K63-linked polyubiquitination of transcription factor IRF1 is essential for IL-1-induced production of chemokines CXCL10 and CCL5.

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) and the kinase that produces it have been implicated in inflammatory bowel diseases in mice and humans; however, little is known about the role of the 2 S1P-specific phosphohydrolase isoforms, SGPP1 and SGPP2, which catalyze dephosphorylation of S1P to sphingosine. To elucidate their functions, we generated specific knockout mice. Deletion of Sgpp2, which is mainly expressed in the gastrointestinal tract, significantly reduced dextran sodium sulfate (DSS)-induced colitis severity, whereas deletion of ubiquitously expressed Sgpp1 slightly worsened colitis. Moreover, Sgpp1 deletion enhanced expression of multifunctional proinflammatory cytokines, IL-6, TNF-α, and IL-1ß, activation of the transcription factor signal transducer and activator of transcription 3, and immune cell infiltration into the colon. Conversely, Sgpp2-null mice failed to mount a DSS-induced systemic inflammatory response. Of interest, Sgpp2 deficiency suppressed DSS-induced intestinal epithelial cell apoptosis and improved mucosal barrier integrity. Furthermore, down-regulation of Sgpp2 attenuated LPS-induced paracellular permeability in cultured cells and enhanced expression of the adherens junction protein E-cadherin. Finally, in patients with ulcerative colitis, SGPP2 expression was elevated in colitis tissues relative to that in uninvolved tissues. These results indicate that induction of SGPP2 expression contributes to the pathogenesis of colitis by promoting disruption of the mucosal barrier function. SGPP2 may represent a novel therapeutic target in inflammatory bowel disease.-Huang, W.-C., Liang, J., Nagahashi, M., Avni, D., Yamada, A., Maceyka, M., Wolen, A. R., Kordula, T., Milstien, S., Takabe, K., Oravecz, T., Spiegel, S. Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

Interstitial Fluid Sphingosine-1-Phosphate in Murine Mammary Gland and Cancer and Human Breast Tissue and Cancer Determined by Novel Methods.

The tumor microenvironment is a determining factor for cancer biology and progression. Sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphKs), is a bioactive lipid mediator that regulates processes important for cancer progression. Despite its critical roles, the levels of S1P in interstitial fluid (IF), an important component of the tumor microenvironment, have never previously been measured due to a lack of efficient methods for collecting and quantifying IF. The purpose of this study is to clarify the levels of S1P in the IF from murine mammary glands and its tumors utilizing our novel methods. We developed an improved centrifugation method to collect IF. Sphingolipids in IF, blood, and tissue samples were measured by mass spectrometry. In mice with a deletion of SphK1, but not SphK2, levels of S1P in IF from the mammary glands were greatly attenuated. Levels of S1P in IF from mammary tumors were reduced when tumor growth was suppressed by oral administration of FTY720/fingolimod. Importantly, sphingosine, dihydro-sphingosine, and S1P levels, but not dihydro-S1P, were significantly higher in human breast tumor tissue IF than in the normal breast tissue IF. To our knowledge, this is the first reported S1P IF measurement in murine normal mammary glands and mammary tumors, as well as in human patients with breast cancer. S1P tumor IF measurement illuminates new aspects of the role of S1P in the tumor microenvironment.

Aberrant ORM (yeast)-like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice.

BACKGROUND: Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known. OBJECTIVES: We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. METHODS: We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice. RESULTS: Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice. CONCLUSIONS: Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma.

Role of sphingosine kinase 1 and sphingosine-1-phosphate in CD40 signaling and IgE class switching.

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen-presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40-mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40-mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF-κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1(-/-) mice, isotype switching to antigen-specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40-mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen-specific IgE production.

Development of a metastatic murine colon cancer model.

