15-deoxy-delta(12-14)-prostaglandin-j2 and Colonic-Neoplasms

There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation of cytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Colonic Neoplasms; Cytoplasmic Granules; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4A; Female; Heterografts; Humans; Mice; Mutation; Neoplasm Transplantation; Pancreatic Neoplasms; Prostaglandin D2; Proto-Oncogene Proteins p21(ras); Up-Regulation

6-Shogaol has been shown to possess many antitumor properties including inhibition of cancer cell growth, inhibition of cancer metastasis, induction of apoptosis in cancer cells and induction of cancer cell differentiation. Despite its prominent antitumor effects, the direct molecular target of 6-shogaol has remained elusive. To identify the direct targets of 6-shogaol, a comprehensive antitumor profile of 6-shogaol (NSC752389) was tested in the NCI-60 cell line in an in vitro screen. The results show that 6-shogaol is COMPARE negative suggesting that it functions via a mechanism of action distinct from existing classes of therapeutic agents. Further analysis using microarray gene profiling and Connectivity Map analysis showed that MCF-7 cells treated with 6-shogaol display gene expression signatures characteristic of peroxisome proliferator activated receptor γ (PPARγ) agonists, suggesting that 6-shogaol may activate the PPARγ signaling pathway for its antitumor effects. Indeed, treatment of MCF-7 and HT29 cells with 6-shogaol induced PPARγ transcriptional activity, suppressed NFκB activity, and induced apoptosis in breast and colon cancer cells in a PPARγ-dependent manner. Furthermore, 6-shogaol is capable of binding to PPARγ with a binding affinity comparable to 15-delta prostaglandin J2, a natural ligand for PPARγ. Together, our findings suggest that the antitumor effects of 6-shogaol are mediated through activation of PPARγ and imply that activation of PPARγ might be beneficial for breast and colon cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Catechols; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Ligands; MCF-7 Cells; NF-kappa B; Oligonucleotide Array Sequence Analysis; PPAR gamma; Prostaglandin D2; Signal Transduction; Transcription, Genetic

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been shown to induce colon cancer cell apoptosis in the presence of interferon-gamma. We hypothesized that co-treatment using TWEAK with other pro-apoptosis agents could sensitize death receptor-resistant colon cancer cells.. The effects of chemopreventive agents and TWEAK on cell death and apoptosis were determined using propidium iodide (PI) exclusion and M30 CytoDEATH.. We found that 15d-PGJ(2) sensitizes colon cancer cells to TWEAK-induced apoptosis. Caspase inhibition reduced 15d-PGJ(2)-, but not 15d-PGJ(2)+TWEAK-induced apoptosis. 15d-PGJ(2) promoted reactive oxygen species (ROS) production and dissipation of mitochondrial potential (DeltaPsi(m)) that were more marked with combined treatment. ROS, DeltaPsi(m) and cell death were partially normalized by the antioxidant N-acetylcysteine. TWEAK induced nuclear factor-kappa B activation, which was attenuated by 15d-PGJ(2). 15d-PGJ(2) reduced the expression of the anti-apoptotic proteins BCL-X(L) and MCL-1, while increasing BAX and translocation of cytochrome c and apoptosis-inducing factor.. 15d-PGJ(2) sensitized cancer cells to TWEAK-induced apoptosis through an ROS-dependent cell death pathway and may have chemotherapeutic utility as an apoptosis-enhancing agent.

