Abstract
5-benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anti-cancer small molecules that kill proliferative and non-proliferative 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 with mitochondrial depolarization with the release and nuclear translocation of apoptotis inducing factor (AIF) followed by irreversible, caspase-independent cell demise. High content quantification of immunocytochemical co-localization studies identified that UCD38B treatment increased endocytosis of 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, non-apoptotic cell death. Following UCD38B treatment the receptor guidance protein LRP-1, required for endosomal recycling of uPAR to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased 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 anti-cancer 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.
- Receptor synthesis/trafficking
- Fluorescence techniques
- Immunocytochemistry
- Structure/function/mechanism
- Apoptosis
- The American Society for Pharmacology and Experimental Therapeutics