Alpha-Lipoic Acid Attenuates Cadmium- and Lead-Induced Neurotoxicity by Inhibiting Both Endoplasmic-Reticulum Stress and Activation of Fas/FasL and Mitochondrial Apoptotic Pathways in Rat Cerebral Cortex.

Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity.

Acetylsalicylic acid rescues the immunomodulation of inflamed gingiva-derived mesenchymal stem cells via upregulating FasL in mice.

BACKGROUND: Gingiva-derived mesenchymal stem cells (GMSCs) obtained multipotent differentiation and immunomodulatory properties. However, collecting healthy gingival tissues may be challenging in the clinical situation. Thus, in our present study, we aim to evaluate whether the immunomodulatory capacity of gingiva-derived mesenchymal stem cells from inflamed gingival tissues (iGMSCs) is impaired and find a way to rescue their deficient properties. METHODS: We compared the immunomodulation capacity of GMSCs and iGMSCs using an in vitro co-culture system and a mouse colitis model. T cell apoptosis, T helper 17 (Th17), and regulatory T (Treg) cell differentiation were detected by flow cytometry analysis. RESULTS: We demonstrated that iGMSCs obtained a decreased immunomodulatory capacity compared with GMSCs. Acetylsalicylic acid (ASA) pretreatment was able to rescue iGMSCs' impaired immunomodulatory properties. Mechanistically, ASA was capable of upregulating the expression of Fas ligand (FasL) in iGMSCs, leading to an improvement in iGMSC-mediated T cell apoptosis and therapeutic efficacy in the treatment in colitis mice. CONCLUSIONS: This study indicates that the deficient immunomodulatory function of iGMSCs could be rescued by ASA pretreatment via upregulating of FasL in mice. This strategy might serve as a practical approach to rescue deficient MSC function for further therapeutic application.

Safety and efficacy of the CD95-ligand inhibitor asunercept in transfusion-dependent patients with low and intermediate risk MDS.

In low risk MDS, increased apoptosis of erythroid progenitors mediated via CD95 (Fas) activation has been described to result in peripheral cytopenia. Blockade of the CD95 system can improve erythropoiesis in MDS. Asunercept (APG101) is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1 blocking the interaction between CD95 and its ligand. Here we report on results from a phase I study in 20 transfusion-dependent low and intermediate risk MDS patients treated with intravenous asunercept (EudraCT 2012-003027-37). Primary objectives were safety and tolerability as well as pharmacodynamic effects. Secondary objectives were hematologic improvement, incidence and time to leukemic progression as well as overall survival. Frequency and severity of adverse events were in range of what could be expected in a patient cohort comprising of elderly MDS patients. Two patients experienced a serious adverse event with a suspected relationship to asunercept. The incidence of disease progression was low. In the 20 patients a decrease of the transfusion need from a mean of 10,8 (±5,1) pRBCs during the 12 weeks treatment phase to a mean of 10,0 (±4,2) pRBCs at the end of the study was observed. In conclusion, asunercept was well tolerated and showed efficacy in transfusion-dependent low and intermediate risk MDS patients. Further clinical investigation is warranted, particularly in combination with erythropoiesis stimulating agents (ESAs).

Inhibition of CD95/CD95L (FAS/FASLG) Signaling with APG101 Prevents Invasion and Enhances Radiation Therapy for Glioblastoma.

CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by in vitro proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as in vivo therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both in vitro and in vivo, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells.Implications: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. Mol Cancer Res; 16(5); 767-76. ©2018 AACR.

Identification of DISE-inducing shRNAs by monitoring cellular responses.

