European Journal of Pharmacology (v.641, #1)
Editorial Board (ii).
Inhibitory effects of baicalin on IL-1β- induced MMP-1/TIMP-1 and its stimulated effect on Collagen-I production in human periodontal ligament cells by Zhengguo Cao; Chengzhang Li; Guangxun Zhu (1-6).
Our previous research has proved that baicalin can inhibit the expression of Matrix metalloproteinases (MMPs) in periodontal ligament cells (PDLC) by cell immunocytochemistry. Therefore, the purpose of this study was to address the effects of baicalin on the total protein amount and Collagen I mRNA expression in PDLC, and the regulatory effects on Matrix metalloproteinase-1/ tissue inhibitors of metalloproteinase-1( MMP-1/ TIMP-1 ) expression. PDLC were incubated with 0–1000 ng/ml baicalin for 1, 3 and 5 days. Coomasie brilliant blue staining was used to detect the synthesis of the total protein, and the collagen I mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). PDLC were treated with phorbol 12-myristate 13-acetate (PMA) or interleukin-1β (IL-1β) with or without 100 ng/ml baicalin and then mRNA levels for MMP-1 and TIMP-1 were detected. Enzyme linked immunosorbent assay (ELISA) was used to assess the MMP-1 protein. The range of 1–1000 ng/ml baicalin can enhance the amount of the total protein of PDL cells and the response had a dose-dependent manner in the range of 1–100 ng/ml baicalin. And 0–1000 ng/ml baicalin also significantly increased the Collagen I mRNA expression of PDLC. 1–100 pmol/ml PMA and 0.01–1 ng/ml IL-1βsignificantly (p < 0.05) stimulated the production of MMP-1 by PDLC at both the transcriptional and the translational level. Different concentration PMA enhanced TIMP-1 mRNA expression, but IL-1βdid not affect the TIMP-1 mRNA expression. Moreover, in the presence of 100 ng/ml baicalin, both the MMP-1 and TIMP-1 mRNA expression were down regulated. The present study suggests that baicalin inhibits IL-1βinduction of MMP-1 by altering the mRNA and protein levels. In addition, baicalin may increase Collagen I mRNA and total protein levels in PDLC.
Keywords: Baicalin; Periodontal ligament cell; MMP-1; TIMP-1; IL-1β;
Extract of Cassiae Semen and its major compound inhibit S100b-induced TGF-β1 and fibronectin expression in mouse glomerular mesangial cells by Dong Ho Jung; Young Sook Kim; Nan Hee Kim; Jun Lee; Dae Sik Jang; Jin Sook Kim (7-14).
Non-enzymatic glycation reactions between reducing sugar and free reactive amino groups of protein lead to the formation of advanced glycation end products, which increase under conditions of aging or diabetes. A previous study showed that extracts of Cassiae Semen (CS), the seed of Cassia tora, had inhibitory activity on advanced glycation end products formation in vitro. To examine the pharmacological effects of a butanol-soluble extract of CS under conditions of diabetic nephropathy, we evaluated the expression of transforming growth factor-β1 (TGF-β1) and fibronectin, key mediators of diabetic nephropathy, in mouse glomerular mesangial cells cultured in the presence of S100b (a specific ligand for receptor of advanced glycation end products). CS inhibited S100b-induced TGF-β1 and fibronectin expression in mouse mesangial cells by suppressing activation of Smad2/3, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), and oxidative stress. Moreover, CS suppressed nuclear factor-kappa B (NF-κB) activation in S100b-stimulated mouse mesangial cells. To identify the active compounds of CS, three major compounds, rubrofusarin-6-O-β-d-gentiobioside (CS-A), toralactone-9-O-β-d-gentiobioside (CS-B), and cassiaside (CS-C), were tested in cells. Of these compounds, CS-A significantly decreased the expression of TGF-β1 and fibronectin and NF-κB DNA binding activity. These findings suggest that CS, especially CS-A, has potential as a preventive agent for advanced glycation end products-related diabetic complications.
