OBJECTIVEEffects of ginsenoside Rb1, Rg1 and Re on neurotrophic factor signal transduction pathway using liposome-mediated transfection of eukaryotic cells approach.

METHODThe injury model was established by treating SH-SY5Y cells with 0.6 mmol x L(-1) of corticosterone (CORT) by 24 h. SH-SY5Y cell were pretreated with CORT for 30 min followed by co-treated with 120,60 and 20 micromol x L(-1) of Rb1, 120, 80 and 40 micromol x L(-1) of Rg1 and 120, 80 and 40 micromol x L(-1) of Re for 24 h. Cells viability was determined by Cell Counting Kit (CCK) assay. CREB expressing Luciferase reporter gene was constructed and transfected with plasmid containing hRaf, hcAMP, hAkt, hCaMK gene into human embryonic kidney (HEK293) cells using liposornal transfection reagent lipofection 2000. The expression of CREB before and after it addion of Rb1, Rg1 and Re was examined by Luc assay system and Western blotting.

RESULTCompared with normal control group, CORT significantly decreased the viability of SH-SY5Y cells to 67.21% (P < 0.01). CCK results show that Rb1 (60 micromol x L(-1)), Rg1 (80 micromol x L(-1)) and Re (80 micromol x L(-1)) on SH-SY5Y cells have significant protective effect (P < 0.01). Lucassay and Western blotting results show that the gene and protein levels of CREB increased significantly through the pathway of Raf and Akt with Rb1 and Rg1 (P < 0.01), Re can increase significantly the gene and protein levels of CREB through the pathway of Raf and CaMK II.

CONCLUSIONRb1, Rg1 and Re protects SH-SY5Y cells from CORT-induced damage and the neuroprotective mechanism may be associated with the Raf-CREB, Akt-CREB and CaMK II -CREB pathways.

Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Cell Line ; Cell Survival ; drug effects ; Cyclic AMP Response Element-Binding Protein ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Genes, Reporter ; Ginsenosides ; pharmacology ; Humans ; Panax ; chemistry ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; drug effects ; raf Kinases ; genetics ; metabolism

In the present study, a new compound named 17-(6-cinnamamido-hexylamino-)-17-demethoxygeldanamycin (CDG) was obtained by introducing the cinnamic acid (CA) group into the 17-site of geldanamycin (GDM). The anti-cancer effects of CDG in vitro and in vivo were evaluated. MTT assay was used to examine the inhibitory effect of CDG on the proliferation of MCF-7, HepG2, H460 and SW1990 cells. Immunofluorescent staining flow cytometry combined with Annexin V-FITC/PI staining were used to detect apoptotic cells. Transwell assay was used to analyze the effect of CDG on cell invasion and migration ability. Western blotting was used to detect the expression levels of RAF-1, EGFR, AKT, CDK4 and HER-2 of MCF-7, HepG2 and H460 cells. The toxicities of CDG and GDM were evaluated in mice. Using the subcutaneously transplanted MCF-7 xenograft in nude mice, inhibitory effect was evaluated in vivo. The results showed that CDG inhibited the proliferation of cancer cells (IC50: 13.6-67.4 microg.mL-1). After exposure to CDG for 48 h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus. The rates of apoptosis of MCF-7, HepG2, H460 and SW1990 cells incubated with 10 microg.mL-1 CDG were 23.16%, 27.55%, 22.21%, 20.47%, respectively. A dose-dependent reduction of migration of four cell lines was found after exposure to CDG. The decreased levels of RAF-1, EGFR, AKT, CDK4 and HER-2 showed that CDG possessed HSP90 inhibitory effect. The result of animal toxicity test on the mice suggested that CDG had lower toxicity than GDM. Meanwhile, CDG inhibited the growth of MCF-7 xenografts of athymic mice.
Animals ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Benzoquinones ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase 4 ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; chemical synthesis ; chemistry ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Proto-Oncogene Proteins A-raf ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

