P38 Extracellular Signal-regulated Kinase Research Articles

Increased SPK1 expression promotes cell growth by activating the ERK1/2 signaling in non-small-cell lung cancer.

Lung cancer remains the leading cause of cancer-associated mortality in China and the world. Increasing numbers of studies have reported that sphingosine kinase 1 (SPK1) is frequently highly expressed in tumors of various origins, including lung cancer, and its high expression contributes toward tumor progression. However, the clinical significance of SPK1 and its role in the growth and metastasis of non-small-cell lung cancer (NSCLC) remain unclear. In the present study, we found that SPK1 expression was expressed highly in NSCLC tissues and cell lines. Knockdown of SPK1 suppressed cell growth, proliferation, migration, and invasion and increased apoptosis. Moreover, knocking down SPK1 expression inhibited the growth of tumors in nude mice. Mechanistically, silencing the expression of SPK1 inhibited the expression of p-extracellular signal-regulated kinase (ERK). Moreover, the ERK-specific inhibitor U0126 suppressed the expression of the epithelial-mesenchymal transition of lung cancer cells. Together, our findings indicated that SPK1 enhanced tumor growth in lung cancer and induced metastasis by activating the ERK1/2 signaling pathway, indicating its potential application in NSCLC diagnosis and therapy.

2,4,5-Trichloro-6-((2,4,6-trichlorophenyl)amino)isophthalonitrile, Exerts Anti-bladder Activities through IGF-1R/STAT3 Signaling.

2,4,5-Trichloro-6-((2,4,6-trichlorophenyl)amino)isophthalonitrile (SYD007) is a small molecule compound that was synthesized according to the structure of diarylamine. In this study, we evaluated the anti-bladder activities of SYD007, and determined its cytotoxic mechanism. We found that SYD007 exerted cytotoxicity to bladder cancer cells. Furthermore, SYD007 induced bladder cancer cell early apoptosis and arrested cell cycle. Mechanistically, SYD007 suppressed phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Tyr705) level in parallel with increases of p-extracellular signal-regulated kinase (ERK) and p-AKT. SYD007 significantly inhibited insulin-like growth factor 1 (IGF-1)-induced STAT3 activation through down-regulation of total IGF-1R level. No dramatic changes in IGF-1R mRNA levels were observed in SYD007-treated cells, suggesting that SYD007 acted primarily at a posttranscriptional level. Using molecular docking analysis, SYD007 was identified as an IGF-1R inhibitor. In summary, we reported that SYD007 exerted anti-bladder activities, and these effects were partially due to inhibition of IGF-1R/STAT3 signaling.

USP34 Regulated Human Pancreatic Cancer Cell Survival <i>via</i> AKT and PKC Pathways

Pancreatic cancer is known to be a fatal disease, which is difficult to be diagnosed in its early stages. Ubiquitin-Specific Protease 34 (USP34) are closely related to human cancers in the development and progression. However, there are rarely studies about the role of USP34 in pancreatic cancer. Thus, we aimed to investigate the effect of USP34 in human pancreatic cancer. Short-hairpin RNA targeting USP34 (USP34-shRNA) and USP34 overexpression lentivirus were used in the current study. The level of USP34 in human pancreatic cancer (PANC-1) cells were then analyzed by quantitative (q)RT-PCR. In addition, Western blotting was used to examine phosphorylated (p)-AKT, p-protein kinase C (PKC) and p-extracellular signal-regulated kinase (ERK) protein levels. CCK-8 assay, flow cytometry, and migration assay were used to detect cell proliferation, apoptosis and migration, respectively in vitro. According to the result of qRT-PCR and Western blotting, USP34-shRNA1 significantly downregulated USP34 gene level in PANC-1 cell. Subsequently, Western blotting assay indicated that USP34 silencing significantly down-regulated the expression of p-AKT and p-PKC in cells. On the other hand, USP34 overexpressing remarkably up-regulated the expression of p-AKT and p-PKC in cells. In addition, USP34 overexpression promoted PANC-1 cell proliferation and migration via up-regulating the proteins of p-AKT and p-PKC. Moreover, USP34 overexpression reversed AKT inhibitor and PKC inhibitor induced PACN-1 cell apoptosis. Our results indicated USP34 regulated h PANC-1 cell survival via AKT and PKC pathways, and which played a pro-survival role in human pancreatic cancer. Therefore, we suggested USP34 could be a potential therapeutic target for pancreatic cancer.

Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells.

Transient receptor potential melastatin 8 (TRPM8) is a major receptor of cold environment. Recently, we found that cigarette smoke extract (CSE) upregulated TRPM8 mRNA and protein expression in bronchial tissues that made them more sensitive to cold stimuli. In our present study, we found that cold temperature (18°C)-induced activation of TRPM8 in 16HBE (human bronchial epithelial) cells facilitated Ca2+ influx and subsequently led to the increased expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α via the upregulation of p-extracellular signal-regulated kinase (ERK) and the activation of NF-κB. In addition, 16HBE cells that co-stimulated with 18°C and CSE were used to explore the synergistic effect of CSE on cold temperature-induced inflammatory cytokine production as well as the possible involved signaling pathway. RT-PCR and western blot analysis revealed that CSE upregulated TRPM8 mRNA and protein level in 16HBE cells. Ca2+ imaging, western blot, and luciferase assay showed more robust increase in intracellular Ca2+ and promoted phosphorylated ERK, P38, and NF-κB activity, respectively, in 16HBE cells co-stimulated with CSE and cold temperature, and such alteration was attenuated by TRPM8 short hairpin RNA (shRNA) transfection and BCTC pretreatment. Furthermore, enhanced levels of IL-6, IL-8, and TNF-α showed by enzyme-linked immunosorbent assay (ELISA) were reduced by specific inhibitors of ERK and NF-κB. Collectively, our results suggest that mitogen-activated protein kinase (MAPK)/NF-κB signaling is involved in TRPM8-mediated cold temperature-induced inflammatory cytokine expression. In addition, CSE synergistically amplifies cold temperature-induced inflammatory factors release via upregulating TRPM8 expression and enhancing MAPK/NF-κB signaling pathway.

Undercarboxylated Osteocalcin Improves Insulin-Stimulated Glucose Uptake in Muscles of Corticosterone-Treated Mice.

Short-term administration of glucocorticoids (GCs) impairs muscle insulin sensitivity at least in part via the reduction of undercarboxylated osteocalcin (ucOC). However, whether ucOC treatment reverses the GC-induced muscle insulin resistance remains unclear. To test the hypothesis that ucOC directly ameliorates impaired insulin-stimulated glucose uptake (ISGU) induced by short-term GC administration in mice muscle and to identify the molecular mechanisms, mice were implanted with placebo or corticosterone (CS) slow-release pellets. Two days post-surgery, insulin-tolerance tests (ITTs) were performed. On day 3, serum was collected and extensor digitorum longus (EDL) and soleus muscles were isolated and treated ex vivo with vehicle, ucOC (30 ng/mL), insulin (60 µU/mL), or both. Circulating hormone levels, muscle glucose uptake, and muscle signaling proteins were assessed. CS administration reduced both serum osteocalcin and ucOC levels, whole-body insulin sensitivity, and muscle ISGU in EDL. Ex vivo ucOC treatment restored ISGU in CS-affected muscle, without increasing non-insulin-stimulated glucose uptake. In CS-affected EDL muscle, ucOC enhanced insulin action on phosphorylated (p-)protein kinase B (Akt)Ser473 and the p-extracellular signal-regulated kinase isoform 2 (ERK2)Thr202/Tyr204 /total (t)ERK2 ratio, which correlated with ISGU. In CS-affected soleus muscle, ucOC enhanced insulin action on p-mammalian target of rapamycin (mTOR)Ser2481 , the p-mTORSer2481 /tmTOR ratio, p-Akt substrate of 160kD (AS160)Thr642 , and p-protein kinase C (PKC) (pan)Thr410 , which correlated with ISGU. Furthermore, p-PKC (pan)Thr410 correlated with p-AktSer473 and p-AS160Thr642 . ucOC exerts direct insulin-sensitizing effects on CS-affected mouse muscle, likely through an enhancement in activity of key proteins involved in both insulin and ucOC signaling pathways. Furthermore, these effects are muscle type-dependent. © 2019 American Society for Bone and Mineral Research.

