P03.07 * RESVERATROL ENHANCES RADIATION EFFECTS ON CLONOGENIC SURVIVAL IN HUMAN MEDULLOBLASTOMA CELL LINES

Abstract

BACKGROUND AND OBJECTIVE: In recent years, the natural plant polyphenol resveratrol (RV) attracted an increasing attention in tumor therapy. Currently, clinical trials for colon cancer and multiple myeloma have been completed. RV has to been shown to exhibit tumor preventive as well as anticancer effects. These are of particular interest for the therapy of pediatric medulloblastoma (MB), as low-risk MB patients (5-year survival up to 95 %) require a therapy focused on the reduction of late adverse effects (i.e. neurological and endocrine disorders), whereas patients belonging to the high-risk group (i.e. such owning MYC amplification; 5-year survival 13 %) urgently need a more intensive therapy to improve survival. In the present study, we analyzed RV-mediated changes on the clonogenic survival and proliferation of irradiated medulloblastoma cells. Furthermore, we investigated if RV may induce double-strand breaks (DSB), and if combined RV/irradiation (IR) treatment affects the number of potential tumor stem cells (TSC). METHODS: Three MB cell lines were treated with RV doses ranged from 9.5 µM to 40 µM (based on preliminary experiments examining metabolic activity or clonogenic survival of each cell line) for three or six days. 2 Gy IR was applied immediately before RV (3 d treatment) or after three days (6 d treatment). Clonogenic assays were conducted at the end of treatment time. DSB were measured by γH2AX assay 1 h and 24 h after IR or RV. To quantify potential TSC, we detected vital CD15+ and CD133+ cells by flow cytometry three days after IR and RV treatment. Effects on proliferation were measured flow cytometrically in CSFE-labeled cells. RESULTS: The post-treatment of irradiated MB cells with RV for three days significantly decreased the clonogenic survival 1.8-fold (DAOY) to 36-fold (MEB-Med8a). The longer RV treatment period reached stronger effects decreasing the clonogenic survival by up to 900-fold in irradiated D283-Med cells. Both, IR and RV alone, induced DSB. An increase of residual, unrepaired IR-induced DSB was observed after RV treatment in two-out-of-three MB cell lines (DAOY, MEB-Med8a). Examining the number of potential TSC, we revealed an enrichment of CD15+ and CD133+ cells after RV or IR alone, which was slightly enhanced by combination of both. We found no differences in proliferation rates between TSC and non-TSC. Therefore, we assume that the enrichment of TSC is caused by a higher treatment resistance of potential TSC compared to non-TSC. CONCLUSION: RV may enhance the radiation effects on clonogenic survival of human MB cells, possibly through an increased induction of additional DSB. Further studies in animal MB models are warranted to clarify if the observed in vitro effects translate into improved survival rates in vivo.