Keywords: Advanced glycation end products; Cassiae Semen; Diabetic nephropathy; Fibronectin; Transforming growth factor-beta (TGF-β1); Mouse mesangial cells;
Cucurbitacin B, a small molecule inhibitor of the Stat3 signaling pathway, enhances the chemosensitivity of laryngeal squamous cell carcinoma cells to cisplatin by Tingyan Liu; Huaguang Peng; Meixia Zhang; Yihui Deng; Zhenghu Wu (15-22).
We have previously shown that the simultaneous exposure of Hep-2 cells to cucurbitacin B and docetaxel significantly enhances anticancer activity of these cells by suppressing Stat3 activation and down-regulating the expression levels of key cell cycle and anti-apoptosis regulators. In order to determine whether cucurbitacin B can also enhance the sensitivity of Hep-2 laryngeal cells to cisplatin, we treated Hep-2 cells with either cucurbitacin B, cisplatin, or the combination and evaluated these cells for proliferation, cell cycle distribution, and apoptosis. Our results demonstrate that, in comparison to single agent cucurbitacin B or cisplatin treated cells, Hep-2 cells treated with a cucurbitacin B/cisplatin combination display synergistic effects on growth inhibition, cell cycle arrest, and apoptosis induction. Western blot analysis using protein extracts from Hep-2 cells treated with cucurbitacin B, cisplatin, or the combination largely recapitulated the observations made when treated with the cucurbitacin B/docetaxel combination. More specifically, Hep-2 cell lines treated with the cucurbitacin B/cisplatin combination demonstrated a significantly reduced level of p-Stat3 in comparison with single agent treated cells. In addition, cucurbitacin B/cisplatin treated Hep-2 cells also demonstrated a significant reduction in Bcl-2 and Cyclin B1 protein levels compared to single agent cucurbitacin B or cisplatin treated cells. Xenograft models containing Hep-2 cells in mice also demonstrated that this cucurbitacin B/cisplatin combination led to the synergistic inhibition of tumor growth. Taken together, these results suggest that the cucurbitacin B/cisplatin combination treatment may be a potentially useful therapeutic option for individuals diagnosed with laryngeal cancer.
Keywords: Cucurbitacin B; Cisplatin; Hep-2; Cell cycle; Apoptosis;
The sigma agonist 1,3-di-o-tolyl-guanidine directly blocks SK channels in dopaminergic neurons and in cell lines by Cédric Lamy; Jacqueline Scuvée-Moreau; Sébastien Dilly; Jean-François Liégeois; Vincent Seutin (23-28).
Small conductance Ca2+-activated K+ (SK) channels are widely expressed in the brain and underlie medium-duration afterhyperpolarizations (mAHPs) in many types of neurons. It was recently reported that the activation of sigma-1 (σ1) receptors inhibits SK currents in rat hippocampus. Because many interactions between σ receptors and brain dopaminergic systems have been reported, we set out to examine putative effects of σ receptor ligands on the SK mediated mAHP in midbrain dopaminergic neurons. We found that 1,3-di-o-tolyl-guanidine (DTG) inhibited the mAHP in a concentration-dependent manner (∼ 60% inhibition at 100 µM), while other σ receptor agonists (carbetapentane, (+)-SKF10047 and PRE-084) had little effect. Moreover, the effect of DTG was not affected by high concentrations of the σ1 receptor antagonist BD 1047. A role for σ2 receptors could also be excluded by the lack of effect of the σ2 receptor ligand 5-bromo-tetrahydroisoquinolinylbenzamide. These results argue against a coupling of σ receptors to SK channels in dopaminergic neurons. We next hypothesized that DTG could directly block the channel. This hypothesis was tested in HEK-293 cells which were transiently transfected with rSK2 or hSK3 subunits. DTG inhibited the current flowing through both subtypes with mean IC50s ∼ 200 µM. This action was also unaffected by BD 1047. Other σ receptor ligands had little or no effect. We conclude that DTG directly blocks SK channels. This pharmacological action may be important to consider in future experimental settings.
Keywords: SK channel; Sigma receptor; DTG; Dopaminergic neuron;
The natural antioxidant alpha-lipoic acid induces p27Kip1-dependent cell cycle arrest and apoptosis in MCF-7 human breast cancer cells by Elena Dozio; Massimiliano Ruscica; Luca Passafaro; Giada Dogliotti; Liliana Steffani; Alessandra Pagani; Germana Demartini; Daniele Esposti; Franco Fraschini; Paolo Magni (29-34).
