Increased expression of FLIP,an inhibitor of Fas-mediated apoptosis,in stomach cancer

Despite the cell surface expression of Fas (Apo-1/CD95), many types of tumor cells, including stomach cancer cells, are resistant to Fas-mediated apoptosis, indicating the presence of inactivating mechanisms of Fas signaling. Expression of FLICE-like inhibitory protein (FLIP), one of the inhibitory proteins of Fas-mediated apoptosis, has been reported in several cancer types, but not in stomach cancer. In the present study, we analyzed the expression of Fas and FLIP in 60 advanced gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Immunopositivity (defined as >/=30% of the neoplastic cells) was observed for Fas in 58 (97%) and FLIP in 54 (90%) of the 60 cancers. All of the tumors with FLIP immunostaining also showed Fas immunostaining. Loss of cell surface Fas immunostaining, another mechanism of Fas resistance, was observed in 45 tumors (75%). By contrast, normal gastric mucosal cells showed no or weak expression of both Fas and FLIP. Taken together, these results indicate that increased expression of FLIP is a frequent event in stomach carcinomas, and suggest that for evading apoptosis stomach carcinoma cells in vivo may need FLIP expression, which might contribute to tumor development.     

Decreased expression of tumour suppressor Bax-interacting factor-1 (Bif-1), a Bax activator, in gastric carcinomas

AIMS: Evasion of apoptosis is one of the hallmarks of cancer. Bax-interacting factor-1 (Bif-1) interacts with both Bax and Bak that are essential for the intrinsic pathway of apoptosis, and enhances apoptosis. The aim of this study was to explore whether alteration of Bif-1 expression might be a characteristic of gastric cancer. METHODS: We analysed the expression of Bif-1 protein in 60 gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. RESULTS: In normal gastric mucosal cells, surface and glandular epithelial cells strongly expressed Bif-1. While Bif-1 expression was detected in 24 cases (40%) of the gastric carcinomas, there was no Bif-1 immunostaining in the remaining 36 cancers. Even in the 24 cases with positive Bif-1 immunostainings, 10 cancers showed decreased intensity of immunostaining compared with the normal mucosal epithelial cells. CONCLUSION: The decreased expression of Bif-1 in malignant gastric epithelial cells compared with the normal mucosal cells suggests that loss of Bif-1 expression may play a role in gastric tumorigenesis, possibly by inhibiting the apoptosis mediated by Bif-1.     

Immunohistochemical analysis of NF‐κB signaling proteins IKKε, p50/p105, p52/p100 and RelA in prostate cancers

Seo SI, Song SY, Kang MR, Kim MS, Oh JE, Kim YR, Lee JY, Yoo NJ, Lee SH. Immunohistochemical analysis of NF‐κB signaling proteins IKKε, p50/p105, p52/p100 and RelA in prostate cancers. APMIS 2009; 117:623–8. Activation of nuclear factor‐kappa B (NF‐κB) signaling is considered an important mechanism in the development of prostate cancers. A recent study revealed that IκB kinase epsilon (IKKε), an activator of NF‐κB, was overexpressed in breast cancers and acted as an oncogene. Expression of NF‐κB members has been reported in prostate cancer tissues, but expression of IKKε has not yet been studied in prostate cancers. In this study, we attempted to explore as to whether expressions of IKKε and NF‐κB members p50/105, p52/p100 and RelA are altered in prostate cancers. We analyzed the expression of IKKε, p50/105, p52/p100 and RelA in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. In the TMA, IKKε is expressed in basal cells, but not in alveolar cells in normal prostate glands. IKKε is expressed in 60.0% of prostate intraepithelial neoplasm (PIN) and 70.1% of the prostate cancers in the cytoplasm. Nuclear immunostainings of NF‐κB members p50/105, p52/p100 and RelA, which are considered activation of NF‐κB signaling, were observed respectively in 28.0%, 18.7% and 37.4% of the cancers. Nuclear staining was detected neither in normal alveolar cells nor in PIN. However, none of the expression of p50/105 nor p52/p100 nor RelA nor IKKε was associated with pathologic characteristics, including size of the cancers, age, Gleason score and stage. The increased cytoplasmic expression of IKKε as well as the increased nuclear expressions of p50/105, p52/p100 and RelA in the prostate cancers compared to normal alveolar cells suggested that overexpression of these proteins may be related to activation of the NF‐κB pathway and might play a role in tumorigenesis of prostate cancers.     

