Fas-associated Death Domain 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.

(2,6-Dimethylphenyl)arsonic Acid Induces Apoptosis through the Mitochondrial Pathway, Downregulates XIAP, and Overcomes Multidrug Resistance to Cytostatic Drugs in Leukemia and Lymphoma Cells In Vitro.

Cancer treatment is greatly challenged by drug resistance, highlighting the need for novel drug discoveries. Here, we investigated novel organoarsenic compounds regarding their resistance-breaking and apoptosis-inducing properties in leukemia and lymphoma. Notably, the compound (2,6-dimethylphenyl)arsonic acid (As2) demonstrated significant inhibition of cell proliferation and induction of apoptosis in leukemia and lymphoma cells while sparing healthy leukocytes. As2 reached half of its maximum activity (AC50) against leukemia cells at around 6.3 µM. Further experiments showed that As2 overcomes multidrug resistance and sensitizes drug-resistant leukemia and lymphoma cell lines to treatments with the common cytostatic drugs vincristine, daunorubicin, and cytarabine at low micromolar concentrations. Mechanistic investigations of As2-mediated apoptosis involving FADD (FAS-associated death domain)-deficient or Smac (second mitochondria-derived activator of caspases)/DIABLO (direct IAP binding protein with low pI)-overexpressing cell lines, western blot analysis of caspase-9 cleavage, and measurements of mitochondrial membrane integrity identified the mitochondrial apoptosis pathway as the main mode of action. Downregulation of XIAP (x-linked inhibitor of apoptosis protein) and apoptosis induction independent of Bcl-2 (B-cell lymphoma 2) and caspase-3 expression levels suggest the activation of additional apoptosis-promoting mechanisms. Due to the selective apoptosis induction, the synergistic effects with common anti-cancer drugs, and the ability to overcome multidrug resistance in vitro, As2 represents a promising candidate for further preclinical investigations with respect to refractory malignancies.

FADD promotes type I interferon production to suppress porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) is an epidemic animal infectious disease worldwide, causing huge economic losses to the global swine industry. Fas-associated death domain (FADD) was previously reported to be an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In the current study, we unravel its unidentified role in promoting type I interferon (IFN) production during PRRS virus (PRRSV) infection. We identified that FADD inhibited PRRSV infection via promotion of type I IFN transcription. Overexpression of FADD suppressed the replication of PRRSV, while knockout of FADD increased viral titer and nucleocapsid protein expression. Mechanistically, FADD promoted mitochondrial antiviral signaling protein (MAVS)-mediated production of IFN-β and some IFN-stimulated genes (ISGs). Furthermore, FADD exerted anti-PRRSV effects in a MAVS-dependent manner and increased the type I IFN signaling during PRRSV infection. This study highlights the importance of FADD in PRRSV replication, which may have implications for the future control of PRRS.

Apoptosis as a therapeutic strategy for breast cancer: the role of Thymax, a gross thymic extract, in modulating cell death pathways

Background Breast cancer is a prevalent disease in women and a leading cause of cancer-related health issues. Thymax, a thymic extract, has shown potential for inducing breast cancer cell apoptosis in vitro. Objective This study aims to investigate how Thymax induces apoptosis and inhibits breast cancer growth and metastasis in vivo. Materials and methods Thymax treatment was divided into five groups: the first group (negative control) − normal rats without tumors. In the second group (positive control), rats were injected subcutaneously in the mammary gland with a single dose of 50 mg/kg b.w. of 7,12-Dimethylbenz(a)anthracene (in 2 ml of corn oil) and allowed to develop tumors for 120 days. Group 3: Thymax was orally administered 6 days a week to tumor-bearing rats (0.4 mg/rat) and continued for 5 weeks. Tumor-bearing rats in group 4 (Thymax injection) received 0.1 ml of Thymax solution through intraperitoneal injection twice weekly for 5 weeks. The last group was Thymax mix (oral and injection); tumor-bearing rats received Thymax solution by dual routes: orally with 0.4 ml six times per week and intraperitoneally with 0.1 ml twice weekly for 5 weeks. Thymax treatment, beginning after 120 days of tumor induction, continued for 5 weeks. Results and conclusion Thymax- induced apoptosis in breast cancer cells by increasing cytochrome c, tumor necrosis factor receptor type 1-associated death domain protein (TRADD), and Fas associated death domain (FADD) levels. It also activated the mitochondrial-dependent pathway with up-regulation of tumor protein gene (P53) expression and cysteine-dependent, aspartate-specific peptidase (caspase-8) activation. Thymax restored normal renal and hepatic cell function and enhanced the immune system by improving total antioxidant levels and inhibiting malondialdehyde levels in treated animals. Histopathological results showed a significant apoptotic effect in the group receiving Thymax injections, demonstrating its capability to induce apoptosis without tumors or atypia in mammary glands. Our findings indicate that Thymax has a significant effect on enhancing tumor cell death and inducing apoptosis in vivo. Thymax may also modulate proapoptotic and antiapoptotic protein expression and activity, regulate the penetrability of the mitochondrial membrane, and release cytochrome c. Furthermore, our findings show that the injection route of Thymax is the fastest and most efficient method to deliver the extract to the tumor site and exert its antitumor effects. These results suggest that Thymax has the potential to be a novel adjuvant in the treatment of breast cancer, as it can enhance the efficacy of conventional therapies and reduce the risk of recurrence and metastasis.