Ras is best known for its ability to regulate cell growth, proliferation and differentiation. Mutations in Ras are associated with the abnormal cell proliferation which can result in incidence of all human cancers. Extracellular signal-regulated kinase (ERK) is a downstream effector of Ras and plays important roles in prognosis of tumors. Recently, evidence has gradually accumulated to demonstrate that there are other effectors between Ras and ERK, these proteins interact each other and constitute the thorough Ras/Raf/MEK/ERK signaling pathway. The pathway has profound effects on incidence of esophageal carcinoma and clinical applications of some chemotherapeutic drugs targeting the pathway. Further understanding of the relevant molecular mechanisms of Ras/Raf/MEK/ERK signaling pathway can be helpful for the development of efficient targeting therapeutic approaches which contribute to the treatment of esophageal cancer. In this article, roles of Ras/Raf/MEK/ERK signaling pathway in esophageal carcinoma as well as pharmacological targeting point in the pathway are reviewed.
Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinoma, Squamous Cell ; drug therapy ; enzymology ; pathology ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Esophageal Neoplasms ; drug therapy ; enzymology ; pathology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-raf ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects ; ras Proteins ; antagonists & inhibitors ; metabolism

Bibenzyl is a type of active compounds abundant in Dendrobium. In the present study, we investigated the inhibitory effects of six bibenzyls isolated from Dendrobium species on vascular endothelial growth factor (VEGF)-induced tube formation in human umbilical vascular endothelial cells (HUVECs). All those bibenzyls inhibited VEGF-induced tube formation at 10 micromol x L(-1) except tristin, and of which moscatilin was found to have the strongest activity at the same concentration. The lowest effective concentration of moscatilin was 1 micromol x L(-1). Further results showed that moscatilin inhibited VEGF-induced capillary-like tube formation on HUVECs in a concentration-dependent manner. Western blotting results showed that moscatilin also inhibited VEGF-induced phosphorylation of VEGFR2 (Flk-1/KDR) and extracellular signal-regulated kinase 1/2 (ERK1/2). Further results showed that moscatilin inhibited VEGF-induced activation of c-Raf and MEK1/2, which are both upstream signals of ERK1/2. Taken together, results presented here demonstrated that moscatilin inhibited angiogenesis via blocking the activation of VEGFR2 (Flk-1/KDR) and c-Raf-MEK1/2-ERK1/2 signals.
Angiogenesis Inhibitors ; administration & dosage ; isolation & purification ; pharmacology ; Animals ; Benzyl Compounds ; administration & dosage ; isolation & purification ; pharmacology ; Bibenzyls ; isolation & purification ; pharmacology ; Cell Count ; Cells, Cultured ; Dendrobium ; chemistry ; Dose-Response Relationship, Drug ; Human Umbilical Vein Endothelial Cells ; Humans ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Kinase 2 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Inbred C57BL ; Neovascularization, Physiologic ; drug effects ; Phosphorylation ; drug effects ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-raf ; metabolism ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVETo explore the prognostic values of Raf-1 kinase (Raf-1), phosphorylated mitogen extracellular kinase 1 (pMEK1), and phosphorylated extracellular signal-regulated protein kinase 1/2(pERK1/2) in hepatocellular carcinoma (HCC) patients.

METHODSWe assessed the expressions of Raf-1, pMEK1, and pERK1/2 in HCC using immunohistochemical techniques. The relationships between the expressions of Raf-1, pMEK1, and pERK1/2 and the prognosis were explored.

RESULTSThe over-expression rates of Raf-1, pMEK1, and pERK1/2 in HCC were 38.3%, 46.7%, and 38.3%, respectively. The over-expressions of Raf-1, pMEK1, and pERK1/2 were positively correlated with each other (P>0.05), but had no significant correlation with sex, age, α-fetoprotein, hepatitis B surface antigen status, the TNM stage, size,differentiation and vascular invasion of tumor, and liver cirrhosis (P>0.05). Univariate survival analysis and COX proportional hazard regression model showed that Raf-1 over-expression was an independent prognostic factor of poor survival (P<0.05).

CONCLUSIONRaf-1 over-expression is an independent marker for the patients of HCC, which may provide new clue in the future targeted therapy.

Adult ; Aged ; Carcinoma, Hepatocellular ; diagnosis ; enzymology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Humans ; Liver Neoplasms ; diagnosis ; enzymology ; MAP Kinase Kinase 1 ; metabolism ; Male ; Middle Aged ; Phosphorylation ; Prognosis ; Proto-Oncogene Proteins c-raf ; metabolism

OBJECTIVETo study the possible role of JNK1, Raf-1 and Livin in the carcinogenesis of sporadic colorectal tubular adenoma.

METHODSImmunohistochemical staining was used to detect the expression of JNK1, Raf-1 and Livin proteins in 65 sporadic colorectal tubular adenomas with dysplasia of varying degrees and 22 colorectal tubular adenoma with cancerous area.

