Fas-associated Death Domain Protein Research Articles

Deciphering the therapeutic potential of trimetazidine in rheumatoid arthritis via targeting mi-RNA128a, TLR4 signaling pathway, and adenosine-induced FADD-microvesicular shedding: In vivo and in silico study.

Rheumatoid arthritis (RA) is a debilitating autoimmune condition characterized by chronic synovitis, joint damage, and inflammation, leading to impaired joint functionality. Existing RA treatments, although effective to some extent, are not without side effects, prompting a search for more potent therapies. Recent research has revealed the critical role of FAS-associated death domain protein (FADD) microvesicular shedding in RA pathogenesis, expanding its scope beyond apoptosis to include inflammatory and immune pathways. This study aimed to investigate the intricate relationship between mi-RNA 128a, autoimmune and inflammatory pathways, and adenosine levels in modulating FADD expression and microvesicular shedding in a Freund's complete adjuvant (FCA) induced RA rat model and further explore the antirheumatoid potency of trimetazidine (TMZ). The FCA treated model exhibited significantly elevated levels of serum fibrogenic, inflammatory, immunological and rheumatological diagnostic markers, confirming successful RA induction. Our results revealed that the FCA-induced RA model showed a significant reduction in the expression of FADD in paw tissue and increased microvesicular FADD shedding in synovial fluid, which was attributed to the significant increase in the expression of the epigenetic miRNA 128a gene in addition to the downregulation of adenosine levels. These findings were further supported by the significant activation of the TLR4/MYD88 pathway and its downstream inflammatory IkB/NFB markers. Interestingly, TMZ administration significantly improved, with a potency similar to methotrexate (MTX), the deterioration effect of FCA treatment, as evidenced by a significant attenuation of fibrogenic, inflammatory, immunological, and rheumatological markers. Our investigations indicated that TMZ uniquely acted by targeting epigenetic miRNA128a expression and elevating adenosine levels in paw tissue, leading to increased expression of FADD of paw tissue and mitigated FADD microvesicular shedding in synovial fluid. Furthermore, the group treated with TMZ showed significant downregulation of TLR4/MYD88 and their downstream TRAF6, IRAK and NF-kB. Together, our study unveils the significant potential of TMZ as an antirheumatoid candidate, offering anti-inflammatory effects through various mechanisms, including modulation of the FADD-epigenetic regulator mi-RNA 128a, adenosine levels, and the TLR4 signaling pathway in joint tissue, but also attenuation of FADD microvesicular shedding in synovial fluid. These findings further highlight the synergistic administration of TMZ and MTX as a potential approach to reduce adverse effects of MTX while improving therapeutic efficacy.

Expression of RIPK1 and FADD are associated with chemosensitivity and survival in head and heck squamous cell carcinoma via tanshinone IIA-mediated modulation of the RIPK1-FADD-Caspase 8 complex.

Tanshinone IIA (Tan IIA), a main active ingredient of salvia miltiorrhiza, has a wide range of antitumor effects, while its specific role and mechanism in head and neck squamous cell carcinomas (HNSCC) is not fully understood. Totally 59 primary HNSCC patients underwent two courses of induction chemotherapy before surgery. The association between expression of Fas-Associated Death Domain (FADD) and receptor interacting protein kinase 1 (RIPK1) and chemotherapy resistance and survival were evaluated. The cell counting kit-8 was used to detect the effect of Tan IIA on the activity of cisplatin in chemoresistant HNSCC cells through a series of in vitro experiments. The quantitative real-time reverse-transcription polymerase chain reaction, Western blot analysis and flow cytometry were used. FADD and RIPK1 expressions were differentially expressed in Chemosensitive and drug-resistant patients. Furthermore, patients with tumors exhibiting high expression of FADD and RIPK1 had significantly greater risk for chemoresistance and mortality than patients with tumors that had low levels of these proteins. Moreover, Tan IIA reduced the expression of RIPK1 and FADD in HNSCC chemoresistant cell lines, which could increase the chemosensitivity of cisplatin and promote apoptosis. Overexpression of RIPK1 led to attenuation of therapeutic effects of Tan IIA, which were mainly realized through regulation of the RIPK1-FADD-Caspase 8 complex. This study is the first to demonstrate the clinical value and role of FADD and RIPK1 in the treatment of HNSCC. This work establishes the proapoptotic effects of Tan IIA and its potential to enhance chemosensitivity in HNSCC by modulating the RIPK1-FADD-Caspase 8 complex.

