mRNA Expression Levels Of Fas Research Articles

GSF3, a polysaccharide from guava (Psidium guajava L.) seeds, inhibits MCF-7 breast cancer cell growth via increasing Bax/Bcl-2 ratio or Fas mRNA expression levels

Guava seed polysaccharide fraction 3 (GSF3) is an immunomodulatory polysaccharide from guava (Psidium guajava L.) seed polysaccharides. However, effects of GSF3 on the growth of breast cancer cells were not understood, yet. To clarify the GSF3 effects on breast cancer cell growth, GSF3 was subjected to treat MCF-7 cells using direct action or indirect immunotherapy using immune cells conditioned media, respectively. The viabilities of MCF-7 cells were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Changes in pro-(Bax)/anti-apoptotic (Bcl-2) and Fas mRNA expression levels in the treated MCF-7 cells were measured using two-step reverse transcription quantitative polymerase chain reaction. Our results showed that GSF3 inhibited MCF-7 cell growth through either direct action or indirect immunotherapy. GSF3 direct action significantly (P < 0.05) decreased Bcl-2 mRNA expression amount but increased pro-(Bax)/anti-apoptotic (Bcl-2) mRNA expression ratios in the treated cells. The splenocytes conditioned media cultured with GSF3 increased Fas mRNA expression amounts in the treated MCF-7 cells. There was a significant negative correlation between Th2-polarized cytokines secreted by immune cells and Fas mRNA expression levels in the corresponding treated MCF-7 cells. Our findings suggested that GSF3 is a potent anti-cancerous polysaccharide by direct action or indirectly modulating immune cell cytokine secretion profiles.

Protective effects of hypericin against infectious bronchitis virus induced apoptosis and reactive oxygen species in chicken embryo kidney cells

Avian infectious bronchitis virus (IBV), a coronavirus, causes infectious bronchitis leading to enormous economic loss in the poultry industry worldwide. Hypericin (HY) is an excellent compound that has been investigated in antiviral, antineoplastic, and antidepressant. To investigate the inhibition effect of HY on IBV infection in chicken embryo kidney (CEK) cells, 3 different experimental designs: pre-treatment of cells prior to IBV infection, direct treatment of IBV-infected cells, and pre-treatment of IBV prior to cell infection were used. Quantitative real-time PCR (qRT-PCR), immunofluorescence assay (IFA), flow cytometry, and fluorescence microscopy were performed and virus titer was determined by TCID50. The results revealed that HY had a good anti-IBV effect when HY directly treated the IBV-infected cells, and virus infectivity decreased in a dose-dependent manner. Furthermore, HY inhibited IBV-induced apoptosis in CEK cells, and significantly reduced the mRNA expression levels of Fas, FasL, JNK, Bax, Caspase 3, and Caspase 8, and significantly increased Bcl-2 mRNA expression level in CEK cells. In addition, HY treatment could decrease IBV-induced reactive oxygen species (ROS) generation in CEK cells. These results suggested that HY showed potential antiviral activities against IBV infection involving the inhibition of apoptosis and ROS generation in CEK cells.

The Protective Role of Selenium Against AFB1-Induced Liver Apoptosis by Death Receptor Pathway in Broilers.

Aflatoxin B1 (AFB1) is the most toxic among the mycotoxins and causes detrimental health effects on the liver of human and animals. Selenium (Se) plays an important role in protection of various animal species against numerous notorious toxic agents. The present study is designed to explore the protective effects of Se against AFB1-induced liver pathogenesis by the methods of histopathology, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and biochemical analysis. A total of 312, 1-day-old healthy Cobb-500 broilers were randomly divided into four groups and fed with basal diet (control group), 0.6mg/kg AFB1 (AFB1 group), 0.4mg/kg Se (+ Se group), and 0.6mg/kg AFB1 + 0.4mg/kg Se (AFB1 + Se group) for 21days, respectively. Our results showed that 0.4mg/kg Se supplement in broiler's diets could alleviate the AFB1-induced histological lesions in the liver. The apoptosis analysis by flow cytometry showed that 0.4mg/kg Se ameliorated the AFB1-induced apoptosis in the liver. Moreover, the mRNA expression levels of Fas, TNF-α, FAS-associated death domain, TNF receptor-associated death domain, TNF receptor-associated factor 2, caspase 10, caspase 8, B cell lymphoma 2, IκB kinase, X-linked inhibitor of apoptosis protein, caspase 9, and caspase 3 analyzed by qRT-PCR demonstrated that 0.4mg/kg Se could relieve the impact caused by AFB1 to these parameters. The biochemical analyses of activities of CAT, GSH-Px and SOD, hydroxyl ion scavenging and contents of MDA and GSH in liver cells also indicated that 0.4mg/kg Se has positive effect on AFB1-induced oxidative stress in the liver. In conclusion, Se could relieve AFB1-induced apoptosis by the molecular regulation of death receptors pathway in the liver of broilers. The outcomes from the present study may lead to a better understanding of the nature of selenium's essentiality and its protective roles against AFB1.

