Protein Kinase B Research Articles

Myristoylated alanine rich protein kinaseC substrate is a potential cancer prognostic factor that regulates cell migration and invasion in glioblastoma.

Myristoylated alanine‑rich C‑kinase substrate (MARCKS) serves an important role in various pathological processes in several malignancies. However, little is known about the specific role and molecular mechanism of MARCKS in glioblastoma (GBM). In the present study, it was found that the expression of MARCKS was significantly upregulated in GBM, and was associated with a poor clinical outcome in patients with GBM. Knockdown of MARCKS suppressed the migration and invasion of GBM cells invitro. Western blotting showed that the knockdown of MARCKS reduced the expression of phosphorylated phosphoinositide3‑kinase and protein kinaseB, as well as zinc finger protein SNAI1 expression, thereby modulating the expression of its downstream epithelial‑mesenchymal transition (EMT)‑associated factors, including E‑cadherin, vimentin, N‑cadherin and β‑catenin in GBM cells. These results indicate that MARCKS functioned in the migration and invasion of GBM, and therefore may provide a potential therapeutic target in GBM therapy.

Anti-colorectal cancer targets of resveratrol and biological molecular mechanism: Analyses of network pharmacology, human and experimental data.

In this study, colorectal cancer (CRC)-diseased targets and resveratrol (Res)-associated targets were combined and constructed by the use of grouped databases for identification of the predicted targets. After production of target-functional protein interaction network of Res anti-CRC, the topological analysis was used to create the core targets of Res anti-CRC. All core targetsperformed the analyses of biological function and pathway enrichment to optimize the biological processes and key signaling pathways of Res anti-CRC. The resultant five core therapeutic targets of Res anti-CRC were identified as protein kinase B1 (AKT1), interleukin 6 (IL6), Tumor protein p53 (TP53), vascular endothelial growth factor, and mitogen-activated protein kinase 1, respectively. Biological processes of Res anti-CRC were predominantly associated with regulating apoptosis, immune response, cellular communication, signal transduction, and metabolism of the nuclide. In addition, the top 10 key signaling pathways were identified, respectively. In human CRC sample assays, CRC histologic sections showed elevated expression of AKT1 and IL6 proteins, accompanied with abnormal changes in blood molecules. In pharmacological experiments of Res anti-CRC in vitro, Res-treated HCT116 cells showed inhibited cell growth, induced cell death. In addition, downregulation of intracellular AKT1 and IL6 expression were checked in Res-treated HCT116 cells. Taken together, these bioinformatic findings and preliminary validated data uncovered pharmacological molecular mechanisms associated with Res anti-CRC, and further identified top five core therapeutic targets. Beneficially, these five predicted targets might serve as potential biomolecules for anti-CRC treatment.

Targeting allosteric site of AKT by 5,7-dimethoxy-1,4-phenanthrenequinone suppresses neutrophilic inflammation.

BackgroundAcute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown.MethodsHuman neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI.FindingsCLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309–313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice.InterpretationOur results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

Neuroprotective and neuroregenerative effects of CRMP-5 on retinal ganglion cells in an experimental in vivo and in vitro model of glaucoma.

PurposeTo analyze the potential neuro-protective and neuro-regenerative effects of Collapsin-response-mediator-protein-5 (CRMP-5) on retinal ganglion cells (RGCs) using in vitro and in vivo animal models of glaucoma.MethodsElevated intraocular pressure (IOP) was induced in adult female Sprague-Dawley (SD) rats by cauterization of three episcleral veins. Changes in CRMP-5 expression within the retinal proteome were analyzed via label-free mass spectrometry. In vitro, retinal explants were cultured under elevated pressure (60 mmHg) within a high-pressure incubation chamber with and without addition of different concentrations of CRMP-5 (4 μg/l, 200 μg/l and 400 μg/l). In addition, retinal explants were cultured under regenerative conditions with and without application of 200 μg/l CRMP-5 after performing an optic nerve crush (ONC). Thirdly, an antibody against Protein Kinase B (PKB) was added to examine the possible effects of CRMP-5. RGC count was performed. Number and length of the axons were determined and compared. To undermine a signal-transduction pathway via CRMP-5 and PKB microarray and immunohistochemistry were performed.ResultsCRMP-5 was downregulated threefold in animals showing chronically elevated IOP. The addition of CRMP-5 to retinal culture significantly increased RGC numbers under pressure in a dose-dependent manner and increased and elongated outgrowing axons in retinal explants significantly which could be blocked by PKB. Especially the number of neurites longer than 400 μm significantly increased after application of CRMP-5. CRMP-5 as well as PKB were detected higher in the experimental than in the control group.ConclusionCRMP-5 seems to play an important role in an animal model of glaucoma. Addition of CRMP-5 exerts neuro-protective and neuro-regenerative effects in vitro. This effect could be mediated via activation of PKB affecting intra-cellular apoptosis pathways.

