Doxorubicin induces Fas-mediated apoptosis in human thyroid carcinoma cells.

Doxorubicin remains the most extensively used drug in the chemotherapy of thyroid cancer. However, drug resistance often limits the efficacy of chemotherapy in clinical practice. Several anticancer drugs exert their cytotoxic effect by triggering Fas-mediated apoptosis in some cell types. However, no investigations have been conducted to determine whether doxorubicin causes apoptosis in thyroid carcinomas. In the present study, we assessed the cytotoxic and apoptotic effects of doxorubicin on two thyroid cancer cell lines (FTC 238 and FTC 133). Cytotoxic effects of doxorubicin were evaluated by a 3-(4,5 dimethylthiazol-2yl) 2-5 diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by fluorescein isothiocyanate-conjugated annexin V/flow cytometric analysis and by DNA fragmentation. Fas expression was measured by flow cytometric analysis. After a 24-hour incubation, doxorubicin induces a dose-dependent cytotoxicity in the two cell lines. Treatment with doxorubicin (0.5 and 1 microM) for 24 hours induced cell apoptosis and upregulated Fas expression. A significant correlation was found between the fluorescence intensity values obtained with annexin V staining and those observed for Fas expression (r = 0.996; p < 0.001 or r = 0.957; 0.02 < p < 0.05 for FTC 238 or FTC 133 cells, respectively). In conclusion, doxorubicin exerts its cytotoxic effects, at least partly, through Fas-mediated apoptosis in thyroid cancer cells. These results may have clinical implications for thyroid cancer therapy.     

The role of apoptosis in thyroid autoimmunity.

There is increasing evidence showing that apoptosis plays a role in the development of the autoimmune thyroid diseases-Hashimoto's (lymphocytic) thyroiditis (HT) and Graves' disease (GD). The immune pathogenesis of HT and GD is not yet fully understood, but evidence points toward several steps. A defect in CD4(+)CD25(+) T regulatory cells breaks the immunological tolerance of the host and induces an abnormal production of cytokines, which facilitates the initiation of apoptosis. Though apoptosis appears to play a role in the pathogenesis of both HT and GD, the mechanisms that mediate these processes appear different. The induction of apoptosis in HT results in the destruction of thyrocytes, while apoptosis in the GD leads to damage of thyroid-infiltrating lymphocytes. The differences in the apoptotic mechanisms produce two very different forms of thyroid autoimmune responses, eventually developing into HT and GD, respectively.     

The role of octreoscan in thyroid eye disease

Until recently there was no imaging technique available which could demonstrate pathological changes in orbital tissues and could be regarded as a reliable measure of inflammation in thyroid eye disease (TED). Pentetreotide (a synthetic derivative of somatostatin) labelled with 111In has been used to localize tumours which possess surface or membrane receptors for somatostatin in vivo using a gamma camera (1). This technique visualizes somatostatin receptors in endocrine-related tumours in vivo and predicts the inhibitory effect of the somatostatin analogue octreotide on hormone secretion by the tumours (1). By applying 111In-DTPA-d-Phe octreotide scintigraphy (octreoscan), accumulation of the radionuclide was also detected in both the thyroid and orbit of patients with Graves' disease (2-4). If peak activity in the orbit 5h after injection of radiolabelled octreotide is set at 100%, a decrease to 40+/-4% is found at 24h, significantly different from the decrease in blood pool radioactivity, which is 15+/-4% at 24h. Accumulation of the radionuclide is most probably due to the presence in the orbital tissue of activated lymphocytes bearing somatostatin receptors (5). Alternative explanations are binding to receptors on other cell types (e.g. myoblasts, fibroblasts or endothelial cells) or local blood pooling due to venous stasis by the autoimmune orbital inflammation.     

