Down-regulation Of FasL Research Articles

MicroRNA-216a-5p Alleviates Acute Kidney Injury of Mice via Suppressing FAS Ligand Expression.

The aim of this present work was to investigate the mechanism of the microRNA (miR)-216a-5p/FASL axis in mice with acute kidney injury (AKI). Mice kidney ischemia/reperfusion (I/R) injury was used as AKI models in this study. I/R mice were injected with miR-216a-5p- and FASL-related constructs to investigate potential mechanisms of kidney protection. Kidney function, inflammation, oxidative stress, and kidney cell apoptosis were assessed after 24 h of reperfusion. In vitro, the hypoxia-reoxygenation (H/R) model was used with kidney tubular epithelial cells (TECs) to mimic kidney I/R injury. H/R-treated TECs were transfected with miR-216a-5p- and FASL-related constructs to detect cell viability, inflammation, and oxidative stress. MiR-216a-5p and FASL expression levels in mouse kidney tissues and in H/R-treated TECs were detected. MiR-216a-5p was downregulated and FASL was upregulated in kidney tissues of I/R mice and H/R-treated TECs. Upregulating miR-216a-5p attenuated kidney cell apoptosis and the damage of kidney function, and reduced inflammatory factor levels and oxidative stress response in kidney tissues of I/R mice. Upregulating miR-216a-5p advanced cell viability and reduced inflammatory factor levels and oxidative stress response in H/R-treated TECs. Downregulation of FASL effectively reversed the influences of downregulation of miR-216a-5p on kidney injury in mice and kidney TEC survival. Our study reveals that miR-216a-5p reduces I/R-induced pathological kidney damage in AKI via suppressing FASL.

Inhibiting Endothelial Cell-Mediated T Lymphocyte Apoptosis with Integrin-Targeting Peptide-Drug Conjugate Filaments for Chemoimmunotherapy of Triple-Negative Breast Cancer.

Tumor-associated endothelial cells (TECs) limit antitumor immunity via inducing apoptosis of infiltrating T lymphocytes through a Fas ligand (FasL) mediated mechanism. Herein, this work creates a peptide-drug conjugate (PDC) by linking 7-ethyl-10-hydroxycamptothecin (SN38) to hydrophilic segments with either RGDR or HKD motif at their C-terminus through a glutathione-responsive linker. The PDCs spontaneously assemble into filaments in aqueous solution. The PDC filaments containing 1% of SN38-RGDR (SN38-HKD/RGDR) effectively target triple-negative breast cancer (TNBC) cells and TECs with upregulated expression of integrin, and induce immunogenic cell death (ICD) of tumor cells and FasL downregulation of TECs. SN38-HKD/RGDR increases infiltration, activity, and viability of CD8+ T cells, and thus inhibits the growth of primary tumors and pulmonary metastasis. This study highlights the synergistic modulation of cancerous cells and TECs with integrin-targeting PDC filaments as a promising strategy for TNBC chemoimmunotherapy.

Upregulation of miR‑128 inhibits neuronal cell apoptosis following spinal cord injury via FasL downregulation by repressing ULK1.

Spinal cord injury (SCI) is characterized by permanent motor deficits followed by inflammation and oxidative stress, causing neuronal cell death. The present study aimed to investigate the role of microRNA (miR)-128 in neuronal cell apoptosis and its underlying mechanism. Targeting relationships among miR-128 and Unc-51 like autophagy activating kinase 1 (ULK1) and Fas ligand (FasL) were verified using dual-luciferase reporter assay and ChIP assays. Loss- and gain-of-function assays were conducted in rat models of SCI to determine the roles of miR-128 and ULK1 in neuronal cell apoptosis, inflammation, and motor function. Apoptosis, motor function and expression of inflammatory factors were respectively determined by Terminal deoxynucleotidyl transferase-mediated dUTp nick end-labeling, Basso, Beattie and Bresnahan (BBB) score and enzyme-linked immunosorbent assay. Hematoxylin and eosin staining, Nissl staining and immunofluorescence were respectively performed to observe morphological changes and number of neurons and nestin-positive cells. The neuronal cells were isolated from neuron injury models and cultured in vitro. MTT and flow cytometry was conducted to determine the neuronal cell viability and apoptosis respectively. miR-128 was downregulated whereas ULK1 was upregulated in rats with SCI. Overexpression of miR-128 or downregulation of ULK1 inhibited neuronal cell apoptosis and inflammation as evidenced by an increased BBB score and more neurons and nestin-positive cells, but reduced expression of inflammatory and apoptosis-related factors. ULK1 was negatively regulated by miR-128, whereas FasL was positively regulated by ULK1. In vitro experiments validated the roles of miR-128 and ULK1 in neuronal cell differentiation and apoptosis. In conclusion, the upregulation of miR-128 depresses neuronal cell apoptosis by downregulating ULK1, thereby attenuating SCI via the downregulation of FasL.

