Mitogen-activated Extracellular Signal-regulated Kinase Research Articles

MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts

There are two major pathways, homology-directed repair (HDR) and nonhomologous end joining (NHEJ), involved in double-strand break (DSB) repair. Single-stranded oligodeoxyribonucleotide (ssODN)-mediated homologous recombination repair is commonly used for animal site-directed genome editing, with great scientific and practical value. To improve ssODN-mediated HDR efficiency in the pig genome, we investigated the effect and molecular mechanism of mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor PD0325901 on the HDR efficiency in porcine fetal fibroblasts (PFFs). The results showed that PD0325901 obviously increased the percentage of G2 and S phase cell populations and reduced the cell population ratio in the G1 phase of PFFs, and promoted the expression of HDR repair factor. At the optimal concentration of 250 nmol/L, PD0325901 increased the repair efficiency of ssODN-mediated GFP reporter vector by 58.8% and the directed editing efficiency of PFF DMD and ROSA26 locus by 48.16% and 17.64%, respectively. The results show that MEK inhibitor PD0325901 significantly promotes the efficiency of ssODN-mediated homologous-directed repair in the porcine genome, thus offering a new idea to generate genetically modified pigs more effectively.

Novel oncogene COPS3 interacts with Beclin1 and Raf-1 to regulate metastasis of osteosarcoma through autophagy

BackgroundExpression of COP9 signalosome subunit 3 (COPS3), an oncogene overexpressed in osteosarcoma, has been demonstrated to be significantly correlated with tumor metastasis. However, the underlying mechanism by which COPS3 promotes metastasis of osteosarcoma and its role in autophagy remain unknown.MethodsThe expression of COPS3 was detected in primary osteosarcoma tissues and matching lung metastasis tissues by immunohistochemistry (IHC). The effect of COPS3 on the metastasis of osteosarcoma cells was investigated by transwell, wound healing assays and animal studies. Indicated proteins was analyzed by western blotting when COPS3 was knockdown or overexpressed. The COPS3 Interacting protein was determined by immunoprecipitation assay. The relationship between COPS3 and autophagy was detected by western blotting and immunofluorescence.ResultsWe found that knockdown of COPS3 significantly reduced the lung metastasis of osteosarcoma cells in a mouse model, coinciding with downregulation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling. The silencing of COPS3 also inhibited the epithelial–mesenchymal transition (EMT) through the 90 kDa ribosomal S6 kinases (RSK), a family of signal transduction proteins downstream of MEK/ERK. Reciprocal immunoprecipitation assays revealed that COPS3 directly interacts with Raf-1, an upstream regulator of MEK/ERK. Surprisingly, Beclin1, an important autophagic protein, appeared in the COPS3-immunoprecipitates, along with the autophagic markers LC3-I and LC3-II. Loss of COPS3 completely inhibited H2O2-induced autophagic flux and reduced Beclin1 expression. Additionally, autophagy inhibitor or silencing of Beclin1 both decreased cell metastasis.ConclusionsTaken together, these data reveal a novel function of COPS3 in the regulation of autophagy and highlight the relationship between autophagy and metastasis in osteosarcoma cells.

Wedelolactone Enhances Osteoblastogenesis through ERK- and JNK-mediated BMP2 Expression and Smad/1/5/8 Phosphorylation.

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 μg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.

A Combination of Chitosan, Cellulose, and Seaweed Polysaccharide Inhibits Postoperative Intra-abdominal Adhesion in Rats.