New naphthalene derivatives induce human lung cancer A549 cell apoptosis via ROS-mediated MAPKs, Akt, and STAT3 signaling pathways

1,4-Naphthoquinone compounds are a class of organic compounds derived from naphthalene. They exert a wide variety of biological effects, but when used as anticancer drugs, have varying levels of side effects. In the present study, in order to reduce toxicity and improve the antitumor activity, we synthesized two novel 1,4-naphthoquinone derivatives, 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BSQ) and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OSQ). We investigated the antitumor effects of BSQ and OSQ in human lung cancer cells and the underlying molecular mechanisms of these effects, focusing on the relationship between these compounds and reactive oxygen species (ROS) production. MTT assay and trypan blue exclusion assay results showed that BSQ and OSQ had significant cytotoxic effects in human lung cancer cells. Flow cytometry results indicated that the number of apoptotic cells and the intracellular ROS levels significantly increased after treatment with BSQ and OSQ. However, cell apoptosis was inhibited by pretreatment with the ROS scavenger N-acetyl-l-cysteine (NAC). Western blotting results showed that BSQ and OSQ increased the expression levels of p-p38 kinase and p-c-Jun N-terminal kinase (p-JNK), and decreased the expression levels of p-extracellular signal-regulated kinase (p-ERK), p-protein kinase B (p-Akt), and p-signal transducer and activator of transcription-3 (p-STAT3). These phenomena were blocked by mitogen-activated protein kinase (MAPK) inhibitors, Akt inhibitors and NAC. In conclusion, BSQ and OSQ induce human lung cancer A549 cell apoptosis by ROS-mediated MAPKs, Akt, and STAT3 signaling pathways. Therefore, BSQ and OSQ may be therapeutic potential agents for the treatment of human lung cancer.

High Glucose-Induced Apoptosis in Human Kidney Cells Was Alleviated by miR-15b-5p Mimics.

MicroRNAs were involved in a wide range of biological processes of diabetic nephropathy (DN). It is reported that miR-15b-5p was downregulated in the patients with DN. However, the mechanisms underlying the regulatory effects of miR-15b-5p on patients with diabetes remain unclear. Thus, this study aimed to investigate the role of miR-15b-5p during high glucose (HG)-induced apoptosis in human kidney cells. Quantitative real-time (qRT)-PCR was used to detect the level of miR-15b-5p. CCK-8 assay, EdU staining assays and flow cytometry were used to detect cell proliferation, apoptosis respectively in vitro. In addition, Western blotting was used to determine active caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), phosphorylated (p)-AKT, p-mammalian target of rapamycin (mTOR), p-S6, p-c-Jun N terminal kinase (JNK), p-p38 and p-extracellular signal-regulated kinase (ERK) proteins levels. The expression of miR-15b-5p in patients with DN were dramatically decreased compared with health persons. Similarly, HG down-regulated the expression of miR-15b-5p in HK-2 cells. In contrast, miR-15b-5p mimics alleviated HG-induced apoptosis in HK-2 cells via decreasing the expressions of active caspase 3 and cleaved PARP. EdU detection further confirmed that miR-15b-5p mimics attenuated the anti-proliferation effect of HG in HK-2 cells. Furthermore, HG-induced Akt/mTOR pathway downregulation and JNK upregulation were markedly reversed by miR-15b-5p mimics in cells. The data suggested that miR-15b-5p mimics protects HK-2 cells from HG-induced apoptosis. The anti-apoptotic effects of miR-15b-5p may due to the activation of the Akt/mTOR pathway as well as inactivation of JNK. Taken together, miR-15b-5p might be a potential therapeutic target for the treatment of patients with DN.

Epigallocatechin-3-gallate inhibits the growth and increases the apoptosis of human thyroid carcinoma cells through suppression of EGFR/RAS/RAF/MEK/ERK signaling pathway

