Fas-associated Death Domain Research Articles

Identification of potential binding pocket on viral oncoprotein HPV16 E6: a promising anti-cancer target for small molecule drug discovery

BackgroundSeveral human cancers, especially cervical cancer are caused by the infection of high risk strains of human papillomaviruses (HPV), notably HPV16. It is implicated that the oncoprotein E6 expressed from HPV, is inhibiting the apoptotic pathway by binding to adaptor molecule FADD (Fas-associated death domain). Inhibiting E6 interactions with FADD could provide a promising treatment for cervical cancer. There are few small molecules reported to inhibit such interactions. However, the FADD binding site information on the HPV E6 is not currently available. This binding site information may provide an opportunity to design new small molecule inhibitors to treat E6 mediated cancers. In this study we report the possible binding pocket on HPV16 E6 oncoprotein by using activity data of reported inhibitors through a stepwise molecular modeling approach.ResultsBlind docking and removing duplicates followed by visual inspection to determine ligand-receptor interactions provided 68 possible binding sites on the E6 protein. Individual docking of all known inhibitors lead to the identification of 28 pockets having some kind of correlation with their activity data. It was also observed that several of these pockets overlapped with each other, having some amino acids in common. Amino acids Leu50 and Cys51 were identified as key E6 residues for high affinity ligand binding which are seen in most of these pockets. In most cases, ligands demonstrated a hydrogen bond interaction with Cys51. Ala61, Arg131 and Gln107 were also frequently observed showing interactions among these pockets. A few amino acids unique to each ligand were also identified representing additional interactions at the receptor site.ConclusionsAfter determining receptor-ligand interactions between E6 oncoprotein and the six known inhibitors, the amino acids Cys51, Leu50, Arg102, Arg131, Leu67, Val62, and Gln107 were identified to have importance in E6 inhibition. It was generally observed that Leu50 and Cys51 are necessary for high binding affinity with Cys51 being essential for hydrogen bonding. This study identified a potential binding pocket for the E6 inhibitors. Identification of the ligand binding pocket helps to design novel inhibitors of HPV16 E6 oncoprotein as a promising treatment for cervical cancer.

Lactobacillus plantarum C88 protects against aflatoxin B1-induced liver injury in mice via inhibition of NF-\u03baB\u2013mediated inflammatory responses and excessive apoptosis

BackgroundProbiotics play an important role in the human and animal defense against liver damage. However, the protective mechanism of Lactobacillus plantarum C88 on chronic liver injury induced by mycotoxin remains unclear.ResultsIn this study, the addition of L. plantarum C88 obviously ameliorated the increased contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol and triglyceride, the diminish contents of total protein and albumin in serum of mice challenged with AFB1. Simultaneously, L. plantarum C88 attenuated the inflammatory response via significantly reducing the levels of pro-inflammatory factors, including interleukin-1β (IL-1β), IL-6, IL-8, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in serum. Furthermore, L. plantarum C88 remarkably down-regulated the nuclear factor kappa B (NF-κB) signaling pathways by weakening the expression of toll-like receptor 2 (TLR2) and TLR4, and inhibited NF-κB nuclear translocation through enhancing the expression of NF-κB inhibitor (IκB). Neutralization experiments confirmed that L. plantarum C88 decreased the levels of some pro-inflammatory factors due to the suppression of the NF-κB signaling pathways. Besides, L. plantarum C88 decreased the levels of Bax and Caspase-3, elevated the level of Bcl-2, and reduced mRNA expressions of Fatty acid synthetase receptor (Fas), FAS-associated death domain (FADD), TNF receptor associated death domain (TRADD) and Caspase-8 in the liver.ConclusionsProbiotic L. plantarum C88 prevented AFB1-induced secretion of pro-inflammatory cytokines by modulating TLR2/NF-κB and TLR4/NF-κB pathways. The molecular mechanisms of L. plantarum C88 in ameliorating AFB1-induced excessive apoptosis included regulating the mitochondrial pathway and cell death receptor pathways.

Detoxification, Apoptosis, and Immune Transcriptomic Responses of the Gill Tissue of Bay Scallop Following Exposure to the Algicide Thiazolidinedione 49.

