p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.     

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS(-/-) mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor N(G)-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca(2+) levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca(2+) levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol- treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca(2+) chelator, calmodulin antagonist, and CaMKKβ siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca(2+)-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca(2+)/CaMKKβ-dependent eNOS phosphorylation and Ca(2+)-dependent eNOS dimerization.     

Lysophosphatidylglycerol inhibits formyl peptide receptor like-1-stimulated chemotactic migration and IL-1�� production from human phagocytes

In this study, we observed that lysophosphatidylglycerol (LPG) completely inhibited a formyl peptide receptor like-1 (FPRL1) agonist (MMK-1)-stimulated chemotactic migration in human phagocytes, such as neutrophils and monocytes. LPG also dramatically inhibited IL-1β production by another FPRL1 agonist serum amyloid A (SAA) in human phagocytes. However, LPG itself induced intracellular calcium increase and superoxide anion production in human phagocytes. Keeping in mind that phagocytes migration and IL-1β production by FPRL1 are important for the induction of inflammatory response, our data suggest that LPG can be regarded as a useful material for the modulation of inflammatory response induced by FPRL1 activation.     

Prunus yedoensis Matsum. stimulates glucose uptake in L6 rat skeletal muscle cells by activating AMP-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways

Prunus yedoensis Matsum. is used as a medicinal plant to alleviate symptoms of diabetes; however, the molecular mechanism underlying its antihyperglycaemic activity is unknown. In this study, we investigated the antihyperglycaemic effects of P. yedoensis and its molecular mechanism. Prunus yedoensis leaf extract (PLE) increased the glucose uptake of phosphorylatinginsulin receptor substrate (IRS)-1, 3'-phosphoinositide-dependent kinase (PDK)-1 and Akt PLE, and also increased the phosphorylation of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK). PLE-stimulated glucose uptake was blocked by an AMPK inhibitor (Compound C) and a p38 MAPK inhibitor (SB203580). Inhibition of AMPK activity reduced p38 MAPK phosphorylation, whereas the inhibition of p38 MAPK activity did not affect AMPK phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 and Compound C reduced PLE-stimulated glucose uptake. Our results demonstrate that PLE stimulated glucose uptake by activating both insulin signalling and AMPK-p38 MAPK pathways. PLE shows potential as a natural antihyperglycaemic agent.     

Akt1-mediated intracellular oxidation after UVB irradiation suppresses apoptotic cell death induced by cell detachment and serum starvation

Apoptosis is an important cell death system that deletes damaged and mutated cells to prevent cancer. We have previously reported that a certain dose of UVB irradiation inhibited the apoptosis induced by serum starvation and cell detachment, leading to cell transformation. This antiapoptotic effect was partially inhibited by phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. UVB irradiation is known to cause the phosphorylation of Akt via the activation of PI3-kinase; however, the Akt isoform-specific relationship has not yet been clarified. Notably, the role in antiapoptotic effect of UVB has yet to be elucidated. In this study, the role of Akt1 in the UVB-induced inhibition of apoptosis was examined by Akt1 knockdown using small interfering RNA (siRNA). NIH3T3 cells showed typical apoptotic cell death by serum starvation and cell detachment, which was significantly inhibited by UVB irradiation. Akt1 knockdown decreased the antiapoptotic effect of UVB. Hydrogen peroxide-induced suppression of cell death was also decreased in Akt1 knockdown cells. An antioxidant, N-acetylcysteine, inhibited the antiapoptotic effect by UVB irradiation, whereas no inhibition was observed in Akt1 knockdown cells. Furthermore, UVB-induced intracellular peroxidation was not observed in the knockdown cells, indicating that Akt1 played an important role in mediating the intracellular redox status. Treatment with insulin had a similar antiapoptotic effect as UVB irradiation involving intracellular peroxidation, which was also attenuated in Akt1 knockdown cells. These findings suggest that appropriate intracellular oxidation after UVB irradiation prevented apoptosis, a process which might be partially regulated by the production of reactive oxygen species mediated by Akt1.     

