Increased Fas Expression Research Articles

Induction of apoptosis by cinobufacini preparation through mitochondria- and Fas-mediated caspase-dependent pathways in human hepatocellular carcinoma cells

Cinobufacini (Huachansu), an aqueous extract from the skins of Bufo bufo gargarizans Cantor, is a well-known traditional Chinese medicine widely used in clinical cancer therapy in China. However, the precise mechanisms induced by cinobufacini in human hepatocellular carcinoma (HCC) cells are still not very clear. The aim of present study was to investigate possible apoptotic mechanisms induced by cinobufacini in HCC cell lines HepG2 and Bel-7402. We found that cinobufacini treatment resulted in a significant decrease in cell proliferation and induced apoptotic cell death with the increase of treatment time. It indicated that cinobufacini-induced apoptosis was associated with mitochondria-mediated pathway including the loss of mitochondrial membrane potential (Δψm), the increase of Bax/Bcl-2 ratio, cytochrome c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) degradation. Additionally, cinobufacini also activated Fas-mediated apoptosis pathway obviously as evident by an increase in Fas expression, and caspase-8 and caspase-10 activation. Moreover, the BH3-only protein Bid was cleaved into a truncated Bid (tBid) after cinobufacini treatment. Taken together, these data suggested cinobufacini could induce apoptosis of HCC cells through mitochondria- and Fas-mediated caspase-dependent pathways with the increase of treatment time, which might provide an experimental evidence for cinobufacini treatment of HCC.

Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-α and IFN-γ reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-α- and IFN-γ-stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased ∼6-fold in an NF-κB- and p38(mapk)-dependent fashion. Prevention of the increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-α and IFN-γ to sensitize fibroblasts to Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal resistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF.

Effects of pneumonia caused by klebsiella pneumoniae on apoptosis of thymocytes in rats

Objective To study the effects of pneumonia caused by Klebsiella pneumoniae on apoptosis of thymocytes in rats and its possible mechanism. Methods A total of 48 Sprague Dawley rats were randomly (random number) divided into 2 groups, namely the control group (n =24) and the infection group ( n = 24). The pneumonia models of rats were made with 0.3 mL Klebsiella pneumoniae suspension administered intratracheally per animal. On the 2nd, 4th, and 6th day after intratracheal instillation of bacteria, 1/ 3 of the rats in each group were sacrificed and TdT-mediated dUTP nick end labeling (TUN EL) method was used to assess the apoptosis of thymocytes. The expressions of cleaved Caspase-3, Bcl-2 and Fas in thympcytes of rats were detected with immunohistochemical staining. Results On each interval, apoptosis index of thymocytes, and the expressions of Cleaved Caspase-3 and Fas in the infection group were all higher than those in the control group (P < 0.01) , while the expressions of Bcl-2 lower than those in the control group (P<0.01). As more time consumed, the apoptotic index of thymocytes and the expressions of cleaved Caspase-3 in the infection group increased significantly (P<0.05). The expressions of Bcl-2 declined gradually (P < 0.05), but the expressions of Fas reached their peak 4th day after infection. There were no significant dynamic changes in all above mentioned variables in control group. Conclusions Pneumonia caused by Klebsiella Pneumoniae can lead to the increase in thymocyte apoptosis in rats. The mechanism may be associated with the decreased expression of Bcl-2 and the increased expression of Fas in thymocytes of rats with pneumonia caused by Klebsiella pneumoniae. The different apoptosis regulation pathways have different effects on different phase of pneumonia, that the effects of Fas decrease 4th day after pneumonia, while the effects of Bcl-2 increase further. Key words: Klebsiella pneumoniae; Pneumonia; Thymocyte; Rat; Apoptosis; Cleaved Caspase-3; Bcl-2; Fas