Topics: Apoptosis; Butyrates; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Cytokine TWEAK; Drug Synergism; HT29 Cells; Humans; Interferon-gamma; Leucine; Ligands; Membrane Potential, Mitochondrial; Mitochondria; PPAR gamma; Prostaglandin D2; Thiazolidinediones; Tumor Necrosis Factors

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) has been shown to possess antineoplastic activity in human cancers of various origins. However, the mechanism of the antineoplastic activity of 15d-PGJ2 remains to be completely elucidated. It has been reported that inhibiting the expression of human telomerase reverse transcriptase (hTERT), a major determinant of telomerase activity, induces rapid apoptosis in cancer cells. In this study, we investigated the effect of 15d-PGJ2 on hTERT expression. Treatment with 30 microM 15d-PGJ2 for 72 h induced apoptosis in the colon cancer cells LS180. 15d-PGJ2 treatment decreased hTERT protein expression in a dose-dependent manner. Down-regulation of hTERT expression by hTERT-specific small inhibitory RNA induced apoptosis. These results indicate that the down-regulation of hTERT expression by 15d-PGJ2 plays an important role in its proapoptotic properties. Since 15d-PGJ2 reduced hTERT mRNA expression, we examined the effect of 15d-PGJ2 on the DNA-binding activity of c-Myc, specificity protein 1 (Sp1) and estrogen receptor (ER) to the hTERT gene promoter using an electrophoretic mobility shift assay. 15d-PGJ2 attenuated the DNA-binding of all three transcriptional factors. Further, we observed that 15d-PGJ2 inhibited the DNA binding of these factors by different mechanisms; suppressed c-Myc mRNA expression, enhanced Sp1 protein degradation via the ubiquitin-proteasome pathway and inhibited ERbeta phosphorylation at serine residues. We conclude that hTERT down-regulation by 15d-PGJ2 plays an important role in its proapoptotic properties. Furthermore, 15d-PGJ2 inhibits the transcriptional activity of c-Myc, Sp1 and ER by three different mechanisms and results in the transcriptional repression of the hTERT gene.

Topics: Apoptosis; Colonic Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Promoter Regions, Genetic; Prostaglandin D2; Protein Binding; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; RNA, Messenger; RNA, Small Interfering; Sp1 Transcription Factor; Telomerase; Tumor Cells, Cultured; Ubiquitination

Although 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) was reported to up-regulate death receptor 5 (DR5) protein expression and sensitize TRAIL-induced cytotoxicity, its action mechanism remains unclear. Using HCT116 colon cancer cells, we found that sensitization of TRAIL-induced cytotoxicity by 15dPGJ(2) resulted from up-regulation of DR5 via gene transcription but was not associated with PPAR-gamma activation. Moreover, 15dPGJ(2) induced GRP78, XBP1, and C/EBP homologous transcription factor (CHOP) expression in HCT116 cells, confirming that 15dPGJ(2) is an endoplasmic reticulum stress inducer. Knockdown of the CHOP gene by siRNA attenuated DR5 up-regulation and the sensitized cytotoxicity in colon cancer HCT116 and SW480. With deletion plasmids of DR5 promoters, we found that the CHOP-binding site was involved in activating the DR5 gene by 15dPGJ(2). A mechanistic study showed the contributions of reactive oxygen species (ROS) and intracellular calcium in CHOP and DR5 gene up-regulation. 15dPGJ(2) was also found to induce DR5 in two prostate cancer cell lines, LNCaP and PC3. Although in LNCaP DR5 up-regulation was accompanied by CHOP expression by 15dPGJ(2), no significant increase in CHOP expression or DR5 promoter activity was observed in PC3 cells. Intriguingly, 15dPGJ(2) induced ROS and calcium production in PC3 cells. This inability to induce CHOP was not due to the p53-null in PC3 cells, as similar extents of increase in CHOP protein were found due to 15dPGJ(2) in both wild-type and p53-null HCT116 cells. In summary, the effect of up-regulation of DR5 by 15dPGJ(2) in colon cancer cells is independent of PPAR-gamma and p53 but relies on CHOP induction through gene transcription involving ROS and calcium.

