In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid, modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB) cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16 alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined by using MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to the addition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showed little effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHA enhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHA and VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signaling pathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in marked suppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, and MAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part by the down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that brain tumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.

Medulloblastoma (MB) is the most common brain tumor in children and often spread to the spinal cord though the cerebrospinal fluid. Understanding the biological mechanism of this disease would provide a better care for these patients. Recently, overexpression of platelet derived growth factor receptor (PDGFR) alpha and beta has been linked to a metastatic stage of the disease. To comprehensively analyze the functions of PDGFRs and find their potential downstream targets in MB, we utilized Daoy cells, a metastatic MB cell line, as a MB model to assess the roles of PDGFR alpha and beta in this disease. The expression levels of PDGFR alpha and beta were determined by real time PCR and immunobloting. It was shown that Daoy cells express both PDGFR alpha and PDGFR beta. By using siRNAs to knockdown PDGFR alpha or beta specifically, and neutralizing antibody specific to PDGFR alpha and PDGFR beta respectively, it was demonstrated that PDGFR alpha signaling hinders cell proliferation and cell migration/invasion while PDGFR beta signaling promotes cell proliferation and migration/invasion. To elucidate the mechanism and find their potential downstream targets, total RNAs from the PDGFR knockdown cells were further analyzed by real time PCR and our results showed that PDGFR alpha and PDGFR beta signaling differentially modulate a set of genes which are important for cell death/surviving and proliferation such as NF kappa B, ATF3, Txnip and cMYC. To elucidate which pathway might govern the different roles of PDGFR alpha and beta in tumor cell proliferation/surviving, an NF kappa B report plasmid was co-transfected with siRNAs of PDGFRs into the MB cells and then the NF kappa B activities in the co-transfected cells were determined using a luciferase assay system. It was demonstrated that the NF kappa B activity was largely reduced in PDGFR beta knockdown cells, which suggested that PDGFR alpha and PDGFR beta may balance the cell proliferation/surviving signaling via cMYC and NF kappa B pathway. Furthermore, by analyzing the expression of cell surface protein on the PDGFR knockdown cells using immunohistochemistry staining, we demonstrated that CD44, a hall marker for cell migration/invasion was down regulated by PDGFR beta knockdown but not by PDGFR alpha knockdown, which implicated PDGFRs balance their migration/invasion signaling via CD44. Taken together, these data suggest that PDGFR alpha and PDGFR beta play different roles in cell proliferation and migration/invasion in MB brain tumor cells and these studies also highlight the potential of targeting PDGFRs signaling therapy in MB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 288.