Intra-abdominal adhesion is a common complication after laparotomy. Conventional therapeutic strategies still cannot safely and effectively prevent this disorder. In this study, a combination of chitosan, cellulose, and seaweed polysaccharide (thereafter referred as CCS) was developed to significantly alleviate the formation of postoperative adhesion in rats with abdominal trauma. Transforming growth factor β1 (TGF-β1, an important promoter of fibrosis) and its downstream factors-namely, alpha-smooth muscle actin and plasminogen activator inhibitor-1 (PAI-1)-were effectively suppressed by CCS in vivo, and as a result, the activation of tissue plasminogen activator (tPA, may generate plasmin that is a fibrinolytic factor capable of breaking down fibrin) was significantly promoted, presenting antifibrosis effects of CCS. In addition, the activity of kinases [e.g., transforming growth factor-activated kinase 1 (TAK1), c-Jun N-terminal kinase (JNK)/Stress-activated Protein Kinase (SAPK), and p38] in the mitogen-activated protein kinase (MAPK) inflammation signaling pathway was also significantly suppressed by CCS in vivo, demonstrating anti-inflammatory functions of CCS. The histologic studies further confirmed the role of CCS in the inhibition of fibrosis, collagen deposition, inflammation, and vascular proliferation. These results indicate the clinical potential of CCS in the treatment of postoperative intra-abdominal adhesion. CCS may induce both antifibrosis and anti-inflammatory effects, potentially inhibiting the postoperative intra-abdominal adhesion. For antifibrosis effects, the expression of PAI-1 (a key factor for the adhesion formation) can be regulated by different TGF-β1-associated signaling pathways, such as the Smads/p53 pathway, metalloproteinase/tissue inhibitor of matrix metalloproteinases pathway, Mitogen-activated Extracellular signal-regulated Kinase (MEK)/extracellular regulated protein kinase (ERK) pathway, and Yes-associated protein/transcriptional coactivator with PDZ-binding motif pathway. Following the downregulation of PAI-1 achieved by CCS, the activation of tPA (which may generate plasmin that is a fibrinolytic factor capable of breaking down fibrin) is significantly promoted. For anti-inflammation effects, CCS may suppress the phosphorylation of classic kinases (e.g., TAK1, JNK, and p38) in the MAPK signaling pathway. In addition to the MAPK pathway, inflammatory pathways, such as Nuclear Factor-κ-gene Binding(NF-κB), MEK/ERK, and Ras homologue protein/Rho associated coiled coil forming protein, are associated with the formation of intra-abdominal adhesion. Therefore, the prevention mechanisms of CCS will be further investigated in the future, with a hope of fully understanding of antiadhesion effects.

Mechanisms for DNA-damaging agent-induced inactivation of ErbB2 and ErbB3 via the ERK and p38 signaling pathways.

Cisplatin (CDDP) and doxorubicin (DOX) are chemotherapeutic drugs that trigger apoptosis by inducing DNA-damage. A previous study using breast cancer cells demonstrated the negative feedback modulation of the epidermal growth factor receptor (EGFR) and receptor tyrosine-protein kinase erbB-2 (ErbB2) via extracellular signal-regulated kinase (ERK)-mediated phosphorylation of conserved Thr-669 and Thr-677 residues, respectively, in the juxtamembrane domain. In addition, CDDP has been identified to cause negative feedback inhibition of activated EGFR in lung cancer cells. In the present study, the role of phosphorylation in the feedback control of the ErbB2/ErbB3 heterodimer in human breast and gastric cancer cells was investigated. Phosphorylation of ErbB2 at Thr-677 was induced by CDDP and DOX, which in turn reduced tyrosine autophosphorylation of ErbB2 and ErbB3. Treatment with trametinib, a mitogen-activated protein kinase inhibitor that blocks ERK-mediated Thr-677 phosphorylation, and substitution of Thr-677 to alanine, blocked the feedback inhibition of ErbB2 and ErbB3. In addition, these agents caused the degradation of ErbB proteins through the activation of p38 mitogen-activated protein kinase (p38) and ERK. These results demonstrate that chemotherapeutic agents trigger ERK- and p38-mediated post-translational downregulation of ErbB receptors.

Dabrafenib plus trametinib for compassionate use in metastatic melanoma: A STROBE-compliant retrospective observational postauthorization study.

The main objective of the study was to evaluate the efficacy and safety of dabrafenib alone or combined with trametinib for compassionate use in patients with metastatic melanoma.This retrospective, observational study involved 135 patients with unresectable stage IIIC or stage IV melanoma from an expanded-access program at 30 Spanish centers.Forty-eight patients received dabrafenib monotherapy and 87 received combination dabrafenib and trametinib; 4.4% and 95.6% of the patients had stage IIIC and IV melanoma, respectively. All patients showed BRAF mutations in their primary or metastatic lesions; 3 were positive for V600K while the remainder had V600E or V600+. A positive response to treatment was reported in 89.3% of the patients. Overall survival rates at 12 and 24 months were 59.6% (95% confidence interval [CI], 52.5–68.9%) and 36.4% (95% CI, 27.8–45%), respectively. Progression-free survival rates at 12 and 24 months were 39.3% (95% CI, 31.1–47.5%) and 21.6% (95% CI, 14.5–28.7%), respectively. Fifty-seven patients (42.2%) reported cutaneous toxicity of any type, mainly hyperkeratosis (14.8%) and rash (11.9%). The most frequent adverse events were pyrexia (27.4%), asthenia (19.3%), arthralgia (16.9%), and diarrhoea (13.2%).Our results suggest that both dabrafenib alone or in combination with trametinib are effective for compassionate use in terms of response and/or survival rates. However, differences in patients’ prognostic features ought to be considered. No new findings were revealed regarding the safety profiles of either regimen. This is the first study to evaluate the efficacy of these 2 selective BRAF and mitogen-activated extracellular signal-regulated kinase inhibitors in a real-world setting in Spain.

