gms | German Medical Science

Objective: We showed increased fatty acid synthase (FASN) expression level in glioblastoma (GBM) compared to astrocytoma WHO grade I to III. We revealed a dose and time dependent decrease in viability and proliferation rate of primary human GBM cell cultures under treatment of Orlistat. With regard to possible clinical use we investigated the effects of chronic exposure to low dose Orlistat (27.8µM) on viability, autophagy, and genes participating in fatty acid metabolism in GBM cell cultures.

Methods: Three primary human GBM cell cultures (G1145, G1273, G1319) were incubated with 24mM ethanol (vehicle control) or 27.8µM Orlistat for 96h in total. We analyzed cell viability by Prestoblue® staining after 0h, 24h, 48h, 72h, and 96h and induction of autophagy by monodansylcadaverine staining after 12h, 24h, and 48h. Furthermore we studied changes in expression levels of genes involved in fatty acid metabolism by quantitative PCR: ATP-citrate-lyase (ACLY), acetyl-CoA-carboxylase 1 (ACACA), FASN, and carnitine-palmitoyltransferase 1A (CPT1A).

Results: All results are in comparison to vehicle control. In all treated cultures we revealed a significant decrease of cell viability up to 79.0%, 96.6% and 253.2% after 96h (p=0.000) incubation with Orlistat with a positive significant correlation to time of exposure in G1319 (p=0.040, r=0.896). The higher the growth rate of the analyzed GBM cells, the more Orlistat affects cell viability negatively. In G1145 and G1273 Orlistat increased significantly autophagy up to 15.8% respectively 16.0% after 48h (p=0.000). The observed autophagic processes may represent an escape mechanism which follows fatty acid deprivation caused by low dose Orlistat. ACLY showed decreased (p=0.025), ACACA and FASN increased mRNA expression levels after 48h, and CPT1A decreased levels after 24h (p=0.004). A positive correlation exists between changes in expression level of ACLY and CPT1A (p=0.007, r=0.665) as well as FASN and CPT1A (p=0.013, r=0.625), both induced by Orlistat.

Conclusion: Orlistat influences the fatty acid metabolism in primary GBM cell cultures even at low concentration. To escape inhibition of Orlistat by binding at thioesterase of FASN, its mRNA is upregulated. This functional inhibition results in malonyl-CoA accumulation and palmitate deprivation so CPT1A for fatty acid transport is needed less. Malonyl-CoA upregulates FASN and inhibits ACLY. Therefore less acetyl-CoA is produced. Furthermore ACACA is upregulated to use the little acetyl-CoA as effectively as possible to synthesize malonyl-CoA which should be used by FASN to produce palmitate. Direct inhibition of FASN by Orlistat is able to down- and upregulate genes taking part in fatty acid metabolism but not in a constant manner. GBM cells may counter these metabolic changes so inhibition of fatty acid metabolism should be performed in more than one way in future.