Protein FADD Research Articles

Cadmium exposure induces oxidative stress-mediated necroptosis via TLR4/NF-κB signaling pathway in pig epididymis.

Cadmium (Cd) can cause reproductive disorders through epididymal injury. However, the specific molecular mechanism of Cd-induced epididymal toxic injury is rarely reported. In this study, the model of Cd poisoning in pig epididymis was established. Ten 6-week-old male piglets were divided into two groups. The control group was fed a basic diet, while the Cd group received a diet supplemented with 20mg/kg CdCl2. After 40 days, All piglets were euthanized, and epididymal tissues were collected to detect morphological changes, trace element contents, oxidative stress (OS) parameters, toll like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway and necroptosis marker genes. This study showed that Cd led to an increased concentration of Cd element in pig epididymis. According to morphological observation, pig epididymal tissue in the Cd group was damaged. Cd decreased the contents of glutathione (GSH), total antioxidant capacity (T-AOC), catalase (CAT), dismutase (SOD), and glutathione peroxidase (GSH-px), but the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were increased. Additionally, Caspase 8 expression was decreased, whereas the expression of TLR4, NF-κB, FADD, RIPK1, RIPK3, MLKL and heat shock proteins (HSPs) were increased after Cd stimulation. We concluded that Cd-triggered TLR4/NF-κB signaling pathway and oxidative stress potentially promoted necroptosis in pig epididymis.

Diversity of Molecular Functions of RNA-Binding Ubiquitin Ligases from the MKRN Protein Family.

Makorin RING finger protein family includes four members (MKRN1, MKRN2, MKRN3, and MKRN4) that belong to E3 ubiquitin ligases and play a key role in various biological processes, such as cell survival, cell differentiation, and innate and adaptive immunity. MKRN1 contributes to the tumor growth suppression, energy metabolism, anti-pathogen defense, and apoptosis and has a broad variety of targets, including hTERT, APC, FADD, p21, and various viral proteins. MKRN2 regulates cell proliferation, inflammatory response; its targets are p65, PKM2, STAT1, and other proteins. MKRN3 is a master regulator of puberty timing; it controls the levels of gonadotropin-releasing hormone in the arcuate nucleus neurons. MKRN4 is the least studied member of the MKRN protein family, however, it is known to contribute to the Tcell activation by ubiquitination of serine/threonine kinase MAP4K3. Proteins of the MKRN family are associated with the development of numerous diseases, for example, systemic lupus erythematosus, central precocious puberty, Prader-Willi syndrome, degenerative lumbar spinal stenosis, inflammation, and cancer. In this review, we discuss the functional roles of all members of the MKRN protein family and their involvement in the development of diseases.

Hippophae Rhamnoides-derived Phytomedicine Nano-System Modulates Bax/Fas Pathways to Reduce Proliferation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most common primary tumor of the breast with limited effectual drug availability. Therefore, the aim of the study is to develop an innovative phyto-nanomedicine (PNM) to cure TNBC with the least genotoxicity. Hereinafter, the sea buckthorn' extracted polyphenols (SBP), combine with metformin (MET), are synthesized as a novel PNM to evaluate its anti-tumor properties, effectiveness, and mechanism of action in TNBC in vitro and in vivo models. The SBP exhibits 16 new kinds of polyphenols that are been reported earlier which regulated cell development, proliferation, and programmed cell death (PCD) effectively. SBP-MET PNM inhibits MDA-MB-231 (47%), MDA-MB-436 (46%), and 4T1 (46%) cell proliferation but does not affect L929 normal murine cell development and successfully induce PCD (73.19%) in MDA-MB-231 cells. Mechanistically, in vivo SBP-MET proteome expression profiling reveals upregulation of proapoptotic Bax protein and activation of Fas signaling pathways convince downstream Daxx and FADD proteins, which further triggers Caspase-3 that prompts apoptosis in human TNBC cells by cleaving PARP-1 protein. Current findings establish innovative highly biocompatible phyto-nanomedicine that has significant potential to inhibit TNBC cell growth and induce regulated cell death (RCD) in vivo model, thereby opening a new arena for TNBC therapy.