Decreased expression of Bax-interacting factor-1 (Bif-1) in invasive urinary bladder and gallbladder cancers

Aims: Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Bax-interacting factor-1 (Bif-1) interacts with both Bax and Bak that are crucial for the intrinsic apoptosis signalling. Functionally, loss of Bif-1 expression has been proven to enhance tumorigenesis. The aim of this study was to explore whether loss of Bif-1 expression occurs in urinary bladder (UB) and gallbladder (GB) cancer tissues. Methods: We analysed Bif-1 protein expression in 41 transitional cell carcinomas of UB and 26 GB adenocarcinomas by immunohistochemistry. Results: In both UB and GB, normal mucosal epithelial cells strongly expressed Bif-1 protein. In the UB cancers, Bif-1 expression was strongly positive in 25 cases (61.0%), but the remaining 16 cases showed no (14.6%) or markedly decreased (24.4%) Bif-1 immunostaining compared with the normal mucosal epithelial cells. Similarly, in the GB cancers, Bif-1 immunostaining was strong in 17 cases (65.4%), while the remaining nine cases showed no (15.4%) or markedly decreased (19.2%) Bif-1 immunostaining compared with the normal mucosal epithelial cells. Conclusion: The decreased expression of Bif-1 in large fractions of both UB and GB cancers (39.0% and 34.6%, respectively) compared with their normal mucosal cells suggested that loss of Bif-1 expression might play a role in tumorigenesis in both UB and GB cancers, possibly by inhibiting apoptosis mediated by Bif-1.     

Immunohistochemical localization of FAP-1, an inhibitor of Fas-mediated apoptosis, in normal and neoplastic human tissues

Fas, a death receptor, is widely expressed in human tissue, but its expression, although a prerequisite for the induction of apoptosis, does not predict its biological function. To understand the mechanisms of Fas resistance in human tissues in vivo, we performed immunohistochemistry using an antibody against Fas-associated phosphatase-1 (FAP-1), which interacts with the cytosolic domain of Fas and inhibits Fas-mediated apoptosis. In normal human tissues, FAP-1 immunostaining was easily detected, for example, in renal tubules, skeletal muscle, myocardiocytes, pituitary gland, parathyroid gland, pancreatic islets, hepatocytes, testicular germ cells, prostatic glands, neurons, epithelium of fallopian tube, endometrial glands, trophoblasts, bronchial epithelial cells, and some types of gastrointestinal epithelial cells. In 123 (78%) of 158 cancers of various origins, including breast carcinomas, stomach carcinomas, colon carcinomas, lung carcinomas and several types of sarcomas, variable intensities of FAP-1 expression were evident. Taken together, these findings demonstrated that FAP-1 is widely expressed in normal human tissues and partly overlapped with Fas expression described in earlier reports, suggesting that FAP-1 may have an important role in the regulation of apoptosis in vivo. In addition, FAP-1 expression in cancers suggests that many cancers may be resistant to Fas-mediated apoptosis through the action of FAP-1 in vivo.     

Fas ligand upregulation is an early event in colonic carcinogenesis

BACKGROUND/AIMS: Fas ligand (FasL) is a mediator of apoptosis via the Fas receptor (Fas/CD95/APO-1). Normal colonic epithelium expresses Fas, and appears to be relatively sensitive to Fas mediated apoptosis. Colonic adenocarcinomas coexpress FasL and Fas without undergoing widespread apoptosis. This study investigates the expression of FasL in colonic carcinogenesis from the earliest stages of the adenoma-carcinoma sequence. METHODS: FasL expression was determined in colonic adenomas (n = 38) of varying degrees of dysplasia and histological type by immunohistochemistry. Adenomas that contained areas of carcinomatous change were included (n = 12 of 38). Normal colonic epithelium (n = 10), hyperplastic polyps (n = 8), and serrated adenomas (n = 3) from patients without colonic adenocarcinomas were used for comparison. Cell death was detected in situ in adenomas using TUNEL (terminal transferase mediated dUTP nick end labelling). RESULTS: In normal colonic epithelium and hyperplastic polyps, FasL expression was restricted to the luminal surface of the crypts, where Fas-FasL coexpression was coincident with a high frequency of TUNEL positive epithelial cells. All adenomas (n = 38) had an altered distribution of positive FasL staining; FasL expression was found in most cells (> 70% of neoplastic cells). Expression of Fas was also detected throughout the adenomas, but coexpression of FasL and Fas was not associated with TUNEL positivity in most cells. CONCLUSIONS: FasL upregulation occurs early in the adenoma-carcinoma sequence of colon carcinogenesis, and is evident at the level of mild dysplasia. The lack of pronounced apoptosis in areas of adenomas coexpressing Fas and FasL suggests that colonocytes acquire resistance to Fas mediated apoptosis early in the transformation process.     

