BACKGROUND AND OBJECTIVEInvasion and metastasis are the primary causes of death in patients with pulmonary carcinoma. The epidermal growth factor (EGF) stimulates A549 cells invasion greatly through activating ERK and PI3K-Akt signaling pathway. The aim of this study is to elucidate the inhibitory effect of Resveratrol on EGF-induced invasive ability of A549 cells in vitro and explore the molecular mechanism.

METHODSThe cytotoxicity of Resveratrol was evaluated by methyl thiazolyltetrazolium (MTT) assay. Then, the A549 cells were treated with EGF and non-cytotoxic concentration of Resveratrol. The cells' invasion were detected by Boyden chamber assay; MMP-2 activity was determined by gelatine zymography assay; the changes of the related proteins were detected by Western blot.

RESULTSResveratrol was not toxic to A549 cells at the concentration between 0 to 30 microM. The invasion ability of EGF-induced A549 cells was decreased after treatment with 20 microM resveratrol for 24 h, accompanied by the inhibition of MMP-2 secretion. And the levels of p-ERK1/2, PI3K (within 6 h) were suppressed too.

CONCLUSION20 microM Resveratrol inhibits A549 cells' invasion possibly through the suppression of the activation of ERK and PI3K-Akt signaling pathways, subsequently exerting inhibitory effect on MMP-2.

Adenocarcinoma ; metabolism ; Anticarcinogenic Agents ; pharmacology ; Blotting, Western ; Cell Line, Tumor ; Epidermal Growth Factor ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; drug effects ; Stilbenes ; pharmacology

Phosphatidylinositol (PI) 3-kinase plays an important role in transducing the signals of various growth factor receptors. However, the regulatory mechanism of PI3-kinase activity by these growth factor receptors is not completely understood. Therefore, we attempted to clarify the regulatory mechanism of PI3-kinase using insulin and 3T3 L1 fibroblasts. Our results showed that insulin stimulated PI3-kinase activity seven-fold and concomitantly phosphorylated a p85 subunit at the tyrosine residue. However, this tyrosine phosphorylation was not significant in the activation of PI3-kinase as the PI3-kinase pulled down by the overexpressed GST-p85 fusion protein showed as high an activity as the immunoprecipitated one. The p110 subunit was phosphorylated at both serine and tyrosine residues without insulin treatment. Since the phosphorylation state was not changed by insulin. The results suggested that phosphorylation of the p110 subunit does not control PI3-kinase activity. Finally, it was shown that the insulin receptor substrate-1 (IRS-1) binding to PI3-kinase was not sufficient for full activation because the amount of IRS-1 pulled down by the GST-p85 fusion protein reached almost maximum, after incubation with insulin-treated cell lysates for 20 min, whereas PI3-kinase activity reached its maximum only after incubation for 5 h. All results suggest that the phosphorylation of p85 subunit at tyrosine residues and phosphorylation of p110 subunit at tyrosine or serine residues are not functionally significant in the regulation of PI3-kinase activity. They also suggest that P13-kinase is needed to bind to other protein(s) as well as the insulin receptor substrate-1 for full activation.
1-Phosphatidylinositol 3-Kinase/metabolism* ; 3T3 Cells ; Animal ; Enzyme Activation ; Fibroblasts/metabolism* ; Human ; Immunoblotting ; Insulin/pharmacology* ; Mice ; Phosphoproteins/metabolism ; Phosphorylation ; Receptor, Insulin/metabolism ; Time Factors