The Involvement of Epithelial Fas in a Human Model of Graft Versus Host Disease

Graft-versus-host disease (GVHD) is an important complication occurring after hematopoietic stem-cell transplantation (HSCT). Animal model studies have shown the involvement of the Fas (APO-1/CD95)/Fas-Ligand pathway in GVHD pathogenesis, but its association with cutaneous GVHD in human remains to be established. In the present study, Fas involvement in skin damage was assessed using a human skin explant model of GVHD. Fas and FasL expression were measured by immunohistochemistry and blockade of Fas pathway was investigated using an antagonistic anti-human Fas monoclonal antibody. In addition, levels of soluble Fas (sFas) were determined in the serum of patients receiving allogeneic HSCT with and without GVHD. The results showed that Fas up-regulation in the epithelium of human skin explants correlated with graft-versus-host reaction (GVHR) in the skin explant model (P<0.001). Decreased GVHR grades were observed by using a Fas blocking monoclonal antibody. Levels of sFas were increased post-HSCT (P<0.001) but rather than being associated with the severity of GVHD, sFas levels differed with the conditioning treatments the patients received before the HSCT. Higher GVHR grades were associated with increased Fas expression in the epithelium of the skin explants. In addition, by blocking Fas-mediated apoptosis, the GVHR grades were decreased. Our study thus shows the involvement of Fas in cutaneous GVHD damage, and supports the potential use of Fas as a therapeutic target.

LMNA E82K Mutation Activates FAS and Mitochondrial Pathways of Apoptosis in Heart Tissue Specific Transgenic Mice

The lamin A/C (LMNA), nuclear intermediate filament proteins, is a basic component of the nuclear lamina. Mutations in LMNA are associated with a broad range of laminopathies, congenital diseases affecting tissue regeneration and homeostasis. Heart tissue specific transgenic mice of human LMNA E82K, a mutation causing dilated cardiomyopathy, were generated. Lmna E82K transgenic mouse lines exhibited thin-walled, dilated left and right ventricles, a progressive decrease of contractile function assessed by echocardiography. Abnormalities of the conduction system, myocytes disarray, collagen accumulation and increased levels of B-type natriuretic peptide (BNP), procollagen type III α1 (Col3α1) and skeletal muscle actin α1 (Actα1) were detected in the hearts of Lmna E82K transgenic mice. The LMNA E82K mutation caused mislocation of LMNA in the nucleus and swollen mitochondria with loss of critae, together with the loss of nuclear envelope integrity. Most interestingly, we found that the level of apoptosis was 8.5-fold higher in the Lmna E82K transgenic mice than that of non-transgenic (NTG) mice. In the presence of the LMNA E82K, both of FAS and mitochondrial pathways of apoptosis were activated consistent with the increase of FAS expression, the release of cytochrome c from mitochondria to cytosol and activation of caspase-8, -9 and -3. Our results suggested that the apoptosis, at least for the LMNA E82K or the mutations in the rod region of Lamin A/C, might be an important mechanism causing continuous loss of myocytes and lead to myocardial dysfunction. It could be a potential therapeutic means to suppress and/or prevent inappropriate cardiac cell death in patients carrying LMNA mutation.

Abstract 1271: In vitro expanded natural killer (NK) cells are more susceptible to Fas-mediated apoptosis compared to fresh and overnight IL-2 activated NK cells