Galangin decreases p‑tau, Aβ42 and β‑secretase levels, and suppresses autophagy in okadaic acid‑induced PC12 cells via an Akt/GSK3β/mTOR signaling‑dependent mechanism.

Okadaic acid (OA)‑induced neurotoxicity may be considered a novel tool used to study Alzheimer's disease (AD) pathology, and may be helpful in the development of a novel therapeutic approach. It has been reported that galangin inhibits β‑site amyloid precursor protein‑cleaving enzyme 1 expression, which is a key enzyme for amyloidβ (Aβ) generation and is a potential drug candidate for AD therapy. However, further studies are required to confirm its neuroprotective effects in other AD models. The present study aimed to explore the neuroprotective effects of galangin on OA‑induced neurotoxicity in PC12 cells. The cells were divided into the following groups: Control group, model group (175nM OA for 48h) and galangin groups (0.25, 0.5 and 1µg/ml). Beclin‑1, phosphorylated (p)‑protein kinaseB (Akt), p‑glycogen synthase kinase (GSK)3β and p‑mechanistic target of rapamycin (mTOR) expression was also measured in the following PC12 cell groups: Control group, model group, 3‑methyladenine group (5nM), rapamycin group (100nM) and galangin group (1µg/ml). The levels of β‑secretase, Aβ42 and p‑tau were detected by ELISA, Beclin‑1 expression was examined by immunohistochemistry and the protein expression levels of p‑Akt, p‑mTOR p‑GSK3β, and Beclin‑1 were detected by western blotting. Galangin treatment enhanced cell viability in cells treated with OA, and decreased β‑secretase, Aβ42 and p‑tau levels. In addition, it suppressed Beclin‑1 and p‑GSK3β expression, but promoted p‑Akt and p‑mTOR expression by regulating the Akt/GSK3β/mTOR pathway. These results indicated that galangin protected PC12 cells from OA‑induced cytotoxicity and inhibited autophagy via the Akt/GSK3β/mTOR pathway, thus suggesting that it may be considered a potential therapeutic agent for AD.

MiR‑625‑5p suppresses inflammatory responses by targeting AKT2 in human bronchial epithelial cells.

Asthma is a common chronic inflammatory airway disease; however, whether microRNAs (miRs) could be used in the treatment of asthma remains unclear. The aim of the present study was to investigate the role of miR‑625‑5p in the inflammatory response of human bronchial epithelial cells(HBECs). Inflammation in the HBEC line, 16HBEC, was induced using different concentrations of lipopolysaccharide(LPS), which demonstrated that 1µg/ml LPS was an appropriate concentration for further experiments. The association between protein kinaseB2(AKT2) and miR‑625‑5p was verified using a luciferase reporter assay. LPS was added to 16HBECs following the administration of miR‑625‑5p mimics or miR‑625‑5p inhibitors, and cells with silenced or overexpressed AKT2 levels. miR‑625‑5p was expressed at a high level in LPS‑activated 16HBECs. Overexpression of miR‑625‑5p inhibited interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α secretion in 16HBECs. Inhibition of miR‑625‑5p enhanced LPS‑induced IL‑6 and TNF‑α secretion. miR‑625‑5p negatively regulated the expression of AKT2 in 16HBECs. A dual‑luciferase reporter assay system confirmed that miR‑625‑5p directly targeted the 3'untranslated region of AKT2. Transfection with a small interfering RNA against AKT2 inhibited inhibitor of κB phosphorylation. In brief, miR‑625‑5p may protect LPS‑induced HBECs by targeting AKT2 and inhibiting the nuclear factor‑κB signaling pathway. Therefore, miR‑625‑5p may function as an inhibitor of asthma airway inflammation in HBECs by targeting AKT2.

Characterization and Hypoglycemic Activity of a Rhamnan-Type Sulfated Polysaccharide Derivative.