Unlike normal cells, tumor cells survive in a specific redox environment where the elevated reactive oxygen species contribute to enhance cell proliferation and to suppress apoptosis. Alpha-lipoic acid, a naturally occurring reactive oxygen species scavenger, has been shown to possess anticancer activity, due to its ability to suppress proliferation and to induce apoptosis in different cancer cell lines. Since at the moment little information is available regarding the potential effects of alpha-lipoic acid on breast cancer, in the present study we addressed the question whether alpha-lipoic acid induces cell cycle arrest and apoptosis in the human breast cancer cell line MCF-7. Moreover, we investigated some molecular mechanisms which mediate alpha-lipoic acid actions, focusing on the role of the PI3-K/Akt signalling pathway. We observed that alpha-lipoic acid is able to scavenge reactive oxygen species in MCF-7 cells and that the reduction of reactive oxygen species is followed by cell growth arrest in the G1 phase of the cell cycle, via the specific inhibition of Akt pathway and the up-regulation of the cyclin-dependent kinase inhibitor p27kip1, and by apoptosis, via changes of the ratio of the apoptotic-related protein Bax/Bcl-2. Thus, the anti-tumor activity of alpha-lipoic acid observed in MCF-7 cells further stresses the role of redox state in regulating cancer initiation and progression.
Keywords: Alpha-lipoic acid; MCF-7 cells; p27Kip1; Akt; Reactive oxygen species;
Pamidronate inhibits antiapoptotic bcl-2 expression through inhibition of the mevalonate pathway in prostate cancer PC-3 cells by Kazuhiro Iguchi; Yoshiki Tatsuda; Shigeyuki Usui; Kazuyuki Hirano (35-40).
Bisphosphonates are expected to be efficacious to prevent the growth of metastatic cancer in bone tissue. Bone metastases often occur in patients with various cancers, such as breast, lung and prostate cancer. Bcl-2 is a potent antiapoptotic protein and its expression is known to be closely related to its function. In this study, to investigate the effect of bisphosphonates on cancer cells, we focused on bcl-2 expression in bisphosphonate-treated prostate cancer cells. First, we observed that bcl-2 mRNA expression in PC-3 was significantly inhibited to 12% of the control level by treatment with 100 μM pamidronate for 12 h. Inhibition was seen in cells treated with nitrogen-containing bisphosphonates, which have the ability to inhibit isoprenoid biosynthesis via the mevalonate pathway, but not in non-nitrogen-containing etidronate. Simultaneous treatment with geranylgeraniol, an intermediate of the mevalonate pathway, significantly blocked inhibition by pamidronate, and treatment with geranylgeranyl transferase inhibitor GGTI-286 also suppressed bcl-2 mRNA expression. Furthermore, pamidronate inhibited the translocation of Rap1 protein to the membrane fraction, suggesting that a change in posttranslational modification of Rap1 occurred in treated cells. Finally, knockdown of Rap1 by siRNA resulted in the inhibition of bcl-2 expression. These results strongly indicate that bcl-2 reduction in bisphosphonate-treated PC-3 cells is dependent on inhibition of the mevalonate pathway. The inhibitory effect of bisphosphonates on bcl-2 expression shown in prostate cancer cell line should be tested in animal experiments and clinical studies.
Keywords: Bcl-2; Pamidronate; Bisphosphonate; Prostate cancer; Mevalonate pathway; Rap1;
α1-Adrenoceptors in the rat cerebral cortex: New insights into the characterization of α1L- and α1D-adrenoceptors by Vanessa Segura; Nicla Flacco; Eduardo Oliver; Domingo Barettino; Pilar D'Ocon; Maria Dolores Ivorra (41-48).
Among the three α1-adrenoceptor subtypes (α1A, α1B and α1D) a peculiar intracellular localization and poor coupling to membrane signals of cloned α1D-adrenoceptor have been reported. In addition, the α1L-adrenoceptor (low affinity for prazosin), a functional phenotype of α1A, has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of α1-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (α1D> α1B ≫ α1A). α1-Adrenoceptors were also detected by immunoblotting, revealing α1A- and α1B-adrenoceptors to be predominantly expressed in the membrane fraction and the α1D-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of α1D-adrenoceptor in tissue homogenates, whereas only α1A- and α1B-subtypes were detected in membranes. The proportion of α1A-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (α1A/L)-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The α1A/L-adrenoceptor was heavily implicated in the inositol phosphate response, while the α1D-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of α1D-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the α1L-adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the α1A-adrenoceptor coupled to G-protein.