Therapeutic efficacy and safety of combined <em>BRAF</em> and MEK inhibition in patients with malignant melanoma: a meta-analysis

BackgroundRecent clinical studies have shown that initial therapy with combined BRAF and mitogen-activated extracellular signal-regulated kinase (MEK) inhibition is more effective in metastatic melanoma than single-agent BRAF inhibitors. However, the response rates with single-agent BRAF are low. Thus, the objective of this study was to conduct a meta-analysis of randomized controlled trials to compare the efficacy and adverse events risk between mono-therapy and combination therapy.Materials and methodsSearches were made in PubMed and EMBASE electronic databases and conference abstracts published by the American Society of Clinical Oncology from 2000 to 2017. Outcomes included overall response, progression-free survival, and overall survival, as well as the incidence rate of adverse events.ResultsEight trials comprising 2,664 patients were included in the meta-analysis. Patients with combined therapies showed superior results compared to those with BRAF inhibitors alone for the following: overall response rate (combined relative risk [RR] =1.34, 95% confidence interval [95% CI]: 1.24–1.45, P<0.00001), progression-free survival (combined hazards ratio [HR] =0.58, 95% CI: 0.52–0.64, P<0.00001), and overall survival rate (combined HR =0.70, 95% CI: 0.62–0.80, P<0.00001). Patients with combination therapies had higher incidence of adverse events including pyrexia (combined RR =2.00, 95% CI: 1.40–2.84), nausea (combined RR =1.41, 95% CI: 1.03–1.94), diarrhea (combined RR =1.50, 95% CI: 1.08–2.06), and vomiting (combined RR =1.87, 95% CI: 01.52–2.31) compared to those with BRAF inhibitors alone.ConclusionThese data suggested that the combined BRAF and MEK inhibition was associated with a significant improvement in overall response, progression-free survival, and overall survival, but increased the incidence of adverse events among the patients with BRAF V600-mutated metastatic melanoma. Further large-scale, high-quality, placebo-controlled, double-blind trials are needed to confirm this conclusion.

Synthetic lethal short hairpin RNA screening reveals that ring finger protein 183 confers resistance to trametinib in colorectal cancer cells

BackgroundThe mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer (CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells, using a synthetic lethal short hairpin RNA (shRNA) screening approach.MethodsWe infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183 (RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA (siRNA) was used to knock down RNF183 transiently. Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the protein abundance. MTT assay, colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.ResultsIn the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8 (IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.ConclusionThe RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.

Association analysis between mitogen-activated protein/extracellular signal-regulated kinase (MEK) gene polymorphisms and depressive disorder in the Han Chinese population

BackgroundRecent research findings suggest that BDNF and BDNF signaling pathways participate in the development of major depressive disorder. Mitogen-activated extracellular signal-regulated kinase (MEK) is the most important kinase in the extracellular signal-regulated kinase pathway, and the extracellular signal-regulated kinase pathway is the key signaling pathway of BDNF, so it may play a role in development of depressive disorder.The aim of this study is to investigate the association between polymorphisms of the MAP2K1 (also known as MEK) gene and depressive disorder. ResultsThree single nucleotide polymorphisms (SNPs), were significantly associated with depressive disorder: rs1549854 (p = 0.006), rs1432441 (p = 0.025), and rs7182853 (p = 0.039). When subdividing the sample by gender, two of the SNPs remained statistically associated with depressive disorder in females: rs1549854 (p = 0.013) and rs1432441 (p = 0.04). ConclusionThe rs1549854 and rs1432441 polymorphisms of the MAP2K1 gene may be associated with major depressive disorder, especially in females. This study is the first to report that the MAP2K1 gene may be a genetic marker for depressive disorder.

Lipopolysaccharide Stimulates Surfactant Protein-A in Human Renal Epithelial HK-2 Cells through Upregulating Toll-like Receptor 4 Dependent MEK1/2-ERK1/2-NF-κB Pathway.

