Gene expression profiling analysis of lung adenocarcinoma

The present study screened potential genes related to lung adenocarcinoma, with the aim of further understanding disease pathogenesis. The GSE2514 dataset including 20 lung adenocarcinoma and 19 adjacent normal tissue samples from 10 patients with lung adenocarcinoma aged 45-73 years was downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between the two groups were screened using the t-test. Potential gene functions were predicted using functional and pathway enrichment analysis, and protein-protein interaction (PPI) networks obtained from the STRING database were constructed with Cytoscape. Module analysis of PPI networks was performed through MCODE in Cytoscape. In total, 535 upregulated and 465 downregulated DEGs were identified. These included ATP5D, UQCRC2, UQCR11 and genes encoding nicotinamide adenine dinucleotide (NADH), which are mainly associated with mitochondrial ATP synthesis coupled electron transport, and which were enriched in the oxidative phosphorylation pathway. Other DEGs were associated with DNA replication (PRIM1, MCM3, and RNASEH2A), cell surface receptor-linked signal transduction and the enzyme-linked receptor protein signaling pathway (MAPK1, STAT3, RAF1, and JAK1), and regulation of the cytoskeleton and phosphatidylinositol signaling system (PIP5K1B, PIP5K1C, and PIP4K2B). Our findings suggest that DEGs encoding subunits of NADH, PRIM1, MCM3, MAPK1, STAT3, RAF1, and JAK1 might be associated with the development of lung adenocarcinoma.

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

BACKGROUND: Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential. RESULTS: The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude. CONCLUSIONS: Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.

Roles of targeting Ras/Raf/MEK/ERK signaling pathways in the treatment of esophageal carcinoma / 药学学报

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.

Bibenzyl from Dendrobium inhibits angiogenesis and its underlying mechanism / 药学学报

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.

The design of muti-target antitumor drugs affecting on FTase and Raf-1 kinase / 药学学报

Ras/Raf/MEK/ERK singal transduction plays an important role in cell proliferation, differentiation, apoptosis, metastasis and metabolism. This investigation focused on this signal pathway and chose farnesyl transferase (FTase) as the main target and Raf-1 kinase as the second target. A lot of compounds were selected to construct the pharmacophore models of farnesyl transferase inhibitors (FTIs) and Raf-1 kinase inhibitors by using computer-aided drug design (CADD). The pharmacophore of FTIs is constituted by a hydrogen bonding acceptor, an aromatic ring, a positive ionizable and two hydrophobic regions; the pharmacophore of Raf-1 kinase is constituted by a hydrogen donor, a hydrogen acceptor, a hydrophobic regions and an aromatic ring. There are some similarities between the two pharmacophores. After analysis of the constructions of these two pharmacophores, some new aminomethylbenzoic acid derivatives with good forecasting activity against both of FTase and Raf-1 kinase were designed with these new pharmacophore models.

Expression and significance of JNK1, Raf-1 and Livin in the carcinogenesis of sporadic colorectal tubular adenoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the possible role of JNK1, Raf-1 and Livin in the carcinogenesis of sporadic colorectal tubular adenoma.</p><p><b>METHODS</b>Immunohistochemical 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>JNK1, Raf-1 and Livin may be involved in the carcinogenesis of sporadic colorectal tubular adenoma.</p>

Expressions of Raf-1 kinase, phosphorylated mitogen extracellular kinase 1, and phosphorylated extracellular signal-regulated protein kinase 1/2 in hepatocellular carcinoma and their relationship with prognosis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>We 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Raf-1 over-expression is an independent marker for the patients of HCC, which may provide new clue in the future targeted therapy.</p>

Effect of N-acetyl-seryl-aspartyl-lysyl-proline on regulation of expression of ras-raf-ERK1/2 signal transduction pathway in lung of rats with silicosis / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>to 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.</p><p><b>METHODS</b>rats 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.</p><p><b>RESULTS</b>compared 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.</p><p><b>CONCLUSION</b>AcSDKP possibly plays an important role in anti-silicotic fibrosis by blocking the TGF-β-induced Ras-Raf-ERK1/2 signal transduction pathway.</p>

Expression of Raf-1 Kinase Inhibitory Protein in Extrahepatic Bile Duct Carcinoma