Dingxian pill alleviates hippocampal neuronal apoptosis in epileptic mice through TNF-α/TNFR1 signaling pathway inhibition

Ethnopharmacological relevanceDingxian Pill (DXP), a famous traditional Chinese medicine prescription, and has been widely proven to have positive therapeutic effects on “Xianzheng” (the name of epilepsy in ancient China). However, the anti-epileptic molecular mechanisms of DXP are not yet fully understood and remain to be further investigated. Aim of the studyTo elucidate the molecular mechanism of DXP's improvement in epileptic neuronal loss, damage and apoptosis by regulating TNF-α/TNFR1 signaling pathway. Materials and methodsSixty Kunming mice were randomly divided in 6 groups: control group (equal volume of normal saline), model group (180 mg kg−1 pilocarpine hydrochloride – used to establish the epilepsy animal model), carbamazepine group (30 mg kg−1), and low, medium, and high-dose Dingxian Pill groups (4.08, 8.16, and 16.32 g kg−1, respectively – oral administration once daily for 2 weeks). Successful establishment of the epileptic mouse model was monitored with electroencephalography. Pathological changes in hippocampal tissue were analyzed with hematoxylin-eosin staining. Hippocampal neuronal apoptosis was analyzed with TUNEL staining. TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein expression levels in hippocampal tissue were analyzed with real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot, respectively. Cleaved caspase-8 protein levels in hippocampal tissue were measured with immunohistochemistry and Western blot. ResultsCompared to control, the model group showed an increase in continuous epileptic discharge waves on EEG, a damaged hippocampal neuron morphological structure, increased hippocampal neuronal apoptosis, and significantly increased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels, and increased caspase-8 cleavage (P < 0.05). Compared to the model group, the carbamazepine group as well as the low-, medium-, and high-dose Dingxian Pill groups showed decreased epileptic discharges on EEG, an obvious hippocampal neuron morphological structure restoration, varying degrees of attenuated hippocampal neuronal apoptosis, and significantly decreased TNF-α, TNFR1, TRADD, FADD, and caspase-8 mRNA and protein levels as well as decreased caspase-8 cleavage (P < 0.05). ConclusionsDingxian Pill exerts an anti-epileptic effect through inhibition of TNF-α/TNFR1 signaling pathway-mediated apoptosis in hippocampal neurons.

Mangiferin, A Naturally Occurring Glucosylxanthone, Induces Apoptosis in Caco-2 Cells In Vitro and Exerts Protective Effects on Acetic Acid-Induced Ulcerative Colitis in Mice through the Regulation of NLRP3.

Inflammatory bowel diseases (IBD), an inflammatory disease, include Crohn's disease and ulcerative colitis. Dysregulated autoimmune response to gut dysbiosis is mainly involved in the pathogenesis of IBD and is triggered by various inciting environmental factors. With its rising prevalence in every continent, IBD has evolved into a global disease, which is on the rise, affecting people of all ages. There is a growing incidence of IBD in the elderly population, as evidenced by epidemiological data. IBD is characterized by an inflammatory process that requires a lifelong treatment. The main challenge in IBD management is the adverse side effects associated with almost all of the currently available drugs. Hence, there is a search for drugs with more efficacy and fewer side effects. Natural products with great structural diversity and ease of modification chemically are being explored, as they were shown to control IBD by safely suppressing pro-inflammatory pathways. The present study aims at understanding the role of mangiferin, a COX-2 inhibitor isolated from tubers of Pueraria tuberosa in the treatment of IBD and colon cancer, in vitro on the Caco-2 human colon cancer cell line and in vivo in the acetic acid-induced IBD mouse model. In the acetic acid-induced colitis model, it prevented the decrease in length of the colon, mucosal erosion, and cellular infiltration in a dose-dependent manner. The expression levels of various pro-inflammatory markers like COX-2, IL1β, TNF-α, INF-γ, IL-6, NLRP3, and caspase-1 were downregulated in an acetic acid-induced mouse model on treatment with mangiferin in a dose dependent manner. Mangiferin also showed anticancer effects on Caco-2 cells by increasing the expression of Fas ligand, Fas receptor, FADD, caspase-8, and caspase-3 proteins, whereas Bid and Bcl-2 proteins showed decreased expression. These data suggest that mangiferin, an inhibitor of COX, induces apoptosis in colon cancer cells in vitro and protects mice from acetic acid-induced colitis in vivo.

