Expression of c-FLIP in peripheral blood mononuclear cells of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway / 浙江大学学报·医学版

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 (DAS285.1). 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.

Effects of c-FLIPL knockdown in cervical uterine carcinoma cell lines / Efectos de la inhibición de C-Flipl en líneas celulares de cáncer de cuello uterino

Overexpression of Short and Raji variants of Cellular FLICE-like inhibitory protein (c-FLIP) is capable of inhibiting apoptosis, while the function of the Long isoform depends of c-FLIPL concentration in cells. The aim of this study was to determine the effects of c-FLIPL knockdown in cervical cell lines. SiHa, C-4I and C-33A cervical cancer cell lines were analyzed. c-FLIPL level expression was determined by quantitative real-time PCR and western blotting. c-FLIPL was transiently downregulated by siRNA. The effects of knockdown of c-FLIPL on cell viability, proliferation and apoptosis were assessed by comparing with scrambled siRNA-transfected cells. SiHa and C-4I c-FLIPL knockdown cells showed increased viability compared with scrambled siRNA-transfected cells (P<0.05), while C-33A cells did not show significant differences. Ki-67 and PCNA immunocytochemistry was performed to evaluate proliferation on these cervical cancer cell lines. SiHa cells with c-FLIPL knockdown showed elevated expression of Ki-67 protein compared with their scrambled counterparts (P<0.0001), while C-33A c-FLIPL knockdown cells showed a significantly lower in PCNA expression (P<0.01) compared with control. All three c-FLIP-transfected cell lines showed a higher level of apoptosis compared with their scrambled controls. Our results suggest that c-FLIPL could have effects in proliferation and apoptosis in cervical cancer cell lines.

Cuando las variantes Short y Raji de la proteína Cellular FLICE-like inhibitory protein (c-FLIP) se encuentran sobrexpresadas son capaces de inhibir la apoptosis, mientras la función de la isoforma Long (c-FLIPL), depende de la concentración de esta molécula en las células. El objetivo de este estudio fue determinar los efectos de la inhibición de c-FLIPL en líneas celulares de cáncer de cuello uterino. Para realizar el estudio fueron utilizadas SiHa, C-4I y C-33A, líneas celulares de cáncer cervical. La expresión de c-FLIPL en estas líneas fue establecida mediante PCR en tiempo real y western blot. Posteriormente la expresión de c-FLIPL fue inhibida, mediante transfeción transiente con siRNA complementario al mRNA mensajero de c.-FLIPL. Los efectos de esta inhibición en la viabilidad celular, proliferación y apoptosis fue comparada con células transfectadas con un siRNA control (scrambled). Una vez reprimido c-FLIPL, las líneas celulares SiHa y C-4I presentaron un aumento de la viabilidad celular (P<0,05). Para evaluar la proliferación celular se utilizó inmunocitoquímica de los marcadores Ki-67 y PCNA. Las células SiHa transfectadas con siRNA c-FLIPL, mostraron una elevada expresión de Ki-67 (P<0,0001), mientras que las células C-33A con c-FLIPL inhibido mostraron una menor expresión de PCNA (P<0,01). Las tres líneas celulares con c-FLIPL reprimido mostraron un mayor nivel de apoptosis que las células control. Estos resultados sugieren que c-FLIPL puede tener efectos en la proliferación y apoptosis de líneas celulares de cáncer de cuello uterino.

Expression levels of heat shock protein 27 and cellular FLICE-like inhibitory protein in prostate cancer correlate with Gleason score sum and pathologic stage

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.

Relationship of c-FLIP(L) protein expression with molecular subtyping and clinical prognosis in invasive breast cancer / 中华病理学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Immunohistochemistry 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.</p><p><b>RESULTS</b>High 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).</p><p><b>CONCLUSIONS</b>C-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.</p>

Hypobaric hypoxia increases the expression of death receptor 5 and inhibits that of FLICE-like inhibitory protein in the rat testis / 中华男科学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Forty 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Simulated hypobaric hypoxia at 4000 m above the sea level increased the expression of DR5 and inhibited that of c-FLIP in the rat testis.</p>

Molecular mechanism of hydroxyurea enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand / 中国医学科学院学报

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>Chronic 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>HU 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.</p>

Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-kappaB-dependent cFLIP expression in HeLa cells

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.

Changes of death receptor regulator expression in the articular cartilage of patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>KBD 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.</p>

Paxilline enhances TRAIL-mediated apoptosis of glioma cells via modulation of c-FLIP, survivin and DR5

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.

Effect of sodium valproate on human myelodysplastic syndrome cell line SKM-1 and its mechanism / 中国实验血液学杂志

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.

