DEDD is a member of the death-effector domain protein family. DEDD inhibits the Smad3 mediated transcriptional activity and participates in the regulation of apoptosis. In this study, how the death-effector domain of DEDD participates in the regulation of Smad3 activity and apoptosis has been further investigated. Immunoblotting, immunofluorescence and immunoprecipitation had been used to detect the effects of the full length DEDD and its two truncated mutants, N-DEDD and C-DEDD on Smad3 subcellular distribution, phosphorylation, and interaction between Smad4. The effects of the full length DEDD and its two truncated mutants on cell apoptosis and proliferation had also been explored by flow cytometry and MTT assay. It showed that DEDD and N-DEDD inhibit TGF-beta1 induced Smad3 nuclear translocation and the formation of Smad3-Samd4 complex. DEDD and its two mutants can induce cell apoptosis and inhibit cell proliferation. These results suggested that DEDD inhibits the activity of Smad3 through its death-effector domain. Both the two truncated mutants of DEDD participate in the regulation of apoptosis and cell proliferation.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; DNA-Binding Proteins ; pharmacology ; Death Domain Receptor Signaling Adaptor Proteins ; pharmacology ; HEK293 Cells ; Hep G2 Cells ; Humans ; Phosphorylation ; drug effects ; Protein Binding ; Smad3 Protein ; metabolism ; Smad4 Protein ; metabolism

OBJECTIVETo investigate the changes in the expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the articular cartilage of patients with Kashin-Beck disease (KBD) and the role of these proteins in the pathogenesis of KBD.

METHODSThe cartilage samples were collected from patients with established diagnosis of KBD and osteoarthritis and from healthy control subjects undergoing amputation due to traffic accidents. The expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the cartilage were detected by immunohistochemistry, and the positive chondrocytes were counted in different layers of the articular cartilage under microscope.

RESULTSThe positivity rates of FADD in the middle layer of articular cartilage from patients with KBD [(28.68∓2.19)%] and osteoarthritis [(35.40∓2.34)%] were significantly higher than that in normal cartilage [(10.51∓5.02)%, F=16.245, P=0.000], but the rates in the upper and deeper layers were comparable among the 3 groups (P=0.206-0.761). In KBD cartilage, FADD expression was the highest in the middle layer [(28.68∓5.38)%] followed by the deeper layer [(17.94∓8.38)%]. Compared with the healthy controls, KBD and osteoarthritis patients showed significantly higher FLIP expression in the upper layer of the cartilage (F=5.929, P=0.018) but similar expressions in middle and deeper layers.

CONCLUSIONSKBD patients have significant increased FADD expression in the middle layer but decreased FLIP expression in the upper layer of the cartilage, suggesting that the death receptor pathway and its regulators play important roles in the pathogenesis of KBD.

CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Cartilage, Articular ; metabolism ; pathology ; Case-Control Studies ; Fas-Associated Death Domain Protein ; metabolism ; Humans ; Immunohistochemistry ; Kashin-Beck Disease ; metabolism ; pathology

OBJECTIVETo investigate the mRNA expressions of the TNF adapter proteins, including TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) of lupus nephritis (LN) patients of various TCM asthenia syndromes. Methods Fifty-one inpatients with LN were differentiated according to TCM syndrome differentiation, 13 cases of yin-deficiency with inner heat syndrome (A); 26 cases of both qi-yin deficiency syndrome (B), 12 cases of Pi-Shen yang-deficiency syndrome (C). Peripheral venous blood samples from the 51 LN patients and 17 healthy subjects were collected to separate PBMCs. The mRNA expressions of TNF adapter molecules (TRADD, FADD, RIP-1 and TRAF-2), as well as Caspase-3 and interleukin-1beta (IL-1beta) were analyzed by quantitative real-time PCR and the differences among them were compared.