Annexin A5 promotes macrophage activation and contributes to pulmonary fibrosis induced by silica particles.

To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis. Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 μg/ml for 6 or 12 h, the expression levels of transforming growth factor β1 (TGF-β1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-β1 secretion into the cell culture medium was measured by ELISA assays or Western blots. Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-β1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-β1, IL-1α, and FasL downstream target molecules. These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis.

Effect of human umbilical cord blood derived lineage negative stem cells transplanted in amyloid-β induced cognitive impaired mice

Alzheimer's disease (AD) is pathologically characterized by extracellular deposition of insoluble amyloid-β (Aβ) plaques and intracellular tangles made up of phosphorylated tau in brain. Several therapeutic approaches are being carried out in animal AD models for testing their safety and efficacy in altering disease pathology and behavioral deficits. Very few studies have examined the effect of human umbilical cord blood (hUCB) derived stem cells in degenerative disease models despite growing number of cord blood banks worldwide. Here we have examined the therapeutic efficacy of hUCB derived lineage negative (Lin −ve) stem cells in alleviating behavioral and neuropathological deficits in a mouse model of cognitive impairment induced by bilateral intrahippocampal injection of Aβ-42. Lin −ve cells were transplanted at two doses (50,000 and 100,000) at the site of injury and examined at 10 and 60 days post transplantation for rescue of memory deficits. These cells were found to ameliorate cognitive impairment in 50,000—60 days and 100,000—10 days groups whereas, 50,000—10 days and 100,000—60 days groups could not exert any significant improvement. Further, mice showing spatial memory improvement were mediated by up-regulation of BDNF, CREB and also by concomitant down regulation of Fas-L in their brain. The transplanted cells were found in the host tissue and survived up to 60 days without expressing markers of neuronal differentiation or reducing Aβ burden in mouse brain. We suggest that these undifferentiated cells could exert neuroprotective effects either through inhibiting apoptosis and/or trophic effects in the brain.

Expression of Fas (CD95/APO-1) and Fas Ligand (FasL) in Experimentally-Induced Acute Pancreatitis

ABSTRACTIntroduction: Acinar cell death is a crucial event in acute pancreatitis (AP) and may occur either by apoptosis or necrosis. The aim of this study was to investigate the expression of the apoptosis associated proteins Fas and FasL in experimentally induced severe AP. Methods: AP was induced in 30 rats by injecting 0.2 ml of 4.5% sodium taurocholate solution into the biliopancreatic duct. Sham operated animals (n = 30) and 10 normal controls were used for comparisons. Animals were killed at 6, 12, 24, 48, 72 hr and 1 week after operation (five animals at each time point) and both serum and pancreatic tissue were obtained. The severity of AP was graded by morphological evaluation and by measuring serum amylase levels. Acinar cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Tissue expressions of Fas and FasL were evaluated by immunohistochemistry. Results: Sodium taurocholate injection resulted in severe acute necrotizing pancreatitis as early as six hr after taurocholate infusion with gradually increasing severity and a peak at 72 hr, and a significant increase of serum amylase at 6 and 12 hr. Apoptotic acinar cells were observed between 48 and 72 hr. The expression of both Fas and FasL in pancreatic tissue was induced in comparison with normal controls. Fas expression in AP was higher and statistically significant at 24 hr whereas FasL expression was consistently lower with a statistical significance observed at 12 hr when compared to sham-operated animals suggesting Fas upregulation and FasL downregulation in this model of AP. Conclusions: Induction and sequential changes in the expressions of Fas and FasL occur during taurocholate induced severe AP in rats and their temporal modulation might associate with acinar cell death by apoptosis.