Keywords: α 1-Adrenoceptor subtype; Rat cerebral cortex; α1D-Intracellular localization; α1L-Adenoceptor; [3H]prazosin binding study; G-protein;
Effect of acute topical application of (+)-pentazocine on the mechanical allodynia in diabetic mice by Masahiro Ohsawa; Shun-suke Hayashi; Junzo Kamei (49-53).
Mechanical allodynia is a major complication in diabetic mellitus. Peripheral σ1 receptors were shown to be involved in nociceptive perception. We therefore investigated the effect of σ1 receptor ligand (+)-pentazocine injected into the dorsal surface of the hindpaw on mechanical allodynia in streptozotocin-induced diabetic mice. Injection of (+)-pentazocine (30 μg) into the dorsal surface of the hindpaw did not affect the mechanical threshold and hindpaw NO contents in non-diabetic mice, whereas the mechanical allodynia and hindpaw contents of NO metabolites in diabetic mice were normalized by (+)-pentazocine. These effects of (+)-pentazocine in diabetic mice were inhibited by pretreatment with an σ receptor antagonist BD1047 into the same area, but not by systemic pretreatment with a κ-opioid receptor antagonist nor-binaltorphimine. These results suggest that (+)-pentazocine injected into the dorsal surface of the hindpaw increases the lowered mechanical threshold in diabetic mice through the activation of peripheral σ1 receptors. This attenuation may be, in part, due to the normalization of increased peripheral NO contents in the hindpaw of diabetic mice.
Keywords: Anti-allodynia; Nitric oxide (NO); Sigma receptor; Sensory nerve; (Diabetic mice);
Possible inhibitory roles of endogenous 2-arachidonoylglycerol during corticotropin-releasing factor-induced activation of central sympatho-adrenomedullary outflow in anesthetized rats by Takahiro Shimizu; Lianyi Lu; Kunihiko Yokotani (54-60).
We previously reported that intracerebroventricularly (i.c.v.) administered corticotropin-releasing factor (CRF) (0.5–3.0 nmol/animal) dose-dependently elevates plasma noradrenaline and adrenaline through brain phospholipase C-, diacylglycerol lipase- and prostanoids-mediated mechanisms in rats. Diacylglycerol produced by phospholipase C from phospholipids can be hydrolyzed by diacylglycerol lipase into 2-arachidonoylglycerol, which may be further hydrolyzed by monoacylglycerol lipase into arachidonic acid, a precursor of prostanoids. Recently, 2-arachidonoylglycerol has been recognized as a major brain endocannabinoid, which can modulate synaptic transmission through presynaptic cannabinoid CB1 receptors. Released 2-arachidonoylglycerol is rapidly deactivated by uptake into cells and enzymatic hydrolysis. In the present study, therefore, we examined (1) the involvement of brain 2-arachidonoylglycerol, (2) the regulatory role of 2-arachidonoylglycerol as a brain endocannabinoid, and (3) the effect of exogenous cannabinoid receptor agonist, on the CRF-induced elevation of plasma noradrenaline and adrenaline using anesthetized rats. The elevation of both catecholamines induced by a submaximal dose of CRF (1.5 nmol/animal, i.c.v.) was reduced by i.c.v. administered MAFP (monoacylglycerol lipase inhibitor) (0.7 and 1.4 µmol/animal), AM 404 (endocannabinoid uptake-inhibitor) (80 and 250 nmol/animal) and ACEA (cannabinoid CB1 receptor agonist) (0.7 and 1.4 µmol/animal), while AM 251 (cannabinoid CB1 receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the response induced by a small dose of CRF (0.5 nmol/animal, i.c.v.). These results suggest a possibility that 2-arachidonoylglycerol is endogenously generated in the brain during CRF-induced activation of central sympatho-adrenomedullary outflow, thereby inhibiting the peptide-induced response by activation of brain cannabinoid CB1 receptors in anesthetized rats.
