Neuroprotective effect of methyl lucidone against microglia-mediated neurotoxicity

Excessive microglial activation-mediated neurotoxicity has been implicated in playing a crucial role in the pathogenesis of stroke and neurodegenerative diseases. Therefore, much attention has been paid to therapeutic strategies aimed at suppressing neurotoxic microglial activation. The microglial regulatory mechanism of methyl lucidone, a cyclopentenedione isolated from the stem bark of Lindera erythrocarpa Makino, was investigated in the present study. Methyl lucidone treatment (0.1-10 μM) significantly inhibited lipopolysaccharide (LPS, 100 ng/ml, 24 h)-stimulated nitric oxide (NO) production in a dose-dependent manner in both primary cortical microglia and BV-2 cell line. Moreover, it strongly inhibited LPS-stimulated secretion of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). Methyl lucidone treatment markedly induced down-regulation of LPS-induced nuclear translocation of nuclear factor κB (NF-κB) through preventing the degradation of the inhibitory protein IκBα. In addition, phosphorylation of Akt and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK) and p38 kinases were also suppressed by methyl lucidone. The cell viabilities of HT-22 neurons were significantly attenuated by treatment of the conditioned media containing neurotoxic secretary molecules from LPS-stimulated microglia. However, methyl lucidone significantly blocked neuronal cell death induced by microglial conditioned media. These neuroprotective effects of methyl lucidone were also confirmed in a neuron-microglia co-culture system using EGFP-transfected B35 neuroblastoma cell line. Taken together, these results suggest that methyl lucidone may have a neuroprotective potential via inhibition of neurotoxic microglial activation implicated in neurodegeneration.     

N-(p-Coumaryol)-Tryptamine Suppresses the Activation of JNK/c-Jun Signaling Pathway in LPS-Challenged RAW264.7 Cells

N-(p-Coumaryol) tryptamine (CT), a phenolic amide, has been reported to exhibit anti-oxidant and anti-inflammatory activities. However, the underlying mechanism by which CT exerts its pharmacological properties has not been clearly demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of CT in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. CT significantly inhibited LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE₂, and protein expressions of iNOS and COX-2. In addition, CT significantly suppressed LPS-induced secretion of pro-inflammatory cytokines such as TNF-α and IL-1β. To elucidate the underlying anti-inflammatory mechanism of CT, involvement of MAPK and Akt signaling pathways was examined. CT significantly attenuated LPS-induced activation of JNK/c-Jun, but not ERK and p38, in a concentration-dependent manner. Interestingly, CT appeared to suppress LPS-induced Akt phosphorylation. However, JNK inhibition, but not Akt inhibition, resulted in the suppression of LPS-induced responses, suggesting that JNK/c-Jun signaling pathway significantly contributes to LPS-induced inflammatory responses and that LPS-induced Akt phosphorylation might be a compensatory response to a stress condition. Taken together, the present study clearly demonstrates CT exerts anti-inflammatory activity through the suppression of JNK/c-Jun signaling pathway in LPS-challenged RAW264.7 macrophage cells.     

Inhibition of glial inflammatory activation and neurotoxicity by tricyclic antidepressants

Glial activation and neuroinflammatory processes play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and HIV dementia. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which may contribute to neuronal cell death. Inhibition of glial activation may alleviate neurodegeneration under these conditions. In the present study, the antiinflammatory and neuroprotective effects of tricyclic antidepressants were investigated using cultured brain cells as a model. The results showed that clomipramine and imipramine significantly decreased the production of nitric oxide or tumor necrosis factor-alpha (TNF-alpha) in microglia and astrocyte cultures. Clomipramine and imipramine also attenuated the expression of inducible nitric oxide synthase and proinflammatory cytokines such as interleukin-1beta and TNF-alpha at mRNA levels. In addition, clomipramine and imipramine inhibited IkappaB degradation, nuclear translocation of the p65 subunit of NF-kappaB, and phosphorylation of p38 mitogen-activated protein kinase in the lipopolysaccharide-stimulated microglia cells. Moreover, clomipramine and imipramine were neuroprotective as the drugs reduced microglia-mediated neuroblastoma cell death in a microglia/neuron co-culture. Therefore, these results imply that clomipramine and imipramine have antiinflammatory and neuroprotective effects in the central nervous system by modulating glial activation.     

Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS

Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxic concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IкB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation.     

