysis revealed that genes involved in lipid metabolism are differentially expressed in hNAG-1 mammary glands. Furthermore, the expression of Cidea/CIDEA that has been shown to regulate milk lipid secretion in the mammary gland was reduced in hNAG-1 mammary glands. This study suggests that expression of hNAG-1 in mice leads to impaired lactation and reduces pup survival due to altered milk quality and quantity.