Paradoxical activation of Raf by a novel Raf inhibitor.

BACKGROUND: Raf is a proto-oncogene that is activated in response to growth factors or phorbol esters, and is thought to activate MAP kinase kinase-1 (MKK1) and hence the classical MAP kinase (MAPK) cascade. RESULTS: The compound ZM 336372 is identified as a potent and specific inhibitor of Raf isoforms in vitro. Paradoxically, exposure of cells to ZM 336372 induces > 100-fold activation of c-Raf (measured in the absence of compound), but without triggering any activation of MKK1 or p42 MAPK/ERK2. The ZM 336372-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras and is not prevented by inhibition of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase. ZM 336372 does not prevent growth factor or phorbol ester induced activation of MKK1 or p42 MAPK/ERK2, or reverse the phenotype of Ras- or Raf-transformed cell lines. The only other protein kinase inhibited by ZM 336372 out of 20 tested was SAPK2/p38. Although ZM 336372 is structurally unrelated to SB 203580, a potent inhibitor of SAPK2/p38, the mutation of Thr106-->Met made SAPK2/p38 insensitive to ZM 336372 as well as to SB 203580. CONCLUSIONS: Raf appears to suppress its own activation by a novel feedback loop, such that inhibition is always counterbalanced by reactivation. These observations imply that some agonists reported to trigger the cellular activation of c-Raf might actually be inhibitors of this enzyme, and that compounds which inhibit the kinase activity of Raf might not be useful as anticancer drugs. The binding sites for ZM 336372 and SB 203580 on Raf and SAPK2/p38 are likely to overlap.     

Involvement of ��PIX in angiotensin II-induced migration of vascular smooth muscle cells

Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a βPIX GEF-dependent manner. βPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of βPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of βPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of βPIX-Rac1. Taken together, our results suggest that βPIX is involved in Ang II-induced VSMC migration.     

Structural variability in Ag(I) and Cu(I) coordination polymers with thioether-functionalized bis(pyrazolyl)methane ligands

We present here two ligand classes based on a bis(pyrazolyl)methane scaffold functionalized with a rigid (-Ph-S-Ph) or flexible (-CH(2)-S-Ph) thioether function: L(R)PhS (R = H, Me) and L(R)CH(2)S (R = H, Me, iPr). The X-ray molecular structures of Ag(I) and Cu(I) binary complexes with L(R)PhS or L(R)CH(2)S using different types of counterions (BF(4)(-), PF(6)(-), and CF(3)SO(3)(-)) are reported. In these complexes, the ligands are N(2) bound on a metal center and bridge on a second metal with the thioether group. In contrast, when using triphenylphosphine (PPh(3)) as an ancillary ligand, mononuclear ternary complexes [M(L)PPh(3)](+) (M = Cu(I), Ag(I); L = L(R)PhS, L(R)CH(2)S) are formed. In these complexes, the more flexible ligand type, L(R)CH(2)S, is able to provide the N(2)S chelation, whereas the more rigid L(R)PhS ligand class is capable of chelating only N(2) because the thioether function preorganized, as it did in the coordination polymers, to point away from the metal center. Rigid potential-energy surface scans were performed by means of density functional theory (DFT) calculations (B3LYP/6-31+G) on the two representative ligands, L(H)PhS and L(H)CH(2)S. The surface scans proved that the thioether function is preferably oriented on the opposite side of the bispyrazole N(2) chelate system. These results confirm that both ligand classes are suitable components for the construction of coordination polymers. Nevertheless, the methylene group that acts as a spacer in L(H)CH(2)S imparts an inherent flexibility to this ligand class so that the conformation responsible for the N(2)S chelation is energetically accessible.     