Keywords: Corticotropin-releasing factor; Sympatho-adrenomedullary outflow; Brain; 2-Arachidonoylglycerol; Monoacylglycerol lipase; Cannabinoid CB1 receptor;
Regulation of NO-dependent acetylcholine relaxation by K+ channels and the Na+–K+ ATPase pump in porcine internal mammary artery by Rosa María Pagán; Dolores Prieto; Medardo Hernández; Carlos Correa; Albino García-Sacristán; Sara Benedito; Ana Cristina Martínez (61-66).
This study was designed to determine whether K+ channels play a role in nitric oxide (NO)-dependent acetylcholine relaxation in porcine internal mammary artery (IMA). IMA segments were isolated and mounted in organ baths to record isometric tension. Acetylcholine-elicited vasodilation was abolished by muscarinic receptor blockade with atropine (10-6 M). Incubation with indomethacin (3 × 10− 6 M), superoxide dismutase (150 U/ml) and bosentan (10− 5 M) did not modify the acetylcholine response ruling out the participation of cyclooxygenase-derivates, reactive oxygen species or endothelin. The relaxation response to acetylcholine was strongly diminished by NO synthase- or soluble guanylyl cyclase-inhibition using l-NOArg (10− 4 M) or ODQ (3 × 10− 6 M), respectively. The vasodilation induced by acetylcholine and a NO donor (NaNO2) was reduced when rings were contracted with an enriched K+ solution (30 mM), by voltage-dependent K+ (Kv) channel blockade with 4-amynopiridine (4-AP; 10− 4 M), by Ca2+-activated K+ (KCa) channel blockade with tetraethylammonium (TEA; 10− 3 M), and by apamin (5 × 10− 7 M) plus charybdotoxin (ChTx; 10− 7 M) but not when these were added alone. In contrast, large conductance KCa (BKCa), ATP-sensitive K+ (KATP) and inwardly rectifying K+ (Kir) channel blockade with iberiotoxin (IbTx; 10− 7 M), glibenclamide (10− 6 M) and BaCl2 (3 × 10− 5 M), respectively, did not alter the concentration–response curves to acetylcholine and NaNO2. Na+−K+ ATPase pump inhibition with ouabain (10− 5 M) practically abolished acetylcholine and NaNO2 relaxations. Our findings suggest that acetylcholine-induced relaxation is largely mediated through the NO-cGMP pathway, involving apamin plus ChTx-sensitive K+ and Kv channels, and Na+−K+-ATPase pump activation.
Keywords: Porcine internal mammary artery; Acetylcholine relaxation; Endothelium; Nitric oxide; K+ channel; Na-K+ ATPase pump;
Beneficial effects of magnolol in a rodent model of endotoxin shock by Yung-Chieh Tsai; Pao-Yun Cheng; Ching-Wen Kung; Yi-Jen Peng; Tzu-Hsuan Ke; Jhi-Joung Wang; Mao-Hsiung Yen (67-73).
Magnolol is a compound extracted from the Chinese medicinal herb Magnolia officinalis. It has multiple pharmacological effects, notably as an anti-oxidant. The aim of this study was to evaluate the effects of magnolol on sepsis induced by intravenous (i.v.) administration of lipopolysaccharide (LPS; 10 mg/kg) in anaesthetized Wistar rats. Magnolol (4 μg/kg, i.v.) was administered at 30 min after LPS injection. Post-treatment with magnolol significantly attenuated the deleterious haemodynamic changes (e.g., hypotension and bradycardia) caused by LPS. Meanwhile, magnolol significantly inhibited the elevation of plasma levels of tumor necrosis factor alpha, glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and blood urine nitrogen caused by LPS. The induction of inducible nitrous oxide (NO) synthase and the overproduction of NO and superoxide anions by LPS were also significantly reduced by post-treatment with magnolol. Moreover, the plasma level of the thrombin–antithrombin complex following administration of LPS was also reduced by post-treatment with magnolol. Thus, the beneficial effects of magnolol on LPS-induced sepsis result from its anti-inflammatory, anti-coagulatory, and anti-oxidant effects.
Keywords: Magnolol; Reactive oxygen species; Sepsis; TNF-α; Nitric oxide; Circulatory failure;