Fas-associated Death Domain Protein Research Articles

Neurodegeneration and neuroinflammation: two processes, one target.

The etiology of neurodegenerative diseases is diverse, however most of them share common characteristics: accumulation of misfolded proteins, chronic and sustained neuroinflammation, and the dysfunction and death of certain populations of neurons. The brain of Alzheimer's disease (AD) patients presents amyloid plaques and aggregation of hyperphosphorylated tau. The latter is also present in neurodegenerative tauopathies and in Parkinson's disease (PD). Aggregates of α-synuclein is the characteristic hallmark of PD. In amyotrophic lateral sclerosis (ALS) the mutation of SOD1 promotes its accumulation; and the polyglutamine expansion in huntingtin protein favors its aggregation in Huntington's disease (HD). Thus, the initial proteinopathy could be responsible for triggering the activation of the immunological defenses in the nervous system, as it has been demonstrated in some cases. Microglia and astrocytes are the main glial cells involved in the innate inflammatory response in the central nervous system (CNS). These cells are capable of detecting danger signals, and when activated they secrete inflammatory mediators to try to protect or prevent damage. However, in some cases the inflammatory response becomes sustained by an amplified feedback of release of factors between microglia and astrocytes that further activate these cells. This promotes the recruitment of more glial cells that prolongs and up-regulates the neuroinflammatory response contributing to the progression of the disease (the review of Glass et al. (2010) outlines in detail the contribution of glial cells in neurodegenerative diseases). Neurodegeneration has a remarkable apoptotic component; sustained neuroinflammatory response along with the deregulation of protective mechanisms trigger neuronal death.

The apoptotic initiator caspase-8: its functional ubiquity and genetic diversity during animal evolution.

The caspases, a family of cysteine proteases, play multiple roles in apoptosis, inflammation, and cellular differentiation. Caspase-8 (Casp8), which was first identified in humans, functions as an initiator caspase in the apoptotic signaling mediated by cell-surface death receptors. To understand the evolution of function in the Casp8 protein family, casp8 orthologs were identified from a comprehensive range of vertebrates and invertebrates, including sponges and cnidarians, and characterized at both the gene and protein levels. Some introns have been conserved from cnidarians to mammals, but both losses and gains have also occurred; a new intron arose during teleost evolution, whereas in the ascidian Ciona intestinalis, the casp8 gene is intronless and is organized in an operon with a neighboring gene. Casp8 activities are near ubiquitous throughout the animal kingdom. Exogenous expression of a representative range of nonmammalian Casp8 proteins in cultured mammalian cells induced cell death, implying that these proteins possess proapoptotic activity. The cnidarian Casp8 proteins differ considerably from their bilaterian counterparts in terms of amino acid residues in the catalytic pocket, but display the same substrate specificity as human CASP8, highlighting the complexity of spatial structural interactions involved in enzymatic activity. Finally, it was confirmed that the interaction with an adaptor molecule, Fas-associated death domain protein, is also evolutionarily ancient. Thus, despite structural diversity and cooption to a variety of new functions, the ancient origins and near ubiquitous distribution of this activity across the animal kingdom emphasize the importance and utility of Casp8 as a central component of the metazoan molecular toolkit.

A long-awaited merger of the pathways mediating host defence and programmed cell death.

Historically, cell death and inflammation have been closely linked, but the necessary divergence of the fields in the past few decades has enriched our molecular understanding of the signalling pathways that mediate various programmes of cell death and multiple types of inflammatory responses. The fields have now come together again demonstrating a surprising level of integration. Intimate interconnections at multiple levels are revealed between the cell death and inflammatory signal transduction pathways that are mobilized in response to the engagement of pattern recognition receptors during microbial infection. Molecules such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, FAS-associated death domain protein (FADD), FLICE-like inhibitory protein (FLIP) and caspase 8 - which are associated with different forms of cell death - are incorporated into compatible and exceedingly dynamic Toll-like receptor, NOD-like receptor and RIG-I-like receptor signalling modules. These signalling modules have a high capacity to switch from inflammation to cell death, or a programmed execution of both, all in an orchestrated battle for host defence and survival.