Investigating the mechanism and efficacy material basis of Xiehuo Xiaoying decoction for treating Graves' disease via thyroid cell apoptosis based on proteomics and molecular docking techniques

Ethnopharmacological relevanceFor numerous years, the Xiehuo Xiaoying decoction (XHXY), a traditional Chinese medicine formula, has demonstrated substantial promise in treating Graves' disease (GD) in clinical settings, showcasing significant potential. However, the therapeutic mechanism and efficacy material basis of XHXY remains obscure. Aim of the studyThis work aims to investigate the underlying mechanisms and to study the efficacy material basis of XHXY in anti-GD effect using a combination of TMT quantitative proteomics and molecular docking method. Materials and methodsGD model was initiated by administering Ad-TSH289. Subsequently, the mice underwent a four-week regimen that included oral gavage of XHXY at doses of 17 g/kg·d and 34 g/kg·d, along with intraperitoneal injections of Gentiopicroside (GPS). Utilizing the principles of pharmacological chemistry in traditional Chinese medicine, we employed high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) to discern prescribed prototype composition of XHXY in serum samples from mouse. TMT proteomics research provided evidence of XHXY's putative targets and important pathways in vivo. The binding activity of probable action targets and prototype composition was detected by molecular docking. Finally, Immunohistochemistry (IHC) and TUNEL staining were used to verify the mechanism of XHXY and GPS in anti-GD. ResultsXHXY and GPS alleviated GD by ameliorating the pathological changes and reducing thyroxine and TRAb levels. In mouse serum, a total of 31 prototypical XHXY ingredients were detected, and the majority of these components were from monarch and minister medicine. Proteomics study results indicated that the XHXY may mainly regulate targets including FAS-associated death domain protein (FADD), Apolipoprotein C-III, etc. and main pathways are Apoptosis, Cholesterol metabolism, TNF signalling pathway, etc. Strong binding activity of the prototypical active ingredient and GPS towards FADD, Caspase 8, and Caspase 3 was demonstrated by molecular docking. XHXY and its primary component, GPS, elevated the expression of FADD, Caspase 8, and Caspase 3, and enhance apoptosis in thyroid cells, as lastly validated by TUNEL and IHC staining. ConclusionsXHXY exhibits a favorable therapeutic effect in treating GD by promoting apoptosis in thyroid cells through the upregulation of FADD, Caspase 8, and Caspase 3 expression. And GPS is the main efficacy material basis for its therapeutic effect in anti-GD.

The Identification of New c-FLIP Inhibitors for Restoring Apoptosis in TRAIL-Resistant Cancer Cells.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Hellebrigenin induces apoptosis by triggering cellular inhibitor of apoptosis 1 and Jun N-terminal kinase pathway in melanoma cells

Abstract Background: Melanoma, the malignancy of melanocytes, is the most fatal form of skin cancer. Although various interventions are currently available, advanced stages of melanoma are still associated with a risk of metastatic recurrence. Objectives: The present study examined the anticancer activity of hellebrigenin, a natural product extracted from toad skin. Methods: Two types of metastatic melanoma cell lines, A2058 and HMY-1, were selected. Results: The findings revealed that hellebrigenin had cytotoxic effects on the two melanoma cell lines by triggering apoptosis: hellebrigenin activated cell arrest during the G2/M phase and downregulated regulators’ expression (e.g., cyclin-dependent kinase [CDK] 1, CDK 4, and cyclins E1, E2, A2, and D3). In intrinsic and extrinsic pathways of apoptosis, hellebrigenin activated the expression of the proapoptotic proteins t-BID, Bak, poly-ADP-ribose polymerase, Fas-associated death domain protein, death receptors 5, and cleaved caspases 3, 8, and 9; it also reduced the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. Mitochondrial membrane depolarization and cell nucleus condensation were also observed. In addition, human apoptosis arrays revealed that hellebrigenin inhibited the expression of cellular inhibitor of apoptosis 1, which is crucial for blocking cell apoptosis. Regarding upstream signaling activity, hellebrigenin suppressed the phosphorylation of the Jun N-terminal kinase 1/2 pathway in both melanoma cell lines. Conclusion: Overall, hellebrigenin can be used as a potential anticancer agent in clinical settings for treating the advanced stages of melanoma.

