FLICE-like Inhibitory Protein Expression Research Articles

Forefront: MiR-34a-Knockout Mice with Wild Type Hematopoietic Cells, Retain Persistent Fibrosis Following Lung Injury.

MicroRNAs (miRs) are known to limit gene expression at the post-transcriptional level and have important roles in the pathogenesis of various conditions, including acute lung injury (ALI) and fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). In this study, we found increased levels of miR-34 at times of fibrosis resolution following injury, in myofibroblasts from Bleomycin-treated mouse lungs, which correlates with susceptibility to cell death induced by immune cells. On the contrary, a substantial downregulation of miR-34 was detected at stages of evolution, when fibroblasts resist cell death. Concomitantly, we found an inverse correlation between miR-34 levels with that of the survival molecule FLICE-like inhibitory protein (FLIP) in lung myofibroblasts from humans with IPF and the experimental model. Forced upregulation of miR-34 with miR-34 mimic in human IPF fibrotic-lung myofibroblasts led to decreased cell survival through downregulation of FLIP. Using chimeric miR-34 knock-out (KO)-C57BL/6 mice with miR34KO myofibroblasts but wild-type (WT) hematopoietic cells, we found, in contrast to WT mice, increased and persistent FLIP levels with a more severe fibrosis and with no signs of resolution as detected in pathology and collagen accumulation. Moreover, a mimic of miR-34a decreased FLIP expression and susceptibility to cell death was regained in miR-34KO fibroblasts. Through this study, we show for the first time an inverse correlation between miR-34a and FLIP expression in myofibroblasts, which affects survival, and accumulation in lung fibrosis. Reprogramming fibrotic-lung myofibroblasts to regain susceptibility to cell-death by specifically increasing their miR34a and downregulating FLIP, may be a useful strategy, enabling tissue regeneration following lung injury.

HnRNPK knockdown alleviates NLRP3 inflammasome priming by repressing FLIP expression in Raw264.7 macrophages

ABSTRACT Objectives: Inflammation is an important predisposing and progressive factor in chronic kidney disease (CKD). Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is associated with many fundamental cellular processes, but in chronic inflammatory pathologies remains unclear. Methods: An in vitro peripheral inflammation model was established using lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages, followed by inflammasome activation by ATP treatment. Knockdown of hnRNPK by sihnRNPK and FLICE-like inhibitory protein (FLIP) by siFLIP transfection were achieved in Raw264.7 macrophages. ELISA was used to determine the expression of IL-1β, IL-18 and TNF-α. Real time PCR was applied to detect the mRNA levels of hnRNPK, NOD-like receptors family pyrin domain-containing 3 (NLRP3), FLIP, Caspase-1, IL-1β and IL-18. Western blot and immunofluorescence were performed to detect relevant protein expressions. Co-immunoprecipitation (Co-IP) was used to assess the interaction of hnRNPK with FLIP. Results: Results showed that LPS plus ATP activated NLRP3 inflammasome, which evidenced by the up-regulation of TNF-α, IL-1β and IL-18. Notably, hnRNPK and FLIP were significantly up-regulated in activated NLRP3 inflammasome of macrophages. HnRNPK or FLIP knockdown significantly suppressed the activation of NLRP3 inflammasome, as reflected by down-regulation of Caspase-1, IL-1β and IL-18. Importantly, hnRNPK could directly bind to FLIP in activated NLRP3 inflammasome. Discussion: Our findings suggest that hnRNPK could promote the activation of NLRP3 inflammasome by directly binding FLIP, which might provide potential new therapeutic targets for CKD.

Caspase-8: A Novel Target to Overcome Resistance to Chemotherapy in Glioblastoma.

