Downregulation Of Raf-1 Research Articles

The Agonist of Adenosine A1 Receptor Induced Desensitization of delta Opioid receptor-mediated Raf-1/MEK/ERK Signaling by Feedback Phosphorylation of Raf-1-Ser289/296/301.

Our previous study found that activation of adenosine A1 receptor (A1R) induced phosphorylation of delta opioid receptor (DOR) and desensitization of its downstream signaling molecules, cAMP and Akt. To further investigate the effect of A1R agonist on DOR signaling and the underlying mechanism, we examined the effect of A1R activation upon binding of its agonist N6-cyclohexyl-adenosine (CHA) on DOR-mediated Raf-1/MEK/ERK activation, and found that prolonged CHA exposure resulted in downregulation of DOR-mediated Raf-1/MEK/ERK signaling pathway. CHA-treatment time dependently attenuated Raf-1-Ser338 phosphorylation induced by [D-Pen2,5] enkephalin (DPDPE), a specific agonist of DOR, and further caused downregulation of the Raf-1/MEK/ERK signaling pathway activated by DOR agonist. Moreover, CHA exposure time-dependently induced the phosphorylation of Raf-1-Ser289/296/301, the inhibitory phosphorylation sites that were regulated by negative feedback, thereby inhibiting activation of the MEK/ERK pathway, and this effect could be blocked by MEK inhibitor U0126. Finally, we proved that the heterologous desensitization of the Raf-1/MEK/ERK cascade was essential in the regulation of anti-nociceptive effect of DOR agonists by confirming that such effect was inhibited by pretreatment of CHA. Therefore, we conclude that the activation of A1R inhibits DOR-mediated MAPK signaling pathway via heterologous desensitization of the Raf-1/MEK/ERK cascade, which is a result of ERK-mediated Raf-1-Ser289/296/301 phosphorylation mediated by activation of A1R.

Arecoline promotes proliferation and migration of human HepG2 cells through activation of the PI3K/AKT/mTOR pathway

BackgroundArecoline is a well-known risk factor for oral submucosal fibrosis and cancer. However, the mechanistic correlation between arecoline and hepatocellular cancer remains elusive. Here, we investigated the effect of arecoline on the proliferation and migration of human HepG2 hepatoma cells and its potential oncogenic mechanisms.MethodsBioinformatic technologies were used to identify the deferentially expressed miRNAs (DE-miRNAs) and hub target genes of arecoline-induced cancers. These DE-miRNAs, hub genes and pathway were proved in arecoline-treated HepG2 cells.ResultsA total of 86 DE-miRNAs and 460 target genes were identified. These target genes are associated with DNA-templated regulation of transcription and other biological processes. Significant molecular functions were protein binding, calcium ion binding, and enrichment in the nucleus and cytoplasm. These genes are involved in the PI3K-AKT pathway. CDK1, CCND1, RAF1, CDKN1B and BTRC were defined as the top 5 hub target genes, and patients with high expression of CDK1 showed poor prognosis. Compared with control group, 2.5 µM arecoline treatment increased the proliferation and migration ability of the HepG2 cells. Treatment with 2.5 µM arecoline increased the levels of miR-21-3p, miR-21-5p and miR-1267, upregulated the expression of PI3K-AKT pathway factors, CDK1, CCND1 but decreased RAF1 expression.ConclusionA low concentration arecoline can induce the proliferation and migration of HepG2 cells, with the potential mechanism of action linked to high levels of exosomal miR-21 and miR-1267, activation of the PI3K-AKT pathway, upregulation of CDK1 and CCND1, and downregulation of RAF1.

Decrease in MAP3Ks expression enhances the cell death caused by hyperthermia

Purpose Hyperthermia is a promising anticancer treatment modality. However, the molecular mechanism underlying the thermal sensitivity of tumor cells is largely unknown. The aim of this study was to clarify how biochemical changes triggered by heat stimulate antitumor activity. Methods and materials The expression levels of various MAPK members in HeLa cells with or without hyperthermia were evaluated by western blotting and RT-PCR. The intracellular Ca2+ concentration [Ca2+]i was monitored by digital imaging using CaTM-2 AM. An in vitro cleavage assay was used to determine whether calcium-dependent protease calpain cleaves MAPK components. Cell proliferation and clonogenicity were assessed in the absence or presence of siRNAs targeting MAPK members. Results Hyperthermia decreased the levels of MAP3K TAK1, RAF1 and MEKK2 but not of the downstream MAP2K and MAPK members. The hyperthermia-induced degradation of TAK1 and MEKK2 was rescued by either the proteasome inhibitor MG132 or the calpain inhibitor ALLN; however, RAF1 was not affected by the inhibitors. Heat induced down regulation of RAF1. Hyperthermia increased [Ca2+]i and calpain I expression. The calcium ionophore A23187 decreased TAK1 and MEKK2 levels. An in vitro cleavage assay demonstrated that TAK1 and MEKK2 are calpain I substrates. Knockdown of TAK1, RAF1 and MEKK2 suppressed cell proliferation and clonogenicity. Conclusions Hyperthermia decreased the levels of MAP3K TAK1, RAF1 and MEKK2, without reduction of the downstream components in the MAP3K-MAP2K-MAPK cascade, by a calpain-dependent degradation pathway or transcriptional regulation. TAK1, RAF1 and/or MEKK2 play crucial roles in cell proliferation and clonogenicity and are potential molecular targets for hyperthermia.