Off-target effects (OTE) are an undesired side effect of RNA interference (RNAi) caused by partial complementarity between the targeting siRNA and mRNAs other than the gene to be silenced. The death receptor CD95 and its ligand CD95L contain multiple sequences that when expressed as either si- or shRNAs kill cancer cells through a defined OTE that targets critical survival genes. Death induced by survival gene elimination (DISE) is characterized by specific morphological changes such as elongated cell shapes, senescence-like enlarged cells, appearance of large intracellular vesicles, release of mitochondrial ROS followed by activation of caspase-2, and induction of a necrotic form of mitotic catastrophe. Using genome-wide shRNA lethality screens with eight different cancer cell lines, we recently identified 651 genes as critical for the survival of cancer cells. To determine whether the toxic shRNAs targeting these 651 genes contained shRNAs that kill cancer cell through DISE rather than by silencing their respective target genes, we tested all shRNAs in the TRC library derived from a subset of these genes targeting tumor suppressors (TS). We now report that only by monitoring the responses of cancer cells following expression of shRNAs derived from these putative TS it was possible to identify DISE-inducing shRNAs in five of the genes. These data indicate that DISE in general is not an undefined toxic response of cells caused by a random OTE but rather a specific cellular response with shared features that points at a specific biological function involving multiple genes in the genome.

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

Purpose: Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT1 and MT2) in 661W cells, then whether MEL can prevent H2O2-induced cell death, and last, through which pathway MEL confers protection. Methods: The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H2O2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H2O2-mediated cell death, a Fas/FasL antagonist was used before the exposure to H2O2. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H2O2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Results: Both MEL receptors (MT1 and MT2) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H2O2-mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H2O2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H2O2, and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H2O2. Conclusions: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H2O2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Resistance to cancer immunotherapy mediated by apoptosis of tumor-infiltrating lymphocytes.

Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy. TiRP tumors recruit and activate tumor-specific CD8+ T cells, but these cells then undergo apoptosis. This does not occur with isogenic transplanted tumors, which are rejected after adoptive T-cell therapy. Apoptosis of tumor-infiltrating lymphocytes can be prevented by interrupting the Fas/Fas-ligand axis, and is triggered by polymorphonuclear-myeloid-derived suppressor cells, which express high levels of Fas-ligand and are enriched in TiRP tumors. Blocking Fas-ligand increases the anti-tumor efficacy of adoptive T-cell therapy in TiRP tumors, and increases the efficacy of checkpoint blockade in transplanted tumors. Therefore, tumor-infiltrating lymphocytes apoptosis is a relevant mechanism of immunotherapy resistance, which could be blocked by interfering with the Fas/Fas-ligand pathway.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism.

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3'UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.

Pentoxifylline attenuates cytokine stress and Fas system in syngeneic liver proteins induced experimental autoimmune hepatitis.

BACKGROUND: Apoptosis is a hallmark in the pathogenesis of autoimmune hepatitis (AIH). Cytokine stresses and extrinsic apoptotic pathway have been implicated in this type of hepatic injury. Pentoxifylline plays an important role in controlling inflammation and apoptosis in different autoimmune diseases. AIM: To assess the protective effect of pentoxifylline for 30days against pro-inflammatory cytokines as tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ) and mediators of extrinsic apoptotic pathway involving TNF receptor 1 (TNFR1) and its ligand TNF-α and Fas receptor and its ligand (FasL) in experimental autoimmune hepatitis (EAH) model. METHODS: EAH was induced by intraperitoneal injection of syngeneic liver antigen emulsified in complete Freund's adjuvant (CFA) in male C57BL/6 mice. Five groups of mice were used: two control groups; Control PBS group and Control CFA group, EAH group and two EAH+pentoxifylline treated groups in doses (100 or 200mg/kg/d, given by oral gavage). Serum transaminase, pro-inflammatory cytokines (TNF-α and interferon-γ) and hepatic caspase-8 and 3 activities were evaluated. Signs of autoimmune hepatitis were confirmed by liver histology. In addition, hepatic TNFR1, Fas and FasL mRNA expression were assayed. RESULTS: Serum transaminase levels and signs of AIH observed in EAH mice were significantly reduced by pentoxifylline. Upregulated serum TNF-α, IFN-γ, hepatic caspase-8 and 3 activities and TNFR1, Fas and FasL mRNA expression in liver tissues in EAH group were significantly downregulated by pentoxifylline. CONCLUSION: Pentoxifylline protects against syngeneic liver antigen induced hepatitis and associating apoptosis through attenuating the exaggerated cytokine release and extrinsic apoptotic pathway. Thus, this may represent a new therapeutic strategy for hepatitis.

Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases.

TNF blockers are highly efficacious at dampening inflammation and reducing symptoms in rheumatic diseases such as rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis, and also in nonrheumatic syndromes such as inflammatory bowel disease. As TNF belongs to a superfamily of 19 structurally related proteins that have both proinflammatory and anti-inflammatory activity, reagents that disrupt the interaction between proinflammatory TNF family cytokines and their receptors, or agonize the anti-inflammatory receptors, are being considered for the treatment of rheumatic diseases. Biologic agents that block B cell activating factor (BAFF) and receptor activator of nuclear factor-κB ligand (RANKL) have been approved for the treatment of systemic lupus erythematosus and osteoporosis, respectively. In this Review, we focus on additional members of the TNF superfamily that could be relevant for the pathogenesis of rheumatic disease, including those that can strongly promote activity of immune cells or increase activity of tissue cells, as well as those that promote death pathways and might limit inflammation. We examine preclinical mouse and human data linking these molecules to the control of damage in the joints, muscle, bone or other tissues, and discuss their potential as targets for future therapy of rheumatic diseases.

Regulation of Intrinsic and Extrinsic Apoptotic Pathways in Osteosarcoma Cells Following Oleandrin Treatment.

Our previous study has reported the anti-tumor effect of oleandrin on osteosarcoma (OS) cells. In the current study, we mainly explored its potential regulation on intrinsic and extrinsic apoptotic pathway in OS cells. Cells apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using fluorescence staining and flow cytometry. Caspase-3 activity was detected using a commercial kit. The levels of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 were detected by Western blotting. z-VAD-fmk was applied to block both intrinsic and extrinsic apoptosis pathways, and cells apoptosis was also tested. Furthermore, we used z-LEHD-fmk and Fas blocking antibody to inhibit intrinsic and extrinsic pathways, separately, and the selectivity of oleandrin on these pathways was explored. Results showed that oleandrin induced the apoptosis of OS cells, which was accompanied by an increase in ROS and a decrease in MMP. Furthermore, cytochrome c level was reduced in mitochondria but elevated in the cytoplasm. Caspase-3 activity was enhanced by oleandrin in a concentration- and time-dependent manner. Oleandrin also down-regulated the expression of bcl-2, but up-regulated bax, caspase-9, Fas, FasL, caspase-8 and caspase-3. In addition, the suppression of both apoptotic pathways by z-VAD-fmk greatly reverted the oleandrin-induced apoptosis. Moreover, the suppression of one pathway by a corresponding inhibitor did not affect the regulation of oleandrin on another pathway. Taken together, we concluded that oleandrin induced apoptosis of OS cells via activating both intrinsic and extrinsic apoptotic pathways.

High-fluoride acitivates the FasL signalling pathway and leads to damage of ameloblast ultrastructure.

OBJECTIVE: High fluoride can induce stress-mediated apoptosis and degradation of ameloblasts. Fas ligand (FasL) has been regarded as a key regulator in intracellular responses for stress-induced apoptosis in reproductive or cancerous cell lineages. The objective of this study is to explore the role of FasL in the regulation of ameloblast ultrastructure damage. DESIGN: Primary ameloblasts were isolated from the molar tooth germ of 4-day-old SD rats. The ameloblasts were incubated with 3.2mM NaF or nothing. After incubation for different time arranging from 12h to 72h, ELISA was used to detected the secretion levels of FasL in the medium. Then at 48h post treatment, the ameloblast ultrastructure was detected with Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), and expression of apoptotic proteins and peroxidative enzymes/products were examined. Finally, a specific FasL inhibitor was applied to co-treat the ameloblasts with NaF, and the ameloblast ultrastructure was detected with TEM and SEM. RESULTS: The secretion of FasL was notably increased by 3.2mM NaF treatment, and the increase reached to the peak after incubation for 48h. High fluoride incubation damaged the ameloblast untrastructure manifesting a series of intracelluar stress responsing cell organelle destruction, and a marked increase in expression of apoptotic genes and oxidative stress. The FasL inhibitor treatment partially mitigated the untrastructure damage caused by high dose NaF. CONLUSION: High-fluoride leads to damage of the ameloblast ultrastructure through paritially acitivating the FasL signalling pathway.