Achillea fragrantissima (Forssk.) Sch.Bip instigates the ROS/FADD/c-PARP expression that triggers apoptosis in breast cancer cell (MCF-7).

Achillea fragrantissima is a shrub plant that belongs to the Asteraceae family in Arabia and Egypt. It is used as folk medicine and is a good source of phenolic acids, flavonoids, and some active compounds. To investigate the anti-cancer effect of A.fragrantissima on breast cancer MCF-7 cells and find the critical mechanism involved in apoptosis. The toxicity and pharmacokinetic studies of ethanolic extract of A.fragrantissima was examined for anti-breast cancer properties. In turn, cytotoxicity and cell viability were achieved by the MTT method. Furthermore, the trypan blue exclusion and microscopy examination proved the presence of apoptotic cells. Again, fluorescent staining such as AO/EtBr, DCFH-DA, Rho-123, and Hoechst-33342 reveals the cellular cytoplasmic disciplines upon A. fragrantissima effect. Moreover, cellular functioning tests like wound healing, colony formation, and Transwell invasion assay were demonstrated. In addition, the qRT-PCR technique authenticates the A. fragrantissima -induced apoptotic network genes (Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, PTEN, PI3K, and Akt) expression were evaluated. Mainly, the Immunoblot technique proved the expressed level of apoptotic proteins such as cleaved PARP, CYCS, and FADD. This study confirmed that the A. fragrantissima exerts cytotoxicity at 20 μg/mL for 24 hrs in MCF-7 cells. Also, decreases cellular viability, producing apoptotic cells and damaged cellular surfaces with dead matter. Consequently, it creates ROS species accumulation, loss of mitochondrial membrane potential, and fragmentation of DNA in MCF-7 cells. Furthermore, it arrests cell migration, induces colony-forming ability loss, and suppresses cell invasion. In addition, A. fragrantissima significantly upregulates genes such as caspase-3, 9, cytochrome c, BID, BAX, and PTEN while downregulating the Pi3K/ Akt signaling. Nonetheless, A.fragrantissima induced cleaved PARP, CYCS, and FADD proteins in MCF-7 cells to avail apoptosis.

A new chink in cancer’s armor: Unleashing cell death by selective PP5 inhibition

Serine/threonine protein phosphatases play a significant role in the survival and propagation of multiple cancers. In this issue of Cell Chemical Biology, Ahanin etal.1 demonstrate that protein phosphatase 5 (PP5) dephosphorylates and inactivates the cell death effector protein FADD independently of Hsp90, and they identify a selective PP5 inhibitor as a new therapeutic strategy for renal cancer.

Catalytic inhibitor of Protein Phosphatase 5 activates the extrinsic apoptotic pathway by disrupting complex II in kidney cancer

Serine/threonine protein phosphatase-5 (PP5) is involved in tumor progression and survival, making it an attractive therapeutic target. Specific inhibition of protein phosphatases has remained challenging because of their conserved catalytic sites. PP5 contains its regulatory domains within a single polypeptide chain, making it a more desirable target. Here we used an in silico approach to screen and develop a selective inhibitor of PP5. Compound P053 is a competitive inhibitor of PP5 that binds to its catalytic domain and causes apoptosis in renal cancer. We further demonstrated that PP5 interacts with FADD, RIPK1, and caspase 8, components of the extrinsic apoptotic pathway complex II. Specifically, PP5 dephosphorylates and inactivates the death effector protein FADD, preserving complex II integrity and regulating extrinsic apoptosis. Our data suggests that PP5 promotes renal cancer survival by suppressing the extrinsic apoptotic pathway. Pharmacologic inhibition of PP5 activates this pathway, presenting a viable therapeutic strategy for renal cancer.