Background:Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.Methods:Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.Results:HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.Conclusions:The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

Metastatic BRAF K601E-mutated melanoma reaches complete response to MEK inhibitor trametinib administered for over 36\xa0months

BackgroundThe BRAF K601E mutation occurs in 5% of patients with melanoma, and is the third most common type of BRAF mutation. However, treatment with BRAF and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors is only approved in patients with BRAF V600-positive melanoma, and patients with K601E-mutated melanoma do not have access to such drugs.Case presentationA female patient was diagnosed with high tumor burden metastatic melanoma harboring the BRAF K601E mutation. After chemotherapy failure, she underwent compassionate treatment with trametinib. Trametinib showed good activity and efficacy, with 48% shrinkage of a metastatic lymphadenopathy after 4 months’ treatment. However, the patient reported treatment-related skin toxicity that required dosage reduction and a personalized intermittent trametinib dosing schedule. After over 36 months from the first trametinib administration, and resection of a metastatic lymphadenopathy, the patient experienced complete response.ConclusionsThis case report shows that trametinib could be a valid therapeutic option in patients with metastatic melanoma harboring the rare BRAF K601E mutation.

The mitogen-activated protein kinase pathway in melanoma part I - Activation and primary resistance mechanisms to BRAF inhibition.

Mitogen-activated protein kinase (MAPK) pathway has an important role in normal cells and can be activated under physiological conditions. MAPK pathway activation is a fundamental step in several intracellular processesrequiring a sequential phosphorylation of the different pathway components. In normal cells, when MAPK pathway activation occurs, it leads to cell growth and differentiation. In order to prevent persistent MAPK pathway activation, physiological upstream negative feedbackalso takes place. In cells harbouring BRAFV600 mutations, the process leading to MAPK pathway activation is different, and the negative physiological feedback does not existthus leading to permanent MAPK pathway activation, which ultimately can lead to uncontrolled proliferation. Targeted therapy with rapidly accelerated fibrosarcoma - B (BRAF) and/or mitogen-activated extracellular signal-regulated kinase kinase (MEK) inhibitors is indicated in patients with metastatic melanoma harboring BRAFV600 mutations. However, several different resistance mechanisms to this therapy were identified. In this review, we focus on primary or intrinsic resistance mechanisms to BRAF and MEK inhibition. In this setting, although a BRAF mutation is identified, there is no response to treatment with either BRAF or MEK inhibitor.

The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic β cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca2+ increase ([Ca2+] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 μm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner.

Combined blockade of Tim-3 and MEK inhibitor enhances the efficacy against melanoma

Insights into the role of the mitogen-activated protein kinase (MAPK) pathway and immune checkpoints have led combined targeted therapy and immunotherapy to be a promising regimen. Trametinib, as a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, has demonstrated effectiveness in patients with advanced melanoma. T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3), an immune checkpoint molecule, participates in multiple negative regulation of antitumor immunity. We for the first time to our knowledge reported the combination of trametinib and anti-Tim-3 monoclonal antibody (mAb) in treating B16-F10 melanoma mice. We discovered that trametinib remarkably promoted apoptosis and inhibited cell proliferation while inhibition of MEK improved the expression of Tim-3 and caused the decrease of CD8+ T cells; to the contrary, anti-Tim-3 mAb enhanced antitumor immunity by stimulating CD8+ T cells, thus the combined therapy produced potent antitumor effect cooperatively. Taken together, our study provides compelling evidence for combining trametinib and anti-Tim-3 mAb as a potential valuable regimen in treating melanoma.