BACKGROUND: It has now become clear that the complex interplay of cancer and the immune responses against it plays a critical role in the tumor microenvironment during cancer progression. As new targets for cancer treatment are being discovered and investigated, murine models used for preclinical studies need to include intact immune responses to provide a closer correlation with human cancer. We have recently developed a modified syngeneic orthotopic murine colon cancer model that mimics human colon cancer progression with consistent results. MATERIALS AND METHODS: Tumors were created using the murine colon adenocarcinoma cell line, CT26, modified to overexpress the firefly luciferase gene (CT26-luc1), which allowed real-time in vivo monitoring of tumor burden when the substrate, D-luciferin, was injected intraperitoneally using the In Vivo Imaging System. Mice are Balb/c (Harlan), syngeneic with the CT26-luc1 cells. Cells are injected submucosally, suspended in Matrigel, into the cecum wall under direct visualization. RESULTS: The model has demonstrated consistent implantation in the cecum. In vivo bioluminescence allowed real-time monitoring of total tumor burden. Perioperative preparation had a significant impact on reproducibility of the model. Finally, total tumor burden quantified with bioluminescence enabled estimation of lymph node metastasis ex vivo. CONCLUSIONS: This method maintains an intact immune response and closely approximates the clinical tumor microenvironment. It is expected to provide an invaluable murine metastatic colon cancer model particularly in preclinical studies for drug development targeting those mechanisms.

Morphology, molecular stacking, dynamics and device performance correlations of vacuum-deposited small-molecule organic solar cells.

The "all carbon" organic solar cells (OSCs) based on the homocyclic molecule tetraphenyldibenzoperiflanthene (DBP) as a donor and C60 as an acceptor were comprehensively characterized. The optimized planar-mixed heterojunction device with a DBP:C60 mixture ratio of DBP : C60 (1 : 2) exhibited a power conversion efficiency of 4.47%. To understand why DBP possesses such advantageous characteristics, the correlations of the morphology, molecular stacking, carrier dynamics and performance of DBP:fullerene-based devices have been systematically studied. First, the face-on stacked DBP molecules could enhance both the absorption of light and the charge carrier mobility. Second, DBP : C60 (1 : 2) thin films with optimized domain sizes and partially interconnected acceptor grains led to the most balanced carrier mobility and the lowest bimolecular recombination in devices. Finally, the DBP molecules were found to stack closely using grazing incidence wide-angle X-ray scattering measurements, with a π-π stacking spacing of 4.58 Å, indicating an effective molecular orbital overlap in DBP. The study not only reveals the promising characteristics of DBP as a donor in OSCs but the clear correlations of the thin-film nano-morphology, molecular stacking, carrier mobility and charge recombination found here could also provide insights into the characterization methodology and optimization of the small molecule OSCs.

Glycogen synthase kinase-3 facilitates con a-induced IFN-γ-- mediated immune hepatic injury.

Immune hepatic injury induced by Con A results primarily from IFN-γ-mediated inflammation, followed by hepatic cell death. Glycogen synthase kinase (GSK)-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. We hypothesized a pathogenic role for GSK-3 in Con A hepatic injury. Con A stimulation caused GSK-3 activation in the livers of C57BL/6 mice. Inhibiting GSK-3 reduced Con A hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of adhesion molecule CD54. Con A induced hepatic injury in an IFN-γ receptor 1-dependent manner. Con A/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced inducible NO synthase, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased Con A-induced IFN-γ production in both wild-type and IFN-γ receptor 1-deficient C57BL/6 mice. In Con A-activated NKT cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. These results demonstrate the dual and indispensable role of GSK-3 in Con A hepatic injury by facilitating IFN-γ-induced hepatopathy.

Unrevealed roles of extracellular enolase­1 (ENO1) in promoting glycolysis and pro­cancer activities in multiple myeloma via hypoxia­inducible factor 1α.

The involvement of enolase­1 (ENO1), intracellularly or extracellularly, has been implicated in cancer development. Moreover, anticancer activities of an ENO1­targeting antibody has demonstrated the pathological roles of extracellular ENO1 (surface or secreted forms). However, although ENO1 was first identified as a glycolytic enzyme in the cytosol, to the best of our knowledge, extracellular ENO1 has not been implicated in glycolysis thus far. In the present study, the effects of extracellular ENO1 on glycolysis and other related pro­cancer activities were investigated in multiple myeloma (MM) cells in vitro and in vivo. Knockdown of ENO1 expression reduced lactate production, cell viability, cell migration and surface ENO1 expression in MM cells. Notably, addition of extracellular ENO1 protein in cancer cell culture enhanced glycolytic activity, hypoxia­inducible factor 1­α (HIF­1α) expression, glycolysis­related gene (GRG) expression and pro­cancer activities, such as cell migration, cell viability and tumor­promoting cytokine secretion. Consistently, these extracellular ENO1­induced cellular effects were inhibited by an ENO1­specific monoclonal antibody (mAb). In addition, extracellular ENO1­mediated glycolysis, GRG expression and pro­cancer activities were also reduced by HIF­1α silencing. Lastly, administration of an ENO1 mAb reduced tumor growth and serum lactate levels in an MM xenograft model. These results suggested that extracellular ENO1 (surface or secreted forms) enhanced a HIF­1α­mediated glycolytic pathway, in addition to its already identified roles. Therefore, the results of the present study highlighted the therapeutic potential of ENO1­specific antibodies in treating MM, possibly via glycolysis inhibition, and warrant further studies in other types of cancer.

