Methylmercury increases N-methyl-D-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity

Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl-D-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to investigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [(3)H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25-1 microM). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25-5 microM). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 microM) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxicity. Intracellular calcium chelator BAPTA-AM (1 microM) partially attenuated the neurotoxicity effect of 1 microM MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs.     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

Glycyrrhizin Attenuates MPTP Neurotoxicity in Mouse and MPP+-Induced Cell Death in PC12 Cells

The present study examined the inhibitory effect of licorice compounds glycyrrhizin and a metabolite 18β-glycyrrhetinic acid on the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse and on the 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death in differentiated PC12 cells. MPTP treatment increased the activities of total superoxide dismutase, catalase and glutathione peroxidase and the levels of malondialdehyde and carbonyls in the brain compared to control mouse brain. Co-administration of glycyrrhizin (16.8 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. In vitro assay, licorice compounds attenuated the MPP⁺-induced cell death and caspase-3 activation in PC12 cells. Glycyrrhizin up to 100µM significantly attenuated the toxicity of MPP⁺. Meanwhile, 18β-glycyrrhetinic acid showed a maximum inhibitory effect at 10µM; beyond this concentration the inhibitory effect declined. Glycyrrhizin and 18β-glycyrrhetinic acid attenuated the hydrogen peroxide- or nitrogen species-induced cell death. Results from this study indicate that glycyrrhizin may attenuate brain tissue damage in mice treated with MPTP through inhibitory effect on oxidative tissue damage. Glycyrrhizin and 18β-glycyrrhetinic acid may reduce the MPP⁺ toxicity in PC12 cells by suppressing caspase-3 activation. The effect seems to be ascribed to the antioxidant effect.     

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl⁺ chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl⁺ cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl⁺ cells.     

Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK,Akt and NADPH Oxidase-mediated Pathways

Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 µg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91^phox, which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways.     

Quantitative relationship between guanine O-alkyl lesions produced by Onrigin™ and tumor resistance by O-alkylguanine-DNA alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O⁶-chloroethyl (Onrigin™ and carmustine) and O⁶-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O⁶-methylguanines/cell and ∼300 O⁶-chloroethylguanines/cell, respectively. AGT repaired O⁶-methylguanines until the AGT pool was exhausted, while its repair of O⁶-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O⁶-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O⁶-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.     

Up-regulation of endothelial monocyte chemoattractant protein-1 by coplanar PCB77 is caveolin-1-dependent

Atherosclerosis, the primary cause of heart disease and stroke is initiated in the vascular endothelium, and risk factors for its development include environmental exposure to persistent organic pollutants. Caveolae are membrane microdomains involved in regulation of many signaling pathways, and in particular in endothelial cells. We tested the hypothesis that intact caveolae are required for coplanar PCB77-induced up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of the atherosclerosis development. Atherosclerosis-prone LDL-R^−/− mice (control) or caveolin-1^−/−/LDL-R^−/− mice were treated with PCB77. PCB77 induced aortic mRNA expression and plasma protein levels of MCP-1 in control, but not caveolin-1^−/−/LDL-R^−/− mice. To study the mechanism of this effect, primary endothelial cells were used. PCB77 increased MCP-1 levels in endothelial cells in a time- and concentration-dependent manner. This effect was abolished by caveolin-1 silencing using siRNA. Also, MCP-1 up-regulation by PCB77 was prevented by inhibiting p38 and c-Jun N-terminal kinase (JNK), but not ERK1/2, suggesting regulatory functions via p38 and JNK MAPK pathways. Finally, pre-treatment of endothelial cells with the aryl hydrocarbon receptor (AhR) inhibitor α-naphthoflavone (α-NF) partially blocked MCP-1 up-regulation. Thus, our data demonstrate that coplanar PCB77 can induce MCP-1 expression by endothelial cells and that this effect is mediated by AhR, as well as p 38 and JNK MAPK pathways. Intact caveolae are required for these processes both in vivo and in vitro. This further supports a key role for caveolae in vascular inflammation induced by persistent organic pollutants.     

c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH₂-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIP_L/S and Bcl₂ proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl₂ which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl₂ at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death.     