Abstract Introduction: NK cells are currently being explored as a treatment modality for many oncologic diseases. However, the small number of NK cells in the circulation, approximately 5-15%, may limit the effectiveness of host NK cell immunity against cancer. Several methods to expand NK cells for subsequent adoptive infusion in cancer patients have been explored, including culturing NK cells in vitro with interleukin-2 (IL-2). Unfortunately, IL-2 stimulation alone results in only a small increase in NK cell proliferation. Recently, a novel in vitro method for large scale NK cell expansions using irradiated EBV-LCL feeder cells has been developed and is currently being utilized in an NHLBI clinical trial. Purpose: The aims of this study included: a) to identify differences in susceptibility to apoptotic pathways between fresh, overnight IL-2 activated, and expanded NK cells by comparing the death receptors DR4, DR5, and Fas; b) to determine when Fas expression is acquired on IL-2 activated NK cells; and c) to determine if Fas expression differs on NK cells expanded with different feeder cell populations. Methods: NK cells from healthy donors were isolated using CD56+ magnetic selection beads. Using flow cytometry, we examined Fas, DR4, and DR5 expression on fresh, overnight IL-2 activated, and NK cells expanded with either EBV-LCL or K562 41bb ligand transfected feeder cells for 7-14 days. To assess susceptibility to Fas mediated apoptosis, each NK cell group was cultured with recombinant FasL (rhFasL) for 12 hours with Annexin V expression by flow cytometry used to measure apoptosis. In addition, Fas expression was examined over multiple days for both IL-2 activated NK cells and expanded NK cells. Results: Fas expression was similar on overnight IL-2 activated NK cells compared to fresh NK cells. In contrast, NK cells maintained in IL-2 for more than 2 days and expanded NK cells had a substantial increase in Fas expression. Similar increases in Fas expression were seen in NK cells expanded with either EBV-LCL or K562 41bb ligand transfected feeder cells. Enhanced Fas expression was associated with increased susceptibility to rhFasL mediated apoptosis; the fold increase in Annexin V induced by rhFasL at 12 hours was significantly higher amongst expanded NK cells (5.6 fold increase; p = 0.0001) compared to either fresh NK cells (1.2 fold increase; p = 0.32) or overnight IL-2 activated NK cells (1.0 fold increase; p = 0.62). In contrast to Fas, surface expression of the TRAIL death receptors DR4 and DR5 were similar amongst all NK cell populations analyzed. Conclusion: These data show for the first time that expanded NK cells are more susceptible to Fas mediated apoptosis compared to fresh and overnight IL-2 activated NK cells. Whether expanded NK cells are more susceptible to apoptosis mediated by tumor cells expressing Fas Ligand in vitro and in vivo is currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1271.

Plasma membrane-derived vesicles (PMVs) released from apoptotic Jurkat cells express upregulated surface Fas and induce apoptosis in recipient viable cells (89.45)

Abstract Apoptosis, distinct from necrosis, is irreversible programmed cell death. Characterized by membrane blebbing, DNA fragmentation and cell shrinkage it plays an important role in infection. During viral infection, depending on the virus, apoptosis may either be enhanced or inhibited. Apoptosis can be induced by dexamethasone (DEX), cychloheximide (CHX) and H2O2 in vitro in various cell types. PMV release from apoptotic cells (isolated by differential centrifugation, expressing surface phosphatidylserine and of 0.2-1μm average diameter) could be inhibited by pretreatment with calpeptin. Such PMVs, which we distinguished from apoptotic bodies and other microvesicle populations, such as exosomes, have increased Fas expression on DEX and CHX- but not H2O2-mediated apoptotic Jurkat cells and an increased capacity to induce apoptosis in recipient healthy Jurkat cells, maximally at a 5:1 PMV-cell ratio. This effect, specific to PMVs from apoptotic cells, as opposed to those from viable cells, occurred after 30 min incubation of recipient, viable cells with PMVs and dose-dependently increased up to a maximum after 5h incubation. Inhibitors of caspase and anti-Fas blocking antibodies have been used to elucidate the importance of these factors in PMV transmission of this apoptotic signal. PMV-mediated apoptosis is currently being investigated for its role in in vitro studies of Coxsackie B3 infection in HeLa cells.

Role of fatty acid chain length on the induction of apoptosis by newly synthesized catechin derivatives

The catechins, a family of polyphenols found in tea, can evoke various responses, including apoptosis. In this study we investigated whether the chemical modification of (-)-epigallocatechin gallate (EGCG) could enhance its apoptosis activity. We found that one of the catechin conjugated with capric acid [(2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl decanoate; catechin-C10] was most potent to induce apoptosis in U937 cells. C10 treatment resulted in a significant increase in reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, cytochrome c release caspase-9 and caspase-3 activation. In addition to this C10 also activated extrinsic pathway significantly as evident by time-dependent increase in Fas expression and caspase-8 activity. C10 mediated cleavage of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Moreover, pre-treatment of cells with anti-oxidant N-acetyl-L-cysteine (NAC) significantly prevented C10-induced apoptosis but did not protect MMP loss. Treatment of cells with pan-caspase inhibitor significantly inhibited apoptosis indicating that caspases are playing key role. In addition to this C10 was found to induce apoptosis in human colon cancer (HCT116) cells while it showed resistance to human keratinocytes (HaCat). In short our results showed that the optimal fatty acid side chain length is required for the apoptosis inducing activity of catechin derivatives in U937 cells.