Association between recently raised anticholinergic burden and risk of acute cardiovascular events: nationwide case-case-time-control study.

OBJECTIVE: To evaluate the association between recently raised anticholinergic burden and risk of acute cardiovascular events in older adults. DESIGN: Case-case-time-control study (ie, incorporating a case crossover design and a control crossover design consisting of future cases). SETTING: Taiwan's National Health Insurance Research Database. PARTICIPANTS: 317 446 adults aged ≥65 who were admitted to hospital because of an incident acute cardiovascular event between 2011 and 2018. Acute cardiovascular events included myocardial infarction, strokes, arrhythmias, conduction disorders, and cardiovascular death. MAIN OUTCOME MEASURES: The anticholinergic burden was measured for each participant by adding up the anticholinergic scores for individual drugs using the Anticholinergic Cognitive Burden Scale. Scores were classified into three levels (0 points, 1-2 points, and ≥3 points). For each participant, anticholinergic burden levels during hazard periods (day -1 to -30 before the cardiovascular event) were compared with randomly selected 30 day reference periods (ie, periods between days -61 and -180). Conditional logistic regression determined odds ratios with 95% confidence intervals to evaluate the association between acute cardiovascular events and recently raised anticholinergic burden. RESULTS: The crossover analyses included 248 579 current cases. Participants' average age on the index date was 78.4 years (standard deviation 0.01), and 53.4% were men. The most frequently prescribed drugs with anticholinergic activity were antihistamines (68.9%), gastrointestinal antispasmodics (40.9%), and diuretics (33.8%). Among patients with varying levels of anticholinergic burden in different periods, more patients carried higher levels of anticholinergic burden during hazard periods than during reference periods. For example, 17 603 current cases had 1-2 points of anticholinergic burden in the hazard period with 0 points in the reference period, while 8507 current cases had 0 points in the hazard period and 1-2 points in the reference period. In the comparison of 1-2 points versus 0 points of anticholinergic burden, the odds ratio was 1.86 (95% confidence interval 1.83 to 1.90) in the case crossover analysis and 1.35 (1.33 to 1.38) in the control crossover analysis, which yielded a case-case-time-control odds ratio of 1.38 (1.34 to 1.42). Similar results were found in the comparison of ≥3 versus 0 points (2.03, 1.98 to 2.09) and ≥3 versus 1-2 points (1.48, 1.44 to 1.52). The findings remained consistent throughout a series of sensitivity analyses (eg, cut-off points for anticholinergic burden categories were redefined and different scales were used to measure anticholinergic burden). CONCLUSIONS: An association was found between recently raised anticholinergic burden and increased risk of acute cardiovascular events. Furthermore, a greater increase in anticholinergic burden was associated with a higher risk of acute cardiovascular events.

Glycogen synthase kinase-3ß is critical for interferon-α-induced serotonin uptake in human Jurkat T cells.

Dysregulation of glycogen synthase kinase (GSK)-3ß contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3ß regulation on IFN-α-induced 5-HTT functions. GSK-3ß short hairpin RNAs (shRNAs) or GSK-3ß inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3ß (Tyr216), which regulated 5-HT uptake. GSK-3ß knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3ß cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3ß activation and GSK-3ß-regulated 5-HT uptake. GSK-3ß signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3ß cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

Albumin prevents reactive oxygen species-induced mitochondrial damage, autophagy, and apoptosis during serum starvation.