Long noncoding RNA ITGB2-AS1 promotes growth and metastasis through miR-4319/RAF1 axis in pancreatic ductal adenocarcinoma.

Long noncoding RNA (lncRNA) has been considered as potentially critical regulators in pancreatic ductal adenocarcinoma (PDAC). In this study, we prospectively investigate the effect and mechanism of lncRNA integrin subunit beta 2-anti-sense RNA 1 (ITGB2-AS1) on regulation of PDAC progression. The expression of ITGB2-AS1 and its target were analyzed by quantitative real-time polymerase chain reaction and in situ hybridization. 3-(4,5-Dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, wound healing, and transwell assays were used to investigate the influence of ITGB2-AS1 on cell proliferation, cell cycle, migration, and invasion, respectively. The interaction between ITGB2-AS1 and its target was determined via luciferase activity assay and RNA immunoprecipitation. The subcutaneous xenotransplanted tumor model was established and employed to detect the tumorigenic function of ITGB2-AS1, which was evaluated by western blot analysis, immunohistochemistry, and hematoxylin and eosin staining. The results showed that ITGB2-AS1 was elevated in both PDAC tumor tissues and cell lines, predicting a poor prognosis in PDAC patients. Knocking down of ITGB2-AS1 suppressed PDAC cell proliferation, invasion, and migration but induced cell apoptosis in vitro. Moreover, ITGB2-AS1 could target and inhibit the expression of miR-4319 and miR-4319-targeted and -suppressed serine/threonine kinase RAF1. ITGB2-AS1 promoted PDAC progression via inhibition of miR-4319. Interference of ITGB2-AS1 could suppress in vivo tumorigenic ability of PDAC via downregulation of RAF1. In conclusion, ITGB2-AS1 promoted PDAC progression via sponging miR-4319 to upregulate RAF1, suggesting the potential therapeutic target ability of ITGB2-AS1 in PDAC.

Inhibition of Raf1 ameliorates bleomycin-induced pulmonary fibrosis through attenuation of TGF-β1 signaling.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with aberrant activation and differentiation of fibroblasts, leading to abnormal extracellular matrix production. Currently, it is still an untreatable disease (except for lung transplantation). Here, we demonstrate that the Raf1 inhibitor GW5074 ameliorates lung fibrosis in bleomycin-induced pulmonary fibrosis. Posttreatment with GW5074 reduced fibronectin (FN) expression, collagen deposition, and inflammatory cell infiltration in bleomycin-challenged mice, suggesting an antifibrotic property of GW5074. To determine the molecular mechanisms by which inhibition of Raf1 ameliorates lung fibrosis, we investigated the role of Raf1 in TGF-β1 signaling in human lung fibroblasts. GW5074 or downregulation of Raf1 by siRNAs significantly attenuated TGF-β1-induced smooth muscle actin, FN, and collagen I expression, whereas overexpression of Raf1 promoted the effects of TGF-β1 in lung fibroblasts. Furthermore, we found that Raf1-promoted TGF-β1 signaling was through the Raf1/ERK/Smad pathway and contributed to the cell proliferation and migration in human lung fibroblasts. This study provides preclinical and mechanistic evidence for development of Raf1 inhibitors as potential antifibrotic drugs for the treatment of IPF.

Mechanistic study of the anti-cancer effect of Gynostemma pentaphyllum saponins in the Apc(Min/+) mouse model.