A Novel Role for IL-27 in Mediating the Survival of Activated Mouse CD4 T Lymphocytes

IL-27, an IL-12 family cytokine, has pleiotropic functions in the differentiation and expansion of CD4(+) T cell subsets. In this study, we discovered a novel function of IL-27. CD4(+)CD45RB(high) T cells from mice deficient for the α-chain of IL-27 receptor failed to induce colitis in Rag(-/-) recipients, because of an inability of activated donor cells to survive. Interestingly, IL-27 was indispensable for the prevention of colitis by regulatory T cells, also because of a defect in long-term cell survival. IL-27 affected the survival of activated T lymphocytes, rather than promoting cell proliferation, by inhibiting Fas-mediated activation-induced T cell death, acting through the STAT3 signaling pathway. The addition of IL-27 during activation resulted in an increased cell number, which was correlated with decreased activation of both caspases 3 and 8. This prosurvival effect was attributed to downregulation of FasL and to the induction of the antiapoptotic protein cFLIP. Although activation induced cell death is an important mechanism for the maintenance of immunological homeostasis, protection of lymphocytes from excessive cell death is essential for effective immunity. Our data indicate that IL-27 has a crucial role in the inhibition of activation-induced cell death, thereby permitting Ag-driven T cell expansion.

Fas Ligand Enhances Malignant Behavior of Tumor Cells through Interaction with Met, Hepatocyte Growth Factor Receptor, in Lipid Rafts

Many late-stage cancer cells express Fas ligand (FasL) and show high malignancy with metastatic potential. We report here a novel signaling mechanism for FasL that hijacks the Met signal pathway to promote tumor metastasis. FasL-expressing human tumor cells express a significant amount of phosphorylated Met. The down-regulation of FasL in these cells led to decreased Met activity and reduced cell motility. Ectopic expression of human FasL in NIH3T3 cells significantly stimulated their migration and invasion. The inhibition of Met and Stat3 activities reverted the FasL-associated phenotype. Notably, FasL variants activated the Met pathway, even though most of their intracellular domain or Fas binding sites were deleted. FasL interacted with Met through the FasL(105-130) extracellular region in lipid rafts, which consequently led to Met activation. Knocking down Met gene expression by RNAi technology reverted the FasL-associated motility to basal levels. Furthermore, treatment with synthetic peptides corresponding to FasL(117-126) significantly reduced the FasL/Met interaction, Met phosphorylation, and cell motility of FasL(+) transfectants and tumor cells. Finally, the transfectants of truncated FasL showed strong anchorage-independent growth and lung metastasis potential in null mice. Collectively, our results establish the FasL-Met-Stat3 signaling pathway and explains the metastatic phenotype of FasL-expressing tumors.

Tu1913 Differential Regulation of Oxidative Stress and Apoptosis Related Genes During Active and Inactive Crohn's Disease (CD)