Characterization of intracellular calcium oscillations induced by extracellular nucleotides in HEp-2 cells

The effect of extracellular nucleotides on the cytosolic calcium concentration of fluo-3-loaded HEp-2 cells was examined using confocal microscopy. Extracellular ATP and UTP at micromolar concentration induced cytosolic calcium oscillations in 42-66% of the cells. Oscillations were usually sinusoid and their frequency depended only slightly on agonist concentration. Oscillations developed in calcium-free medium but were diminished by depletion of intracellular calcium stores with thapsigargin, indicating periodic calcium release from internal stores. Inhibition of phospholipase C with U73122 prevented the development of oscillations, while ryanodine did not abolish the response to extracellular nucleotides. Activation of protein kinase C with 4beta-phorbol 12-myristate 13-acetate also prevented the development of oscillations. These results indicate that extracellular nucleotides induce periodic calcium release from inositol 1,4,5-trisphosphate-sensitive pools in HEp-2 cells and that the inhibitory effect of protein kinase C on the phosphatidylinositol signaling pathway can contribute to the development of intracellular calcium oscillations.     

Identification of novel peptides that stimulate human neutrophils

Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we identified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified peptides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-dependent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leukocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimulated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide receptor (FPR)1 while the other peptide (MMHWAM) acted on FPR2. Since the three novel peptides were specific agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling.     

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.     

The effect of staurosporine on Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells

We examined the effect of staurosporine on cytosolic calcium response in rat basophilic leukemia (RBL-2H3) cells using fura-2 as a fluorescent indicator of calcium ion. Staurosporine at a dose of 30 nM inhibited antigen-stimulated Ca2(+)-influx into the cells from the extracellular environment. In contrast, the drug at this concentration inhibited neither the mobilization of Ca2+ from intracellular stores nor inositol 1,4,5-trisphosphate (IP3) formation. At a high concentration (300 nM), however, staurosporine completely inhibited the cytosolic calcium responses as well as IP3 formation. These results indicate that staurosporine, if used at an appropriate concentration, can be used to discriminate Ca2(+)-influx from extracellular environment from mobilization of the ion from intracellular stores. These results also suggest that protein kinases, possibly protein kinase C, are involved in the calcium influx of RBL-2H3 cells from the extracellular environment. Serotonin release was strongly inhibited by the drug at 30 nM staurosporine. Since the inhibition of serotonin release and suppression of cytosolic calcium increase in response to the antigen were in parallel, we concluded that the inhibition of serotonin release from RBL-2H3 cells caused by the drug was elicited by the suppression of Ca2(+)-influx into the cells.     

Theasaponin E1 Inhibits Platinum-Resistant Ovarian Cancer Cells through Activating Apoptosis and Suppressing Angiogenesis

Novel therapeutic strategies for ovarian cancer treatment are in critical need due to the chemoresistance and adverse side effects of platinum-based chemotherapy. Theasaponin E₁ (TSE1) is an oleanane-type saponin from Camellia sinensis seeds. Its apoptosis-inducing, cell cycle arresting and antiangiogenesis activities against platinum-resistant ovarian cancer cells were elucidated in vitro and using the chicken chorioallantoic membrane (CAM) assay. The results showed that TSE1 had more potent cell growth inhibitory effects on ovarian cancer OVCAR-3 and A2780/CP70 cells than cisplatin and was lower in cytotoxicity to normal ovarian IOSE-364 cells. TSE1 significantly induced OVCAR-3 cell apoptosis via the intrinsic and extrinsic apoptotic pathways, slightly arresting cell cycle at the G2/M phase, and obviously inhibited OVCAR-3 cell migration and angiogenesis with reducing the protein secretion and expression of vascular endothelial growth factor (VEGF). Western bolt assay showed that Serine/threonine Kinase (Akt) signaling related proteins including Ataxia telangiectasia mutated kinase (ATM), Phosphatase and tensin homolog (PTEN), Akt, Mammalian target of rapamycin (mTOR), Ribosome S6 protein kinase (p70S6K) and e IF4E-binding protein 1(4E-BP1) were regulated, and Hypoxia inducible factor-1α (HIF-1α) protein expression was decreased by TSE1 in OVCAR-3 cells. Moreover, TSE1 treatment potently downregulated protein expression of the Notch ligands including Delta-like protein 4 (Dll4) and Jagged1, and reduced the protein level of the intracellular domain (NICD) of Notch1. Combination treatment of TSE1 with the Notch1 signaling inhibitor tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate (DAPT), or the Akt signaling inhibitor wortmannin, showed a stronger inhibition toward HIF-1α activation compared with single compound treatment. Taken together, TSE1 might be a potential candidate compound for improving platinum-resistant ovarian cancer treatment via Dll4/Jagged1-Notch1-Akt-HIF-1α axis.     

