The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways.

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis.

Apocynum venetum leaf aqueous extract inhibits voltage-gated sodium channels of mouse neuroblastoma N2A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Apocynum venetum Linn. (Apocynaceae family), also called Luobuma, is a shrub which grows widely in the Xinjiang Autonomous Region of China. Its leaves are used in herbal tea for the treatment of hypertension, anxiety and depression. Animal studies have also shown that Apocynum venetum leaf extract (AVLE) also exerts anti-depressant and anti-anxiety activities. The effects of AVLE on neuronal tissues in vitro are not fully understood. MATERIALS AND METHODS: Using the whole-cell voltage-clamp method, we studied the effects of AVLE on ion channels in cultured mouse neuroblastoma N2A cells. RESULTS: AVLE inhibited voltage-gated inward Na(+) current in a reversible and concentration-dependent manner (half-inhibitory concentration was 18 µg/ml and maximum inhibition at 100 µg/ml). AVLE specifically promoted steady-state inactivation of Na(+) channels but did not affect voltage-dependence of activation. The inhibitory effect was not use-dependent and was not affected by 300µM L-NAME, suggesting that NO was not involved in the action of AVLE in neuronal cells. AVLE also had a mild inhibitory effect on voltage-gated K(+) channels, but did not affect ATP-sensitive K(+) channels. CONCLUSIONS: Since voltage-gated Na(+) and K(+) channels are associated with neuronal excitability and therefore affect neurotransmission, the modulation of neuronal ion channels by AVLE may exert neuropharmacological effects. In particular, the inhibition of voltage-gated Na(+) currents by AVLE may in part account for the psychopharmacological effects of this herbal remedy.

Arylnaphthalene lignans from Taiwania cryptomerioides as novel blockers of voltage-gated K+ channels.

Lignans are natural phytochemicals which exhibit multiple pharmacological effects such as anti-inflammation, antivirus and anti-tumor activities. Whether they have effects on neural tissues and ion channels is still unknown. The effects of several arylnaphathalene lignans purified from Taiwania cryptomerioides on voltage-gated K(+) (Kv) channels in mouse neuroblastoma N2A cells were examined. These lignans included Taiwanin E, helioxanthin (HXT) and diphyllin. All lignans showed inhibitory effects on Kv channels and HXT was the most potent compound (IC(50)=1.7 µM). The mechanism of HXT block was further investigated. Its action was found to be extracellular but not intracellular. HXT accelerated current decay, caused a left-shift in steady-state inactivation curve but had no effect on voltage-dependence of activation. HXT block was unaffected by intracellular K(+) concentrations. Further, it did not affect ATP-sensitive K(+) channels. Our data therefore suggest that HXT is a potent and specific blocker of Kv channels, possibly with an inhibitory mechanism involving acceleration of slow inactivation.

Docosahexaenoic acid suppresses neuroinflammatory responses and induces heme oxygenase-1 expression in BV-2 microglia: implications of antidepressant effects for ω-3 fatty acids.

Accumulating evidence suggests that the pathophysiology of depression might be associated with neuroinflammation, which could be attenuated by pharmacological treatment for depression. Omega-3 polyunsaturated fatty acids (PUFAs) are anti-inflammatory and exert antidepressant effects. The aim of this study was to identify the molecular mechanisms through which docosahexaenoic acid (DHA), the main omega-3 PUFA in the brain, modulates oxidative reactions and inflammatory cytokine production in microglial and neuronal cells. The results of this study showed that DHA reduced expressions of tumor necrosis factor-α, interleukin-6, nitric oxide synthase, and cyclo-oxygenase-2, induced by interferon-γ, and induced upregulation of heme oxygenase-1 (HO-1) in BV-2 microglia. The inhibitory effect of DHA on nitric oxide production was abolished by HO-1 inhibitor zinc protoporphyrin IX. In addition, DHA caused AKT and ERK activation in a time-dependent manner, and the DHA-induced HO-1 upregulation could be attenuated by PI-3 kinase/AKT and MEK/ERK inhibitors. DHA also increased IKKα/ß phosphorylation, IκBα phosphorylation, and IκBα degradation, whereas both nuclear factor-κB and IκB protease inhibitors could inhibit DHA-induced HO-1 expressions. The other major n-3 PUFA, eicosapentaenoic acid, showed similar effects of DHA on inflammation and HO-1 in repeated key experiments. In connecting with inflammation hypothesis of depression and clinical studies supporting the antidepressant effects of omega-3 PUFAs, this study provides a novel implication of the antidepressant mechanisms of DHA.