Thiazolidinedione 49 (TD49), a newly synthesized algicide, shows strong toxicity at low concentrations of 0.1–2.0 μM. However, its potential effects on non-target species at the transcript level were not investigated. Differentially expressed genes (DEGs) in the gills of the bay scallop, Argopecten irradians, were accessed after treatment with 0.68 μM TD49 for up to 48 h. Following exposure, it was observed that 5214 genes were upregulated and 3497 were downregulated. Functional enrichment analysis revealed that the apoptosis pathway was activated. The extrinsic apoptosis pathway was activated and the survival factors related pathway was suppressed. Furthermore, gene expressions related to ATP-binding cassette, nuclear factor erythroid 2-related factor, B cell lymphoma-2 family protein, glutathione reductase, glutathione peroxidase, catalase, NADPH2:quinone reductase, and superoxide dismutase were decreased. Conversely, gene expressions related to FAS-associated death domain protein, glutathione S-transferase, caspase 6, 8, cytochrome P450 1A1, and 2C8 were increased. These results comprehensively demonstrated the toxicity of the novel algicide TD49, and should draw the attention of researchers to the importance of analyzing the potential impact of chemical compounds as algicides to control the proliferation of harmful algae, due to the secondary pollution caused by their application.

PEA-15 C-Terminal Tail Allosterically Modulates Death-Effector Domain Conformation and Facilitates Protein-Protein Interactions.

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) exerts its regulatory roles on several critical cellular pathways through protein–protein interactions depending on its phosphorylation states. It can either inhibit the extracellular signal-regulated kinase (ERK) activities when it is dephosphorylated or block the assembly of death-inducing signaling complex (DISC) and the subsequent activation of apoptotic initiator, caspase-8, when it is phosphorylated. Due to the important roles of PEA-15 in regulating these pathways that lead to opposite cellular outcomes (cell proliferation vs. cell death), we proposed a phosphostasis (phosphorylation homeostasis) model, in which the phosphorylation states of the protein are vigorously controlled and regulated to maintain a delicate balance. The phosphostasis gives rise to the protective cellular functions of PEA-15 to preserve optimum cellular conditions. In this article, using advanced multidimensional nuclear magnetic resonance (NMR) techniques combined with a novel chemical shift (CS)-Rosetta algorithm for de novo protein structural determination, we report a novel conformation of PEA-15 death-effector domain (DED) upon interacting with ERK2. This new conformation is modulated by the irregularly structured C-terminal tail when it first recognizes and binds to ERK2 at the d-peptide recruitment site (DRS) in an allosteric manner, and is facilitated by the rearrangement of the surface electrostatic and hydrogen-bonding interactions on the DED. In this ERK2-bound conformation, three of the six helices (α2, α3, and α4) comprising the DED reorient substantially in comparison to the free-form structure, exposing key residues on the other three helices that directly interact with ERK2 at the DEF-docking site (docking site for ERK, FxF) and the activation loop. Additionally, we provide evidence that the phosphorylation of the C-terminal tail leads to a distinct conformation of DED, allowing efficient interactions with Fas-associated death domain (FADD) protein at the DISC. Our results substantiate the allosteric regulatory roles of the C-terminal tail in modulating DED conformation and facilitating protein–protein interactions of PEA-15.

Effect of enalapril on apoptosis of renal tubular epithelial cells in renal interstitial fibrosis in rats

To observe the effect of enalapril on the apoptosis of renal tubular epithelial cells in renal interstitial fibrosis rats and to explore the mechanism of enalapril on renal interstitial fibrosis. Methods: Twenty-four SD male rats were randomly divided into a sham operation group, a model group and an enalapril group (n=8 in each group). The rats in the model group and the enalapril group underwent the operation of left urethral obstruction to establish the animal model of unilateral urethral obstruction (UUO). Fourteen days later after the operation, all rats were sacrificed and their obstructed kidneys were collected for HE and Masson staining to observe the pathological change of renal tissues. Terminal deoxynucleotidyl transferase-mediated (dUTP) nick end-labeling (TUNEL) staining was used to detect the apoptosis of renal tubular epithelial cells. Immunohistochemistry and Western blotting were used to detect the protein expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (APAF-1) and C/EBP homologous protein (CHOP). Results: Compared with the sham operation group, the renal interstitial injury index and renal interstitial fibrosis index were significantly increased in the model group (P<0.05). Compared with the model group, the renal interstitial injury index and renal interstitial fibrosis index were both significantly decreased in the enalapril group (P<0.05). Compared with the sham group, the apoptosis rate of renal tubular epithelial cells was increased in the model group (P<0.05); compared with the model group, the apoptosis rate of renal tubular epithelial cells was significantly reduced in the enalapril group (P<0.05). The protein levels of FADD, APAF-1 and CHOP in the model group were significantly elevated than those in the sham group (all P<0.05), which were reversed in presence of enalapril (all P<0.05). Conclusion: Enalapril can alleviate renal interstitial fibrosis through inhibiting apoptosis of renal tubular epithelial cells in UUO rats.