Abstract 5545: FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot

Abstract The heterogenicity of hepatocellular carcinoma (HCC) remains a key obstacle in turning the majority of ‘immune-cold’ tumors ‘hot’ for effective immune checkpoint blockade (ICB) therapy. Through studying the naturally-existed ‘hot’ HCC variant lymphoepithelioma-like (LEL)-HCC by RNA and whole exome sequencing analysis, we identified fas-associated death domain (FADD) as one of the top differentially expressed genes that associated with high tumoral CD8+T cell abundance. Of note, FADD upregulation was also observed in HCC patients with better ICB responsiveness. As FADD was mainly expressed in tumor cells from HCC by scRNAseq analysis, we constructed Fadd knockout stable cells using murine HCC cell lines. Interestingly, CRISPR knockout of tumoral Fadd led to increased tumor weights in immunocompetent but not immunodeficient mice, accompanied with decreased tumoral CD8+T cells. Mechanistically, FADD phosphorylation promoted NF-κB transcriptional activity to increase CCL5 secretion from HCC cells via interacting with its nuclear binding partners Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) and lysine (K)-specific methyltransferase 2D (KMT2D), thus directing CD8+T cell tumor infiltration. In addition, anti-PD1 triggered FADD phosphorylation via IFN-γ and TNF-ɑ release from CD8+T cells in ICB-sensitive but not resistant tumors. Most importantly, sequential FADD activation via genetic and pharmacologic approaches could re-sensitize the therapeutic efficacy of anti-programmed death 1 (PD1) antibody in ICB-resistant orthotopic and spontaneous HCC models by turning cold tumors hot. Taken together, our findings reveal a key molecular feature of ICB-responsive hot tumors that may guide the development of sequential combinatory immunotherapies for the majority of HCC patients with low responsiveness to ICB monotherapy. This project is supported by the University Grants Committee through General Research Fund (24110323, 14107622, 14104820) and the Health and Medical Research Fund (07180556). Citation Format: Jingying Zhou, Jiahuan Lu. FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5545.

Cellular Dynamics of Fas-Associated Death Domain in the Regulation of Cancer and Inflammation.

Fas-associated death domain (FADD) is an adaptor protein that predominantly transduces the apoptosis signal from the death receptor (DR) to activate caspases, leading to the initiation of apoptotic signaling and the coordinated removal of damaged, infected, or unwanted cells. In addition to its apoptotic functions, FADD is involved in signaling pathways related to autophagy, cell proliferation, necroptosis, and cellular senescence, indicating its versatile role in cell survival and proliferation. The subcellular localization and intracellular expression of FADD play a crucial role in determining its functional outcomes, thereby highlighting the importance of spatiotemporal mechanisms and regulation. Furthermore, FADD has emerged as a key regulator of inflammatory signaling, contributing to immune responses and cellular homeostasis. This review provides a comprehensive summary and analysis of the cellular dynamics of FADD in regulating programmed cell death and inflammation through distinct molecular mechanisms associated with various signaling pathways.

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.

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.

Semilicoisoflavone B induces oral cancer cell apoptosis by targeting claspin and ATR-Chk1 signaling pathways.

The prevalence of oral squamous cell carcinoma (OSCC) is increasing worldwide mainly due to poor oral hygiene and unrestricted lifestyle. Advanced-stage OSCC is associated with poor prognosis and a 5-year survival rate of only 30%-50%. The present study was designed to investigate the anticancer effect and mode of action of Glycyrrhiza-derived semilicoisoflavone B (SFB) in 5-fluorourasil (5FU)-resistant human OSCC cell lines. The study findings revealed that SFB significantly reduces OSCC cell viability and colony formation ability by arresting cell cycle at the G2/M and S phases and reducing the expressions of key cell cycle regulators including cyclin A, cyclin B, CDC2, and CDK2. The compound caused a significant induction in the percentage of nuclear condensation and apoptotic cells in OSCC. Regarding pro-apoptotic mode of action, SFB was found to increase Fas-associated death domain and death receptor 5 expressions and reduce decoy receptor 2 expression, indicating involvement of extrinsic pathway. Moreover, SFB was found to increase pro-apoptotic Bim expression and reduce anti-apoptotic Bcl-2 and Bcl-xL expressions, indicating involvement of intrinsic pathway. Moreover, SFB-mediated induction in cleaved caspases 3, 8, and 9 and cleaved poly(ADP-ribose) polymerase confirmed the induction of caspase-mediated apoptotic pathways. Regarding upstream signaling pathway, SFB was found to reduce extracellular signal regulated kinase 1/2 (ERK) phosphorylation to execute its pro-apoptotic activity. The Human Apoptotic Array findings revealed that SFB suppresses claspin expression, which in turn caused reduced phosphorylation of ATR, checkpoint kinase 1 (Chk1), Wee1, and CDC25C, indicating disruption of ATR-Chk1 signaling pathway by SFB. Taken together, these findings indicate that SFB acts as a potent anticancer compound against 5FU-resistant OSCC by modulating mitogen-activated protein kinase (MAPK) and ATR-Chk1 signaling pathways.