BackgroundThyroid cancer is the most common type of endocrine malignancy and the incidence rate is rapidly increasing worldwide. Epigallocatechin-3-gallate (EGCG) could suppress cancer growth and induce apoptosis in many types of cancer cells. However, the mechanism of action of EGCG on the growth of human thyroid carcinoma cells has not been fully illuminated.MethodsCell proliferation and viability were detected by EdU and MTS assays. Cell cycle distribution was measured by flow cytometry. Migration and invasion were evaluated by scratch and transwell assays. Apoptotic levels were detected by TUNEL staining and western blotting. The protein levels of EGFR/RAS/RAF/MEK/ERK signaling pathway were detected by western blotting. The in vivo results were determined by tumor xenografts in nude mice. The in vivo proliferation, tumor microvessel density, and apoptosis were detected by immunohistochemistry.ResultsEGCG inhibited the proliferation, viability, and cell cycle progression in human thyroid carcinoma cells. EGCG decreased the migration and invasion, but increased the apoptosis of human thyroid carcinoma cells. EGCG reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), H-RAS, p-RAF, p-MEK1/2, and p-extracellular signal-regulated protein kinase 1/2 (ERK1/2) in human thyroid carcinoma cells. EGCG inhibited the growth of human thyroid carcinoma xenografts by inducing apoptosis and down-regulating angiogenesis.ConclusionsEGCG could reduce the growth and increase the apoptosis of human thyroid carcinoma cells through suppressing the EGFR/RAS/RAF/MEK/ERK signaling pathway. EGCG can be developed as an effective therapeutic agent for the treatment of thyroid cancer.

Paeonol inhibits the development of 1‑chloro‑2,4‑dinitrobenzene‑induced atopic dermatitis via mast and T cells in BALB/c mice.

Our previous studies suggested that paeonol, the active constituent of the traditional Chinese medicine Cortex Moutan, may be an effective treatment for inflammatory disorders. In the present study, the therapeutic potential of paeonol on atopic dermatitis (AD) was investigated using animal and cell experiments. AD-like lesions were induced by repeated application of 1-chloro-2,4-dinitrobenzene (DNCB) to the shaved dorsal skin of BALB/c mice, and P815 cells were used for in vitro assays. The skin lesions, serum and spleens of the mice were analyzed using lesion severity scoring, histological analysis, flow cytometry, reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA, in order to investigate the anti-AD effects of paeonol. In addition, western blotting and ELISA were conducted for in vitro analysis of P815 cells. The results demonstrated that oral administration of paeonol inhibited the development of DNCB-induced AD-like lesions in the BALB/c mice by reducing severity of the lesions, epidermal thickness and mast cell infiltration; this was accompanied by reduced levels of immunoglobulin E and inflammatory cytokines [interleukin (IL)-4, histamine, IL-13, IL-31 and thymic stromal lymphopoietin], along with regulation of the T helper (Th) cell subset (Th1/Th2) ratio. Application of paeonol also reduced the protein expression levels of phosphorylated (p)-p38 and p-extracellular signal-regulated kinase (ERK) in skin lesions. In vitro, paeonol reduced the expression levels of tumor necrosis factor-α and histamine in P815 cells, and inhibited p38/ERK/mitogen-activated protein kinase signaling. The present findings indicated that paeonol may relieve dermatitis by acting on cluster of differentiation 4+ T and mast cells; therefore, paeonol may represent a potential therapeutic strategy for the treatment of allergic inflammatory conditions via immunoregulation.

AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids.

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d-aspartate ( d-Asp) and N-methyl- d-aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l-Glu, d-Glu, d-Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l-Glu-, d-Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l-Glu and NMDA, but not by d-Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.

Peptide V3 Inhibits the Growth of Human Hepatocellular Carcinoma by Inhibiting the Ras/Raf/MEK/ERK Signaling Pathway.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. Peptide V3 has shown anti-angiogenic and anti-tumor effects on S180 and H22 xenografts in nude mice. However, the detailed mechanism of action of peptide V3 has not yet been fully elucidated. In the present study, the effects of peptide V3 on the growth of human HCC cells were examined both in vitro and in vivo. Our results showed that peptide V3 inhibited the proliferation, viability, migration, and invasion of human HCC cells. However, no obvious effect was observed in HL-7702 cells. Peptide V3 increased the apoptosis and decreased the protein levels of H-RAS, phospho (p)-RAF, p-MEK, and p-extracellular signal-regulated protein kinase (ERK) in human HCC cells. Peptide V3 suppressed the growth of human HCC xenografts by down-regulating angiogenesis and up-regulating apoptosis. In conclusion, peptide V3 could inhibit the growth of human HCC by inhibiting the Ras/Raf/MEK/ERK signaling pathway. Novel peptides and modification strategies could be designed and applied for the treatment of different types of cancer.

JAX2, an ethanol extract of Hyssopus cuspidatus Boriss, can prevent bronchial asthma by inhibiting MAPK/NF-κB inflammatory signaling

BackgroundHyssopus cuspidatus Boriss has been used to treat bronchial asthma for many years in Uighur medicine. JAX2, an ethanol extract from this plant, has effectiveness against bronchial asthma. However, the molecular basis for the anti-inflammatory effects of JAX2 remains unclear. PurposeThis study aimed to evaluate the mechanism of JAX2 against bronchial asthma. MethodsWe established an asthma model in rats using ovalbumin (OVA), and an inflammatory model in RAW264.7 cells using lipopolysaccharide (LPS) stimulation. To evaluate the anti-inflammatory effects of JAX2, the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-6, IL-17, eotaxin and immunoglobulin (Ig)E were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was investigated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay. Further, nitric oxide (NO) and reactive oxygen species (ROS) were determined using Griess reagent and 2,7-dichlorofluorescein diacetate. The phosphorylation of p-extracellular signal-regulated kinase (ERK), p-Jun-NH2-terminal kinase (JNK), p38 kinases (p38) and p-inhibitor of nuclear factor kappa-B kinase (IKK), and nuclear translocation of p-p65 kinases (p-p65) were determined by immunofluorescence to uncover the effects of JAX2 on the Mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways. ResultsAfter JAX2 administration to rats, Interferon (IFN)-γ concentrations in BALF increased significantly. Further, the concentrations of TNF-α, IL-4, IL-6, IL-17 and eotaxin in BALF, and IgE in serum decreased. JAX2 decreased TNF-α, IL-6 and NO in cell supernatant, and reduced ROS intracellularly. Concurrently, IFN-γ concentrations increased in cell supernatant significantly. In LPS-induced RAW264.7 cells, JAX2 inhibited phosphorylation of p-ERK, p-JNK and p-38 MAPK. The subsequent phosphorylation of p-IKK and nuclear translocation of the p-p65 subunit of NF-κB were also suppressed. ConclusionBased on these findings, we believe that JAX2 has both preventive and treatment effects in bronchial asthma. Furthermore, in the RAW264.7 cell inflammatory model, JAX2 also inhibited NF-κB and MAPK signaling pathways.

Acetaminophen disrupts memory in object recognition and increases extracellular signal-regulated kinase phosphorylation in male mice.

Acetaminophen (also known as paracetamol) is a commonly used over-the-counter pain medication, but recent evidence suggests that a single exposure or prenatal exposure may have significant behavioral effects. This investigation aimed to determine whether acetaminophen could disrupt memory formation in an object-recognition task and to quantify potential changes in memory-related signaling cascades in the hippocampus of mice after acetaminophen administration. Using male mice, we examined the effect of a single subcutaneous injection of acetaminophen on the object-recognition task, a single-trial, hippocampus-dependent memory task. We also investigated potential changes in the activation of extracellular signal-regulated kinase (ERK) in the dorsal mouse hippocampus 1 hr after a subcutaneous injection of acetaminophen. We found that 50 mg/kg and 100 mg/kg interfered with performance in the object-recognition memory task, whereas 10 mg/kg did not. We also found that a single 50 mg/kg injection of acetaminophen significantly increased p42 ERK phosphorylation in the dorsal mouse hippocampus. Overall, these results suggest that a single dose of acetaminophen can have significant effects on memory and alters signaling kinases critical for memory consolidation. Further work is needed to determine the involved mechanisms. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

MicroRNA-30e inhibits adhesion, migration, invasion and cell cycle progression of prostate cancer cells via inhibition of the activation of the MAPK signaling pathway by downregulating CHRM3.

Prostate cancer (PCa) testing is currently based on measurement of serum prostate-specific antigen levels and digital rectal examination, which are limited by a low predictive value and the adverse effects associated with overdiagnosis and overtreatment. Recent studies have reported that the abnormal expression of microRNAs (miRNAs) is associated with the mechanism underlying the development of PCa. Thus, the aim of the present study was to investigate the effects of miR-30e and its target gene, M3 muscarinic acetylcholine receptor (CHRM3), on the adhesion, migration, invasion and cell cycle distribution of PCa cells via the mitogen-activated protein kinase (MAPK) signaling pathway. The differentially expressed genes were screened in the Gene Expression Omnibus database from a gene expression microarray (GSE55945) of PCa. PCa tissues and adjacent tissues were collected from patients with PCa. The PC-3 and DU145 human PCa cell lines were treated with activator, inhibitor and siRNAs. The effects of miR-30e on cell adhesion, migration, invasion and cell cycle distribution with the involvement of CHRM3 and the MAPK signaling pathway were investigated. The bioinformatics results demonstrated that the CHRM3 gene and the MAKP signaling pathway were involved in the progression of PCa, and has-miR-30e was selected for further study. The levels of miR-30e were significantly downregulated, while the levels of CHRM3 were obviously upregulated in PCa. CHRM3 was verified as a target gene of miR-30e. Upregulation of miR-30e and downregulation of CHRM3 decreased the levels of p-P38, p-extracellular signal-regulated kinase, p-c-Jun N-terminal kinase, p-c-fos and p-c-JUN, cell adhesion, migration and invasion ability, and the number of cells in the S phase, while they increased the number of cells in the G0 and G1 phases. The findings of the present study suggest that miR-30e inhibited the adhesion, migration, invasion and cell cycle entry of PCa cells by suppressing the activation of the MAPK signaling pathway and inhibiting CHRM3 expression. Thus, miR-30e may serve as a candidate target for the treatment of PCa.

Sevoflurane Postconditioning Inhibits Autophagy Through Activation of the Extracellular Signal-Regulated Kinase Cascade, Alleviating Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Hypoxic-ischemic brain injury (HIBI) in neonates is one of the major contributors of newborn death and cognitive impairment. Numerous animal studies have demonstrated that autophagy is substantially increased in HIBI and that sevoflurane postconditioning (SPC) can attenuate HIBI. However, if SPC-induced neuroprotection inhibits autophagy in HIBI remains unknown. To investigate if cerebral protection induced by SPC is related to decreased autophagy in the setting of HIBI. Postnatal rats at day 7 (P7) were randomly assigned to 7 different groups: Sham, HIBI, SPC-HIBI, HIBI + rapamycin, SPC-HIBI + rapamycin, HIBI + p-extracellular signal-regulated kinase (p-ERK) inhibitor, and SPC-HIBI + p-ERK inhibitor. To induce HIBI, neonatal rats underwent left common carotid artery ligation, followed by 2h of hypoxia (8% O2). Rats in the SPC groups were treated with 1 minimum alveolar concentration ([MAC], 2.4%) SPC for 30min after HIBI induction. Markers of autophagy and expression of ERK cascade components were measured in the rat brains after 24h. Spatial learning and memory function were examined 29-34days after administration of an autophagy agonist or a p-ERK inhibitor. The expression of microtubule-associated proteins 1A/1B, light chain 3B II (LC3-II) and tuberous sclerosis complex 2 (TSC2) were decreased in the SPC-HIBI group compared to the HIBI group. Expression of the p62 sequestosome 1 (P62/SQSTM1) protein, p-ERK/ERK, phospho-mammalian target of rapamycin (p-mTOR) and phospho-p70S6 were increased in SPC-HIBI group. Rats within the SPC-HIBI groups that also received the p-ERK inhibitor or autophagy inhibitor demonstrated reduced cross platform times and increased escape latency. Approximately 30min of 2.4% SPC treatment in the P7 rat HIBI model attenuated excessive autophagy in the brain by elevating the ERK cascade. This finding provides additional insight into HIBI and identifies new targets for therapeutic approaches to treat HIBI.

MiR-142-5p suppresses proliferation and promotes apoptosis of human osteosarcoma cell line, HOS, by targeting PLA2G16 through the ERK1/2 signaling pathway.