Gynostemma pentaphyllum saponins (GpS) have been shown to have anti-cancer activity. However, the underlying mechanisms remain unclear. In this study, we used the Apc(Min) (/+) colorectal cancer (CRC) mouse model to investigate the anti-cancer effect of GpS and we demonstrated that GpS treatment could significantly reduce the number and size of intestinal polyps in Apc(Min) (/+) mice. In order to identify the potential targets and mechanisms involved, a comparative proteomics analysis was performed and 40 differentially expressed proteins after GpS treatment were identified. Bioinformatics analyses suggested a majority of these proteins were involved in processes related to cellular redox homeostasis, and predicted Raf-1 as a potential target of GpS. The upregulation of two proteins known to be involved in redox homeostasis, peroxiredoxin-1 (Prdx1) and peroxiredoxin-2 (Prdx2), and the downregulation of Raf-1 were validated using Western blot analysis. After further investigation of the associated signaling networks, we postulated that the anti-cancer effect of GpS was mediated through the upregulation of Prdx1 and Prdx2, suppression of Ras, RAF/MEK/ERK/STAT, PI3K/AKT/mTOR signaling and modulation of JNK/p38 MAPK signaling. We also examined the potential combinatorial effect of GpS with the chemotherapeutic 5-fluorouracil (5-FU) and found that GpS could enhance the anti-cancer efficacy of 5-FU, further suppressing the number of polyps in Apc(Min/+) mice. Our findings highlight the potential of GpS as an anti-cancer agent, the potential mechanisms of its anti-cancer activities, and its effect as an adjuvant of 5-FU in the chemotherapy of CRC.

Upregulation of miR-195 increases the sensitivity of breast cancer cells to Adriamycin treatment through inhibition of Raf-1

Chemotherapy is an important option for the treatment of advanced breast cancer, but multidrug resistance is one of the major obstacles in the clinical control of breast cancer. The present study investigated the effects of the miR‑195-led gene pathway in the sensitization of breast cancer cells to treatment with the chemotherapeutic drug Adriamycin. Breast cancer cell lines and tissue specimens (obtained from chemotherapy-sensitive or resistant patients) as well as a normal breast cell line were used to assess expression of miR-195, Raf-1, Bcl-2 and P-glycoprotein mRNA and/or mRNA. miR-195 mimics, inhibitor and Raf-1 siRNA were used to transfect breast cancer MCF-7 and MCF-7/ADR cells (an Adriamycin-resistant MCF-7 subline) for cell viability, apoptosis and gene expression analysis. The data showed that miR-195 expression was low in breast cancer cells and multidrug-resistant breast cancer tissues, which was associated with reduced Raf-1 expression invitro and exvivo. Induction of miR-195 expression promoted tumor cell apoptosis and inhibited breast cancer cell viability, but induced the sensitivity of breast cancer cells to Adriamycin treatment and was associated with inhibition of Raf-1 expression in breast cancer cells. Moreover, knockdown of Raf-1 expression had similar effects of miR-195 mimics on breast cancer cells, both of which were able to suppress Bcl-2 and P-glycoprotein expression in breast cancer cells. The data from the current study demonstrated that expression of miR-195 was inversely associated with Raf-1 expression in breast cancer cell lines and tissue specimens, and that Raf-1 is the target gene of miR-195. Thus, expression of miR-195 or knockdown of Raf-1 can similarly reduce tumor cell survival but increase apoptosis through downregulation of Raf-1 and Bcl-2 and P-glycoprotein expression. In conclusion, this gene pathway mediated the sensitivity of breast cancer cells to Adriamycin treatment.

Abnormal activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in eutopic endometrial stromal cells of patients with endometriosis

BACKGROUND Enhanced proliferation and survival of eutopic endometrial cells from patients with endometriosis compared with healthy women is associated with abnormal activation of extra-cellular signal-regulated kinases 1 and 2 (ERK1/2). Given the role of Ras/Raf/mitogen-activated protein kinase (MAPK) and RhoA/ROCKII signalling pathways in the regulation of cell proliferation and migration, we analysed their possible roles in endometriosis. METHODS Primary eutopic endometrial stromal cells of patients with endometriosis (Eu-hESC, n= 16) and endometriosis-free controls (Co-hESC, n= 14) were harvested and subjected to proliferation and migration assays as well as kinase activity assays and immunoblot analysis of proteins from the Ras/Raf/MAPK and RhoA/ROCKII signalling pathways. Effects of ROCKII (Y-27632) and MAPK (U0126) inhibitors or siRNA knockdown of ROCKII, Raf-1 and B-Raf were analysed. RESULTS The proliferation rate of Eu-hESC was 54% higher than Co-hESC. Eu-hESC also displayed a 75% higher migration rate than Co-hESC. Eu-hESC displayed higher levels of ERK phosphorylation (83%) and p27 expression (61%) and lower levels of Raf-1 protein (47%) compared with controls. In addition to an inhibitory effect on cell proliferation, ROCKII knockdown led to significant down-regulation of cyclinD1 and p27 but did not affect ERK phosphorylation. Down-regulation of Raf-1 by siRNA was dispensable for cell proliferation control but led to an increase in ROCKII activity and a decrease in cell migration. B-Raf was shown to act as a regulator of hESC proliferation by modulating cellular ERK1/2 activity and cyclinD1 levels. Eu-hESC displayed 2.4-fold higher B-Raf activity compared with Co-hESC and therefore exhibit abnormally activated Ras/Raf/MAPK signalling. CONCLUSIONS We show that the same molecular mechanisms operate in Co- and Eu-hESC. The differences in cell proliferation and migration between both cell types are likely due to increased activation of Ras/Raf/MAPK and RhoA/ROCKII signalling pathways in cells from endometriosis patients.

Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity

Poliovirus (PV) and enterovirus 71 (EV71) cause severe neurological symptoms in their infections of the central nervous system. To identify compounds with anti-PV and anti-EV71 activities that would not allow the emergence of resistant mutants, we performed drug screening by utilizing a pharmacologically active compound library targeting cellular factors with PV and EV71 pseudoviruses that encapsidated luciferase-encoding replicons. We have found that metrifudil (N-[2-methylphenyl]methyl)-adenosine) (an A2 adenosine receptor agonist), N(6)-benzyladenosine (an A1 adenosine receptor agonist) and NF449 (4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriyl bis(carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt) (a Gs-alpha inhibitor) have anti-EV71 activity, and that GW5074 (3-(3, 5-dibromo-4-hydroxybenzylidine-5-iodo-1,3-dihydro-indol-2-one)) (a Raf-1 inhibitor) has both anti-PV and anti-EV71 activities. EV71 mutants resistant to metrifudil, N(6)-benzyladenosine and NF449 were isolated after passages in the presence of these compounds, but mutants resistant to GW5074 were not isolated for both PV and EV71. The inhibitory effect of GW5074 was not observed in Sendai virus infection and the treatment did not induce the expression of OAS1 and STAT1 mRNA. Small interfering RNA treatment against putative cellular targets of GW5074, including Raf-1, B-Raf, Pim-1, -2, and -3, HIPK2, GAK, MST2 and ATF-3, did not consistently suppress PV replication. Moreover, downregulation of Raf-1 and B-Raf did not affect the sensitivity of RD cells to the inhibitory effect of GW5074. These results suggest that GW5074 has strong and selective inhibitory effect against the replication of PV and EV71 by inhibiting conserved targets in the infection independently of the interferon response.

Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) is essential for viral replication. Here, the kinase Raf-1 was identified as a novel cellular binding partner of NS5A, binding to the C-terminal domain of NS5A. Raf-1 colocalizes with NS5A in the HCV replication complex. The interaction of NS5A with Raf-1 results in increased Raf-1 phosphorylation at serine 338. Integrity of Raf-1 is crucial for HCV replication: inhibition of Raf-1 by the small-molecule inhibitor BAY43-9006 or downregulation of Raf-1 by siRNA attenuates viral replication.

Depletion of p185erbB2, Raf-1 and mutant p53 proteins by geldanamycin derivatives correlates with antiproliferative activity.

Recently, it has been shown that geldanamycin (GA), a benzoquinone ansamycin, is able to deplete mutant p53, p185erbB2 and Raf-1 proteins in cancer cells. However, the relationship between these activities of GA and its antiproliferative activity is not clear. Here we investigated the effects of 28 GA derivatives in SKBr3, a human breast cancer cell line. We performed Western blot analysis of Raf-1, p185erbB2 and mutant p53 proteins following drug treatment and correlated these findings with the cytotoxicity of the various GA derivatives. We found that downregulation of Raf-1, p185erbB2 and mutant p53 proteins was correlated. Thus, a drug that was active against one oncoprotein was equally active against the two others. Inactive derivatives were identified by their inability to downregulate these oncoproteins, even at a high dose (2 microM). These inactive drugs also had no or minimal antiproliferative activity (IC50 > 3 microM). All other analogs (at a concentration of 2 microM) downregulated p53, p185erbB2, and Raf-1, and also displayed cytotoxicity (IC50 in the range 6-600 nM). This category of drugs was further divided into more- and less-active agents by testing at lower doses (40 nM). The drugs that remained active against their molecular targets had an IC50 for antiproliferative activity of less than 40 nM. Maximal effects on mutant p53, p185erbB2 and Raf-1 were observed at doses that were 4-5 times greater than the cytotoxic IC50. These findings suggest that GA and its derivatives are cytostatic/cytotoxic at concentrations that also downregulate Raf-1, p185erbB2 and mutant p53, and raise the possibility that depletion of these proteins and the antiproliferative activities of GA have a common mechanism.