RESULTSIn normal colorectal mucosa, colorectal tubular adenoma with dysplasia and colorectal tubular adenoma with cancerous area, the positive rate of JNK1, Raf-1 and Livin expression was increased gradually. The positive expression of JNK1, Raf-1 and Livin was all significantly higher in the cases of colorectal tubular adenoma with dysplasia or with cancerous area than that in normal colorectal mucosa (P < 0.05), and the positive expression of JNK1, Raf-1 and Livin was significantly higher in colorectal tubular adenoma with cancerous area than that in colorectal tubular adenoma with dysplasia of different degrees (P < 0.05). In the cases of colorectal tubular adenoma with dysplasia of varying degrees, the positive expression of Raf-1 was increased along with the increasing dysplasia degree of colorectal tubular adenoma (P < 0.05). Coexpression of JNK1, Raf-1 and Livin increased gradually in the carcinogenesis of sporadic colorectal tubular adenoma, while positive correlation was found among the expressions of JNK1, Raf-1 and Livin.

CONCLUSIONJNK1, Raf-1 and Livin may be involved in the carcinogenesis of sporadic colorectal tubular adenoma.

Adaptor Proteins, Signal Transducing ; metabolism ; Adenoma ; metabolism ; pathology ; Adult ; Carcinoma ; metabolism ; pathology ; Cell Transformation, Neoplastic ; Colorectal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Male ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Neoplasm Proteins ; metabolism ; Precancerous Conditions ; metabolism ; pathology ; Proto-Oncogene Proteins c-raf ; metabolism

OBJECTIVEto investigate the effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on the expressions of c-Raf, ERK1/2 and TGF-β1 in the lung of rats with silicosis, thus to investigate the regulating of AcSDKP on the Ras-Raf-ERK1/2 signal transduction pathway.

METHODSrats were instilled with silica through trachea as silicotic models and administered AcSDKP in the experiment. Rats were divided into 6 groups randomly, 10 rats in each group: Control 1 and 2 of silicotic model: each rat was intratracheally instilled with 1.0 ml normal sodium and was killed after 4 or 8 weeks; Silicotic model 1 and Silicotic model 2: each rat was intratracheally instilled with 1ml silica suspension and was killed after 4 or 8 weeks; Anti-fibrosis treatment of AcSDKP: after each rat was intratracheally instilled with 1ml silica suspension for 4 weeks, AcSDKP 800 microg × kg(-1) × d(-1) was administered into every rat and rats were killed at the eighth week; Preventing fibrosis treatment of AcSDKP: after AcSDKP 800 microg × kg(-1) × d(-1) was administered into every rat for 48 hours, each rat was intratracheally instilled with 1.0 ml silica suspension and rats were killed at the eighth week. The expression of c-Raf, phospho-c-Raf, ERK1/2, phospho-ERK1/2 and TGF-β1 was measured by immunohistochemistry and western blot assay.

RESULTScompared with the corresponding control groups, the expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-β1 increased in the lung tissue of the silicotic models. Compared with the corresponding model groups, after administration AcSDKP, the expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-β1 in the lung tissue reduced obviously. In anti-fibrosis treatment of AcSDKP group, expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-β1 decreased to 52.25%, 51.72% and 67.74% compared with those of the silicotic model 1, and expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-β1 decreased to 49.37%, 55.76%, 65.63% compared with those of the silicotic model 2; In preventing fibrosis treatment of AcSDKP group, expressions of phospho-c-Raf, phospho-ERK1/2 and TGF-β1 decreased to 54.64%, 55.76% and 78.91% compared with those of the silicotic model 2 (P < 0.05) while the expressions of c-Raf and ERK1/2 were not different significantly among each groups.

CONCLUSIONAcSDKP possibly plays an important role in anti-silicotic fibrosis by blocking the TGF-β-induced Ras-Raf-ERK1/2 signal transduction pathway.

Animals ; Lung ; metabolism ; Male ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Oligopeptides ; pharmacology ; Proto-Oncogene Proteins c-raf ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Silicosis ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo investigate the effects of caveolin-1 on the biologic behavior of pancreatic carcinoma cell line panc1 cells in vitro.