Inhibitory effects of enalaprilat on rat cardiac fibroblast proliferation via ROS/P38MAPK/TGF-β1 signaling pathway

Enalaprilat (Ena.), an angiotensin II (Ang II) converting enzyme inhibitor (ACEI), can produce some therapeutic effects on hypertension, ventricular hypertrophy and myocardial remodeling in clinic, but its precise mechanism, especially its signaling pathways remain elusive. In this study, cardiac fibroblasts (CFb) was isolated by the trypsin digestion method; a BrdU proliferation assay was adopted to determine cell proliferation; an immunofluorescence assay was used to measure intracellular reactive oxygen species (ROS); immunocytochemistry staining and Western blotting assay were used to detect phosphorylated p38 mitogen activated protein kinase (p-p38MAPK) and transforming growth factor-β(1) (TGF-β(1)) protein expression, respectively. The results showed that Ang II (10(-7) M) stimulated the cardiac fibroblast proliferation which was inhibited by NAC (an antioxidant), SB203580 (a p38MAPK inhibitor) or enalaprilat; Ang II caused an burst of intracellular ROS level within thirty minutes, an increase in p-p38MAPK (3.6-fold of that in the control group), as well as an elevation of TGF-β(1) meantime; NAC, an antioxidant, and enalaprilat treatment attenuated cardiac fibroblast proliferation induced by Ang II and decreased ROS and p-p38MAPK protein levels in rat cardiac fibroblast; SB203580 lowered TGF-β(1) protein expression in rats' CFb in a dose-dependent manner. It could be concluded that enalaprilat can inhibit the cardiac fibroblast proliferation induced by Ang II via blocking ROS/P38MAPK/TGF-β(1) signaling pathways and the study provides a theoretical proof for the application of ACEIs in treating myocardial fibrosis and discovering the primary mechanism through which ACEIs inhibit CFb proliferation.     

Anti-migration effects of Gekko sulfated glycopeptide on human hepatoma SMMC-7721 cells

Gekko swinhonis Guenther has been used as an anti-cancer drug in traditional Chinese medicine for hundreds of years. Previous studies showed that the Gekko sulfated polysaccharide-protein complex suppressed the proliferation and migration of hepatoma cells. Gekko sulfated glycopeptide α was obtained from Gekko sulfated polysaccharide-protein complex using papain hydrolysis. Gekko sulfated glycopeptide α inhibited the proliferation and migration of SMMC-7721 cells. The secretion of IL-8 and the concentration of intracellular calcium were decreased after Gekko sulfated glycopeptide α exposure. SMMC-7721 cells in the control group showed abnormal features, with a polygonal shape, whereas this changed to a spindle shape after the treatment with Gekko sulfated glycopeptide α. Actin ?laments were distributed diffusely along the cell membrane in control cells, whereas those were polymerized and preferentially accumulated in the cytoplasm of treated cells. Microtubules distributed in the cytoplasm of untreated cells were located diffusely whereas those in treated cells were polymerized. Therefore, Gekko sulfated glycopeptide α inhibit the migration of hepatoma cells via reducing the secretion of IL-8 and the concentration of intracellular calcium, as well as regulating the reorganization of cytoskeleton.     