A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral-CA1 synapses in hippocampus

N-methyl-d-aspartate glutamate receptors (NMDARs) are a key route for Ca²⁺ influx into neurons important to both activity-dependent synaptic plasticity and, when uncontrolled, triggering events that cause neuronal degeneration and death. Among regulatory binding sites on the NMDAR complex is a glycine binding site, distinct from the glutamate binding site, which must be co-activated for NMDAR channel opening. We developed a novel glycine site partial agonist, GLYX-13, which is both nootropic and neuroprotective in vivo. Here, we assessed the effects of GLYX-13 on long-term synaptic plasticity and NMDAR transmission at Schaffer collateral-CA1 synapses in hippocampal slices in vitro. GLYX-13 simultaneously enhanced the magnitude of long-term potentiation (LTP) of synaptic transmission, while reducing long-term depression (LTD). GLYX-13 reduced NMDA receptor-mediated synaptic currents in CA1 pyramidal neurons evoked by low frequency Schaffer collateral stimulation, but enhanced NMDAR currents during high frequency bursts of activity, and these actions were occluded by a saturating concentration of the glycine site agonist d-serine. Direct two-photon imaging of Schaffer collateral burst-evoked increases in [Ca²⁺] in individual dendritic spines revealed that GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca²⁺ influx. Examining the rate of MK-801 block of synaptic versus extrasynaptic NMDAR-gated channels revealed that GLYX-13 selectively enhanced activation of burst-driven extrasynaptic NMDARs, with an action that was blocked by the NR2B-selective NMDAR antagonist ifenprodil. Our data suggest that GLYX-13 may have unique therapeutic potential as a learning and memory enhancer because of its ability to simultaneously enhance LTP and suppress LTD.     

Excitatory and inhibitory synaptic transmission is differentially influenced by two ortho-substituted polychlorinated biphenyls in the hippocampal slice preparation

Exposure to polychlorinated biphenyls impairs cognition and behavior in children. Two environmental PCBs 2,2',3,3',4,4',5-heptachlorobiphenyl (PCB170) and 2,2',3,5',6-pentachlorobiphenyl (PCB95) were examined in vitro for influences on synaptic transmission in rat hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region using a multi-electrode array. Perfusion with PCB170 (10 nM) had no effect on fEPSP slope relative to baseline period, whereas (100 nM) initially enhanced then depressed fEPSP slope. Perfusion of PCB95 (10 or 100 nM) persistently enhanced fEPSP slope > 200%, an effect that could be inhibited by dantrolene, a drug that attenuates ryanodine receptor signaling. Perfusion with picrotoxin (PTX) to block GABA neurotransmission resulted in a modest increase in fEPSP slope, whereas PTX + PCB170 (1-100 nM) persistently enhanced fEPSP slope in a dose dependent manner. fEPSP slope reached > 250% of baseline period in the presence of PTX + 100 nM PCB170, conditions that evoked marked epileptiform after-potential discharges. PCB95 and PCB170 were found to differentially influence the Ca²⁺-dependence of [³H]ryanodine-binding to hippocampal ryanodine receptors. Non-coplanar PCB congeners can differentially alter neurotransmission in a manner suggesting they can elicit imbalances between inhibitory and excitatory circuits within the hippocampus. Differential sensitization of ryanodine receptors by Ca²⁺ appears to mediate, at least in part, hippocampal excitotoxicity by non-coplanar PCBs.     