Identifying prognostic genes related PANoptosis in lung adenocarcinoma and developing prediction model based on bioinformatics analysis

Cell death-related genes indicate prognosis in cancer patients. PANoptosis is a newly observed form of cell death that researchers have linked to cancer cell death and antitumor immunity. Even so, its significance in lung adenocarcinomas (LUADs) has yet to be elucidated. We extracted and analyzed data on mRNA gene expression and clinical information from public databases in a systematic manner. These data were utilized to construct a reliable risk prediction model for six regulators of PANoptosis. The Gene Expression Omnibus (GEO) database validated six genes with risk characteristics. The prognosis of LUAD patients could be accurately estimated by the six-gene-based model: NLR family CARD domain-containing protein 4 (NLRC4), FAS-associated death domain protein (FADD), Tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), Receptor-interacting serine/threonine-protein kinase 1 (RIPK1), Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), and Mixed lineage kinase domain-like protein (MLKL). Group of higher risk and Cluster 2 indicated a poor prognosis as well as the reduced expression of immune infiltrate molecules and human leukocyte antigen. Distinct expression of PANoptosis-related genes (PRGs) in lung cancer cells was verified using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we evaluated the relationship between PRGs and somatic mutations, tumor immune dysfunction exclusion, tumor stemness indices, and immune infiltration. Using the risk signature, we conducted analyses including nomogram construction, stratification, prediction of small-molecule drug response, somatic mutations, and chemotherapeutic response.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome

The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

#3220 Z-DNA BINDING PROTEIN 1 AGGRAVATED THE SEPSIS-INDUCED ACUTE KIDNEY INJURY VIA PANOPTOSIS

Abstract Background and Aims Sepsis-induced acute kidney injury (SI-AKI) is a severe complication of critical patients with high morbidity and mortality. Apoptosis, pyroptosis, and necroptosis, regulated by a common pathway - PANoptosis, play essential roles in SI-AKI without the clarified mechanisms. Method We first established the SI-AKI model by intraperitoneal injection of the lipopolysaccharide (LPS) (O111:B4) in the C57B/L6J mice with increased Serum creatinine (Scr), blood urea nitrogen (BUN), and kidney injury biomarkers (NGAL, KIM-1) in renal tissue. The expression of crucial molecules in PANoptosis were examined by quantitative real-time PCR (qPCR) and Western blotting. RIPK1 inhibitor-necrostatin-1(Nec-1) (1.65mg/kg) and pan-caspase inhibitor- Emricasan (10mg/kg) were further employed to mice with LPS. Potential interacting proteins of panoptosome were detected by co-immunoprecipitation. We also analyzed the methylation level of ZBP1 in cell-free DNA among sepsis-non-AKI and SI-AKI patients. Results The expression of crucial molecules in PANoptosis, including IL-1β(0.82 vs. 0.35, P = 0.003), mixed lineage kinase domain-like (MLKL) (0.67 vs. 0.41, P = 0.014), protein kinase 1 (RIPK1) (1.07 vs. 0.72, P = 0.002), cleaved-caspase 3 (1.13 vs. 0.59, P < 0.001), and FAS-associated death domain protein (FADD) (0.78 vs. 0.43 P < 0.001) elevated in the SI-AKI group. Z-DNA binding protein 1 (ZBP1), a typical sensor of PANoptosis, is dramatically activated in the SI-AKI mice. Co-immunoprecipitation showed that caspase 8, ZBP1, peptidoglycan recognition protein 1 (pglyrp1), and RIPK1 are potential components of PANoptosome. Nec-1 and Emricasan could attenuate the LPS-induced increases in the concentrations of BUN and inflammatory cytokine (TNF-a and IL-6), as well as the methylation level of ZBP1 in cell-free DNA decreased in SI-AKI patients verse sepsis-none-AKI (P = 1.60E-06). Conclusion ZBP1 exacerbated SI-AKI via activating PANoptosis with the potencial PANoptosome of ZBP1, caspase 8, pglyrp1, and RIPK1.

Semilicoisoflavone B Induces Apoptosis of Oral Cancer Cells by Inducing ROS Production and Downregulating MAPK and Ras/Raf/MEK Signaling.

Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer worldwide. Despite advancement in treatment, advanced-stage OSCC is associated with poor prognosis and high mortality. The present study aimed to investigate the anticancer activities of semilicoisoflavone B (SFB), which is a natural phenolic compound isolated from Glycyrrhiza species. The results revealed that SFB reduces OSCC cell viability by targeting cell cycle and apoptosis. The compound caused cell cycle arrest at the G2/M phase and downregulated the expressions of cell cycle regulators including cyclin A and cyclin-dependent kinase (CDK) 2, 6, and 4. Moreover, SFB induced apoptosis by activating poly-ADP-ribose polymerase (PARP) and caspases 3, 8, and 9. It increased the expressions of pro-apoptotic proteins Bax and Bak, reduced the expressions of anti-apoptotic proteins Bcl-2 and Bcl-xL, and increased the expressions of the death receptor pathway protein Fas cell surface death receptor (FAS), Fas-associated death domain protein (FADD), and TNFR1-associated death domain protein (TRADD). SFB was found to mediate oral cancer cell apoptosis by increasing reactive oxygen species (ROS) production. The treatment of the cells with N-acetyl cysteine (NAC) caused a reduction in pro-apoptotic potential of SFB. Regarding upstream signaling, SFB reduced the phosphorylation of AKT, ERK1/2, p38, and JNK1/2 and suppressed the activation of Ras, Raf, and MEK. The human apoptosis array conducted in the study identified that SFB downregulated survivin expression to induce oral cancer cell apoptosis. Taken together, the study identifies SFB as a potent anticancer agent that might be used clinically to manage human OSCC.

RNA sequencing provides insights into the effect of dietary ingestion of microplastics and cadmium in the sea cucumber Apostichopus japonicus

IntroductionMicroplastics (MPs) and cadmium (Cd) are persistent pollutants in aquatic environments. Sea cucumbers are susceptible to MPs and Cd due to their feeding behavior.MethodsThis study, based on Illumina sequencing, compared the transcriptomes of A. japonicus before and after Cd and/or MPs exposure. Additionally, we detected the changes of catalase (CAT), and superoxide dismutase (SOD) activity, glutathione (GSH), and malondialdehyde (MDA) content in sea cucumbers.Results and DiscussionHigh concentration of MPs caused the increase of SOD activity. High concentration combined treatment resulted in significant up regulation of these four indicators in A. japonicus and had the largest number of differential expression genes (DEGs) reaching 1,618 DEGs, consisting of 789 up regulated along with 829 down regulated DEGs. Transcriptome results showed that Cd induced up regulation of intestinal FAS associated death domain protein (FADD) expression, which may cause apoptosis and inflammation. The increase of intestinal putative heparan sulfate 2-O-sulfotransferase in cadmium treatment groups provided a mechanism for host defense. The imbalance of expression of the NOD-like receptor (NLR) family inflammatory bodies and caspase 6 in the microplastic treatment group also led to the inflammatory reaction in the intestine of sea cucumber. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that in the process of fatty acid metabolism, MPs and Cd showed antagonistic effects, mainly in the inconsistent expression of Stearoyl CoA Ddesaturase (SCD1) protein. The significant changes of Toll interacting protein (TOLLIP) and E-selectin (SELE) in all Cd and MPs treatment groups may indicate the key immune response genes of sea cucumber to Cd exposure and MPs exposure. These genes were involved in the immune defense of sea cucumber exposed to different levels of Cd and MPs. This study provided insights into the mechanism of dietary MPs and Cd intake in an economically and ecologically important invertebrate species.

Differentially Expressed Inflammatory Cell Death-Related Genes and the Serum Levels of IL-6 are Determinants for Severity of Coronaviruses Diseases-2019 (COVID-19).

Inflammatory cell death, PANoptosis, has been suggested to orchestrate the lymphocyte decrement among coronavirus disease-2019 (COVID-19) patients. The main aim of this study was to examine the differences in the expression of key genes related to inflammatory cell death and their correlation with lymphopenia in the mild and severe types of COVID-19 patients. Eighty-eight patients (36 to 60 years old) with mild (n = 44) and severe (n = 44) types of COVID-19 were enrolled. The expression of key genes related to apoptosis (FAS-associated death domain protein, FADD), pyroptosis (ASC (apoptosis-associated speck-like protein containing caspase activation and recruitment domains (CARD)), the adapter protein ASC binds directly to caspase-1 and is critical for caspase-1 activation in response to a broad range of stimuli), and necroptosis (mixed lineage kinase domain-like, MLKL) genes were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay, and compared between the groups. The serum levels of interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay (ELISA) assay. A major increase in the expression of FADD, ASC, and MLKL-related genes in the severe type of patients was compared to the mild type of patients. The serum levels of IL-6 similarly indicated a significant increase in the severe type of the patients. A significant negative correlation was detected between the three genes' expression and the levels of IL-6 with the lymphocyte counts in both types of COVID-19 patients. Overall, the main regulated cell-death pathways are likely to be involved in lymphopenia in COVID-19 patients, and the expression levels of these genes could potentially predict the patients' outcome.