Expression of PBMC apoptosis-related factors in patients with chronic hepatitis B and their relationships with clinical prognosis.

The present study was conducted to investigate the expression of apoptosis-related factors in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B and their correlation to clinical prognosis. Sixty-two patients that were diagnosed with chronic hepatitis B were admitted to Xuzhou Hospital from March 2015 to February 2016 and were enrolled as the observation group, while 60 healthy subjects who were examined in the health examination center were selected as the control group. The PBMC of patients were collected, and mRNA expression levels of the apoptotic molecules (FAS, CASP3, CASP8 and CASP9) were measured using real-time fluorescence quantitative PCR. The protein expression levels of apoptosis-related genes in the plasma were detected using enzyme-linked immunosorbent assay (ELISA) and the serum HBV-DNA was quantitatively measured using real-time fluorescence quantitative PCR. The mRNA and protein expression levels of FAS, CASP3, CASP8 and CASP9 in the observation group were significantly higher than those in the control group (P<0.05). The positive rate and log value of the copy amount of HBV DNA in the observation group were significantly higher than those in the control group (P<0.05). The Pearson correlation coefficient analysis showed that FAS, CASP3, CASP8 and CASP9 were positively correlated with HBV DNA (P<0.05). The mRNA expression levels of FAS, CASP3, CASP8 and CASP9 in patients with negative HBV-DNA were significantly lower than those with positive HBV-DNA (P<0.05). Apoptosis of PBMCs play an important role in the occurrence and development of chronic hepatitis B, and is closely correlated to the level of serum virus replication and prognosis.

Chrysotile Causes Human Bronchial Epithelial Cell Apoptosis in Response to the Fas-Mediated Apoptosis Pathway

Aims: Asbestos is harmful to human health. However, the pathogenicity of chrysotile is a controversial matter. This study aimed to investigate the apoptosis of a human bronchial epithelial cell line (BEAS-2B) exposed to chrysotile that may function in part through the Fas death receptor pathway. Methods: Cultured human BEAS-2B cells were treated with chrysotile and cell viability was evaluated by CCK-8 assay. The induction of cell apoptosis was evaluated by FACS analysis. mRNA expression levels of Fas, caspase-3, and caspase-8 were evaluated quantitatively by real-time PCR. The expression of Fas, caspase-3, and caspase-8 proteins were evaluated by Western blot. Meanwhile, cells were preincubated with various concentrations of anti-Fas antibody (CH11) and antagonistic anti-Fas antibody (ZB4). Results: Chrysotile inhibits proliferation, induces apoptosis, and upregulates the expression of Fas, caspase-3, and caspase-8. The role of Fas as a regulator of chrysotile-induced apoptosis in BEAS-2B cells was tested by the prominent increase in and partial blockade of the apoptotic rate with CH11 and ZB4. When CH11 was pretreated, a synergistic effect was apparent on chrysotile-induced apoptosis and the mRNA and protein expression levels of Fas and cleaved caspase-3. Conclusion: Chrysotile causes the apoptosis of BEAS-2B cells via the Fas death receptor pathway. The Fas-mediated apoptosis pathway plays an important role in chrysotile-induced apoptosis of BEAS-2B cells in vitro.

Nickel chloride (NiCl2) in hepatic toxicity: apoptosis, G2/M cell cycle arrest and inflammatory response.

