sulindac and Glioblastoma

NSAIDs are known to be inhibitors of cyclooxygenase-2 (COX-2) accounting for their anti-inflammatory and anti-tumor activities. However, the anti-tumor activity cannot be totally attributed to their COX-2 inhibitory activity as these drugs can also inhibit the growth and tumor formation of COX-2-null cell lines. Several potential targets aside from COX-2 for NSAIDs have been proposed. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH), a key prostaglandin catabolic enzyme, was recently shown to be a tumor suppressor. Effects of NSAIDs on 15-PGDH expression were therefore studied. Flurbiprofen, indomethacin and other NSAIDs stimulated 15-PGDH activity in colon cancer HT29 cells as well as in lung cancer A549 cells and glioblastoma T98G cells. (R)-flurbiprofen and sulindac sulfone, COX-2 inactive analogs, also stimulated 15-PGDH activity indicating induction of 15-PGDH is independent of COX-2 inhibition. Stimulation of 15-PGDH expression and activity by NSAIDs was examined in detail in colon cancer HT29 cells using flurbiprofen as a stimulant. Flurbiprofen stimulated 15-PGDH expression and activity by increasing transcription and translation and by decreasing the turnover of 15-PGDH. Mechanism of stimulation of 15-PGDH expression is not clear. Protease(s) involved in the turnover of 15-PGDH remains to be identified. However, flurbiprofen down-regulated matrix metalloproteinase-9 (MMP-9) which was shown to degrade 15-PGDH, but up-regulated tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of MMP-9 contributing further to a slower turnover of 15-PGDH. Taken together, NSAIDs may up-regulate 15-PGDH by increasing the protein expression as well as decreasing the turnover of 15-PGDH in cancer cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Enzyme Activation; Flurbiprofen; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Hydroxyprostaglandin Dehydrogenases; Indomethacin; Kinetics; Lung Neoplasms; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Signal Transduction; Sulindac; Tissue Inhibitor of Metalloproteinase-1; Up-Regulation

Glioblastoma tumors (GBM) are very heterogeneous, being comprised of several cell subtypes, including glioblastoma stem cells (GSC). These tumors have a high rate of recurrence after initial treatment and one of the most prevalent theories to explain this is the cancer stem cell theory, which proposes that glioblastomas arise from mutations that transform normal neural stem cells (NSC) into GSC, which are highly resistant to oxidative stress and anti-cancer therapies. Sulindac is a non-steroidal anti-inflammatory drug (NSAID) that has been shown to protect the normal cells against oxidative damage by initiating a preconditioning response, but selectively sensitizes several cancer cell lines to agents that affect mitochondrial respiration, resulting in enhanced killing of the cancer cells. These effects of sulindac are independent of its NSAID activity. There is little information on the effect of sulindac on normal and cancer stem cells. To study the effect of sulindac on both normal and cancer stem cells, we have isolated normal neural stem cells (NSC), from mice hippocampi and glioblastoma stem cells (GSC) from a glioma cell line, U87. As expected from previous studies sulindac can protect normal astrocytes against oxidative stress. Sulindac induces differentiation of both NSC and GSC cells and sulindac upregulates neurogenesis in NSC. The differentiated NSC are also protected from oxidative stress damage, whereas the differentiation of GSC by sulindac increases the sensitivity of these cells to agents that cause oxidative stress. The S epimer of sulindac is more effective than the R epimer in inducing neuronal differentiation in both NSC and GSC. These results indicate that the ability of sulindac to induce GSC differentiation may have therapeutic value in preventing tumour recurrence.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Glioblastoma; Humans; Mice; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Oxidative Stress; Sulindac

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

Non-steroidal anti-inflammatory drug (NSAID), sulindac has chemopreventive and anti-tumorigenic properties, however, the molecular mechanism of this inhibitory action has not been clearly defined. The Akt/protein kinase B, serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. In the present study, we demonstrate that down-regulation of Akt is a major effect of anti-invasiveness property of sulindac and its metabolites in glioblastoma cells. Myristoylated Akt (MyrAkt) transfected U87MG glioblastoma cells showed increase invasiveness, whereas DN-Akt transfected cells showed decrease invasiveness indicating that Akt potently promoted glioblastoma cell invasion. MMP-2 promoter and enzyme activity were up-regulated in Akt kinase activity dependent manner. Sulindac and its metabolites down-regulated Akt phosphorylation, inhibited MMP-2 production, and significantly inhibited invasiveness of human glioblastoma cells. In addition, sulindac and LY294002, a selective inhibitor of phosphoinositide 3-kinase (PI3K), synergistically inhibited the invasion of glioblastoma cells. Furthermore, only celecoxib showed Akt phosphorylation reduction and an anti-invasivness in glioblastoma cells, whereas aspirin, ketoprofen, ketorolac, and naproxen did not. In conclusion, our results provide evidence that down-regulation of Akt pathway and MMP-2 may be one of the mechanisms by which sulindac and its metabolites inhibit glioblastoma cell invasion.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Cell Line, Tumor; Down-Regulation; Glioblastoma; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sulindac

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

It is well established that traditional NSAIDs, which inhibit cyclooxygenase (COX) 1 and COX-2, have the potential to reduce the risk of colorectal cancer. New generation COX inhibitors have been developed that selectively inhibit COX-2, which might cause less side effects while still retaining their therapeutic potential. As patients with brain tumors, such as glioblastoma, exhibit a very poor prognosis, we began to explore whether COX inhibitors could be useful for the treatment of this type of tumor. We found that celecoxib inhibited the proliferation of various glioblastoma cell lines in vitro much more potently than traditional NSAIDs. In addition, although several different selective COX-2 inhibitors potently reduced PGE2 levels in these cells, none of them exerted anti-proliferative effects that were comparable to celecoxib. The addition of external PGE2 to celecoxib-treated cells did not restore proliferation, indicating that growth inhibition by celecoxib was not mediated via the blockage of PGE2 production. In an effort to determine the underlying molecular processes that might mediate celecoxib's potent anti-proliferative effects, we found a loss of the activity of cyclin-dependent kinases, the essential regulators of cell proliferation, which was due to the transcriptional downregulation of cyclin A and cyclin B expression. Taken together, our results show that celecoxib exerts COX-2-independent anti-proliferative effects on glioblastoma cell growth, which are more potent than those of other selective COX-2 inhibitors or traditional NSAIDs, and which are mediated via the transcriptional inhibition of two essential components of the cell cycle machinery, cyclin A and cyclin B.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Celecoxib; Cell Cycle; Cell Division; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Glioblastoma; Humans; Indomethacin; Isoenzymes; Isoxazoles; Lactones; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Sulfonamides; Sulfones; Sulindac