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.
Cell Line ; Drug Resistance ; Hand ; Melanoma ; Proto-Oncogene Proteins c-raf

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.
Humans ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Pertussis Toxin ; Phenotype* ; Phosphorylation ; Protein Kinase C ; Proto-Oncogene Proteins c-raf ; Thrombin

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.
Bile Duct Neoplasms ; Bile Ducts, Extrahepatic ; Humans ; Immunohistochemistry ; Logistic Models ; Lymphatic Metastasis ; Neoplasm Metastasis ; Proto-Oncogene Proteins c-raf

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.
Cell Proliferation ; Fibroblasts ; Humans ; Lipids ; NIH 3T3 Cells ; Parents ; Phosphorylation ; Phosphotransferases ; Platelet-Derived Growth Factor ; Protein Kinase C ; Protein Kinases ; Proteins ; Proto-Oncogene Proteins c-raf ; Transfection

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.
Cell Line ; Cell Proliferation ; Genes, ras ; Humans ; Hydroquinones ; Lipids ; NIH 3T3 Cells ; Parents ; Phenotype ; Proteins ; Proto-Oncogene Proteins c-raf ; RNA, Small Interfering ; src-Family Kinases ; Transfection ; Tyrosine

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

OBJECTIVEIt 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.

METHODSThe 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).

RESULTSThe 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.

CONCLUSIONSGenistein 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.

Animals ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Genes, fos ; Genes, jun ; Genistein ; pharmacology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Ovarian Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-jun ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-raf ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Signal Transduction ; drug effects

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.
3T3 Cells ; Animal ; Cell Transformation, Neoplastic* ; Genes, ras* ; Mice ; Mitogen-Activated Protein Kinase Kinases/analysis ; Mitogen-Activated Protein Kinases/analysis ; Phosphorylation ; Protein-Serine-Threonine Kinases/analysis* ; Proto-Oncogene Proteins c-raf/analysis ; p42 MAP Kinase/analysis

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 silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique.

METHODSThe 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.

RESULTSVector-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.

CONCLUSIONRaf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.

Apoptosis ; genetics ; Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-raf ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; Transfection