PP60. EVALUATING THE EFFICACY OF CURRENT AND NOVEL CHEMOTHERAPEUTIC AGENTS FOR THE TREATMENT OF GLIOMA UNDER NORMOXIC AND HYPOXIC CONDITIONS
Authors: Mrs Joud Sabouni, Dr Tim Snape, Dr Gail Welsby, Dr Philip Welsby
Journal: Neuro-Oncology (2017)

INTRODUCTION: Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumour in adults. Use of the chemotherapeutic agents, temozolomide (TMZ) and cisplatin, is one of the standard practices in treatment of GBM alongside surgical resection and radiotherapy. However, GBM prognosis remains poor with a median survival time of less than 16 months. In addition to the therapeutic challenge of the BBB, hypoxia plays a central role in resistance and progression of GBM. However, studies on GBM are rarely undertaken in hypoxic conditions which are present in vivo and could contribute to resistance. Research has suggested a role for aspirin in the treatment or prevention of glioma. Several compounds with structural similarity to aspirin have been developed and tested to avoid the side-effects of aspirin with a recent focus on the novel aspirin analogue (PN517). METHODS: U-87-MG (Grade IV human glioblastoma) and SVG-p12 (human foetal astroglial) cell lines were maintained in EMEM in a humidified normoxic (95% air/5% CO2), and hypoxic (1% O2 / 5% CO2) atmosphere. Both cell lines were treated with the aspirin analogue PN517, aspirin, temozolomide, cisplatin or combinations of aspirin/PN517 with cisplatin or temozolomide at IC50 concentrations to examine synergism for 24, 48 and 72 hr under both normoxic and hypoxic conditions. Subsequently, cell viability was assessed by PrestoBlue® assay, and apoptosis was measured by annexin V/PI staining with flow cytometry. All experiments were performed in triplicate and results were expressed as mean ± SEM. A two-way ANOVA test was used to identify any statistical significance between treatments, with significance set at p<0.05. RESULTS: The IC50 for cisplatin was similar between U87-MG and SVG-p12 cells under normoxic conditions (3µM), however, it was different under hypoxic conditions (10µM). Both aspirin and its analogue PN517 induced a decrease in cell viability for both cell lines in a concentration- and time- dependent manner, and the IC50 were 3mM and 1mM in normoxia and hypoxia, respectively. PN517 was marginally more potent than aspirin and produced cytotoxic effect after 3 days of incubation of about 24% of cell viability. TMZ did not significantly reduce viability in either cell line at IC50 of 1mM in both conditions, with lowest efficacy observed under hypoxia. Mono-therapies reduced the U87MG cell viability to 38 ± 2% with cisplatin and 24 ± 2% with TMZ after three days of treatment under normoxia, whereas the combination of PN517 and cisplatin or TMZ led to enhanced reduction of viability to 18 ± 1% or 10 ± 1%, respectively. Similar results were found under hypoxia. Initial findings indicate that the percentage of U87MG apoptotic cells after 72hr was 9% with cisplatin and 17% with TMZ, whereas the combinatorial treatment with PN517 induced greater apoptosis (17% and 50%, respectively). CONCLUSION: These results show that the combination of aspirin or PN517 with cisplatin or TMZ may have therapeutic potential in GBM. Furthermore, the combinations were effective under hypoxia, an environment normally associate with chemotherapy resistance.