The Cyclooxygenase Inhibitor Sulindac Sulfide Inhibits EP4 Expression and Suppresses the Growth of Glioblastoma Cells

Abstract

EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase inhibitor sulindac sulfide first altered specificity protein-1 (Sp-1) and early growth response gene-1 expression, then increased the expression of nonsteroidal antiinflammatory drug-activated gene 1 and activating transcription factor 3, and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase kinase-1/extracellular signal-regulated kinase inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide-induced Erk activation. Chromatin immunoprecipitation assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and seems to require not only the inhibition of cyclooxygenase activity but also increased expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity.

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

“…The human EP4 promoter region contains two Sp1 sites [57] overlapping with an EGR-1 site [56], and mutations of these sites in luciferase promoter studies confirmed that Sp1/EGR-1 sites are important in the regulation of EP4 expression and the response to sulindac sulfide treatment. In addition, the ChIP assay and expression studies with EGR-1 and Sp1 proteins confirmed that the EGR-1 sites are involved in the increased expression of EP4, while Sp1 sites are important for sulindac sulfide suppression of EP4 expression [56, 58]. Phosphorylated threonine residues in Sp1 activated by sulindac sulfide-induced Erk were detected.…”

“…Phosphorylated threonine residues in Sp1 activated by sulindac sulfide-induced Erk were detected. The ChIP assay experiment revealed that sulindac sulfide decreases DNA-binding activity of responsible Sp1 binding sites in the human EP4 promoter [56, 58]. Taken together, these data suggest that phosphorylation of Sp1 is critical and results in a decrease in Sp1 DNA binding, and hence suppression of transcription activation of target genes like EP4 is observed.…”

“…Phosphorylation of Sp1 critically alters the binding to DNA, and a decrease in Sp1 DNA binding can occur. Furthermore, the phosphorylated Sp1 can effectively block the binding of either EGR-1 or Sp1 to the Egr-1/Sp1 binding sites on the NAG-1 promoter, and hence suppression of transcription activation of target genes is observed [56, 58]. Figure 4 summarizes the transcriptional regulation of the EP4 receptor by two mechanisms and shows that COX inhibitors, as illustrated by sulindac sulfide, can either increase (a) or decrease (b) gene expression by altering the balance and phosphorylation status of the EGR-1 and Sp1 transcription factors.…”

“…This conclusion is illustrated by a study of the growth of glioblastoma T98G cells on soft agar to estimate the antitumorigenic activity. Sulindac sulfide inhibited T98G cell colony growth on soft agar in a concentration-dependent manner [58]. Sulindac sulfide concentrations as high as 60–120 μM were required for the inhibition of cell growth and inhibition of prostaglandin formation.…”

Analysis of how the production and activity of PGD2 affects glioma cell lines.

“…Also, when Kambe et al inhibited EP4 expression, suppression in glioma cell growth was observed (Kambe et al, 2009). The presence of EP2 and EP4 is significant because it implies that even if PGD 2 is capable of anti-tumorigenic activity through its DP1 receptor, the abundance of PGE 2 could easily mask PGD 2 's impact by activating EP2 and EP4 signaling pathways.…”