Role of phosphoinositide 3-kinase in the pathogenesis of acute pancreatitis

A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity.Thus,research has recently focused on molecules that can regulate the inflammatory processes,such as phosphoinositide 3-kinases(PI3Ks),a family of lipid and protein kinases involved in intracellular signal transduction.Studies using genetic ablation or pharmacologic inhibitors of different PI3 K isoforms,in particular the class I PI3Kδ and PI3Kγ,have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses.Recent data suggest that PI3 Ks are also involved in the pathogenesis of acute pancreatitis.Activation of the PI3K signaling pathway,and in particular of the class IB PI3Kγ isoform,has a significant role in those events which are necessary for the initiation of acute pancreatic injury,namely calcium signaling alteration,trypsinogen activation,and nuclear factor-κB transcription.Moreover,PI3Kγ is instrumental in modulating acinar cell apoptosis,and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis.The availability of PI3 K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease.This article presents a brief summary of PI3 K structure and function,and highlights recent advances that implicate PI3 Ks in the pathogenesis of acute pancreatitis.

Effects of small interfering RNA inhibit Class Ⅰ phosphoinositide 3-kinase on human gastric cancer cells

AIM:To investigate the effects of small interfering RNA(siRNA)-mediated inhibition of Class Ⅰ phosphoinositide 3-kinase(Class Ⅰ PI3K) signal transduction on the proliferation,apoptosis,and autophagy of gastric cancer SGC7901 and MGC803 cells.METHODS:We constructed the recombinant replication adenovirus PI3K(I)-RNA interference(RNAi)-green fluorescent protein(GFP) and control adenovirus NCRNAi-GFP,and infected it into human gastric cancer cells.MTT assay was used to determine the growth rate of the gastric cancer cells.Activation of autophagy was monitored with monodansylcadaverine(MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment.Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3).Mitochondrial membrane potential was measured using the fluorescent probe JC-1.The expression of autophagy was monitored with MDC,LC3 staining,and transmission electron microscopy.Western blotting was used to detect p53,Beclin-1,Bcl-2,and LC3 protein expression in the culture supernatant.RESULTS:The viability of gastric cancer cells was inhibited after siRNA targeting to the Class Ⅰ PI3K blocked Class Ⅰ PI3K signal pathway.MTT assays revealed that,after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP,the rate of inhibition reached 27.48% ± 2.71% at 24 h,41.92% ± 2.02% at 48 h,and 50.85% ± 0.91% at 72 h.After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAiGFP,the rate of inhibition reached 24.39% ± 0.93% at 24 h,47.00% ± 0.87% at 48 h,and 70.30% ± 0.86% at 72 h(P < 0.05 compared to control group).It was determined that when 50 MOI,the transfection efficiency was 95% ± 2.4%.Adenovirus PI3K(I)RNAi-GFP(50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells,and the results described here prove that RNAi of Class Ⅰ PI3K induced apoptosis in SGC7901 cells.The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity,indicating increased formation of autophagosomes.The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low.After incubating with adenovirus PI3K(I)-RNAi-GFP(50 MOI),Beclin-1,LC3,and p53 protein expression was significantly increased from 24 to 72 h.We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP(50 MOI).A number of isolated membranes,possibly derived from ribosomefree endoplasmic reticulum,were seen.These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles.We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)RNAi-GFP(50 MOI) treatment.Control cells showed a round shape and contained normal-looking organelles,nucleus,and chromatin,while adenovirus PI3K(I)-RNAiGFP(50 MOI)-treated cells exhibited the typical signs of autophagy.CONCLUSION:After the Class Ⅰ PI3K signaling pathway has been blocked by siRNA,the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase- B/mechanistic target of rapamycin pathway

BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma(HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHPassociated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIM To confirm the effect of XHP on HCC and the possible mechanisms involved.METHODS The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS). Cellbased experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP(0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay.Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction(RT-qPCR), respectively.Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway.Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTS The following 12 compounds were identified in XHP using high-resolution mass spectrometry:Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose-and timedependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract(0.625mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins(e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSION XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3.Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Phosphoinositide 3-kinase dependent modulation of morphine versus cocaine dependence involves activation of nischarin