The polychlorinated biphenyl mixture aroclor 1254 induces death of rat cerebellar granule cells: the involvement of the N-methyl-D-aspartate receptor and reactive oxygen species

Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants that display a complex spectrum of toxicological properties, including neurotoxicity. The present study investigates the effects of the PCB mixtures Aroclor 1242 (A1242) and Aroclor 1254 (A1254), and the PCB congeners 126 (3,3',4,4',5,-PeCB) and 153 (2,2',4,4',5,5'-HxCB) on formation of reactive oxygen species (ROS) and cell death in cultured rat cerebellar granule cells. The increase of ROS and induction of cell death were assayed using the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) and the trypan blue exclusion assay, respectively. A1242 and A1254 and PCB 153 induced a concentration-dependent increase in cell death and ROS formation. A1254 was selected for mechanistic studies. When the cerebellar granule cells were exposed to 15 microM A1254 for 12 h, 95% of the cells died. Both PCB-mediated cell death and the increase of the ROS formation were inhibited by MK-801, demonstrating the importance of the N-methyl-D-aspartate receptor. Inhibitors of nitric oxide synthase and phospholipase A2 led to a significant reduction of the DCF fluorescence and cell death. The mitochondrial permeability transition pore blocker cyclosporin A and the antioxidant vitamin E also increased survival and reduced ROS formation. The results show a connection between cell death and free radical formation.     

Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells

In this report, we investigated the role of oxidative stress in Physalis angulata-induced apoptosis of human oral cancer cells. P. angulata-induced apoptosis was characterized by nuclear morphological changes, membrane blebbing and activation of caspase-9. Exposure of HSC-3 cells to P. angulata caused production of reactive oxygen species and up-regulation of oxidative stress markers heme oxygenase-1 (HO-1), superoxide dismutase (SOD), heat shock protein 70 (HSP70) and caspase-4. Down-regulation of HO-1, SOD and HSP70 proteins expression by attenuation of oxidative stress, pretreatment with glutathione or N-acetylcysteine, significantly decreased P. angulata-triggered cell death. The present study also demonstrated that the mitochondria and the endoplasmic reticulum are the targets of P. angulata in HSC-3 cells. Our results revealed that: (1) reactive oxygen species may play a dominant role in this process, (2) P. angulata induces oxidative stress in HSC-3 cells, (3) P. angulata-initiated apoptosis is caused through oxidative stress-dependent induction of heme oxygenase-1, Cu/Zn SOD and HSP70 proteins expression and (4) antioxidants inhibited P. angulata-induced cell death through inhibition of the proteins expression of HO-1, Cu/Zn SOD and HSP70.     

Evaluation of the mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay

The minor tobacco alkaloid myosmine is implicated in DNA damage through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA). In contrast to TSNA, occurrence of myosmine is not restricted to tobacco. Myosmine is genotoxic to human cells in the comet assay. In this study, the mutagenic effect of myosmine was evaluated using the cloning hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. Four hour exposure of isolated peripheral blood lymphocytes from 14 subjects homozygous for the Leu84 wild-type of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene to 1 mM of myosmine increased mutant frequency from 0.73 ± 0.58 × 10⁻⁶ in control to 1.14 ± 0.89 × 10⁻⁶ lymphocytes (P < 0.05). These new data further confirm the mutagenic effects of myosmine.     

Activation of distinct P2Y receptor subtypes stimulates insulin secretion in MIN6 mouse pancreatic β cells

Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y₁ and P2Y₆ receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7 mM glucose with or without P2Y₁ and P2Y₆ agonists, 2-MeSADP and Up₃U, respectively. Both the agonists increased insulin secretion with EC₅₀ values of 44.6 ± 7.0 nM and 30.7 ± 12.7 nM respectively. The insulin secretion by P2Y₁ and P2Y₆ agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y₁ receptor antagonist radioligand [¹²⁵I]MRS2500 in MIN6 cell membranes was saturable (K_D 4.74 ± 0.47 nM), and known P2Y₁ ligands competed with high affinities. Inflammation and glucose toxicity lead to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up₃U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y₁ and P2Y₆ receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes.     