Polysaccharide chromium (III) derivatives are gaining increasing attention in improving type 2 diabetes. In this study, the sulfated polysaccharide from Enteromorpha prolifera (SPE) with 4.8 kDa was prepared by specific enzymatic hydrolysis. The obtained SPE was used to prepare a rhamnan-type sulfated polysaccharide derivative (SPED). Results indicated that O-H, C=O, and S=O were effectively involved in the chelation of SPED (chromium content 20.26%). Acute (half lethal dose > 2.38 g/kg) and sub-acute toxicity showed that SPED had no damaging effects on mice. Anti-diabetic experiment demonstrated that SPED improved glucose metabolism. Moreover, SPED promoted the PI3K/PKB/GSK-3β signaling pathway by regulating mRNA expression of insulin receptors (IR), insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3β (GSK-3β). In conclusion, the SPED might represent a novel marine-derived candidate against hyperglycemia, which may undergo further pharmaceutical development as a hypoglycemic agent.

Autophagy induced by temozolomide combined with radiotherapy in human glioma U251 cells

Objective To investigate the autophagy and related mechanisms of human glioma U251 cells induced by temozolomide (TMZ) combined with radiotherapy. Methods MTT assay was used to detect the inhibitory effect of TMZ treatments with different concentrations (0~64 μmol/L) on U251 cells for 24 h and 48 h respectively. The clone formation assay was used to detect the survival fraction of U251 cells under different concentrations of TMZ combined with radiotherapy, and the radiosensitization effect of TMZ was evaluated. The effect of the combined treatment on the apoptosis of U251 cells was detected by flow cytometry. The expression of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin-1 and phosphated protein kinase B (p-Akt) was detected by Western Blot. Results The inhibitory effect of TMZ on the proliferation of U251 cells was concentration- and time-dependent, and the IC50 values were 42.25 μmol/L and 25.13 μmol/L, respectively for the 24 h and 48 h treatment. TMZ has a radiosensitizing effect on U251 cells. TMZ combined with radiotherapy can induced apoptosis of U251 cells, and significantly up-regulate the expression levels of LC3-I, LC3-I and Beclin-1, and the differences were statistically significant (all P<0.01). This combined treatment can down-regulate the expression of p-Akt protein, and the difference was statistically significant (P<0.05). Conclusions TMZ combined with radiotherapy can activate autophagy on U251 cells. The mechanism may involve the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by inhibiting Akt phosphorylation, the promotion of the expression of autophagy marker proteins (LC3-II, LC3-I and Beclin-1), and subsequent autophagy activation playing an anti-tumor effects. Key words: Malignant glioma; U251 cell; Temozolomide; Apoptosis; Autophagy

The effect of resolvin D2 on radicular pain caused by lumbar disc herniation

Objective To explore the effect of resolvin D2 (RvD2) on radicular pain induced by intervertebral disc herniation. Methods Thirty-six male Sprague-Dawley rats were randomly divided into a sham operation group, a model group and an RvD2 group, each of 12. Non-compressive lumbar disc herniation was induced in the rats in the model and RvD2 groups using the autologous nucleus pulposus filling method. Those in the sham group had the surgical site exposed without any other treatment. After the modeling, 10 μl of phosphate-buffered saline solution was administered intrathecally to the rats in the sham and model groups for 3 days, while the rats in the RvD2 group received 10 ng/10 μl of RvD2 intrathecally as well. Paw withdrawal thresholds (50%PWT) were observed 1 day before modeling and 7 days afterward for the rats of all three groups. On the 7th day after modeling, the L4 to L6 spinal dorsal horns on the surgery side were resected to detect the protein expression of phosphorylated protein kinase B (p-AKT), protein kinase B (t-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK-3β) and glycogen synthase kinase 3β (GSK-3β) using western blotting. The protein levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) were determined using enzyme-linked immunosorbent assays. Results On the 1st and 7th day after modeling, significant differences were observed between the model and sham groups in terms of the 50%PWT. From the 3rd day the average 50%PWT in the RvD2 group was significantly higher than that of the model group at the same time points. On the 7th day after the modeling the average p-AKT and p-GSK-3β protein levels of the model and RvD2 groups were significantly different from that of the sham group, and the model group′s average was also of significantly different from that of the RvD2 group. The average protein levels of the pro-inflammatory cytokines TNF-α and IL-6, as well as of the anti-inflammatory factors TGF-β1 and IL-10 in the dorsal horns of the model group and the RvD2 group were also significantly different on the 7th day, and both were significantly different from the sham group′s average. Conclusion RvD2 can alleviate radicular pain in rats with non-compressive disc herniation. The mechanisms might involve inhibition of GSK-3β activity, down-regulation of pro-inflammatory factors and up-regulation of anti-inflammatory factors. Key words: Resolvin D2; Lumbar disc herniation; Radicular pain; Inflammation; Mediators

The novel long non‑coding RNA PRNCR1‑2 is involved in breast cancer cell proliferation, migration, invasion and cell cycle progression.

