PP46. THE PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR GAMMA AGONIST PIOGLITAZONE REDUCES ELECTRICALLY DIRECTED GLIOBLASTOMA MULTIFORME CELL MIGRATION

Abstract

AbstractINTRODUCTION: Glioblastoma Multiforme (GBM) is a malignancy characterised by diffuse infiltration of brain matter with heterogeneous populations of differentiated and cancer stem cells. Electrical fields (EF) guided migration (electrotaxis) has been shown to occur in GBM cells and neural stem cells, with the implicated pathways frequently dysregulated in GBM. One such pathway is the EGFR/PI3k/AKt pathway, which is downregulated by peroxisome proliferator activated receptor gamma (PPARγ) agonists. We investigated the effect of EFs on GBM differentiated and stem cell migration, and whether this was affected by treatment with the PPARγ agonist pioglitazone. METHODS: The primary GBM cell line HROG02 was cultured as a differentiated (HROG02-Diff) and stem cell line (HROG02-GSC). Immunocytochemistry was performed, using CD133, Nestin, GFAP and Sox-2 primary antibodies. Electrotactic assays exposing cells to 0, 50, 100 and 200mV/mm for 3 hours were completed and recorded using time-lapse microscopy to determine cell migration parameters. Further 200mV/mm electrotactic experiments were completed following 12–24 hour treatments of either 15µM pioglitazone + 5µMDMSO (pioglitazone), 20µM DMSO (DMSO), or 15µM pioglitazone + 5µM GW9662 (Pioglitazone/GW9662) (PPARγ antagonist) treatments. Drug concentrations were guided by XTT viability assay. Western blots were performed for PPARγ expression with and without an EF. All p values for migratory data represent one-way ANOVA, and western blot; T-test. RESULTS: Immunocytochemistry confirmed differentiated and stem cell phenotypes of HROG02-Diff and HROG02-GSC. With an increase in exogenous EF, more HROG02-Diff cells migrated towards the anode; 47% with no EF, to 66%, 81% and 84% at 50, 100 and 200mV/mm respectively. Directedness of this anodal migration was significantly increased (p<0.0001). Velocity and accumulated distance also increased with EF applied (p<0.0001), whilst Euclidean distance was highest at 100mV/mm (p=0.0033). In comparison, HROG02-GSC migrated with increased directedness towards the cathode with increasing EF strength (p<0.0001), as well as increased velocity and accumulated distance with increased EF applied (p<0.0001); however there was no significance difference in Euclidean distance (p=0.4136). Pioglitazone treatment resulted in decreased anodal directedness compared to DMSO and pioglitazone/GW9662 (p=0.0280). There was no significant difference in velocity, accumulated distance or Euclidean distance (p=0.3592, p=0.3592 and p=0.6430 respectively). HROG02-GSC cells treated with pioglitazone showed a statistically significant decrease in cathodally directed migration (p=0.0002). There was no significant difference in velocity, accumulated distance or Euclidean distance (p=0.4484, p=4484, p=0.4927 respectively). Western blots showed trends for increased and decreased expression of PPARγ after EF exposure in HROG02-Diff and HROG02-GSC respectively, however this was not statistically significant due to high variability (p=0.2101 and p=0.4301). CONCLUSION: Here we demonstrate for the first time opposing migratory responses to an EF in differentiated and stem cell GBM cells. The presence of endogenous EFs within the brain may mean electrotaxis of GBM cells occurs in vivo; potentially implicated in disease recurrence. Furthermore, pioglitazone decreases directedness in both cell lines, and is recoverable with GW9662 treatment suggesting a PPARγ dependent pathway. Therefore, PPARγ agonists may offer a novel therapeutic approach in mitigating GBM recurrence, warranting further investigation.