CD3/CD28 costimulation-induced NF-KB activation, is not mediated by protein metallothionein 2A and FAS associated death domain

CD3/CD28 costimulation-induced NF-KB activation, is not mediated by protein metallothionein 2A and FAS associated death domain

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.

Multiple TLRs elicit alternative NLRP3 inflammasome activation in primary human monocytes independent of RIPK1 kinase activity.

The canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) pathway involves a priming step to induce pro-IL-1β followed by a secondary signal such as K+ efflux to activate inflammasome formation. This then leads to the maturation of IL-1β and the formation of gasdermin D (GSDMD) pores that initiate pyroptosis and mediate IL-1β release. In contrast, primary human monocytes also engage an alternative pathway in response to toll-like receptor (TLR) 4 activation, without the need for a secondary signal. Data from a monocyte-like cell line suggest that the alternative pathway functions via the TLR adaptor protein TIR-domain-containing adapter-inducing interferon-β (TRIF), receptor-interacting protein kinase 1 (RIPK1), FAS-associated death domain (FADD) and caspase-8 upstream of NLRP3 activation, but in the absence of K+ efflux or pyroptosis. Usage of the alternative pathway by other members of the TLR family that induce IL-1β but do not signal through TRIF, has yet to be explored in primary human monocytes. Furthermore, the mechanism by which IL-1β is released from monocytes remains unclear. Therefore, this study investigated if the alternative NLRP3 inflammasome pathway is initiated following activation of TLRs other than TLR4, and if GSDMD was necessary for the release of IL-1β. Monocytes were stimulated with ligands that activate TLR1/2, TLR2/6, TLR4 and TLR7 and/or TLR8 (using a dual ligand). Similar to TLR4, all of the TLRs investigated induced IL-1β release in a NLRP3 and caspase-1 dependent manner, indicating that TRIF may not be an essential upstream component of the alternative pathway. Furthermore, inhibition of RIPK1 kinase activity had no effect on IL-1β release. Although IL-1β was released independently of K+ efflux and pyroptosis, it was significantly reduced by an inhibitor of GSDMD. Therefore, it is feasible that low level GSDMD pore formation may facilitate the release of IL-1β from the cell, but not be present in sufficient quantities to initiate pyroptosis. Together these data suggest that the alternative pathway operates independently of RIPK1 kinase activity, downstream of diverse TLRs including TLR4 in primary human monocytes and supports the potential for IL-1β release via GSDMD pores alongside other unconventional secretory pathways.

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.

The diagnostic or prognostic values of FADD in cancers based on pan‑cancer analysis.

Previous studies have determined that aberrant expression of the fas-associated death domain (FADD) contributes to the development of cancer. However, no pan-cancer analysis has been reported to explore the relationship between FADD and various cancers. Multiple databases were screened to identify cancer datasets for the present study and to validate the expression of FADD in various tumors. The association of FADD alteration with cancer prognosis, clinical features and tumor immunity was also evaluated. Reverse transcription-quantitative PCR (RT-qPCR) was utilized to confirm the expression of FADD in breast, colon, liver and gastric cancer cells. Analysis of Gene Expression Omnibus database and The Cancer Genome Atlas database indicated that FADD was highly expressed in breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD) and prostate adenocarcinoma, whereas RT-qPCR results revealed that FADD was highly expressed in breast cancer and colon cancer. Further analyses demonstrated that FADD expression was significantly altered in ESCA, head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma and BRCA. FADD expression was observed to be a risk factor of the overall survival in patients with HNSC, LIHC and LUAD as demonstrated by Kaplan-Meier and Cox regression analyses. The results of the present study demonstrated that FADD is highly expressed in numerous malignancies and can be utilized as a biomarker for the diagnosis of BRCA, COAD, LIHC and stomach adenocarcinoma. Moreover, FADD expression is a predictive risk factor for the development of HNSC, LIHC and LUAD and can potentially be used as a prognostic marker for these cancers.