Aberrant levels of reactive oxygen species (ROS) rapidly generated from NADPH oxidase (NOX) activation can be cytotoxic due to activating pro-apoptotic signals. However, ROS also induce pro-survival autophagy through the engulfment of damaged mitochondria. This study is aimed at investigating the cytoprotective role of albumin against NOX/ROS-induced autophagy and apoptosis under serum starvation. Serum starvation induced apoptosis following a myeloid cell leukemia sequence 1 (Mcl-1)/Bax imbalance, loss of the mitochondrial transmembrane potential, and caspase activation accompanied by pro-survival autophagy following canonical inhibition of mammalian target of rapamycin complex 1 (mTORC1). Aberrant ROS generation, initially occurring through NOX, facilitated mitochondrial damage, autophagy, and apoptosis. Autophagy additionally regulated the accumulation of ROS-generating mitochondria. NOX/ROS permitted p38 mitogen-activated protein kinase (p38 MAPK)-regulated mitochondrial apoptosis, accompanied by non-canonical induction of autophagy. In addition, activation of glycogen synthase kinase (GSK)-3ß by NOX/ROS-inactivated Akt facilitated a decrease in Mcl-1, followed by mitochondrial apoptosis as well as autophagy. Restoring albumin conferred an anti-oxidative effect against serum starvation-deregulated NOX, p38 MAPK, and Akt/GSK-3ß/Mcl-1/caspase-3 signaling. Albumin also prevented autophagy by sustaining mTORC1. These results indicate an anti-oxidative role for albumin via preventing NOX/ROS-mediated mitochondrial signaling to stimulate apoptosis as well as autophagy. Autophagy, initially induced by canonical inhibition of mTORC1 and enhanced by non-canonical mitochondrial damage, acts physically as a pro-survival mechanism.

Inhibiting glycogen synthase kinase-3 decreases 12-O-tetradecanoylphorbol-13-acetate-induced interferon-γ-mediated skin inflammation.

Glycogen synthase kinase-3 (GSK-3) facilitates interferon (IFN)-γ signaling. Because IFN-γ is involved in inflammatory skin diseases, such as psoriasis, the aim of this study was to investigate the pathogenic role of GSK-3 in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IFN-γ-mediated ear skin inflammation. TPA (3 µg per ear) induced acute skin inflammation in the ears of C57BL/6 mice, including edema, infiltration of granulocytes but not T cells, and IFN-γ receptor 1-mediated deregulation of intercellular adhesion molecule 1 (CD54). TPA/IFN-γ induced GSK-3 activation, which in turn activated signal transducer and activator of transcription 1. Inhibiting GSK-3 pharmacologically, by administering 6-bromoindirubin-3'-oxime (1.5 µg per ear), and genetically, with lentiviral-based short-hairpin RNA, reduced TPA-induced acute skin inflammation but not T-cell infiltration. It is noteworthy that inhibiting GSK-3 decreased TPA-induced IFN-γ production and the nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, in CD3-positive T cells. In chronic TPA-induced skin inflammation, inhibiting GSK-3 attenuated epidermis hyperproliferation and dermis angiogenesis. These results demonstrate the dual role of GSK-3 in TPA-induced skin inflammation that is not only to facilitate IFN-γ signaling but also to regulate IFN-γ production. Inhibiting GSK-3 may be a potential treatment strategy for preventing such effects.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.

Anesthetic propofol causes glycogen synthase kinase-3ß-regulated lysosomal/mitochondrial apoptosis in macrophages.