Gene Expression Changes in Bone Marrow of MDS Patients with Del(5q) During Lenalidomide Treatment.

Abstract 2622Poster Board II-598 Introduction:Genetic alterations reported in myelodysplastic syndromes (MDS) are not disease-specific and the underlying molecular causes of the disease remain poorly understood. It has been suggested that one or more of the genes mapping within the commonly deleted region of the 5q syndrome, together with other distant genes, may be critical to the development of the 5q syndrome. Potential candidate genes have been identified including the tumor suppressor gene SPARC, and the ribosomial protein gene, RPS14. Haploinsufficiency of RPS14 has been demonstrated and recent evidence indicates RPS14 as a causal gene for the 5q syndrome. Lenalidomide has proven efficacy in MDS patients with del(5q). Rapid and durable responses include transfusion-independence, with a rise in Hb, suppression of the 5q-deletion clone and improvement in bone marrow morphologic features. Methods:In a multicenter Italian phase II trial to evaluate safety, changes in quality of life and efficacy of lenalidomide in primary MDS patients with del(5q) and low or Int-1 risk IPSS, we investigate changes in bone marrow cytogenetics and gene expression patterns during treatment. The starting dose of lenalidomide is 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Dosing is based upon clinical and laboratory findings. Bone marrow cytogenetics and gene expression profiling are performed on study entry and every 12 weeks up to end of study (week 52). Gene expression assays of 51 candidate genes from the published literature and genomic databases have been selected and are carried out with TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative dd CT method, according to manufacturer's instructions. Using an 18S mRNA gene pre-designed assay from Applied Biosystems to detect the expression of the housekeeping gene 18S in each sample, target gene expression is normalized with 18S gene expression derived from a bone marrow pool of normal healthy subjects and for each sample the ratio between the target and 18S are expressed. Results:Baseline values for 23 patients (mean age 73 ± 10 years) are available and 16 have been re-evaluated after 12 weeks. Mean Hb was 8.6 ± 0.9 g/dL and 20 patients were transfusion-dependent. Seven patients had additional cytogenetic abnormalities. At baseline, RPS14 was under-expressed in 19 out of 21 patients evaluated. After 12 weeks RPS14 was re-evaluated in 13 patients: all had erythroid responses and RPS14 increased significantly from 0.07 (IQ Range 0.03–0.13) to 76.1 (0.73– 304.0, p=0.002). SPARC expression was under-expressed in 15/23 patients and variations during treatment were not significant. Baseline FAS gene was under-expressed in all patients and increased above reference values (p=0,006) after 12 weeks in 7/14 cases. IL7R was over-expressed in all patients at baseline (median 3263.3, IQ range 1998.3–5027.1) and was significantly reduced after 12 weeks (median 0.17, IQ range 0.05–2.20, p<0.0001). TINAGL1 gene expression was very high at baseline (mean 5214 ± 3661) and significantly reduced below normal values during treatment (0.63±0.87, p<0.0001). WTI was over-expressed in almost all patients (median 3246, IQ range 1590–7785) and, though reduced during treatment, still remained highly over reference values at 12 weeks (p=0.148). The expression of the other genes did not vary significantly during treatment. Conclusions:A preliminary study evaluating short-term effects of lenalidomide in low and Int-1 IPSS risk MDS and del(5q) demonstrates that gene expression is abnormal and varies during treatment. RPS14 is down-expressed and a significant increase in its expression is observed during treatment with lenalidomide in responsive patients. There is also an increase in FAS expression; the protein encoded by FAS gene is a member of the TNF-receptor superfamily and its interaction with its ligand leads to apoptosis.The IL7R gene on chromosome 5 (5p13) codifies for the IL7 receptor, which blocks apoptosis during differentiation and activation of T lymphocytes. TINAGL1 gene on chromosome 1 codifies for a Cathepsin B-like protein implicated in a number of human diseases such as cancer. During lenalidomide treatment, the expression of IL7R and TINAGL1 are reduced. Further results and repeated testing during this trial may develop new insights in the pathogenesis of MDS with del(5q) and in long-term effects of lenalidomide on bone marrow changes. Disclosures:Oliva:Celgene: Consultancy. Balleari:Celgene: Consultancy. Finelli:Celgene: Consultancy.

