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

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 caspase-independent 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

Amiloride kills malignant glioma cells independent of its inhibition of the sodium-hydrogen exchanger. J Pharmacol Exp Ther 2004;310:67–74

ABSTRACT Previously, we demonstrated that malignant glioma cell lines have increased intracellular pH (pH i ) as a result of increased activities of the type I sodium/hydrogen exchanger (NHE1). This alkalotic pH i of 7.2 to 7.4 is favorable for augmented glycolysis, DNA synthesis, and cell cycle progression. Conversely, reductions in pH i have been associated with reduced rates of proliferation in transformed cell types. The effects of reducing pH i directly and by NHE1 inhibition on human malignant glioma cells were systematically compared with those on primary rat astrocytes. Neither cariporide, nor direct acidification to pH i 6.9 altered the proliferative rates or viabilities of human U87 or U118 malignant glioma cell lines. However, amiloride significantly impaired glioma cell proliferation and viability while not affecting astrocytes at concentrations (500 M) that exceeded its inhibition of NHE1 in glioma cells (IC 50 ϭ 17 M). Preventing a reduction of pH i did not alter the drug's antiproliferative and cytotoxic effects on glioma cells. These findings indicated that amiloride's cytotoxic effects on glioma cells are independent of its ability to inhibit NHE1 or to reduce intracellular pH i . The amiloride derivative 2,4 dichlorobenzamil (DCB) inhibits the sodium-calcium exchanger (NCX) and was both antiproliferative and cytotoxic to glioma cells at low doses (20 M). preferentially blocks sodiumdependent calcium influx by NCX (reverse mode) and was nontoxic to glioma cells. It is proposed that DCB (20 M) and amiloride (500 M) impair calcium efflux by NCX, leading to elevations of intracellular calcium that initiate a morphologically necrotic, predominantly caspase-independent glioma cell death. High-grade malignant gliomas are the most common, lethal primary brain tumor in adults Our earlier investigation of four human and rat malignant glioma cell lines revealed an alkaline intracellular pH (pH i of 7.31-7.48) compared with primary rat astrocytes (pH i of 6.98 Ϯ 0.01) Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.065029. ABBREVIATIONS: pH i , intracellular pH; NHE1, sodium hydrogen exchanger type 1; NCX, sodium calcium exchanger; DCB, 2,4 dichlorobenzamil; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PBS-CMF, phosphate-buffered saline-Ca 2ϩ and Mg 2ϩ free; TBS, Tris-buffered saline; BCECF, 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM; HR, HEPES Ringer; pH e , extracellular (buffer) pH; [Ca 2ϩ ] i , intracellular calcium concentration

Progress Toward Cariporide Analogs for Sodium-Proton Exchange Inhibition

The sodium proton exchanger (NHE) is particularly important in maintaining the intracellular pH in human heart and brain. Under anaerobic conditions (i.e., ischemia), a shift from oxidative to nonoxidative glycolysis occurs. The resultant decrease in the intracellular pH activates NHE, which increases the intracellular sodium, initiating the sequence of physiological events that lead to cell death. Thus, there has been great interest in the development of compounds that inhibit NHE. Indeed, potent NHE inhibitors are available. However, a fundamental impediment to the field is the delivery of these compounds to poorly vascularized tissues during the early phases of ischemia when NHE inhibition is most beneficial. We have synthesized analogs of cariporide, a potent (e.g., nanomolar IC50 activity) NHE inhibitor, to address these temporal and delivery challenges. The preparation and biological activities of our cariporide analogs will be discussed

Review of evidence implicating the plasminogen activator system in blood-brain barrier dysfunction associated with Alzheimer’s disease

Elucidating the pathogenic mechanisms of Alzheimer’s disease (AD) to identify therapeutic targets has been the focus of many decades of research. While deposition of extracellular amyloid-beta plaques and intraneuronal neurofibrillary tangles of hyperphosphorylated tau have historically been the two characteristic hallmarks of AD pathology, therapeutic strategies targeting these proteinopathies have not been successful in the clinics. Neuroinflammation has been gaining more attention as a therapeutic target because increasing evidence implicates neuroinflammation as a key factor in the early onset of AD disease progression. The peripheral immune response has emerged as an important contributor to the chronic neuroinflammation associated with AD pathophysiology. In this context, the plasminogen activator system (PAS), also referred to as the vasculature’s fibrinolytic system, is emerging as a potential factor in AD pathogenesis. Evolving evidence suggests that the PAS plays a role in linking chronic peripheral inflammatory conditions to neuroinflammation in the brain. While the PAS is better known for its peripheral functions, components of the PAS are expressed in the brain and have been demonstrated to alter neuroinflammation and blood-brain barrier (BBB) permeation. Here, we review plasmin-dependent and -independent mechanisms by which the PAS modulates the BBB in AD pathogenesis and discuss therapeutic implications of these observations

Review of evidence implicating the plasminogen activator system in blood-brain barrier dysfunction associated with Alzheimer's disease.

Elucidating the pathogenic mechanisms of Alzheimer's disease (AD) to identify therapeutic targets has been the focus of many decades of research. While deposition of extracellular amyloid-beta plaques and intraneuronal neurofibrillary tangles of hyperphosphorylated tau have historically been the two characteristic hallmarks of AD pathology, therapeutic strategies targeting these proteinopathies have not been successful in the clinics. Neuroinflammation has been gaining more attention as a therapeutic target because increasing evidence implicates neuroinflammation as a key factor in the early onset of AD disease progression. The peripheral immune response has emerged as an important contributor to the chronic neuroinflammation associated with AD pathophysiology. In this context, the plasminogen activator system (PAS), also referred to as the vasculature's fibrinolytic system, is emerging as a potential factor in AD pathogenesis. Evolving evidence suggests that the PAS plays a role in linking chronic peripheral inflammatory conditions to neuroinflammation in the brain. While the PAS is better known for its peripheral functions, components of the PAS are expressed in the brain and have been demonstrated to alter neuroinflammation and blood-brain barrier (BBB) permeation. Here, we review plasmin-dependent and -independent mechanisms by which the PAS modulates the BBB in AD pathogenesis and discuss therapeutic implications of these observations