The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.

Effects of vigorous isometric muscle contraction on titin stiffness-related contractile properties in rat fast-twitch muscles

This study was conducted to examine the effects of an acute bout of vigorous isometric contractions on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ until the force was reduced to ~50% of the initial force. Immediately after cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. The stimulation resulted in a decrease in the titin-based passive force. The amounts of fragmented titin were unchanged by the stimulation. Protein kinase Cα-treatment increased the passive force in stimulated fibers to resting levels. The stimulation had no effect on the maximum Ca2+-activated force (max Ca2+ force) at a sarcomere length (SL) of 2.4 μm and decreased myofibrillar (my)-Ca2+ sensitivity at 2.6-μm SL. Stretching the SL to 3.0 μm led to the augmentation of the max Ca2+ force and my-Ca2+ sensitivity in both rested and stimulated fibers. For the max Ca2+ force, the extent of the increase was smaller in stimulated than in rested fibers, whereas for my-Ca2+ sensitivity, it was higher in stimulated than in rested fibers. These results suggest that vigorous isometric contractions decrease the titin-based passive force, possibly because of a reduction in phosphorylation by protein kinase Cα, and that the decreased titin stiffness may contribute, at least in part, to muscle fatigue.

Identification of Activated Protein Kinase Cα (PKCα) in the Urine of Orthotopic Bladder Cancer Xenograft Model as a Potential Biomarker for the Diagnosis of Bladder Cancer

Bladder cancer has a high recurrence rate; therefore, frequent and effective monitoring is essential for disease management. Cystoscopy is considered the gold standard for the diagnosis and continuous monitoring of bladder cancer. However, cystoscopy is invasive and relatively expensive. Thus, there is a need for non-invasive, relatively inexpensive urinary biomarker-based diagnoses of bladder cancer. This study aimed to investigate the presence of activated protein kinase Cα (PKCα) in urine samples and the possibility of PKCα as a urinary biomarker for bladder cancer diagnosis. Activated PKCα was found to be present at higher levels in bladder cancer tissues than in normal bladder tissues. Furthermore, high levels of activated PKCα were observed in urine samples collected from orthotopic xenograft mice carrying human bladder cancer cells compared to urine samples from normal mice. These results suggest that activated PKCα can be used as a urinary biomarker to diagnose bladder cancer. To the best of our knowledge, this is the first report describing the presence of activated PKCα in the urine of orthotopic xenograft mice.

Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA

Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs) causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients virtually every PMEC is infected, however the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase, RhoA, in primary human PMECs causing VE-Cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA, but co-precipitates RhoGDI (Rho GDP-dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C-terminus (69-204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N-terminus (1-69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and VEGF increase RhoA induced PMEC permeability by directing Protein Kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing Protein Kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV induced capillary permeability, edema and HPS. Importance HPS causing hantaviruses infect pulmonary endothelial cells causing vascular leakage, pulmonary edema and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses don't lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34 phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC phosphorylated RhoGDI, synergistically enhancing hypoxia directed RhoA activation and PMEC permeability. Our data suggests inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.

Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA

Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs) causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients virtually every PMEC is infected, however the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase, RhoA, in primary human PMECs causing VE-Cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA, but co-precipitates RhoGDI (Rho GDP-dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C-terminus (69-204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N-terminus (1-69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and VEGF increase RhoA induced PMEC permeability by directing Protein Kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing Protein Kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV induced capillary permeability, edema and HPS.ImportanceHPS causing hantaviruses infect pulmonary endothelial cells causing vascular leakage, pulmonary edema and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses don’t lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34 phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC phosphorylated RhoGDI, synergistically enhancing hypoxia directed RhoA activation and PMEC permeability. Our data suggests inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.