Propofol depresses angiotensin II-induced cell proliferation in rat cardiac fibroblasts.

BACKGROUND: Propofol may have beneficial effects on the prevention of angiotensin II (Ang II)-induced cardiac fibroblast proliferation via its antioxidative properties. The authors hypothesized that propofol may alter Ang II-induced cell proliferation and aimed to identify the putative underlying signaling pathways in rat cardiac fibroblasts. METHODS: Cultured rat cardiac fibroblasts were pretreated with propofol then stimulated with Ang II; cell proliferation and endothelin-1 gene expression were examined. The effect of propofol on Ang II-induced nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity were also examined. The effect of propofol on nitric oxide production and protein kinase B and endothelial nitric oxide synthase phosphorylations were also tested to elucidate the intracellular mechanism of propofol in proliferation. RESULTS: Ang II (100 nm) increased cell proliferation and endothelin-1 expression, which were partially inhibited by propofol (10 or 30 microm). Propofol also inhibited Ang II-increased nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity. Propofol was also found to increase nitric oxide generation and protein kinase B and nitric oxide synthase phosphorylations. Nitric oxide synthase inhibitor (N-nitro-L-arginine methylester) and the short interfering RNA transfection for protein kinase B or endothelial nitric oxide synthase markedly attenuated the inhibitory effect of propofol on Ang II-induced cell proliferation. CONCLUSIONS: The authors' results suggest that propofol prevents cardiac fibroblast proliferation by interfering with the generation of reactive oxygen species and involves the activation of the protein kinase B-endothelial nitric oxide synthase-nitric oxide pathway.

Osthol is a use-dependent blocker of voltage-gated Na+ channels in mouse neuroblastoma N2A cells.

Osthol, a Chinese herbal compound, has been shown to possess vasorelaxant and neuroprotective properties. Not much is known about the effects of osthol on ionic channels, activities of which are implicated in vasorelaxation and neuroprotection. In this work we report that osthol could inhibit voltage-gated Na (+) currents with state-dependence in mouse neuroblastoma N2A cells (IC (50) = 12.3 microM and 31.5 microM at holding potentials of - 70 mV and - 100 mV, respectively). Current blockade was equally effective in both extracellular and intracellular application of osthol. Osthol (18 microM) did not significantly affect the kinetics and voltage-dependence of Na (+) channel activation, but left-shifted the steady-state inactivation curve (V (1/2) = - 60.5 mV and - 78.7 mV in the absence and presence of osthol, respectively). Osthol also mildly but significantly retarded channel recovery from inactivation (recovery time constant = 19.9 ms and 35.6 ms in the absence and presence of osthol, respectively). In addition, osthol blocked Na (+) currents in a frequency-dependent fashion: blockades of 17 %, 34 % and 49 % when currents were triggered at 0.33 Hz, 1 Hz and 3.33 Hz, respectively. Taken together, our results therefore suggest that osthol blocked voltage-gated Na (+) channels intracellularly with state- and frequency-dependence.

Rhynchophylline from Uncaria rhynchophylla functionally turns delayed rectifiers into A-Type K+ channels.

Rhynchophylline (1), a neuroprotective agent isolated from the traditional Chinese medicinal herb Uncaria rhynchophylla, was shown to affect voltage-gated K(+) (Kv) channel slow inactivation in mouse neuroblastoma N2A cells. Extracellular 1 (30 microM) accelerated the slow decay of Kv currents and shifted the steady-state inactivation curve to the left. Intracellular dialysis of 1 did not accelerate the slow current decay, suggesting that this compound acts extracellularly. In addition, the percent blockage of Kv currents by this substance was independent of the degree of depolarization and the intracellular K(+) concentration. Therefore, 1 did not appear to directly block the outer channel pore, with the results obtained suggesting that it drastically accelerated Kv channel slow inactivation. Interestingly, 1 also shifted the activation curve to the left. This alkaloid also strongly accelerated slow inactivation and caused a left shift of the activation curve of Kv1.2 channels heterologously expressed in HEK293 cells. Thus, this compound functionally turned delayed rectifiers into A-type K(+) channels.