Long non‑coding RNAs (lncRNAs) have recently been reported to act as important mediators of tumor initiation and progression. The present study aimed to investigate the expression and pathogenic roles of the lncRNA prostate cancer‑associated non‑coding RNA (PRNCR)1‑2 in breast cancer. The expression levels of PRNCR1‑2 were detected in breast cancer tissues and numerous breast cancer cell lines using reverse transcription‑quantitative polymerase chain reaction. Depletion of PRNCR1‑2 expression in breast cancer cells was conducted through small interfering RNA‑mediated silencing. Subsequently, cell proliferation was assessed by MTS assay, cell migration and invasion capacities were evaluated using the Transwell culture system, and cell cycle progression and apoptosis were analyzed by flow cytometry. Protein expression levels of the signaling components checkpoint kinase2 (CHK2), protein kinaseB (AKT), phosphorylated (p)‑CHK2 and p‑AKT were measured by western blotting. The results demonstrated that PRNCR1‑2 expression was significantly elevated in breast cancer tissues compared with in adjacent normal tissues. Furthermore, depletion of PRNCR1‑2 in HS‑578T and MDA‑MB‑231 breast cancer cells markedly suppressed their proliferation rates, migration and invasion capacities, and cell cycle progression; however, it had no effect on cell apoptosis. In addition, PRNCR1‑2 depletion increased CHK2 phosphorylation and decreased AKT phosphorylation in HS‑578T and MDA‑MB‑231 cells. In conclusion, the lncRNA PRNCR1‑2 may promote breast cancer cell proliferation, migration, invasion and cell cycle progression.

PI3K-PKB-mTOR hyperactivation in relation to nasopharyngeal carcinoma progression and prognosis.

Nasopharyngeal carcinoma (NPC) has a unique and complex etiology, which is not completely understood. The aim of this study is to investigate the expression patterns of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and mammalian target of rapamycin (mTOR) proteins in patients with NPC and their relationship with NPC progression and prognosis. Between January 2008 and March 2010, PI3K, PKB, and mTOR protein expressions were detected using immunohistochemistry among 119 patients with NPC and 30 healthy people. A 5-year follow-up was conducted for all patients. Correlations of PI3K, PKB, and mTOR proteins with the clinicopathological features and prognosis of NPC were evaluated using Spearman's rank correlation coefficient and Kaplan-Meier curve. Cox's regression analysis was performed to analyze the risk factors for the prognosis of NPC. First, PI3K, PKB, and mTOR were highly expressed in patients with NPC. The expressions of PI3K, PKB, and mTOR proteins were associated with T stage, N stage, clinical stage, relapse, and distant metastasis. Meanwhile, PI3K is positively correlated with PKB and PKB is positively correlated with mTOR in NPC. Higher PI3K, PKB, and mTOR protein expressions were related to a shorter survival time and a lower survival rate in NPC. Cox regression analysis revealed that age, T stage, N stage, PI3K, PKB, and mTOR were independent risk factors for NPC patient survival. Altogether, our data suggest that overexpression of PI3K, PKB, and mTOR proteins is an important indicator of poor survival in NPC. In addition, inhibition of PI3K-PKB-mTOR signaling may also contribute to the development of new therapeutic strategies for NPC.

