Killing Glioma 'Stem-like' Cells via Drug-Induced Relocation of Endosomal Urokinase Proteins.

High grade gliomas (HGGs) are primary CNS cancers with more than 95% of patients experiencing tumor recurrence following radiation therapy, chemotherapy, and/or an anti-angiogenic therapy. Populations of glioma 'stem-like' cells (GSCs) exist in both proliferative and non-proliferative states and are capable of tumor regrowth. These GSCs survive within hypoxic tumor regions and avascular tumor margins, while retaining the capability to regenerate. Successful treatment of HGGs depends on therapeutic targeting of GSCs to avert tumor regeneration. Here, we review novel intracellular mechanisms by which 3-amino-5-arylamino-6-chloro-N-(diaminomethylene) pyrazine-2-carboximide (UCD38B) and the much more potent 5'-substituted arylamino compounds (cmpd 10357) irreversibly kill GSCs utilizing caspase-independent, programmed necrotic cell death. Drug-induced relocation of a subset of endosomes to perinuclear mitochondria triggers the mitochondrial release and nuclear translocation of apoptosis inducible factor (AIF) that is followed by nuclear condensation and cancer cell demise. This drug-induced endosomal 'mis-trafficking' affects a subset of endosomes containing proteins belonging to the urokinase plasminogen activator system (uPAS) and guided by lipoprotein receptor protein type 1 (LRP-1). UCD38B and congeners act intracellularly and bind to intracellular urokinase plasminogen activator (uPA) to disrupt uPA binding to PAI-1 and the endosomal LRP-1 guidance protein. These small molecules are cytotoxic to persistently hypoxic and acidotic HGG cell lines and to high grade gliomas from patient derived xenografts (PDX). Immunodeficient mice with intracerebral PDX glial tumors demonstrate drug-specific, AIF- mediated necrosis after 24h of treatment. The propensity of these small molecules to kill non-proliferating and proliferating hypoxic GSCs, suggests a potential synergistic therapeutic role with radiotherapy, anti-mitotic and anti-angiogenic therapies.

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.

A novel carboline derivative inhibits nitric oxide formation in macrophages independent of effects on tumor necrosis factor α and interleukin-1ß expression.

Neuropathic pain is a maladaptive immune response to peripheral nerve injury that causes a chronic painful condition refractory to most analgesics. Nitric oxide (NO), which is produced by nitric oxide synthases (NOSs), has been implicated as a key factor in the pathogenesis of neuropathic pain. ß-Carbolines are a large group of natural and synthetic indole alkaloids, some of which block activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), a predominant transcriptional regulator of NOS expression. Here, we characterize the inhibitory effects of a novel 6-chloro-8-(glycinyl)-amino-ß-carboline (8-Gly carb) on NO formation and NF-κB activation in macrophages. 8-Gly carb was significantly more potent than the NOS inhibitor NG-nitro-L-arginine methyl ester in inhibiting constitutive and inducible NO formation in primary rat macrophages. 8-Gly carb interfered with NF-κB-mediated gene expression in differentiated THP1-XBlue cells, a human NF-κB reporter macrophage cell line, but only at concentrations severalfold higher than needed to significantly inhibit NO production. 8-Gly carb also had no effect on tumor necrosis factor α (TNFα)-induced phosphorylation of the p38 mitogen-activated protein kinase in differentiated THP1 cells, and did not inhibit lipopolysaccharide- or TNFα-stimulated expression of TNFα and interleukin-1ß. These data demonstrate that relative to other carbolines and pharmacologic inhibitors of NOS, 8-Gly carb exhibits a unique pharmacological profile by inhibiting constitutive and inducible NO formation independent of NF-κB activation and cytokine expression. Thus, this novel carboline derivative holds promise as a parent compound, leading to therapeutic agents that prevent the development of neuropathic pain mediated by macrophage-derived NO without interfering with cytokine expression required for neural recovery following peripheral nerve injury.

A cell-permeant amiloride derivative induces caspase-independent, AIF-mediated programmed necrotic death of breast cancer cells.

Amiloride is a potassium-sparing diuretic that has been used as an anti-kaliuretic for the chronic management of hypertension and heart failure. Several studies have identified a potential anti-cancer role for amiloride, however the mechanisms underlying its anti-tumor effects remain to be fully delineated. Our group previously demonstrated that amiloride triggers caspase-independent cytotoxic cell death in human glioblastoma cell lines but not in primary astrocytes. To delineate the cellular mechanisms underlying amiloride's anti-cancer cytotoxicity, cell permeant and cell impermeant derivatives of amiloride were synthesized that exhibit markedly different potencies in cancer cell death assays. Here we compare the cytotoxicities of 5-benzylglycinyl amiloride (UCD38B) and its free acid 5-glycinyl amiloride (UCD74A) toward human breast cancer cells. UCD74A exhibits poor cell permeability and has very little cytotoxic activity, while UCD38B is cell permeant and induces the caspase-independent death of proliferating and non-proliferating breast cancer cells. UCD38B treatment of human breast cancer cells promotes autophagy reflected in LC3 conversion, and induces the dramatic swelling of the endoplasmic reticulum, however these events do not appear to be the cause of cell death. Surprisingly, UCD38B but not UCD74A induces efficient AIF translocation from the mitochondria to the nucleus, and AIF function is necessary for the efficient induction of cancer cell death. Our observations indicate that UCD38B induces programmed necrosis through AIF translocation, and suggest that its cytosolic accessibility may facilitate drug action.