Delineating the functions of RIPK1 in vivo through CRISPR/Cas9-mediated gene editing in mice

Abstract RIPK1 is originally identified as a protein associated with Fas and TNFR1, which can trigger apoptosis. However, initial studies indicated that RIPK1 is dispensable for apoptotic signaling. Instead, cells lacking RIPK1 are defective in TNFR1-induced activation of NF-kB, a critical player involved in pro-survival signaling. Recent studies indicate that a certain form of necrosis-like death, necroptosis, requires RIPK1. In contrast, the Fas-associated death domain (FADD) protein appears to play an obligatory role in apoptosis induced by Fas and TNFR1. Fadd−/− mice die in utero due to RIPK1 mediated necroptosis. More recently, studies including ours demonstrated that only when both FADD and RIPK3 were deleted will Ripk1−/− mice survive to adulthood. This result indicates perinatal lethality in Ripk1−/− mice is due to not only FADD-mediated apoptosis but also RIPK3-dependent necrosis. Interestingly, we found that in adult T cells RIPK1 only suppresses apoptosis. To facilitate the dissection of the multiple functions of RIPK1, we performed in vivo gene editing using the CRISPR/Cas9 technology. This allows selective interruption of one functional domain/motif of RIPK1. Using these novel mouse models, our data reveals a previously unappreciated function for RIPK1 in promoting apoptosis.

Antiapoptotic and mitochondrial biogenetic effects of exercise training on ovariectomized hypertensive rat hearts.

This study was to investigate the effects of exercise training on antiapoptotic pathways and mitochondrial biogenesis in ovariectomized hypertensive rats. Histopathological analysis, TUNEL assay, and Western blotting were performed on the excised hearts from female spontaneously hypertensive rats (SHR), which were divided into a sham-operated sedentary hypertensive (SHR-S), a sedentary hypertensive ovariectomized (SHR-O), and hypertensive ovariectomized rats that underwent treadmill exercise training (SHR-OT; 60 min/day, 5 days/wk) for 8 wk, along with normotensive Wistar Kyoto rats (WKY). When compared with the WKY group, the SHR-S group exhibited decreased protein levels of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial OPA-1 (mitochondrial biogenesis) and decreased further in the SHR-O group. The protein levels of p-PI3K, p-Akt, Bcl-2, Bcl-xL (prosurvival pathways), and the protein levels of PGC-1α and mitochondrial OPA1 (mitochondrial biogenesis) were increased in the SHR-OT group, but estrogen receptor (ER)α and ERβ were not changed when compared with the SHR-O group. The protein levels of t-Bid, Bad, Bax, cytosolic cytochrome c, activated caspase 9, and activated caspase 3 (mitochondria-dependent apoptotic pathways), as well as Fas ligand, TNF-α, Fas receptors, Fas-associated death domain, activated caspase 8 (Fas receptor-dependent apoptotic pathways) were decreased in the SHR-OT group, when compared with the SHR-O group. Exercise training protection on the coexistence of hypertension and ovariectomy-induced cardiac mitochondria-dependent and Fas receptor-dependent apoptotic pathways by enhancing the Bcl2-related and mitochondrial biogenetic prosurvival pathways might provide a new therapeutic effect on cardiac protection in oophorectomized early postmenopausal hypertensive women. NEW & NOTEWORTHY Widely dispersed cardiac apoptosis was found in the coexistence of hypertension and ovariectomy. Exercise training on a treadmill could prevent ovariectomized hypertension-induced widely dispersed cardiac apoptosis via mitochondria-dependent apoptotic pathway (t-Bid, Bad, Bax, cytosolic cytochrome c, activated caspase 9, and activated caspase 3) and Fas receptor-dependent apoptotic pathway (Fas ligand, tumor necrosis factor-α, Fas receptors, Fas-associated death domain, activated caspase 8, and activated caspase 3) through enhancing the Bcl2-related (p-PI3K, p-Akt, Bcl-2, Bcl-xL) and mitochondrial biogenetic (PGC-1α and mitochondrial optic atrophy 1) prosurvival pathways.

