LAUSR

Abstract Introduction: Glioblastoma (GBM), the primary brain tumour derived from astroglial cells, is the most frequently diagnosed intracranial malignancy in adults. Poor prognosis for GBM patients results from high invasiveness of neoplastic cells related with faulty angiogenesis and persistent hypoxia states. In such conditions glioma cells develop radio- and chemotherapy-resistant phenotype. It is postulated that application of hyperbaric oxygen (HBO) may improve therapeutic response via refinement of tumour tissue oxygenation. The more effective therapeutic strategies are also required. One of the novel compounds that exhibit anti-tumour properties are pentabromobenzylisothioureas. The aim of this study was to examine whether HBO may enhance anti-tumour efficacy of selected pentabromobenzylisothiourea when both agents were administered as combined ZKK-3/HBO therapy. Materials and Methods: Human glioblastoma T98G cell line was cultured in medium supplemented with novel isothiourea derivative - ZKK-3. The proliferation and viability of glioma cells cultured in hypoxia or hyperbaric oxygen conditions were examined. Cell proliferation was tested 24 hours after ZKK-3 addition using Nikon Inverted Microscope and Multisizer 3 Beckman Coulter. The viability assay was taken 24 and 48 hours post ZKK-3 supplementation by CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega). Hypoxia state of T98G cells was determined via the evaluation of HIF-1α protein expression using HIF-1A ELISA Kit (Thermo Scientific). Tests were performed on cells treated with ZKK-3 and cultured in various oxygen conditions: 1/ normoxia (24 hours), 2/ hypoxia (24 hours), 3/ HBO (2 ATA, 1 hour followed by 23 hours of normoxia), 4/ double hypoxia, 5/ hypoxia/HBO. Results: Proliferation of T98G cells treated with ZKK-3 was significantly decreased after hyperbaric oxygenation in comparison to hypoxia conditions. Administration of HBO resulted also in statistically significant diminution of glioma cells’ viability. Expression of HIF-1α in T98G cells strongly depended on oxygen conditions: in hypoxia and double hypoxia the level of protein was significantly elevated compared with normoxia, while after hyperbaric oxygenation no changes or even decrease of HIF-1α level was observed. Comparison of double hypoxia and hypoxia/HBO groups showed that HBO addition after ZKK-3 supplementation caused significant reduction of HIF-1α expression. Conclusions: Treatment of malignant glioma cells with selected isothiourea derivative is significantly more efficient when combined with hyperbaric oxygen administration. Beneficial effect of HBO on decreasing HIF-1α expression allows to consider hyperbaric oxygenation as the way to overcome tumour hypoxia.ACKNOWLEDGEMENT: The research was supported by the KNOW-MMRC project and Foundation for the Development of Diagnostic and Therapy.