Association among Fas expression in leucocytes, serum Fas and Fas-ligand concentrations and hepatic inflammation and fibrosis in chronic hepatitis C

Replication of the hepatitis C virus (HCV) in peripheral blood mononuclear cells (PBMC) may impair immune functions and establish persistent infection. The aim of this study was to assess the influence of HCV on PBMC and their susceptibility to apoptosis in relation to liver inflammation and fibrosis. Eighty-one patients with chronic hepatitis C (CHC) were enrolled in this study. Flow cytometry was used to determine the amount of T cells (CD4(+), CD8(+)), B cells (CD19(+)), monocytes (CD14(+)) and natural killer cells (CD16(+)) in the peripheral blood and the expression of CD95(+) (CD95/APO-1) in each subset. Serum concentrations of sFas and sFasL were assessed by the enzyme-linked immunosorbent assay method. An increased expression of Fas was observed in CD4(+) and CD8(+) cells in CHC. There was a more prominent expression of Fas on CD4(+) cells in HCV genotype 1b in contrast to 3a. Increased Fas expression on CD4(+) cells was seen in advanced stages of liver disease. Fas expression on monocytes was lower in advanced stages of liver inflammation and fibrosis. Serum sFas concentration was higher in CHC compared with the control group. There was an association between sFasL concentration and inflammatory activity in the liver. Serum sFasL concentration correlated positively with the mean intensity of fluorescence of the Fas receptor in CD4(+) and CD8(+) cells, granulocytes and monocytes. These findings indicate that there is an increased susceptibility of PBMC to apoptosis, which can be attributed to the constant contact of leucocytes with the inflamed liver tissue, or from direct HCV influence.

Double role of Fas ligand in the apoptosis of intervertebral disc cells &amp;lt;italic&amp;gt;in vitro&amp;lt;/italic&amp;gt;

Fas ligand (FasL) may play an important role in maintaining the immune privilege of intervertebral disc (IVD). Besides, it is closely related to the apoptosis of degenerative disc cells. Nowadays, lots of reports have described about the paradoxical effects of FasL, although the effect of FasL on IVD cells is still under debate. In this study, we tried to investigate the effects of FasL on Fas expression and on the apoptosis of nucleus pulposus (NP) cells in Sprague-Dawley rats. The results showed that the expression of Fas in NP cells was significantly increased by the recombinant FasL. Meanwhile, the apoptosis of NP cells increased markedly in a FasL dose-dependent manner. Interestingly, RNA interference results indicated that the increase of Fas expression and the NP cell apoptosis described previously were inhibited by Fas siRNA, suggesting that RNA interference might be one of novel strategies to prevent IVD cells from apoptosis.

Effects of the levonorgestrel-releasing intrauterine system on cell proliferation, Fas expression and steroid receptors in endometriosis lesions and normal endometrium

The objectives of this study were: (i) to evaluate the effects of the levonorgestrel-releasing intrauterine system (LNG-IUS) on both proliferation and apoptosis markers and hormone receptors of the eutopic and ectopic endometrium of women experiencing pain related to endometriosis and (ii) to compare the results with those obtained with GnRH agonist (GnRHa) injections. Pre- and post-treatment endometrium and endometriosis specimens were obtained from 22 women experiencing pain related to endometriosis who were treated with LNG-IUS (n = 11) or GnRHa (n = 11) for 6 months. Changes in the expression of proliferating cell nuclear antigen, Fas, progesterone receptor (PRA) and estrogen receptor alpha (ER-alpha) were analyzed by immunohistochemistry. The cell proliferation index was significantly reduced in the epithelium and stroma of both the eutopic and the ectopic endometrium after treatment with the LNG-IUS and GnRHa. Only LNG-IUS users showed an increased H-score for Fas in the epithelium of the eutopic and ectopic endometrium (P < 0.05). Expression of ER-alpha and PRA by the glandular epithelium was lower in the eutopic endometrium after both treatments, but this reduction was noted in the ectopic endometrium only after LNG-IUS treatments (P < 0.05). No difference was detected between groups for any of the markers. LNG-IUS reduced both cell proliferation and the expression of PRA and ER-alpha and increased Fas expression in the eutopic and ectopic endometrium of patients with endometriosis. Some of these actions were not observed with GnRHa.