Topics: Calcium; Cell Death; Colonic Neoplasms; Drug Screening Assays, Antitumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Intracellular Space; PPAR gamma; Prostaglandin D2; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation

PPAR-gamma has been known to induce suppression, differentiation and reversal of malignant changes in colon cancer in vitro. However, there are several reports that PPAR-gamma ligands enhance colon polyp development in APCmin mice in vivo. These contradictory results have not yet been thoroughly explained. To explain the contradictory results, we analyzed the effects of different concentrations of the PPAR-gamma agonist, 15-deoxy-D12, 14-prostaglandin (15-d Delta PGJ2) and pioglitazone, on APC gene-mutated colon cancer cell lines (HT-29). We measured cell growth and suppression by cell count and MTT assay and analyzed the expression of beta-catenin and c-Myc protein by Western blot. In addition, we inoculated HT-29 cells into APCmin mice to compare tumor size. High concentrations (10-100 microM/L 15-d Delta PGJ2 and pioglitazone) of PPAR-gamma ligand suppressed growth, while low concentrations (0.01-1 microM/L 15-d Delta PGJ2 and pioglitazone) of PPAR-gamma ligand promoted growth. In particular, the effects of 0.1 microM/L 15-d Delta PGJ2 and pioglitazone on cell growth were statistically significant (P = 0.003, P = 0.001, respectively). Tumor growth was associated with an increase in beta-catenin and c-Myc expression. The growth of xenograft tumors was greater in PPAR-gamma ligand-treated mice than in control mice (control vs day 14: P = 0.024, control vs day 28: P = 0.007). The expression of beta-catenin and c-Myc protein were also elevated in PPAR-gamma-treated mouse tissues. PPAR-gamma ligand can promote the growth of APC-mutated HT-29 colon cancer cells in vitro and in vivo. In addition, the tumor promoting effect seems to be associated with an increase in beta-catenin and c-Myc expression. We think that well-controlled clinical trials should be conducted to confirm our results and to verify clinical applications.

Topics: Animals; beta Catenin; Cell Proliferation; Colonic Neoplasms; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Genes, APC; HT29 Cells; Humans; Ligands; Mice; Mice, Nude; Mutation; Pioglitazone; PPAR gamma; Prostaglandin D2; Proto-Oncogene Proteins c-myc; Thiazolidinediones; Xenograft Model Antitumor Assays

Prostaglandin (PG) E(2) (PGE(2)) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE(2) is accomplished by conversion of the cyclooxygenase (COX) product PGH(2) by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Delta(12,14)-PGJ(2) and PGA(2) downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor gamma or PGD(2) receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE(2). Our data suggest that there is a feedback mechanism between PGE(2) and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.

Topics: Caco-2 Cells; Colonic Neoplasms; Dinoprostone; Down-Regulation; HCT116 Cells; Humans; Intramolecular Oxidoreductases; Microsomes; PPAR gamma; Prostaglandin D2; Prostaglandin-E Synthases; Prostaglandins A; Receptors, Immunologic; Receptors, Prostaglandin

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical albeit poorly defined role in the development and progression of several cancer types including those of the breast, colon, and lung. A PPAR response element (PPRE) reporter assay was utilized to evaluate the selective transactivation of PPARgamma in 10 different cell lines including normal mammary epithelial, breast, lung, and colon cancer cells. Cells were treated with one of four compounds including rosglitizone (Ros), ciglitizone (Cig), 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2), or GW 9662 (GW). We observed differences in transactivation between cell lines from different tissue origin, across cell lines from a single tissue type, and selective modulation of PPARgamma within a single cell line by different ligands. Interestingly, GW, a PPARgamma antagonist in adipocytes, enhanced PPRE reporter activation in normal mammary epithelial cells while it had virtually no effect in any of the cancer cell lines tested. Within each cancer type, individual cell lines were found to respond differently to distinct PPARgamma ligands. For instance, Ros, Cig, and PGJ2 were all potent agonist of PPARgamma transactivation in lung adenocarcinoma cell lines while these same ligands had no effect in squamous cell or large cell carcinomas of the lung. Message levels of PPARgamma and retinoid X receptor alpha (RXRalpha) in the individual cell lines were quantitated by real time-polymerase chain reaction (RT-PCR). The ratio of PPARgamma to RXRalpha was predictive of how cells responded to co-treatment of Ros and 9-cis-retinoic acid, an RXRalpha agonist, in two out of three cell lines tested. These data indicate that PPARgamma can be selectively modulated and suggests that it may be used as a therapeutic target for individual tumors.