5-Benzylglycinyl-amiloride kills proliferating and nonproliferating malignant glioma cells through caspase-independent necroptosis mediated by apoptosis-inducing factor.

5'-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, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation.

2-Amidino analogs of glycine-amiloride conjugates: inhibitors of urokinase-type plasminogen activator.

The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC(50) ranging from 3 to 7 µM and were cytotoxic to human U87 malignant glioma cells.

Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function.

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.

The role of the cysteine residue in the chaperone and anti-apoptotic functions of human Hsp27.

The small heat shock protein Hsp27 is a molecular chaperone and an anti-apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti-apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27(C137A)) and comparing it to wild-type protein (Hsp27(WT)). Both proteins were multi-subunit oligomers, but subunits of Hsp27(WT) were disulfide-linked unlike those of Hsp27(C137A), which were monomeric. Hsp27(C137A) was indistinguishable from Hsp27(WT) with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S-thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27(WT). The anti-apoptotic function of Hsp27(C137A) and Hsp27(WT) was studied by overexpressing them in CHO cells. No difference in the caspase-3 or -9 activity was observed in staurosporine-treated cells. The rate of apoptosis between Hsp27(C137A) and Hsp27(WT) overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild-type protein in preventing caspase-3 and caspase-9 activation and apoptosis induced by 1 mM H(2)O(2) in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti-apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti-apoptotic function against oxidative stress.

The small chromatin-binding protein p8 coordinates the association of anti-proliferative and pro-myogenic proteins at the myogenin promoter.

Quiescent muscle progenitors called satellite cells persist in adult skeletal muscle and, upon injury to muscle, re-enter the cell cycle and either undergo self-renewal or differentiate to regenerate lost myofibers. Using synchronized cultures of C2C12 myoblasts to model these divergent programs, we show that p8 (also known as Nupr1), a G1-induced gene, negatively regulates the cell cycle and promotes myogenic differentiation. p8 is a small chromatin protein related to the high mobility group (HMG) family of architectural factors and binds to histone acetyltransferase p300 (p300, also known as CBP). We confirm this interaction and show that p300-dependent events (Myc expression, global histone acetylation and post-translational acetylation of the myogenic regulator MyoD) are all affected in p8-knockdown myoblasts, correlating with repression of MyoD target-gene expression and severely defective differentiation. We report two new partners for p8 that support a role in muscle-specific gene regulation: p68 (Ddx5), an RNA helicase reported to bind both p300 and MyoD, and MyoD itself. We show that, similar to MyoD and p300, p8 and p68 are located at the myogenin promoter, and that knockdown of p8 compromises chromatin association of all four proteins. Thus, p8 represents a new node in a chromatin regulatory network that coordinates myogenic differentiation with cell-cycle exit.

Glyoxalase I activity and immunoreactivity in the aging human lens.

Glyoxalase I (GLOI) is the first enzyme of the glyoxalase system that catalyzes the metabolism of reactive dicarbonyls, such as methylglyoxal (MGO). During aging and cataract development, human lens proteins are chemically modified by MGO, which is likely due to inadequate metabolism of MGO by the glyoxalase system. In this study, we have determined the effect of aging on GLOI activity and the immunoreactivity and morphological distribution of GLOI in the human lens. A monoclonal antibody was developed against human GLOI. GLOI immunoreactivity was strongest in the anterior epithelial cells and weaker in rest of the lens. Cultured human lens epithelial cells showed immunostaining throughout the cytoplasm. In the human lens, GLOI activity and immunoreactivity both decreased with age. We believe that this would lead to promotion of MGO-modification in aging lens proteins.

Role of cysteine residues in the enhancement of chaperone function in methylglyoxal-modified human alpha A-crystallin.

We have previously demonstrated that the reaction of a physiological dicarbonyl, methylglyoxal (MGO) enhances the chaperone function of human alpha A-crystallin. MGO can react with cysteine, arginine, and lysine residues in proteins. Although the role of arginine and lysine residues in the enhancement of chaperone function has been investigated, the role of cysteine residues is yet to be determined. In this study, we have investigated the effect of MGO modification on the structure and chaperone function of alpha A-crystallin mutant proteins in which C131 and C142 were replaced either individually or simultaneously with isoleucine. MGO-modification resulted in improved chaperone function in all three alpha A-crystallin mutants, including the cysteine-free double mutant. The enhanced chaperone function was due to increased surface hydrophobicity and increased binding of client proteins. These results suggest that the two cysteine residues, even though they could be modified, do not take part in the MGO-induced improvement in the chaperone function of human alpha A-crystallin.