FasL Protein Expression Research Articles

Piceatannol induces Fas and FasL up-regulation in human leukemia U937 cells via Ca 2+/p38α MAPK-mediated activation of c-Jun and ATF-2 pathways

To verify whether piceatannol-induced death of leukemia cells was associated with Fas-mediated death pathway, the present study was conducted. Piceatannol-induced apoptotic death of human leukemia U937 cells was characterized by increase in intracellular Ca 2+ concentration ([Ca 2+]i), ERK inactivation, p38 MPAK activation, degradation of procaspase-8 and production of t-Bid. Piceatannol treatment increased Fas and FasL protein expression, and up-regulated transcription of Fas and FasL mRNA. Down-regulation of FADD blocked piceatannol-induced procaspase-8 degradation and rescued viability of piceatannol-treated cells. Abolition of piceatannol-induced increase in [Ca 2+]i abrogated p38 MAPK activation and up-regulation of Fas and FasL expression, but restored ERK activation and viability of piceatannol-treated cells. Suppression of p38α MAPK or transfection of constitutively active MEK1 abolished piceatannol-induced Fas and FasL up-regulation. Piceatannol treatment repressed ERK-mediated c-Fos phosphorylation but evoked p38α MAPK-mediated c-Jun and ATF-2 phosphorylation. Knockdown of c-Fos, c-Jun and ATF-2 by siRNA reflected that c-Fos attenuated the effect of c-Jun and ATF-2 on Fas/FasL up-regulation. Taken together, our data indicate that Fas/FasL up-regulation in piceatannol-treated U937 cells is elicited by Ca 2+/p38α MAPK-mediated activation of c-Jun and ATF-2, and suggest that autocrine Fas-mediated apoptotic mechanism is involved in piceatannol-induced cell death.

Anticancer Effects of Flavonoid Derivatives Isolated from Millettia reticulata Benth in SK-Hep-1 Human Hepatocellular Carcinoma Cells

Millettia reticulata Benth is cultivated in Asian countries. M. reticulata Benth has multiple biological functions and is one of the oldest tonic herbs in traditional Chinese medicine. It has been elevated to one of the most commonly used herbs in modern Chinese medicine. The aims of this work were to study the in vitro anticancer activity of flavonoid derivatives isolated from the stems of M. reticulata Benth. Six flavonoid derivatives including (-)-epicatechin (1), naringenin (2), 5,7,3',5'-tetrahydroxyflavanone (3), formononetin (4), isoliquiritigenin (5), and genistein (6) were isolated from the stems of M. reticulata Benth. The structures of 1-6 were determined by spectroscopic methods. The effects of flavonoid derivatives (1-6) on the viability of human cancer cells (including HepG2, SK-Hep-1, Huh7, PLC5, COLO 205, HT-29, and SW 872 cells) were investigated. The results indicated that genistein (6) had the strongest inhibitory activity with an IC(50) value of 16.23 microM in SK-Hep-1 human hepatocellular carcinoma cells. Treatment of SK-Hep-1 cells with genistein (6) caused loss of mitochondrial membrane potential. Western blot data revealed that genistein (6) stimulated an increase in the protein expression of Fas, FasL, and p53. Additionally, treatment with genistein (6) changed the ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members and subsequently induced the activation of caspase-9 and caspase-3, which was followed by cleavage of poly(ADP-ribose) polymerase (PARP). These results demonstrate that genistein (6) induces apoptosis in SK-Hep-1 cells via both Fas- and mitochondria-mediated pathways.