Expression of FLICE-inhibitory protein in synovial tissue and its association with synovial inflammation in juvenile idiopathic arthritis / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To examine the expression of FLICE-inhibitory protein (FLIP) in juvenile idiopathic arthritis (JIA) and analyze its correlation with synovial inflammation.</p><p><b>METHODS</b>The expression of FLIP was assessed in 11 JIA and 3 normal synovial tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The cell types expressing FLIP were further characterized, and the correlation of FLIP expression with the degree of synovial inflammation, as well as the activity of caspase 8 was then analyzed.</p><p><b>RESULTS</b>RT-PCR revealed the expression of FLIP mRNA in all 11 JIA samples, but not in 3 normal synovial tissues. In JIA, FLIP expression could be found in both the lining and sublining layers, mainly in the macrophage-like cells. Moreover, the expression of FLIP in JIA synovial tissues was positively correlated with the degree of synovial inflammation (r = 0.563, P < 0.05).</p><p><b>CONCLUSION</b>The expression of antiapoptotic FLIP in JIA synovial tissue and its correlation to accumulation of inflammatory cells in synovial tissue suggests that FLIP potentially extends the lifespan of synovial cells and thus contributes to the progression of joint destruction.</p>

Gene expression in patients with myelodysplastic syndrome / 中国实验血液学杂志

This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in the patients with myelodysplastic syndrome (MDS) and its clinical significance. The mRNA expression of c-FLIPL, c-FLIPS and DLK1 in bone marrow mononuclear cells (BMMNC) of 16 patients with MDS and 3 controls were detected by RT-PCR. The results indicated that the expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls (p < 0.05). There was no significant difference in expression of DLK1 between RA and RAEB patients (p > 0.05); the expression of c-FLIPL mRNA both in RA and RAEB patients was higher than that in controls (p < 0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB patients (p > 0.05); the expression of c-FLIPS mRNA was not significantly different between MDS patients and controls (p > 0.05), but its expression in RAEB patients was significantly higher as compared with RA patients and controls (p < 0.05). It is concluded that the mRNA expressions of DLK1, c-FLIPL and c-FLIPS in MDS patients are abnormal, some of which may be useful as an important indicator for the evaluation of development in MDS.

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression

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.

FLIP as an Anti-Cancer Therapeutic Target

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.

Cytotoxic Effects of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)and its Molecular Mechanism in Human Gastric Cancer Cells / 대한해부학회지

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.

Clinical significance and expression of FLIP and PTEN protein in laryngeal squamous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

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.

Expression of c-FLIP in Gastric Cancer and its Relation to Tumor Cell Proliferation and Apoptosis

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.

Synergistic induction of apoptosis by the combination of TRAIL and low dose adriamycin in human osteosarcoma cell line U2OS / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate whether TRAIL can synergize with adriamycin (ADM) to kill osteosarcoma cells (U2OS) in vitro, and its possible molecular mechanism.</p><p><b>METHODS</b>MTT was used to evaluate the cytotoxic effect of TRAIL and ADM either used alone or in combination at 24 hours after treatment to U20S cells. Cell apoptosis and its proportion were detected by flow cytometry assay. Acridine orange fluorescence microscopy and transmission electron microscopy were used to examine cellular and ultrastructural changes of apoptosis. The changes of cFLIP in mRNA and protein level were semi-quantified by RT-PCR and Western blot analysis.</p><p><b>RESULTS</b>(1) U2OS cells were not sensitive to TRAIL (IC(50) > 1 mg/L); the cells were relatively more responsive to ADM in an apparent dose-effect fashion. (2) The combination of TRAIL and ADM presented a synergistic effect on U2OS cells. Subtoxic concentration of TRAIL (0.1 mg/L) combined with subtoxic concentration of ADM (1.0 micromol/l) killed (49.54 +/- 2.79)% of U2OS cells. (3) The cytotoxicity was mainly attributed to cell apoptosis as demonstrated by flow cytometry assay, fluorescence microscopy and electron microscopy.</p><p><b>CONCLUSION</b>Subtoxic dose of TRAIL can effectively kill osteosarcoma cells (U2OS) in combination with subtoxic dose of ADM, but not effective when used alone. Apoptosis is the main mechanism of this killing effect induced by combination of TRAIL and ADM. Down regulation of cFLIP at mRNA and protein level is involved in this apoptosis pathway.</p>

Adenovirus containing c-FLIPs gene induces antiapoptosis activation of lymphocyte in vitro / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy and mechanisms of recombinant adenovirus containing c-FLIPs (cellular FLICE-inhibitory protein short) in inducing antiapoptosis activation of lymphocyte in vitro.</p><p><b>METHODS</b>c-FLIPs gene was cloned from total RNA of lymphocyte with RT-PCR methods, then constructed recombined adenovirus Ad.c-FLIPs by pAdeasy system. H9 cells infected with Ad.c-FLIPs produced antiapoptosis derived from anti-Apo-1 antibody, and those antiapoptosis evaluated with PI stain flow cytometry (FCM) and Hoechst stain.</p><p><b>RESULTS</b>c-FLIPs gene was successfully cloned from lymphocytes and incorporated into recombinant adenovirus Ad.c-FLIPs.After exposure to anti-Apo-1 for 24 h, the apoptosis of H9 cells preconditioned by Ad.c-FLIPs for 24 h as measured by Hochst stain or FCM significantly decreased, compared with non-preconditioned cells, 3.60% +/- 0.21% vs. 48.33% +/- 7.41%, respectively, indicating that Ad.c-FLIPs preconditioning protected H9 cells against apoptosis induced by anti-Apo-1.</p><p><b>CONCLUSION</b>Recombinant adenovirus Ad.c-FLIPs effectively induced anti-apoptosis activation of lymphocyte.</p>