Background: An increase of oxidative stress (OxS) and a decrease of apoptosis have been reported as key factors in Crohn's disease (CD) pathogenesis. We have previously shown that OxS in CD depends on an increased production of H2O2 which detoxification is independent of catalase (CAT) enzyme because CAT showed permanently inhibited in CD patients1. CAT could be more implicated in regulating cellular processes, as apoptosis, than in detoxifying H2O2. Aims: To characterize the genetic expression of CAT and other OxS and apoptosis related genes in CD at beginning of the disease and when remission has been achieved. Methods: Blood samples were obtained from 18 healthy subjects (H), 20 patients at onset of CD and yet to begin any specific medication (active CD, aCD) and from 10 patients who achieved clinical, analytical and morphological remission (inactive CD, iCD). Patients were diagnosed according to Leonard-Jones criteria and disease activity was scored based on the Harvey-Bradshaw index, serological markers of inflammation (CRP, fibrinogen) and evaluation of morphological lesions (magnetic resonance enterography, colonoscopy or capsule endoscopy). PWMC were isolated by Histopaque sedimentation. Total RNA from leukocytes was extracted according to manufacturer's protocol (LeukoLOCKTM; Ambion). mRNA expression was measured by Sequenom's MassARRAY® quantitative gene expression analysis application. The genes analyzed were: CAT, SOD2, NOS2A, STAT1, NFKB1, PKCgamma, PKCzeta, PSKH1, PPID, ABCB1, ASK1, FASR, FASLG, TERT, IL-2, IL23R. Results: Genes differentially expressed in CD are summarized in the table. Specific upregulated genes during flair were SOD2, STAT1, and PSKH1, and down-regulated were FASL and ABCB1. In iCD, FASR and NFKB1 were up-regulated. The down-regulation of CAT gene expression was persistent. Conclusions: Up regulation of SOD2, STAT1 and PSKH1 only during a flare, indicates the relation between OxS and aCD. Down regulation of FASL and ABCB1 shows an apoptotic and multidrug resistance alteration at onset of the disease. Increased expression of FASR and NFKB1 in iCD could indicate that apoptosis are genetically affected in CD. Permanent inhibition of CAT activity in CD patients correlates to a persistent down regulation of the CAT-gene mRNA. The implications of CAT inhibition in regulating cellular processes such as apoptosis should be studied. 1) Beltran B, et al. Inflamm Bowel Dis. 2010 16:76-86.

Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

BackgroundThrombolytic therapy with tissue plasminogen activator (tPA) benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS) protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA) excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer) receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult.ResultsWe show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL) production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant AktK179A and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM), we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates phosphorylation of FHKRL1 that is required for PS-mediated neuronal protection after tPA/NMDA-induced injury.ConclusionsPS blocks the extrinsic apoptotic cascade through a novel mechanism mediated by Tyro3-dependent FKHRL1 phosphorylation which inhibits FasL-dependent caspase-8 activation and can control tPA-induced neurotoxicity associated with pathologic activation of NMDA receptors. The present findings should encourage future studies in animal stroke models to determine whether PS can increase the therapeutic window of tPA by reducing its post-ischemic neuronal toxicity.

Senescence regulates macrophage activation and angiogenic fate at sites of tissue injury in mice

Abnormal angiogenesis plays a key role in diseases of aging such as heart disease, certain cancers, and eye diseases including age-related macular degeneration. Macrophages have been shown previously to be both anti- and proangiogenic, and their role in regulating angiogenesis at sites of tissue injury is critical and complex. In this study, we analyzed cytokine gene expression patterns of mouse macrophages by real-time quantitative PCR and tested the functional effects of senescence on gene expression and macrophage polarization. Following laser injury to the retina, IL-10 was upregulated and Fas ligand (FasL), IL-12, and TNF-alpha were downregulated in ocular macrophages of old mice (>18 months of age). Downregulation of FasL on macrophages led to a loss of the antiangiogenic phenotype, as evidenced by the inability of these macrophages to inhibit vascular endothelial cells. Our results demonstrate that senescence, FasL, and IL-10 are key determinants of macrophage function that alter the growth of abnormal postdevelopmental blood vessels in disease processes including blinding eye disease.