FasL Gene–Deficient Mice Display a Limited Disruption in Spermatogenesis and Inhibition of Mono-(2-ethylhexyl) Phthalate–Induced Germ Cell Apoptosis

FasL (TNFSF6, CD95L) is hypothesized to trigger testicular germ cell apoptosis that normally occurs during a distinct peripubertal period as well as in response to toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the testis of FasL gene–deficient mice (FasL−/−) at two distinct developmental ages (postnatal day [PND] 28 and 44) and after toxicant-induced Sertoli cell injury. Testicular cross sections from peripubertal (PND 28) FasL−/− mice showed significant increases in the basal germ cell apoptotic index (AI; 20.58 ± 4.59) as compared to the testis of C57BL/6J wild-type mice (5.16 ± 0.08) and closely correlated with increased expression of TRAIL protein in the testis of FasL−/− mice. A limited, but significant, number of seminiferous tubules in the testis of PND 28 FasL−/− mice showed a severe loss of germ cells with only Sertoli cells present. In contrast, no apparent gross histological changes were observed in the testis of adult (PND 44) FasL−/− mice. However, PND 44 FasL−/− mice did show a 51% reduction in homogenization-resistant elongate spermatids as compared to age-matched C57BL/6J mice. Exposure of PND 28 FasL−/− mice to mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant, unexpectedly caused a rapid decrease in the germ cell AI that paralleled increased levels of the CFLAR (c-FLIP) protein, a known inhibitor of death receptor signaling. In contrast, MEHP treatment did not decrease c-FLIP levels in PND 28 C57BL/6J mice. Taken together, these findings indicate that FasL protein expression is required during the peripubertal period for the proper regulation of germ cell apoptosis that occurs normally during this period. The influence of FasL on the cellular regulation of c-FLIP protein levels appears to be a unique mechanism for modulating germ cell apoptosis after toxicant-induced Sertoli cell injury.

Clinical significance of Fas and FasL protein expression in gastric carcinoma and local lymph node tissues

To investigate the relation of Fas and Fas ligand (FasL) protein expression with carcinogenesis and metastasis of gastric carcinoma. Immunohistochemistry was used to detect Fas and FasL protein expression in 64 gastric carcinoma tissue samples and 20 normal gastric tissue samples. Relation between FasL and Fas expression, age and gender of gastric cancer patients, and pathological subtype and lymph node metastasis of gastric cancer was analyzed. The Fas expression level was significantly higher in normal gastric tissue samples than in gastric carcinoma tissue samples (85.0% vs 25.0%, P < 0.001), while the FasL expression level was significantly lower in normal gastric tissue samples than in gastric carcinoma tissue samples (30.0% vs 81.3%, P < 0.001). The Fas expression level was significantly higher in invasive lymph nodes than in non-invasive lymph nodes (82.9% vs 56.5%, P < 0.003) and in well-differentiated gastric carcinoma tissue samples than in poorly-differentiated gastric carcinoma tissue samples (50.0% vs 18.0%, P = 0.015). The FasL expression level was significantly lower in well-differentiated gastric carcinoma tissue samples than in poorly- differentiated gastric carcinoma tissue samples (42.9% vs 84.0%, P = 0.021). The Fas and FasL expression levels (25.0% and 81.3%) were significantly different in gastric carcinoma tissue samples (P < 0.001), but had a non-linear correlation (P = 0.575). Abnormal Fas and FasL expressions in gastric carcinoma and lymph node tissues are involved in carcinogenesis and metastasis of gastric cancer.

Adipocyte Apoptosis, a Link between Obesity, Insulin Resistance, and Hepatic Steatosis

Adipocyte death has been reported in both obese humans and rodents. However, its role in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity has not been studied. We now show using real-time reverse transcription-PCR arrays that adipose tissue of obese mice display a pro-apoptotic phenotype. Moreover, caspase activation and adipocyte apoptosis were markedly increased in adipose tissue from both mice with diet-induced obesity and obese humans. These changes were associated with activation of both the extrinsic, death receptor-mediated, and intrinsic, mitochondrial-mediated pathways of apoptosis. Genetic inactivation of Bid, a key pro-apoptotic molecule that serves as a link between these two cell death pathways, significantly reduced caspase activation, adipocyte apoptosis, prevented adipose tissue macrophage infiltration, and protected against the development of systemic insulin resistance and hepatic steatosis independent of body weight. These data strongly suggest that adipocyte apoptosis is a key initial event that contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis associated with obesity in both mice and humans. Inhibition of adipocyte apoptosis may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.