Melatonin Induces Akt Phosphorylation through Melatonin Receptor- and PI3K-Dependent Pathways in Primary Astrocytes

Melatonin has been reported to protect neurons from a variety of neurotoxicity. However, the underlying mechanism by which melatonin exerts its neuroprotective property has not yet been clearly understood. We previously demonstrated that melatonin protected kainic acid-induced neuronal cell death in mouse hippocampus, accompanied by sustained activation of Akt, a critical mediator of neuronal survival. To further elucidate the neuroprotective action of melatonin, we examined in the present study the causal mechanism how Akt signaling pathway is regulated by melatonin in a rat primary astrocyte culture model. Melatonin resulted in increased astrocytic Akt phosphorylation, which was significantly decreased with wortmannin, a specific inhibitor of PI3K, suggesting that activation of Akt by melatonin is mediated through the PI3K-Akt signaling pathway. Furthermore, increased Akt activation was also significantly decreased with luzindole, a non-selective melatonin receptor antagonist. As downstream signaling pathway of Akt activation, increased levels of CREB phoshorylation and GDNF expression were observed, which were also attenuated with wortmannin and luzindole. These results strongly suggest that melatonin exerts its neuroprotective property in astrocytes through the activation of plasma membrane receptors and then PI3K-Akt signaling pathway.     

Caffeine suppresses lipopolysaccharide-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB activation and ERK phosphorylation

Since the anti-inflammatory effect of caffeine is unclear in microglial cells, we performed whether caffeine attenuates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Caffeine substantially suppressed the LPS-induced pro-inflammatory mediators nitric oxide (NO), prostaglandin E₂ (PGE₂) and tumor necrosis factor-α (TNF-α) in BV2 microglial cells. These effects resulted from the inhibition of their regulatory genes inducible NO synthase (iNOS), cycloxygenase-2 (COX-2) and TNF-α. In addition, caffeine significantly decreased LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing the nuclear translocation of p50 and p65 subunits. A specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated the LPS-induced expression of iNOS, COX-2 and TNF-α genes. In addition, we elucidated that inhibition of Akt phosphorylation plays a crucial role in caffeine-mediated NF-κB regulation in LPS-stimulated BV2 microglial cells. Caffeine also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and a specific inhibitor of ERK, PD98059, subsequently downregulated the expression of the pro-inflammatory genes iNOS, COX-2 and TNF-α. Taken together, our data indicate that caffeine suppresses the generation of pro-inflammatory mediators, such as NO, PGE₂ and TNF-α as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting Akt-dependent NF-κB activation and the ERK signaling pathway.     

Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4): Protein kinase modulation and reactive oxygen species generation

While it has been reported that genistein induces differentiation in multiple tumour cell models, the signalling and regulation of isoflavone-provoked differentiation are poorly known. We here demonstrate that genistein causes G₂/M cycle arrest and expression of differentiation markers in human acute myeloid leukaemia cells (HL60, NB4), and cooperates with all-trans retinoic acid (ATRA) in inducing differentiation, while ATRA attenuates the isoflavone-provoked toxicity. Genistein rapidly stimulates Raf-1, MEK1/2 and ERK1/2 phosphorylation/activation, but does not stimulate and instead causes a late decrease in Akt phosphorylation/activation which is attenuated by ATRA. Both differentiation and G₂/M arrest are attenuated by MEK/ERK inhibitors (PD98059, U0126) and ERK1-/ERK2-directed small interfering RNAs (siRNAs), and by the PI3K inhibitor LY294002, but not by the p38-MAPK inhibitor SB203580. Genistein stimulates p21^waf1/cip1 and cyclin B1 expression, phosphorylation/activation of ATM and Chk2 kinases, and Tyr15-phosphorylation/inactivation of Cdc2 (Cdk1) kinase, and these effects are attenuated by MEK/ERK inhibitors, while LY294002 also attenuates ERK and ATM phosphorylation. Caffeine abrogates the genistein-provoked G₂/M blockade and alterations in cell cycle regulatory proteins, and also suppresses differentiation. Finally, genistein causes reactive oxygen species (ROS) over-accumulation, but the antioxidant N-acetyl-l-cysteine fails to prevent ERK activation, G₂/M arrest, and differentiation induction. By contrast, N-acetyl-l-cysteine and p38-MAPK inhibitor attenuate the apoptosis-sensitizing (pro-apoptotic) action of genistein when combined with the antileukaemic agent arsenic trioxide. In summary, genistein-induced differentiation in acute myeloid leukaemia cells is a ROS-independent, Raf-1/MEK/ERK-mediated and PI3K-dependent response, which is coupled and co-regulated with G₂/M arrest, but uncoupled to the pro-apoptotic action of the drug.     

