sulindac-sulfide and Glioblastoma

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β 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.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Azacitidine; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Decitabine; DNA Methylation; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Growth Differentiation Factor 15; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Sulindac; Transfection

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.

Topics: Activating Transcription Factor 3; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Brain Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Chromatin Immunoprecipitation; Colony-Forming Units Assay; Cyclooxygenase Inhibitors; Early Growth Response Protein 1; Flavonoids; Glioblastoma; Humans; Immunoprecipitation; Luciferases; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Phosphorylation; Promoter Regions, Genetic; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP4 Subtype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sp1 Transcription Factor; Sulindac; Tumor Cells, Cultured

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Blotting, Western; Cell Line, Tumor; Cyclooxygenase Inhibitors; Densitometry; Dose-Response Relationship, Drug; Etoposide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Indomethacin; Kinetics; Laminin; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; Sulindac