Structural study of medium-long chain fatty acyl-CoA ligase FadD8 from Mycobacterium tuberculosis

In mycobacteria, lipids are important components of the cell wall and play a critical role for pathogenic activities. Lipids need to be activated before participating in many biological pathways. FadD proteins are members of the adenylate-forming superfamily, catalyzing activation of fatty acids. FadD8 is one of the 34 Mycobacterium tuberculosis FadD proteins, which was reported to be a putative medium-long chain fatty acyl-CoA ligase. Previous studies showed FadD8 from Mycobacterium smegmatis exhibited higher activity with oxidized cholesterol than fatty acids. However, the catalytic mechanism of the FadD8 is still exclusive. Here, we reported the crystal structure of FadD8 from Mycobacterium tuberculosis, which forms homodimer. Structural analysis revealed presence of a relatively narrow pocket compared to other FadD proteins and a novel alternative pocket, implying distinct substrate binding preference. We propose that FadD8 plays a vital role in cholesterol utilization and metabolism by catalyzing activation of cholesterol. Collectively, our findings provide novel information for the further studies of the inhibitor and drug development.

Impaired Death Receptor Signaling Mediates Cross-Resistance to Immunotherapy in MM

The treatment landscape in various cancers is currently shifting from genotoxic drugs to immunotherapy, such as monoclonal antibodies, immunoconjugates, T-cell engaging antibodies (TCE) or CAR T-cells (CARTs). This is in particular true for multiple myeloma (MM), in which unprecedented treatment efficiencies are induced by these novel therapies; however, patients continue to relapse and a subset even faces primary resistance, with the underlying mechanisms poorly understood. The role of the death receptor signaling proteins FADD and BID in MM resistance towards immunotherapy has not yet been explored. Therefore, we first generated FADD and BID CRISPR-Cas9 knock out (KO) models in two independent MM cell lines, AMO1 and L363. We confirmed the functional impact of the KO genes on the apoptotic activity using apoptosis inducing, CD95 activating antibody (anti-FAS Ab). As hypothesized, no apoptosis was observed in our FADD KO clones following 24 hours treatment with 15µg/ml Fas antibody, compared to a 50% increase of apoptosis in the WT cells via Annexin-V PI staining. Activity of Caspase 3/7 and 8 increased 3-4 folds in the WT cells and no increase was seen in our KO models by using commercially available fluorometric assays. Detection of cleaved PARP in WT and its absence in KO clones via Western blotting further validated the inactivation of the apoptotic pathways in our KO models. Next, we treated our KO cells with 0,1µg/ml Daratumumab (Dara) along with 10:1 ratio of PBMCs from healthy donors and 5:1 and 10:1 effector to target ratio of BCMA-CARTs: Of note, killing efficacy by Daratumumab was significantly lower in our KO models compared to the WT cells (22% vs 47% cell death) after 24 hours of treatment. Similarly, BCMA-CARTs efficiency was dramatically reduced with 58% survival in our KO models compared to around 10% in WT cells. Strikingly, we did not observe such a difference in IC50 between the KO and the WT cells when we treated for 3-5 days with Bortezomib, Lenalidomide, Doxorubicin, Melphalan, or the approved ADC Belantamab mafodotin. To determine the functional profile of the CART cells we performed a commercially available cytokine quantification assay (Human IL-2, Human IFN- γ) in response to 20 hours of antigen stimulation with WT and KO cells and no difference in IL-2 and IFN-γ production was observed. In addition, we excluded CD38 or BCMA antigen loss in our KO model by Direct Stochastic Optical Reconstruction Microscopy (dSTORM) and confirmed no significant differences in receptor density /µm2 or antigen surface distribution between the WT and the KO cells. To determine the clinical relevance of our findings we screened for decreased FADD and BID expression levels in a pilot cohort of 24 newly diagnosed (NDMM) and 54 relapsed refractory MM (RRMM) patients and normalized expression levels with 26 CD138+ bone marrow plasma cell samples from healthy donors. We did not find significant expression differences between the cohorts (that did not include patients post TCE or CARTs). However, when we ranked the patients according to their gene expression of FADD and BID the ten patients with the lowest values were predominantly Daratumumab resistant (7/10 and 9/10, respectively), whereas in the patients with highest FADD BID expression Daratumumab resistance was less pronounced (3/10 and 4/10 respectively). In conclusion, our study highlights induction of apoptosis as the main mode of action of T-cell based immunotherapies in MM. Furthermore, we provide first evidence, that gene expression of FADD and BID is essential for the action of Daratumumab and BCMA CARTs but may be dispensable for the anti-tumor effects in MM of ADC, IMiD, PI or chemotherapeutic agents. In our in vivo screen, we identified patients with low expression of FADD and BID being resistant to Daratumumab, suggesting a potential resistance mechanism.

