5-Benzylglycinyl-Amiloride Kills Proliferating and Nonproliferating Malignant Glioma Cells through Caspase-Independent Necroptosis Mediated by Apoptosis-Inducing Factor

Abstract

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.

Mis-Trafficking of Endosomal Urokinase Proteins Triggers Drug-Induced Glioma Nonapoptotic Cell Death

“…AIF-mediated glioma necrosis produced by UCD38B is independent of canonical apoptosis Pasupuleti et al, 2013) and appears to be distinct from ripoptosomemediated necroptosis (Feoktistova et al, 2012). RIP-1 inhibition by necrostatin does not alter the anticancer cytotoxicity of UCD38B .…”

“…types Pasupuleti et al, 2013). Previously, we demonstrated that UCD38B is a cell-permeant molecule that rapidly initiates a set of intracellular events causing mitochondrial depolarization with the release and nuclear translocation of AIF.…”

“…Previously, we demonstrated that UCD38B is a cell-permeant molecule that rapidly initiates a set of intracellular events causing mitochondrial depolarization with the release and nuclear translocation of AIF. Nuclear AIF mediates irreversible programmed necrotic cancer cell death, independent of caspase and poly ADP-ribose polymerase activation Pasupuleti et al, 2013). The cell-impermeant congener of UCD38B, 5-glycinylamiloride (UCD74A), is a comparably competitive uPA inhibitor, inhibits cancer cell migration, and is cytostatic but not cytotoxic (Table 1) (Massey et al, 2012;Pasupuleti et al, 2013).…”

“…Nuclear AIF mediates irreversible programmed necrotic cancer cell death, independent of caspase and poly ADP-ribose polymerase activation Pasupuleti et al, 2013). The cell-impermeant congener of UCD38B, 5-glycinylamiloride (UCD74A), is a comparably competitive uPA inhibitor, inhibits cancer cell migration, and is cytostatic but not cytotoxic (Table 1) (Massey et al, 2012;Pasupuleti et al, 2013). Our initial reports describing the anticancer cytotoxicity of UCD38B against proliferating and nonproliferating well characterized human glioblastoma and human breast cancer cell lines produced several unanswered questions about its cellular mechanisms, which are better understood as a consequence of this investigation.…”

“…This unusual drug mechanism of action explains why the antiglioma cytotoxicity of UCD38B was identified as being independent of the cell cycle in human glioma and breast cancer cell lines Pasupuleti et al, 2013) and appears to be restricted to cancer cell types having altered uPAS expression, notably uPA and PAI-1.…”