Fas-mediated apoptosis in acute alcoholic hepatitis

BACKGROUND/AIMS: The Fas-mediated apoptosis pathway has been implicated in liver diseases. The aim of the study was to investigate the role of this system in alcoholic hepatitis. METHODOLOGY: The expression of Fas, Fas ligand, and intracellular signaling molecules for apoptosis were determined by immunoblot analysis in fresh frozen liver samples from 19 patients with alcoholic liver disease. RESULTS: Fas and Fas ligand expression was significantly increased in the liver of patients with alcoholic hepatitis (n = 11) as compared with alcoholic liver disease patients without hepatitis (n = 8). Similarly, there were significant differences in the expression of FADD, ICE, and CPP32 in the liver between the two groups. There were significant positive correlations between the Fas ligand and the FADD, ICE, or CPP32 levels in the liver. The expression of Fas, Fas ligand, FADD, ICE, or CPP32 correlated with serum markers of hepatic injury. Plasma soluble Fas levels in patients with alcoholic hepatitis (median: 15.1 U/mL, range: 9.7-19.2 U/mL) were significantly higher than in normal controls (n = 9) (median: 2.8 U/mL, range: 1.9-3.7 U/mL; p < 0.001). There was a significant positive correlation between plasma soluble Fas levels and the hepatic expression of FADD in these patients. CONCLUSIONS: These results indicate that Fas-mediated apoptosis may play an important role in alcoholic hepatitis.     

Fas-mediated apoptosis in accelerated graft arteriosclerosis

Pathological conditions have been recognized where vessel destruction is a prominent feature of the pathogenic process. One such condition consists of the chronic rejection of blood vessels in transplanted solid organs. Accelerated graft arteriosclerosis (AGA) is a multifactorial process characterized by the concentric proliferation of smooth muscle cells (SMCs) within the intima of the vessel wall of transplanted organs. Proliferation of SMCs within the intima corresponds to a response of these cells to injury. In situations like restenosis post-angioplasty, the mechanism of injury: the mechanical disruption of the tunica media, is evident. However, in the case of AGA, the mechanism of injury has remained elusive. In this report, we provide evidence that injury to SMCs in AGA vessels requires an intact Fas pathway. The resulting damage to the tunica media and internal elastic lamina, in turn, might trigger the proliferation of intimal smooth muscle cells that appear to be less sensitive to Fas mediated killing, particularly when supported by a favorable context of inflammatory cytokines and growth factors, as it is the case in AGA. This pathogenic process results in a absolute loss of functional blood vessels that is not being compensated by an efficient angiogenic response. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential regulation of Fas-mediated apoptosis in both thyrocyte and lymphocyte cellular compartments correlates with opposite phenotypic manifestations of autoimmune thyroid disease.

Several mechanisms are probably involved in determining the evolution of autoimmune thyroid disease (AITD) towards either hypothyroidism and the clinical syndrome known as Hashimoto's thyroiditis (HT) or toward hyperthyroidism and the symptoms of Graves' disease (GD). To gain further insight into such mechanisms we performed an exhaustive comparative analysis of the expression of key molecules regulating cell death (Fas, Fas ligand [FasL], Bcl-2) and apoptosis in both thyrocytes and thyroid infiltrating lymphocytes (TILs) from patients with either GD or HT. GD thyrocytes expressed less Fas/FasL than HT thyrocytes, whereas GD TILs had higher levels of Fas/FasL than HT TILs. GD thyrocytes expressed increased levels of the antiapoptotic molecule Bcl-2 compared to the low levels detected in HT thyrocytes. The opposite pattern was observed in GD (low Bcl-2) and HT (high Bcl-2) TILs. The patterns of apoptosis observed were consistent with the regulation of Fas, FasL, and Bcl-2 described above. Our findings suggest that in GD thyroid the regulation of Fas/FasL/Bcl2 favors apoptosis of infiltrating lymphocytes, possibly limiting their autoreactive potential and impairing their ability to mediate tissue damage. Moreover, the reduced levels of Fas/FasL and increased levels of Bcl-2 should favor thyrocyte survival and favor the thyrocyte hypertrophy associated with immunoglobulins stimulating the thyrotropin (TSH) receptor. In contrast, the regulation of Fas/FasL/Bcl2 expression in HT promotes thyrocyte apoptosis, tissue damage, and a gradual reduction in thyrocyte numbers leading to hypothyroidism. These findings help define key molecular mechanisms contributing to the clinical outcome of thyroid autoimmunity.     

Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts

OBJECTIVE: Fas-mediated apoptosis is preferentially observed in synoviocytes of patients with rheumatoid arthritis (RA) and is associated with the pathophysiological process of RA. To clarify the molecular mechanisms of Fas-mediated apoptosis of RA synoviocytes, we investigated the role of the mitochondrial pathway and tumour suppressor p53 in this process. METHODS: Cultured synovial fibroblasts were prepared from RA patients. After treatment of RA synovial fibroblasts with anti-Fas monoclonal antibody, the expression levels of activated caspase-9 and -3, Bid cleavage, cytochrome c release and phosphorylation of p53 at Ser15 were assessed using immunoblot analysis. The mitochondrial membrane potential (DeltaPsim) was evaluated with a fluorescence-based detection assay. Apoptotic cells were determined by a DNA fragmentation assay in the presence or absence of caspase inhibitors. Expression of p53-regulated apoptosis-inducing protein 1 (p53AIP1) was measured by real-time PCR. RA synovial fibroblasts stably transfected with a dominant-negative (DN) p53 were prepared in order to investigate the role of p53 during Fas-induced apoptosis. RESULTS: Fas ligation induced Bid cleavage, loss of DeltaPsim, cytochrome c release to the cytosol and activation of caspase-9 and -3 in RA synovial fibroblasts. Treatment with a caspase-9-specific inhibitor almost completely inhibited Fas-mediated apoptosis. Moreover, p53 activation after Fas ligation was evidenced by its phosphorylation at Ser15 and up-regulation of the p53 target gene p53AIP1. Fas-mediated apoptosis was significantly suppressed by anti-sense p53 oligonucleotides and by p53DN. CONCLUSION: Our findings strongly suggest the involvement of mitochondria and p53 in Fas-mediated apoptosis of RA synovial fibroblasts.     

The role of T lymphocytes in autoimmune thyroid disease

Abnormalities in cellular mechanisms of immunoregulation are important factors in the initiation and/or propagation of autoimmune thyroid disease and Graves' ophthalmopathy. The primary immunologic lesion, however, remains elusive despite increasingly sophisticated investigations in the areas of antigen presentation and subsequent T-cell responses. Emphasis on functional evaluations of relevant cell populations and advances in lymphokine analysis, interpretation of DR antigen expression, and T-cell cloning offer promise for our future understanding of this complex process.     

COX-2 inhibits Fas-mediated apoptosis in cholangiocarcinoma cells

Fas expression has been shown to negatively regulate the progression of cholangiocarcinoma cells in xenografts. However, many human cholangiocarcinomas express Fas, suggesting these cancers have developed mechanisms to inhibit Fas-mediated apoptosis. Cyclooxygenase-2 (COX-2), which generates prostanoids, is expressed by many cholangiocarcinomas. Therefore, our aim was to determine whether COX-2 expression inhibits death receptor--mediated apoptosis in KMBC cells, a cholangiocarcinoma cell line. These cells express messenger RNA for the death receptors Fas, tumor necrosis factor receptor 1 (TNF-R1), death receptor 4 (DR4), and DR5. Agonists for these death receptors, CH-11, TNF-alpha, and TRAIL all induced apoptosis. However, COX-2, whether induced by proinflammatory cytokines or transient transfection, only significantly inhibited Fas-mediated apoptosis. The COX-2 inhibitor NS-398 restored Fas-mediated apoptosis in COX-2 transfected cells. Prostaglandin E2 reduced apoptosis and mitochondrial depolarization after treatment with the Fas agonist CH-11. Of a variety of antiapoptotic proteins examined, COX-2/prostaglandin E2 only increased expression of Mcl-1, an antiapoptotic member of the Bcl-2 family. In conclusion, these data suggest that prostanoid generation by COX-2 specifically inhibits Fas-mediated apoptosis, likely by up-regulating Mcl-1 expression. Pharmacologic inhibition of COX-2 may be useful in augmenting Fas-mediated apoptosis of cholangiocarcinoma cells.     

The role of apoptosis in aging and age-related disease: update

Summary Programmed death of cells by apoptosis is regarded as a protective mechanism of the organism against an accumulation and spread of defective cells. The rate of apoptosis is elevated in most types of aging cell populations. However, there are also findings about a decreased susceptibility of senescent cells in vivo and in vitro, particularly to apoptosis induced by oxidative and energetic stress. Mitochondria appear to have a key function in apoptosis regulation. Thus, apoptosis can be induced by defective mitochondrial oxidative phosphorylation. The role of apoptosis in aging and age-related disease was outlined for different organs (brain, cardio-vascular system, immune system, intestine, macula of the eye, Langerhans islets, prostate gland, oocytes of ovaries). The age-related intensification of this dismantling system of cells seems to highlight the deterioration of tissue and organ structure and function in aging. Received: 24 October 2001/Accepted: 28 October 2001     