OBJECTIVE Phosphoinositide 3-kinase(PI3K) activation was reported to participate in the development of effect of some drugs,such as morphine and cocaine dependence.We previous found nischarin is associated with the activation of PI3K.It is our great interest to investigate the involvement of nischarin in PI3K dependent modulation of morphine versus cocaine dependence.METHODS In order to study the role of nischarin in drug dependence and tolerance,nischarin knockout mice were used for our research.Effect of psychological dependence was studied by conditioned place preference(CPP),and the effect of physical dependence was tested by naloxone-precipitated withdrawal signs.Some brain tissues were harvested 24 h after the behavioral experiment for the further measurement.RESULTS PI3K specific inhibitor LY294002 significantly blocked the acquisition of morphine-induced CPP in wild-type mice,but had no effect on its expression.In comparison,LY294002 failed to block the acquisition of cocaine-induced CPP but inhibited the expression.Furthermore,we found naloxoneprecipitated withdrawal signs in the morphine dependent mice was inhibited by LY294002.Nischarin knockout in mice could abolish the effect of LY294002 on blocking the effects of morphine,but had no effect on cocaine.CONCLUSION PI3K activation is involved in the different phases of morphine and cocaine dependence,and nischarin plays an important role in the process.

Phosphoinositide and phospholipid phosphorylation and hydrolysis pathways<br/>—Organophosphate and organochlorine pesticides effects<br>

Phospholipid and phosphoinositide phosphorylation pathways have been shown to be of crucial importance on producing lipid mediators. The earlier findings reported on lipid molecules playing roles in different metabolic pathways used to assign them the exclusive role of second messenger generators. Several researchers have recently described how direct interaction of phospholipids and phosphoinositides with molecules or organelles, without the need for producing second messenger molecules, is responsible for their mechanism of action. Organophosphate and organochlorine pesticide toxicity mechanisms have been extensively studied in relation to their well known effects on cholinesterase activities and on the alterations of electric activity in the nervous system of different organisms respectively. There is little but consistent evidence that some compounds, including in both groups of pesticides, are also able to interact with phospholipid and phosphoinositide phosphorylation pathways in several organisms and tissues. The present review consists of an actualization of basic research on phospholipid and phosphoinositide phosphorylation and hydrolysis pathways, as well as a description of some reported evidences for the effects of the above mentioned pesticides on them.

Role of lipids in the control of autophagy and primary cilium signaling in neurons

The brain is,after the adipose tissue,the organ with the greatest amount of lipids and diversity in their composition in the human body.In neurons,lipids are involved in signaling pathways controlling autophagy,a lysosome-dependent catabolic process essential for the maintenance of neuronal homeostasis and the function of the primary cilium,a cellular antenna that acts as a communication hub that transfers extracellular signals into intracellular responses required for neurogenesis and brain development.A crosstalk between primary cilia and autophagy has been established;however,its role in the control of neuronal activity and homeostasis is barely known.In this review,we briefly discuss the current knowledge regarding the role of autophagy and the primary cilium in neurons.Then we review the recent literature about specific lipid subclasses in the regulation of autophagy,in the control of primary cilium structure and its dependent cellular signaling in physiological and pathological conditions,specifically focusing on neurons,an area of research that could have major implications in neurodevelopment,energy homeostasis,and neurodegeneration.

Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions

Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.

Emerging roles of phosphoinositide-associated membrane trafficking in plant stress responses

Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell.These barriers are formed by assembly of lipids and protein in aqueous environments.Lipids not only serve as building blocks for membrane construction,but also possess regulatory functions in cellular activities.These regulatory lipids are non-uniformly distributed in membrane systems;their temporal and spatial accumulation in specific membranes decodes environmental cues and changes cellular activity accordingly.Phosphoinositides(PIs)are phospholipids that exert regulatory effects.In recent years,research on PIs roles in regulating plant growth,development,and responses to environmental stress is increasing.Several reviews have been published on the composition of PIs,intermolecular transferring of PIs by lipid kinases(phosphatases)or PI-PLCs,subcellular localization,and specially their functions in plant developments.Herein,we review the crucial regulatory functions of PIs in plant stress responses,with a particular focus on PIs involved in membrane trafficking.

