trichostatin-a and sulindac-sulfide

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

Growth arrest and DNA damage inducible 45 alpha (GADD45alpha) is a central player in mediating apoptosis induced by a variety of stress stimuli and genotoxic agents. Regular usage of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and sulindac is associated with reduced risk for various cancers, including colon cancer. The role of GADD45alpha in NSAID-induced colon cancer cell cytotoxicity is unknown. In this study, we report that indomethacin and sulindac sulfide treatments up-regulate GADD45alpha mRNA expression and protein levels in colon cancer HT-29, RKO and Caco-2 cells. This up-regulation of GADD45alpha is accompanied by necrotic cell death and apoptosis. Anti-sense suppression of GADD45alpha expression inhibited indomethacin and sulindac sulfide-induced necrotic cell death and apoptosis. These findings confirm a role for GADD45alpha in NSAID-induced cytotoxicity, a mechanism for the anti-neoplastic effect of NSAIDs in colon tumorigenesis and cancer growth.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caco-2 Cells; Cell Cycle Proteins; Colonic Neoplasms; Humans; Hydroxamic Acids; Indomethacin; Necrosis; Nuclear Proteins; Oligonucleotides, Antisense; RNA, Messenger; Sulindac; Up-Regulation