Protective role of estrogen receptor-alpha on lower chlorinated PCB congener-induced DNA damage and repair in human tumoral breast cells

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants. Much of the research has focused on the carcinogenic potential of higher chlorinated PCBs, but accumulative evidence has shown that lower chlorinated PCB congeners have initiating and promoting activities. The goal of this study was to examine the potential of lower chlorinated PCBs, including 2,2′,5,5′-tetrachlorobiphenyl (PCB52) and 3,3′,4,4′-tetrachlorobiphenyl (PCB77), to induce DNA damage and apoptosis in human MDA-MB-231 (MDA) and MCF-7 breast cancer cells. Results confirmed that treatment of cells with PCB52 and PCB77 resulted in oxidative stress and caspase-dependent apoptosis in both MDA and MCF-7 cells. We noticed that at non-cytotoxic concentrations PCB52 and PCB77-induced decreases in intracellular NAD(P)H in MDA cells but not in MCF-7 cells. Further investigation confirmed that decreases in intracellular NAD(P)H in PCB-treated MDA cells are primarily due to reduction in intracellular NAD⁺ pool mediated by poly(ADP-ribose)polymerase-1 activation through formation of DNA strand breaks. Antagonism was observed between PCB52 and PCB77 for the effect on induction of DNA strand breaks in MDA cells. Overall, this evidence demonstrates that at non-cytotoxic concentrations, lower chlorinated PCB congeners are capable of inducing oxidative DNA lesions in ERα(−)/MDA cells but not in ERα(+)/MCF-7 cells and that functional ERα plays a protective role in modulating the PCB-induced DNA damage in human breast cancer cells.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

Search for Compounds with Hypoglycemic Activity in the Series of 1-[2-(1H-Tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-Tetrazolo[1,5-c]quinazolin-5(6H)-ones

Methods of 1-[2-(1H-tetrazol-5-yl)-R¹-phenyl]-3-R²-phenyl(ethyl)ureas and R¹-tetrazolo[1,5-c]quinazolin-5(6H)-ones synthesis were designed. IR, LC-MS, ¹H NMR, and elemental analysis data evaluated the structure and purity of the obtained compounds. Different products, depending on the reaction conditions, were distinguished and discussed. The preliminary hypoglycemic activity of 36 synthesized compounds was revealed. Docking studies to 11β-hydroxysteroid dehydrogenase 1, γ-peroxisome proliferator-activated receptor, and dipeptidyl peptidase-4 were conducted. Eight of these substances were further tested on glucocorticoid-induced insulin resistance models, namely glucose tolerance, oral rapid insulin, and adrenalin tests. One of the most active compounds turned out to be tetrazolo[1,5-c]quinazolin-5(6H)-one 3.1, exceeding the reference drugs Metformin (50 and 200 mg/kg) and Gliclazide (50 mg/kg).     

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca²⁺ productions as well as loss of mitochondrial membrane potential (ΔΨ_m) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca²⁺ release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.     

Morphological alterations of Vero cell exposed to coplanar PCB 126 and noncoplanar PCB 153

Polychlorinated biphenyls (PCBs) are widespread, persistent environmental contaminants that display a complex spectrum of toxicological properties. Exposure to PCBs has been associated with morphological anomalies in cell cultures. However, most mechanistic studies of PCBs' toxic activity have been focused on coplanar congeners. It is of importance to determine whether PCB treatment would influence cell configuration and whether these changes would depend on the structural characteristics of PCBs. In this study, we investigated cell morphological alteration in Vero cell cultures after exposure to coplanar PCB 126 and noncoplanar PCB 153. The survival of Vero cells was measured through the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) test. Cytotoxicity results suggested that PCB congeners had a toxic, antiproliferative effect on Vero cells. Morphological studies described structural modifications and provided evidence that apoptosis might be the main cell death pathway in PCB 153‐treated cells. The comparison between PCB 126 and PCB 153 indicated that the cell death mechanisms involved in coplanar or noncoplanar PCB congener exposure were different in Vero cells. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.