Caspase-8 was originally identified as a central player of programmed cell death triggered by death receptor stimulation. In that context, its activity is tightly regulated through several mechanisms, with the best established being the expression of FLICE-like inhibitory protein (FLIP) family proteins and the Src-dependent phosphorylation of Caspase-8 on Tyr380. Loss of apoptotic signaling is a hallmark of cancer and indeed Caspase-8 expression is often lost in tumors. This event may account not only for cancer progression but also for cancer resistance to radiotherapy and chemotherapy. Intriguingly, other tumors, such as glioblastoma, preferentially retain Caspase-8 expression, and high levels of Caspase-8 expression may correlate with a worse prognosis, suggesting that in this context this protease loses its apoptotic activity and gains additional functions. Using different cellular systems, it has been clearly shown that in cancer Caspase-8 can exhibit non-canonical functions, including promotion of cell adhesion, migration, and DNA repair. Intriguingly, in glioblastoma models, Caspase-8 can promote NF-κB-dependent expression of several cytokines, angiogenesis, and in vitro and in vivo tumorigenesis. Overall, these observations suggest that some cancer cells may hijack Caspase-8 function which in turn promote cancer progression and resistance to therapy. Here we aim to highlight the multiple functions of Caspase-8 and to discuss whether the molecular mechanisms that modulate the balance between those functions may be targeted to dismantle the aberrant activity of Caspase-8 and to restore its canonical apoptotic functionality.

Expression and mutation analyses of Fas, FLIP and Bcl-2 in granulosa cell tumor of ovary.

Mounting evidence indicates that evasion of apoptosis contributes to tumor pathogenesis. Although both Fas and Bcl-2 are crucial in apoptosis of normal ovarian cells, their roles in ovarian tumors, especially stromal tumors, are largely unknown. The aim of this study was to explore mutation of the Fas gene and expression of the apoptosis-related proteins Fas, FLICE-like inhibitory protein (FLIP) and Bcl-2 in granulosa cell tumor (GCT) of the ovary. We analyzed the expression of Fas, FLIP and Bcl-2 in 20 GCT tissues by immunohistochemistry. We also analyzed somatic mutations of the Fas gene in the same GCT tissues by polymerase chain reaction and single-strand conformation polymorphism assay. Expression of Fas was evident in 12 GCTs (60%), but the remaining 8 GCTs showed no or markedly decreased Fas immunostaining. Expression of FLIP was identified in 30% of the GCT samples and expression of Bcl-2 in 75%. All GCTs with positive Fas expression (n = 12) showed either FLIP or Bcl-2 expression. The GCTs were found to carry no somatic Fas mutations. Our data show that alterations of the apoptosis-related proteins Fas, FLIP and Bcl-2 are common in GCT, and suggest that expression of FLIP and Bcl-2 and loss of Fas expression might play role in the pathogenesis of GCT, possibly by inhibiting apoptosis.

Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation.

High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells.

Delaying mitotic exit downregulates FLIP expression and strongly sensitizes tumor cells to TRAIL

Many of the current antitumor therapeutic strategies are based on the perturbation of the cell cycle, especially during mitosis. Antimitotic drugs trigger mitotic checkpoint activation, mitotic arrest and eventually cell death. However, mitotic slippage represents a major mechanism of resistance to these treatments. In an attempt to circumvent the process of slippage, targeting mitotic exit has been proposed as a better strategy to kill tumor cells. In this study, we show that treatments that induce mitotic checkpoint activation and mitotic arrest downregulate FLICE-like inhibitory protein (FLIP) levels and sensitize several tumor cell lines to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. Interestingly, we also demonstrate that in absence of mitotic checkpoint activation, mitotic arrest induced either by Cdc20 knockdown or overexpression of nondegradable cyclin B is sufficient to induce both FLIP downregulation and sensitivity to TRAIL. In summary, our data suggest that a combination of antimitotic drugs targeting cyclin B degradation and TRAIL might prevent mitotic slippage and allow tumor cells to reach the threshold for apoptosis induction, thereby facilitating tumor suppression.

Expression and Mutation Analyses of Fas, FLIP and Bcl-2 in Granulosa Cell Tumor of Ovary

