P04.48 Role of the expression of protein kinase D1 and its phosphorylated forms in glioma-derived cell line exposed to modified isothiourea derivative ZKK-3 combined with hyperbaric oxygen therapy

Abstract

Glioblastoma (GBM) is the most malignant primary tumour of the central nervous system. Its standard treatment includes surgery, radiotherapy and chemotherapy using temozolomide. Despite this, therapies still seems to be insufficient and median survival of the patients with GBM is less than 16 months. One of the main factors involved in glioma progression is the high level of tumour hypoxia, which contributes also to its resistance to treatment. In our previous studies we demonstrated that combination of hyperbaric oxygenation with novel pentabromobenzylisothiourea ZKK-3 resulted in an improvement of GBM cells oxygenation status and the reduction of their proliferation and viability in vitro. There is a high structural similarity between isothioureas and known casein kinase 2 (CK2) inhibitors. Nevertheless, ZKK-3 does not suppress CK2 expression, while it inhibits other protein kinases, including protein kinase D1 (PKD1), which promotes tumour growth and mediates detoxification of mitochondrial reactive oxygen species. The aim of this study was to assess the changes of the expression of PKD1 and its phosphorylated forms - pPKD1 (Ser 916) and pPKD1 (Ser 744/748), in glioma-derived cells treated with ZKK-3 under various oxygen conditions in vitro. Studies were performed on human glioblastoma T98G cell line (ATCC). Cells were cultured in MEM medium supplemented with N,N’-dimethyl-S-(2,3,4,5,6-pentabromobenzyl)-isothiouronium bromide (ZKK-3) in concentrations of 10 μM, 25 μM and 50 μM. Cultures were sustained in various oxygen conditions: normoxia, hypoxia, HBO, double hypoxia, hypoxia/HBO for 24 hour and then lysed with RIPA Lysis Buffer System (Santa Cruz). Using Western Blot protocol and appropriate primary and secondary antibodies, levels of tested protein kinases were determined. As the loading control β-actin was used. Administration of ZKK-3 did not affect PKD1 expression in glioblastoma cell line. Nevertheless, treatment with 50 µM of selected agent resulted in significant changes in the level of phosphorylated form of tested protein: diminution of pPKD1 (Ser 916) level and elevation of pPKD1 (Ser 744/748) level. Exposition of T98G cells to various oxygen conditions only slightly altered the expression of PKD1 and its phosphorylated forms in comparison to standard conditions, in both control and ZKK-3-treated groups, but observed differences were generally statistically insignificant. Obtained results suggested that PKD1-inhibiting properties of ZKK-3 were related with blocking the phosphorylation of that kinase at Ser 916. Unchanged expression of PKD1 as well as its phosphorylated forms under the influence of different oxygen conditions allowed to exclude PKD1 as a key protein for antitumour properties of ZKK-3/HBO combination strategy. The research was supported by KNOW-MMRC project.