Immunohistochemical analysis of Fas ligand expression in normal human tissues

Cross-linking of Fas and Fas ligand (FasL) induces apoptosis in Fas-bearing cells and regulates apoptosis. Fas is widely expressed in normal human tissues, but FasL expression has been considered to be restricted to lymphoid tissues. Recent studies have demonstrated that FasL is also expressed in some nonlymphoid tissues. To screen the in situ expression of FasL in normal human tissues, immunohistochemistry was performed using paraffin-embedded human tissues. FasL immunostaining was easily detected in testis, neurons, trophoblasts, tonsil, lymph node, Paneth cells, hepatocytes, renal tubular epithelium and bronchial epithelium, consistent with previous reports. Surprisingly, FasL was also expressed in many other cell types, including thymic medulla, skeletal muscle, cardiac muscle, pituitary gland, parathyroid gland, prostate glands, oocytes, epithelium of fallopian tube, endometrial glands, and gastric parietal cells. These findings demonstrate that FasL is widely expressed in human tissues and suggest that wide but cell-type specific expression of FasL may not only be implicated in the regulation of immune homeostasis but also in the regulation of cell death and life in many cell types in vivo.     

Expressional and mutational analyses of ATG5 gene in prostate cancers

Autophagy is an evolutionarily conserved mechanism that plays important roles in both cell death and cell survival. ATG5 is an essential constituent for autophagosome formation, which sequesters cytoplasmic materials before lysosomal delivery. Although both cell death and survival are important in cancer development, the role of autophagy in prostate cancer development remains unclear. The aim of this study was to see whether alterations of ATG5 protein expression and somatic mutations of the ATG5 gene are found in prostate cancers. In the present study, we analyzed ATG5 protein expression in 107 prostate carcinomas by immunohistochemistry; additionally, we assayed the presence of ATG5 somatic mutations in 45 prostate carcinomas by single-strand conformation polymorphism. Immunostaining of ATG5 in normal prostate cells was observed in 44.9% of the cases, whereas in prostate intraepithelial neoplasm (PIN) and prostate cancer cells, ATG5 was observed in 100% and 89.7% of the cases, respectively. Cytoplasmic expression of ATG5 that might be related to autophagy was seen in PIN (100%) and cancers (83.2%), but not in normal cells (0%). ATG5 expression was not associated with any of the pathologic characteristics, including size of the cancers, age, Gleason score, and stage. As for the ATG5 gene, we found no somatic mutations in the prostate cancers. In this study, we analyzed ATG5 expression and mutation in prostate cancers, and found that ATG5 expression was altered in prostate cancers. The expression of ATG5, especially in the cytoplasm, in the prostate cancers compared with normal prostate cells suggested that overexpression of this protein may be related to autophagy and might play a role in prostate tumorigenesis.     

Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis

We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis.     

乙型肝炎肝组织Fas，FasL和HBV抗原的表达

目的 评价Fas和FasL介导细胞凋亡在乙型肝炎肝损伤中的作用及其与HBV抗原的关系。方法 应用免疫组化方法检测了62例乙型肝炎患者肝组织内的Fas、FasL和HBsAg、HBcAg,并以6例下正常肝组织为对照。结果 Fas/FasL在正常肝组织内无表达;Fas主要在乙型肝炎患者肝细胞质内表达,58例阳性（93．5％）;FasL在肝内浸润的单个核细胞和肝细胞质内均见到,37例阳性（59．7％）。结论 Fas/FasL的表达程度与肝组织活动性炎症程度密切相关;Fas/FasL介导的细胞凋亡可能在乙型肝炎时肝损伤中起重要作用;Fas/FasL表达程度与肝内HBV抗原HBsAg、HBcAg无明显相关性。     

Mitochondria-mediated apoptosis of lung epithelial cells in idiopathic interstitial pneumonias