BACKGROUND: Raf-1 kinase inhibitory protein (RKIP) recently has been identified as a metastasis suppressor in a variety of human carcinomas. The prognostic significance of RKIP expression in extrahepatic bile duct (EBD) carcinoma has not been studied. The aims of the current study were to evaluate RKIP expression and to determine the prognostic significance of RKIP expression in EBD carcinoma. METHODS: Immunohistochemical staining for RKIP was performed for 131 cases of EBD carcinoma. The associations of RKIP expression with clinicopathologic parameters and patient outcomes were examined. Multivariate logistic regression analysis was used to identify independent predictive parameters for lymphovascular invasion and nodal and distant metastases. RESULTS: Loss of RKIP expression was observed in 55.0% (72/131) of cases. EBD carcinoma had significantly lower RKIP immunoreactivity than normal EBD (p < 0.001). Loss of RKIP expression was significantly associated with lymphatic invasion (p = 0.030) and nodal metastasis (p = 0.036), but it was not found to be a significant prognostic predictor for overall, disease-free or distant metastasis-free survival. In addition, loss of RKIP expression was an independent predictor for lymphatic invasion (p = 0.027). CONCLUSIONS: These results suggest that RKIP may play a role in the suppression of lymphatic invasion and nodal metastasis in EBD carcinoma.

Over-expression of caveolin-1 inhibits proliferation and invasion of pancreatic carcinoma cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of caveolin-1 on the biologic behavior of pancreatic carcinoma cell line panc1 cells in vitro.</p><p><b>METHODS</b>Eukaryotic 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.</p><p><b>RESULTS</b>Three 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.</p><p><b>CONCLUSION</b>Over-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.</p>

Molecular targeting for treatment of advanced hepatocellular carcinoma / 대한간학회지

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.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. RESULTS: The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. CONCLUSION: Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.

Effects of electromagnetic radiation on RAF/MEK/ERK signaling pathway in rats hippocampus / 中国应用生理学杂志

<p><b>AIM</b>To 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.</p><p><b>METHODS</b>Rats 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Raf/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.</p>

Influence of electromagnetic radiation on raf kinase inhibitor protein and its related proteins of hippocampus / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the development of changes for Raf kinase inhibitor protein (RKIP) and its mRNA in rats hippocampus after electromagnetic radiation.</p><p><b>METHODS</b>Rats were exposed to X-band high power microwave (X-HPM), S-band high power microwave (S-HPM) and electromagnetic pulse (EMP) radiation source respectively. The animal model of electromagnetic radiation was established. Western blot was used to detect the expression of RKIP, and RT-PCR was applied to detect the expression of RKIP mRNA. The interaction of RKIP and Raf-1 was measured with co-immunoprecipitation method, and the expression of cerebral choline acetyltransferase (CHAT) was measured by immunohistochemistry.</p><p><b>RESULTS</b>The expression of RKIP significantly down-regulated at 6 h after radiation, and recovered at 1 d in group EMP, but the down-regulation continued during 1 approximately 7 d after radiation in the two microwave groups. The expression of RKIP mRNA changed wavily during 6 h approximately 7 d after radiation, which showed down-regulation at 6 h, and up-regulation at 3 d. The interaction of RKIP and Raf-1 decreased during 6 h approximately 7 d after radiation, most significantly at 7 d, and the two microwave groups were more significant. The expression of CHAT decreased continuously during 6 h approximately 7 d after radiation, and generally recovered on 14 d.</p><p><b>CONCLUSION</b>The down-regulation of RKIP and its related proteins of hippocampus is induced by electromagnetic radiation.</p>

Differential Physiological Effects of Raf-1 Kinase Pathways Linked to Protein Kinase C Activation Depending on the Stimulus in v-H-ras-transformed Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We investigated the molecular mechanism by which the Raf-1 kinase pathways that are linked to protein kinase C induce differential physiological effects, depending on the stimulus, by employing the pharmacological PKC activator PMA. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems and these cells were transiently transfected with the pMTH vector that encodes dominant-negative (DN) PKC-epsilon with using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. The Raf-1 kinase activity was measured by assessing the phosphorylation of recombinant MEK with using the immunoprecipitated Raf-1 proteins. The phosphorylated MEK protein bands were quantified by using Quantity One analysis software. RESULTS: The pharmacological PKC activator phorbol-12-myristate-13-acetate (PMA) and platelet-derived growth factor (PDGF) were able to induce the activation of Raf-1 kinase in the v-H-ras-transformed NIH3T3 fibroblasts. However, PMA was found to be much less sensitive PI3 kinase inhibitor or the chemical antioxidant than is PDGF. Especially, PMA mediated growth arrest while PDGF induced mitogenic signaling through the PKC-epsilon activation. Thus, the regulation of the Raf-1 cascade by both PDGF and PMA is likely to be intimately linked and they converge at the PKC level through different upstream pathways, as was shown by the inhibition of PDGF-induced Raf-1 kinase activation by the transient transfection with a dominant-negative mutant of PKC-epsilon. CONCLUSIONS: Taken together, these results imply that, depending on the stimulus, Raf-1 kinase leads to different physiological effects.