Knockdown of inhibitor of differentiation 1 suppresses proliferation and induces apoptosis by inactivating PI3K/Akt/mTOR signaling in hemangioma-derived endothelial cells

Hemangioma (HA) is one of the commonest benign vascular neoplasms of infancy. Inhibitor of differentiation 1 (ID-1) has been reported to be an oncogene in multiple cancers. However, the role of ID-1 and its molecular mechanism in HA progression have not been elucidated. In the present study, we found that ID-1 expression at mRNA and protein levels was up-regulated in HA-derived endothelial cells (HDECs). Knockdown of ID-1 inhibited proliferation, facilitated apoptosis, and enhanced propranolol cytotoxicity in HDECs. Knockdown of ID-1 decreased the protein levels of phospholyrated protein kinase-B (Akt) and phospholyrated mammalian target of rapamycin (mTOR). Inhibition of PI3K/Akt/mTOR pathway by LY294002 abrogated ID-1-mediated pro-proliferation and anti-apoptosis effects in HDECs. In conclusion, knockdown of ID-1 suppressed proliferation and promoted apoptosis by inactivating phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling in HDECs, shedding light on the function of ID-1 in HA progression and highlighting the therapeutic value of ID-1 for HA.

Targeting Allosteric Site of AKT by 5,7-Dimethoxy-1,4-Phenanthrenequinone Suppresses Neutrophilic Inflammation

Background: Acute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown. Methods: Human neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI. Findings: CLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice. Interpretation: Our results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation. Funding Statement: This research was financial supported by the grants from the Ministry of Science Technology (MOST 106-2320-B-255-003-MY3 and MOST 104-2320-B-255-004-MY3), Ministry of Education (EMRPD1G0231 and EMRPD1H0381), and Chang Gung Memorial Hospital (CMRPF1F0011~3, CMRPF1F0061~3, CMRPF1G0241~3, and BMRP450), Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The authors declare that no competing interests exist. Ethics Approval Statement: The procedure of neutrophil isolation was approved by the Institutional Review Board at Chang Gung Memorial Hospital. ALI was induced by intra-tracheal spray of 2 mg/kg LPS (Escherichia coli 0111:B4) in seven to eight weeks old C57BL/6 male mice, according to the guidelines and approved by Institutional Animal Care and Use Committee of Chang Gung University, Taiwan.

Effect of ulinastatin on brain injury after asphyxiating cardiac arrest and resuscitation in rats

Objective Investigating the effect of ulinastatin on brain injury after asphyxiating cardiac arrest and cardiopulmonary resuscitation (CA/CPR) in rats. Methods A total of sixty-three clean male SD rats were randomly allocated into sham operation group (sham group), CA/CPR group (CA group) and ulinastatin administration group (UTI group), 21 rats per group. Rats did not receive CA/CPR in sham group. The model of cardiac arrest induced by asphyxia was established and cardiopulmonary resuscitation was performed in CA group. Ulinastatin with the dose of 105 U/kg was intravenously injected at the onset of resuscitation in UTI group. The same volume of saline was administrated to rats by intravenous injection in sham group and CA group at the same time point. Brain tissue including hippocampus was collected after the experiment. Subsequently, the wet/dry weight ratio (W/D) of brain tissue was determined. The concentration of interleukin (IL)-6 and IL-8 in the hippocampus was tested by enzyme-linked immunosorbent assay (ELISA). The expression of extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (Akt) mRNA in the hippocampus were detected by reverse transcriptive polymerase chain reaction (RT-PCR). The expression of phosphorylate-ERK1/2 (p-ERK1/2) and phosphorylate-Akt (p-Akt) protein in the hippocampus were measured by Western bolt. The morphological changes of brain of rats were observed under light microscopy. The number of normal pyramidal neurons was determined. The ultrastructural changes of brain of rats were observed under light microscopy and the average score of mitochondrial damage was tested. Results Compared with sham group, W/D ratio of brain tissue, the average score of mitochondrial damage and the concentration of IL-6 and IL-8, the expression of ERK1/2 and Akt mRNA, p-ERK1/2 and p-Akt protein in the hippocampus were obviously significantly higher(P<0.05), while the number of normal pyramidal neurons was lower in CA group and UTI group (P<0.05). Compared with CA group, W/D of brain tissue, the average score of mitochondrial damage and the concentration of IL-6 and IL-8, the expression of ERK1/2 and Akt mRNA, and p-ERK1/2 and p-Akt protein in the hippocampus were lower (P<0.05), while the number of normal pyramidal neurons was higher in UTI group (P<0.05). Morphology and ultrastructure of rat hippocampus showed no obvious damage in sham group. But significant damage occurred in CA group while the damage was significantly reduced in UTI group. Conclusions Ulinastatin can attenuate brain injury after asphyxiating CA/CPR in rats via inhibiting the expressions of IL-6 and IL-8, which may be related to suppressing activation of ERK1/2 and Akt. Key words: Ulinastatin; Brain injury; Extracellular signal-regulated kinase; Serine-threonine kinase; Cardiac arrest; Cardiopulmonary resuscitation

Experimental study of silencing hypoxia inducible factor-1 and protein kinase B joint intervention on the proliferation of human hepatocellular carcinoma cell line SMMC-7721

Experimental study of silencing hypoxia inducible factor-1 and protein kinase B joint intervention on the proliferation of human hepatocellular carcinoma cell line SMMC-7721

Coptisine Suppresses Mast Cell Degranulation and Ovalbumin-Induced Allergic Rhinitis.