Aims and background Mounting evidence indicates that evasion of apoptosis contributes to tumor pathogenesis. Although both Fas and Bcl-2 are crucial in apoptosis of normal ovarian cells, their roles in ovarian tumors, especially stromal tumors, are largely unknown. The aim of this study was to explore mutation of the Fas gene and expression of the apoptosis-related proteins Fas, FLICE-like inhibitory protein (FLIP) and Bcl-2 in granulosa cell tumor (GCT) of the ovary. Methods We analyzed the expression of Fas, FLIP and Bcl-2 in 20 GCT tissues by immunohistochemistry. We also analyzed somatic mutations of the Fas gene in the same GCT tissues by polymerase chain reaction and single-strand conformation polymorphism assay. Results Expression of Fas was evident in 12 GCTs (60%), but the remaining 8 GCTs showed no or markedly decreased Fas immunostaining. Expression of FLIP was identified in 30% of the GCT samples and expression of Bcl-2 in 75%. All GCTs with positive Fas expression (n = 12) showed either FLIP or Bcl-2 expression. The GCTs were found to carry no somatic Fas mutations. Conclusions Our data show that alterations of the apoptosis-related proteins Fas, FLIP and Bcl-2 are common in GCT, and suggest that expression of FLIP and Bcl-2 and loss of Fas expression might play role in the pathogenesis of GCT, possibly by inhibiting apoptosis.

Abstract 351: 2-Methoxyestradiol targets RON kinase and FLIP to inhibit epithelial-mesenchymal transition in prostate cancer

Abstract Prostate Cancer (PCA) is the second leading cause of cancer-related deaths among men in the United States. Despite significant progress in our understanding of prostate biology, early detection and treatment, the five-year survival for hormone refractory metastatic prostate cancer (HRPCA) patients is less than 30%. Current therapies targeting PCA are not effective against the metastatic stage. 2-Methoxyestradiol (2-ME2) is a metabolic product of 17-β-estradiol and is found in trace amounts in the body; it has shown anti-tumorigenic properties in multiple cancer models. Previous studies from our lab have shown that 2-ME2 can induce apoptosis in PCA cells by blocking cell cycle progression, and also inhibits PCA growth and progression in TRAMP mice. However, the precise mechanism of action of 2-ME2 has not yet been elucidated. In order to classify the mechanism behind PCA inhibition by 2-ME2, we tested its effectiveness on an array containing genes associated with epithelial-mesenchymal transition (EMT). We identified Recepteur d'Origine Nantais (RON) as one of the most significantly downregulated genes in response to 2-ME2 treatment. RON has been shown to promote cell invasiveness, migration, survivability, and proliferation in many cancers such as breast, colon, lung, and pancreatic. However, very little is known about RON's signaling mechanism in PCA. We've found RON to be highly increased in advanced stage PCA cell lines. RON knockdown shows a concomitant decrease in the expression of FLICE-like inhibitory protein (FLIP). FLIP is an anti-apoptotic protein which prevents death receptor-induced apoptotic signaling. FLIP has been found to promote EMT as well as androgen-independent PCA growth. Stimulation with the RON ligand HGF yielded elevated expression of RON and FLIP in PCA cell lines while treatment with 2-ME2 caused downregulation. Immunoprecipitation experiments showed RON to be associated with the androgen-induced proliferation inhibitor (APRIN) in PCA cells. Our data suggest that RON/FLIP crosstalk plays a role in prostate cancer. Therefore, targeting RON/FLIP signaling could have potential therapeutic benefit for metastatic PCA. Supported by NIH CA 135451 (APK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 351. doi:1538-7445.AM2012-351

α-Tocopheryl succinate induces apoptosis in erbB2-expressing breast cancer cell via NF-κB pathway

to study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells. the annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM(TM) NF-κB p65 kit was used to assess NF-κB activation. α-TOS (100 μmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 μmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 μmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 μmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB . our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.

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.

Effects of phosphatidylinositol 3-kinase inhibitor on human cervical carcinoma cells in vitro

Phosphatidylinositol 3-kinase (PI3K) is a crucial cell survival pathway implicated in tumorigenesis because of its role in stimulating cell proliferation and suppressing apoptosis. This study was to investigate the regulation of proliferation and apoptosis by LY294002, an inhibitor of PI3K in cervical cancer cells and the expression of FLICE-like inhibitory protein (c-FLIP) in vitro. Human cervical cancer HeLa cells were used in this experiment and cultured. The cultured cells were treated with LY294002 at different concentrations (10, 25, 50 and 100 μmol/L) for 6, 12, 24, and 48 h before harvesting for evaluation. Cell viability was measured by 3-(4,5)-dimethylthiazol(-2-y1)-3,5-di-phenyltetrazoliumbromide (MTT) assay. Apoptosis was analyzed by flow cytometry. The expression of c-FLIP was detected by Western blot. Cell viability was inhibited by LY294002 significantly (P<0.05). Flow cytometry analysis revealed that cell apoptosis was significantly increased in the presence of LY294002 as compared with the control group. Although the expression of c-FLIP was increased in a short time, the expression of c-FLIP was markedly suppressed after the treatment of LY294002 for 48 h. These results suggested that the PI3K/Akt signal pathway might be involved in the regulation of cell apoptosis in cervical cancer cells. Moreover, the regulation of c-FLIP expression through PI3K/Akt signal pathway in cervical cancer cells was observed in vitro.