The role of 131I in the apoptosis of thyrocytes of patient with Graves' disease

The changes of Fas,FasL and Bcl-2 expression in thyrocytes of patients with Graves＇disease were investigated before and 2 weeks after ^131 I administration. The results showed that ^131 I could induce thyrocytes to express the apoptotic protein Fas, FasL and the anti-apoptotic protein Bcl-2 in patients with Graves＇disease. A statistically significant correlation was found between the dose of ^131I and the expression levels of Fas and FasL but not Bcl-2,suggesting that early onset of hypothyroid after ^131I administration may be due to the increased expression of Fas and FasL in thyrocytes. Key words: Graves＇disease; ^131 I therapy; Apoptosis; Apoptosis-related proteins

Increased expression of CD154 and FAS in SLE patients’ lymphocytes

An increased level of apoptotic material and B cell activation leading to autoantibody production are hallmarks of systemic lupus erythematoses (SLE). Increased FAS expression, apoptosis, and CD154-mediated signaling, enabling T–B cell interaction are involved in the pathogenesis of SLE. This study addresses the expression profile of CD154 and FAS in the peripheral blood of patients with SLE, rheumatoid arthritis (RA) and normal healthy control donors. Surface markers on peripheral blood T and B cells from patients and healthy control donors were assessed using flow cytometry. The expression of CD154 and FAS were significantly increased in T and B cells of SLE patients as compared to healthy control donors and RA patients. In SLE and RA patients, FAS expression strongly correlated with CD154 expression on T cells, which was not found in healthy control donors. FAS expression was also associated with the occurrence of anti-DNA antibodies. We demonstrate high CD154 and FAS expression as a characteristic feature of SLE. This pattern may reflect simultaneous activation of apoptosis and activation of B–T cell interaction in SLE.

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

FAPP2 gene downregulation increases tumor cell sensitivity to Fas-induced apoptosis

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.

HCV Induces Oxidative and ER Stress, and Sensitizes Infected Cells to Apoptosis in SCID/Alb-uPA Mice

Hepatitis C virus (HCV) is a blood-borne pathogen and a major cause of liver disease worldwide. Gene expression profiling was used to characterize the transcriptional response to HCV H77c infection. Evidence is presented for activation of innate antiviral signaling pathways as well as induction of lipid metabolism genes, which may contribute to oxidative stress. We also found that infection of chimeric SCID/Alb-uPA mice by HCV led to signs of hepatocyte damage and apoptosis, which in patients plays a role in activation of stellate cells, recruitment of macrophages, and the subsequent development of fibrosis. Infection of chimeric mice with HCV H77c also led an inflammatory response characterized by infiltration of monocytes and macrophages. There was increased apoptosis in HCV-infected human hepatocytes in H77c-infected mice but not in mice inoculated with a replication incompetent H77c mutant. Moreover, TUNEL reactivity was restricted to HCV-infected hepatocytes, but an increase in FAS expression was not. To gain insight into the factors contributing specific apoptosis of HCV infected cells, immunohistological and confocal microscopy using antibodies for key apoptotic mediators was done. We found that the ER chaperone BiP/GRP78 was increased in HCV-infected cells as was activated BAX, but the activator of ER stress–mediated apoptosis CHOP was not. We found that overall levels of NF-κB and BCL-xL were increased by infection; however, within an infected liver, comparison of infected cells to uninfected cells indicated both NF-κB and BCL-xL were decreased in HCV-infected cells. We conclude that HCV contributes to hepatocyte damage and apoptosis by inducing stress and pro-apoptotic BAX while preventing the induction of anti-apoptotic NF-κB and BCL-xL, thus sensitizing hepatocytes to apoptosis.