Anticancer Effects of Alpinia pricei Hayata Roots

The leaves and roots of Alpinia pricei Hayata are used as a traditional wrapping for food and as a cooking substitute for fresh ginger. Our previous study showed that ethanol extracts from the roots of A. pricei Hayata (EEAP) and its phenolic compounds have anti-inflammatory effects. The aims of this work were to further study the in vitro anticancer activity of EEAP and its active compounds with respect to various cancer cells. The results from an MTT assay demonstrated that EEAP decreased the cell population growth of CH27, HL-60, and A549 cells. Flow cytometric analysis of HL-60 cells exposed to EEAP showed that the number of apoptotic cells increased in a time- and dose-dependent manner. Western blot data revealed that EEAP stimulated an increase in the level of protein expression of Fas, FasL, caspase-8, and tBid. Moreover, the ratio of the expression levels of pro- and anti-apoptotic Bcl-2 family members was changed after treatment with EEAP. EEAP-induced apoptosis involved the release of mitochondrial cytochrome c and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase (PARP). The results also demonstrated that phenolic compounds (caffeic acid, apigenin, curcumin, and pinocembrin) from EEAP decreased the rate of population growth of HL-60 cells. Treatment of HL-60 cells with these phenolic compounds caused the loss of mitochondrial membrane potential. Our finding could provide critical information regarding the chemopreventive potential of ethanol extracts from A. pricei Hayata. These results also demonstrate that the EEAP-induced apoptotic ability in HL-60 cells might be related to the phenolic compounds.

Notexin upregulates Fas and FasL protein expression of human neuroblastoma SK-N-SH cells through p38 MAPK/ATF-2 and JNK/c-Jun pathways

Notechis scutatus scutatus notexin induced an increase in Fas and FasL protein expression of human neuroblastoma SK-N-SH cells in a dose- and time-dependent manner. Moreover, notexin treatment upregulated transcription of Fas/FasL mRNA. Downregulation of FADD blocked notexin-induced procaspase-8 degradation and cleavage of Bid and rescued viability of notexin-treated cells. Upon exposure to notexin, activation of JNK and p38 MAPK was observed in SK-N-SH cells. Notexin-induced upregulation of Fas and FasL was suppressed by SB202190 (p38 MAPK inhibitor) and S600125 (JNK inhibitor). Downregulation of p38α MAPK and JNK1 by siRNA proved that upregulation of Fas/FasL was related to p38α MAPK and JNK1 activation. Notexin treatment evoked p38α MAPK-mediated ATF-2 phosphorylation and JNK1-mediated c-Jun phosphorylation. Knockdown of c-Jun and ATF-2 by siRNA or overexpression of dominant-negative c-Jun and ATF-2 revealed that both c-Jun and ATF-2 were crucial for Fas/FasL upregulation. Taken together, our data indicate that notexin-induced upregulation of Fas and FasL is triggered by p38 MAPK/ATF-2 and JNK/c-Jun signaling pathways in SK-N-SH cells.

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A(2) (PLA(2)) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl-2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA(2)-treated cells. Moreover, PLA(2) treatment increased Fas and FasL protein expression. Upon exposure to PLA(2), activation of p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun NH(2)-terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA(2) and led to prolonged JNK activation, but failed to affect PLA(2)-induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria-dependent death pathway, downregulated Bcl-2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA(2) and PLA(2)-induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38alpha MAPK proved that ASK1 pathway was responsible for PLA(2)-induced p38 MAPK and JNK activation and p38alpha MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA(2)-induced procaspase-8 degradation and rescued viability of PLA(2)-treated cells. Taken together, our results indicate that JNK-mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl-2 family proteins are involved in PLA(2)-induced death of K562 cells.