Phosphoprotein enriched in astrocytes (PEA)-15: A potential therapeutic target in multiple disease states

Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse cellular processes, such as proliferation and apoptosis, dependent upon stimulation. Regulation of these processes occurs by virtue of the unique interaction of PEA-15 with other signalling proteins. PEA-15 acts as a cytoplasmic tether for the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) preventing nuclear localisation. In order to release ERK1/2, PEA-15 requires to be phosphorylated via several potential pathways. PEA-15 (and its phosphorylation state) therefore regulates many ERK1/2-dependent processes, including proliferation, via regulating ERK1/2 nuclear translocation. In addition, PEA-15 contains a death effector domain (DED) which allows interaction with other DED-containing proteins. PEA-15 can bind the DED-containing apoptotic adaptor molecule, Fas-associated death domain protein (FADD) which is also dependent on the phosphorylation status of PEA-15. PEA-15 binding of FADD can inhibit apoptosis as bound FADD cannot participate in the assembly of apoptotic signalling complexes. Through these protein–protein interactions, PEA-15-regulated cellular effects have now been investigated in a number of disease-related studies. Changes in PEA-15 expression and regulation have been observed in diabetes mellitus, cancer, neurological disorders and the cardiovascular system. These changes have been suggested to contribute to the pathology related to each of these disease states. As such, new therapeutic targets based around PEA-15 and its associated interactions are now being uncovered and could provide novel avenues for treatment strategies in multiple diseases.

FADD Is Upregulated in Relapsing Remitting Multiple Sclerosis

Objective: To elucidate the role of tumor necrosis factor (TNF) receptor signal transduction in multiple sclerosis (MS). Methods: We performed a cross-sectional analysis of the gene expression of TNF receptor-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD) in peripheral blood leukocytes of 23 relapsing remitting (RR), 19 secondary progressive (SP) and 12 primary progressive (PP) MS patients, as well as of 29 healthy controls by quantitative RT-PCR. Additionally, we monitored a subgroup of 15 RR MS patients longitudinally every 3 months over the time period of 9 months. Results: FADD expression was significantly elevated in RR MS patients compared to the other disease courses (p < 0.048). The median of FADD expression was elevated in the RR MS patient groups compared to the healthy group, but this was not significant (p < 0.053). The median of TRADD expression was elevated in the patient groups compared to the healthy group, but this was not significant (p < 0.14). Neither variable changed significantly over the time course of 9 months. Conclusion: FADD elevation in leukocytes might be interpreted as the molecular equivalent of an elevated general inflammatory activity in RR MS patients compared to other disease courses. FADD elevation in RR MS reinforces the concept that different pathophysiological and immunological processes sustain RR MS and SP or PP MS.

Phosphodiesterase 5 Inhibitors Enhance Chemotherapy Killing in Gastrointestinal/Genitourinary Cancer Cells

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill gastrointestinal/genitourinary cancer cells. In bladder cancer cells, regardless of H-RAS mutational status, at clinically achievable doses, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/mitomycin C/gemcitabine/cisplatin/paclitaxel to cause cell death. In pancreatic tumor cells expressing mutant active K-RAS, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/gemcitabine/paclitaxel to cause cell death. The most potent PDE5 inhibitor was sildenafil. Knock down of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of cellular FLICE-like inhibitory protein-short did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with sildenafil. Overexpression of B-cell lymphoma-extra large suppressed individual and combination drug toxicities. Knock down of CD95 or Fas-associated death domain protein suppressed drug combination toxicity. Combination toxicity was also abolished by necrostatin or receptor interacting protein 1 knock down. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy, which was maximal at ∼24 hour posttreatment, and 3-methyl adenine or knock down of Beclin1 suppressed drug combination lethality by ∼50%. PDE5 inhibitors enhanced and prolonged the induction of DNA damage as judged by Comet assays and γhistone 2AX (γH2AX) and checkpoint kinase 2 (CHK2) phosphorylation. Knock down of ataxia telangiectasia mutated suppressed γH2AX and CHK2 phosphorylation and enhanced drug combination lethality. Collectively our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for gastrointestinal/genitourinary cancers represents a novel modality.

Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains

The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 μM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

Ethanol-induced apoptosis in human liver adenocarcinoma cells (SK-Hep1): Fas- and mitochondria-mediated pathways and interaction with MAPK signaling system

For studying molecular mechanisms regulating the fate of ethanol-treated hepatocytes, involvement of Fas in ethanol-induced apoptosis was examined in human liver adenocarcinoma (SK-Hep1) cells in which the function of Fas-associated death domain (FADD) protein was knocked down by transfection. In FADD-knocked down cells, while ethanol-induced increase in generation of reactive oxygen species (ROS) was unaffected, apoptosis was significantly suppressed, demonstrating the involvement of Fas in ethanol-induced hepatocyte apoptosis more directly than in the past reports. On the other hand, effects of mitogen-activated protein kinase (MAPK), which is well known to determine the fate of various cells, on ethanol-induced apoptosis have not been examined in SK-Hep1 cells. Of three major MAPKs, only p38 MAPK and JNK were found activated by 200mM ethanol treatment. When cells were incubated with inhibitors of p38 MAPK and JNK, ethanol-induced apoptosis was decreased while ROS generation was unaffected, and examination of pro-apoptotic Bax and anti-apoptotic Bcl-2 levels showed decrease of the former and increase of the latter. We concluded that oxidative stress inflicted by ROS triggered Fas-mediated and mitochondria-mediated apoptotic pathways in ethanol-treated SK-Hep1 cells, and that p38 MAPK and JNK were promoting mitochondrial pathway, suggesting interaction between apoptosis and MAPK signaling systems.

FAS-associated death domain and tripartite motif-containing protein 21 negatively regulate virus-induced interferon production (P6126)

Abstract While induction of type I Interferon (IFN) during the host antiviral response is imperative for efficient viral clearance, regulation of this response is crucial for immune system homeostasis. We have reported that FADD, a well characterized apoptotic protein, is involved in negatively regulating RNA virus-induced IFN-α production through a novel interaction with the E3 ubiquitin ligase, Tripartite Motif-Containing Protein 21 (TRIM21). Interaction between FADD and TRIM21 cooperatively represses IFN-α activation in Sendai virus infected cells. This occurs through TRIM21 targeted ubiquitin-mediated degradation of transcription factor IRF7. Recently, it was also determined that FADD and TRIM21 can be found in 2 different complexes within cells: one containing FADD/TRIM21/IRF7 and a second containing FADD/TRIM21/RIP1 (receptor-interacting protein 1). We hypothesized that TRIM21 may have different functions dependent on complex composition and that its activity could be regulated via its autoubiquitination status. Interaction of TRIM21 with FADD yields increased autoubiquition while interaction with RIP1 yields decreased autoubiqtuitination suggesting a regulation mechanism. However, studies involving mutant forms of TRIM21 which lack autoubiquitination demonstrate that autoubiquitination status does not regulate TRIM21’s ability to target IRF7 for degradation. We are currently investigating the function of TRIM21 in other FADD/RIP1 dependent processes like cell death.

Overexpression and Biological Characterization of the Death Domain Complex between TRADD and FADD

The tumor necrosis factor-receptor 1 (TNFR1)-associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain. TRADD is known to interact directly with TNF receptor 2 (TNFR2) and the Fas-associated death domain protein (FADD), which are signal transducers that activate NF-<TEX>${\kappa}B$</TEX> and induce apoptosis, respectively. To date, there has been no structural information on the TRADD and FADD death domain (DDs) complex. In this study, the death domains of TRADD and FADD were co-expressed and purified from Escherichia coli for structural characterization. We found that human TRADD (hTRADD) interacted strongly with mouse FADD (mFADD) via their DDs and interacted weakly with human FADD (hFADD)-DD. Moreover, the structures of the TRADD-DD:FADD-DD complexes were separately modeled from predicted structures in the protein data bank (PDB). The results of this study will have important applications in human diseases such as cancer, AIDS, degenerative and autoimmune diseases, and infectious diseases.

Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by tannic acid and resveratrol

Tannic acid (TA), a plant polyphenol, has been described as having anticarcinogenic, antioxidant, antimutagenic, antimicrobial, antiallergic, antiinflammatory and astringent properties. Resveratrol (RES), a phytoalexin phenolic compound, has been known as an antioxidant, anticarcinogenic, antiinflammatory, antimutagenic, antiproliferative, antiviral, antibacterial, estrogenic and vasodilator agent. Although molecular mechanisms of TA and RES responsible for anticancer activity are yet to be known, there are in vitro studies indicating that these substances contribute to anticancer activity by inducing apoptosis. In this study, the effects of TA and RES in MCF-7 breast cancer cells on apoptotic index, FAS-associated death domain (FADD) and Bak proteins were investigated. Terminal transferase-mediated dUTP nick end-labeling (TUNEL) method was applied for apoptosis and immunohistochemical staining for FADD and Bak proteins. Our study results show that TA and RES increased apoptotic index, percentage ratios of apoptotic proteins Bak and FADD in MCF-7 cell line. Keywords : Bak, Fas-associated death domain (FADD), MCF-7, resveratrol, tannic acid African Journal of Biotechnology Vol. 12(12), pp. 1431-1437

AXL Mediates TRAIL Resistance in Esophageal Adenocarcinoma

The overexpression of AXL receptor tyrosine kinase is a frequent finding that has been associated with poor prognosis in esophageal adenocarcinoma (EAC). As the majority of EAC are intrinsically resistant to DNA-damaging therapies, an alternative therapeutic approach based on the activation of death receptors may be warranted. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been evaluated in clinical trials and found promising as anticancer agent with mild side effects; unfortunately, resistance to TRAIL remains a major clinical problem. Herein, we explored the role of AXL in TRAIL resistance and elucidated the underlying mechanism. Overexpression of AXL in OE33 and OE19 cells promoted cell survival and attenuated TRAIL-induced cellular and molecular markers of apoptosis. In contrast, knockdown of endogenous AXL sensitized FLO-1 cells to TRAIL. The mechanism by which AXL regulates TRAIL resistance was examined. Protein and mRNA expression of DR4 and DR5 death receptors was not downregulated by AXL. In addition, the possible involvement of FLICE-inhibitory protein (FLIP) in regulating the interaction of caspase-8 with Fas-associated death domain protein (FADD) was excluded, as AXL did not enhance FLIP expression or FLIP/FADD association. Alternatively, protein association of AXL with DR5, independent of TRAIL, was confirmed, suggesting that AXL could regulate DR5 receptor activity. The AXL/DR5 association had no negative effect on TRAIL-induced interaction with FADD. However, the AXL/DR5 interaction blocked the recruitment of caspase-8 to the death-inducing signal complex (DISC). Collectively, our findings uncover a novel mechanism of TRAIL resistance mediated by AXL through regulation of the DISC and provide strong evidence that AXL could be exploited as a therapeutic target to circumvent TRAIL resistance.

Phosphorylation Status of Fas-associated Death Domain Protein Is Associated With Biochemical Recurrence After Radical Prostatectomy

To assess whether the phosphorylated Fas-associated death domain protein (FADD) at 194 serine (p-FADD) is valuable as a marker of biochemical recurrence in hormone-naive patients who had undergone radical prostatectomy. We used radical prostatectomy specimens from 106 patients. None of the patients had received neoadjuvant or adjuvant therapy. The percentage of positive p-FADD cells (nuclear staining) was immunohistochemically evaluated. The correlation between FADD phosphorylation and the clinicopathologic parameters was assessed. The correlation between the biochemical recurrence-free rate and the p-FADD expression level was analyzed using the Kaplan-Meier method. Overall, 39 patients developed biochemical recurrence. We investigated the expression of p-FADD in 106 patients with prostate cancer using immunohistochemistry. We compared our findings with the clinicopathologic parameters, including the follow-up data. Patients with a greater positive p-FADD rate had a significantly lower biochemical recurrence rate than those with a lower positive p-FADD rate (P < .001). A significant inverse correlation was found between the positive p-FADD rate and the Gleason score. A low expression of p-FADD could be a predictor of biochemical recurrence in hormone-naive patients who have undergone radical prostatectomy.