METHODSEukaryotic expression vectors containing human caveolin-1 gene was stably transfected into panc1 cells with Lipofectamine2000. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western plotting. The cell growth activity was examined by MTT assay. Anchorage-independent growth was detected by colony formation assay in soft agar. Flow cytometry was used to analyze the cell cycle and apoptosis. Cell invasion assay was used for evaluating cell invasion capacity. The relative phosphorylation level of EGFR, c-Raf, Mek, Erk, p38 and SAPK/JNK were detected by Western blotting.

RESULTSThree transfected cell clones overexpressing caveolin-1 were obtained. Comparing with the panc1 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of flow cytometry showed that over-expression of caveolin-1 resulted in the cell cycle arrest at G(0)/G(1) phase and increased the apoptotic cell fraction. Cell invasion assay showed that overexpression of caveolin-1 significantly inhibited the panc1 cell invasion. Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR, c-Raf, Mek and Erk while did not affect the activity of p38 and SAPK/JNK.

CONCLUSIONOver-expression of caveolin-1 inhibits the growth and invasion of pancreatic carcinoma cells in vitro. These phenotypes may be correlated with the inhibition of EGFR-c-Raf-Mek-Erk signaling pathway.

Apoptosis ; Caveolin 1 ; genetics ; metabolism ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Neoplasm Invasiveness ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; Proto-Oncogene Proteins c-raf ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; Transfection

Hepatocellular carcinoma (HCC) is a major global health problem, which has a grave morbidity and mortality. Over the past few decades, no effective systemic therapeutic modalities have been established for patients with the unresectable HCC in advanced stage. Sorafenib is a small molecule that blocks cancer cell proliferation by targeting the intracellular signaling pathway at the level of Raf-1 and B-Raf serine-threonine kinases, and exerts an anti-angiogenic effect by targeting the vascular endothelial growth factor receptor-1, 2 and 3, and platelet-derived growth factor receptor-beta tyrosine kinases. Recently, two clinical successful applications, SHARP and Asia-Pacific trial, of multikinase inhibitor sorafenib represent a significant advance in the treatment of advanced HCC patients without a curative chance. However, because the results of clinical trials show diverse responses in a subset of HCC patients, a molecular classification of HCC through the excavation of specific biomarkers related to its biological behavior is necessary for sorting HCC patients to each group with a biological homogeneity, ultimately leading to the most suitable individualization of molecular targeted therapy in HCC.
Antineoplastic Agents/therapeutic use ; Benzenesulfonates/therapeutic use ; Carcinoma, Hepatocellular/pathology/secondary/*therapy ; Humans ; Liver Neoplasms/blood supply/pathology/*therapy ; Neovascularization, Pathologic ; Proto-Oncogene Proteins B-raf/antagonists & inhibitors/metabolism ; Proto-Oncogene Proteins c-raf/antagonists & inhibitors/metabolism ; Pyridines/therapeutic use ; Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors/metabolism ; Signal Transduction

AIMTo study the development of changes for signaling molecules related to Raf/MEK/ERK pathway in hippocampus of rats after electromagnetic radiation, and investigate the mechanisms of radiation injury.

METHODSRats were exposed to X-HPM, S-HPM and EMP radiation source respectively, and animal model of electromagnetic radiation was established. Western blot was used to detect the expression of Raf-1, phosphorylated Raf-1 and phospholylated ERK.

RESULTSThe expression of Raf-1 down-regulated during 6 h-14 d after radiation, most significantly at 7 d, and recovered at 28 d. There was no significant difference between the radiation groups. The expression of phosphorylated Raf-1 and phosphorylated ERK both up-regulated at 6 h and 7 d after radiation, more significantly at 6 h, and the two microwave groups were more serious for phosphorylated ERK. During 6 h-14 d after S-HPM radiation, the expression of phosphorylated Raf-1 increased continuously, but phosphorylated ERK changed wavily, 6 h and 7 d were expression peak.

CONCLUSIONRaf/MEK/ERK signaling pathway participates in the hippocampus injury induced by electromagnetic radiation. The excessive activation of ERK pathway may result in the apoptosis and death of neurons, which is the important mechanism of recognition disfunction caused by electromagnetic radiation.

Animals ; Apoptosis ; Electromagnetic Radiation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hippocampus ; metabolism ; physiopathology ; radiation effects ; MAP Kinase Kinase Kinases ; metabolism ; MAP Kinase Signaling System ; radiation effects ; Male ; Phosphorylation ; Proto-Oncogene Proteins c-raf ; metabolism ; Random Allocation ; Rats ; Rats, Wistar