Human and Mouse Colon Cancer Utilizes CD95 Signaling for Local Growth and Metastatic Spread to Liver

Analysis of clinical colon cancer specimens show alterations in the CD95 (Fas Ag/Fas L) pathway as tumors progress from local to metastatic disease, suggesting that this pathway may play a role in invasive behavior of colon cancer. However, direct causality between these alterations and clinical disease progression has not been shown. Surgically resected metastatic colon cancer samples were evaluated for Fas Ag/L and apoptosis. Alterations in the Fas-signaling pathway found in human samples were recreated through a series of staged transfection experiments in the MC38 mouse colon cancer cell line and the effects on growth tested in vitro and in vivo. Expression of FLICE-like inhibitory protein confers apoptosis resistance, increasing the incidence of primary tumors through a survival advantage by avoiding apoptosis and inducing Fas-mediated proliferation. Coexpression of Fas L enables colon cancer cells to metastasize to the liver from local tumors as well as from intravenous injection of cells. MC38-FasL/FLICE-like inhibitory protein colon cancer cells induce apoptosis in hepatocytes via activation of type II Fas Ag signaling, thus creating a niche conducive to tumor growth and fueling their own growth via Fas proliferative signaling. Alterations in the Fas Ag pathway which inhibit apoptosis and increase Fas-mediated proliferation directly increase local colon cancer growth, and enhance metastasis to the liver. Delineating points in the pathway responsible for growth and metastasis will offer targets that may be exploited for therapy.

Activation-induced Degradation of FLIPL Is Mediated via the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Macrophages

Cellular FLIP (Flice-like inhibitory protein) is critical for the protection against death receptor-mediated cell apoptosis. In macrophages, FLIP long (FLIP(L)) and FLIP short (FLIP(S)) mRNA was induced by tumor necrosis factor (TNF) alpha, mediated through NF-kappaB. However, we observed TNFalpha reduced the protein level of FLIP(L), but not FLIP(S), at 1 and 2 h. Similar results were observed with lipopolysaccharide. The reduction of FLIP(L) by TNFalpha was not mediated by caspase 8, or through JNK or Itch, but was suppressed by inhibition of the phosphatidylinositol 3-kinase/Akt pathway employing chemical inhibitors, a dominant negative Akt-1, or Akt-1 small interfering RNA. The reduction of FLIP(L) resulted in the short term induction of caspase 8-like activity, which augmented NF-kappaB activation. A co-immunoprecipitation assay demonstrated that Akt-1 physically interacts with FLIP(L). Moreover, TNFalpha enhanced FLIP(L) serine phosphorylation, which was increased by activated Akt-1. Serine 273, a putative Akt-1 phosphorylation site in FLIP(L), was critical for the activation-induced reduction of FLIP(L). Thus, these observations document a novel mechanism where by TNFalpha facilitates the reduction of FLIP(L) protein, which is dependent on the phosphatidylinositol 3-kinase/Akt signaling.

Immunohistochemical analysis of Fas and FLIP in prostate cancers

Fas-mediated apoptosis is considered a principal pathway for apoptosis induction in normal and cancer cells. Expression of Fas has been reported in prostate tissues several times, but the data were not consistent. Expression of FLICE-like inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, has not been studied by immunohistochemistry in prostate tissues. The aim of this study is to explore whether alterations of Fas and FLIP expression occur in prostate cancer tissues. We analyzed the expression of Fas and FLIP in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray approach. Normal glandular cells of the prostates strongly expressed both Fas and FLIP proteins. Prostate intraepithelial neoplasm also showed a strong Fas immunoreactivity. Fas expression was strongly positive in 60 cancers (56.1%), but the remaining 47 cancers showed no (6.5%) or markedly decreased (37.4%) Fas immunostaining compared with the normal glandular cells of the same patients. By contrast, FLIP expression was strong in most (103/107; 96.3%) of the cancers, and only four cancers (3.7%) showed decreased immunoreactivities compared with the normal cells. The decreased expression of Fas was not associated with pathologic characteristics, including FLIP expression, size of the cancers, age, Gleason score and stage. The decreased expression of Fas in a large fraction of prostate cancers compared with their normal cells suggested that loss of Fas expression might play a role in tumorigenesis in some prostate cancers possibly by inhibiting apoptosis mediated by Fas.