Synergistic induction of cancer cell migration regulated by Gβγ and phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is essential for both G protein-coupled receptor (GPCR)- and receptor tyrosine kinase (RTK)-mediated cancer cell migration. Here, we have shown that maximum migration is achieved by full activation of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) in the presence of Gβγ and PI3K signaling pathways. Lysophosphatidic acid (LPA)- induced migration was higher than that of epidermal growth factor (EGF)-induced migration; however, LPA-induced activation of Akt was lower than that stimulated by EGF. LPA-induced migration was partially blocked by either Gβγ or RTK inhibitor and completely blocked by both inhibitors. LPA-induced migration was synergistically increased in the presence of EGF and vice versa. In correlation with these results, sphingosine-1-phosphate (S1P)-induced migration was also synergistically induced in the presence of insulin-like growth factor-1 (IGF-1). Finally, silencing of P-Rex1 abolished the synergism in migration as well as in Rac activation. Moreover, synergistic activation of MMP-2 and cancer cell invasion was attenuated by silencing of P-Rex1. Given these results, we suggest that P-Rex1 requires both Gβγ and PI3K signaling pathways for synergistic activation of Rac, thereby inducing maximum cancer cell migration and invasion.     

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

Anti-inflammatory effect of myristicin on RAW 264.7 macrophages stimulated with polyinosinic-polycytidylic acid

Myristicin (1-allyl-5-methoxy-3,4-methylenedioxybenzene) is an active aromatic compound found in nutmeg (the seed of Myristica fragrans), carrot, basil,cinnamon, and parsley. Myristicin has been known to have anti-cholinergic, antibacterial,and hepatoprotective effects, however, the effects of myristicin on virus-stimulated macrophages are not fully reported. In this study, the anti-inflammatory effect of myristicin on double-stranded RNA (dsRNA)-stimulated macrophages was examined. Myristicin did not reduce the cell viability of RAW 264.7 mouse macrophages at concentrations of up to 50 μM. Myristicin significantly inhibited the production of calcium, nitric oxide (NO),interleukin (IL)-6, IL-10, interferon inducible protein-10, monocyte chemotactic protein(MCP)-1, MCP-3, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α, MIP-1β, and leukemia inhibitory factor in dsRNA[polyinosinic-polycytidylic acid]-induced RAW 264.7 cells (P < 0.05). In conclusion,myristicin has anti-inflammatory properties related with its inhibition of NO, cytokines,chemokines, and growth factors in dsRNA-stimulated macrophages via the calcium pathway.     

Harmine Hydrochloride Mediates the Induction of G2/M Cell Cycle Arrest in Breast Cancer Cells by Regulating the MAPKs and AKT/FOXO3a Signaling Pathways

Breast cancer (BC) is one of the most common causes of death among women worldwide. Recently, interest in novel approaches for BC has increased by developing new drugs derived from natural products with reduced side effects. This study aimed to treat BC cells with harmine hydrochloride (HMH) to identify its anticancer effects and mechanisms. HMH treatment suppressed cell growth, migration, invasion, and colony formation in MCF-7 and MDA-MB-231 cells, regardless of the hormone signaling. It also reduced the phosphorylation of PI3K, AKT, and mTOR and increased FOXO3a expression. Additionally, HMH treatment increased p38 phosphorylation in MCF-7 cells and activated c-Jun N-terminal kinase (JNK) phosphorylation in MDA-MB-231 cells in a dose-dependent manner, where activated p38 and JNK increased FOXO3a expression. Activated FOXO3a increased the expression of p53, p21, and their downstream proteins, including p-cdc25, p-cdc2, and cyclin B1, to induce G2/M cell cycle arrest. Furthermore, HMH inhibited the PI3K/AKT/mTOR pathway by significantly reducing p-AKT expression in combination with LY294002, an AKT inhibitor. These results indicate that mitogen-activated protein kinases (MAPKs) and AKT/FOXO3a signaling pathways mediate the induction of cell cycle arrest following HMH treatment. Therefore, HMH could be a potential active compound for anticancer bioactivity in BC cells.