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.

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.

L-Carnitine attenuates angiotensin II-induced proliferation of cardiac fibroblasts: role of NADPH oxidase inhibition and decreased sphingosine-1-phosphate generation.

The heart is unable to synthesize L-carnitine and is strictly dependent on the L-carnitine provided by the blood stream; however, additional studies are needed to better understand the mechanism of L-carnitine supplementation to the heart. The aim of this study was to evaluate the effects of L-carnitine on angiotensin II (Ang II)-induced cardiac fibroblast proliferation and to explore its intracellular mechanism(s). Cultured rat cardiac fibroblasts were pretreated with L-carnitine (1-30 mM) then stimulated with Ang II (100 nM). Ang II increased fibroblast proliferation and endothelin-1 expression, which were partially inhibited by L-carnitine. L-carnitine also attenuated Ang II-induced NADPH oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, activator protein-1-mediated reporter activity and sphingosine-1-phosphate generation. In addition, L-carnitine increased prostacyclin (PGI(2)) generation in cardiac fibroblasts. siRNA transfection of PGI(2) synthase significantly reduced L-carnitine-induced PGI(2) and its anti-proliferation effects on cardiac fibroblasts. Furthermore, blockading potential PGI(2) receptors, including immunoprecipitation (IP) receptors and peroxisome proliferator-activated receptors alpha (PPAR alpha) and delta, revealed that siRNA-mediated blockage of PPAR alpha considerably reduced the anti-proliferation effect of L-carnitine. In summary, these results suggest that L-carnitine attenuates Ang II-induced effects (including NADPH oxidase activation, sphingosine-1-phosphate generation and cell proliferation) in part through PGI(2) and PPAR alpha-signaling pathways.

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.

Tetramethylpyrazine inhibits angiotensin II-increased NAD(P)H oxidase activity and subsequent proliferation in rat aortic smooth muscle cells.

Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb, Chuanxiong, which is widely used in China for the treatment of cardiovascular problems. The aims of this study were to examine whether TMP may alter angiotenisn II (Ang II)-induced proliferation and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with TMP and then stimulated with Ang II, [3H]-thymidine incorporation and the ET-1 expression was examined. Ang II increased DNA synthesis which was inhibited by TMP (1-100 microM). TMP inhibited the Ang II-induced ET-1 mRNA levels and ET-1 secretion. TMP also inhibited Ang II-increased NAD(P)H oxidase activity, intracellular reactive oxygen species (ROS) levels, and the ERK phosphorylation. Furthermore, TMP and antioxidants such as Trolox and diphenylene iodonium decreased Ang II-induced ERK phosphorylation, and activator protein-1 reporter activity. In summary, we demonstrate for the first time that TMP inhibits Ang II-induced proliferation and ET-1, partially by interfering with the ERK pathway via attenuation of Ang II-increased NAD(P)H oxidase and ROS generation. Thus, this study delivers important new insight in the molecular pathways that may contribute to the proposed beneficial effects of TMP in cardiovascular disease.

Isosteviol as a potassium channel opener to lower intracellular calcium concentrations in cultured aortic smooth muscle cells.

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, and is commonly used as a non-caloric sugar substitute in Japan and Brazil. The present study attempted to elucidate the role of potassium (K (+)) channels in the action of isosteviol on intracellular calcium concentrations ([Ca (2+)]i) in cultured vascular smooth muscle (A7r5) cells using the Ca (2+)-sensitive dye Fura-2 as an indicator. The increase of [Ca (2+)]i in A7r5 cells produced by vasopressin (1 micromol/L) or phenylephrine (1 micromol/L) was attenuated by isosteviol from 0.01 micromol/L to 10 micromol/L. The attenuation by isosteviol of the vasopressin- and phenylephrine-induced increase in [Ca (2+)]i was inhibited by glibenclamide, apamin and 4-aminopyridine but not by charybdotoxin. Furthermore, the inhibitory action of isosteviol on [Ca (2+)]i was blocked when A7r5 cells co-treated with glibenclamide and apamin in conjunction with 4-aminopyridine were present. Therefore, not only did the ATP-sensitive potassium (K (ATP)) channel affect the action of isosteviol on [Ca (2+)]i modulation in A7r5 cells, but also those on the small conductance calcium-activated potassium (SK (Ca)) channels and voltage-gated (Kv) channels. However, the blockers of large-conductance Ca (2+)-activated potassium channels failed to modify the inhibitory action of isosteviol on [Ca (2+)]i. The obtained results indicated that a decrease of [Ca (2+)]i in A7r5 cells by isosteviol is mainly mediated by the selective opening of K (ATP) channel or/and SK (Ca) channel. Alteration in the Kv channel also plays a critical role in the inhibitory action of isosteviol.