Effects of 2( RS)- n-propylthiazolidine-4( R)-carboxylic acid on 4-hydroxy-2-nonenal-induced apoptotic T cell death

4-Hydroxy-2-nonenal (HNE), the aldehydic product of lipid peroxidation, is associated with multiple immune dysfunctions, such as HIV and hepatitis C virus infection. HNE-induced immunosuppression could be due to a decrease in CD4 + T lymphocyte activation or proliferation. Glutathione (GSH) is the most abundant endogenous antioxidant in cells, and an adduct between HNE and GSH has been suggested to be a marker of oxidative stress. Our earlier studies showed that HNE induced cytotoxicity and Akt inactivation, which led to the enhancement of FasL expression and concomitantly decreased cellular FLICE-like inhibitory protein (c-FLIP S) levels. In this study, we found that HNE caused intracellular GSH depletion in Jurkat T cells, and we further investigated the role of 2( RS)- n-propylthiazolidine-4( R)-carboxylic acid (PTCA), a GSH prodrug, in attenuating HNE-induced cytotoxicity in CD4 + T lymphocytes. The results show that PTCA protected against HNE-induced apoptosis and depletion of intracellular GSH. PTCA also suppressed FasL expression through increasing levels of Akt kinase as well as antiapoptotic c-FLIP S and decreasing the activation of type 2 protein serine/threonine phosphatase. Taken together, these data demonstrate a novel correlation between GSH levels and Akt activation in T lymphocyte survival, which involves FasL down-regulation and c-FLIP S expression through increasing intracellular GSH levels. This suggests that PTCA could potentially be used in the treatment of oxidative stress-induced immunosuppressive diseases.

Down-regulation of Fas-ligand mRNA in Sjögren's syndrome patients with enlarged exocrine glands

We have reported that Sjögren's syndrome (SS) patients with enlarged exocrine glands (EEG) formerly referred to as Mikulicz's disease were defective with Fas-ligand (FasL) expression in PBL and lacrimal glands (LGs). To investigate the mechanisms of reduced FasL expression in SS patients with EEG, FasL mRNA expression level was determined using real-time PCR. The FasL gene promoter region (from − 1197 to − 3) was also amplified using PCR and specific primers. Expression of the FasL mRNA in the LGs and PBLs of three SS patients with EEG was significantly decreased. Direct sequencing revealed a heterozygous point mutation ( − 259T/C) in the FasL gene promoter region in one SS patient with EEG. A luminescent β-galactosidase (β-gal) reporter assay using a pβgal Enhancer Vector demonstrated that β-gal activity from the vector including the mutant ( − 259C) FasL (pβgal/mFasL) gene promoter region (735 ± 42) was similar (p = 0.13) to that from a pβgal Enhancer Vector without the gene promoter region (603 ± 66). On the other hand, the β-gal activity was significantly lower (p < 0.0001) than that from a vector including the wild-type ( − 259T) FasL (pβgal/wFasL) (3226 ± 148). In conclusion, the down-regulation of FasL in SS patients with EEG may be due to transcriptional regulation, and the point mutation at − 259T/C in the FasL gene promoter region may lead to the down-regulation of FasL mRNA expression and the lymphoproliferative process observed in SS patients with EEG.

Peroxisome proliferator-activated receptor-γ-agonist, rosiglitazone, promotes angiogenesis after focal cerebral ischemia

Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, not only improves insulin resistance in patients with type II diabetes but also exerts a broad spectrum protective effects in variable animal models of neurologic or cardiovascular diseases. We studied the effect of rosiglitazone on angiogenesis and neurological recovery after focal cerebral ischemia. Rosiglitazone (3 mg/kg or 0.3 mg/kg, p.o.) was administered for 7 days prior to and 3 days after the induction of focal ischemia (total 10 days) in adult rats. The rosiglitazone-treated group showed the enhanced neurologic improvement, the reduced infarction volume compared to the ischemia-vehicle group with dose dependency, and the reduced hemispheric atrophy. Rosiglitazone treatment reduced TUNEL +/activated caspase-3 + cells, MPO +/Ox-42 + inflammatory cell infiltrations, caspase-3 activity, and Bax + cells, as compared to the ischemia-vehicle group. The vascular surface area, the vascular branch points, the vascular length, and the number of BrdU + endothelial cells were significantly increased in the rosiglitazone group compared with the ischemia-vehicle group. Rosiglitazone increased eNOS expression around the ischemic margin with downregulation of FasL. Here, we show that rosiglitazone treatment enhances angiogenesis and functional recovery with dose-dependent induction of ischemic tolerance.