Coptisine is one of the main components of isoquinoline alkaloids in the coptidis rhizome. The effect of coptisine on allergic rhinitis has not been investigated. In this study, we report the effects and mechanisms of coptisine using monoclonal anti-2,4,6-dinitrophenyl-immunoglobulin (Ig) E/human serum albumin (DNP-IgE/HSA)-stimulated rat basophilic leukemia cells (RBL-2H3 cells) in vitro and an ovalbumin (OVA)-induced allergic rhinitis (AR) in mice. The results showed that coptisine markedly decreased the levels of β-hexosaminidase, histamine, interleukin (IL)-4, and tumor necrosis factor (TNF)-α. Coptisine also prevented morphological changes, such as restoring an elongated shape, inhibiting granule release on toluidine blue staining, and reorganizing inhibited filamentous actins (F-actin). Additionally, coptisine blocked the phosphorylation of phosphoinositide3-kinase (PI3K)/Akt (as known as protein kinase B(PKB)) in RBL-2H3 cell. Furthermore, the results showed that coptisine suppressed OVA-induced allergic rhinitis symptoms, such as nasal rubbing and OVA-specific IgE, and histamine, IL-4 and TNF-α levels in the serum of AR mice. These data suggested that coptisine should have inhibitory effects on the inflammatory responses of mast cells, and may be beneficial for the development of coptisine as a potential anti-allergic drug.

HOXA5 inhibits the proliferation and induces the apoptosis of cervical cancer cells via regulation of protein kinase B and p27.

Homeobox A5 (HOXA5) is a member of the homeobox gene (HOX) family, which plays an important role in the development of various malignant tumors. Here, we speculated that HOXA5 has an effect on cervical cancer development. In our study, we aimed to explore the role and molecular mechanism of HOXA5 in regards to the cell proliferation and apoptosis in cervical cancer. We found that expression levels of HOXA5 measured by RT‑qPCR and western blot assays in cervical cancer cell lines and tissues were both significantly downregulated. We performed a gain‑of‑function experiment by the transfection with pcDNA.3.1‑HOXA5 in ME‑180 and HT‑3 cells to overexpress HOXA5, and the caspase‑3 activity measured by caspase‑3 activity assay kit and cell apoptosis detected by flow cytometry were obviously promoted. Meanwhile, cell proliferation tested by BrdU assay, invasion determined by Transwell and cell viability tested by MTT were inhibited. Moreover, protein kinaseB (AKT) was activated by incubation with SC79 (AKT activator; 1µg/ml) after HOXA5 overexpression, and reversed the effect of HOXA5 overexpression on p27 expression. Additionally, significant elevation of AKT activation measured by western blot analysis abrogated the effect of HOXA5 on caspase‑3 activity, cell apoptosis, proliferation, invasion and cell viability. Taken together, this study revealed that HOXA5 inhibits cervical cancer progression by regulating AKT/p27, proposing the potential role of HOXA5 in the prevention and treatment of cervical cancer.

Protective effect and mechanism of quercetin on learning and memory function after global ischemia/reperfusion injury in rats