Effects of somatostatin on the apoptosis of colorectal cancer cells

Objective To investigate the effects of somatostatin on the apoptosis of colorectal cancer cells. Methods The expression of somatostatin mRNA in colorectal cancer tissues from 79 patients who had been admired to Yijishan Hospital from January 2004 to October 2006 was detected by nested RT-PCR. The apoptotic index of colorectal cancer cells was detected by TUNEL, and the protein expressions of somatostatin, Fas, FasL, caspase-3 and caspase-8 in colorectal cancer tissues were detected by immunohistochemistry. All data were analyzed by chi-square test, q test and Spearman rank correlation coefficient. Results There was a positive correlation between the mRNA and protein expression of somatostatin (r = 0.98, P < 0.05). The mRNA and protein expression of somatostatin in poorly and moderately differentiated colorectal cancers were significantly lower than that in well differentiated colorectal cancers (χ2 = 10.78, 11.24, 5.27, 5.24, P < 0.05). The positive expression rates of mRNA and protein of somatostatin in papillary adenocarcinoma were significantly higher than those in mucinous adenocarcinoma and signet ring cell carcinoma and undifferentiated carcinoma (χ2= 6.56, 6.99, 5.44, 7.39, P < 0.05). The mRNA and protein expression of somatostatin in colorectal cancer in Dukes A and B were significantly higher than that in Dukes C and D (χ2 =5.17, 4.06, P <0.05). The apoptotic index in high or moderate somatostatin expression group was significantly higher than that in low somatostain expression group (q = 5.66, 4.21, P < 0.05), and the positive expression rates of Fas, caspase-8 and caspase-3 in high or moderate somatostatin expression group were significantly higher than those in low somatostafin expression group (χ2= 5.48, 5.62, 6.89, 4.32, 4.19, 3.91, P <0.05). Conclusion Somatostatin plays an important role in the regulation of cell apoptosis in colorectal cancer, and the mechanism may be related to the aberrant expression of Fas/FasL. Key words: Colorectal neoplasms; Somatostatin; Fas/FasL; Caspase

JNK1/c‐Jun and p38α MAPK/ATF‐2 pathways are responsible for upregulation of Fas/FasL in human chronic myeloid leukemia K562 cells upon exposure to Taiwan cobra phospholipase A2

Fas and FasL expression upregulation was found in human leukemia K562 cells upon exposure to Naja naja atra phospholipase A(2) (PLA(2)). PLA(2) treatment induced an increase in intracellular Ca(2+) ([Ca(2+)]i) and ROS generation levels, leading to activation of p38 MAPK and JNK. Suppression of both p38 MAPK and JNK abrogated Fas and FasL upregulation. Unlike PLA(2), catalytically inactive PLA(2) treatment did not markedly increase Fas and FasL protein expression, and p38 MAPK activation was exclusively responsible for catalytically inactive PLA(2)-induced increase in Fas and FasL protein expression. Knockdown of p38 alpha MAPK and JNK1 by siRNA proved that p38 alpha MAPK and JNK1 were involved in ATF-2 and c-Jun phosphorylation, respectively. Compared with the p38 alpha MAPK/ATF-2 pathway, the JNK1/c-Jun pathway played a crucial role in Fas/FasL upregulation. Unlike arachidonic acid, lysophosphatidylcholine mimicked the PLA(2) action in inducing Fas/FasL upregulation. Together with the previous finding that c-Jun and ATF-2 are involved in transcriptional regulation of Fas and FasL, our data suggest that PLA(2) induces Fas and FasL upregulation through p38 alpha MAPK/ATF-2 and JNK1/c-Jun pathways in K562 cells, and PLA(2) catalytic activity is involved in this action.

Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells

Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.