Porphyromonas gingivalis enhances FasL expression via up-regulation of NFκB-mediated gene transcription and induces apoptotic cell death in human gingival epithelial cells

The interaction between epithelial cells and micro-organisms is often a crucial initiating event in infectious diseases. Infection with Porphyromonas gingivalis, a Gram-negative anaerobe, is strongly associated with severe periodontal disease. This bacterium possesses an array of virulence factors, some of which can induce apoptosis. The tumour necrosis factor (TNF) receptor family is involved in the regulation of cellular homeostasis, cell surface molecules involved in phagocytosis, Fas ligand (L) expression and activation of the caspase cascade resulting in DNA fragmentation and cell blebbing. The current study examined the role of nuclear factor-kappaB (NFkappaB) in FasL-mediated apoptotic cell death in primary human gingival epithelial cells (HGEC) induced by heat-killed P. gingivalis, probably through TLR signalling pathways. A marked up-regulation of TLR2 and Fas-FasL was detected in HGEC stimulated with P. gingivalis. Activation of NFkappaB by P. gingivalis in HGEC was demonstrated by an NFkappaB promoter luciferase assay as well as by phosphorylation of p65 as detected by Western blotting. Activation of cleaved caspase-3 and caspase-8 resulted in apoptotic cell death of HGEC. The survival proteins c-IAP-1/c-IAP-2 were decreased in HGEC exposed to P. gingivalis. HGEC apoptosis induced by P. gingivalis was inhibited by an anti-human FasL monoclonal antibody. Blockade of NFkappaB by helenalin resulted in down-regulation of FasL whereas a caspase-8 inhibitor did not decrease FasL. Taken together, these studies show that P. gingivalis can induce epithelial cell apoptosis through Fas-FasL up-regulation and activation of caspase-3 and caspase-8.

Amelioration of murine lupus by a peptide, based on the complementarity determining region-1 of an autoantibody as compared to dexamethasone: Different effects on cytokines and apoptosis

A peptide (hCDR1) based on the sequence of the complementarity-determining region-1 of an anti-DNA autoantibody ameliorates clinical manifestations of lupus. We analyzed the beneficial effects of hCDR1 when given alone or in combination with dexamethasone, while comparing the mechanisms of action of the latter. Treatment with either hCDR1 or dexamethasone, or a combination of the latter significantly reduced titers of dsDNA-specific autoantibodies, levels of proteinuria, and intensity of glomerular immune complex deposits. Both drugs down-regulated the secretion and expression of IFN-γ and IL-10, but only treatment with hCDR1 up-regulated TGF-β. While both drugs reduced the expression of Fas ligand (FasL) and caspase 8, treatment with hCDR1 resulted in reduced whereas dexamethasone administration resulted in increased rate of apoptosis. Furthermore, down-regulation of FasL appeared to play a role in cytokine modulation. We conclude that specific treatment with hCDR1 ameliorates murine lupus via distinct mechanisms of action than those of dexamethasone.

Fas-ligand gene silencing in basal cell carcinoma tissue with small interfering RNA

Basal cell carcinoma (BCC) is the most frequent cancer in the Caucasian population. Cells of BCC strongly express Fas-ligand (FasL), a member of the tumor necrosis family, which induces apoptosis in Fas receptor-expressing cells. It has been suggested that by expression of FasL, BCC cells may evade the attack of Fas-positive immune effector cells allowing the tumor to expand. Thus, downregulation of FasL should prime BCC to the assault of immune effector cells. Recently, it has been shown that RNA interference is a highly successful approach to specifically silence a gene of interest in single cells and some animal models. However, RNAi in human tissues has not been shown so far. Here, we provide evidence that small interfering RNAs (siRNAs) efficiently transfect tumor tissue ex vivo and silence the gene of interest. We demonstrate that a specific siRNA efficiently downregulates FasL not only in FasL-positive indicator cells but also in surgically excised BCC tissue at both the protein and the mRNA level. The successful transfection of tumor tissues with siRNAs now allows to test the function of the molecule under study and opens up the investigation of other target genes in the tumor.