P38 MAPK/PP2Acα/TTP pathway on the connection of TNF-α and caspases activation on hydroquinone-induced apoptosis

This study investigated tumor necrosis factor-α (TNF-α)-mediated death pathway contribution to hydroquinone (HQ) cytotoxicity in human leukemia U937 cells. HQ-induced apoptosis of human leukemia U937 cells was characterized by the increase in mitochondrial membrane depolarization, procaspase-8 degradation and tBid production. Downregulation of Fas-associated death domain protein (FADD) blocked HQ-induced procaspase-8 degradation and rescued the viability of HQ-treated cells, suggesting the involvement of a death receptor-mediated pathway in HQ-induced cell death. HQ induced increased TNF-α mRNA stability led to TNF-α protein expression upregulation, whereas HQ suppressed TNF-α-mediated NFκB pathway activation. HQ elicited protein phosphatase 2A catalytic subunit α (PP2Acα) upregulation via p38 mitogen-activated protein kinase (MAPK)-mediated CREB/c-Jun/ATF-2 phosphorylation, and PP2Acα upregulation was found to promote tristetraprolin (TTP) degradation. Suppression of p38 MAPK activation and protein phosphatase 2A (PP2A) activity abrogated TNF-α upregulation and procaspase degradation in HQ-treated cells. Overexpression of TTP suppressed HQ-induced TNF-α upregulation and restored the viability of HQ-treated cells. Moreover, TTP overexpression increased TNF-α mRNA decay in HQ-treated cells. Taken together, our data indicate that HQ elicits TNF-α upregulation via p38 MAPK/PP2A-mediated TTP downregulation, and suggest that the TNF-α-mediated death pathway is involved in HQ-induced U937 cell death. The same pathway was also proven to be involved in the HQ-induced death of human leukemia HL-60 and Jurkat cells.

The expression pattern of antiapoptotic protein c-FLIP in psoriatic epidermis

Background Psoriasis is a chronic, relapsing, inflammatory, and hyperproliferative disease. The pathological changes affecting skin in psoriasis are mainly driven by abnormal differentiation secondary to activation of T cells. Keratinocytes may undergo apoptosis by loss of cell-cell contact after crosslinking of the FAS (CD95) molecule. The epidermal hyperplasia in psoriasis has been implicated to be a result of suppression of apoptosis. A key inhibitor of death receptor signaling is cellular FLICE inhibitory protein (c-FLIP), which interacts with FAS-associated death domain protein (FADD) and procaspase-8 preventing it from binding to caspase-8, thereby inhibiting the initiation of the apoptotic cascade. Aim of work This study was designed to investigate the expression pattern of antiapoptotic molecule c-FLIP in psoriasis vulgaris patients. Materials and methods Twenty psoriasis vulgaris patients were enrolled in this study. All patients were subjected to assessment of the degree of severity using PASI score. A 2-mm punch biopsy was taken from each patient from lesional and nonlesional areas for evaluation of c-FLIP mRNA gene expression using RT-PCR. Results We found that there was a direct significant relationship between psoriasis and c-FLIP protein levels measured. The study also showed that the discrimination of the degree of severity of psoriasis using psoriatic skin c-FLIP mRNA level can be reliable, with 100% sensitivity, 81.2% specificity, and 85% accuracy. Conclusion Increased levels of c-FLIP might induce upregulation of keratinocyte proliferation and downregulation of the apoptotic signaling pathway in psoriatic epidermis. c-FLIP might be a potential new target for preventing keratinocyte proliferation in psoriasis in the future.