BACKGROUND: Overdose propofol treatment with a prolong time causes injury to multiple cell types; however, its molecular mechanisms remain unclear. Activation of glycogen synthase kinase (GSK)-3ß is proapoptotic under death stimuli. The authors therefore hypothesize that propofol overdose induces macrophage apoptosis through GSK-3ß. METHODS: Phagocytic analysis by uptake of Staphylococcus aureus showed the effects of propofol overdose on murine macrophages RAW264.7 and BV2 and primary human neutrophils in vitro. The authors further investigated cell apoptosis in vitro and in vivo, lysosomal membrane permeabilization, and the loss of mitochondrial transmembrane potential (MTP) by propidium iodide, annexin V, acridine orange, and rhodamine 123 staining, respectively. Protein analysis identified activation of apoptotic signals, and pharmacologic inhibition and genetic knockdown using lentiviral-based short hairpin RNA were further used to clarify their roles. RESULTS: A high dose of propofol caused phagocytic inhibition and apoptosis in vitro for 24 h (25 µg/ml, in triplicate) and in vivo for 6 h (10 mg/kg/h, n = 5 for each group). Propofol induced lysosomal membrane permeabilization and MTP loss while stabilizing MTP and inhibiting caspase protected cells from mitochondrial apoptosis. Lysosomal cathepsin B was required for propofol-induced lysosomal membrane permeabilization, MTP loss, and apoptosis. Propofol decreased antiapoptotic Bcl-2 family proteins and then caused proapoptotic Bcl-2-associated X protein (Bax) activation. Propofol-activated GSK-3ß and inhibiting GSK-3ß prevented Mcl-1 destabilization, MTP loss, and lysosomal/mitochondrial apoptosis. Forced expression of Mcl-1 prevented the apoptotic effects of propofol. Decreased Akt was important for GSK-3ß activation caused by propofol. CONCLUSIONS: These results suggest an essential role of GSK-3ß in propofol-induced lysosomal/mitochondrial apoptosis.

A Novel Enolase-1 Antibody Targets Multiple Interacting Players in the Tumor Microenvironment of Advanced Prostate Cancer.

Prostate cancer is one of the most common causes of cancer death in men worldwide, and the treatment options are limited for patients with advanced stages of prostate cancer. Upon oncogenic or inflammatory stimulation, tumor cells or immune cells express cell surface enolase-1 (ENO1) as plasminogen receptor to facilitate their migration via plasmin activation. Little is known about the roles of ENO1 in prostate cancer, especially in the tumor microenvironment (TME). We hypothesized that targeting surface ENO1 with specific mAbs would exert multifactorial therapeutic potentials against prostate cancer. In vivo, we showed ENO1 mAb (HuL227) reduced the growth of subcutaneous PC-3 xenograft, monocytes recruitment, and intratumoral angiogenesis. In a PC-3 intratibial implantation model, HuL227 reduced tumor growth and osteoclast activation in the bone. To investigate the antitumor mechanism of ENO1 mAb, we found that blocking surface ENO1 significantly reduced VEGF-A-induced tube formation of endothelial cells in vitro. Furthermore, HuL227 inhibited inflammation-enhanced osteoclasts activity and the secretion of invasion-related cytokines CCL2 and TGFß from osteoclasts. In addition, inflammation-induced migration and chemotaxis of androgen-independent prostate cancer cells were dose-dependently inhibited by HuL227. In summary, we showed that, ENO1 mAb targets multiple TME niches involved in prostate cancer progression and bone metastasis via a plasmin-related mechanism, which may provide a novel immunotherapy approach for men with advanced prostate cancer.

The Combined Effects of Dietary Diversity and Frailty on Mortality in Older Taiwanese People.

OBJECTIVE: To assess the prospective association between frailty and dietary diversity on mortality. METHOD: This prospective cohort study used the 2005-2008 Nutrition and Health Survey in Taiwan (N = 330; age ≥ 65 years) and this was linked to the Death Registry where we used the data that was recorded up to 31 January 2020. Dietary intake information was assessed using a 24-h dietary recall and food-frequency questionnaire, which were calculated a dietary diversity score (DDS; range, 0-6) and food consumption frequency. Assessment of frailty phenotypes was based on FRAIL scale which was proposed by the International Academy on Nutrition and Aging. RESULTS: Frail older adults had a higher risk of all-cause mortality when they were compared to those with robust physiologies (hazard ratio [HR]: 3.73, 95% confidence interval [CI]: 2.13-6.52). Frailty and a lower DDS were associated with a higher risk of mortality (joint adjusted HR: 2.30, 95% CI: 1.11-4.75) which, compared with a robust physiology and higher DDS, were associated with a lower risk of mortality. CONCLUSIONS: Frailty and a lower DDS were associated with a higher mortality. Prefrailty and frailty with a higher DDS were associated with a lower risk of mortality when compared with those with prefrailty and frailty and a lower DDS. These results suggest that eating a wide variety of foods might reduce the risk of mortality in older adults with prefrailty and frailty.

Autophagy facilitates IFN-gamma-induced Jak2-STAT1 activation and cellular inflammation.

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.