To maintain tissue homeostasis, apoptosis is functionally linked to the cell cycle through the retinoblastoma (Rb)/E2F pathway. When the Rb tumor suppressor protein is functionally inactivated, E2F1 elicits an apoptotic response through both intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) apoptotic pathways in order to eliminate hyperproliferative cells. Rb/E2F-associated apoptosis has been demonstrated to be associated with the loss of constitutive transcriptional repression by Rb/E2F complexes and mediated by caspase-8. Protein tyrosine phosphatases (PTPs) PTP-1B and SHP-2 have been previously shown to be directly activated by loss of Rb/E2F repression during Rb/E2F-associated apoptosis. In this current study, we demonstrate that the PTEN tumor suppressor is also directly activated by loss of Rb/E2F repression. We also demonstrate that PTP-1B, SHP-2, and PTEN play a functional role in Rb/E2F-associated apoptosis. Knockdown of PTP1B, SHP2, or PTEN expression with small interfering RNA (siRNA) in apoptotic cells increases cell viability and rescues cells from the Rb/E2F-associated apoptotic response. Furthermore, rescue from apoptosis coincides with inhibition of caspase-8 and caspase-3 cleavage (activation). Our results indicate PTP-1B, SHP-2, and PTEN all play a functional role in Rb/E2F-associated apoptotic signal transduction and provide further evidence that PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism.
Prostate cancer (PCa) is the major cause of cancer-related death in males; however, effective treatments to prevent aggressive progression remain an unmet need. We have previously demonstrated that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator that promotes PCa invasion. In the current study, we further investigate the therapeutic effects of an eNAMPT-neutralizing humanized monoclonal antibody (ALT-100 mAb) in preclinical PCa orthotopic xenograft models. We utilized human aggressive PCa cells (DU145 or PC3) for prostate implantation in SCID mice receiving weekly intraperitoneal injections of either ALT-100 mAb or IgG/PBS (control) for 12 weeks. Prostatic tumors and solid organs were examined for tumor growth, invasion, and metastasis and for biochemical and immunohistochemistry evidence of NFκB activation. ALT-100 mAb treatment significantly improved overall survival of SCID mice implanted with human PCa orthotopic prostate xenografts while inducing tumor necrosis, decreasing PCa proliferation and reducing local invasion and distal metastases. The ALT-100 mAb inhibits NFκB phosphorylation and signaling in PCa cells both in vitro and in vivo. This study demonstrates that eNAMPT neutralization effectively prevents human PCa aggressive progression in preclinical models, indicating its high potential to directly address the unmet need for an effective targeted therapy for patients with aggressive PCa.
Lipopolysaccharide (LPS), a product of gram‐negative bacilli, exerts its effect on the endothelium by inducing Toll‐like receptor 4 (TLR‐4)‐mediated release of pro‐inflammatory mediators, including barrier disruptive agents such as histamine and platelet activating factor (PAF) from dendritic cells and monocytes, as well as leading to generation of thrombin in fibrin clots. Many of these mediators signal through G‐protein coupled receptors (GPCR), which directly activate the Gαq/11 family of heterotrimeric G proteins. We propose that G αq/11 –mediated signaling in the endothelium plays a critical role in the development of pulmonary edema in the context of inflammation. To address this hypothesis we utilized mice with endothelial‐specific Gαq/Gα11 deficiency for an LPS inhalation model of lung inflammation and injury. Lungs isolated from control group demonstrated two fold increase in capillary filtration coefficient (Kf,c) value, a measure of vessel permeability, at five hours after LPS inhalation. Gαq/Gα11 deficiency significantly inhibited the increased permeability of lung vessels, suggesting that endothelial‐specific events downstream of Gαq/Gα11 activation play a critical role in the mechanism of lung edema and injury. In the future study we will determine whether Gαq/Gα11 signaling acts via PLCβ to induce acto‐myosin contractility and cell shape change in endothelial cells.