Apoptotic effect of sonodynamic therapy mediated by hematoporphyrin monomethyl ether on C6 glioma cells in vitro

Sonodynamic therapy (SDT) is a new method for treating cancer by inducing cell necrosis and/or apoptosis. However, the molecular mechanisms of cell apoptosis after SDT treatment remain unclear. In this study we investigated the apoptotic effect and mechanisms of SDT mediated by hematoporphyrin monomethyl ether (HMME) on C6 glioma cells in vitro. The time of ultrasound irradiation was optimized by MTT. The apoptotic rate, generation of ROS (reactive oxygen species), and mitochondrial membrane potential (MMP) were detected by flow cytometry, and expressions of apoptosis proteins were measured by Western blot after SDT treatment. The results displayed that treatment of C6 glioma cells with SDT resulted in the occurrence of apoptosis, which was associated with the production of ROS and loss of MMP. The results also showed that protein expression of Bax, caspase-9, and caspase-3 significantly increased, protein expression of Bcl-2 and Fas-L significantly decreased, and Fas protein expression was unchanged after SDT treatment. Our results suggested that the mitochondrial signal pathway may play a pivotal role in the apoptosis induced by SDT in C6 glioma cells.

The regulatory effect of the p38 signaling pathway on valdecoxib-induced apoptosis of the Eca109 cell line

Valdecoxib is a second generation selective COX-2 inhibitor that can induce cell apoptosis in a variety of cell types, but its precise regulatory mechanism is unknown. Apoptosis of Eca109 cells and p38 mRNA expression were investigted. The expression of p-p38MAPK, Fas and FasL proteins were detected by immunohistochemical staining and FCM. Valdecoxib increased the apoptosis rate of Eca109 cells. Fas and FasL protein expression was up-regulated in the valdecoxib groups, while SB203580 partly inhibited the valdecoxib-induced overexpression. Valdecoxib increased p38MAPK expression, while SB203580 inhibited the overexpression of this protein and the apoptosis rate decreased. The expression of Fas, FasL and p38MAPK protein were positively correlated with the apoptotic rate. In conclusion, valdecoxib activates the p38MAPK pathway, thus up-regulating expression of the Fas and FasL proteins, which may be one of the mechanisms through which valdecoxib induces apoptosis.

Expression of TRAIL and the death receptors DR4 and DR5 correlates with progression of degeneration in human intervertebral disks

Intervertebral disks degenerate far earlier than other musculoskeletal tissues and apoptosis has been suggested to have a vital function in promoting the degeneration process that is strongly associated with back pain. However, the molecular mediators of apoptosis in the intervertebral disk are poorly understood. Fas/FasL, TRAIL/DR4, TRAIL/DR5 and TNF-α/TNFR1 are ligand/receptor pairs of the tumor necrosis factor/nerve growth factor family, which are able to induce apoptosis by trimerization of the receptor by its corresponding ligand. We investigated which of these molecules are expressed in intervertebral disks and whether their expression correlates to disk degeneration. Intervertebral disks from 28 donors (age 12–70 years) suffering from scoliosis, vertebrae fracture or disk degeneration were scored histologically for degeneration and analyzed for gene expression of FasL/Fas, TRAIL/DR4, TNF-α/TNFR1 and caspase 8. Protein expression of FasL and TRAIL was assessed by immunohistology and apoptotic cell death was quantified by poly(ADP-ribose) polymerase (PARP) p85 staining. Isolated disk cells were analyzed by flow cytometry for Fas, FasL, TRAIL, DR4 and DR5 expression. Gene expression of TRAIL (P=0.002) and caspase 8 (P=0.027) significantly correlated with degeneration. TRAIL expression further correlated with cellularity (P=0.04), muccoid matrix changes (P=0.009) and tears and cleft formation (P=0.019). FasL and TRAIL expression was confirmed by immunohistology and PARP cleavage was significantly associated with degeneration (P=0.027). Flow cytometry on isolated disk cells revealed correlations between DR4 and degeneration (P=0.014), DR4/DR5 double-positive cells and degeneration (P=0.019), as well as DR5 and changes in tissue granularity (P=0.03). This is the first study that shows that intervertebral disk cells express TRAIL, DR4 and DR5, which correlate to the degenerative state of the disk. Therefore, disk cells inherit the molecular machinery to induce and undergo cellular apoptosis, and the frequency of cytokine expression suggests that the TRAIL/DR4/DR5 axis is an important molecular mediator of apoptosis induction in disk tissue.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from 1 h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly.