Topics: Alitretinoin; Anilides; Breast Neoplasms; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; HT29 Cells; Humans; Ligands; Lung Neoplasms; PPAR gamma; Prostaglandin D2; Retinoid X Receptor alpha; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Transfection; Tretinoin

Cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) are significantly associated with tumor growth and metastasis. Here we show that phorbol ester-mediated induction of VEGF and COX-2 expression in colon carcinoma cells is inhibited by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This cyclopentenone was able to inhibit activator protein1 (AP-1)-dependent transcriptional induction of COX-2 and VEGF promoters induced by phorbol 12-myristate 13-acetate (PMA) or c-Jun overexpression. 15d-PGJ(2) interfered with at least two steps within the signaling pathway leading to AP-1 activation. First, 15d-PGJ(2) impaired AP-1 binding to a consensus DNA sequence. Second, 15d-PGJ(2) selectively inhibited c-Jun NH(2) terminal kinase (JNK) but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase activation induced by PMA. This led to a decreased ability of JNK to phosphorylate c-Jun and to activate its transactivating activity. Inhibition of AP-1 activation and COX-2 or VEGF transcriptional induction by this cyclopentenone was found to be independent of peroxisome proliferator-activated receptor-gamma (PPARgamma) because it was not affected by either expression of a dominant negative form of PPARgamma or the use of a PPARgamma antagonist. In contrast, we have found that the effects of 15d-PGJ(2) on AP-1 activation may occur through its ability to induce intracellular oxidative stress. The antioxidant N-acetylcysteine significantly reversed the inhibition by 15d-PGJ(2) of AP-1 activity and COX-2 or VEGF transcriptional induction. Together, these findings provide new insight into the antitumoral properties of 15d-PGJ(2) through the inhibition of the induction of AP-1-dependent genes involved in tumor progression, such as COX-2 and VEGF.

Topics: Cell Survival; Colonic Neoplasms; Consensus Sequence; Cyclooxygenase 2; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Genes, jun; Humans; Immunologic Factors; Isoenzymes; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Protein Binding; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo. PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer. Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation. Caveolin-1 has growth-inhibitory activities and its disruption is sufficient to induce transformation in fibroblasts. Herein we have tested the hypothesis that caveolins are transcriptional target genes for PPARgamma. In human HT-29 colon carcinoma cells, thiazolidinedione PPARgamma ligands increased the levels of caveolin-1 and caveolin-2 proteins two to fivefold in a concentration-dependent manner within 24 h. In human MCF-7 breast adenocarcinoma cells, nonthiazolidinedione PPARgamma ligands elevated caveolin-2 protein three to fourfold, while the thiazoli-dinediones were less effective. Caveolin-1 mRNA levels were found to be upregulated by PPARgamma ligands already after 3 h in both the cell lines. Ectopic expression of a dominant-negative PPARgamma construct attenuated ligand-induced upregulation of caveolins in both HT-29 and HEK-293T cells, indicating that ligand action is mediated by PPARgamma. Ligand-treated MCF-7 cells exhibited a differentiated phenotype, as evinced by analysis of cell-specific differentiation markers: protein levels of maspin were elevated and perinuclear lipid droplets accumulated. In contrast, in HT-29 cells, caveolin expression was not correlated with differentiation. Interestingly, PPARgamma partially cofractionated in lipid rafts and could be coimmunoprecipitated from cell lysates with caveolin-1, indicating that PPARgamma and caveolin-1 may coexist in a complex. Our data indicate that PPARgamma participates in the regulation of caveolin gene expression in human carcinoma cells and suggest that caveolin-1 may mediate some of the phenotypic changes induced by this nuclear receptor in cancer cells. These findings may have potentially important functional implications in the context of cancer differentiation therapy and multidrug resistance.