We previously demonstrated that the up-regulation of p53, Fas, and DNA damage are present in lung epithelial cells from patients with idiopathic interstitial pneumonias (IIP). Fas ligation induces apoptosis of lung epithelial cells predominantly through the direct activation of the caspase cascade via caspase-8 activation, whereas the up-regulation of p53 and other cellular stresses can induce mitochondria-mediated apoptosis. In this study, we investigated the incidence of mitochondria-mediated apoptosis of epithelial cells in IIP. We performed TUNEL staining to detect apoptotic cells and western blot analysis and immunohistochemistry to assess the expression and activation of caspases and the cytochrome c release from mitochondria in lung tissues from eight patients with usual interstitial pneumonia, five patients with nonspecific interstitial pneumonia, and eight patients with normal lung parenchyma. The expressions of pro- and cleaved caspase-8, 9, 3, and cytochrome c release from the mitochondria were all significantly increased in the lung tissues of IIP compared with normal lung parenchyma. The positive signals for caspases in epithelial cells were increased in IIP compared with normal lung parenchyma by immunohistochemistry. The results of TUNEL and electron microscopy suggested that apoptotic cells were predominantly epithelial cells. TUNEL-positive cells in % of epithelial cells were significantly increased in IIP compared with normal lung parenchyma, and significantly correlated with cytochrome c release from the mitochondria and with the expression of cleaved caspase-3 in epithelial cells. We conclude that mitochondria-mediated apoptosis may be involved in the pathophysiology of IIP.     

Fas介导的细胞凋亡在乙型肝炎肝组织中分布状况初探

细胞凋亡可能是某些因素通过基因发动的一种细胞死亡过程。据认为,该过程主要通过Ｆａｓ抗原介导。为调查Ｆａｓ抗原表达与慢性乙型肝炎组织损伤的关系,本研究分别用免疫组化、原位末端标记法观察Ｆａｓ抗原表达及核ＤＮＡ断裂在慢性乙型肝炎肝组织中的分布状况。结果发现,Ｆａｓ抗原表达在活动性炎症区明显多于非炎症区,且主要位于汇管区及小叶内的炎症浸润区,５６例慢性乙型肝炎患者中,Ｆａｓ抗原阳性者４６例,阳性率８２％。直线相关回归分析提示,核ＤＮＡ断裂与Ｆａｓ表达明显相关。结果表明,Ｆａｓ介导的细胞凋亡在慢性乙型肝炎发病机制中具有重要作用     

Fas-induced apoptosis is a rare event in Sjögren's syndrome

The aim of this study was to perform a controlled in situ analysis on the incidence of apoptosis, investigate the expression of apoptosis-mediating proteins, and determine the frequency of apoptotic CD4+ and CD8+ T cells in Sj?gren's syndrome (SS). The study was extended to patients with atrophy-fibrosis (AF) not related to SS, as well as to a control group. Immunohistochemistry and the terminal deoxynucleotidyl transferase mediated dUTP digoxigenin nick end labeling (TUNEL) method were applied to study the Fas and FasL expression and the incidence of apoptosis in salivary glands (SG) from patients with primary and secondary SS, AF, and controls. These methods were also combined to enable simultaneous detection of apoptotic and CD4+ or CD8+ T cells. Despite abundant expression of Fas and FasL in SS SG, apoptotic cells were not exceeding 1% in the foci of infiltrating mononuclear cells (IMC). Double staining showed that the frequency of apoptosis was low among both CD4+ and CD8+ T cells. Only a few TUNEL+ epithelial cells were found in all patient groups. Fas was expressed predominantly on SS IMC, single SS epithelial cells, and a few normal acinar cells, but not in AF SG. Although FasL was present on SS and AF IMC and epithelial cells, it was rarely detected in normal tissue. Consequently we demonstrate that Fas-induced apoptosis among SS SG is a rare event. Our findings support an earlier hypothesis indicating that IMC seem to be able to escape apoptosis, resulting in foci of inflammatory cells. Notably, however, no obvious correlation can be drawn to previous studies where a high incidence of apoptosis of epithelial cells was proposed as an important mechanism leading to decreased glandular function, which is a hallmark of SS.     

