Abstract
Glioblastoma Multiforme is the most aggressive type of brain tumour, accounting for 13.9% of all brain tumours and is associated with a poor 5-year survival rate. The current standard of care involves an initial debulking surgery, followed by treatment with the alkylating agent temozolomide alongside radiation. Vitamin C (ascorbate) is taken up by sodium-dependant vitamin C transporters, SVCT1 and SVCT2. The function of ascorbate involves antioxidant as well as prooxidant effects intra- and extracellularly, respectively. This project aimed to characterise two CRISPR/Cas knockout glioblastoma cell lines (U87 3.19 and U251 1.8) harbouring a non-functional SVCT2, and to compare them to their two parental cell lines (U87-MG and U251-MG). Cells were tested for cell authenticity (STR profiling), ascorbate uptake (HPLC-ECD), cell growth (cell count), viability (MTT assay), life/death assays (ImageXpress Pico), and DNA damage response (γH2AX by western blotting). Statistics were based on three biological repeats using the student’s t-test and ANOVA.
The knockout cell lines were authentic and showed no difference in growth behaviour compared to their parental cells. U87 3.19 and U251 1.8 showed a significant reduction in ascorbate uptake (p = 0.02 and 0.03, respectively) compared to their parental cells. Upon exposure to a range of ascorbate concentrations (0.2 - 4 mM), cell viability assays showed no significant difference in metabolic activity between the knockout and parental cells. Live/death analysis showed reduced growth for all cells, and cell death at 4 mM ascorbate for all except U251 1.8 cells. Temozolomide exposure (0.001 - 0.5 mM) had limited effect on cell death across all cell lines. Upon exposure to both agents, ascorbate and temozolomide toxicity appeared additive. The DNA damage response to ascorbate was complex. At baseline, U87 3.19 cells had significantly reduced levels of H2AX phosphorylation compared to their parental cells (p < 0.05). When exposed to 2 mM of ascorbate, U251 1.8 cells showed significantly increased levels of γH2AX (p = 0.04 (2 h)). U87 3.19 cells showed a significant reduction in γH2AX over time (p = 0.01), whereas U251 1.8 cells, exposed to 0.5 mM of ascorbate, increased γH2AX over time (p< 0.05). This data shows no evidence that ascorbate hindered temozolomide toxicity in vitro. Our results give valuable insights into the effects of intra- and extracellular ascorbate. Further research is required to determine the impact of these findings on the clinical use of ascorbate.