ROS‐mediated p38α MAPK activation and ERK inactivation responsible for upregulation of Fas and FasL and autocrine Fas‐mediated cell death in Taiwan cobra phospholipase A2‐treated U937 cells

The aim of the present study is to explore the signaling pathway associated with Naja naja atra phospholipase A(2) (PLA(2))-induced apoptotic death of human leukemia U937 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, and cytochrome c release were observed in PLA(2)-treated cells. PLA(2) treatment increased Fas and FasL protein expression, and upregulated transcription of Fas and FasL mRNA. Upon exposure to PLA(2), ROS generation, p38 MAPK activation, and ERK inactivation were found in U937 cells. Abolition of PLA(2)-induced ROS generation abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored ERK activation and viability of PLA(2)-treated cells. Block of p38 MAPK by SB202190 abolished PLA(2)-induced Fas/FasL upregulation and ERK inactivation, but not ROS generation. Activated ERK suppressed p38 MAPK activation and Fas/FasL protein expression. Selective inactivation or overexpression of p38alpha MAPK proved that upregulation of Fas/FasL and ERK inactivation were related to p38alpha MAPK activation. Deprivation of catalytic activity with PLA(2) blocked completely PLA(2)-induced Fas/FasL upregulation. Downregulation of FADD abolished PLA(2)-induced procaspase-8 degradation and rescued viability of PLA(2)-treated cells. Taken together, our results indicate that Fas/FasL upregulation in PLA(2)-treated U937 cells is elicited by ROS-mediated p38alpha MAPK activation and ERK inactivation, and suggest that autocrine Fas/FasL apoptotic mechanism is involved in PLA(2)-induced cell death. J. Cell. Physiol. 219: 642-651, 2009. (c) 2009 Wiley-Liss, Inc.

Elevated serum soluble Fas ligand is a promising marker of testicular toxicity induced by epirubicin in rats

To investigate the role of the Fas/Fas ligand (Fas/FasL) system in testicular toxicity induced by epirubicin (Epi) and to correlate the system with the serum levels of soluble Fas and Fas ligand (sFas/sFasL), epirubicin was intraperitoneally administered to male Sprague-Dawley male rats at doses of 1.2mg/kg once a week for 10 weeks, and genital organ weights and histopathology were examined. Fas and FasL expression in rat testis were examined by immunohistochemistry. Apoptosis was assessed by TUNEL assay. Expression levels of Fas and FasL were analyzed by RT-PCR and Western blotting. Serum sFas/sFasL levels were determined by ELISA. The results show that the testicular toxicity of Epi involved germ cell apoptosis. Fas and FasL protein expression levels were markedly increased in Epi-treated rat testes, as was expression of sFasL. In particular, increasing serum sFasL levels were positively correlated with elevated expression levels of FasL and sFasL in the testes of Epi-treated rats, revealing serum sFasL to be a promising marker of testicular toxicity after cytotoxic chemotherapy.