RESULTS(1) As compared with the healthy subjects, expression of TRADD mRNA in patients of syndrome A, B and C was lowered to 0.54, 0.32, and 0.38-fold, respectively (P < 0.05, P < 0.01), showing insignificant difference among the three syndromes; (2) FADD mRNA lowered to 0.79, 0.62, and 0.72-fold respectively, only with significance shown in syndrome B (P < 0.05); (3) RIP-1 mRNA lowered to 0.79, 0.50, and 0.60-fold respectively with significance shown in syndrome B and C (P < 0.01, P < 0.05), and insignificant difference was shown among the three syndromes; (4) TRAF-2 lowered to 0.70, 0.52, and 0.50-fold respectively (P < 0.01, P < 0.01, P = 0.07), significance shown in syndrome B and C (P < 0.01), but with insignificant difference among the three; (5) Caspase-3 elevated in all patients of the three syndromes (all P < 0.01); (6) IL-1beta in syndrome A was apparently lower ed to the normal range and also lower than that in the other two syndromes (both P < 0.05).

CONCLUSIONSExpressions of TRADD, FADD, RIP-1 and TRAF-2 mRNA decreased in all the patients of various TCM asthenia syndromes, the decrement in patients of syndrome B and C was lesser than that in syndrome A. These abnormal low expressions of signal proteins might be the substantial bases for asthenia syndromes of LN patients, and the apoptotic signal mediated by them may involve in the formation of asthenia syndrome in LN.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Adolescent ; Adult ; Case-Control Studies ; Child ; Fas-Associated Death Domain Protein ; genetics ; metabolism ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lupus Nephritis ; blood ; Male ; Medicine, Chinese Traditional ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; metabolism ; TNF Receptor-Associated Death Domain Protein ; genetics ; metabolism ; TNF Receptor-Associated Factor 2 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Yang Deficiency ; blood ; Yin Deficiency ; blood ; Young Adult

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induces apoptosis in some cancer cells such as breast,prostate,lung,and colon cancer cells,but not normal cells.However,because the effects of TRAIL in gastric cancer cells is unclear,we undertook this study to clarify the effects of TRAIL and its mechanism. To assess the cytotoxicity of TRAIL,two human gastric cancer cell lines,SNU-484 and SNU601,were treated with TRAIL (0-200 ng/mL)in the presence or absence of cycloheximide (1 microgram/mL)for 24 h.Both SNU-484 and SNU-601 were sensitive to TRAIL-induced cell death in a dose-dependent manner.The combination of TRAIL (100 ng/mL)and cycloheximide (1 microgram/mL)for 24 h enhanced cell death and PARP cleavage by promoting activations of caspase-8, caspase-9,and caspase-3,relative to that of TRAIL alone.We further examined the expressions of death receptor 4 (DR4),death receptor 5 (DR5),and FLICE inhibitory protein (FLIP).Although DR4 and DR5 were expressed in both cell lines,the expression of long form (FLIPL )and short form (FLIPS )of FLIP were detected at the low levels. Overexpression of FLIPL or FLIPS in both cell lines rendered the cells resistant to TRAIL.Taken together,our results suggest that FLIP promotes human gastric cancer cell survival against TRAIL-induced apoptosis and is important modulator for TRAIL-induced cell death in human gastric cancer cells.
Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein ; Caspase 8 ; Cell Death ; Cell Line ; Cell Survival ; Colonic Neoplasms ; Cycloheximide ; Humans* ; Necrosis* ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Stomach Neoplasms*

Suppression of apoptosis is one of the hallmarks of carcinogenesis. Tumor cells endure apoptotic pressure by overexpressing several antiapoptotic proteins, and FLICE inhibitory protein (FLIP) is one of the important antiapoptotic proteins that have been shown to be overexpressed in various primary tumor cells. FLIP has two death-effector domains in tandem, mimicking the prodomain of procaspase-8. It is recruited to Fadd in death-inducing signaling complex, thereby preventing the activation of procaspase-8. To date, three isoforms of human cytosolic FLIP (c-FLIP) and six viral homologs (v-FLIP) have been identified. Recently, the crystal structure of v-FLIP MC159 was determined for the first time as an atomic-detail FLIP structure, which revealed that two death effector domains are packed tightly against each other mainly through conserved hydrophobic interactions. The overexpression of c-FLIP in tumor cells has been shown to be the determinant of the tumor's resistance to death ligands such as FasL and TRAIL. It has also been shown that the down-regulation of c-FLIP results in sensitizing resistant tumor cells. Therefore, the agents directly targeting c-FLIP at mRNA and protein levels are expected to be developed in near future and tested for the potential as a new class of anti-cancer drugs.
Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein/antagonists & inhibitors/chemistry/*metabolism ; Caspases/antagonists & inhibitors/metabolism ; Humans ; Neoplasms/*metabolism/pathology/*therapy ; Signal Transduction

OBJECTIVETo evaluate the effects of hypobaric hypoxia on the expressions of death receptor 5 (DR5) and cellular FLICE-like inhibitory protein (c-FLIP) and the distribution of c-FLIP in the rat testis.