The heterotrimeric G protein α unit Gα13 transmits signals from G protein coupled receptors (GPCRs) to effectors to regulate cell spreading, differentiation, migration, and cell polarity. Both conventional and endothelial cell‐specific Gα13 gene knockouts are embryonically lethal because of impaired vasculogenesis. Here we describe a new pro‐inflammatory function of Gα13 through its displacement from the GPCR protease‐activated receptor 1 (PAR1) to the endothelial adherens junction (AJ) protein VE‐cadherin. H 2 O 2 production induced by multiple mediators (thrombin, LPS or TNFα) induced a shift in Gα13 binding to VE‐cadherin and disassembly of AJs. Gα13/VE‐cadherin interaction activated c‐Src resulting in phosphorylation of VE‐cadherin at Tyr 658, the p120‐ catenin binding site responsible for VE‐cadherin endocytosis from the membrane. Preventing Gα13/VE‐cadherin interaction by Cre recombinase‐mediated Gα13 knockout in mice or an interfering myristoylated peptide derived from VE‐cadherin, inhibited AJ disruption and inflammatory response to sepsis. These studies establish a crucial Gα13 switch mechanism activated by H 2 O 2 generation that mediates Gα13 binding to VE‐cadherin and thereby disrupts AJ integrity. Therefore, blocking Gα13 translocation and Gα13/VE‐cadherin interaction represents a potential anti‐inflammatory target.
BackgroundThere remains a serious need to prevent the progression of invasive prostate cancer (PCa). We previously showed that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator via TLR4 ligation which has been implicated in PCa progression. Here we investigate the role of eNAMPT as a diagnostic biomarker and therapeutic target in the progression of PCa.MethodsTumor NAMPT expression and plasma eNAMPT level were evaluated in human subjects with various PCa tumor stages and high risk subjects followed-up clinically for PCa. The genetic regulation of NAMPT expression in PCa cells and the role of eNAMPT in PCa invasion were investigated utilizing in vitro and in vivo models.FindingsMarked NAMPT expression was detected in human extraprostatic-invasive PCa tissues compared to minimal expression of organ-confined PCa. Plasma eNAMPT levels were significantly elevated in PCa subjects compared to male controls, and significantly greater in subjects with extraprostatic-invasive PCa compared to subjects with organ-confined PCa. Plasma eNAMPT levels showed significant predictive value for diagnosing PCa. NAMPT expression and eNAMPT secretion were highly upregulated in human PCa cells in response to hypoxia-inducible factors and EGF. In vitro cell culture and in vivo preclinical mouse model studies confirmed eNAMPT-mediated enhancement of PCa invasiveness into muscle tissues and dramatic attenuation of PCa invasion by weekly treatment with an eNAMPT-neutralizing polyclonal antibody.InterpretationThis study suggests that eNAMPT is a potential biomarker for PCa, especially invasive PCa. Neutralization of eNAMPT may be an effective therapeutic approach to prevent PCa invasion and progression.
Abstract Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.
Abstract Background The COVID‐19 pandemic exerted an unprecedented impact on the blood supply from 2020 through 2022. As a result, throughout 2021 there were months our hospital had less than one‐day supply of type O RBCs. To meet transfusion needs, whole RBC units were split into half units and issued to stable, non‐bleeding patients. This single‐institution, retrospective study examines time intervals to subsequent transfusion and total numbers of RBC units subsequently transfused after the first half or whole RBC unit. Study Design and Methods Patients who were transfused RBC between May 21, 2021 and November 1, 2021 were divided into in‐ and outpatient groups, then based on whether they received at least 1 half RBC unit or only whole RBC units during the study period. The time interval between this first half unit transfusion, or first whole unit transfusion in those who did not receive half units, and the subsequent RBC transfusion within 90 days was calculated and compared, as well as the total number of RBC units transfused 30 days after the first unit. Results In general, patients transfused with half units received a subsequent transfusion significantly earlier than those transfused with whole units. Additionally, receiving an index half unit was associated with more RBC transfusions in the following 30 days ( p = .001). Conclusion Transfusion of half RBC units during a severe RBC blood shortage can temporarily decrease RBC usage but will result in a shorter interval to the next transfusion and greater total number of RBC units transfused in subsequent days.
