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

Abstract

5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40-50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal "mis-trafficking" caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspaseindependent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4-to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA-PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death.

Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

“…We focused on mitochondria, which act as the control unit of the cell being involved in the cell response to a wide range of insults [34], showing that in HMA-treated colon cancer cells they appeared to be relocalised to the perinuclear region (as recently reported in breast cancer cells [9]) and functionally impaired due to mitochondrial membrane depolarization. This is in agreement with the observations obtained in erythroleukemia cells treated with 5-(N-ethyl-N-isopropyl) amiloride [35] and in glioma cells with 5-benzylglycinyl-amiloride [36] and with the well-known inhibition of mitochondrial complex I activity by amilorides [37]. …”

Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells

“…While some have identified specific perturbations in cell cycle progression and DNA integrity [35], the majority of studies have focused on plasma membrane associated ion and proton transporters [11,12,41], and/or uPA as the therapeutic targets (reviewed in [11]). Along these lines, a recent study suggested that UCD38B-induced necrosis depends upon mis-trafficking of endocytosed uPAR [15]. Specifically, the aberrant perinuclear accumulation of uPAR containing endosomes in close proximity to mitochondria may induce mitochondrial membrane depolarization and release of the pro-death factor AIF [15].…”

“…Along these lines, a recent study suggested that UCD38B-induced necrosis depends upon mis-trafficking of endocytosed uPAR [15]. Specifically, the aberrant perinuclear accumulation of uPAR containing endosomes in close proximity to mitochondria may induce mitochondrial membrane depolarization and release of the pro-death factor AIF [15]. While our data support mitochondrial dysfunction in mediating HMA associated cell death, evidenced by increased mitochondrial swelling, aggregation and ROS production, it is probable that endosomal trafficking may only be a consequence of amiloride derivative action and not required for cell death.…”

“…In both cases, UCD38B cytotoxicity was attributed to the induction of a caspase-independent, non-apoptotic cell death resembling programmed necrosis [13,14]. Further investigation suggested that UCD38B-induced cytotoxicity toward glioma cells correlates with the mis-trafficking of components of the uPA system following drug treatment [15]. Taken together, these data point to UCD38B as a potential treatment for refractory cancers.…”

Disruption of endolysosomal trafficking pathways in glioma cells by methuosis-inducing indole-based chalcones

“…In one example, the vacuole-inducing anti-cancer compound, 5-benzylglycinyl-amiloride, has been reported to cause necrotic cell death in glioblastoma cells by redirecting endosomes containing the LRP-1 receptor and its associated urokinase plasminogen activator system to perinuclear mitochondrial regions, where release of mitochondrial AIF (apoptosis-inducing factor) is triggered (Pasupuleti et al 2015). In another study, leelamine, a tricyclic diterpene that induces caspase-independent death in melanoma cells, was shown to impair endosomal trafficking and autophagic flux, with consequent accumulation of cholesterol in endosomal compartments and disruption of multiple pro-survival receptor tyrosine kinase signaling pathways (Kuzu et al 2014).…”