Torularhodin Alleviates Hepatic Dyslipidemia and Inflammations in High-Fat Diet-Induced Obese Mice via PPARα Signaling Pathway.

Torularhodin is a β-carotene-like compound from Sporidiobolus pararoseus, and its protective effect against high-fat diet (HFD)-induced hepatic dyslipidemia and inflammation was investigated. Compared to mice of C57BL/6J fed on HFD, the addition of Torularhodin into the HFD (HFD-T) significantly reduced body weight, serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and the inflammatory mediators of TNF-α, IL-6, IL-1β, and lipopolysaccharide (LPS). A significant increase of high-density lipoprotein cholesterol (HDL-c), which is beneficial to cholesterol clearance, was also observed in HFD-T group. Proteomic analysis showed HDL-C-c is highly correlated with proteins (e.g., CPT1A and CYP7A1) involved in lipid β-oxidation and bile acid synthesis, whereas the other phenotypic parameters (TC, TG, LDL, and inflammatory cytokines) are highly associated with proteins (e.g., SLC27A4) involved in lipid-uptake. The up-regulated anti-inflammation proteins FAS, BAX, ICAM1, OCLN, GSTP1, FAF1, LRP1, APEX1, ROCK1, MANF, STAT3, and INSR and down-regulated pro-inflammatory proteins OPTN, PTK2B, FADD, MIF, CASP3, YAP1, DNM1L, and NAMPT not only demonstrate the occurrence of HFD-induced hepatic inflammation, but also prove the anti-inflammatory property of Torularhodin. KEGG signaling pathway analysis revealed that the PPARα signaling pathway is likely fundamental to the health function of Torularhodin through up-regulating genes related to fatty acid β-oxidation, cholesterol excretion, HDL-Cc formation, and anti-inflammation. Torularhodin, as a new food resource, may act as a therapeutic agent to prevent hepatic dyslipidemia and related inflammation for improved health.

Heritability Estimation of Multiple Sclerosis Related Plasma Protein Levels in Sardinian Families with Immunochip Genotyping Data.

This work aimed at estimating narrow-sense heritability, defined as the proportion of the phenotypic variance explained by the sum of additive genetic effects, via Haseman–Elston regression for a subset of 56 plasma protein levels related to Multiple Sclerosis (MS). These were measured in 212 related individuals (with 69 MS cases and 143 healthy controls) obtained from 20 Sardinian families with MS history. Using pedigree information, we found seven statistically significant heritable plasma protein levels (after multiple testing correction), i.e., Gc ( = 0.77; 95%CI: 0.36, 1.00), Plat ( = 0.70; 95%CI: 0.27, 0.95), Anxa1 ( = 0.68; 95%CI: 0.27, 1.00), Sod1 ( = 0.58; 95%CI: 0.18, 0.96), Irf8 ( = 0.56; 95%CI: 0.19, 0.99), Ptger4 ( = 0.45; 95%CI: 0.10, 0.96), and Fadd ( = 0.41; 95%CI: 0.06, 0.84). A subsequent analysis was performed on these statistically significant heritable plasma protein levels employing Immunochip genotyping data obtained in 155 healthy controls (92 related and 63 unrelated); we found a meaningful proportion of heritable plasma protein levels’ variability explained by a small set of SNPs. Overall, the results obtained, for these seven MS-related proteins, emphasized a high additive genetic variance component explaining plasma levels’ variability.