Topics: Adenocarcinoma; Antigens, Differentiation; Antigens, Neoplasm; Breast Neoplasms; Caveolin 1; Caveolin 2; Caveolins; Cell Differentiation; Cell Line; Chromans; Colonic Neoplasms; Dimerization; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Humans; Kidney; Ligands; Macromolecular Substances; Membrane Microdomains; Neoplasm Proteins; Phenotype; Phenylacetates; Prostaglandin D2; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; RNA, Neoplasm; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Troglitazone; Tumor Cells, Cultured

Peroxisome-proliferator activated receptor-gamma (PPARgamma) has been demonstrated to exert an inhibitory effect on cell growth in most cell types studied, but its role in colon cancer is still uncertain. The molecular mechanism between the activation of PPARgamma and its consequence is unknown. In the present report, we show that the expression of PPARgamma was significantly increased in tumor tissues from human colon cancer compared with non-tumor tissues and that PPARgamma ligands, 15-Deoxy-delta(12,14)prostaglandin J2 or ciglitizone, induced apoptosis in HT-29 cells, a human colon cancer cell line. The occurrence of apoptosis induced by PPARgamma ligands was sequentially accompanied by reduced levels of NF-kappaB and Bcl-2. Over-expression of Bcl-2 significantly protected the cells from apoptosis. This study suggested that a PPARgamma-Bcl-2 feedback loop may function to control the life-death continuum in colonic cells and that a deficiency in generation of PPARgamma ligands may precede the development of human colon cancer.

Topics: Apoptosis; Cell Division; Cell Survival; Colon; Colonic Neoplasms; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; HT29 Cells; Humans; Hypoglycemic Agents; Immunoenzyme Techniques; Immunologic Factors; Ligands; NF-kappa B; Promoter Regions, Genetic; Prostaglandin D2; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic

Involvement of peroxisome proliferator activated receptor gamma (PPARgamma) in the growth response of colon cancer cells has been suggested.. To investigate the characteristics of PPARgamma induced apoptosis in colon cancer cells.. The effects of ligands for each of the PPAR subtypes (alpha, delta, and gamma) on DNA synthesis and cell viability were examined in HT-29 colon cancer cells. Modulation of apoptosis related gene expression by PPARgamma ligands was screened with cDNA arrays, and the results were confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis.. PPARalpha, PPARdelta, and PPARgamma were all expressed in HT-29 cells. PPARgamma ligands, 15-deoxy-delta(12,)(14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of HT-29 cells whereas ligands for PPARalpha and PPARdelta had no significant effects. Both 15d-PGJ2 and TGZ induced HT-29 cell death in a dose dependent manner which was associated with an increase in fragmented DNA and was sensitive to a caspase inhibitor. Among several genes selected by cDNA array screening, quantitative RT-PCR analysis confirmed downregulation of c-myc expression and upregulation of c-jun and gadd153 expression by 15d-PGJ2 and TGZ. PPARgamma induced apoptosis was antagonised by the presence of serum in the culture medium, and interaction between PPARgamma signalling and cell survival signalling through the phosphatidylinositol 3-kinase pathway was suggested.. As c-myc is an important target gene of the adenomatous polyposis coli (APC)/beta-catenin and/or APC/gamma-catenin pathway, activation of PPARgamma signalling appears to compensate for deregulated c-myc expression caused by mutated APC. The present results suggest the potential usefulness of PPARgamma ligands for chemoprevention and treatment of colon cancers.