Sorafenib inhibits 5-fluorouracil-resistant gastric cancer cell growth

Background: Sorafenib is a multi-kinase inhibitor used in the treatment of various cancers. This study investigated the inhibitory effect of sorafenib on xenograft models of gastric cancer cells and 5-fluorouracil (5-FU)-resistant cells.Methods: The half-maximal inhibitory concentration (IC50) of sorafenib in NCI-N87 cells was determined. Xenograft models were established using BALB/c nude mice and were divided into four groups treated with vehicle, sorafenib (20 mg kg−1 day−1), 5-FU (50 mg kg−1 week−1), or a combination of sorafenib (20 mg kg−1 day−1) plus 5-FU (50 mg kg−1 week−1). 5-FU-resistant NCI-N87 cells were established by repeated exposure to 5-FU.Results: Sorafenib inhibited NCI-N87 cell growth in a concentration-dependent manner with a mean IC50 of 16.345 ± 5.391 μM. Phosphorylation levels of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase in these cells decreased in a dose-dependent manner after exposure to sorafenib. Sorafenib induced the activation of caspase-3, and its combination with 5-FU more effectively inhibited the growth of xenograft tumors than either sorafenib or 5-FU alone (p < 0.05). Sorafenib markedly inhibited 5-FU-resistant NCI-N87 cell growth as well as sphere formation in both parental and 5-FU-resistant NCI-N87 cells.Conclusions: The sorafenib and 5-FU combination exhibited enhanced antitumor effects in a gastric cancer xenograft model and inhibited 5-FU-resistant cell proliferation and sphere formation. These findings suggest that sorafenib is useful in overcoming gastric cancer resistance to conventional chemotherapy.

Puerarin inhibits M2 polarization and metastasis of tumor-associated macrophages from NSCLC xenograft model via inactivating MEK/ERK 1/2 pathway.

Non-small cell lung carcinoma (NSCLC) metastasis is responsible for most of cancer-related mortality. The tumor associated macrophages (TAMs) are known to be crucial cells in lung cancer and are usually divided into two antagonistic types, M1 and M2. Puerarin has a wide spectrum of pharmacological properties. The present study explores puerarin on macrophage polarization and metastasis of NSCLC. The results demonstrated that puerarin inhibited tumor growth and tumor volumes in NSCLC xenograft model, increased M1 markers [CD197+, inducible nitric oxide synthase (iNOS)+, CD40+)] and reduced M2 markers (CD206+, Arg-1+ and CD163+). Besides, puerarin elevated the level of pro-inflammatory cytokine interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-12, decreased the expression of pro-tumor cytokines IL-10, IL-4 and transforming growth factor (TGF)-β. To explore whether puerarin directly acts on macrophages, we purified macrophages from NSCLC model, the results showed that puerarin inhibited macrophages polarized to M2 phenotype and did not require the auxiliary of other cells. In addition, puerarin suppressed the invasion and migration of NSCLC macrophages, restrained the expression of angiogenesis factors. Puerarin also inhibited the activation of mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) 1/2 pathway through inhibition of ERK nucleus translocation. Finally, IL-4 induced M2 macrophage polarization and metastasis were partially offset by puerarin through inactivating the MEK/ERK 1/2 pathway. Taken together, this study validated that puerarin is able to skew macrophage populations back to M1 subsets to stimulate antitumor effects and suggests puerarin is a negative metastatic regulator of NSCLC.

Moxibustion eases chronic inflammatory visceral pain through regulating MEK, ERK and CREB in rats.

AIMTo investigate the effects of herb-partitioned moxibustion (HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase (MEK)1, extracellular signal-regulated kinase (ERK)1/2 and cAMP response element binding protein (CREB) in spinal cord of rats with chronic inflammatory visceral pain (CIVP), and to explore the central mechanism of HPM in treating CIVP.METHODSMale Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide (DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu (ST25) and Qihai (CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor (p)MEK1, pERK1/2 and pCREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB mRNA in rat spinal cord were detected using real-time polymerase chain reaction.RESULTSCompared with the normal group, the AWR scores were increased significantly (P < 0.01) and the MWT and TWL scores were decreased significantly (P < 0.05) in the model, sham-HPM and DMSO groups. Compared with the model group, the AWR scores were decreased significantly (P < 0.01) and the MWT and TWL scores were increased significantly in the HPM and MEK-inhibitor groups (P < 0.05). Compared with the sham-HPM and DMSO groups, the AWR scores were decreased significantly (P < 0.01) and the MWT and TWL scores were increased significantly (P < 0.05) in the HPM and MEK-inhibitor groups. Compared with the normal group, the expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups (P < 0.01 or < 0.05). Compared with the model group, the expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups (P < 0.01 or < 0.05). Compared with the sham-HPM and DMSO groups, expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups (P < 0.01 or < 0.05).CONCLUSIONHPM down-regulates protein phosphorylation of MEK1, ERK1/2 and CREB, and mRNA expression of MEK, ERK and CREB, inhibiting activation of the MEK/ERK/CREB signaling pathway in the spinal cord of CIVP rats, which is possibly a critical central mechanism of the analgesic effect of HPM.