Transforming growth factor ß1 induces apoptosis by suppressing FLICE‐like inhibitory protein in DU145 prostate epithelial cells

Transforming growth factor beta (TGFbeta) is a paracrine mediator of prostate epithelial cell apoptosis. In rodents, castration induces production of TGFbeta by stromal cells, which leads to apoptosis of epithelial cells. To identify potential mediators of this cell death pathway, we developed a model using DU145 cells, a tumorigenic human prostate epithelial cell line. We discovered that at low density, in low mitogen media, DU145 cells apoptose when treated with TGFbeta1. Prior to the onset of death, TGFbeta1 treatment downregulated the expression of the caspase inhibitor FLICE-like inhibitory protein (FLIP), at both the mRNA and protein level, suggesting a causal role between FLIP downregulation and cell death. To confirm the importance of FLIP in TGFbeta1-induced apoptosis, we employed small interfering RNA (siRNA) to silence FLIP expression. Doing so led to apoptosis, which is consistent with the hypothesis that FLIP prevents death in these cells. Furthermore, inhibition of caspase-8 by siRNA knockdown partially rescued the apoptotic effects of TGFbeta1, suggesting a role for death receptor signaling components in TGFbeta-mediated death of prostate epithelial cells.

FLICE-Like Inhibitory Protein Blocks Transforming Growth Factor β1–Induced Caspase Activation and Apoptosis in Prostate Epithelial Cells

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.

Augmentation of Proliferation of Vascular Smooth Muscle Cells by Plasminogen Activator Inhibitor Type 1

Proliferation of vascular smooth muscle cells (VSMCs) contributes to restenosis after coronary intervention. We have shown previously that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits VSMC apoptosis. Because apoptosis and proliferation appear to be linked, we sought to determine whether increased PAI-1 would affect VSMC proliferation. VSMCs were explanted from control and transgenic mice (SM22-PAI+) in which VSMC expression of PAI-1 was increased. Increased growth of SM22-PAI+-VSMCs (2.3+/-0.4-fold) reflected, at least partially, increased proliferation. Greater expression of FLICE-like inhibitory protein (FLIP; 2.7-fold) and its cleaved active form were seen in SM22-PAI+-VSMCs. The balance between caspase-8 and FLIP favored proliferation in SM22-PAI+-VSMCs. Increased expression of NF-kappaB and activation of extracellular signal-regulated kinase (ERK) were demonstrated in SM22-PAI+-VSMCs (fold=NF-kappaB=2.2+/-0.1, fold=phosphorylated-ERK=1.6+/-0.1). Results were confirmed when expression of PAI-1 was increased by transfection. Inhibition of NF-kappaB and ERK attenuated proliferation in SM22-PAI+-VSMCs. Increased expression of PAI-1 promoted proliferation when VSMCs were exposed to tumor necrosis factor (TNF). Increased expression of PAI-1 is associated with greater activity of FLIP that promotes VSMC proliferation through NF-kappaB and ERK. Thus, when vascular wall expression of PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMC and resistance to apoptosis.

Sequencing, chromosomal mapping, and functional characterization of bovine FLICE-Like Inhibitory Protein (FLIP)