The role of apoptosis in aging and age-related disease: update

Programmed death of cells by apoptosis is regarded as a protective mechanism of the organism against an accumulation and spread of defective cells. The rate of apoptosis is elevated in most types of aging cell populations. However, there are also findings about a decreased susceptibility of senescent cells in vivo and in vitro, particularly to apoptosis induced by oxidative and energetic stress. Mitochondria appear to have a key function in apoptosis regulation. Thus, apoptosis can be induced by defective mitochondrial oxidative phosphorylation. The role of apoptosis in aging and age-related disease was outlined for different organs (brain, cardio-vascular system, immune system, intestine, macula of the eye, Langerhans islets, prostate gland, oocytes of ovaries). The age-related intensification of this dismantling system of cells seems to highlight the deterioration of tissue and organ structure and function in aging.     

Resistance to Fas-mediated apoptosis in human lung fibroblast.

The current authors have demonstrated previously that epithelial cell apoptosis, induced by the Fas-Fas ligand pathway, might be involved in fibrosing lung diseases. Whereas lung epithelial cells are sensitive to the Fas-mediated apoptosis, lung fibroblasts may be resistant to Fas-mediated apoptosis and replace damaged epithelial cells. The WI-38 lung fibroblast cell line and primary lung fibroblasts were used to examine the resistant to Fas-mediated apoptosis and the association of anti-apoptotic proteins with this resistance. The administration of agonistic anti-Fas antibody (CH-11) or cycloheximide alone did not induce apoptosis, whereas the co-administration of CH-11 with cycloheximide induced apoptosis in WI-38 cells, in which caspase-8 and -3, but not -9, were activated, and X chromosome-linked inhibitor of apoptosis (ILP) and FLICE-like inhibitor protein (FLIP(L)), but not bcl-xL and bcl-2, were remarkably down regulated. Primary lung fibroblasts were also resistant to Fas-mediated apoptosis, and ILP and FLIP appeared to be involved in this resistance. Furthermore, the results of immunohistochemistry demonstrated that fibroblasts expressed ILP and FLIP(L) proteins in lung tissues from patients with idiopathic pulmonary fibrosis. These results suggest that anti-apoptotic proteins such as X chromosome-linked inhibitor of apoptosis and FLICE-like inhibitor protein may play an important role in preventing Fas-mediated apoptosis in lung fibroblasts, and participate in the development of pulmonary fibrosis.     

溶酶体在新城疫病毒诱导肿瘤细胞凋亡和自噬中的作用

溶瘤病毒(oncolytic virus,OV)具有对肿瘤细胞选择性杀伤而对正常细胞无影响的特性,在针对肿瘤治疗方面有着巨大的潜力.凋亡与自噬在溶瘤病毒杀伤肿瘤细胞的过程中扮演者重要的角色,而溶酶体与二者有着密切的联系.新城疫病毒(newcastle disease virus,NDV)在感染肿瘤细胞后,溶酶体不仅会触...     

Homocysteine enhances endothelial apoptosis via upregulation of Fas-mediated pathways

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However, the underlying mechanism of endothelial cell injury in hyperhomocysteinemia has not been elucidated. In this study, we examined the effect of homocysteine (Hcy) on Fas-mediated apoptosis in endothelial cells. Hcy-induced upregulation of Fas in endothelial cells (ECs) in a dose-dependent manner. At the same time, Hcy increased intracellular peroxide in ECs. Hcy-induced Fas expression was inhibited by the treatment with catalase. Hcy increased NF-kappaB DNA binding activity, and adenovirus-mediated transfection of a Ikappa-B mutant (Ikappa-B mt) gene inhibited Hcy-induced Fas expression. ECs were sensitive to Fas-mediated apoptosis when exposed to Hcy. Under these condition, Ikappa-B mt protected ECs from Fas-mediated apoptosis. In addition, Hcy inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP). Adenovirus-mediated transfection of constitutively active Akt gene abolished the Hcy-mediated downregulation of FLIP. These data suggest that upregulation of Fas expression and downregulation of FLIP is a mechanism through which Hcy induces EC apoptosis.