The first cloned sea cucumber FADD from Holothuria leucospilota: Molecular characterization, inducible expression and involvement of apoptosis

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5′-untranslated region (UTR), a 1334-bp 3′-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.

Phosphorylation of PEA‐15 in HIV‐TAT Mediated Disruption of Blood‐Brain Barrier

Trans‐activator of transcription, TAT, one of the proteins that is expressed by the HIV genome, has been heavily associated with HIV‐associated cognitive morbidities. In vitro experiments have shown that the presence of TAT disrupts blood‐brain barrier (BBB) tight junctions and overall integrity. The mechanisms for TAT associated BBB disruption remain largely unknown.Phosphoprotein enriched in astrocytes (PEA‐15) is a ubiquitously expressed protein that is best known for its ability to regulate critical cellular pathways via protein‐protein interactions. PEA‐15 gains multiple protein binding affinities depending on the phosphorylation states of its residues Ser104 and Ser116. When PEA‐15 is phosphorylated at both Ser104 and Ser116 it gains high affinity to the Fas‐associated death domain (FADD). When protein‐protein binding with FADD, PEA‐15 blocks FADD from inducing apoptosis by preventing it from activating Caspase‐8.By blocking FADD effector functions via transfecting a phosphomimetic mutant of PEA‐15, in which both Ser104 and Ser116 are replaced with Aspartic Acid (PEA‐15‐DD), into primary human brain microvascular endothelial cells, we set to explore the mechanisms of HIV‐TAT mediated disruption of BBB tight junctions.Support or Funding InformationThis research is supported by NIH Grant R21DA046223This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The classical NLRP3 inflammasome controls FADD unconventional secretion through microvesicle shedding

Fas-associated death domain (FADD) is a key adaptor molecule involved in numerous physiological processes including cell death, proliferation, innate immunity and inflammation. Therefore, changes in FADD expression have dramatic cellular consequences. In mice and humans, FADD regulation can occur through protein secretion. However, the molecular mechanisms accounting for human FADD secretion were still unknown. Here we report that canonical, non-canonical, but not alternative, NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion. NLRP3 inflammasome activation by the bacterial toxin nigericin led to the proinflammatory interleukin-1β (IL-1β) release and to the induction of cell death by pyroptosis. However, we showed that FADD secretion could occur in absence of increased IL-1β release and pyroptosis and, reciprocally, that IL-1β release and pyroptosis could occur in absence of FADD secretion. Especially, FADD, but not IL-1β, secretion following NLRP3 inflammasome activation required extracellular glucose. Thus, FADD secretion was an active process distinct from unspecific release of proteins during pyroptosis. This FADD secretion process required K+ efflux, NLRP3 sensor, ASC adaptor and CASPASE-1 molecule. Moreover, we identified FADD as a leaderless protein unconventionally secreted through microvesicle shedding, but not exosome release. Finally, we established human soluble FADD as a new marker of joint inflammation in gout and rheumatoid arthritis, two rheumatic diseases involving the NLRP3 inflammasome. Whether soluble FADD could be an actor in these diseases remains to be determined. Nevertheless, our results advance our understanding of the mechanisms contributing to the regulation of the FADD protein expression in human cells.

The Protective Role of Selenium Against AFB1-Induced Liver Apoptosis by Death Receptor Pathway in Broilers.

Aflatoxin B1 (AFB1) is the most toxic among the mycotoxins and causes detrimental health effects on the liver of human and animals. Selenium (Se) plays an important role in protection of various animal species against numerous notorious toxic agents. The present study is designed to explore the protective effects of Se against AFB1-induced liver pathogenesis by the methods of histopathology, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and biochemical analysis. A total of 312, 1-day-old healthy Cobb-500 broilers were randomly divided into four groups and fed with basal diet (control group), 0.6mg/kg AFB1 (AFB1 group), 0.4mg/kg Se (+ Se group), and 0.6mg/kg AFB1 + 0.4mg/kg Se (AFB1 + Se group) for 21days, respectively. Our results showed that 0.4mg/kg Se supplement in broiler's diets could alleviate the AFB1-induced histological lesions in the liver. The apoptosis analysis by flow cytometry showed that 0.4mg/kg Se ameliorated the AFB1-induced apoptosis in the liver. Moreover, the mRNA expression levels of Fas, TNF-α, FAS-associated death domain, TNF receptor-associated death domain, TNF receptor-associated factor 2, caspase 10, caspase 8, B cell lymphoma 2, IκB kinase, X-linked inhibitor of apoptosis protein, caspase 9, and caspase 3 analyzed by qRT-PCR demonstrated that 0.4mg/kg Se could relieve the impact caused by AFB1 to these parameters. The biochemical analyses of activities of CAT, GSH-Px and SOD, hydroxyl ion scavenging and contents of MDA and GSH in liver cells also indicated that 0.4mg/kg Se has positive effect on AFB1-induced oxidative stress in the liver. In conclusion, Se could relieve AFB1-induced apoptosis by the molecular regulation of death receptors pathway in the liver of broilers. The outcomes from the present study may lead to a better understanding of the nature of selenium's essentiality and its protective roles against AFB1.