Identification of a novel prognostic signature related to PANoptosis and its regulatory mechanism as well as targeted treatment of active ingredients and traditional Chinese medicine in lung adenocarcinoma

Lung adenocarcinoma (LUAD) with a poor prognosis is the most common type of lung cancer, and one of the most aggressive and rapidly fatal tumor types. PANoptosis is a newly identified pro-inflammatory programmed cell death pathway controlled by the recently discovered cytoplasmic polymeric protein complex is called a PANoptosome. PANoptosome Components are linked to a variety of human diseases, including autoinflammatory diseases, cancer, etc. However, the specific effect of PANoptosis regulatory factors in regulating the prognosis and potential mechanism in lung adenocarcinoma is unclear. Here, we ultimately identified 14 differentially expressed PANoptosis related regulators by comparing unpaired and paired lung adenocarcinoma samples and summarized the genetic mutations and molecular correlations of PANoptosis regulators in LUAD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these PANoptosis factors were mainly involved in the NOD-like receptor signaling pathway, necroptosis, TNF signaling pathway, apoptosis, and IL-17 signaling pathway. PANoptosis related prognostic signature based on three genes (FADD, MAP3K7, and NLRP3) has a good performance in prognostic prediction models of LUAD patients. The validation results showed that the expression trend of the FADD gene and protein was in agreement with the previous analysis results of the Cancer Genome Atlas (TCGA) database. In addition, we found that the expression of FADD was involved in most immune infiltrating cell types. We also found the regulatory axis of lncRNA TUG1/ miR-128–3p /FADD in the progression of LUAD. Besides, we found that resveratrol, quercetin, capsaicin, as well as Silybum marianum (L.) Gaertn. (Shui Fei Ji), Schisandra chinensis (Turcz.) Baill. (Wu Wei Zi), Mangifera indica L. (Mang Guo), Panax ginseng C.A.Mey. (Ren Shen) are potential therapeutic drugs targeting PANoptosis prognostic signature FADD. In a word, we analyzed and identified a novel PANoptosis related prognostic signature FADD as a potential biomarker for LUAD, and revealed its role in regulating immune microenvironment, as well as found its potential lncRNA TUG1/ miR-128–3p/FADD regulatory axis and targeted therapy traditional Chinese medicine (TCM) and active ingredients, which provides a basis for the development of new targeted immunotherapy drugs.

Molecular and cellular evidence for hsa-miR-1254 suppressor effect against HER2 signaling in breast cancer.

HER2 signaling upregulation is a hallmark of breast cancer which is the second cause of cancer-related death in women. Here, we were looking for the candidate microRNAs which is capable of regulating the HER2 receptor and the genes of its downstream. To this aim, preliminary bioinformatics analysis introduced hsa-miR-1254 (miR-1254) as a potential common regulator of HER2, HER3, PIK3R2, and AKT1 genes. Then, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated a lower expression level of miR-1254 in breast cancer specimens, compared to their normal pairs. Furthermore, overexpression of miR-1254 resulted in HER2, HER3, PIK3R2, and AKT1 genes downregulation, detected by RT-qPCR and confirmed by western blot analysis and enzyme-linked immunosorbent assay test against AKT1, BAX, FADD, and HER2 protein levels in SKBR3 cells. Dual-luciferase assay also supported direct interaction of miR-1254 with MREs within 3' untranslated region sequences of HER2, HER3, PIK3R2, and AKT1 target genes. Overexpression of miR-1254 in SKBR3 cells was followed by increased BAX/BCL2 expression ratio, detected by RT-qPCR, and increased proportion of G1 cell population, detected by flow cytometry. Corroborated by cell cycle analysis, MTT, Annexin V-FITC, and Live-Dead cell staining assays, overexpression of miR-1254 in MDA-MB-231 cells showed opposing results following the overexpression of miR-1254. Taken together, results indicated that miR-1254 acts as cell-type-specific tumor suppressor that targets HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-1254 as a potential therapeutic candidate for breast cancer subtypes.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Molecular Markers of MDR of Chemotherapy for HSCC: Proteomic Screening With High-Throughput Liquid Chromatography-Tandem Mass Spectrometry.