Objective To observe the effect of quercetin (Que) on neurological behavior, cell apoptosis and apoptosis-related protein expression in hippocampus after global cerebral ischemia/reperfusion (I/R) injury. Exploring the mechanism of quercetin-induced neuroprotective effect against global cerebral I/R injury. Methods Seventy two Male SD rats were randomly divided into 4 groups (n=18): sham+vehicle group (S group), I/R+vehicle group (I/R group), I/R+Que 5 mg·kg-1·d-1 group (Q5 group) and I/R+Que 10 mg·kg-1·d-1 group (Q10 group). The global cerebral ischemia reperfusion model was established by a four-vessel occlusion (4-VO) ligation method, and the duration of ischemia was 15 min. Sham-operated animals were treated similarly to those in the ischemic group, but neither the vertebral arteries nor the common carotid arteries were occluded. The rats in Q5 group and Q10 group were administered with 0.05%, 0.10% of the quercetin solution by garage at the dose of 5 mg·kg-1·d-1 and 10 mg·kg-1·d-1 respectively. S group and I/R group were given equal volume of vehicle. The quercetin and vehicle were administrated by gavage once per day 3 d before I/R and continued till the end of observation. The Morris maze test was performed 8 d after reperfusion. At 24 h after reperfusion, TUNEL staining and Annexin V-FITC/PI double labeled flow cytometry were used to evaluate apoptosis in hippocampus tissue, the expressions of Bcl-2, Bcl-xl, survivin, cleaved caspase-3, phosphorylated protein kinase B(p-Akt), Bad, p-Bad protein were determined by Western blot. Results In Morris water maze test, escape latency results showed that compared with I/R group, the escape latency in Q5 group and Q10 group was obviously reduced (P<0.05). Space probe test showed that the active time in the target quadrant in Q5 group and Q10 group were significantly longer than active time in the target quadrant (P<0.05). The frequency of crossing the original position platform was significantly increased (P<0.05). TUNEL assay showed that a large number of apoptotic cells in I/R group, while less positive cells could be seen in Q5 group and Q10 group. Annexin V-FITC/PI double staining flow cytometry detection results showed that the proportion of apoptotic cells in Q5 group and Q10 group were significantly reduced (P<0.05). Western blot results showed that Bcl-2, Bcl-xl, survivin, p-Akt and p-Bad expression levels were significantly increased in Q5 group and Q10 group (P<0.05) while cleaved caspase-3 expression level was significantly decreased (P<0.05). Conclusions Quercetin can up-regulate the expression of p-Akt, p-Bad, Bcl-2, Bcl-xl and survivin while down-regulate the expression of cleaved caspase-3, which leads to the inhibition of apoptosis, eventually protects neuron cells from global cerebral I/R injury and improves the learning and memory function. Key words: Quercetin; Cerebral ischemia-reperfusion; Learning; Memory; Hippocampus; Apoptosis

Nicotine promotes lymph node metastasis and cetuximab resistance in head and neck squamous cell carcinoma.

Epidermal growth factor(EGF) is overexpressed in many cancers and is associated with worse prognosis. EGF binds to its cell surface receptor(EGFR), which induces EGFR phosphorylation. Phosphorylated EGFR(p‑EGFR) is translocated into the nucleus, which increases cancer cell activity. Nicotine, which is one of the main components of tobacco, is absorbed through pulmonary alveoli and mucosal epithelia in the head and neck region by smoking and moves into the blood. Nicotine in blood binds to nicotinic acetylcholine receptor(nAChR) in the central nervous system and serves a crucial role in tobacco addiction. Although nAChR localization is thought to be limited in the nervous system, nAChR is present in a wide variety of non‑neuronal cells, including cancer cells. Recent studies suggest that nicotine contributes to the metastasis and resistance to anti‑cancer drugs of various cancer cells. However, it remains unknown whether head and neck squamous cell carcinoma(HNSCC) cells can utilize nicotine‑nAChR signaling to metastasize and acquire resistance to anti‑cancer drugs, even though the mucosal epithelia of the head and neck region are the primary sites of exposure to tobacco smoke. To the best of our knowledge, the present study is the first to demonstrate the role of nicotine in metastasis and anti‑EGFR‑therapy resistance of HNSCC. The present findings demonstrated that nicotine increased proliferation, migration, invasion, p‑EGFR nuclear translocation and protein kinaseB(Akt) phosphorylation in HNSCC cells. It was also demonstrated that nicotine restored cetuximab‑inhibited proliferation, migration and invasion of HNSCC cells. Finally, an invivo experiment revealed that nicotine increased lymph node metastasis of xenografted tumors, whereas an nAChR inhibitor suppressed lymph node metastasis and p‑EGFR nuclear localization of xenografted tumors. Taken together, these results demonstrated that nicotine induced nuclear accumulation of p‑EGFR, and activation of Akt signaling. These signaling pathways elevated the activities of HNSCC cells, causing lymph node metastasis and serving a role in cetuximab resistance.

Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

Lysyl oxidase‐like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial‐to‐mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial‐to‐mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor‐β (TGF‐β)‐mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity.