BRAF V600E mutation mediates invasive and growth features in ameloblastoma.

OBJECTIVES: Ameloblastoma (AM), a locally aggressive tumor with extensive growth capacity, causes significant damage to the jaw and affects facial appearance. Although the high prevalence of BRAF V600E mutation in AM is known, its specific impacts on patients with AM remain unclear. Thus, the present study investigated the role of BRAF V600E mutation, thereby focusing on its impact on AM invasion and growth. MATERIALS AND METHODS: Immunohistochemical analysis was used to compare BRAF V600E, MMP2, MMP9, and Ki-67 expressions in AM (n = 49), normal oral mucosa (NOM) (n = 10), and odontogenic keratocyst (OKC) (n = 15) tissues. AM was further classified according to the presence or absence of BRAF V600E. The relationship between BRAF V600E and invasion as well as growth was evaluated. In addition, correlation analysis was performed using immunohistochemistry and confirmed via double-labeling immunofluorescence. Finally, comparative analyses using mass spectrometry, immunohistochemistry, and immunofluorescence were performed to explore and identify underlying mechanisms. RESULTS: AM exhibited a higher incidence of BRAF V600E mutation than NOM and OKC. BRAF V600E expression was positively correlated with the invasion-associated proteins MMP2 and MMP9 and the growth-related protein Ki-67. Proteomic data revealed that BRAF V600E primarily activates the MAPK signaling pathway in AM, particularly driving the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). CONCLUSIONS: In summary, the findings suggested that the BRAF V600E mutation enhances the invasion and growth abilities of AM via the MAPK/ERK signaling pathway. Thus, targeting BRAF V600E or the MAPK/ERK pathway may be a potential AM therapy.

Role of miR-106-mediated mitogen-activated protein kinase signaling pathway in oxidative stress injury and inflammatory infiltration in the liver of the mouse with gestational hypertension.

We aim to investigate the role of miR-106-mediated mitogen-activated protein kinase (MAPK) signaling pathway in oxidative stress (OS) injury and inflammatory infiltration in the liver of the mouse with gestational hypertension (GH). Ninety specific pathogen-free mice (Kunming species) during middle to late gestation were selected for the study. Fifteen mice were used as control, while the rest were used for establishing the GH model. The mice were assigned to six groups: normal group (normal gestation), model group (GH model), negative control group (GH model, intravenously injected with negative control vector), miR-106a-mimic group (GH model, intravenously injected with vector overexpressing miR-106a, which mimics the overexpression of endogenous mature miR-106a), SB203580 group (GH model, intravenously injected with MAPK pathway inhibitor SB203580), and miR-106a-mimic+SB203580 group (GH model, intravenously injected with SB203580 and vector overexpressing miR-106a). Fourteen days after electrical stimulation, all the groups except for the normal group had elevated blood pressure vs those on day 0 and 7. Compared with the normal group, the other groups had lower levels of miR-106a expression, nitric oxide, nitric oxide synthase, catalase, superoxide dismutase, S cell ratio, and interleukin-4 (IL-4) and IL-10 in the serum and liver as opposed to increased levels of blood pressure, p38MAPK mRNA expression, p-p38MAPK positive expression rate, protein expressions of p-p38MAPK, p-ERK, and p-JNK, H2 O2 and malondialdehyde in liver, G0/G1 cell ratio, apoptosis rate, and IL-6, interferon-γ (IFN-γ), and IFN-α in the serum and liver (all P < .05). The miR-106 overexpression or inhibiting MAPK signaling pathway can attenuate OS injury and inflammatory response in the liver of the mouse with GH, and the effect can be even better if both miR-106a overexpression and inhibiting MAPK pathway are applied. In conclusion, miR-106a overexpression can inhibit OS injury and inflammatory infiltration in the liver of the mouse with GH by mediating MAPK signaling pathway.

Curcumin attenuates renal ischemia reperfusion injury via JNK pathway with the involvement of p300/CBP-mediated histone acetylation.

Apoptosis is proved responsible for renal damage during ischemia/reperfusion. The regulation for renal apoptosis induced by ischemia/reperfusion injury (IRI) has still been unclearly characterized to date. In the present study, we investigated the regulation of histone acetylation on IRI-induced renal apoptosis and the molecular mechanisms in rats with the application of curcumin possessing a variety of biological activities involving inhibition of apoptosis. Sprague-Dawley rats were randomized into four experimental groups (SHAM, IRI, curcumin, SP600125). Results showed that curcumin significantly decreased renal apoptosis and caspase-3/-9 expression and enhanced renal function in IRI rats. Treatment with curcumin in IRI rats also led to the decrease in expression of p300/cyclic AMP response element-binding protein (CBP) and activity of histone acetyltransferases (HATs). Reduced histone H3 lysine 9 (H3K9) acetylation was found near the promoter region of caspase-3/-9 after curcumin treatment. In a similar way, SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), also attenuated renal apoptosis and enhanced renal function in IRI rats. In addition, SP600125 suppressed the binding level of p300/CBP and H3K9 acetylation near the promoter region of caspase-3/-9, and curcumin could inhibit JNK phosphorylation like SP600125. These results indicate that curcumin could attenuate renal IRI via JNK/p300/CBP-mediated anti-apoptosis signaling.

Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling.

BACKGROUND: Cardiac kinases play a critical role in the development of heart failure, and represent potential tractable therapeutic targets. However, only a very small fraction of the cardiac kinome has been investigated. To identify novel cardiac kinases involved in heart failure, we used an integrated transcriptomics and bioinformatics analysis and identified Homeodomain-Interacting Protein Kinase 2 (HIPK2) as a novel candidate kinase. The role of HIPK2 in cardiac biology is unknown. METHODS: We used the Expression2Kinase algorithm for the screening of kinase targets. To determine the role of HIPK2 in the heart, we generated cardiomyocyte (CM)-specific HIPK2 knockout and heterozygous mice. Heart function was examined by echocardiography, and related cellular and molecular mechanisms were examined. Adeno-associated virus serotype 9 carrying cardiac-specific constitutively active MEK1 (TnT-MEK1-CA) was administrated to rescue cardiac dysfunction in CM-HIPK2 knockout mice. RESULTS: To our knowledge, this is the first study to define the role of HIPK2 in cardiac biology. Using multiple HIPK2 loss-of-function mouse models, we demonstrated that reduction of HIPK2 in CMs leads to cardiac dysfunction, suggesting a causal role in heart failure. It is important to note that cardiac dysfunction in HIPK2 knockout mice developed with advancing age, but not during development. In addition, CM-HIPK2 knockout mice and CM-HIPK2 heterozygous mice exhibited a gene dose-response relationship of CM-HIPK2 on heart function. HIPK2 expression in the heart was significantly reduced in human end-stage ischemic cardiomyopathy in comparison to nonfailing myocardium, suggesting a clinical relevance of HIPK2 in cardiac biology. In vitro studies with neonatal rat ventricular CMscorroborated the in vivo findings. Specifically, adenovirus-mediated overexpression of HIPK2 suppressed the expression of heart failure markers, NPPA and NPPB, at basal condition and abolished phenylephrine-induced pathological gene expression. An array of mechanistic studies revealed impaired extracellular signal-regulated kinase 1/2 signaling in HIPK2-deficient hearts. An in vivo rescue experiment with adeno-associated virus serotype 9 TnT-MEK1-CA nearly abolished the detrimental phenotype of knockout mice, suggesting that impaired extracellular signal-regulated kinase signaling mediated apoptosis as the key factor driving the detrimental phenotype in CM-HIPK2 knockout mice hearts. CONCLUSIONS: Taken together, these findings suggest that CM-HIPK2 is required to maintain normal cardiac function via extracellular signal-regulated kinase signaling.

Autosomal dominant polycystic kidney disease and pioglitazone for its therapy: a comprehensive review with an emphasis on the molecular pathogenesis and pharmacological aspects.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited chronic kidney disorder (CKD) that is characterized by the development of numerous fluid-filled cysts in kidneys. It is caused either due to the mutations in the PKD1 or PKD2 gene that encodes polycystin-1 and polycystin-2, respectively. This condition progresses into end-stage renal disorder if the renal or extra-renal clinical manifestations remain untreated. Several clinical trials with a variety of drugs have failed, and the only Food and Drugs Administration (FDA) approved drug to treat ADPKD to date is tolvaptan that works by antagonizing the vasopressin-2 receptor (V2R). The pathology of ADPKD is complex and involves the malfunction of different signaling pathways like cAMP, Hedgehog, and MAPK/ERK pathway owing to the mutated product that is polycystin-1 or 2. A measured yet substantial number of preclinical studies have found pioglitazone to decrease the cystic burden and improve the renal function in ADPKD. The peroxisome proliferator-activated receptor-gamma is found on the epithelial cells of renal collecting tubule and when it gets agonized by pioglitazone, confers efficacy in ADPKD treatment through multiple mechanisms. There is only one clinical trial (ongoing) wherein it is being assessed for its benefits and risk in patients with ADPKD, and is expected to get approval from the regulatory body owing to its promising therapeutic effects. This article would encompass the updated information on the epidemiology, pathophysiology of ADPKD, different mechanisms of action of pioglitazone in the treatment of ADPKD with preclinical and clinical shreds of evidence, and related safety updates.

S100A12 promotes inflammation and apoptosis in ischemia/reperfusion injury via ERK signaling in vitro study using PC12 cells.

S100A12 is a member of S100 calcium-binding proteins with effect to promote inflammation in brain damage and stroke. However, the role of S100A12 in ischemia/reperfusion (I/R) remains to be clarified. This study aimed to explore the effect of S100A12 on I/R and discover the possible mechanism. Oxygen-glucose deprivation and reperfusion (OGD/R) was used to induce I/R injury model in vitro. Knockdown or overexpression of S100A12 was utilized to explore the role of S100A12 in I/R-induced inflammation and apoptosis. Results indicated that S100A12 expression was dramatically upregulated after OGD/R. Knockdown of S100A12 inhibited, while overexpression of S100A12 enhanced, the activation of ERK1/2 protein. OGD/R also triggered the occurrence of inflammation and oxidative stress, while these effects were blunted by S100A12 silencing and aggravated by S100A12 overexpression, and the presence of MAP kinase signaling system (ERK) inhibitor MK-8353 counteracted the effect of S100A12 overexpression. Besides, S100A12 silencing abolished, while its overexpression restored, the OGD/R-induced increased apoptosis rate and pro-apoptotic proteins expression. Similarly, ERK inhibitor MK-8353 reversed the effects of S100A12 overexpression. In conclusion, S100A12 promoted OGD/R-induced inflammation, oxidative stress and apoptosis via activation of ERK signaling in vitro.

IL-15 and IFN-γ signal through the ERK pathway to inhibit HCV replication, independent of type I IFN signaling.

Despite effective new treatments for Hepatitis C virus (HCV) infection, development of drug resistance, safety concerns and cost are remaining challenges. More importantly, there is no vaccine available against hepatitis C infection. Recent data suggest that there is a strong correlation between spontaneous HCV clearance and human NK cell function, particularly IFN-γ production. Further, IL-15 has innate antiviral activity and is also one of the main factors that activates NK cells to produce IFN-γ. To examine whether IL-15 and IFN-γ have direct antiviral activity against HCV, Huh7.5 cells were treated with either IFN-γ or IL-15 prior to HCV infection. Our data demonstrate that IFN-γ and IL-15 block HCV replication in vitro. Additionally, we show that IL-15 and IFN-γ do not induce anti-HCV effects through the type I interferon signaling pathway or nitric oxide (NO) production. Instead, IL-15 and IFN-γ provide protection against HCV via the ERK pathway. Treatment of Huh7.5 cells with a MEK/ERK inhibitor abrogated the anti-HCV effects of IL-15 and IFN-γ and overexpression of ERK1 prevented HCV replication compared to control transfection. Our in vitro data support the hypothesis that early production of IL-15 and activation of NK cells in the liver lead to control of HCV replication.

Fibroblast growth factor receptor 3-mediated reactivation of ERK signaling promotes head and neck squamous cancer cell insensitivity to MEK inhibition.

Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has been a longstanding challenge for head and neck oncologists, and current treatments still have limited efficacy. ERK is aberrantly overexpressed and activated in HNSCC. Herein, we aimed to investigate the cause of the limited therapeutic effect of selumetinib, a selective inhibitor of MEK in HNSCC, as MEK/ERK reactivation inevitably occurs. We assessed the effects of combining selumetinib with fibroblast growth factor receptor 3 (FGFR3) inhibitor (PD173074) on tumor growth. Selumetinib transiently inhibited MAPK signaling and reactivated ERK signaling in HNSCC cells. Rebound in the ERK and Akt pathways in HNSCC cells was accompanied by increased FGFR3 signaling after selumetinib treatment. Feedback activation of FGFR3 was a result of autocrine secretion of the FGF2 ligand. The FGFR3 inhibitor PD173074 prevented MAPK rebound and sensitized the response of HNSCC cells to selumetinib. These results provided rational therapeutic strategies for clinical studies of this subtype of patients that show a poor prognosis with selumetinib. Our data provide a rationale for combining a MEK inhibitor with inhibitors of feedback activation of FGFR3 signaling in HNSCC cells. ERK rebound as a result of the upregulation of FGFR3 and the ligand FGF2 diminished the antitumor effects of selumetinib, which was overcome by combination treatment with the FGFR3 inhibitor.

WNT5A promotes migration and invasion of human osteosarcoma cells via SRC/ERK/MMP-14 pathway.

WNT5A, a representative ligand of activating several non-canonical WNT signal pathways, plays significant roles in oncogenesis and tumor inhibition. It has been shown that the non-receptor tyrosine kinase SRC is required for WNT5A-induced invasion of osteosarcoma cells. However, the precise molecular mechanism underlying WNT5A/SRC-mediated osteosarcoma cells invasion remains poorly defined. The study was designed to explore the role of ERK1/2 in WNT5A/SRC-induced osteosarcoma cells invasion and the downstream target of the SRC/ERK1/2 signalings. We found that WNT5A (100 ng/mL) remarkably stimulated migration and invasion of human osteosarcoma MG-63 cells, whereas inhibiting either SRC kinase activity by siRNA-mediated SRC silence or ERK1/2 phosphorylation by PD98059 treatment suppressed these effects, which suggested that the activation of SRC and ERK1/2 is essential for WNT5A-induced MG-63 cells migration and invasion. Furthermore, ERK1/2 phosphorylation induced by WNT5A was dramatically blocked by SRC siRNA. Additionally, our study further demonstrated that MMP-14 was upregulated after exposure to WNT5A in MG-63 cells, and the increased expression was blocked by SRC siRNA or PD98059. Collectively, these results indicate that WNT5A activates SRC/ERK1/2 signal pathway, leading to the upregulation of MMP-14 expression and MG-63 cells migration and invasion.

The MEK1/2 Inhibitor AZD6244 Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

BACKGROUND [i]BRAF[/i]V600E mutation occurs in approximately 45% of papillary thyroid cancer (PTC) cases, and 25% of anaplastic thyroid cancer (ATC) cases. Vemurafenib/PLX4032, a selective BRAF inhibitor, suppresses extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) signaling and shows beneficial effects in patients with metastatic melanoma harboring the [i]BRAFV600E[/i] mutation. However, the response to vemurafenib is limited in BRAF-mutant thyroid cancer. The present study evaluated the effect of vemurafenib in combination with the selective MEK1/2 inhibitor AZD6244 on cell survival and explored the mechanism underlying the combined effect of vemurafenib and AZD6244 on thyroid cancer cells harboring BRAFV600E. MATERIAL AND METHODS Thyroid cancer 8505C and BCPAP cells harboring the [i]BRAFV600E[/i] mutation were exposed to vemurafenib (0.01, 0.1, and 1 µM) and AZD6244 (0.01, 0.1, and 1 µM) alone or in the indicated combinations for the indicated times. Cell viability was detected by the MTT assay. Cell cycle distribution and induction of apoptosis were detected by flow cytometry. The expression of cyclin D1, P27, (P)-ERK1/2 was evaluated by Western blotting. The effect of vemurafenib or AZD6244 or their combination on the growth of 8505C cells was examined in orthotopic xenograft mouse models [i]in vivo[/i]. RESULTS Vemurafenib alone did not increase cell apoptosis, whereas it decreased cell viability by promoting cell cycle arrest in BCPAP and 8505C cells. AZD6244 alone increased cell apoptosis by inducing cell cycle arrest in BCPAP and 8505C cells. Combination treatment with AZD6244 and vemurafenib significantly decreased cell viability and increased apoptosis in both BCPAP and 8505C cells compared with the effects of each drug alone. AZD6244 alone abolished phospho-ERK1/2 (pERK1/2) expression at 48 h, whereas vemurafenib alone downregulated pERK1/2 at 4-6 h, with rapid recovery of expression, reaching the highest level at 24-48 h. Combined treatment for 48 h completely inhibited pERK1/2 expression. Combination treatment with vemurafenib and AZD6244 inhibited cell growth and induced apoptosis by causing cell-cycle arrest, with the corresponding changes in the expression of the cell cycle regulators p27Kip1 and cyclin D1. Co-administration of vemurafenib and AZD6244 [i]in vivo[/i] had a significant synergistic antitumor effect in a nude mouse model. CONCLUSIONS Vemurafenib activated pERK1/2 and induced vemurafenib resistance in thyroid cancer cells. Combination treatment with vemurafenib and AZD6244 inhibited ERK signaling and caused cell cycle arrest, resulting in cell growth inhibition. Combination treatment in patients with thyroid cancer harboring the [i]BRAFV600E[/i] mutation may overcome vemurafenib resistance and enhance the therapeutic effect.

Jnk1 Deficiency in Hematopoietic Cells Suppresses Macrophage Apoptosis and Increases Atherosclerosis in Low-Density Lipoprotein Receptor Null Mice.

OBJECTIVE: The c-Jun NH2-terminal kinases (JNK) are regulated by a wide variety of cellular stresses and have been implicated in apoptotic signaling. Macrophages express 2 JNK isoforms, JNK1 and JNK2, which may have different effects on cell survival and atherosclerosis. APPROACH AND RESULTS: To dissect the effect of macrophage JNK1 and JNK2 on early atherosclerosis, Ldlr(-/-) mice were reconstituted with wild-type, Jnk1(-/-), and Jnk2(-/-) hematopoietic cells and fed a high cholesterol diet. Jnk1(-/-)→Ldlr(-/-) mice have larger atherosclerotic lesions with more macrophages and fewer apoptotic cells than mice transplanted with wild-type or Jnk2(-/-) cells. Moreover, genetic ablation of JNK to a single allele (Jnk1(+/-)/Jnk2(-/-) or Jnk1(-/-)/Jnk2(+/-)) in marrow of Ldlr(-/-) recipients further increased atherosclerosis compared with Jnk1(-/-)→Ldlr(-/-) and wild-type→Ldlr(-/-) mice. In mouse macrophages, anisomycin-mediated JNK signaling antagonized Akt activity, and loss of Jnk1 gene obliterated this effect. Similarly, pharmacological inhibition of JNK1, but not JNK2, markedly reduced the antagonizing effect of JNK on Akt activity. Prolonged JNK signaling in the setting of endoplasmic reticulum stress gradually extinguished Akt and Bad activity in wild-type cells with markedly less effects in Jnk1(-/-) macrophages, which were also more resistant to apoptosis. Consequently, anisomycin increased and JNK1 inhibitors suppressed endoplasmic reticulum stress-mediated apoptosis in macrophages. We also found that genetic and pharmacological inhibition of phosphatase and tensin homolog abolished the JNK-mediated effects on Akt activity, indicating that phosphatase and tensin homolog mediates crosstalk between these pathways. CONCLUSIONS: Loss of Jnk1, but not Jnk2, in macrophages protects them from apoptosis, increasing cell survival, and this accelerates early atherosclerosis.

[Inhibitory effect of insulin-like growth factor binding protein-related protein 1 on retinal angiogenesis via ERK signaling pathway].

Objective: To explore the inhibitory effect of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), a novel anti-angiogenic factor, on retinal angiogenesis and its underlying molecular mechanisms. Methods: Experimental study. C57BL/6J mice were classified into three groups: control group (n=24), oxygen-induced retinopathy (OIR) non-intervention group (n=24) and OIR intervention group (n=72). The OIR mouse model was established using improved Smith's methods (n= 96). Twelve-day-old mice in the OIR intervention group were randomly assigned into three groups receiving intravitreal injection of recombinant mouse IGFBP-rP1 (50 µg/L, 100 µg/L and 200 µg/L, respectively). Five days later, the proliferative neovascular responses were estimated by quantifying the new vessel area relative to the total retinal area in flattening retinas stained by high molecular FITC-Dextran and counting the number of neovascular cell nuclei breaking through the internal limiting membrane (ILM) in cross-sections. Retinal phosphor-ERK1/2 (p-ERK1/2), ERK1/2 and vascular endothelial growth factor (VEGF) protein expression was assessed by Western blot. Results: In the fluorescence angiograms, irregular neovascularization and fluorescence leakage were observed in the OIR model. In the OIR non-intervention group, the expression of p-ERK1/2 and VEGF was significantly up-regulated in comparison with the control group (t=100.068, P=0.000. t=6.526, P=0.003). The area ratios of new retinal vessels and the number of neovascular cell nuclei in mice receiving intravitreal injection of recombinant mouse IGFBP-rP1 both decreased significantly (F=1920, P=0.000. F=852.387, P=0.000), following the down-regulation of retinal p-ERK1/2 protein expression (F=859.587, P=0.000) and VEGF protein expression (F=24.301, P= 0.000) in a dose-dependent manner (P<0.05). There was no significant difference in ERK1/2 protein expression (P>0.05). Conclusions: IGFBP-rP1 inhibits retinal angiogenesis by blocking ERK signaling pathway and down-regulating VEGF expression. This highlights the potential importance of IGFBP-rP1 serving as a target of gene therapy for retinal neovascularization. (Chin J Ophthalmol, 2017, 53: 207-211).

[The protective effect of the activation of NMDAR1/ERK1/2 signal pathway induced by levodopa on visual cortical neurons in monocular deprivation rats].

Objective: To investigate the protection effects of the activation of NMDAR1(NMDA receptor 1)/ERK1/2 signal pathway on visual cortex nerve cells induced by levodopa in amblyopia rats. Methods: SD rats of SPF grade, were randomly divided into for groups of 9, group A. Control, B. MD group, C. L-levodopa(20 mg·kg(-1))+MD group, D. H-levodopa (80 mg·kg(-1))+MD group, E. H-levodopa+MD+MK801 group, F. H-levodopa+MD+PD98059 group, G. MD+MK801 group, H. MD+PD98059 group, I. MD+DMSO group. Amblyopia rats were made by suture of the right eye. Levodopa was used to treatment amblyopia by gavage, and intervened by intracerebroventricular injection of MK801 and PD98059 respectively. The expression of NR1, p-ERK1/2, ERK1/2, NGF and c-FOS were detected by Western blotting. Nissl staining was used to detect morphological changes of neurons. Neuronal apoptosis was detected by TUNEL method, and detected the expression of Caspase-3, NGF and c-FOS by immunohistochemical staining. One/Two way Chi-square analysis was used for data analysis. LSD-t test was used as comparison between every two groups. Results: The morphology of Nissl bodies in neurons was complete and clear in A group, and the size of Nissl bodies got smaller, and caused karyopyknosis and loss of neurons in visual cortex of B group. Compared with A group, the apoptosis of visual cortical neurons(23.09±2.00 vs. 2.20±0.35, t=12.120, P=0.000) and the number of Caspase-3 postive cells (22.70±1.50 vs. 3.30±0.54, t=12.120, P=0.000)were significantly increased, the expression of NGF(0.31±0.04 vs. 0.74±0.09, t=7.674, P=0.000) and c-FOS(0.25±0.03 vs. 0.57±0.07, t=5.919, P=0.000) and the rats of NGF(8.30±0.82 vs. 35.18±2.01, t=12.37, P=0.0000) and c-FOS (10.84±1.02 vs. 35.68±2.55, t=9.056, P=0.0001) postive cell were decreased significantly in B group. After treatment with levodopa, the morphology of neurons recovered, the apoptosis of visual cortical neurons relieved, the expression of NR1(0.75±0.09 vs. 0.40±0.05, t=8.528, P=0.001) and p-ERK1/2(2.13±0.26 vs. 0.68±0.17, t=3.488, P=0.008) were increased significantly, and the rats of NGF (18.07±0.87 vs. 8.30±0.82, t=8.18, P=0.0000) and c-FOS (19.78±0.91 vs. 10.84±1.02, t=6.543, P=0.0001) postive cells were significantly increased. MK-801 or PD98059 intervention could effectively attenuate the effect of levodopa. It could effectively down-regulated the expression of NR1 (0.53±0.06 vs. 0.95±0.12, t=5.647, P=0.005) and p-ERK1/2(1.52±0.18 vs. 2.58±0.30, t=3.091, P=0.013) interference with MK801 or PD98059 in MD rats. MK-801 or PD98059 intervention further promote the Nissl body volume reduced, neurons karyopyknosis, the apoptosis of visual cortical neurons and Caspase-3 expression, and restrain the expression of NGF and c-FOS. Conclusion: Levodopa played a protective role in visual cortex nerve cells of amblyopia rats at least partially through activation of NMDA-ERK1/2 signal pathway. (Chin J Ophthalmol, 2017, 53: 931-940).

MAPK7 gene controls proliferation, migration and cell invasion in osteosarcoma.

Osteosarcomas (OS) are the most common malignant bone tumors, and the identification of useful tumor biomarkers and target proteins is required to predict the clinical outcome of patients and therapeutic response as well as to develop novel therapeutic strategies. In our previous study, MAPK7 has been identified as a candidate oncogene, and a promising prognostic marker for OS. Sequential activation of protein kinases within the mitogen-activated protein kinase (MAPK) cascades is a common mechanism of signal transduction in many cellular processes. In this study, we investigated the behavior of MAPK7 gene in OS cell lines. Technical viability, proliferation, migration, invasion, and apoptosis were used to evaluate the function of the MAPK7 gene. We evaluated the behavior of the OS cells with MAPK7 gene silenced, not silenced, and exposed to the main chemotherapy drugs used in OS treatment. We found that silenced MAPK7 gene is effective at suppressing cell proliferation, inhibiting cell migration, and invasion. Furthermore, MAPK7 is an important activator of transcription factors and is the main expression modulator of other key genes in the MAPK pathway. In summary, our study suggests that MAPK7 might be a promising therapeutic target for OS. © 2015 Wiley Periodicals, Inc.

The green tea polyphenol EGCG potentiates the antiproliferative activity of sunitinib in human cancer cells.

Sunitinib is a promising drug for clinical applications; however, the efficacy is reduced by the feedback activation of many signaling cascades. In this study, we investigated the ability of (-)-epigallocatechin-3-gallate (EGCG) to synergize with sunitinib and inhibit insulin receptor substrate (IRS)/mitogen-activated protein kinase (MAPK) pathway activation. MCF-7, H460, and H1975 cell lines with PIK3CA mutations were treated with sunitinib or mock treated 0-24 h and then pulsed with 0-50 µM EGCG for another 12 h; cell proliferation and vascular endothelial growth factor (VEGF) secretion were then evaluated. To analyze angiogenesis and VEGF levels in vivo, MCF-7 and H460 xenograft tumors were established. Cell growth signaling cascades were assessed via western blotting in vitro, and tumors were subjected to immunohistochemical analyses to evaluate signaling cascades in vivo. EGCG enhanced the antiproliferation and VEGF secretion-reducing effects of sunitinib in the three tested cell lines. In vivo, EGCG administration at 4 h after sunitinib treatment resulted in greater tumor shrinkage and antiangiogenesis than with sunitinib alone. We further demonstrated that sunitinib exposure induces insulin receptor substrate-1 (IRS-1) upregulation and activation of MAPK signaling. More strikingly, EGCG treatment downregulated IRS-1 levels and suppressed mitogenic effects. In vivo, immunohistochemical analyses demonstrated marked suppression of the IRS/MAPK/p-S6K1 signaling cascade by EGCG, especially after sunitinib treatment. EGCG potentially synergizes with sunitinib due to its ability to suppress the IRS/MAPK signaling induced by sunitinib. We conclude that administration of EGCG after sunitinib treatment represents a promising strategy for the treatment of cancer.

MIR4435-2HG: A novel biomarker for triple-negative breast cancer diagnosis and prognosis, activating cancer-associated fibroblasts and driving tumor invasion through EMT associated with JNK/c-Jun and p38 MAPK signaling pathway activation.

BACKGROUND: Breast cancer has the highest incidence rate and causes the most fatalities among all female cancers worldwide. Triple-negative breast cancer (TNBC) is known for its strong invasiveness and higher rates of recurrence. In this research, we aimed to identify MIR4435-2HG as a promising long non-coding RNA (lncRNA) biomarker and therapeutic target for TNBC. METHODS: Utilizing clinicopathological information and transcriptome data from The Cancer Genome Atlas (TCGA) database, we assessed the clinical relevance of MIR4435-2HG in breast cancer through univariate and multivariate COX regression, receiver operating characteristic (ROC) analysis, as well as Kaplan-Meier survival analysis. To investigate the biological role of MIR4435-2HG in TNBC, we conducted gene set enrichment analysis (GSEA), as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Additionally, we constructed and validated a nomogram to predict disease-free survival (DFS). Both the R package "pRRophetic" and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were employed to forecast the sensitivity to different therapeutics between the high- and low-MIR4435-2HG groups. We employed single-cell RNA sequencing analysis and tumor microenvironment infiltration analysis to investigate the potential involvement of MIR4435-2HG in the TNBC tumor microenvironment. Cellular biological behaviors were assessed utilizing CCK-8, transwell assays, and wound-healing assays. Furthermore, we performed RNA-seq, qRT-PCR, and western blotting analyses to elucidate and confirm the specific mechanisms underlying the role of MIR4435-2HG in TNBC. RESULTS: In our study, we have identified MIR4435-2HG as a significant diagnostic and prognostic factor for TNBC. We observed that MIR4435-2HG is widely expressed and might have a significant impact on the reshaping of the TNBC tumor microenvironment. Patients with TNBC in the high-MIR4435-2HG group may show reduced sensitivity to cisplatin, doxorubicin, and gemcitabine and have an increased propensity for immune escape. Knockdown of MIR4435-2HG inhibits cancer-associated fibroblasts (CAFs) activation. Notably, MIR4435-2HG predominantly enhances the migratory and invasive capabilities of TNBC cells through the epithelial-mesenchymal transition (EMT) process. Mechanistically, we validated that MIR4435-2HG activates the JNK/c-Jun and p38 non-classical MAPK signaling pathway in TNBC cells. CONCLUSIONS: Our findings highlight the significant potential of MIR4435-2HG as a highly promising biomarker for TNBC. Targeting MIR4435-2HG could represent an appealing therapeutic approach for TNBC.

Anti-inflammatory effects of Peucedanum japonicum Thunberg leaves extract in Lipopolysaccharide-stimulated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Peucedanum japonicum Thunberg are perennial herbaceous plants known to be cultivated for food and traditional medicinal purposes. P. japonicum has been used in traditional medicine to soothe coughs and colds, and to treat many other inflammatory diseases. However, there are no studies on the anti-inflammatory effects of the leaves. AIM OF THE STUDY: Inflammation plays an important role in our body as a defense response of biological tissues to certain stimuli. However, the excessive inflammatory response can lead to various diseases. This study aimed to investigate the anti-inflammatory effects of P. japonicum leaves extract (PJLE) in LPS-stimulated RAW 264.7 cells. MATERIAL AND METHODS: Nitric Oxide (NO) production assay measured by NO assay. Inducible NO synthase (iNOS), COX-2, MAPKs, AKT, NF-κB, HO-1, Nrf-2 were examined by western blotting. PGE2, TNF-α, and IL-6 were analyzed by ELSIA. Nuclear translocation of NF-κB was detected by immunofluorescence staining. RESULTS: PJLE suppressed inducible nitric oxygen synthase (iNOS) and prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2, COX-2) expression, increased heme oxygenase 1 (HO-1) expression, and decreased nitric oxide production. And PJLE inhibited the phosphorylation of AKT, MAPK, and NF-κB. Taken together, PJLE down-regulated inflammatory factors such as iNOS and COX-2 by inhibiting the phosphorylation of AKT, MAPK, and NF-κB. CONCLUSIONS: These results suggest that PJLE can be used as a therapeutic material to modulate inflammatory diseases.

The high-intensity interval training mitigates the cardiac remodeling in spontaneously hypertensive rats.

AIM: To evaluate the influence of high-intensity interval training (HIIT) on cardiac structural and functional characteristics and myocardial mitogen-activated protein kinase (MAPK) signaling in hypertensive rats. METHODS: Male rats (12 months old) were divided into three groups: Wistar Kyoto rats (WKY, n = 8); sedentary spontaneously hypertensive rats (SED-SHR, n = 10), and trained spontaneously hypertensive rats (HIIT-SHR, n = 10). Systolic blood pressure (SBP), functional capacity, echocardiography, isolated papillary muscle, and gene expression of MAPK gene-encoding proteins associated with Elk1, cJun, ATF2, MEF2 were analyzed. KEY FINDINGS: HIIT decreased SBP and increased functional capacity, left ventricular diastolic diameter, posterior wall thickness-left ventricle, relative wall thickness-left ventricle, and resting tension of the papillary muscle. In hypertensive rats, we observed a decrease in the gene-encoding ATF2 protein; this decrease was reversed by HIIT. SIGNIFICANCE: The influence of HIIT in the SHR model in the compensated hypertension phase generated an increase in cardiac hypertrophy, attenuated myocardial diastolic dysfunction, lowered blood pressure, improved functional capacity, and reversed the alteration in gene-encoding ATF2 protein.

Imipramine alleviates memory impairment and hippocampal apoptosis in STZ-induced sporadic Alzheimer's rat model: Possible contribution of MAPKs and insulin signaling.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, associated with several pathophysiological complaints. Impaired insulin signaling in the brain, is one of the important characteristic features of AD which is accompanied by cognitive deficits. According to the multifactorial and complicated pathology of AD, no modifying therapy has been approved yet. Imipramine is a kind of tricyclic antidepressant with reported anti-inflammatory and anti-oxidant effects in the brain. There are controversial studies about the effect of this drug on spatial memory. This study investigates the effect of imipramine on streptozotocin (STZ) induced memory impairment in rats. Pursuing this objective, rats were treated with imipramine 10 or 20 mg/kg i.p. once a day for 14 days. 24 h after the last injection, memory function was evaluated by the Morris water maze (MWM) test in 4 consecutive days. Then, hippocampi were removed and the activity of caspase-3, mitogen activated protein kinases (MAPKs) family and inhibitory phosphorylation of insulin receptor substrate-1 (IRS-1ser307) were analyzed using Western blotting. Results showed that imipramine prevents memory impairment in STZ induced rats and this improvement was accompanied with an increase in ERK activity, reduction of caspase-3 and JNK activity, as well as partial restoration of P38 and IRS-1 activity. In conclusion, our study demonstrated that at least some members of the MAPK family are involved in the neuroprotective effect of imipramine.

A Convergent Functional Genomics Analysis to Identify Biological Regulators Mediating Effects of Creatine Supplementation.

Creatine (Cr) and phosphocreatine (PCr) are physiologically essential molecules for life, given they serve as rapid and localized support of energy- and mechanical-dependent processes. This evolutionary advantage is based on the action of creatine kinase (CK) isozymes that connect places of ATP synthesis with sites of ATP consumption (the CK/PCr system). Supplementation with creatine monohydrate (CrM) can enhance this system, resulting in well-known ergogenic effects and potential health or therapeutic benefits. In spite of our vast knowledge about these molecules, no integrative analysis of molecular mechanisms under a systems biology approach has been performed to date; thus, we aimed to perform for the first time a convergent functional genomics analysis to identify biological regulators mediating the effects of Cr supplementation in health and disease. A total of 35 differentially expressed genes were analyzed. We identified top-ranked pathways and biological processes mediating the effects of Cr supplementation. The impact of CrM on miRNAs merits more research. We also cautiously suggest two dose-response functional pathways (kinase- and ubiquitin-driven) for the regulation of the Cr uptake. Our functional enrichment analysis, the knowledge-based pathway reconstruction, and the identification of hub nodes provide meaningful information for future studies. This work contributes to a better understanding of the well-reported benefits of Cr in sports and its potential in health and disease conditions, although further clinical research is needed to validate the proposed mechanisms.