Curcumin and its emerging intraocular benefits

I read with great interest the recent article “Curcumin inhibits proliferation of human lens epithelial cells: a proteomic analysis” by Hu et al. (2012), published in Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology). Curcumin may be of significant benefit in other optic disorders besides cataracts. Curcumin exerts anti-neoplastic effects in eye tumors such as retinoblastomas. It mediates these anti-cancer effects by altering the microRNAs (miRNAs) expression profile. Overall, sixteen miRNAs are down-regulated. Simultaneously five miRNAs, especially miR-22, are up-regulated in the retinoblastomas cells (Sreenivasan et al., 2012). The target gene of miR-22 is erythoblastic leukemia viral oncogene homolog 3 (Erbb3). Curcumin attenuates reactive oxygen species (ROS) levels and thereby affords protection to retinal pigment cells from oxidation injury. These protective effects are mediated by curcumin induced up-regulation of heme oxygenase-1 (HO-1) (Woo et al., 2012). The highest HO-1 expression is seen at a concentration of 15 μmol/L (Mandal et al., 2009). Diabetic retinopathy can be attenuated by curcumin by virtue of its anti-oxidant properties. It also indices hypoglycemia thus proving to be of further benefit in mitigating the development of diabetic retinopathy (Gupta et al., 2011). Curcumin decreases the elevations in vascular endothelial growth factor noted in diabetic retinopathy. Ischemia/reperfusion injury in the retina is attenuated by curcumin administration. It mediates these protective effects by decreasing the activation of signal transducers and activators of transcription 3 (STAT3) (Wang et al., 2011). Simultaneously, activation of nuclear factor-κB (NF-κB) is also inhibited and further augments the protective effects of curcumin. As a result, it decreases vascular as well as neuronal degeneration in the retina. Up-regulation of p-IκBα and MCP-1 is also inhibited. These protective effects are even seen 48 h following the ischemic injury. Curcumin also may be effective in the therapeutic management of retinitis pigmentosa. Retinitis pigmentosa secondary to P23H rhodopsin mutation may especially be amenable to curcumin therapy. Curcumin mediates these effects by decreasing stress on the endoplasmic reticulum and by improving gene expression in the retina as well as by improving retinal morphology (Vasireddy et al., 2011). Curcumin also dissociates protein aggregates and restores the expression of NF-κB thus further improves the pathology in retinitis pigmentiosa. These protective effects are reversed by the application of NF-κB inhibitory peptides. Curcumin is also of benefit in proliferative vitreoretinopathy (Alex et al., 2010). It mediates these effects by inducing caspase 3 dependent apoptosis. Curcumin has an attenuatory effect on Bcl-2 and augments Bax levels (Lu et al., 2009). It also accentuates Fas-associated death domain protein (FADD) and Fas levels at the same time. As a result there is increased G2/M phase arrest. The above examples clearly illustrate the significant benefits of curcumin in intraocular pathologies and the need for further studies in this regard.

톳(Hizikia fusiformis) 당단백질에 의한 HepG2 세포 증식 억제기전

Hizikia fusiformis, a brown alga that is widely consumed in Korea, Japan, and China, possesses a number of potentially beneficial compounds, including antioxidants and anticoagulants. However, the molecular mechanisms of H. fusiformis in hepatoma cells have not been elucidated. This study investigated the antiproliferative effect and mechanism of action of a glycoprotein from H. fusiformis (HFGP) in HepG2 human hepatoma cells. In an MTS assay, 25 HFGP inhibited the proliferation of HepG2 cells by . HFGP caused the dose-dependent growth inhibition of HepG2 cells by inducing apoptosis and a sub-G1 phase arrest. The antiproliferative activity of HFGP was confirmed based on the expression of several apoptosis-related proteins, which was assessed by Western blot analysis. The expressions of Fas, Fas-associated death domain protein, Bax, and Bad was significantly up-regulated in HFGP-treated cells, and HFGP induced the translocation of Bax to mitochondria and the release of cytochrome c into the cytosol. Therefore, HFGP might be useful in the treatment of liver cancer.

Postexposure Application of Fas Receptor Small-Interfering RNA to Suppress Sulfur Mustard–Induced Apoptosis in Human Airway Epithelial Cells: Implication for a Therapeutic Approach

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 µM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.

Vesicles Released by Activated T Cells Induce Both Fas-Mediated RIP-Dependent Apoptotic and Fas-Independent Nonapoptotic Cell Deaths

Activated T cells secrete Fas ligand (FasL)-containing vesicles (secreted vesicles) that induce death of target cells. We provide evidence that secreted vesicles from culture supernatants (Csup) of various origins are able to generate both Fas-dependent apoptotic and Fas-independent, nonapoptotic cell death. In the absence of Fas, the nonapoptotic, Fas-independent pathway could still induce cell death. In contrast to RIP-independent classical Fas-induced cell death triggered by cross-linked or membrane-bound FasL, CSup-derived stimuli-induced apoptosis exhibited unique molecular and enzymatic characteristics. It could be partially inhibited by blocking cathepsin D enzyme activity and required the presence of RIP. Whereas stimulation with CSup, derived from both FasL-overexpressing Jurkat cells and PBMC, could induce cell death, the requirements for Fas-associated death domain protein and caspase-9 were different between the two systems. Our study highlights an important distinction between cell contact-mediated and secreted vesicle-generated activation-induced cell death and also demonstrates that the type of the secreted vesicles can also modify the cell death route. We propose that besides cell-to-cell interaction-mediated Fas triggering, stimuli induced by secreted vesicles can mediate important additional cell death signals regulating activation-induced cell death under physiological conditions.