Specific inhibition of Raf-1 gene expression in HNE1 by RNA interference / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique.</p><p><b>METHODS</b>The vector containing the human U6 promoter was used for targeted gene silencing when a dsDNA oligonucleotide encoding an appropriate shRNA was ligated into the vector, and 67nt oligonucleotide fragment was inserted into the downstream of the U6 promoter. Plasmids containing different Raf-1 target sequences [ (1) pshuttle-Raf-1-a( 225), (2) pshattle-Raf-1-b ( 358) and (3) pshuttle-Raf-1-c(474)], were transfected into HNE1 cells. Expression of Raf-1 mRNA was assayed by RT-PCR. Apoptosis were determined by cytometry.</p><p><b>RESULTS</b>Vector-based RNAi had advantages over antisense RNA because it could be delivered to the target cell more efficiently, and effect could last longer. Raf-1 expression could be inhibited by plasmid-expressed shRNA. Three different targeting sequences were selected from Raf-1 gene, and the inhibitory effect of pSIREN shuttle-Raf-1-b (358) was biggest.</p><p><b>CONCLUSION</b>Raf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.</p>

The effects of genistein on epidermal growth factor receptor mediated signal transduction pathway in human ovarian carcinoma cells lines SKOV3 and its xenograft in nude mice / 中华病理学杂志

<p><b>OBJECTIVE</b>It was reported previously that genistein could inhibit proliferation of human ovarian carcinoma cell line SKOV(3), but mechanism was not clear. There is a close relationship between EGFR mediated signal transduction pathway and the development of ovarian tumor. This study aimed to investigate the effects of genistein on the EGFR mediated signal transduction pathway and to clarify its mechanisms of proliferation inhibition on human ovarian carcinoma cell line SKOV(3) and its xenograft in nude mice.</p><p><b>METHODS</b>The expression of c-erbB-2 protein was determined using immunocytochemistry. The expressions of c-jun and c-fos protein were determined using Western blotting. The expression of c-erbB-2, c-raf-1, c-jun and c-fos mRNA were tested by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The expression of c-erbB-2, c-raf-1 and its downstream gene c-jun and c-fos were decreased at mRNA level in the 20 micromol/L genistein group. The expression of c-erbB-2 protein were decreased, its average absorbency (A) were decreased after treatment of SKOV(3) with 20 micromol/L genistein for 48 h, reached at 0.42 +/- 0.02 (P < 0.05). Western blotting demonstrated that the expressions of c-jun and c-fos protein were decreased gradually after being treated with 20 micromol/L genistein for 12 - 72 h.</p><p><b>CONCLUSIONS</b>Genistein could down-regulate the expression of two key genes, c-erbB-2 and c-raf-1 at mRNA and protein level in the EGFR mediated signal transduction pathway, and down-regulate the expression of its downstream nuclear transcription factors c-jun and c-fos at mRNA and protein level. It is suggested that interfering the expressions of some key signal molecules in EGFR mediated signal transduction system by genistein may account for its molecular foundation of proliferation inhibition in ovarian carcinoma.</p>

Cellular Proliferative Effect of Dexamethasone in Immortalized Trabecular Meshwork Cell (TM5) Line

Dexamethasone (DEX), one of the corticosteroid hormones, is one of the most common therapeutic strategies in ophthalmological treatment. Despite its widespread use and clinical efficiency, little is known regarding the specific effects of DEX on cell growth, differentiation and cell death in human trabecular meshwork cells. The presence of the glucocorticoid receptor (GR, dexamethasone receptor) in TM-5 cell line, which was derived from the primary human trabecular meshwork cells, was verified by RT-PCR and western blot analysis. The effects of DEX on the cellular proliferation of TM5 cells were measured by a BrdU incorporation assay. Western blot analysis were used to examine the effects of DEX on the Ras/MEK/ERK signaling pathway. The total Ras, MEK1/2 and ERK1/2 protein levels as well as the levels of activated (phosphorylated) form were both significantly increased by the DEX treatment for 5 days. Both MEK1/2 and ERK1/2 were significantly activated by phosphorylation after 10 minutes. The dependence of this increased cell proliferation on GR activation by DEX and the sustained activation of ERK was examined using RU486 (a GR inhibitor) and U0126 (a MEK inhibitor). Both RU486 and U0126 prevented the induction of cell proliferation by the DEX treatment in the TM5 cells. In conclusion this study demonstrated that GR is expressed in TM5 cells. Secondly, DEX treatment for 5 days stimulates cell proliferation in TM5 cells, and that this increased proliferation effect is mediated by the Ras/MEK/ERK pathway.