METHODSForty adult male SD rats were randomly divided into four groups of equal number: normoxia control, 3 d hypoxia, 15 d hypoxia and 30 d hypoxia. The control rats were raised at 300 m above the sea level, while the latter three groups of rats in a hypobaric chamber at a simulated altitude of 4000 m for 5, 15 and 30 days, respectively. Then the expressions of DR5 and c-FLIP were detected by immunoblotting and the distribution of c-FLIP in the testis observed by immunofluorescence.

RESULTSThe expressions of DR5 were 2.04 +/- 0.11, 1.97 +/- 0.12 and 2.34 +/- 0.11 in the 3 d, 15 d and 30 d hypoxia groups, respectively, significantly higher than 1.78 +/- 0.09 in the normoxia group (P < 0.05). The expressions of c-FLIP were 0.87 +/- 0.03 and 0.74 +/- 0.07 in the 15 d and 30 d hypoxia groups, respectively, significantly lower than 1.03 +/- 0.02 in the normoxia group (P < 0.05).

CONCLUSIONSimulated hypobaric hypoxia at 4000 m above the sea level increased the expression of DR5 and inhibited that of c-FLIP in the rat testis.

Animals ; CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Hypoxia ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Testis ; metabolism

OBJECTIVE: To investigate the expression of apoptosis related protein cellular Fas associated death domain like interleukin 1 converting enzyme inhibitory protein (c-FLIP) in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway. METHODS: Sixty patients with rheumatoid arthritis were collected from Zhangzhou Affiliated Hospital of Fujian Medical University during January 2014 and June 2015, including 22 patients with low activities (DAS28<3.2), 20 patients with middle activities (3.2 ≤ DAS28 ≤ 5.1), and 18 patients with high activities (DAS28>5.1). And 25 healthy controls were also collected. The mRNA and protein expression levels of c-FLIP and the extrinsic apoptotic pathway related proteins Fas-associated protein with death domain (FADD), caspase-8 in PBMCs were detected by real-time RT-PCR and Western blot, respectively. Correlations between c-FLIP and FADD, caspase-8 in PBMCs were analyzed by pearson test. RESULTS: mRNA expression levels of c-FLIP, FADD and caspase-8 in PBMCs of patients with rheumatoid arthritis were all higher than those of healthy controls (all <0.05). mRNA expression levels of FADD and caspase-8 in patients with middle activities were significantly higher than those in patients with low activities (all <0.05), but the mRNA expression level of c-FLIP was not significantly higher than that in patients with low activities. mRNA expression level of c-FLIP in patients with high activities was higher than those in patients with middle or low activities (all <0.05), while the mRNA expression level of caspase-8 was lower than those in patients with middle or low activities (all <0.05). mRNA expression level of FADD in patients with high activities was higher than those in patients with low activities (<0.05). Pearson analysis showed that there was a positive correlation between c-FLIP and FADD mRNA expression (=0.323, <0.05), and negative correlation between c-FLIP and caspase-8 mRNA expression (-1.104, <0.05). The protein expression levels of c-FLIP and FADD in patients with middle activities were significantly higher than those in control group and patients with low or high activities (<0.05 or 0.01). The protein expression levels of caspase-8 in patients with middle and high activities were significantly higher than those in control group and patients with low activities (<0.05 or <0.01), and the protein expression level of caspase-8 in patients with high activities was higher than that in patients with middle activities (<0.05). CONCLUSIONS c-FLIP may be involved in the extrinsic apoptotic pathway in rheumatoid arthritis, and can provide reference for the evaluation of disease activities.
Apoptosis ; genetics ; Arthritis, Rheumatoid ; blood ; CASP8 and FADD-Like Apoptosis Regulating Protein ; genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Leukocytes, Mononuclear ; metabolism