Antioxidant activity of Ganoderma lucidum in acute ethanol-induced heart toxicity.

The hot water extract of the mushroom Ganoderma lucidum was shown to have antioxidative effect against heart toxicity. Investigations into the mechanisms of action, level of lipid peroxidation level in vivo, and superoxide scavenging activity were also conducted. The mice were divided into six groups with ten animals in each group. Ganoderma lucidum, at doses of 10, 25 and 50 mg/kg (p.o.) was administered. Superoxide anions were assayed by UV spectrophotometer using the cytochrome C reduction method. The results of this study showed that Ganoderma lucidum exhibited a dose-dependent antioxidative effect on lipid peroxidation and superoxide scavenging activity in mouse heart homogenate. Additionally, this result indicated that heart damage induced by ethanol shows a higher malonic dialdehyde level compared with heart homogenate treated with Ganoderma lucidum. It is concluded that the antioxidative activity may therefore contribute to the cardioprotective effect of Ganoderma lucidum, and may therefore protect the heart from superoxide induced damage.

Protection by hot water extract of Panax notoginseng on chronic ethanol-induced hepatotoxicity.

The purpose of this study was to investigate the antioxidant effects of a hot water extract of Panax notoginseng (PNG) against chronic ethanol-induced hepatotoxicity. Fifty mice were divided into fi ve equal groups with 10 in each group. Group 1 (control) received saline, whereas group 2 received ethanol (70%, 0.1 mL, p.o.) once daily for 4 weeks, which induced hepatotoxicity, manifested biochemically by a significant elevation of serum enzyme activities, such as SGOT and SGPT. Hepatotoxicity was further evidenced by a significant increase in the hepatic lipid peroxidation measured. Groups 3-5 were administered a hot water extract of PNG at doses of 10, 25 and 50 mg/kg 2 weeks after initiating oral administration of ethanol, for a further 2 weeks. PNG ameliorated the rise in serum sGOT and sGPT induced by chronic ethanol administration. The mice were killed after PNG administration. In a separate study, PNG inhibited the lipid peroxidation in the mouse liver homogenate induced by ethanol in a dose-dependent manner. The findings indicate that PNG is an efficient cytoprotective agent against chronic ethanol-induced hepatotoxicity, possibly through inhibition of the production of oxygen-free radicals that cause lipid peroxidation.

Tetramethylpyrazine as potassium channel opener to lower calcium influx into cultured aortic smooth muscle cells.

In the present study, the effect of tetramethylpyrazine (TMP) on calcium (Ca 2+) influx was investigated in cultured vascular smooth muscle (A7r5) cells using Fura-2 as an indicator. The increase of Ca 2+ concentration in A7r5 cells produced by vasopressin or phenylephrine was attenuated by TMP from 0.01 micromol/L to 1 mmol/L. The decrease in the intracellular potassium concentration in A7r5 cells by TMP from 0.01 micromol/L to 10 micromol/L was characterized using PBFI/AM. Inhibitors specific to the small conductance calcium-activated potassium (SKCa ) channel or the ATP-sensitive potassium (K ATP ) channel abolished the actions of TMP. The obtained results indicate that the decrease of Ca 2+ influx into A7r5 cells by TMP is mainly mediated by the opening of potassium channels.