Up to now, the precise mechanism of Ni toxicology is still indistinct. Our aim was to test the apoptosis, cell cycle arrest and inflammatory response mechanism induced by NiCl2 in the liver of broiler chickens. NiCl2 significantly increased hepatic apoptosis. NiCl2 activated mitochondria-mediated apoptotic pathway by decreasing Bcl-2, Bcl-xL, Mcl-1, and increasing Bax, Bak, caspase-3, caspase-9 and PARP mRNA expression. In the Fas-mediated apoptotic pathway, mRNA expression levels of Fas, FasL, caspase-8 were increased. Also, NiCl2 induced ER stress apoptotic pathway by increasing GRP78 and GRP94 mRNA expressions. The ER stress was activated through PERK, IRE1 and ATF6 pathways, which were characterized by increasing eIF2α, ATF4, IRE1, XBP1 and ATF6 mRNA expressions. And, NiCl2 arrested G2/M phase cell cycle by increasing p53, p21 and decreasing cdc2, cyclin B mRNA expressions. Simultaneously, NiCl2 increased TNF-α, IL-1β, IL-6, IL-8 mRNA expressions through NF-κB activation. In conclusion, NiCl2 induces apoptosis through mitochondria, Fas and ER stress-mediated apoptotic pathways and causes cell cycle G2/M phase arrest via p53-dependent pathway and generates inflammatory response by activating NF-κB pathway.

Intrahepatic mRNA Expression of FAS, FASL, and FOXP3 Genes Is Associated with the Pathophysiology of Chronic HCV Infection.

This study aimed to evaluate the relative mRNA expression of Fas receptor (FAS), Fas ligand (FASL), and forkhead box protein 3 (FOXP3) in liver biopsy specimens obtained from patients with viral and non-viral chronic hepatitis and correlate their expression with the fibrosis stage. A total of 51 liver biopsy specimens obtained from HBV (n = 6), HCV (n = 28), and non-viral hepatic disease (NVHD) (n = 9) patients and from individuals with normal liver histology (n = 8) (control—CT) were analyzed. Quantifications of the target genes were assessed using qPCR, and liver biopsies according to the METAVIR classification. The mRNA expression levels of FAS and FASL were lower in the CT group compared to the groups of patients. The increase in the mRNA expression of FAS and FASL was correlated with higher levels of inflammation and disease progression, followed by a decline in tissues with cirrhosis, and it was also associated with increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Higher mRNA expression of FOXP3 was observed in the HCV and NVHD groups, with the peak observed among patients with cirrhosis. The increased FOXP3 mRNA expression was positively correlated with increased FAS and FASL mRNA expression and the AST and ALT levels in all patients. Conclusions: These results suggest that regardless of the cause, the course of chronic liver disease may be modulated by the analyzed genes and correlated with an increase in regulatory T cells during the liver damage followed by hepatocyte destruction by Fas/FasL system and subsequent non specific lymphocytic infiltrate accumulation.

Croton Tiglium Extract Induces Apoptosis via Bax/Bcl-2 Pathways in Human Lung Cancer A549 Cells

Objective:To investigate the impact of a Croton tiglium extract on cellular proliferation and apoptosis in a non-small cell lung cancer cell line (A549) in vitro.Methods:A Croton tiglium seed methanol extract was prepare and assessed for effects on A549 cells regarding cellular proliferation, apoptotic rates, and expression of apoptosis related genes and proteins using real-time PCR and immunofluorescence.Results:The tested Croton tiglium extract inhibited A549 cell proliferation in a dose- and time-dependent manner, with significant elevation of apoptotic indexes at various concentrations after 24 h. In addition, rates in both early and late stages were higher in treated than untreated groups, the 100 μg/ml dose causing the highest levels of apoptosis. RT-PCR showed that A549 cells treated with 100 μg/ml Croton tiglium extract for 24 h has markedly higher Bax mRNA expression levels and obviously lower Bcl-2 expression levels than controls, equivalent results being observed for proteins by immunofluorescence. However, the mRNA expression levels of Fas and caspase-8 were not significantly altered.Conclusion:A Croton tiglium extract can inhibit proliferation of A549 cells and promote apoptosis though Bax/Bcl-2 pathways.

Differential expression of peroxisome proliferator-activated receptor γ, fatty acid synthase, and hormone-sensitive lipase in fat-tailed and thin-tailed sheep breeds

Tail fat content affects meat quality, and it varies in different sheep breeds. Theoretically, lipid metabolism contributes to variation in tail fat content. Tail length, tail width, and tail girth were measured in live Tong sheep (with both short fat tail and long fat tail), Shaanbei fine wool sheep (long thin tail), Tan sheep (short fat tail), Kazakh sheep (hip fat tail), and Tibetan sheep (short thin tail). The expression levels of genes related to tail adipose tissue lipid metabolism were investigated, which included lipogenetic genes (PPARγ and FAS) and lipolytic gene (HSL). Differences were observed (P < 0.05) in PPARγ mRNA expression levels in the different breeds; FAS mRNA expression levels did not differ (P > 0.05) in Tong sheep with short fat tail, Tong sheep with long fat tail, Shaanbei fine wool sheep, and Tibetan sheep; HSL mRNA expression levels were not different (P > 0.05) in Tong sheep. PPARγ and HSL protein expression levels differed (P < 0.05) between the different breeds; FAS protein expression levels were different (P < 0.05) in Tong sheep with long fat tails, Tan sheep, Kazakh sheep, and Tibetan sheep, but did not differ (P > 0.05) in Tong sheep with short fat tails and Shaanbei fine wool sheep. These results provide useful information to further understand the function of PPARγ, FAS, and HSL in sheep tail lipid metabolism, which should be applicable to studies on the regulation of fat deposition and improvement of meat quality.

Molecular markers of programmed cell death in donor hearts before transplantation

In this study we investigate whether pro-apoptotic, pro-inflammatory and other early signaling markers indicative of increased propensity for cell death processes were evident in human donor heart allografts immediately before transplantation, and whether there is an association with primary graft failure. A prospective study was performed utilizing donor left atrial myocardium collected at the time of implantation of hearts from brain-dead donors (BDD, n = 29). In addition, to explore the potential of donor hearts from donation after circulatory death (DCD), myocardial samples were obtained during transplantation of lungs from DCD donors (n = 6). A comparator reference group (n = 7) consisted of left atrial specimens from patients undergoing mitral valve surgery. Significantly raised levels of caspase-3 specific activity, activated hypoxia inducible factor-1 (HIF-1α) and 8-hydroxy-2'-deoxyguanosine were evident in the transplanted hearts (from BDD) that developed primary graft failure (n = 11). DCD hearts did not differ from BDD with regard to mRNA expression levels of FAS, Bax, IL-6 and caspase-3. Although DCD hearts exhibited lower caspase-3 specific activity and activated hypoxia-inducible factor-1 protein, they had higher levels of mRNA for NF-κB, Bnip3 and caspase-1 mRNA. Increased 8-hydroxy-2'-deoxyguanosine levels reflected greater oxidative stress and reactive oxygen species-related DNA fragmentation. Our data indicate a significant role of pro-apoptotic and pro-inflammatory activity in allografts that subsequently exhibit primary graft failure. The relatively lower levels of apoptotic and inflammatory activity in DCD hearts suggest they may represent a potentially usable donor cardiac allograft pool. This possibility requires further detailed molecular and clinical research.

Lanostane triterpenes from Ganoderma lucidum suppress the adipogenesis in 3T3-L1 cells through down-regulation of SREBP-1c

Several lanostane triterpenes [butyl ganoderate A ( 1), butyl ganoderate B ( 2), butyl lucidenate N ( 3), and butyl lucidenate A ( 4)] bearing a butyl ester side chain from the fruiting bodies of Ganoderma lucidum exhibited considerable inhibitory effects on adipogenesis in 3T3-L1 cells. The inhibitory mechanism of 1 and 3 on adipogenesis in 3T3-L1 cells was investigated; we found that the mRNA and protein expression levels of SREBP-1c were reduced by treatment with 1 and 3 versus the untreated control. Furthermore, compounds 1 and 3 suppressed the mRNA expression levels of FAS and ACC. These results demonstrate that inhibition of adipogenesis in 3T3-L1 cells by treatment with 1 and 3 may be mediated in part through down-regulation of the adipogenic transcription factor SREBP-1c and its target genes, such as FAS and ACC.

Characterization of mature vs aged rabbit articular cartilage: analysis of cell density, apoptosis-related gene expression and mechanisms controlling chondrocyte apoptosis

The prevalence of osteoarthritis (OA) is increased in aged individuals and a direct correlation between chondrocyte apoptosis and cartilage degradation secondary to OA has been demonstrated. To address the question of whether age predisposes articular cartilage to apoptosis, the objective of the present study was to characterize and compare in aged and mature non-OA rabbit articular cartilage, cell density and expression levels of specific genes associated with apoptosis. Mechanistic studies on the inhibition of induced apoptosis were also carried out. Grade I (non-OA) femoral condyles and tibial plateaus from mature and aged rabbits were taken for assessment of viable cell density (VCD) and mRNA (reverse transcription-polymerase chain reaction) expression levels of the pro-apoptotic genes, Fas, Fas ligand (FasL), caspase-8, inducible nitric oxide synthase (iNOS) and p53. In vitro insulin-like growth factor (IGF-1)-mediated inhibition of nitric oxide (NO)-induced apoptosis was also examined using sodium nitroprusside (SNP) as NO donor. VCD was decreased 50-70% in aged articular cartilage relative to mature cartilage. mRNA expression levels of Fas, FasL, caspase-8 and p53 were higher in aged cartilage than in mature cartilage. iNOS expression was unchanged. IGF-1-mediated inhibition of NO-induced apoptosis was dose-dependent and reversed with addition of phosphatidylinositol-3 kinase inhibitor. This controlled animal model study demonstrates that age predisposes articular cartilage to changes in VCD and expression levels of specific pro-apoptotic genes. It is significant that these findings were demonstrated on cartilage that showed no prior signs of OA; it is also possible that such changes are a prelude to the age-related development of OA.

Anti-CD25 therapy affects the death signals of activated T-cells after clinical heart transplantation

IL-2 was initially defined as the T-cell growth factor mediating allograft rejection. However, recent experimental studies have shown that IL-2 also regulates the mechanism by which the immune system eliminates activated T-cells. IL-2 is required for the apoptosis of antigen specific T-cells by upregulating FAS receptors. To define whether inhibition of the IL-2 pathway by immunosuppressive agents prevents activation induced cell death (AICD) of activated T-cells, we measured intragraft mRNA expression of IL-2, IL-15, CD25, FAS, FASL, and characterized the phenotype of graft infiltrating T-cells (CD3, CD25) by real time RT-PCR and immunohistochemistry, respectively. Analysis was performed in EMB (n536) from cardiac allograft recipients who were treated with anti-CD25 mAb induction therapy (Daclizumab, n58) or with placebo (n56), in combination with CsA, steroids, and mycophenolate mofetil. Treatment with anti-CD25 mAb significantly affected the IL-2 pathway. In the presence of circulating anti-CD25 mAb, the first 4 months post-transplant, extremely low intragraft IL-2, and CD25 mRNA transcription levels and low numbers of activated CD25 positive T-cells were found compared to the levels measured in EMB from placebo treated patients (5-10 fold, p, 0.0001). The inhibition of the IL-2 pathway during anti-CD25 mAb therapy was associated with reduced FASL mRNA expression and low numbers of CD3 positive T-cells in the graft. Significantly lower FASL mRNA expression levels and CD3 positive T-cells were measured in EMB of anti-CD25 mAb treated patients than in EMB from patients who received placebo (p50.01 and p50.02, respectively). No effect of anti-CD25 therapy was found on intragraft IL-15 and FAS mRNA expression levels. In conclusion, our data suggest that immune activation in the absence of IL-2 is associated with impaired apoptosis of activated T-cells in the graft. Manipulation of the immune system by immunosuppressive agents such as anti-CD25 mAb might prevent the induction of activation induced cell death (AICD) of alloreactive T-cells by hindering the IL-2 dependent FASL expression.