FLICE-like inhibitory protein (FLIP) has been shown in both humans and mice to inhibit apoptosis and NF-ĸB activation induced by pro-inflammatory mediators. The activation of NF-ĸB and the induction of apoptosis are critical events in the pathogenesis of a variety of disease states in cattle, including mastitis. Since FLIP is known to moderate these events in other species, we mapped the bovine FLIP gene, sequenced bovine FLIP cDNA, and characterized its expression in cultured primary bovine endothelial cells. Sequencing of bovine FLIP revealed approximately 83, 74, and 68% amino acid sequence identity to its porcine, human, and murine orthologs, respectively. Bovine FLIP was mapped to chromosome 2 by radiation hybrid mapping. Interestingly the region to which bovine FLIP maps contains a putative quantitative trait locus for functional herd life which is an indicator of a cow’s ability to survive involuntary culling due primarily to mastitis and infertility. In addition to sequencing and mapping, the function of bovine FLIP was studied. Over-expression of bovine FLIP protected against bacterial lipopolysaccharide (LPS)- and TNF-α-induced apoptosis in bovine endothelial cells consistent with previous studies of human FLIP. In addition, elevated expression of bovine FLIP blocked LPS- and TNF-α-induced upregulation of NF-ĸB-dependent gene products as assayed by E-selectin expression. Only the full-length bovine FLIP protein could inhibit NF-ĸB activation induced by LPS, whereas the death effector domain region alone was able to inhibit TNF-α-induced NF-ĸB activation. Together, these data demonstrate the conservation of FLIP’s ability to inhibit apoptosis and to downregulate NF-ĸB activation across species.

911 POSTER Expression of FLICE-like inhibitory protein (c-FLIPL) is associated with ovarian cancer patient's chemoresistance

Gastro-intestinal neuroendocrine tumours (GI-NETs) are chemotherapy-resistant tumours. Bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF), has shown promising results in several phase II trials of gastro-entero-pancreatic-NETs. We assessed bevacizumab combined with capecitabine, specifically in GI-NET patients.BEvacizumab in The Treament of neuroEndocrine tumoRs (BETTER) was a multicentre, open-label, non-randomised, two-group phase II trial. Here we present the group of patients with progressive, metastatic, well-differentiated GI-NETs. Patients Eastern Cooperative Oncology Group-performance status (ECOG-PS) ⩽ 2, Ki-67 proliferation rate <15% and no prior systemic chemotherapy were treated with bevacizumab (7.5 mg/kg/q3w) and capecitabine (1000 mg/m2 twice daily, orally d1–14, resumed on d22) for 6–24 months. The primary end-point was progression-free survival (PFS); secondary end-points included overall survival (OS), response rate, safety and quality of life.Of the 49 patients included, 53% were men, median age was 60 years (41–82), primary tumour site was ileal in 82% patients and Ki-67 was <15% in 48 patients and not available for one patient. After a maximum of 24 month follow-up per patient, the median PFS by investigator assessment was 23.4 months [95% confidence interval (CI): 13.2; not reached] and the overall disease control rate was 88% (18% partial response, 70% stable disease). The 2-year survival rate was 85%. Median OS was not reached. The most frequent grade 3–4 adverse events were hypertension (31%), diarrhoea (14%) and hand-foot syndrome (10%).The combination of bevacizumab and capecitabine showed clinical activity and a manageable safety profile in the treatment of GI-NETs that warrant confirmation in a randomised phase III trial.

PKCδ-mediated regulation of FLIP expression in human colon cancer cells

FLICE-like inhibitory protein (FLIP), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Decreased FLIP levels sensitize tumor cells to Fas- and TRAIL-mediated apoptosis; however, the cellular mechanisms regulating FLIP expression have not been defined. Here, we examined the roles of the PKC and NF-kappaB pathway in the regulation of FLIP in human colon cancers. FLIP mRNA levels were increased in Caco-2 cells by treatment with PMA; actinomycin D completely inhibited the induction of FLIP by PMA, indicating transcriptional regulation. PKC inhibitors Gö6983 and Ro-31-8220 blocked PMA-stimulated FLIP expression. Pretreatment with the PKCdelta-selective inhibitor rottlerin or transfection with PKCdelta siRNA inhibited PMA-induced FLIP expression, which identifies a role for PKCdelta in FLIP induction. Treatment with the proteasome inhibitor, MG132, or the NF-kappaB inhibitor (e.g., PDTC and gliotoxin), or overexpression of the superrepressor of IkappaB-alpha inhibited PMA-induced upregulation of FLIP. Moreover, PMA-induced NF-kappaB transactivation was blocked by GF109203x. In conclusion, our results demonstrate a critical role for PKCdelta/NF-kappaB in the regulation of FLIP in human colon cancer cells.