Sodium Selenite-Induced Apoptosis Mediated by ROS Attack in Human Osteosarcoma U2OS Cells

Sodium selenite (Na₂SeO₃, SSE) is an inorganic Se compound that is widely used in cancer chemoprevention studies. SSE has been shown to have anti-proliferative effects on several types of human cancer cells, but its effect on osteosarcoma cells has thus far not been reported. In this study, the cytotoxic effect of SSE on osteosarcoma cells U2OS was investigated in vitro and found to be higher than on comparable non-cancer cell lines 293 and L6. Treatment with SSE decreased cell growth in a dose- and time-dependent manner and altered cellular morphology. SSE also inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and accumulation of cells during the advanced phase of apoptosis. SSE-induced apoptosis correlated with the activation of CASP 3, downregulation of BCL-2, and upregulation of P53 and PTEN in U2OS cells. These results indicated that SSE induces apoptosis in U2OS cells mainly through an ROS-mediated caspase pathway. This is the first report to show a possible mechanism of the anti-proliferative effect of SSE for the prevention of osteosarcoma in cell culture models.     

Coumarin Therapy and Platelet Aggregation

Platelet aggregation has been related to blood coagulation studies in patients on nicoumalone, a coumarin anticoagulant. Aggregation studies were performed by means of Chandler''s tube and the adenosine diphosphate (A.D.P.)-induced optical density method. Platelet aggregation in Chandler''s tube has been shown to be quite different from A.D.P. aggregation and to be dependent on the “intrinsic” (blood) clotting system. When the intrinsic system was depressed by coumarin anticoagulant, aggregation was delayed in Chandler''s tube, but patients with a predominantly “extrinsic” (tissue) system defect gave normal results even when their prothrombin time was excessively prolonged. In contrast there was an increased response to A.D.P. in the anticoagulated patients.The study emphasizes the different mechanisms of platelet aggregation, which we have referred to as coagulation-induced and A.D.P.-induced aggregation. It also shows the limitations of routine control of oral anticoagulants by prothrombin time alone, as the coagulation-induced platelet aggregation appears to be quantitatively related to the overall level of clotting factors in the intrinsic system and independent of the extrinsic system.     

Cigarette Smoking and Platelet Aggregation

The influence of cigarette smoking on platelet aggregation was studied in habitual and non-habitual smokers. The results indicate that habitual smokers have a greater tendency to platelet aggregation than do non-habitual smokers. Acute effects of cigarette smoking were, however, not significant. The nucleotide content and the serotonin content of the platelets were analyzed. The adenosine nucleotide and serotonin contents were similar in smokers and non-smokers in the control state and neither showed significant changes on cigarette smoking. There were significant correlations between the control concentrations of the various nucleotides in both groups and there were even higher correlations after smoking. Platelet aggregation bore no demonstrable relationship to the nucleotide or serotonin contents of the platelet. We conclude that the long-term effect of smoking is probably more important than the acute effect.     

JJK694, a Synthesized Obovatol Derivative,Inhibits Platelet Activation by Suppressing Cyclooxygenase and Lipoxygenase Activities

Three triterpenes having the 6/6/5-fused tri- and 6/6/6/5-fused tetracyclic skeletons were isolated from an incubation mixture of the mutated F601A enzyme, these products being in accordance with a Markovnikov closure. Successful trapping of the tricyclic cationic intermediate by using the squalene analog having a highly nucleophilic hydroxyl group leads us to propose that the ring expansion process of the 5-membered C-ring is involved in the squalene cyclization cascade.     

A Novel Labdane-Type Trialdehyde from Myoga (Zingiber mioga Roscoe) That Potently Inhibits Human Platelet Aggregation and Human 5-Lipoxygenase

We screened myoga extracts for inhibitors of human platelet aggregation and human 5-lipoxygenase. We identified a novel labdane type of diterpene, together with three known diterpenes (miogadial and galanals A and B) from the flower buds of myoga. Spectroscopic data indicated the structure of the new compound to be 12(E)-labdene-15,16,(8β)17-trial (miogatrial). Miogatrial and miogadial were potent inhibitors of human platelet aggregation and human 5-lipoxygenase (5-LOX). The sesquiterpene, polygodial, also exhibited strong inhibitory activity against human platelet aggregation and 5-LOX. On the other hand, galanals A and B did not have inhibitory activity in either experimental system. It thus appears that a 3-formyl-3-butenal structure was essential for the potent inhibition of human platelet aggregation and human 5-LOX.     

白藜芦醇甙对血小板聚集功能及内钙水平的影响

为了研究白藜芦醇甙(polydatin,PD)的抗血小板聚集作用及对细胞内钙水平的影响,并探讨其抗血栓形成作用的机制,应用Bom比浊法和Grynkiewicz方法分别测定PD对兔血小板聚集功能和血小板内钙水平的影响。PD在体外显著抑制花生四烯酸(arachidonic acid,AA)和腺苷二磷酸(adenosine diplrasphate,ADP)诱导的富血小板血浆中的血小板聚集,其半数抑制浓度(medium inhibitory concentration,IC50)分别为5．13及10．0μmol／L;5、10和20mg／kg的PD静注均明显降低兔血小板聚集率,且呈明显的剂量一效应关系;PD明显减少兔洗涤血小板内钙释放及外钙内流,本实验说明PD体内、外均有明显的抗血小板聚集作用,其机制与其降低血小板内钙浓度密切相关。     

钙离子通道A23187对血小板聚集和蛋白质磷酸化的影响

以 ³²P-Na₂HPO₄标记猪血小板,在阿斯匹林阻断花生四烯酸代谢,Apyrase去除分泌的ADP情况下,以A23187和PMA为血小板激动剂,staurosporine为PKC抑制剂,研究Ca²⁺和蛋白激酶C在血小板聚集中的作用.结果表明,a.A23187在1～20 μmol/L引起血小板聚集,相应地,明显地引起40 ku、20 ku蛋白质磷酸化,且存在剂量和时间效应关系.b.A23187和PMA在血小板聚集和蛋白质磷酸化上都存在着协同效应.c.1 μmol/L staurosporine可大部分抑制20 μmol/L A23187诱导的血小板聚集和20 ku、40 ku蛋白质磷酸化.结果提示,Ca²⁺激活血小板是建立在激活PKC的基础上,Ca²⁺通过激活PKC诱导血小板聚集,这是Ca²⁺激活血小板的主要途径.     

细胞调亡是半胱天冬酶参与的复杂过程

自从发现线虫(Caenorhabditis elegan)的CED蛋白与哺乳动物的白介素-1β转换酶(ICE)有相似作用以来[1],已有13种半胱天冬酶家族成员先后被发现或克隆[2](表1).半胱天冬酶(caspase)分为启动型和执行型,前者接受死亡信号、启动凋亡或激活下游的分子,后者则降解细胞骨架、蛋白质、核酸等.     

血小板聚集功能障碍与梅尼埃病的关系

目的探讨血小板聚集功能障碍与梅尼埃病的关系。方法对３２例梅尼埃病患者血小板聚集功能进行检测。结果血小板最大聚集率均数为（６１．４２±１９．４）％，与对照组（４７．２７±２０．４）％比较，有统计学差异（Ｐ＜０．０１）。结论血小板聚集功能的异常，可能与梅尼埃病的发病有密切关系。     

A New Ribityllumazine Derivative

Lipase-catalyzed enantioselective lactonization of racemic methyl 6-phenyl-4-hydroxy-5-hexynoate worked well to afford (R)-6-phenyl-5-hexyn-4-olide (42% yield, 81% e.e.). The e.e. of the product was enhanced to 97% by the repetition of enzymatic reaction. From this lactone, (4R,5R)-isomer of 5-hydroxy-6-phenyl-4-hexanolide, a lateral root inducing substance from Erwinia quercina was synthesized via a selective epoxidation of the intermediate and subsequent hydrogenolysis as the keystep.     

Hepatitis C Virus Nonstructural Protein 5A Inhibits Thapsigargin-Induced Apoptosis

Background

We previously reported that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) down-regulates TLR4 signaling and lipopolysaccharide-induced apoptosis of hepatocytes. There have been several reports regarding the association between HCV infection and endoplasmic reticulum (ER) stress. Here, we examined the regulation of HCV NS5A on the apoptosis of hepatocytes induced by thapsigargin, an inducer of ER stress.

Methods

The apoptotic response to thapsigargin and the expression of molecules involved in human hepatocyte apoptotic pathways were examined in the presence or absence of HCV NS5A expression.

Results

HCV JFH1 infection induced ER stress in the Huh7 cell line. HCV NS5A protected HepG2 cells against thapsigargin-induced apoptosis, the effect of which was linked to the enhanced expression of the 78-kDa glucose-regulated protein/immunoglobulin heavy-chain binding protein (GRP78). Consistent with a conferred pro-survival advantage, HCV NS5A reduced poly(adenosine diphosphate-ribose) polymerase cleavage and activation of caspases-3, -7 and -9, and Bax expression, while increasing the expressions of the anti-apoptotic molecules XIAP and c-FLIP. HCV NS5A weakly interacts with GRP78 and enhances GRP78 expression in hepatocytes.

Conclusion

HCV NS5A enhances GRP78 expression, resulting in the inhibition of apoptotic properties, and inhibits thapsigargin-induced apoptotic pathways in human hepatocytes, suggesting that disruption of ER stress-mediated apoptosis may have a role in the pathogenesis of HCV infection. Thus, HCV NS5A might engender the survival of HCV-infected hepatocytes contributing to the establishment of persistent infection.     

Glaucocalyxin A Inhibits Platelet Activation and Thrombus Formation Preferentially via GPVI Signaling Pathway

Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01μg/ml, 0.1μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.     

Platelet Aggregation Inhibitory Activity of Selective A2 Adenosine Receptor Agonists

Abstract

A series of new 2-alkynyl, 2-cycloalkynyl, and 2-aralkynyl derivatives of adenosine-5′-ethyluronamide (NECA) were synthesized and evaluated in binding studies and functional assays to assess their potency and selectivity at A₂ vs A₁ receptors. The new derivatives were also tested as inhibitors of rabbit platelet aggregation induced by ADP. While the presence of an aromatic or heteroaromatic ring conjugated to the triple bond decreased antiplatelet activity, the introduction of a hydroxyl group or a heterocyclic ring on the alkynyl side chain increased the antiaggregatory activity in comparison with NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. However, the presence of an α-quaternary carbon markedly reduced the antiaggregatory potency without affecting the A₂ binding affinity, suggesting that the platelet receptor is not a typical A_2a site.     

Platelet Aggregation Inhibitors from Philippine Marine Invertebrate Samples Screened in a New Microplate Assay

A new microplate assay for Ca²⁺-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 µg/ml, and epinephrine-induced aggregation by 78% at 20 µg/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 µg/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity.     

Blood Clotting and Platelet Aggregation during Oral Progestogen Contraception: A Follow-up Study

A two-year follow-up study of progestogen-only contraception with chlormadinone acetate indicates no increase of the level of factors VII and X, as found after three cycles with all oestrogen-progestogen oral contraceptives. Clotting factors which were raised with combined preparations became normal after the sixth monthly cycle of progestogen and remained normal during the two-year period of study.From 12 months onwards significant changes in the thromboelastograph pattern were recorded, but not to the same extent as with combined preparations. At two years platelet aggregation was significantly accelerated with chlormadinone acetate but was not as rapid as with combined preparations.     

Fas介导细胞凋亡及相关免疫调节作用

Fas/CD95作为肿瘤坏死因子(TNF)/神经生长因子(NGF)分子受体超家族成员,在细胞凋亡及体内免疫调节方面发挥重要的作用。细胞在接收到经Fas传递的凋亡信号后,主要经胞浆caspase和线粒体两种途径引发细胞程序性死亡。Fas介导T/B淋巴细胞的凋亡,在这些细胞的早期分化发育和维持机体免疫平衡中起主要作用。CTL和NK等杀伤细胞也通过Fas-FasL介导的细胞凋亡而发挥对靶细胞的杀伤效应。因此,Fas-FasL系统在肿瘤、自身免疫性疾病、艾滋病等疾病过程中发挥重要的作用。     

Exploring New Biological Functions of Amyloids: Bacteria Cell Agglutination Mediated by Host Protein Aggregation

Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.     

Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G₀/G₁ phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21^Cip1 and p27^Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.