Previous studies have revealed that miR-142-5p serves a critical role in human cancer progression. However, the biological function of miR-142-5p in osteosarcoma (OS) development remains unclear. In the present study, the role of miR-142-5p in human OS HOS cells was determined, and the underlying mechanism involved was examined. Compared with the adjacent healthy tissues, the expression level of miR-142-5p was downregulated and the expression level of group XVI phospholipase A2 (PLA2G16) protein was upregulated in human OS tissues. The aforementioned results were also indicated in human OS HOS cells when compared with human fetal osteoblastic hFOB1.19 cells. Additionally, the results demonstrated that PLA2G16 was a direct target of miR-142-5p. miR-142-5p transfection upregulated the expression level of miR-142-5p and suppressed the expression level of PLA2G16 protein in HOS cells. MTT assays indicated a time-dependent decrease by miR-142-5p transfection in the proliferation of HOS cells. 5-bromo-2'-deoxyuridine incorporation assays confirmed that miR-142-5p transfection inhibited DNA synthesis in HOS cells. In addition, miR-142-5p transfection increased the Caspase-3 (CASP3) activity and apoptotic rate. Western blot analysis indicated that miR-142-5p transfection reduced BCL2, apoptosis regulator expression and upregulated the expression of CASP3 and BCL2 associated X, apoptosis regulator in HOS cells. Furthermore, miR-142-5p transfection decreased the expression levels of phosphorylated (p)-proto-oncogene, serine/threonine kinase, p-mitogen-activated protein kinase kinase, and p-extracellular signal-regulated kinase (ERK) 1/2 proteins in HOS cells. PLA2G16 overexpression restored the expression level of p-ERK 1/2 protein, which was reduced by miR-142-5p overexpression. MTT and CASP3 activity assays indicated that restoration of PLA2G16 reversed the tumour-suppressive role of miR-142-5p transfection in HOS cells. In conclusion, the results of the present study indicated that miR-142-5p suppressed proliferation and promoted apoptosis in human OS HOS cells by targeting PLA2G16 through ERK1/2 signaling pathway.

The neuroprotective effect of deep brain stimulation at nucleus basalis of Meynert in transgenic mice with Alzheimer's disease

BackgroundAlzheimer's disease (AD) is the most common type of dementia and mainly treated by drugs, while the therapeutic outcomes are very limited. This study aimed to determine the optimized parameters of deep brain stimulation (DBS) which was applied to the treatment of AD and propose the involved mechanisms. MethodsAmyloid-β precursor protein/Presenilin1 (APP/PS1) transgenic mice were used and received DBS at nucleus basalis of Meynert (NBM). The optimized parameters of DBS were determined by using different stimulation frequencies, durations and ages of mice under Morris water maze test. The involved mechanisms and the possible signal pathways were also investigated. ResultsThe optimized parameters for DBS were high frequency (100 Hz) for 21 days starting from early age (4 months old). Under the above parameters, the soluble Aβ40 and Aβ42 in the hippocampus and cortex were down-regulated significantly. DBS increased survival neurons and reduced apoptotic cells in the hippocampus and cortex. Meanwhile, the apoptosis-related proteins caspase-3, caspase-8 and Bid were down-regulated. Moreover, DBS caused a significant increase of superoxide dismutase, glutathione peroxidase and choline acetyltransferase activity as well as a decrease of methane dicarboxylic aldehyde content and acetylcholine esterase activity. Phosphorylation of Akt (p-Akt)/total Akt (t-Akt) was up-regulated while p-extracellular signal-regulated kinase 1/2 (ERK1/2)/t-ERK1/2 was down-regulated. The neuroprotective effect of DBS was attenuated by their inhibitors. ConclusionsNBM-DBS starting from 4 months of age for 21 days at a high frequency (100 Hz) has therapeutic effects on AD through activating phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and inhibiting ERK1/2 pathway.

Aloperine inhibits proliferation, migration and invasion and induces apoptosis by blocking the Ras signaling pathway in human breast cancer cells.