Mitochondria Influence Fas Expression in GP120-Induced Apoptosis of Neuronal Cells

The human immunodeficiency virus-1 (HIV-1) destroys the immune system and also induces neurological disease culminating into dementia (HIV-associated dementia). Though the HIV viral protein gp120 induces apoptosis in neuronal cells, the mechanism of action is still poorly defined. Recent studies show that cells die during apoptosis by Fas aggregation aided by the mitochondrial proapoptotic proteins. Our studies show an increase in expression of Fas and its associated downstream proteins after treatment of the neuroblastoma cells, SH-SY5Y, with gp120. Fas and its associated death proteins, FADD and caspase-8 (DISC), are downregulated when treated with the caspase inhibitors. The results indicate that mitochondrial-death proteins like caspases may influence the upregulation of the death receptor Fas, and the inhibition of caspases prevents gp120-induced apoptosis.

Mechanistic Study on Growth Suppression and Apoptosis Induction by Targeting Hepatoma-derived Growth Factor in Human Hepatocellular Carcinoma HepG2 Cells

Hepatoma-derived growth factor (HDGF) is frequently overexpressed in human cancer. The growth factor was previously demonstrated to be a survival factor as knock-down of HDGF suppresses the growth and induces apoptosis in human cancer cells through the Bad-mediated intrinsic apoptotic pathway. However, inactivation of Bad cannot completely repress the apoptosis induced upon HDGF knock-down, indicating the presence of other unidentified pathways. In the present study, HDGF knock-down was shown to trigger the Fas-mediated extrinsic apoptotic pathway in human hepatocellular carcinoma HepG2 cells through NF-κB signaling pathway. Increases in Fas expression and fas promoter activity were detected upon HDGF knock-down by Western blot analysis and luciferase reporter assay. Knock-down of fas inhibited HDGF knock-down effect on apoptosis induction and growth suppression as revealed by annexin V binding assay and soft agar assay. Down-regulation of IκBα was also observed upon HDGF knock-down. Overexpression of IκBα by transient transfection or inhibition of NF-κB by BAY11-7082 suppressed HDGF knock-down effect on fas promoter activation, Fas up-regulation, apoptosis induction and growth suppression. Furthermore, the interaction of Fas-mediated extrinsic and Bad-mediated intrinsic apoptotic pathways was demonstrated as a stronger inhibition on apoptosis induction and growth suppression upon HDGF knock-down was observed when both pathways were inactivated. The results therefore suggested that, through both intrinsic and extrinsic apoptotic pathways, HDGF may function as a survival factor and be a potential target for cancer therapy.

Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas–FasL and mitochondria-dependent pathways

Lindane, an organochlorine pesticide, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underpinning the gonadal effects of lindane, we sought to investigate the levels of apoptosis-related proteins, namely cytochrome c, caspase-3 and-9, Fas and FasL in the testis of adult rats. Furthermore, the study aims to delineate whether nuclear factor kappa B (NF-κB) is involved in meditating the testicular effects of lindane. Animals were administered with a single dose of lindane (5 mg/kg body weight) and sacrificed at specific post-treatment intervals (0, 3, 6, 12, 24 and 72 h). Significant elevations in the levels of cytosolic cytochrome c with a parallel increase in pro-caspase-9 were observed as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL and caspase-3 were observed. Immunofluorescence studies revealed increased colocalization of Fas and caspase-3 in peritubular germ cells. FasL levels were increased in Sertoli and peritubular germ cells. The cytoplasmic levels of NF-κB p65 decreased from 3 h following exposure with a maximal decline at 12 and 24 h. Changes in the localization of NF-κB were observed with maximal nuclear translocation in germ cells at 12 and 24 h. Terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL) assay revealed a time-dependent increase in the number of apoptotic cells. Taken together, the data illustrate induction of testicular apoptosis in adult rats following exposure to a single dose of lindane. Early activation of NF-κB in contrast to late increase in Fas expression suggests a pro-apoptotic role of NF-κB in testicular response to lindane.