Apoptotic CD8 T-lymphocytes disable macrophage-mediated immunity to Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi. CD8 T-lymphocytes help to control infection, but apoptosis of CD8 T cells disrupts immunity and efferocytosis can enhance parasite infection within macrophages. Here, we investigate how apoptosis of activated CD8 T cells affects M1 and M2 macrophage phenotypes. First, we found that CD8 T-lymphocytes and inflammatory monocytes/macrophages infiltrate peritoneum during acute T. cruzi infection. We show that treatment with anti-Fas ligand (FasL) prevents lymphocyte apoptosis, upregulates type-1 responses to parasite antigens, and reduces infection in macrophages cocultured with activated CD8 T cells. Anti-FasL skews mixed M1/M2 macrophage profiles into polarized M1 phenotype, both in vitro and following injection in infected mice. Moreover, inhibition of T-cell apoptosis induces a broad reprogramming of cytokine responses and improves macrophage-mediated immunity to T. cruzi. The results indicate that disposal of apoptotic CD8 T cells increases M2-macrophage differentiation and contributes to parasite persistence.

B cell-targeted immunotherapy for type 1 diabetes: What can make it work?

Immunotherapy has revolutionized treatment of cancers and autoimmune diseases. Bucking the trend, however, is type 1 diabetes (T1D), although it is one of best understood autoimmune diseases and individuals at genetic risk are identifiable with high certainty. Here we review the major obstacles associated with pan-B-cell-depletion using rituximab (RTX) and discuss the notion that B cell-directed therapy may be most effective as a preventive measure. We suggest that it will be more productive to aim at identifying and targeting autoreactive B cells rather than making adjustments to pan-B cell depletion and that non-conventional alternative therapies such as antibody blockade of FasL to bolster IL-10-producing Breg cells, which work successfully in mice, should be considered.

PDZ1 inhibitor peptide protects neurons against ischemia via inhibiting GluK2-PSD-95-module-mediated Fas signaling pathway.

Respecting the selective inhibition of peptides on protein-protein interactions, they might become potent methods in ischemic stroke therapy. In this study, we investigated the effect of PDZ1 inhibitor peptide on ischemic neuron apoptosis and the relative mechanism. Results showed that PDZ1 inhibitor peptide, which significantly disrupted GluK2-PSD-95 interaction, efficiently protected neuron from ischemia/reperfusion-induced apoptosis. Further, PDZ1 inhibited FasL expression, DISC assembly and activation of Caspase 8, Bid, Caspase 9 and Caspase 3 after global brain ischemia. Based on our previous report that GluK2-PSD-95 pathway increased FasL expression after global brain ischemia, the neuron protection effect of PDZ1 inhibitor peptide was considered to be achieved by disrupting GluK2-PSD-95 interaction and subsequently inhibiting FasL expression and Fas apoptosis pathway.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells.

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

Advanced glycation end-product (AGE) induces apoptosis in human retinal ARPE-19 cells via promoting mitochondrial dysfunction and activating the Fas-FasL signaling.