OBJECTIVE: To elucidate the underlying mechanism of Panax notoginseng saponin (PNS) on gastric epithelial cell injury and barrier dysfunction induced by dual antiplatelet (DA). METHODS: Human gastric mucosal epithelial cell (GES-1) was cultured and divided into 4 groups: a control, a DA, a PNS+DA and a LY294002+PNS+DA group. GES-1 apoptosis was detected by flow cytometry, cell permeability were detected using Transwell, level of prostaglandins E2 (PGE2), 6-keto-prostaglandin F1α (6-keto-PGF1α) and vascular endothelial growth factor (VEGF) in supernatant were measured by enzyme linked immunosorbent assay (ELISA), expression of phosphatidylinositide 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), Akt, phosphorylated-Akt (p-Akt), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), glycogen synthase kinase-3β (GSK-3β) and Ras homolog gene family member A (RhoA) were measured by Western-blot. RESULTS: DA induced apoptosis and hyper-permeability in GES-1, reduced supernatant level of PGE2, 6-keto-PGF1α and VEGF (P<0.05). Addition of PNS reduced the apoptosis of GES-1 caused by DA, restored the concentration of PGE2, 6-keto-PGF1α and VEGF (P<0.05). In addition, PNS attenuated the alteration of COX-1 and COX-2 expression induced by DA, up-regulated p-PI3K/p-Akt, down-regulated RhoA and GSK-3β. LY294002 mitigated the effects of PNS on cell apoptosis, cell permeability, VEGF concentration, and expression of RhoA and GSK-3β significantly. CONCLUSIONS PNS attenuates the suppression on COX/PG pathway from DA, alleviates DA-induced GES-1 apoptosis and barrier dysfunction through PI3K/Akt/ VEGF-GSK-3β-RhoA network pathway.
Cyclooxygenase 1 ; Epithelial Cells/metabolism* ; Glycogen Synthase Kinase 3 beta ; Humans ; Panax notoginseng ; Phosphatidylinositol 3-Kinases/metabolism* ; Platelet Aggregation Inhibitors ; Proto-Oncogene Proteins c-akt/metabolism* ; Saponins/pharmacology* ; Vascular Endothelial Growth Factor A ; rhoA GTP-Binding Protein

PURPOSE: Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. MATERIALS AND METHODS: Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. RESULTS: We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. CONCLUSION: Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.
Animals ; Caspase 3/metabolism ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Flavonoids/*pharmacology ; Glucose/*metabolism/pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 1 ; Male ; Mitogen-Activated Protein Kinase 3 ; Muscle, Smooth, Vascular/cytology/*drug effects/enzymology ; Myocytes, Smooth Muscle/metabolism ; NF-kappa B/metabolism ; Phosphatidylinositol 3-Kinases ; Protein Kinase Inhibitors/*pharmacology ; Rats ; Rats, Inbred OLETF ; Rats, Long-Evans ; Reactive Oxygen Species/metabolism ; Rutin/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

Interleukin 1 beta (IL-1 beta), a proinflammatory cytokine, is related with inflammatory diseases and it up-regulates MUC2 gene expression and mucin secretion. This study was designed to investigate the signal transduction pathway of the IL-1 beta-mediated MUC2 gene expression and mucin secretion in human airway epithelial cells. In cultured human airway NCI-H292 epithelial cells, the steady state of the mRNA level of MUC2 gene expression and mucin secretion induced by IL-1 were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunoblot analysis. To observe the signal pathway of the IL-1 beta-mediated MUC2 gene expression and mucin secretion, we used several specific inhibitors. PD98059 (MEK/ERK inhibitor) suppressed IL-1 beta-mediated MUC2 gene expression and mucin secretion, while SB203580 (p38 inhibitor) did not. Ro31-8220 (PKC inhibitor) inhibited IL-1 beta-mediated MUC2 gene expression and mucin secretion. It inhibited ERK phosphorylation, but did not inhibit p38 phosphorylation. LY294002 (PI3K inhibitor) also suppressed MUC2 expression, but did not inhibit any MAPKs phosphorylation. These results suggest that the IL-1 -mediated MUC2 gene expression and mucin secretion in NCI-H292 cells are regulated through activation of the PKC-MEK/ERK pathway, and that PI3K is also involved in the IL-1 beta-mediated MUC2 gene expression and mucin secretion.
1-Phosphatidylinositol 3-Kinase/*metabolism ; Cell Line ; Chromones/pharmacology ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Inhibitors/pharmacology ; Epithelium/*enzymology ; Flavonoids/pharmacology ; Humans ; Imidazoles/pharmacology ; Immunoassay ; Immunoblotting ; Indoles/pharmacology ; Interleukin-1/metabolism/*physiology ; Lung/cytology/*metabolism ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase Kinases/*metabolism ; Morpholines/pharmacology ; Mucin-2 ; Mucins/*biosynthesis/metabolism ; Phosphorylation ; Protein Kinase C/*metabolism ; Protein Structure, Tertiary ; Pyridines/pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Time Factors

