High grade astrocytomas (, malignant gliomas) are the most commonly occurring type of lethal adult brain-tumor with an individual's average life expectancy being less than 2 years from the time of diagnosis. Neither radiation therapy nor chemotherapy has significantly improved quality or length of survival. Since Warburg's initial observation, it has been recognized that most transformed tumor cells have high rates of glycolytic metabolism and consequent H+ production. Given the optimal alkaline pH dependence of key glycolytic enzymes, such as phosphofructokinase and hexokinase, it is essential that tumor cells employ an effective means of removing free cytosolic H+ to maintain metabolism. We have determined that intracellular pH in rat and human gliomas are significantly above that of normal astrocytes (0.2-0.6 pH units) despite the tumor's high rates of metabolic H+ production. This intracellular alkalosis appears to result from persistent activation of the type 1 isoform of the Na+-H+ exchanger (NHE 1). Our preliminary investigations have determined that this altered regulation of NHE 1 is most probably posttranslational and does not result from alterations of the NHE1 gene or proteins expressed in these highly malignant astrocytomas. Unexpectedly, we found that inhibition of NHE I in rat and human glioma cell lines with the diuretic drug, amiloride, or with its derivatives, HOE 694 and EIPA, cause a 70-100 percent cell death within 48-72 hours. By, contrast, primary astrocyte cultures were unaffected by NIHE 1 inhibition. Cell culture and in vivo analyses indicate that glioma death following NHE 1 inhibition appears to be predominantly non-apoptotic and independent of preceding caspase activation. Rat C6 gliomas were implanted into rat brains and allowed to establish for 4 days. Amiloride infusion into the cerebrospinal fluid for 8 days produced a 73 percent reduction in tumor volume. Amiloride is an oral diuretic that is approved for human use. Preliminarily, this novel intrathecal administration of amiloride in rats does not appear to cause behavioral or neuropathological alterations. Amiloride produced a dose-dependent decrease in intracellular pH in malignant gliomas, but not astrocytes. We have pilot data indicating that this ApHi initiates the selective tumor death. We propose to (1) identify the intracellular mechanisms mediating glioma death; (2) use brain implanted tumor models to more thoroughly delineate selective glioma death by NIlE I inhibitors; and (3) study the pharmacological and H+ regulatory properties of surviving gliomas. NHE I inhibitors may represent a new class of pharmacological agents that are useful for treatment of these highly aggressive and lethal brain tumors.
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