DED Interaction of FADD and Caspase-8 in the Induction of Apoptotic Cell Death.

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.

HS3ST1 Promotes Non-Small-Cell Lung Cancer Progression by Targeting the SPOP/FADD/NF-κB Pathway.

Heparan sulfate proteoglycan is a key component of cell microenvironment and plays an important role in cell-cell interaction, adhesion, migration, and signal transduction. Heparan sulfate 3-O-sulfotransferase 1 (HS3ST1) is a metabolic-related gene of HS. The present study was aimed at exploring the role of HS3ST1 in the progress of non-small-cell lung cancer (NSCLC). Our results illustrated that HS3ST1 promoted the malignant behaviors of NSCLC cells both in vitro and in vivo. HS3ST1 was found to inhibit spot-type zinc finger protein (SPOP) expression, which might inhibit the NF-κB pathway activation through mediating the degradation of Fas-associated death domain protein (FADD). By analyzing NSCLC patient samples, we also found increased HS3ST1 expression and decreased SPOP expression in tumor tissues in contrast with those in adjoining normal tissues. In conclusion, HS3ST1 promotes NSCLC tumorigenesis by regulating SPOP/FADD/NF-κB pathway.

The influence of venetoclax, used alone or in combination with cladribine (2-CdA), on CLL cells apoptosis in vitro: Preliminary results

Venetoclax (VEN), a highly selective BCL-2 inhibitor, is successfully used in the treatment of chronic lymphocytic leukemia (CLL). The purine analogue - cladribine (2-CdA) - is also administered to CLL patients, especially as a part of chemoimmunotherapy. To compare the effects of the VEN+2-CdA regimen with that of the 2 drugs used alone on the apoptosis of CLL lymphocytes in vitro. Mononuclear cells were collected from 103 previously untreated CLL patients. They were incubated with VEN (40 nM) or/and 2-CdA (16 μM) for 48 h. Cytotoxicity, overall apoptosis, mitochondrial transmembrane potential changes (ΔΨm), and expression of selected apoptosis-involved proteins were measured. The cytotoxicity, overall apoptosis, caspase-3 or caspase-9 expression, and ΔΨm were significantly higher after VEN+2-CdA addition compared to both drugs used alone, with a very strong synergistic effect observed. The percentage of BCL-2-positive cells decreased after VEN and VEN+2-CdA addition compared to controls. The TP53-expressing cells increased under the influence of all tested regimens. The VEN+2-CdA increased the expression of BIM, BAX and NOXA compared to either controls or VEN or 2-CdA alone. Similar increases in PUMA expression were observed after VEN, 2-CdA and VEN+2-CdA addition. The FAS-associated death-domain protein (FADD) expression was significantly higher after 2-CdA and 2-CdA+VEN addition as compared to control. Our results confirm the involvement of both VEN and 2-CdA in the intrinsic apoptotic pathway. They also demonstrate that these agents have a synergistic effect on CLL cells in vitro. Further studies are needed to assess the influence of VEN+2-CdA on the expression of apoptosis-involved genes.

PEA-15 engages in allosteric interactions using a common scaffold in a phosphorylation-dependent manner

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein. We delineated that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED. Upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly.

Long-chain PUFA ameliorate enterotoxigenic Escherichia coli-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in porcine intestinal epithelial cells.

This study was aimed to investigate whether EPA and arachidonic acid (ARA), the representative n-3 or n-6 PUFA, could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signalling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance, decreased permeability of fluorescein isothiocyanate-labelled dextran, increased membrane protein expression of occludin, ZO-1 and claudin-1 and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of TNF-α, IL-6 and IL-8 and decreased mRNA abundances of intestinal toll-like receptors 4 and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18 and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein, caspase 8, receptor-interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility-related protein 1 (Drp1) and high mobility protein 1 (HMGB1) and inhibited protein expression of phosphorylated-RIP1, p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signalling pathways.