BackgroundHypopharyngeal squamous cell cancer (HSCC) is a head and neck tumor with a poor prognosis. Chemotherapy lacks effectiveness because of multidrug resistance (MDR), which has increased toxic side effects. Thus, there is an urgent need to identify the molecular markers of MDR of chemotherapy for HSCC.MethodsFifty clinical samples of HSCC were derived from patients including 12 sensitive or resistant to chemotherapy drugs. Proteomic screening was performed using liquid chromatography-tandem mass spectrometry (LC-MS), which was based on data-independent acquisition. Molecular markers of MDR of chemotherapy in patients with HSCC were identified with clinical data and validated with ELISA.ResultsA total of 673 differentially expressed proteins were identified in HSCC samples, where 172 were upregulated and 501 were downregulated. A total of 183 differentially expressed proteins including 102 upregulated and 81 downregulated proteins, were identified by comparing cancer sensitive to chemotherapy with cancer resistant to chemotherapy. Clinical HSCC samples had significantly higher expression of FADD and significantly lower expression of RIPK1. Expressions of FADD and RIPK1 proteins were significantly lower in the chemotherapy-sensitive group. These expression differences were not correlated with clinical data. RIPK1 and FADD are involved in necroptosis and the signaling pathway of PRRs. Using ELISA, the low expression of RIPK1 and FADD was found in the patients sensitive to chemotherapy.ConclusionLC-MS proteomics is an effective method to identify the molecular markers of HSCC. FADD and RIPK1 can act as molecular markers of MDR of chemotherapy in patients with HSCC and may function through necroptosis and the PRR signaling pathway.

Comprehensive Sequencing Analysis in Subcutaneous Panniculitis-like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphoma (SPTL) is a rare subtype of peripheral T-cell lymphomas predominantly affecting younger individuals. Typically, SPTL presents with multifocal subcutaneous nodules, and nearly 20% of the patients have autoimmune disorders, including systemic lupus erythematosus, juvenile rheumatoid arthritis and Sjögren disease. Unlike other lymphomas, which usually require chemotherapy for long-term survival, immunosuppressive therapy alone yields favorable outcomes in SPTL. A previous study showed allelic NAV3 gene aberrations (LOH or deletion by FISH) in 44% of the SPTL samples and comparative genomic hybridization (CGH) revealed numerous DNA copy number changes. In another study, recurrent mutations were identified in various epigenetic modifiers (72%) and components of the PI3K/AKT/mTOR signaling pathway (44%). However, molecular pathogenesis of this disease has not been clearly understood.To elucidate the causative genetic lesions of SPTL, we collected 13 patients from Thailand (n=12) and Japan (n=1). Histopathology was reviewed by hematopathologists showing karyorrhexis of tumor cells infiltrating fat lobules. In all cases, lymphoma cells expressed CD3, CD5, CD8 and TIA1 but no CD56 suggesting the cytotoxic T-cells in origin. Tumor cells expressed T-cell receptor alpha/beta. The monoclonality was confirmed by southern blot (n=12). The mean percentage of SPTL cells in tissue blocks was 38% (range 20-80%). The majority of cases (75%) showed a high proliferative index (Ki-67 expression > 70%).Regarding the clinical data, 10 of 13 patients (80%) were female. The median age at diagnosis was 30 years old. Five cases (38.5%) showed systemic symptoms and 2 cases (20%) were associated with hemophagocytic syndromes. Seven cases received cyclosporine and prednisolone. All showed complete response but 2 cases relapsed. Six cases received anthracycline-based chemotherapy but 3 cases relapsed. All relapses responded to immunosuppressive therapy.We collected paired samples from each of cases (n=13). Tumor samples were collected from Formaldehyde Fixed-Paraffin Embedded (FFPE) tissues whereas germline samples were collected from either bone marrow FFPE or blood which showed no tumor involvement. We performed whole-exome and targeted-capture sequencing in those paired tumor/germline DNA samples by SureSelect v5 kit and custom kit, respectively. To identify somatic mutations and structural variations, sequencing data was analyzed using Genomon (ver. 2.3.0) and Genomon-SV. Copy number was analyzed based on sequencing data as described previously (ASH 2016 abstract by Saeki, et al.).By whole exome sequencing, 149 mutation calls were identified in 8 samples. The somatic mutations and copy number variations were grouped into 4 biological pathways which are commonly affected in cancer.1. Apoptotic associated pathway (FADD , ACIN1 , and PPM1L) : Interestingly, a nonsense mutation of FADD was found and validated. FADD protein is critical for promoting apoptosis of T-cell progenitors at the pre-TCR checkpoint.2. Epigenetic modifier pathway (GFIB , SAB130 and CHD9) : Notably, GFI1B mutation was found to be frameshift deletion causing protein truncation. Gfi1b repressed the Gata3, a transcription factor required for survival of T-lymphocyte progenitors.3. Translation initiation pathway (DHX29 and CTIF) : Missense mutation of DHX29 was identified whereas copy number analysis revealed one case possessed focal amplification of CTIF , a component of the CBP80/CBP20 translation initiation complex and associated with nonsense-mediated decay of mRNA.4. Transcription factors (MEF2B and MGA ): Targeted sequence detected mutations in MEF2B, MGA and PMS2 , which are associated with regulation of BCL6, MYC-MAX pathway and DNA mismatch repair, respectively.At least one mutation involved in either of such 4 pathways was identified in 5/8cases (62.5%). In addition, Sequence based T-cell receptor repertoire analysis proved monoclonal expansion in 5 cases.In conclusion, the identifications of novel genetic alterations provide an insight into the lymphomagenesis of SPTL. Heterogeneous mutations in different candidate pathways suggest complexpathogenesis of this rare tumor. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Benzofuranyl-2-imidazoles as imidazoline I2 receptor ligands for Alzheimer's disease.