Herbal cake-partitioned moxibustion improves intestinal epithelial barrier dysfunction by suppressing TNF-α-mediated apoptosis pathway of intestinal epithelium in rats with Crohn's disease

To observe the effect of herbal cake-partitioned moxibustion (Moxi) on tumor necrosis factor (TNF)-α/ TNF receptor 1 (TNFR1)-associated death domain (TRADD) / Fas-associated death domain (FADD) pathway-mediated apoptosis of intestinal epithelial cells in Crohn's disease (CD) rats, so as to explore its underlying mechanisms in the treatment of CD. Forty-eight SD male rats were randomly divided into normal, model, Moxi and medication groups (n＝12 rats in each). The CD model was established by intra-annual perfusion of 2，4，6-trinitrobenzene sulfonic acid (TNBS) solution (TNBS∶50% alcohol＝2∶1, 3 mL/kg), once every 7 days, 4 times altogether. For rats of the Moxi group, moxibustion was given to "Tianshu" (ST25) and "Qihai" (CV6), two moxa-cones every time, once daily for 10 days. For rats of the medication group, intragastric perfusion of mesalazine solution was given twice daily for 10 days. After the treatment, the colonic epithelium tissue was sampled. The epithelial cells were purified and cultured to establish an in vitro intestinal epithelial barrier, and added with TNF-α (a pro-inflammatory factor, 100 ng/mL) in the culture medium for 24 h for making an increased epithelial permeability model. The permeability of intestinal epithelial cell barrier was evaluated by detecting the fluorescence yellow transmittance of the TNF-α-incubated cell medium. Western blot was used to detect the expression levels of TNFR1, TRADD, receptor-interacting protein 1 (RIP1), FADD and zinc finger protein A20 (A20, a ubiquitination enzyme for inhibiting activation of TRADD and RIP1) of the cultured intestinal epithelium cells. The apoptosis of the TNF-α-incubated intestinal epithelial cells was detected by flow cytometry. After modeling and compared with the normal group, the fluorescence yellow transmittance of intestinal epithelia cells, apoptosis rate, and expression levels of TNFR1, TRADD, and RIP1 proteins were significantly increased (P<0.001, P<0.01), and the expression of A20 was significantly decreased (P<0.01) in the model group. In comparison with the model group, the fluorescence yellow transmittance of intestinal epithelial cells, the apoptosis rate and expression levels of TRADD, RIP1 and FADD were remarkably down-regulated (P<0.001, P<0.01), and the expression of A20 was significantly up-regulated (P<0.01) in both the Moxi and medication groups. Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.

Foxo3a-dependent miR-633 regulates chemotherapeutic sensitivity in gastric cancer by targeting Fas-associated death domain

ABSTRACTThe development of chemotherapeutic drugs resistance such as doxorubicin (DOX) and cisplatin (DDP) is the major barrier in gastric cancer therapy. Emerging evidences reveal that microRNAs (miRNAs) contribute to chemosensitivity. In this study, we investigated the role of miR-633, an oncogenic miRNA, in gastric cancer chemoresistance. In gastric cancer tissue and cell lines, miR-633 expression was highly increased and correlated with down regulation of Fas-associated protein with death domain (FADD). Inhibition of miR-633 significantly increased FADD protein level and enhanced DOX/DDP induced apoptosis in vitro. MiR-633 antagomir administration remarkably decreased tumor growth in combination with DOX in vivo, suggesting that miR-633 targets FADD to block gastric cancer cell death. We found that the promoter region of miR-633 contained putative binding sites for forkhead box O 3 (Foxo3a), which can directly repress miR-633 transcription. In addition, we observed that DOX-induced nuclear accumulation of Foxo3a leaded to the suppression of miR-633 transcription. Together, our study revealed that miR-633/FADD axis played a significant role in the chemoresistance and Foxo3a regulated this pathway in gastric cancer. Thus, miR-633 antagomir resensitized gastric cancer cells to chemotherapy drug and had potentially therapeutic implication.

Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

Effect of IRAK1 on Apoptosis and Necroptosis of Hepatoma Cell Line SK-Hep1

Interleukin I receptor associated kinase 1 (IRAK1) is a downstream signal molecule of activated MyD88 recruitment, which can activate Fas associated death domain protein (FADD) to induce apoptosis. IRAK1 can also activate tumor necrosis factor-related factor 6 (TRAF6) and induce the expression of a series of downstream specific genes. IRAK1 is an essential factor in the induction of mitochondrial division and necroptosis. In the current study, RNAi technique was used to silence IRAK1, and the apoptosis and necroptosis rate of SK-Hep1 cells were detected by flow cytometry. The apoptosis and the necroptosis pathway of hepatoma SK-Hep1 cells were blocked separately, and the expressions of FADD, RIP1 and TRAF6 genes were silenced separately. The results showed when the expression of IRAK1 was down-regulated, the apoptosis and necroptosis rate of SK-Hep1 cells were significantly increased. With silenced FADD, RIP1 and TRAF6, respectively, the expression of IRAK1 protein had no significant change. However, the expression of IRAK1 mRNA decreased significantly (p < 0.01) after the silencing of RIP1 and TRAF6 genes, while the IRAK1 mRNA did not change significantly after the silencing of FADD genes; when z-VAD-FMK was interfered, the expression of IRAK1 mRNA decreased significantly after the silencing of TRAF6 genes, while the IRAK1 mRNA did not change significantly after the silencing of FADD and RIP1genes. The study shows that RAK1 gene inhibits apoptosis and necroptosis in SK-Hep1 cells. TRAF6 gene affected the role of IRAK1 in apoptosis and necroptosis, RIP1 gene affected the role of IRAK1 in apoptosis, while FADD gene did not affect the role of IRAK1 in apoptosis and necroptosis.

C-FLIP, a Novel Biomarker for Cancer Prognosis, Immunosuppression, Alzheimer's Disease, Chronic Obstructive Pulmonary Disease (COPD), and a Rationale Therapeutic Target.

Dysregulation of c-FLIP (cellular FADD-like IL-1β-converting enzyme inhibitory protein) has been shown in several diseases including cancer, Alzheimer’s disease, and chronic obstructive pulmonary disease (COPD). c-FLIP is a critical anti-cell death protein often overexpressed in tumors and hematological malignancies and its increased expression is often associated with a poor prognosis. c-FLIP frequently exists as long (c-FLIPL) and short (c-FLIPS) isoforms, regulates its anti-cell death functions through binding to FADD (FAS associated death domain protein), an adaptor protein known to activate caspases-8 and -10 and links c-FLIP to several cell death regulating complexes including the death-inducing signaling complex (DISC) formed by various death receptors. c-FLIP also plays a critical role in necroptosis and autophagy. Furthermore, c-FLIP is able to activate several pathways involved in cytoprotection, proliferation, and survival of cancer cells through various critical signaling proteins. Additionally, c-FLIP can inhibit cell death induced by several chemotherapeutics, anti-cancer small molecule inhibitors, and ionizing radiation. Moreover, c-FLIP plays major roles in aiding the survival of immunosuppressive tumor-promoting immune cells and functions in inflammation, Alzheimer’s disease (AD), and chronic obstructive pulmonary disease (COPD). Therefore, c-FLIP can serve as a versatile biomarker for cancer prognosis, a diagnostic marker for several diseases, and an effective therapeutic target. In this article, we review the functions of c-FLIP as an anti-apoptotic protein and negative prognostic factor in human cancers, and its roles in resistance to anticancer drugs, necroptosis and autophagy, immunosuppression, Alzheimer’s disease, and COPD.

Combined effects of 17β-estradiol and exercise training on cardiac apoptosis in ovariectomized rats.