肾综合征出血热患者外周血淋巴细胞Fas/FasL表达及与血清sFas/sFasL的相关性研究

目的　探讨Fas FasL系统介导的细胞凋亡在肾综合征出血热 (HFRS)致病机理中的作用。方法　采用SABC免疫组化法检测HFRS病人外周血淋巴细胞Fas FasL的表达 ;采用ELISA法测定HFRS病人血清sFas sFasL的浓度。结果　HFRS病人发热期、低血压休克期、少尿期及多尿期外周血淋巴细胞Fas的表达明显增强 ,与正常对照组比较差异有极显著性 (P <0 .0 0 1) ;发热期、低血压休克期及少尿期淋巴细胞FasL的表达明显增强 ,与正常对照组比较差异有极显著性 (P <0 .0 0 1) ;HFRS病人各期血清sFas的浓度增高 ,与正常对照组比较差异有显著性 (P <0 .0 1) ,其中少尿期sFas的浓度最高 ,与其它各期比较差异有显著性 (P <0 .0 1) ;发热期与低血压休克期血清sFasL的浓度增高 ,与其它各期及正常对照组比较差异有显著性 (P <0 .0 1) ;淋巴细胞Fas及FasL的表达经直线相关分析(r=0 .885 5 ,P <0 .0 0 1) ,呈高度正相关 ;淋巴细胞FasL的表达与血清sFasL浓度经直线相关分析(r=0 .480 3,P <0 .0 1) ,呈高度正相关。结论　HFRS的致病过程中存在Fas FasL介导的细胞凋亡 ,sFas浓度的高低与机体损害的程度有关 ;淋巴细胞FasL的高表达及血清sFasL的浓度增高 ,由此而引起的细胞凋亡是机体清除病毒的主要机制之一     

Hydroxychloroquine potentiates Fas-mediated apoptosis of rheumatoid synoviocytes

Inadequate apoptosis may contribute to the synovial hyperplasia associated with rheumatoid arthritis (RA). The Fas-associated death domain protein (FADD)-like interleukin (IL)-1beta-converting enzyme (FLICE)-inhibitory protein (FLIP), which is an apoptotic inhibitor, has been implicated in the resistance to Fas-mediated apoptosis of synoviocytes. This study investigated whether hydroxychloroquine (HCQ), an anti-rheumatic drug, induces the apoptosis of rheumatoid synoviocytes, and modulates the expression of FLIP. Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and were cultured with various concentrations of HCQ in the presence or absence of the IgM anti-Fas monoclonal antibodies (mAb) (CH11). Treatment with HCQ, ranging from 1 to 100 microM, induced the apoptosis of FLS in a dose- and time-dependent manner. The increase in synoviocytes apoptosis by HCQ was associated with caspase-3 activation. A combined treatment of HCQ and anti-Fas mAb increased FLS apoptosis and caspase-3 activity synergistically, compared with either anti-Fas mAb or HCQ alone. The Fas expression level in the FLS was not increased by the HCQ treatment, while the FLIP mRNA and protein levels were decreased rapidly by the HCQ treatment. Moreover, time kinetics analysis revealed that the decreased expression of FLIP by HCQ preceded the apoptotic event that was triggered by HCQ plus anti-Fas mAb. Taken together, HCQ increases the apoptosis of rheumatoid synoviocytes by activating caspase-3, and also sensitizes rheumatoid synoviocytes to Fas-mediated apoptosis. Our data suggest that HCQ may exert its anti-rheumatic effect in rheumatoid joints through these mechanisms.     

Expression of ECRG4 is associated with lower proliferative potential of esophageal cancer cells

We have shown that ECRG4 suppressed Fas‐induced apoptosis in Jurkat cells. ECRG4 mRNA expression was ubiquitously detected in normal adult human tissues, suggesting that ECRG4 plays a major role in human tissues. ECRG4 mRNA expression was down‐regulated in tumor cells. Expression of ECRG4 suppressed cell growth. We established an anti‐ECRG4 monoclonal antibody. Our immunohistochemical analysis demonstrated that ECRG4‐positive cells tended to be distributed in the region that was negative for Ki‐67 in esophageal squamous cell carcinoma tissues. There was a significant inverse correlation between ECRG4 expression and Ki‐67 labeling index in esophageal squamous cell carcinoma. This study provides the first functional evidence for an association of endogenous expression of ECRG4 with cell proliferation. ECRG4 is a candidate tumor suppressor gene that might be involved in the proliferation of esophageal squamous cell carcinoma.