Recent findings unveil the pharmacological modulation of imidazoline I2 receptors (I2-IR) as a novel strategy to face unmet medical neurodegenerative diseases. In this work, we report the chemical characterization, three-dimensional quantitative structure-activity relationship (3D-QSAR) and ADMET in silico of a family of benzofuranyl-2-imidazoles that exhibit affinity against human brain I2-IR and most of them have been predicted to be brain permeable. Acute treatment in mice with 2-(2-benzofuranyl)-2-imidazole, known as LSL60101 (garsevil), showed non-warning properties in the ADMET studies and an optimal pharmacokinetic profile. Moreover, LSL60101 induced hypothermia in mice while decreased pro-apoptotic FADD protein in the hippocampus. Invivo studies in the familial Alzheimer's disease 5xFAD murine model with the representative compound, revealed significant decreases in the protein expression levels of antioxidant enzymes superoxide dismutase and glutathione peroxidase in hippocampus. Overall, LSL60101 plays a neuroprotective role by reducing apoptosis and modulating oxidative stress.

The Mechanisms of the Anti-Inflammatory and Anti-Apoptotic Effects of Omega-3 Polyunsaturated Fatty Acids during Methotrexate-Induced Intestinal Damage in Cell Line and in a Rat Model.

Background: The aim of this study was to examine the anti-inflammatory and anti-apoptotic patterns of omega-3 polyunsaturated fatty acids (n-3 PUFAs) during methotrexate (MTX) induced intestinal damage in cell culture and in a rat model. Methods: Non-treated and treated with MTX HT 29 and HCT116cells were exposed to increasing doses of n-3 PUFAs and cell viability was evaluated using PrestoBlue® assay. Male Sprague-Dawley rats were divided into 4 experimental groups: Control rats, CONTR+n-3 PUFA rats that were treated with oral n-3 PUFA, MTX rats were treated with MTX given IP, and MTX+n-3 PUFA rats were treated with oral n-3 PUFA before and following injection of MTX. Intestinal mucosal parameters and mucosal inflammation, enterocyte proliferation and apoptosis, TNF-α in mucosal tissue and plasma (ELISA), NF-κB, COX-2, TNF-α, Fas, FasL, Fadd, Bid, Bax and Bcl-2gene and protein levels were determined 72 h following MTX injection. Results: Exposure of HT 29 and HCT116cells to n-3 PUFA attenuated inhibiting effects of MTX on cell viability. MTX-n-3 PUFA rats demonstrated a lower intestinal injury score and enhanced intestinal repair. A significant decrease in enterocyte apoptosis in MTX+n-3 PUFA rats was accompanied by decreased TNF-α, FAS, FasL, FADD and BID mRNA levels. Decreased NF-κB, COX-2 and TNF-α levels in mucosa was accompanied by a decreased number of IELs and macrophages. Conclusions: n-3 PUFAs inhibit NF-κB/COX-2 induced production of pro-inflammatory cytokines and inhibit cell apoptosis mainly by extrinsic pathway in rats with MTX-induced intestinal damage.

Neuron-specific activation of necroptosis signaling in multiple sclerosis cortical grey matter

Sustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II–III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I–III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.