Aloperine (Alo), as a quinolizidine alkaloid extracted from S. alopecuroide, has the positive activities of anti-inflammatory, anti-allergenic, antitumor and anti-viral. However, the role and mechanism of Alo in breast cancer have not been studied yet. In the present study, Alo markedly inhibited the proliferation and suppressed the colony formation ability of the breast cancer cell lines MCF-7 and MDA-MB-231 in a dose-dependent manner by Cell Counting kit-8 and colony formation assays, respectively. In addition, the results of confocal microscopy analysis and flow cytometry detection revealed that Alo induced the apoptosis of MCF-7 and MDA-MB-231 cells, and western blotting indicated that Alo upregulated the protein levels of Bax, caspase-3 and caspase-9, and downregulated the expression of Bcl-2. Furthermore, the results of wound healing, Transwell migration and invasion assays demonstrated that Alo inhibited the migration and invasion of MCF-7 and MDA-MB-231 cells, and reduced the protein levels of matrix metalloproteinase (MMP)-2 and MMP-9. Alo also downregulated the protein expressions of Ras, phosphorylated (p)-Raf proto-oncogene, serine/threonine kinase 1 and p-extracellular signal-regulated kinase 1/2. Furthermore, ISIS 2503, a Ras inhibitor, inhibited colony formation, induced apoptosis, and suppressed the migration and invasion of MCF-7 and MDA-MB-231 cells. These effects were more marked in the presence of ISIS 2503 and Alo, when compared with those of either agent alone. In conclusion, the present study reported a novel use of Alo in inhibiting the proliferation, migration and invasion, and inducing the apoptosis of human breast cancer cells by blocking the Ras signaling pathway.

Chrysin suppresses proliferation, migration, and invasion in glioblastoma cell lines via mediating the ERK/Nrf2 signaling pathway.

BackgroundChrysin, an active natural bioflavonoid, has been proven to protect against carcinogenesis. However, the role of chrysin in glioblastoma and the potential molecular mechanisms remain to be elucidated. In our previous study, we found that nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is highly expressed in a variety of glioblastoma cell lines associated with the mitogen-activated protein kinase (MAPK) pathway. The aim of this study was to evaluate the antitumor effects of chrysin in glioblastoma cells and how chrysin is related to the MAPK/Nrf2 signaling pathway.MethodsA Cell Counting Kit-8 assay and a plate colony formation assay were performed to evaluate cell proliferation. Cell migration ability was tested by a wound-healing assay. Transwell migration and Matrigel invasion assay were used to test the migration and invasion potential of cells. Nrf2 was knocked down by shRNA transfection. Protein expression was determined by Western blotting and immunofluorescence staining. The in vivo anticancer effect was measured using tumor xenografts in nude mice.ResultsChrysin inhibited the proliferation, migration, and invasion capacity of glioblastoma cells in dose- and time-dependent manners. Mechanistically, chrysin deactivated the Nrf2 signaling pathway by decreasing the translocation of Nrf2 into the nucleus and suppressing the expression of hemeoxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1, meanwhile, Nrf2 shRNA attenuated the anticancer activity of chrysin. Furthermore, chrysin downregulated the protein expression of p-extracellular signal-regulated kinase 1 and 2 (ERK1/2), but did not significantly affect p-JNK and p-P38 expression levels. However, the downregulated level of Nrf2 and the antitumor effect of chrysin in glioblastoma cell lines were partially abrogated by the ERK1/2 signaling inhibitor (U0126). Finally, chrysin inhibited tumor growth in U87 xenografts.ConclusionOur results show that chrysin exerts anticancer activity in glioblastoma cell lines possibly via the ERK/Nrf2 signaling pathway and indicate the potential application of chrysin as a natural sensitizer in chemotherapy.

Telocinobufagin inhibits the epithelial-mesenchymal transition of breast cancer cells through the phosphoinositide 3-kinase/protein kinase B/extracellular signal-regulated kinase/Snail signaling pathway.

Telocinobufagin (TBG), an active ingredient of Venenumbufonis, exhibits an immunomodulatory activity. However, its antimetastatic activity in breast cancer remains unknown. The present study investigated whether TBG prevents breast cancer metastasis and evaluated its regulatory mechanism. TBG inhibited the migration and invasion of 4T1 breast cancer cells. Furthermore, TBG triggered the collapse of F-actin filaments in breast cancer. The epithelial-mesenchymal transition (EMT) markers, vimentin and fibronectin, were downregulated following TBG treatment. However, E-cadherin was upregulated following TBG treatment. Snail, a crucial transcriptional factor of EMT, was downregulated following TBG treatment. Signaling pathway markers, including phosphorylated protein kinase B (P-Akt), p-mechanistic target of rapamycin (mTOR) and p-extracellular signal-regulated kinase (ERK), were decreased following TBG treatment. The same results were obtained from in vivo experiments. In conclusion, in vitro and in vivo experiments reveal that TBG inhibited migration, invasion and EMT via the phosphoinositide 3-kinase (PI3K)/Akt/ERK/Snail signaling pathway in breast cancer.