BackgroundThe purpose of this study was to investigate the combined 17β-estradiol (E2) and exercise training on cardiac pro-survival and anti-apoptotic pathways in ovariectomized rats.MethodsFifty-six female Sprague–Dawley rats were divided into a sham-operated (Sham), a bilaterally ovariectomized (OVX), an OVX treated with E2 (OVX-E2; 10μg/kg/day), and an OVX with E2 and treadmill exercise training (OVX-E2-EX; 60 min/day, 5 days/week) for 10 weeks. Following 10 weeks of exercise training, rat hearts were isolated for the evaluation of Histopathological analysis, TUNEL assay, and Western blotting.ResultsThe protein levels of estrogen receptor α (ERα), estrogen receptor β (ERβ), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-phosphatidylinositol 3-kinase (p-PI3K) (estrogen receptors/IGF-1-related survival pathway) were significantly increased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. The protein levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL) and phosphorylated-Bad (p-Bad) (Bcl-2 family survival pathway) were significantly increased in the OVX-E2-EX group when compared with the OVX group. Only the p-Bad was significantly increased in the OVX-E2 group when compared with the OVX group. The protein levels of truncation of Bid (t-Bid), Bcl-2-associated death promotor (Bad), Bcl-2-associated X protein (Bax), Cytochrome c, caspases-9, and caspases-3 (mitochondria-dependent apoptotic pathway), as well as the protein levels of tumor necrosis factor-α (TNF-α), Fas ligand, Fas receptors, Fas-associated death domain (FADD), activated caspase-8 and activated caspase-3 (Fas receptor–dependent apoptotic pathway) were significantly decreased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. Furthermore, when compared with the OVX-E2 group, the protein levels of ERβ, IGF-1, IGF-1R, Bcl-2 and Bcl-xL were further enhanced in the OVX-E2-EX group as well as the protein levels of Cytochrome c, Fas receptors, FADD, activated caspase-8, activated caspase-9 and activated caspase-3 were further decreased in the OVX-EX-E2 group.ConclusionsCombined E2 and exercise training exhibited a positive effect of protection on ovariectomy-induced cardiac apoptosis by enhancing ERβ-related survival pathways, which might provide a more effective therapeutic effect on cardiac protection in bilaterally oophorectomized or menopausal women than E2 treatment only.

An RNA Aptamer Targeting the Receptor Tyrosine Kinase PDGFRα Induces Anti-tumor Effects through STAT3 and p53 in Glioblastoma

Human glioblastoma (GBM) is the most aggressive malignancy of the CNS, with less than 5% survival. Despite great efforts to find effective therapeutics, current options remain very limited. To develop a targeted cancer therapeutic, we selected RNA aptamers against platelet-derived growth factor receptor α (PDGFRα), which is a receptor tyrosine kinase. One RNA aptamer (PDR3) with high affinity (0.25 nM) showed PDGFRα specificity and was internalized in U251-MG cells. Following treatment with the PDR3 aptamer, expression of the transcription factor STAT3 (signal transducer and activator of transcription 3) was inhibited, whereas the expression of the histone demethylase JMJD3 and the tumor suppressor p53 were upregulated. PDR3 also upregulated serine phosphorylation of p53, which subsequently mediated apoptosis through the death receptors: tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptors 1/2 (TRAIL-R1/R2), Fas-associated via death domain (FADD), and Fas. PDR3 significantly decreased cell viability in a dose-dependent manner. Furthermore, translocation of PDR3 into the nucleus induced hypomethylation at the promoters of cyclin D2. To assess the feasibility of targeted delivery, we conjugated PDR3 aptamer with STAT3-siRNA for a chimera. The PDR3-siSTAT3 chimera successfully inhibited the expression of target genes and showed significant inhibition of cell viability. In summary, our results show that well-tailored RNA aptamers targeting the PDGFRα-STAT3 axis have the potential to act as anti-cancer therapeutics in GBM.

Dimethyl Oxalyl Glycine a Prolyl Hydroxylase Inhibitor Decreases Early Gastrointestinal Gvhd Via Two Independent Pathways of Apoptosis after Allogeneic Hematopoietic Cell Transplantation