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2high/HER3 and the HER2low/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin-beta1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral-MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.
Breast Neoplasms/enzymology/*genetics/*metabolism ; Butadienes/pharmacology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Enzyme Inhibitors/pharmacology ; Female ; Gene Expression ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; MAP Kinase Signaling System ; MCF-7 Cells ; Matrix Metalloproteinase 1/*genetics/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Neuregulin-1/*pharmacology ; Nitriles/pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Protein Kinase Inhibitors/pharmacology ; Protein Multimerization ; Proto-Oncogene Proteins c-akt/metabolism ; Quinazolines/pharmacology ; Receptor, erbB-2/genetics/*metabolism ; Receptor, erbB-3/*metabolism

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.
1-Phosphatidylinositol 3-Kinase/metabolism ; Androstadienes/pharmacology ; Calcium/metabolism ; Cell Line ; Chemokines, CXC/*pharmacology ; Chemotaxis, Leukocyte/drug effects/*physiology ; Dose-Response Relationship, Drug ; Genistein/pharmacology ; Human ; Jurkat Cells ; Oligopeptides ; Peptide Fragments/chemical synthesis/metabolism/*physiology ; Pertussis Toxin ; Phospholipase C/metabolism ; Protein-Tyrosine Kinase/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes/*drug effects ; Virulence Factors, Bordetella/pharmacology

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.
1-Phosphatidylinositol 3-Kinase/metabolism ; Androstadienes/pharmacology ; Calcium/metabolism ; Cell Line ; Chemokines, CXC/*pharmacology ; Chemotaxis, Leukocyte/drug effects/*physiology ; Dose-Response Relationship, Drug ; Genistein/pharmacology ; Human ; Jurkat Cells ; Oligopeptides ; Peptide Fragments/chemical synthesis/metabolism/*physiology ; Pertussis Toxin ; Phospholipase C/metabolism ; Protein-Tyrosine Kinase/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes/*drug effects ; Virulence Factors, Bordetella/pharmacology

Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with betaVEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to betaVEGF(165) , eNOS activity and cell growth were increased by approximately two-fold in the betaVEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, betaVEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway.
1-Phosphatidylinositol 3-Kinase/*antagonists & inhibitors ; Cell Division/drug effects ; Cell Line ; Endothelial Growth Factors/*metabolism/pharmacology ; Endothelium, Vascular/cytology ; *Gene Expression Regulation, Enzymologic ; Lymphokines/*metabolism/pharmacology ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 1/*antagonists & inhibitors ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases/*antagonists & inhibitors ; Nitric Oxide Synthase/*genetics/metabolism ; Nitric Oxide Synthase Type III ; Signal Transduction ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

The aim of the present study was to investigate the protective effects and underlying mechanisms of Garcinia xanthochymus, a perennial medicinal plant native to Yunnan, China, against HO-induced oxidative damage in rat pheochromacytoma PC12 cells. Preincubation of PC12 cells with fruit EtOAc fraction (fruit-EFr., 12.5-50 µmol·L) of G. xanthochymus for 24 h prior to HO exposure markedly improved cell viability and increased the activities of antioxidant enzymes (superoxide dismutase, catalase, and heme oxygenase-1 [HO-1]), prevented lactate dehydrogenase release and lipid peroxidation malondialdehyde production, attenuated the decrease of matrix metalloproteinases (MMP), and scavenged reactive oxygen species (ROS). Fruit-EFr. also reduced BAX and cytochrome C expression and improved BCL-2 expression, thereby decreasing the ratio of BAX to BCL-2. Fruit-EFr. activated the nuclear translocation of NRF2 to increase HO-1 and induced the phosphorylation of AKT. Its cytoprotective effect was abolished by LY294002, a specific inhibitor of PI3K. Taken together, the above findings suggested that fruit-EFr.of G. xanthochymus could enhance cellular antioxidant defense capacity, at least in part, through upregulating HO-1 expression and activating the PI3K/AKT pathway and that it could suppress HO-induced oxidative damage via PI3K/AKT and NRF2/HO-1 signaling pathways.
Animals ; Antioxidants ; metabolism ; pharmacology ; Apoptosis ; drug effects ; Biological Transport ; Cell Survival ; Cytochromes c ; metabolism ; Fruit ; Garcinia ; Heme Oxygenase-1 ; metabolism ; Hydrogen Peroxide ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; drug effects ; PC12 Cells ; Phosphatidylinositol 3-Kinase ; metabolism ; Phosphatidylinositol 3-Kinases ; Phosphorylation ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Signal Transduction ; bcl-2-Associated X Protein ; metabolism