The neuroprotective effects of Lonicera japonica THUNB. against hydrogen peroxide-induced apoptosis via phosphorylation of MAPKs and PI3K/Akt in SH-SY5Y cells

We investigated the neuroprotective effects of Lonicera japonica THUNB. (Caprifoliaceae) (LJ) extract against hydrogen peroxide (H₂O₂), a toxin created by oxidative stress and implicated in neurodegenerative diseases, in human SH-SY5Y neuroblastoma cells. We examined the effects of LJ against H₂O₂-induced cytotoxicity, apoptosis, the production of reactive oxygen species (ROS), the proteolysis of cleaved poly-ADP-ribose polymerase (PARP), and the expression of Bcl-2, Bcl-xL, and cleaved caspase-3. Moreover, we attempted to determine whether LJ suppressed the phosphorylation of Akt, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). We found that LJ improved cell viability, inhibited cytotoxicity and apoptosis, and attenuated elevations in ROS and nuclear condensation. In addition, LJ showed radical scavenging ability in 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Western blot data revealed that LJ inhibited H₂O₂-induced up- and down-regulation of cleaved PARP, cleaved caspase-3, Bcl-2, and Bcl-xL. Furthermore, LJ significantly attenuated the H₂O₂-induced phosphorylation of Akt, JNK, p38 MAPK, and ERK1/2. These results demonstrate that LJ possesses potent neuroprotective activity. Its potential to treat neurodegenerative diseases warrants further research.     

Chlorpyrifos induces neuronal cell death via both oxidative stress and Akt activation downstream-regulated CHOP-triggered apoptotic pathways

Chlorpyrifos (CPF) is one of the most abundant and widely used organophosphate pesticides for agricultural, industrial, and household purposes in the world. Epidemiological studies have reported that CPF can induce neurotoxic impairments in mammalian, which is linked to an important risk factor for development of neurodegenerative diseases (NDs). However, limited information is available on CPF-induced neurotoxicity, with the underlying exact mechanism remains unclear. In this study, CPF exposure (10–400 μM) significantly reduced Neuro-2a cell viability and induced apoptotic events, including the increase in caspase-3 activity, apoptotic cell population, and cleavage of caspase-3/−7 and PARP. Exposure of Neuro-2a cells to CPF also triggered CHOP activation. Transfection with CHOP-specific siRNA markedly suppressed the expression of CHOP, and attenuated cytotoxicity and apoptotic events in CPF-exposed Neuro-2a cells. Furthermore, CPF exposure obviously evoked the phosphorylation of Akt as well as ROS generation in a time-dependent manner. Pretreatment with LY294002 (an Akt inhibitor) effectively attenuated the CPF-induced Akt phosphorylation, CHOP activation, and apoptotic events, but not that ROS production. Of note, buffering the ROS generation with antioxidant N-acetylcysteine effectively prevented the CPF-induced ROS generation, CHOP activation, and apoptotic events, but not that the Akt phosphorylation. Collectively, these findings indicate that CPF exposure exerts neuronal cytotoxicity via the independent pathways of ROS generation and Akt activation downstream-regulated CHOP-triggered apoptosis, ultimately leading to neuronal cell death.     

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.     

Effect of lysophosphatidylglycerol on several signaling molecules in OVCAR-3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca(2+)](i)) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca(2+)](i) increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca(2+)](i) increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-kappaB-driven luciferase activity in exogenously LPA(1), LPA(2), or LPA(3)-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA(1), LPA(2), and LPA(3)), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca(2+)](i) increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.     

