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 anti-inflammatory 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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Activating Transcription Factor 3 / metabolism
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Anti-Inflammatory Agents, Non-Steroidal / pharmacology*
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Blotting, Western
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Brain Neoplasms / pathology*
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Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
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Chromatin Immunoprecipitation
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Colony-Forming Units Assay
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Cyclooxygenase Inhibitors / pharmacology*
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Early Growth Response Protein 1 / metabolism
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Flavonoids / pharmacology
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Glioblastoma / pathology*
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Humans
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Immunoprecipitation
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Luciferases / metabolism
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Mitogen-Activated Protein Kinase 3 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 3 / metabolism
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Mitogen-Activated Protein Kinases / antagonists & inhibitors
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Mitogen-Activated Protein Kinases / metabolism
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Neoplasm Proteins / metabolism
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Phosphorylation
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Promoter Regions, Genetic / genetics
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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RNA, Small Interfering / pharmacology
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Receptors, Prostaglandin E / antagonists & inhibitors*
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Receptors, Prostaglandin E / genetics
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Receptors, Prostaglandin E / metabolism
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Receptors, Prostaglandin E, EP4 Subtype
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Reverse Transcriptase Polymerase Chain Reaction
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Sp1 Transcription Factor / metabolism
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Sulindac / analogs & derivatives*
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Sulindac / pharmacology
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Tumor Cells, Cultured
Substances
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Activating Transcription Factor 3
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Anti-Inflammatory Agents, Non-Steroidal
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Cyclooxygenase Inhibitors
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Early Growth Response Protein 1
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Flavonoids
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NBAS protein, human
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Neoplasm Proteins
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PTGER4 protein, human
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RNA, Messenger
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RNA, Small Interfering
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Receptors, Prostaglandin E
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Receptors, Prostaglandin E, EP4 Subtype
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Sp1 Transcription Factor
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Sulindac
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sulindac sulfide
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Luciferases
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one