59-Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspaseindependent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation.

Malignant glioma cells maintain an elevated intracellular pH (pH i ) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH i of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na + -dependent calcium efflux by isoform 1.1 of the sodium calcium exchanger (NCX1.1) , which increases [Ca 2+ ] i and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na + ] i ) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca 2+ ] i ) also are increased 5-fold. 2′, 4′-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1uM, while dually inhibiting both NHE1 and NCX1.1 at ≥20uM. DCB (1uM) was not cytotoxic to glioma cells, while DCB (20μM) further increased basal elevated levels of [Ca 2+ ] i in glioma cells that was followed by cell demise. Cariporide and © 2010 Elsevier B.V. All rights reserved. * To whom correspondence should be sent (fagorin@ucdavis.edu).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest:Fredric Gorin, Michael Nantz, and the University of California have filed patents pertaining to the use of amiloride and novel derivatives as potential anti-cancer agents. Fredric Gorin is CEO of D3G, which contracts with publically owned and private labs to perform pre-clinical drug discovery, as it pertains to advance the treatment of primary and metastatic cancers affecting the central nervous system (brain and spinal cord). SEA0400 are more specific inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase-activation. NIH Public Access

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