Inhibition of methionine adenosyltransferase II induces FasL expression, Fas-DISC formation and caspase-8-dependent apoptotic death in T leukemic cells

Methionine adenosyltransferase II (MAT II) is a key enzyme in cellular metabolism and catalyzes the formation of S-adenosylmethionine (SAMe) from L-methionine and ATP. Normal resting T lymphocytes have minimal MAT II activity, whereas activated proliferating T lymphocytes and transformed T leukemic cells show significantly enhanced MAT II activity. This work was carried out to examine the role of MAT II activity and SAMe biosynthesis in the survival of leukemic T cells. Inhibition of MAT II and the resultant decrease in SAMe levels enhanced expression of FasL mRNA and protein, and induced DISC (Death Inducing Signaling Complex) formation with FADD (Fas-associated Death Domain) and procaspase-8 recruitment, as well as concomitant increase in caspase-8 activation and decrease in c-FLIP(s) levels. Fas-initiated signaling induced by MAT II inhibition was observed to link to the mitochondrial pathway via Bid cleavage and to ultimately lead to increased caspase-3 activation and DNA fragmentation in these cells. Furthermore, blocking MAT 2A mRNA expression, which encodes the catalytic subunits of MAT II, using a small-interfering RNA approach enhanced FasL expression and cell death, validating the essential nature of MAT II activity in the survival of T leukemic cells.

Cytotoxic Effects of New Geranyl Chalcone Derivatives Isolated from the Leaves of Artocarpus communis in SW 872 Human Liposarcoma Cells

Breadfruit (Artocarpus communis Moraceae) is cultivated in tropical and subtropical regions as a traditional starch crop and also has potential medicinal properties. The aim of this work was to study the in vitro anticancer activity of compounds isolated from the leaves of Artocarpus communis. Three new geranyl chalcone derivatives including isolespeol (1), 5'-geranyl-2',4',4-trihydroxychalcone (2), and 3,4,2',4'-tetrahydroxy-3'-geranyldihydrochalcone (3), together with two known compounds lespeol (4) and xanthoangelol (5), were isolated from the leaves of Artocarpus communis. The structures of 1- 5 were elucidated by spectroscopy and through comparison with data reported in the literature. The effects of geranyl chalcone derivatives (1- 5) on the viability of human cancer cells (including SW 872, HT-29, COLO 205, Hep3B, PLC5, Huh7, and HepG2 cells) were investigated. The results indicate that isolespeol (1) showed the highest inhibitory activity with an IC 50 value of 3.8 muM in SW 872 human liposarcoma cells. Treatment of SW 872 human liposarcoma cells with isolespeol (1) caused the loss of mitochondrial membrane potential (DeltaPsim). Western blotting revealed that isolespeol (1) stimulated increased protein expression of Fas, FasL, and p53. The expression ratios of pro- and antiapoptotic Bcl-2 family members were also changed by isolespeol (1) treatment to subsequently induce the activation of caspase-9 and caspase-3, which was followed by cleavage of poly (ADP-ribose) polymerase (PARP). These results demonstrate that isolespeol (1) induces apoptosis in SW 872 cells through Fas- and mitochondria-mediated pathways.

Dexamethasone Effects on Fas Ligand Expression in Mouse Testicular Germ Cells

The aim of this study is to investigate the effect of Dex, a widely used GC, on apoptosis and expression of FasL protein in the mouse testicular germ cells. The effects of dexamethasone (Dex) on expression of Fas ligand (FasL), an important proapoptotic protein, in the mouse testicular germ cells were investigated. Six groups, each of 8 male NMRI mice were chosen for the experiment. Experimental groups received one of the following treatments daily for 7 days: 4, 7 and 10 mg kg(-1) Dex. Control groups were treated with equivalent volumes of saline. Experimental and control animals were sacrificed 24 h after the last injection. Immunohistochemical procedure was used to evaluation of FasL expression and the deoxyuridine nick-end labeling (TUNEL) was applied to assessment of the apoptotic germ cells. FasL expression of testicular germ cells were significantly increased in 10 mg kg(-1) Dex treated mice (p < 0.05), particularly at stages VII-VIII of spermatogenic cycle. Apoptotic indexes (AIs) of germ cells were significantly increased in 7 and 10 mg kg(-1) Dex treated mice.