Ectopic overexpression of filamin C scaffolds MEK1/2 and ERK1/2 to promote the progression of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) invasion and metastasis are mediated by a complicated signal transduction network and downstream cytoskeletal and adhesion molecules. In this study, a microarray-based analysis revealed a dramatic increase in filamin C (FLNC), which is commonly expressed in muscle rather than in liver cells, in the two metastatic HCC cell lines MHCC97L and HCCLM3. Clinicopathological studies showed that increased FLNC expression was associated with microvascular invasion and poor prognosis. Specific hypomethylation was identified within the FLNC promoter region in HCC cell lines and patient tumor samples, which might contribute to the ectopic overexpression of FLNC. FLNC downregulation inhibited cell migration and impaired cell proliferation and promoted apoptosis. Mechanistic studies suggested that FLNC interacts with mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) and that FLNC downregulation inhibited MEK1/2 and ERK1/2 activation. Xenographic tumor transplantation experiments in nude mice further confirmed the role of FLNC in HCC progression and metastasis. Our results reveal a novel mechanism by which the cytoskeletal protein FLNC enhances the mitogen-activated protein kinase signaling pathway during tumorigenesis.

Sensitivity Analysis of the NPM-ALK Signalling Network Reveals Important Pathways for Anaplastic Large Cell Lymphoma Combination Therapy

A large subset of anaplastic large cell lymphoma (ALCL) patients harbour a somatic aberration in which anaplastic lymphoma kinase (ALK) is fused to nucleophosmin (NPM) resulting in a constitutively active signalling fusion protein, NPM-ALK. We computationally simulated the signalling network which mediates pathological cell survival and proliferation through NPM-ALK to identify therapeutically targetable nodes through which it may be possible to regain control of the tumourigenic process. The simulations reveal the predominant role of the VAV1-CDC42 (cell division control protein 42) pathway in NPM-ALK-driven cellular proliferation and of the Ras / mitogen-activated ERK kinase (MEK) / extracellular signal-regulated kinase (ERK) cascade in controlling cell survival. Our results also highlight the importance of a group of interleukins together with the Janus kinase 3 (JAK3) / signal transducer and activator of transcription 3 (STAT3) signalling in the development of NPM-ALK derived ALCL. Depending on the activity of JAK3 and STAT3, the system may also be sensitive to activation of protein tyrosine phosphatase-1 (SHP1), which has an inhibitory effect on cell survival and proliferation. The identification of signalling pathways active in tumourigenic processes is of fundamental importance for effective therapies. The prediction of alternative pathways that circumvent classical therapeutic targets opens the way to preventive approaches for countering the emergence of cancer resistance.

Epithelial-mesenchymal transition of ovarian cancer cells is sustained by Rac1 through simultaneous activation of MEK1/2 and Src signaling pathways.

Epithelial-mesenchymal transition (EMT) is regarded as a crucial contributing factor to cancer progression. Diverse factors have been identified as potent EMT inducers in ovarian cancer. However, molecular mechanism sustaining EMT of ovarian cancer cells remains elusive. Here, we show that the presence of SOS1/EPS8/ABI1 complex is critical for sustained EMT traits of ovarian cancer cells. Consistent with the role of SOS1/EPS8/ABI1 complex as a Rac1-specific guanine nucleotide exchange factor, depleting Rac1 results in the loss of most of mesenchymal traits in mesenchymal-like ovarian cancer cells while expressing constitutively active Rac1 leads to EMT in epithelial-like ovarian cancer cells. With the aid of clinically tested inhibitors targeting various EMT-associated signaling pathways, we show that only combined treatment of MEK1/2 and Src inhibitors can abolish constitutively active Rac1-led EMT and mesenchymal traits displayed by mesenchymal-like ovarian cancer cells. Further experiments also reveal that EMT can be induced in epithelial-like ovarian cancer cells by co-expressing constitutively active MEK1 and Src rather than either alone. As the activities of Erk and Src are higher in ovarian cancer cells with constitutively active Rac1, we conclude that Rac1 sustains ovarian cancer cell EMT through simultaneous activation of MEK1/2 and Src signaling pathways. Importantly, we demonstrate that combined use of MEK1/2 and Src inhibitors effectively suppresses development of intraperitoneal xenografts and prolongs the survival of ovarian cancer-bearing mice. This study suggests that cocktail of MEK1/2 and Src inhibitors represents an effective therapeutic strategy against ovarian cancer progression.