Candesartan,an Angiotensin II AT1-Receptor Blocker and PPAR-γ Agonist,Reduces Lesion Volume and Improves Motor and Memory Function After Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) results in complex pathological reactions, the initial lesion worsened by secondary inflammation and edema. Angiotensin II (Ang II) is produced in the brain and Ang II receptor type 1 (AT₁R) overstimulation produces vasoconstriction and inflammation. Ang II receptor blockers (ARBs) are neuroprotective in models of stroke but little is known of their effect when administered in TBI models. We therefore performed controlled cortical impact (CCI) injury on mice to investigate whether the ARB candesartan would mitigate any effects of TBI. We administered candesartan or vehicle to mice 5 h before CCI injury. Candesartan treatment reduced the lesion volume after CCI injury by approximately 50%, decreased the number of dying neurons, lessened the number of activated microglial cells, protected cerebral blood flow (CBF), and reduced the expression of the cytokine TGFβ1 while increasing expression of TGFβ3. Candesartan-treated mice also showed better motor skills on the rotarod 3 days after injury, and improved performance in the Morris water maze 4 weeks after injury. These results indicate that candesartan is neuroprotective, reducing neuronal injury, decreasing lesion volume and microglial activation, protecting CBF and improving functional behavior in a mouse model of TBI. Co-treatment with a peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist significantly reduced some of the beneficial effects of candesartan after CCI, suggesting that PPARγ activation may contribute to part or to all of the neuroprotective effect of candesartan. Overall, our data suggest that ARBs with dual AT₁R-blocking and PPARγ activation properties may have therapeutic value in treating TBI.     

Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation

Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox™) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X_L and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.     

Notoginsenoside Ft1 activates both glucocorticoid and estrogen receptors to induce endothelium-dependent,nitric oxide-mediated relaxations in rat mesenteric arteries

Panax notoginseng (Burk.) F.H. Chen has been used traditionally for the treatment of cardiovascular diseases. Notoginsenoside Ft1 (Ft1) is a bioactive saponin from the leaves of P. notoginseng. Experiments were designed to determine whether or not Ft1 is an endothelium-dependent vasodilator. Rat mesenteric arteries were suspended in organ chambers for the measurement of isometric tension during phenylephrine-induced contractions. The cyclic guanosine monophosphate (cGMP) level was assessed using enzyme immunoassay. The phosphorylation and protein expressions of endothelial nitric oxide synthase (eNOS), glucocorticoid receptors (GR), estrogen receptors beta (ERß), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) were determined by Western blotting. The localization of GR and ERß were determined by immunofluorescence staining. Ft1 caused endothelium-dependent relaxations, which were abolished by l-NAME (inhibitor of nitric oxide synthases) and ODQ (inhibitor of soluble guanylyl cyclase). Ft1 increased the cGMP level in rat mesenteric arteries. GR and ERß were present in the endothelial layer and their antagonism by RU486 and PHTPP, respectively, inhibited Ft1-induced endothelium-dependent relaxations and phosphorylations of eNOS, Akt and ERK1/2. Inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin and ERK1/2 by U0126 reduced Ft1-evoked relaxations and eNOS phosphorylation. Taken in conjunction, the present findings suggest that Ft1 stimulates endothelial GRs and ERßs with subsequent activation of the PI3K/Akt and ERK1/2 pathways in rat mesenteric arteries. This results in phosphorylation of eNOS and the release of NO, which activates soluble guanylyl cyclase in the vascular smooth muscle cells leading to relaxations.     

Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury

Methylmercury (MeHg) is well-known for causing irreversible damage in the central nervous system as well as a risk factor for inducing neuronal degeneration. However, the molecular mechanisms of MeHg-induced neurotoxicity remain unclear. Here, we investigated the effects and possible mechanisms of MeHg in the mouse cerebrum (in vivo) and in cultured Neuro-2a cells (in vitro). In vivo study showed that the levels of LPO in the plasma and cerebral cortex significantly increased after administration of MeHg (50 μg/kg/day) for 7 consecutive weeks. MeHg could also decrease glutathione level and increase the expressions of caspase-3, -7, and -9, accompanied by Bcl-2 down-regulation and up-regulation of Bax, Bak, and p53. Moreover, treatment of Neuro-2a cells with MeHg significantly reduced cell viability, increased oxidative stress damage, and induced several features of mitochondria-dependent apoptotic signals, including increased sub-G1 hypodiploids, mitochondrial dysfunctions, and the activation of PARP, and caspase cascades. These MeHg-induced apoptotic-related signals could be remarkably reversed by antioxidant NAC. MeHg also increased the phosphorylation of ERK1/2 and p38, but not JNK. Pharmacological inhibitors NAC, PD98059, and SB203580 attenuated MeHg-induced cytotoxicity, ERK1/2 and p38 activation, MMP loss, and caspase-3 activation in Neuro-2a cells. Taken together, these results suggest that the signals of ROS-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic pathways that are involved in MeHg-induced neurotoxicity.     

Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway.