Biochemical evidence of a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, including Blastocystis spp. The distinct differences in the phenotypic characterization reported previously have prompted us to compare the rate of apoptosis in Blastocystis spp. isolated from individuals who were symptomatic and asymptomatic. In the current study, we analysed the caspase activation involved in PCD mediated by a cytotoxic drug, (metronidazole) in both symptomatic & asymptomatic isolates.
The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-fluoromethyl ketone (z-FA-FMK) readily inhibits anti-CD3-induced human T cell proliferation, whereas the analogue benzyloxycarbonyl-phenylalanine-alanine-diazomethyl ketone (z-FA-DMK) had no effect. In contrast, benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was toxic. The inhibition of T cell proliferation mediated by z-FA-FMK requires not only the FMK moiety, but also the benzyloxycarbonyl group at the N-terminal, suggesting some degree of specificity in z-FA-FMK-induced inhibition of primary T cell proliferation. We showed that z-FA-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-induced T cell proliferation mediated by z-FA-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. The inhibition of anti-CD3-induced up-regulation of CD25 and CD69 expression mediated by z-FA-FMK was also attenuated in the presence of exogenous GSH. Similar to cell proliferation, GSH, NAC and L-cysteine but not D-cysteine, completely restored the processing of caspase-8 and caspase-3 to their respective subunits in z-FA-FMK-treated activated T cells. Our collective results demonstrated that the inhibition of T cell activation and proliferation mediated by z-FA-FMK is due to oxidative stress via the depletion of GSH.
A series of novel carbonyl compounds was synthesized by a simple, eco-friendly and efficient method. These compounds were screened for anti-oxidant activity, in vitro cytotoxicity and for inhibitory activity for acetylcholinesterase and butyrylcholinesterase. The effect of these compounds against amyloid β-induced cytotoxicity was also investigated. Among them, compound 14 exhibited strong free radical scavenging activity (18.39 μM) while six compounds (1, 3, 4, 13, 14, and 19) were found to be the most protective against Aβ-induced neuronal cell death in PC12 cells. Compounds 4 and 14, containing N-methyl-4-piperidone linker, showed high acetylcholinesterase inhibitory activity as compared to reference drug donepezil. Molecular docking and QSAR (Quantitative Structure-Activity Relationship) studies were also carried out to determine the structural features that are responsible for the acetylcholinesterase and butyrylcholinesterase inhibitory activity.
The caspase inhibitor benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and l-cysteine, whereas d-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.
Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T-cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases-3 and -7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP-ribose) polymerase cleavage (PARP). Pre-treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin-induced apoptosis in Jurkat T-cells.
This study aimed to examine the anti-candidal efficacy of a novel ketone derivative isolated from Diaporthe sp. ED2, an endophytic fungus residing in medicinal herb Orthosiphon stamieus Benth. The ethyl acetate extract of the fungal culture was separated by open column and reverse phase high-performance liquid chromatography (HPLC). The eluent at retention time 5.64 min in the HPLC system was the only compound that exhibited anti-candidal activity on Kirby-Bauer assay. The structure of the compound was also elucidated by nuclear magnetic resonance and spectroscopy techniques. The purified anti-candidal compound was obtainedas a colorless solid and characterized as 3-hydroxy-5-methoxyhex-5-ene-2,4-dione. On broth microdilution assay, the compound also exhibited fungicidal activity on a clinical strain of Candida albicans at a minimal inhibitory concentration of 3.1 μg/ml. The killing kinetic analysis also revealed that the compound was fungicidal against C. albicans in a concentration- and time-dependent manner. The compound was heat-stable up to 70°C, but its anti-candidal activity was affected at pH 2.
Limonene oxidation products (LOPs) have gained interest on their harmful health effects over time. Recently, studies have shown that the selected LOPs: 4-oxopentanal (4-OPA), 3-isopropenyl-6-oxo-heptanal (IPOH) and 4-acetyl-1-methylcyclohexene (4-AMCH) have sensory irritation effects in mice and inflammatory effects in human lung cells. This study was therefore undertaken to investigate the potential capacity of 4-OPA, IPOH and 4-AMCH to cause cell membrane damage, oxidative stress and inflammation in human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines. Overall results suggest that 4-OPA, IPOH have cytotoxic effects on human lung cells that might be mediated by ROS: the highest concentration applied of IPOH [500 μM] enhanced ROS generation by 100-fold ± 7.7 (A549) and 230-fold ± 19.9 (16HBE14o-) compared to the baseline. 4-OPA [500 μM] increased ROS levels by 1.4-fold ± 0.3 (A549) and by 127-fold ± 10.5 (16HBE14o-), while treatment with 4-AMCH [500 μM] led to 0.9-fold ± 0.2 (A549) and 49-fold ± 12.8 (16HBE14o-) increase. IPOH [500 μM] caused a decrease in the thiol-state balance (e.g. after 2 h, GSH:GSSG was reduced by 37% compared to the untreated 16HBE14o-cells). 4-OPA [500 μM] decreased the GSH:GSSG by 1.3-fold change in A549 cells and 1.4-fold change in 16HBE14o-cells. No statistically significant decrease in the GSH:GSSG in A549 and 16HBE14o-cell lines was observed for 4-AMCH [500 μM]. In addition, IPOH and 4-OPA [31.2 μM] increased the amount of the inflammatory markers: RANTES, VEGF and EGF. On the other hand, 4-AMCH [31.2 μM] did not show inflammatory effects in A549 or 16HBE14o-cells. The 4-OPA, IPOH and 4-AMCH treatment concentration and time-dependently induce oxidative stress and/or alteration of inflammatory markers on human bronchial and alveolar cell lines.
The antinociceptive effects produced by intraperitoneal administration of a novel synthetic chalcone, 3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMFP), were investigated in several mouse models of induced nociception. The administration of DMFP (0.1, 0.5, 1.0 and 5.0 mg/kg) produced significant attenuation on the acetic acid-induced abdominal-writhing test. It also produced a significant increase in response latency time in the hot-plate test and a marked reduction in time spent licking the injected paw in both phases of the formalin-induced paw-licking test. In addition, it was also demonstrated that DMFP exhibited significant inhibition of the neurogenic nociceptive response induced by intraplantar injections of capsaicin and glutamate. Moreover, the antinociceptive effect of DMFP in the acetic acid-induced abdominal-writhing test and the hot-plate test was not antagonized by pretreatment with a non-selective opioid receptor antagonist, naloxone. Finally, DMFP did not show any toxic effects and/or mortality in a study of acute toxicity and did not interfere with motor coordination during the Rota-rod test. Our present results show that DMFP exhibits both peripheral and central antinociceptive effects. It was suggested that its peripheral antinociceptive activity is associated with attenuated production and/or release of NO and various pro-inflammatory mediators, while central antinociceptive activity seems to be unrelated to the opioidergic system, but could involve, at least in part, an interaction with the inhibition of capsaicin-sensitive fibers and the glutamatergic system.
Goniothalamin (GN), a styryl-lactone isolated from Goniothalamus andersonii, has been demonstrated to possess antirestenostic properties by inducing apoptosis on coronary artery smooth muscle cells (CASMCs). In this study, the molecular mechanisms of GN-induced CASMCs apoptosis were further elucidated. Apoptosis assessment based on the externalization of phosphatidylserine demonstrated that GN induces CASMCs apoptosis in a concentration-dependent manner. The GN-induced DNA damage occurred with concomitant elevation of p53 as early as 2 h, demonstrating an upstream signal for apoptosis. However, the p53 elevation in GN-treated CASMCs was independent of NAD(P)H: quinone oxidoreductase 1 and Mdm-2 expression. An increase in hydrogen peroxide and reduction in free thiols confirmed the role for oxidative stress in GN treatment. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) that significantly abrogated GN-induced CASMCs apoptosis suggested the involvement of caspase(s). The role of apical caspase-2, -8, and -9 was then investigated, and sequential activation of caspase-2 and -9 but not caspase-8 leading to downstream caspase-3 cleavage was observed in GN-treated CASMCs. Reduction of ATP level and decrease in oxygen consumption further confirmed the role of mitochondria in GN-induced apoptosis in CASMCs. The mitochondrial release of cytochrome c was seen without mitochondrial membrane potential loss and was independent of cardiolipin. These data provide insight into the mechanisms of GN-induced apoptosis, which may have important implications in the development of drug-eluting stents.
Asthma is associated with increased pulmonary inflammation and airway hyperresponsiveness. The interaction between airway epithelium and inflammatory mediators plays a key role in the pathogenesis of asthma. In vitro studies evaluated the inhibitory effects of 3-(2,5-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMPF-1), a synthetic chalcone analogue, upon inflammation in the A549 lung epithelial cell line. DMPF-1 selectively inhibited TNF-α-stimulated CC chemokine secretion (RANTES, eotaxin-1, and MCP-1) without any effect upon CXC chemokine (GRO-α and IL-8) secretion. Western blot analysis further demonstrated that the inhibitory activity resulted from disruption of p65NF-κB nuclear translocation without any effects on the mitogen-activated protein kinase (MAPK) pathway. Treatment of ovalbumin-sensitized and ovalbumin-challenged BALB/c mice with DMPF-1 (0.2-100 mg/kg) demonstrated significant reduction in the secretion and gene expression of CC chemokines (RANTES, eotaxin-1, and MCP-1) and Th2 cytokines (IL-4, IL-5, and IL-13). Furthermore, DMPF-1 treatment inhibited eosinophilia, goblet cell hyperplasia, peripheral blood total IgE, and airway hyperresponsiveness in ovalbumin-sensitized and ovalbumin-challenged mice. In conclusion, these findings demonstrate the potential of DMPF-1, a nonsteroidal compound, as an antiasthmatic agent for further pharmacological evaluation.
The hydroquinone and catechol like metabolites, NCQ344 and NCQ436 respectively, of the antipsychotic remoxipride have recently been demonstrated to induce apoptosis in myeloperoxidase (MPO)-rich human bone marrow progenitor and HL-60 cells [S.M. McGuinness, R. Johansson, J. Lundstrom, D. Ross, Induction of apoptosis by remoxipride metabolites in HL-60 and CD34+/CD19- human bone marrow progenitor cells: potential relevance to remoxipride-induced aplastic anemia, Chem. Biol. Interact. 121 (1999) 253-265]. In the present study, we determined the molecular mechanisms of apoptosis induced by these remoxipride metabolites in HL-60 cells. Our results show that apoptosis was accompanied by phosphatidylserine (PS) exposure, activation of caspases-9, -3, -7 and DNA cleavage. In HL-60 cells treated with the hydroquinone NCQ344 and catechol NCQ436, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp. fluoromethyl ketone (Z-VAD.FMK) blocked DNA cleavage and activation of caspases-9, -3/-7. In addition, PS exposure was significantly but not completely inhibited by Z-VAD.FMK. These results demonstrate that although Z-VAD.FMK inhibitable caspases are necessary for maximal apoptosis induced by NCQ344 and NCQ436, additional caspase-independent processes may orchestrate changes leading to PS exposure during apoptosis induced by the remoxipride polyphenolic metabolites.
Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and downregulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.