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

PURPOSE: Heat shock protein (HSP) 27 protects the cell by controlling apoptosis and immune reactions, and c-FLIP (cellular-FLICE inhibitory protein) inhibits apoptosis by inhibiting caspase-8 activity. We investigated the relationship of HSP27 and c-FLIP expression to prostate-specific antigen, Gleason score sum (GSS), and pathologic stage. MATERIALS AND METHODS: Samples from 163 patients between May 2004 and April 2010 were analyzed: 83 from patients that had underwent a radical prostatectomy, and 80 from those that underwent transurethral resection of the prostate to alleviate urinary symptoms from benign prostate hyperplasia. c-FLIP and HSP27 expression were observed by immunohistochemistry staining. Samples with less than 5% expression-positive cells were scored as 1, with 5%-50% were scored as 2, and with more than 50% were scored as 3. Local reactions were identified as 0.5 and evaluated. RESULTS: Both the presence of HSP27 within the tumor and the number of cancer cells positive for HSP27 were significantly correlated to GSS and pathologic stage (p<0.001, p=0.001, p<0.001, p<0.001). The same was true for c-FLIP expression (p<0.001). GSS was more highly correlated to HSP27 expression than to c-FLIP expression (r=0.814 for HSP27, r=0.776 for c-FLIP), as was pathologic stage (r=0.592 for HSP27, r=0.554 for c-FLIP). CONCLUSIONS: In prostate cancer, higher GSS and a more advanced pathologic stage were associated with a higher likelihood of having a HSP27-positive tumor and more HSP27-positive tumor cells. HSP27 expression was correlated with GSS and prostate cancer stage. A more advanced pathologic stage corresponded to a higher likelihood of having a c-FLIP-positive tumor and more c-FLIP-positive tumor cells. HSP27 expression had a higher correlation with prostate cancer stage and GSS than c-FLIP expression did.
Aged ; Biomarkers, Tumor/*metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/*metabolism ; HSP27 Heat-Shock Proteins/*metabolism ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Proteins/metabolism ; Neoplasm Staging ; Prostatectomy/methods ; Prostatic Hyperplasia/metabolism/surgery ; Prostatic Neoplasms/*metabolism/pathology/surgery ; Transurethral Resection of Prostate

OBJECTIVETo investigate the expression of apoptotic regulator c-FLIP(L) in invasive breast carcinoma tissues, and to evaluate its correlation with molecular subtyping and clinical prognosis.

METHODSImmunohistochemistry using EnVision staining for c-FLIP(L) was performed in 264 cases of invasive breast carcinomas and matched adjacent normal breast tissue samples from January 1996 to December 1999. ER, PR, HER2, Ki-67, CK5/6 and EGFR were evaluated by immunohistochemistry in order to classify the tumors into five molecular subtypes and the difference of c-FLIP(L) expression in these molecular subtypes was also analyzed. The influence of c-FLIP(L) expression on prognosis was evaluated by Kaplan-Meier curves and multi-factor Cox proportional risk model.

RESULTSHigh expression of c-FLIP(L) was observed in 84.5% (223/264) of cases of invasive breast carcinomas which were significantly higher than the 45.1% (119/264) of cases in adjacent normal epithelium of breast (χ² = 89.78, P = 0.000). The expression of c-FLIP(L) in luminal B (HER2 positive) and basal-like breast cancers was 78.1% (25/32) and 46.2% (18/39), respectively, with significant difference (P < 0.05). Moreover, the expression of c-FLIP(L) in luminal B (HER2 positive) was higher than in luminal A cancers (P < 0.05), and the expression of c-FLIP(L) in HER2 positive cancers was higher than in basal-like cancers (P < 0.01). C-FLIP(L) showed deep yellow staining in node positive breast cancer with a high-expression rate of 93.1% (134/144); whereas the expression was sporadic and light yellow in node negative breast cancer with a lower high-expressed rate of 72.5% (87/120, P < 0.01). C-FLIP(L) expression had significant influence on disease-free survival time, with c-FLIP(L)-positive patients showing poor prognosis (P < 0.01). Multi-factor Cox proportional risk model analysis showed that expression of c-FLIP(L), lymph nodes status and molecular subtypes were independent prognostic factors for invasive breast carcinomas (P < 0.05).

CONCLUSIONSC-FLIP(L) is highly expressed in invasive breast carcinomas, and its expression level is closely related to the molecular subtypes and clinical prognosis of breast cancer patients. Thus, c-FLIP(L) could be used as an important tumor marker for personalized cancer therapy and prognostic prediction.

Aged ; Biomarkers, Tumor ; metabolism ; Breast ; metabolism ; Breast Neoplasms ; classification ; metabolism ; mortality ; CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Disease-Free Survival ; Female ; Humans ; Immunohistochemistry ; Prognosis ; Receptor, ErbB-2 ; metabolism

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

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

This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspase-dependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the death-inducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IkappaB degradation and nuclear translocation of NF-kappaB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-kappaB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-kappaB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics/metabolism ; Caspase 3/metabolism ; Caspase 8/metabolism ; Drug Synergism ; Enzyme Activation/drug effects ; Flavonoids/*pharmacology ; Gene Expression/drug effects ; Gene Knockdown Techniques ; HeLa Cells ; Humans ; NF-kappa B/antagonists & inhibitors/*metabolism ; Protein Transport/drug effects ; RNA, Small Interfering/genetics ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand/*physiology ; Up-Regulation/drug effects

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.
Antineoplastic Agents/*pharmacology ; Apoptosis/*drug effects ; Astrocytes/metabolism ; CASP8 and FADD-Like Apoptosis Regulating Protein/genetics/*metabolism ; Caspase 3/metabolism ; Cell Line, Tumor ; Drug Discovery ; Flow Cytometry ; Glioma/*metabolism/pathology ; Humans ; Indoles/*pharmacology ; Inhibitor of Apoptosis Proteins/metabolism ; RNA, Small Interfering ; Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand/metabolism/*pharmacology ; Transcription Factor CHOP/analysis

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

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.
Animals ; *Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein/*genetics/metabolism ; Cells, Cultured ; Forkhead Transcription Factors/genetics/*metabolism ; Macrophages/metabolism ; Mice ; Mice, Inbred C57BL ; Oligodeoxyribonucleotides/metabolism ; Oncogene Protein v-akt/metabolism ; RNA, Small Interfering/metabolism ; Signal Transduction ; Toll-Like Receptor 9/genetics/*metabolism

This study was aimed to investigate the effect of sodium valproate(VPA) on human myelodysplastic syndrome cell line SKM-1 and its mechanism. The cell proliferation was determined by MTT assay, cell apoptosis was analyzed by flow cytometry. The expressions of c-flipl, c-flips and dlk1 mRNA were detected by RT-PCR. The results showed that VPA could inhibited the growth of SKM-1 cells in dose- and time-dependent manners. The flow cytometric analysis indicated that VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner. The expressions of c-flipl, c-flips and dlk1 mRNA in SKM-1 cell treated with VPA decreased using of VPA. It is concluded that VPA can induce apoptosis and inhibited proliferation of SKM-1 cells. In this process, the decreasing of c-flipl, c-flips and dlk1 mRNA expression may play important roles.
Apoptosis ; drug effects ; CASP8 and FADD-Like Apoptosis Regulating Protein ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Membrane Proteins ; metabolism ; Myelodysplastic Syndromes ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Valproic Acid ; pharmacology