OBJECTIVE: To investigate the expression of FLIP and PTEN in laryngeal Squamous cell carcinoma (LSCC), and the relationship between FLIP and PTEN. METHOD: The protein expression of FLIP and PTEN were examined by using immunohistochemical method in 45 cases of LSCC and 15 cases of para-carcinoma tissues. RESULT: FLIP protein positive expressive rate in the laryngeal squamous cell carcinoma cases (77.8%) was higher than that in the para-carcinoma tissues cases (33.3%, P < 0.05). The protein expression of FLIP was correlated with cervical lymph node metastasis, clinical stage and prognosis. On the other hand, PTEN positive expressive rate in the laryngeal squamous cell carcinoma (65.0%) was higher than that in the para-carcinoma tissues (0%, P < 0.01). The protein expression of PTEN was associated with tumor differentiation grade, clinical stage, cervical lymph node metastasis and prognosis. There was a negative relationship between the expression of FLIP and PTEN in LSCC. CONCLUSION The protein expression of FLIP may be an important prognostic marker for LSCC. The protein expression of PTEN was correlated with clinical stage, tumor differentiation grade, and cervical lymph node metastasis . Consequently, it could be used as a valuable marker for the prognosis of LSCC.
CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Neoplasm Staging ; PTEN Phosphohydrolase ; metabolism ; Prognosis

OBJECTIVETo explore the molecular mechanism via which the chemotherapeutic drug hydroxyurea (HU) enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

METHODSChronic myelogenous leukemia-derived K562 and SVT-35 cells were treated with recombinant soluble TRAIL (rsTRAIL) alone or combined with HU for a time course, and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl-2H-tetrazolium-phenazine methosulphate assay. Western blot was performed to analyze the activation of apoptosis-related protein kinases and the expression of apoptosis inhibitor molecules.

RESULTSThe survival rates of SVT-35 and K562 cells treated with 1 μg/ml rsTRAIL for 24 hours were 32% and 93%, respectively. HU significantly increased the sensitivity of K562 cells to rsTRAIL cytotoxicity. Combination of rsTRAIL and HU resulted in the phosphorylation of rat sarcoma (RAS), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase and in the significant reduction of apoptosis-inhibited molecule Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 in K562 cells.

CONCLUSIONSHU enhanced K562 cell sensitivity to rsTRAIL is mediated by Ras-MEK-ERK signaling pathway. Expression of antiapoptotic proteins cellular Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 is also down-regulated during this process. These results may through light on the therapeutic study of human chronic myelogenous leukemia.

Apoptosis ; drug effects ; physiology ; CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Humans ; Hydroxyurea ; pharmacology ; Inhibitor of Apoptosis Proteins ; metabolism ; K562 Cells ; MAP Kinase Signaling System ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

BACKGROUND: The expression of c-FLIP (cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme (FLICE)-inhibitory protein), which is a member of the family of inhibitors of apoptosis, has been associated with tumor development and progression. The aim of this study was to evaluate the expression of c-FLIP in gastric cancer and its correlation with tumor cell proliferation, apoptosis and the clinicopathologic features. METHDOS: Immunohistochemical staining with anti-c-FLIP antibody was performed in 98 tissue samples obtained from gastric cancer patients who underwent surgical treatment. The apoptotic cells were visualized by terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL), and the proliferative cells were visualized by staining with Ki-67 antibody. RESULTS: The positive expression of c-FLIP in the gastric cancer tissues was demonstrated in 57.1% of the cases. The expression of c-FLIP was increased in the gastric cancer tissues compared with the matched normal gastric mucosa. The expression of c-FLIP was significantly associated with histologic differentiation (p=0.038). However, there was no association between the c-FLIP expression and the other clinicopathological parameters, including patient survival. The Ki-67 labeling index (KI) for the 98 tumors ranged from 7.6 to 85.0 with a mean KI of 50.4+/-15.7. The mean KI value of the c-FLIP positive tumors was 54.1+/-15.3 and this was significantly higher than that of the c-FLIP negative tumors (p=0.005). The apoptotic index (AI) for the 98 tumors ranged from 0.0 to 10.0 with a mean AI of 7.4+/-2.3. There was no significant difference between the c-FLIP expression and the AI (p=0.347). CONCLUSIONS: These results suggest that the c-FLIP expression may be associated with tumor cell proliferation of gastric cancer.
Aged ; *Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics ; *Cell Proliferation ; Disease Progression ; Female ; Health Status Indicators ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; Male ; Middle Aged ; Pilot Projects ; Stomach Neoplasms/*genetics/pathology/physiopathology