Introduction: Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges as they significantly contribute to treatment related morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that use of the PHI dimethyl oxalyl glycine (DMOG) reduces GVHD after murine allogeneic HCT. We already showed that DMOG treatment of allogeneic recipients resulted in significantly improved survival by day +50 compared to allogeneic controls, and was associated with significant decrease in organ pathology and proinflammatory cytokines (data not shown). We now expanded our study to better understand the underlying mechanisms for this observation.Methods: Acute GVHD was induced in lethally irradiated BALB/c mice followed by the infusion of 5 million bone marrow cells mixed with 4 million splenocytes from allogeneic C57BL/6 donors. DMOG dissolved in PBS was given at 8mg per mice intraperitoneally while controls received PBS only. Animals were monitored for clinical GVHD and survival and analyzed at day +7 and at day +50 post HCT. Formalin-fixed paraffin sections were processed for immunohistochemistry with CD3, Hif-1 alpha, or FADD pSer191 antibodies. Western blot analysis was performed after CD3/CD28 antibody activation of T cells to see the effect of DMOG treatment on Hif-1 α expression. Effect of DMOG on apoptosis and CD137 expression was analyzed by flow cytometry.Results: Our results demonstrate that the protective effect of DMOG is mainly caused by two independent pathways of apoptosis. DMOG treatment of allogeneic recipients resulted in decreased T cells infiltration (p=0.0534) and (Fas associated death domain) FADDpSer191 level (p<0.05) in small intestine sections at day +50. Decreased FADD pSer191 indicates decreased TNF-a induced epithelial cell apoptosis. This was associated with increased Hif1- α expression at day+7 (p<0.05) as well day+50 (p<0.05) as shown by histochemical analysis of small intestine sections. DMOG treatment of activated T cells showed increased expression of CD137 (p<0.005) and Annexin V FITC (p<0.005) staining suggesting that DMOG subjects T cells to activation induced cell death (AICD) via induced CD137 expression. Microscopic scanning of DMOG treated T cells showed condensed chromatin along with numerous fragmented nuclei, indicative of apoptotic cell. In vitro proliferative T cell responses were suppressed (p<0.05) in the presence of DMOG in a dose-dependent manner along with increase in Hif1- α level (p<0.05) as analyzed by western blot.Conclusion: Our data demonstrates protective effects of DMOG on the GI tract early after allogeneic HCT which is associated with improved survival and reduced lung injury at later time point. DMOG treatment results in the induction of CD137 on T cells subjecting them to AICD and represses FADD level in epithelial cells regulating their apoptosis. Taken together, DMOG treatment reduces GVHD via a two pronged mechanism, possibly allowing for novel strategies to prevent and treat this deleterious disease. DisclosuresPalaniyandi:Pharmacyclics: Research Funding; Takeda Pharmaceutical International, Inc.: Research Funding; JAZZ: Research Funding.

Hepatitis B Spliced Protein (HBSP) Suppresses Fas-Mediated Hepatocyte Apoptosis via Activation of PI3K/Akt Signaling.

Hepatitis B spliced protein (HBSP) is known to associate with viral persistence and pathogenesis; however, its biological and clinical significance remains poorly defined. Acquired resistance to Fas-mediated apoptosis is thought to be one of the major promotors for hepatitis B virus (HBV) chronicity and malignancy. The purpose of this study was to investigate whether HBSP could protect hepatocytes against Fas-initiated apoptosis. We showed here that HBSP mediated resistance of hepatoma cells or primary human hepatocytes (PHH) to agonistic anti-Fas antibody (CH11)- or FasL-induced apoptosis. Under Fas signaling stimulation, expression of HBSP inhibited Fas aggregation and prevented recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 (or FADD-like interleukin-1β-converting enzyme [FLICE]) into the death-inducing signaling complex (DISC) while increasing recruitment of cellular FLICE-inhibitory protein L (FLIPL) into the DISC. Those effects may be mediated through activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway as evidenced by increased cellular phosphatidylinositol (3,4,5)-trisphosphate (PIP3) content and PI3K activity and enhanced phosphorylation of mTORC2 and PDPK1 as well as Akt itself. Confirmedly, inhibition of PI3K by LY294002 reversed the effect of HBSP on Fas aggregation, FLIPL expression, and cellular apoptosis. These results indicate that HBSP functions to prevent hepatocytes from Fas-induced apoptosis by enhancing PI3K/Akt activity, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.IMPORTANCE Our study revealed a previously unappreciated role of HBSP in Fas-mediated apoptosis. The antiapoptotic activity of HBSP is important for understanding hepatitis B virus pathogenesis. In particular, HBV variants associated with hepatoma carcinoma may downregulate apoptosis of hepatocytes through enhanced HBSP expression. Our study also found that Akt is centrally involved in Fas-induced hepatocyte apoptosis and revealed that interventions directed at inhibiting the activation or functional activity of Akt may be of therapeutic value in this process.