ET-17. ANTI-NEOPLASTON AS2-1 AFFECTS CELL CYCLE CHECKPOINTS, LEADING TO APOPTOSIS IN HUMAN GLIOBLASTOMA CELLS
Sonali Patil1, Stanislaw Burzynski2, Sridhar Chittur3, Emilia Mrowczynski4, and Krzysztof Grela1; 1Burzynski Research Institute, Stafford, TX, USA; 2Houston, TX, USA; 3Microarray Core Facility, Center for Functional Genomics, Rensselaer, NY, USA; 4Burzynski Research Institute, TX, USA.
Brain tumors form because of abnormal, unregulated growth of brain cells that normally would have entered a quiescent resting stage. These cells re-enter the cell cycle due to aberrations in many genes that control cell growth and division. The typical therapy for high-grade tumors includes surgery, radiation, or chemotherapy, but unfortunately these standard treatment options are not curative. Anti-neoplastons are naturally occurring peptides and amino acid derivatives currently being used with positive results in phase II clinical trials for the treatment of several brain tumor types. Phenylacetate (PN) and phenylacetylglutaminate (PG) are the two major components of anti-neoplaston AS2-1 (AS) and are metabolites of phenylbutyrate (PB). Though PN has been well studied, the mechanism of action of PG is not well understood. PN has been reported to cause G1 arrest in several tumor cell lines. Here we report that PG also exerts its effect as an anti-proliferative agent by a similar mechanism. We provide evidence that PG causes G1 blockade and apoptosis. This effect is enhanced when PG and PN are used in combination, as in AS. We performed a screen to detect changes in gene expression in response to PG and PN in U87 glioblastoma cells using the Affymetrix Human Genome plus 2.0 oligonucleotide arrays. Pathway analysis was performed using tools such as DAVID, Onto-express, Genespring, and GenMAPP to identify pathways that show fold enrichment of genes based on the expression data. A significant inhibition of major components of the cell cycle was seen in cells treated with a combination of PG and PN. Prominent genes that were suppressed include CDCs 25A and 25B, cyclins D3 and E, and CDKs 3, 4, and 6. It was clear that the down-regulation of all these genes played an important role in the G1 blockade we observed in U87 cells. Genes of the origin recognition complex (ORC), such as ORC1L and CDC6, were suppressed. Also, several genes of the minichromosome maintenance (MCM) complex, including MCMs 2, 3, 4, 5, 6, and 7, and CDC7 were down-regulated. In addition, we saw suppression of several genes of the G2/M checkpoint, such as cyclins A, B1, and B2, polokinase 1, and CDKs 1 and 2,. PG and PN also affected genes encoding proteins necessary for spindle assembly, such as cohesion complex, securin, MAD2L1, BUB1, and CDC20. This would lead to disruption of mitosis. On the other hand, expression of tumor suppressors such as p21, p53, and GADD45A was activated. Exposure of U87 cells to PG alone caused fewer but significant changes in the expression of critical genes. Expression of GADD45A, p21/CDKN1A, and PPM1A was up-regulated. These genes are involved in the induction of cell cycle arrest and apoptosis. Based on pathway analysis, it was observed that anti-neoplastons affected the expression of more than 40 genes instrumental in the cell cycle in GBM cells. Anti-neoplastons may target multiple levels in the cell cycle and enhance the anti-cancer effect of tumor suppressor genes.