Roles of TNF receptor-associated and Fas-associated death domain proteins in the apoptosis of Eimeria tenella host cells

The present study aimed to investigate the effects of death receptor adapter proteins, namely, TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD) proteins, on Eimeria tenella-induced host cell apoptosis. Gene silencing, culture technique for primary chick embryo cecal epithelial cells, enzyme-linked immunosorbent assay, Hoechst–Annexin V/PI apoptosis staining, fluorescence quantitative PCR, and flow cytometry were used to detect the E. tenella host cell apoptotic rate, RIP1 and FADD protein expression levels, and caspase-8 activity of the TRADD siRNA-treated and FADD siRNA-treated groups. Results showed that the apoptotic rate in the TRADD siRNA group was significantly higher than that in the NC siRNA group at 4 h post-infection with E. tenella (P < 0.05). The RIP1 protein expression level in the TRADD siRNA group was significantly lower than that in the NC siRNA group at 4–24 h (P < 0.05). The FADD expression and apoptotic rates in the TRADD siRNA group were significantly lower than those in the NC siRNA group at 24–120 h (P < 0.05). The caspase-8 activity and apoptotic rates in the FADD siRNA group were significantly lower than those in the NC siRNA group (P < 0.05) at 24–120 h. These findings indicated that E. tenella inhibited the host cell apoptosis through the TRADD-RIP1 pathway at the early developmental stage and promoted host cell apoptosis via the TRADD-FADD-caspase-8 apoptotic pathway at the middle and late developmental stages.

Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity

AbstractThe expression of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8+ T cells. Resting NK and CD8+ T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8+ T cells were activated by phytohemagglutinin (PHA) + IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8+ T cells, remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8+ T cells. Both resting and activated NK and CD8+ T cells were found to express high levels of the 2 isoforms of c-FLIP (cellular Fas-associated death domain protein [FADD]–like IL-1–converting enzyme [FLICE]–inhibitory protein). Small interference RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble TRAIL. Thus, once activated the major cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically protected from its apoptotic action by intracellular level of c-FLIP.

Ameliorative effects of alginate oligosaccharide on tumour necrosis factor-α-induced intestinal epithelial cell injury

Alginate oligosaccharide (AOS), produced by the depolymerisation of alginate (a polysaccharide naturally present in certain species of brown algae), has been shown to have versatile biological functions. In the present study, the porcine small intestinal epithelial cell line IPEC-J2 was used to assess the ameliorative effects of AOS on tumour necrosis factor-α (TNF-α)-induced intestinal epithelial cell injury. IPEC-J2 cells were pre-treated with or without AOS (600 μg/mL) in the presence or absence of TNF-α (50 ng/mL) for 24 h. AOS pre-treatment increased (P < 0.05) the occludin protein abundance and decreased (P < 0.05) the cytokine (interleukin-6 and TNF-α) concentrations, apoptosis rate and cysteinyl aspartate-specific protease-3 (caspase-3) and caspase-8 activities in TNF-α-treated IPEC-J2 cells. In addition, AOS pre-treatment increased (P < 0.05) the content of cellular inhibitor of apoptosis protein 2 and decreased (P < 0.05) the expression levels of TNF receptor 1 (TNFR1), TNFR-associated death domain protein and Fas-associated death domain protein in TNF-α-treated IPEC-J2 cells. These results demonstrate that AOS can reduce TNFR1-mediated apoptosis, thereby alleviating TNF-α-induced inflammatory injury in intestinal epithelial cells.

Cell-Penetrable Peptide-Conjugated FADD Induces Apoptosis and Regulates Inflammatory Signaling in Cancer Cells.

Dysregulated expression of Fas-associated death domain (FADD) is associated with the impediment of various cellular pathways, including apoptosis and inflammation. The adequate cytosolic expression of FADD is critical to the regulation of cancer cell proliferation. Importantly, cancer cells devise mechanisms to suppress FADD expression and, in turn, escape from apoptosis signaling. Formulating strategies, for direct delivery of FADD proteins into cancer cells in a controlled manner, may represent a promising therapeutic approach in cancer therapy. We chemically conjugated purified FADD protein with cell permeable TAT (transactivator of transcription) peptide, to deliver in cancer cells. TAT-conjugated FADD protein internalized through the caveolar pathway of endocytosis and retained in the cytosol to augment cell death. Inside cancer cells, TAT-FADD rapidly constituted DISC (death inducing signaling complex) assembly, which in turn, instigate apoptosis signaling. The apoptotic competency of TAT-FADD showed comparable outcomes with the conventional apoptosis inducers. Notably, TAT-FADD mitigates constitutive NF-κB activation and associated downstream anti-apoptotic genes Bcl2, cFLIPL, RIP1, and cIAP2, independent of pro-cancerous TNF-α priming. In cancer cells, TAT-FADD suppresses the canonical NLRP3 inflammasome priming and restricts the processing and secretion of proinflammatory IL-1β. Our results demonstrate that TAT-mediated intracellular delivery of FADD protein can potentially recite apoptosis signaling with simultaneous regulation of anti-apoptotic and proinflammatory NF-κB signaling activation in cancer cells.