Topics: Apoptosis; Cell Division; Cell Survival; Chromans; Colonic Neoplasms; DNA, Complementary; DNA, Neoplasm; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Ligands; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone

The efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is considered to be a result of their inhibitory effect on cyclooxygenase (COX) activity. Here, we report that flufenamic acid shows two opposing effects on COX-2 expression; it induces COX-2 expression in the colon cancer cell line (HT-29) and macrophage cell line (RAW 264.7); conversely, it inhibits tumor necrosis factor alpha (TNFalpha)- or lipopolysaccharide (LPS)-induced COX-2 expression. This inhibition correlates with the suppression of TNFalpha- or LPS-induced NFkappaB activation by flufenamic acid. The inhibitor of extracellular signal-regulated protein kinase, p38, or NFkappaB does not affect the NSAID-induced COX-2 expression. These results suggest that the NSAID-induced COX-2 expression is not mediated through activation of NFkappaB and mitogen-activated protein kinases. An activator of peroxisome proliferator-activated receptor gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2), also induces COX-2 expression and inhibits TNFalpha-induced NFkappaB activation and COX-2 expression. Flufenamic acid and 15-deoxy-Delta(12,14)-prostaglandin J(2) also inhibit LPS-induced expression of inducible form of nitric-oxide synthase and interleukin-1alpha in RAW 264.7 cells. Together, these results indicate that the NSAIDs inhibit mitogen-induced COX-2 expression while they induce COX-2 expression. Furthermore, the results suggest that the anti-inflammatory effects of flufenamic acid and some other NSAIDs are due to their inhibitory action on the mitogen-induced expression of COX-2 and downstream markers of inflammation in addition to their inhibitory effect on COX enzyme activity.

Topics: Adenocarcinoma; Animals; Anti-Inflammatory Agents, Non-Steroidal; Colonic Neoplasms; Cyclooxygenase 2; Enzyme Induction; Flufenamic Acid; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Lipopolysaccharides; Macrophages; Membrane Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; Sulindac; Transcription Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We previously reported that cyclooxygenase (COX)-2 was predominantly expressed in macrophages of human colonic adenomas (Int. J. Cancer 83, 470-475.). The role of prostaglandins (PGs) produced by COX-2-expressing macrophages in colon carcinogenesis is still unclear. Here we show that PGs up-regulate vascular endothelial growth factor (VEGF) production by activated macrophages through their specific receptors. mRNAs of both PGE-specific receptors and peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily of ligand-dependent transcription factors, were expressed in phorbol 12-myristate 13-acetate-differentiated U937, a human macrophage model (H-Mac). Prostaglandin E(1) (PGE(1)) and 15-deoxy-Delta(12,14)-PGJ(2) (a potent PPARgamma ligand, 15d-PGJ(2)) dramatically increased VEGF production. The combination of PGE(1) and 15d-PGJ(2) additively increased VEGF production. In addition, PGE(1) significantly increased cAMP formation, whereas 15d-PGJ(2) did not affect cAMP formation. The effect of the combination of PGE(1) and 15d-PGJ(2) on cAMP formation was similar to that of PGE(1) alone. Unexpectedly, 15d-PGJ(2) also drastically increased IL-1beta production, an indicator of macrophage activation, although PGE(1) only mildly increased it. Additional enhancement of IL-1beta production was observed in the combination of PGE(1) and 15d-PGJ(2). These results suggest that PGs dramatically increased VEGF production by activated macrophages through specific PGE receptor and PPARgamma-mediated processes and that PGs may thereby promote tumor growth through VEGF production.

Topics: Alprostadil; Base Sequence; Colonic Neoplasms; Cyclic AMP; Cyclooxygenase 2; DNA Primers; Endothelial Growth Factors; Humans; Interleukin-1; Isoenzymes; Lymphokines; Macrophage Activation; Macrophages; Membrane Proteins; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; Receptors, Prostaglandin E; RNA, Messenger; Transcription Factors; U937 Cells; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors