GTP Cyclohydrolase I Confers Metabolic Protection Against Lipid Peroxidation in Glioblastoma
Guanosine triphosphate cyclohydrolase I (GCH1) was previously identified as a regulator of reactive oxygen species (ROS) in brain tumor initiating cells (BTICs). Elucidating the GCH1/Tetrahydrobiopterin (BH4) pathway in BTICs has unveiled the necessity for GCH1 to protect BTICs from lipid peroxidation. Within this study, we observed differences in lipid pathways and lipid metabolites during GCH1 modulation. Alteration to polyunsaturated fatty acids (PUFAs), a species of oxidatively vulnerable lipids, were altered during GCH1 knockdown. Pharmacological inhibition of BH4 with drugs known to cross the blood brain barrier (sulfasalazine and pyrimethamine) successfully reduced growth in GCH1 overexpressed BTICs, induced lipid peroxidation. Overall design: GBM 456 were genetically modified to overexpress (OE) GCH1 or knocked down (KD) with GCH1 short hairpin (Sh) RNA. Empty vector (EV) and nontargeting controls (NT) were utilized throughout the study.  Cells were transiently co-transfected with psPAX2, pCMV-VSVG and shRNA constructs using FuGENE® HD Transfection Reagent (Promega) as previously reported(67). Virus titer was determined using Lenti-X qRT-PCR Titration Kit (Takara). Lentivirus pLenti6.3/V5 GCH1 (HsCD00941202) was purchased from DNASU while pLenti6.3 EV was purchased from Addgene. For lentivirus expressing GCH1 shRNAs (TRCN0000050744 and TRCN0000050745) and non-targeting control shRNA were purchased from Sigma. Both shRNA constructs were designed against the GCH1 coding sequence. The shRNA sequences were as follows: GCH1 Sh4:5'CCATATTGGTTATCTTCCT'3' and GCH1 Sh5: 5'GATGTCCTAAACGATGCTA3'