Advanced glycation end-products (AGEs) are extremely accumulated in the retinal vascular and epithelial cells of diabetes mellitus (DM) patients, particularly with diabetic retinopathy (DR). To elucidate the pathogenesis of the AGE-induced toxicity to retinal epithelial cells, we investigated the role of Fas-Fas ligand (FasL) signaling and mitochondrial dysfunction in the AGE-induced apoptosis. Results demonstrated that the AGE-BSA- induced apoptosis of retinal ARPE-19 cells. And the AGE-BSA treatment caused mitochondrial dysfunction, via deregulating the B-cell lymphoma 2 (Bcl-2) signaling. Moreover, the Fas/FasL and its downstreamer Caspase 8 were promoted by the AGE-BSA treatment, and the exogenous α-Fas exacerbated the activation of Caspase 3/8. On the other side, the siRNA-mediated knockdown of Fas/FasL inhibited the AGE-BSA-induced apoptosis. Taken together, we confirmed the activation of Fas-FasL signaling and of mitochondrial dysfunction in the AGE-BSA-promoted apoptosis in retinal ARPE-19 cells, implying the important role of Fas-FasL signaling in the DR in DM.

Annexin A5 promotes macrophage activation and contributes to pulmonary fibrosis induced by silica particles.

OBJECTIVE: To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis. METHODS: Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 µg/ml for 6 or 12 h, the expression levels of transforming growth factor ß1 (TGF-ß1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-ß1 secretion into the cell culture medium was measured by ELISA assays or Western blots. RESULTS: Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-ß1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-ß1, IL-1α, and FasL downstream target molecules. CONCLUSIONS: These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis.

The Antiviral Effect of Baicalin on Enterovirus 71 In Vitro.

Baicalin is a flavonoid compound extracted from Scutellaria roots that has been reported to possess antibacterial, anti-inflammatory, and antiviral activities. However, the antiviral effect of baicalin on enterovirus 71 (EV71) is still unknown. In this study, we found that baicalin showed inhibitory activity on EV71 infection and was independent of direct virucidal or prophylactic effect and inhibitory viral absorption. The expressions of EV71/3D mRNA and polymerase were significantly blocked by baicalin treatment at early stages of EV71 infection. In addition, baicalin could decrease the expressions of FasL and caspase-3, as well as inhibit the apoptosis of EV71-infected human embryonal rhabdomyosarcoma (RD) cells. Altogether, these results indicate that baicalin exhibits potent antiviral effect on EV71 infection, probably through inhibiting EV71/3D polymerase expression and Fas/FasL signaling pathways.

Decoy receptor 3 suppresses FasL-induced apoptosis via ERK1/2 activation in pancreatic cancer cells.

Resistance to Fas Ligand (FasL) mediated apoptosis plays an important role in tumorigenesis. Decoy receptor 3 (DcR3) is reported to interact with FasL and is overexpressed in some malignant tumors. We sought to investigate the role of DcR3 in resistance to FasL in pancreatic cancer. We compared expression of apoptosis related genes between FasL-resistant SW1990 and FasL-sensitive Patu8988 pancreatic cell lines by microarray analysis. We explored the impact of siRNA knockdown of, or exogenous supplementation with, DcR3 on FasL-induced cell growth inhibition in pancreatic cancer cell lines and expression of proteins involved in apoptotic signaling. We assessed the level of DcR3 protein and ERK1/2 phosphorylation in tumor and non-tumor tissue samples of 66 patients with pancreatic carcinoma. RNAi knockdown of DcR3 expression in SW1990 cells reduced resistance to FasL-induced apoptosis, and supplementation of Patu8988 with rDcR3 had the opposite effect. RNAi knockdown of DcR3 in SW1990 cells elevated expression of caspase 3, 8 and 9, and reduced ERK1/2 phosphorylation (P < 0.05), but did not alter phosphorylated-Akt expression. 47 tumor tissue specimens, but only 15 matched non-tumor specimens stained for DcR3 (χ(2) = 31.1447, P < 0.001). The proliferation index of DcR3 positive specimens (14.26  ±  2.67%) was significantly higher than that of DcR3 negative specimens (43.58  ±  7.88%, P < 0.01). DcR3 expression positively correlated with p-ERK1/2 expression in pancreatic cancer tissues (r = 0.607, P < 0.001). DcR3 enhances ERK1/2 phosphorylation and opposes FasL signaling in pancreatic cancer cells.