#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.

Ginsenoside Rb1 promotes the activation of PPARα pathway via inhibiting FADD to ameliorate heart failure

PurposeGinsenoside Rb1 (GRb1), a dammarane-type triterpene saponin compound mainly distributed in ginseng (Panax ginseng), has been demonstrated to ameliorate cardiovascular diseases. However, it remains unclear whether GRb1 alleviates heart failure (HF) by maintaining cardiac energy metabolism balance. Therefore, this work aimed to investigate the cardiac benefits of GRb1 against cardiac energy deficit and explore its mechanism of action. Methods and resultsIsoproterenol (ISO) induced HF Sprague–Dawley rats were administrated with GRb1 or fenofibrate for 6 weeks. ISO-induced primary neonatal rat cardiomyocytes (NRCMs) were used as the in vitro model. In vivo, GRb1 significantly improved the structural and metabolic disorder, as demonstrated by the restoration of cardiac function, inhibition of cardiac hypertrophy and fibrosis, and increased adenosine triphosphate (ATP) generation. In vitro, GRb1 effectively protected mitochondrial function and scavenged excessive reactive oxygen species. Moreover, in ISO-induced NRCMs, GRb1 significantly inhibited the abnormal upregulation of Fas-associated death domain (FADD), promoted transcriptional activation of peroxisome proliferator-activated receptor-alpha (PPARα), improved the aberrant expression of cardiac energy metabolism-related enzymes and cardiac fatty acid oxidation, and subsequently increased the synthesis of ATP. Noticeably, GRb1 could inhibit the increased binding between FADD and PPARα, which contributed to the activation of PPARα. Furthermore, GRb1 strengthened the thermal stabilization of FADD and might bind to FADD directly. ConclusionsCollectively, it’s part of the in-depth mechanism of GRb1’s cardio-protection that GRb1 could directly bind to FADD and counteract its negative role in the transcription of PPARα thus ameliorating cardiac energy derangement and HF.

Effect of penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on expression of TRAIL and its receptors in rats with premature ovarian insufficiency

To observe the effect of penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on the expressions of death receptor pathway-related protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, as death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) in premature ovarian insufficiency (POI) rats, so as to explore its mechanisms underlying improvement of POI. Forty female SD rats were randomly divided into blank control, model, penetrative needling and medication (estradiol valerate) groups, with 10 rats in each group. The POI model was established by intraperitoneal injection of cyclophosphamide (D1: 50 mg·kg-1·d-1, D2 to D15: 8 mg·kg-1·d-1, for a total of 15 d). After successful modeling, the rats in the penetrative needling group received penetrative needling of BL54 through ST28, with the needle retained for 30 min, once a day for a total of 4 weeks. Rats of the medication group received gavage of estradiol valerate (0.09 mg·kg-1·d-1) once daily for 4 weeks. After the intervention, the content of serum follicles of stimulation hormone (FSH),lateinizing hormone (LH),estradiol (E2) and vascular endothelial growth factor (VEGF) were assayed using enzyme-linked immunosorbent assay, and histopathological changes of ovarian tissue and the number of follicles were observed under light microscope after H.E. staining. The expression levels of TRAIL, DR4, DR5, DcR1, DcR2, and Fas-associated death domain (FADD) in ovarian tissues were detected using quantitative real-time PCR, and the immunoactivity of ovarian TRAIL, DR4 and DR5 detected using immunohistochemistry. The body weight and the damp weight of ovary were measured for calculating the ovarian coefficient. Compared with the blank control group, the E2 and VEGF contents, ovarian coefficient, and the number of the primary, secondary and sinus follicles were significantly decreased (P<0.01) in the model group, whereas FSH and LH contents, the atretic follicle number, TRAIL, DR4 and DR5 immunoactivity, and the expression levels of TRAIL, DR4, DR5 and FADD mRNAs considerably increased in the model group (P<0.01). In comparison with the model group, the decrease of the VEGF content, ovarian coefficient, and the number of the primary, secondary and sinus follicles, and the increase of the atretic follicle number, TRAIL, DR4 and DR5 immunoactivity, and expression levels of TRAIL, DR4, DR5 and FADD mRNAs were reversed in both penetrative needling and medication groups (P<0.01, P<0.05). The number of primary follicles was significantly more in the medication group than in the penetrative needling group (P<0.01). Penetrative needling of BL54 and ST28 can improve ovarian weight and promote follicular development in POI rats, which may be associated with its function in down-regulating the expression of pro-apoptotic proteins TRAIL, DR4, DR5 and FADD of the death receptor pathway to inhibit apoptosis of ovarian granulosa cells.

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.