Regulation of Fas ligand expression by vascular endothelial growth factor in endometrial stromal cells in vitro.

When Fas ligand (FasL) interacts with the Fas receptor, it induces apoptosis through autocrine and/or paracrine signalling. Vascular endothelial growth factor (VEGF) is a potent mitogenic cytokine. VEGF plays a role during remodelling of the endometrium following menstruation. We hypothesized that, by regulating FasL expression, VEGF may play a role in endometrial stromal cell survival by decreasing autocrine apoptotic signalling. We aimed to determine the expression of FasL in cultured endometrial stromal cells and its modulation by VEGF. VEGF induced a decrease in both FasL-positive cell number and FasL intensity as determined by immunocytochemistry and western blot respectively (P < 0.05). These effects of VEGF were observed in a concentration-dependent manner (10-42%; P < 0.05). Anti-VEGF neutralizing antibody alone resulted in an increase in the FasL expression. When combined with VEGF, anti-VEGF reversed the VEGF-induced decrease in FasL level up to 100% (P < 0.05). In addition, western blot analysis showed that FasL expression in endometrial stromal cells demonstrated a cyclic change every 12 h during 48 h of incubation. These results suggest that down-regulation of FasL by VEGF may affect endometrial stromal cell survival in an autocrine or paracrine manner. The decrease in FasL level may be due to a stimulation of its degradation. Our results show that FasL in endometrial stromal cells in culture has a cyclic expression model, suggesting that there may be a regulation at the translation level.

Fas Ligand Downregulation with Antisense Oligonucleotides in Cells and in Cultured Tissues of Normal Skin Epidermis and Basal Cell Carcinoma

Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis upon interaction with Fas-receptor-expressing cells. FasL normally plays an important immune regulatory role, but it can also cause severe skin diseases if overexpressed and it may serve some tumors for immune evasion. Thus, in situ inhibition of FasL expression with antisense oligonucleotides in patients may be a novel approach to overcome its pathogenic role. We designed and evaluated 15 phosphorothioate antisense oligonucleotides directed against different regions of the human FasL mRNA. They exhibited different inhibitory activities on FasL expression in HEK293 cells. The most potent antisense oligonucleotide, ASO8, specifically downregulated 90% FasL expression at the protein level and 80% at the mRNA level. FasL downregulation reduced the effector function of HEK293 cells toward Fas receptor positive target cells. Further studies demonstrated that ASO8 efficiently inhibited FasL synthesis in split skin and basal cell carcinoma tissue. Our results show that the modulation of FasL expression by antisense oligonucleotides is possible in cells as well as tissue and indicate that antisense oligonucleotides may provide a promising strategy for the therapy of FasL-mediated disorders.

Cisplatin resistance in an ovarian carcinoma is associated with a defect in programmed cell death control through XIAP regulation.

Chemoresistance is a major impediment to the successful treatment of cancer. It involves various mechanisms, including defects in the apoptosis program that is induced by anticancer drugs. To further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin (CDDP) treatment, we compared the effect of CDDP on expression of X-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, -7, and -9, Fas, Fas-ligand (Fas-L), and pro- and antiapoptotic proteins in a CDDP-sensitive human ovarian carcinoma cell line (2008) and its CDDP-resistant subclone (2008C13). In this article, we show that cisplatin treatment led to a differential expression of distinct apoptotic targets in the CDDP-sensitive cell line (2008) and its CDDP-resistant subclone (2008C13). The acquisition of cisplatin resistance was associated with the ability of the treated cells to enhanced expression of XIAP, whereas the death inducer Fas-L was abrogated in 2008C13 following treatment with CDDP. However, the CDDP-sensitive cells failed to activate XIAP but increased Fas-L expression, indicating that distinct regulatory mechanisms are operative. These findings suggest that the expression of XIAP and downregulation of Fas-L are linked to chemoresistance in ovarian carcinoma cells and may represent one of the potential antiapoptotic mechanisms involved during this process.