Type 1 sodium hydrogen exchanger NHE1 in human glioblastoma cell lines: NHE1 inhibition results in selective glioblastoma cell death

The focus of this doctoral research was to determine whether genetic, transcriptional, or translational differences in the type 1 Na+/H + exchanger (NHE1) could account for the abnormal alkaline intracellular pH (pH_i) of four human glioma cell lines, and to determine whether the differences in pH regulation between the tumor and primary rat astrocytes could be used to kill glioma cells. Nucleotide sequencing of the NHE1 gene from the U87, U118, U251, and U373 human glioblastoma cell lines revealed no differences from previously published sequences. NHE1 mRNA levels from C6 rat glioma cells and the four human glioma cell lines did not correspond with the elevated pH; seen among the rat and human tumor cell lines. Immunoblots further established that there was no relationship between increased pH and amount of NHE1 protein present. Together, these results indicated that the increased steady state pH_i and altered pH regulation found in the glioma cell lines was more likely the result of changes in posttranslational regulation of either NHE1 or an NHE1 accessory protein. Ion transport studies determined that under CO₂/HCO₃−-free conditions the U118 glioma cell line was highly dependent upon NHE1 for pH regulation, whereas primary astrocytes were not. This dependence upon NHE1 was explored as a means of selectively killing the glioma cells. The diuretic drug amiloride, a known NHE 1 inhibitor, produced an immediate and prolonged acidification in U118 glioma cells that was not observed in similarly treated primary rat astrocytes. Within 24h of amiloride treatment, U118 cells showed caspase-3 activation and morphological signs of an apoptotic cell death. Following 48h of treatment, U118 cells showed caspase-mediated apoptotic cell death, as well as caspase-independent cell death. Primary rat astrocytes exhibited caspase activation and morphological signs of an apoptotic cell death only following 72h of amiloride treatment. These studies demonstrate that the abnormal steady-state alkaline pH; and tonic activation of NHE1 in U118 glioblastoma cells is most likely the result of regulation on a posttranslational level, and that NHE1 inhibitors can selectively acidify and kill glioma cells. Thus, NHE1 inhibitors may prove to be a useful adjuvant to current glioblastoma tumor therapy.