Dopamine and cyclic adenosine monophosphate-regulated phosphoprotein with an apparent Mr of 32000 promotes colorectal cancer growth

BACKGROUND Dopamine and cyclic adenosine monophosphate(cAMP)-regulated phosphop-rotein with an apparent Mr of 32000(DARPP-32)is a protein that is involved in regulating dopamine and cAMP signaling pathways in the brain.However,recent studies have shown that DARPP-32 is also expressed in other tissues,including colorectal cancer(CRC),where its function is not well understood.AIM To explore the effect of DARPP-32 on CRC progression.METHODS The expression levels of DARPP-32 were assessed in CRC tissues using both quantitative polymerase chain reaction and immunohistochemistry assays.The proliferative capacity of CRC cell lines was evaluated with Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine assays,while apoptosis was measured by flow cytometry.The migratory and invasive potential of CRC cell lines were deter-mined using wound healing and transwell chamber assays.In vivo studies involved monitoring the growth rate of xenograft tumors.Finally,the underlying molecular mechanism of DARPP-32 was investigated through RNA-sequencing and western blot analyses.RESULTS DARPP-32 was frequently upregulated in CRC and associated with abnormal clinicopathological features in CRC.Overexpression of DARPP-32 was shown to promote cancer cell proliferation,migration,and invasion and reduce apoptosis.DARPP-32 knockdown resulted in the opposite functional effects.Mechanistically,DARPP-32 may regulate the phosphoinositide 3-kinase(PI3K)/AKT signaling pathway in order to carry out its biological function.CONCLUSION DARPP-32 promotes CRC progression via the PI3K/AKT signaling pathway.

PI3K/SHIP2/PTEN pathway in cell polarity and hepatitis C virus pathogenesis

Hepatitis C virus(HCV) infects hepatocytes, polarized cells in the liver. Chronic HCV infection often leads to steatosis, fibrosis, cirrhosis and hepatocellular carcinoma, and it has been identified as the leading cause of liver transplantation worldwide. The HCV replication cycle is dependent on lipid metabolism and particularly an accumulation of lipid droplets in host cells. Phosphoinositides(PIs) are minor phospholipids enriched in different membranes and their levels are tightly regulated by specific PI kinases and phosphatases. PIs are implicated in a vast array of cellular responses that are central to morphogenesis, such as cytoskeletal changes, cytokinesis and the recruitment of downstream effectors to govern mechanisms involved in polarization and lumen formation. Important reviews of the literature identified phosphatidylinositol(Ptd Ins) 4-kinases, and their lipid products Ptd Ins(4)P, as critical regulators of the HCV life cycle. SH2-containing inositol polyphosphate 5-phosphatase(SHIP2), phosphoinositide 3-kinase(PI3K) and their lipid products Ptd Ins(3,4)P2 and Ptd Ins(3,4,5)P3, respectively, play an important role in the cell membrane and are key to the establishment of apicobasal polarity and lumen formation. In this review, we will focus on these new functions of PI3 K and SHIP2, and their deregulation by HCV, causing a disruption of apicobasal polarity, actin organization and extracellular matrix assembly. Finally we will highlight the involvement of this pathway in the event of insulin resistance and nonalcoholic fatty liver disease related to HCV infection.

Involvement of PI3K and ERK1/2 pathways in hepatocyte growth factor-induced cholangiocarcinoma cell invasion

AIM:To investigate the role of hepatocyte growth factor(HGF) in cholangiocarcinoma(CCA) cell invasiveness and the mechanisms underlying such cellular responses. METHODS:Effects of HGF on cell invasion and motility were investigated in two human CCA cell lines,HuCCA-1 and KKU-M213,using Transwell in vitro assay.Levels of proteins of interest and their phosphorylated forms were determined by Western blotting.Localization of E-cadherin was analyzed by immunofluorescence staining and visualized under confocal microscope. Activities of matrix degrading enzymes were determined by zymography. RESULTS:Both CCA cell lines expressed higher Met levels than the H69 immortalized cholangiocyte cell line.HGF induced invasion and motility of the cell lines and altered E-cadherin from membrane to cytoplasm localization,but did not affect the levels of secreted matrix metalloproteinase(MMP) -2,MMP-9 andurokinase plasminogen activator,key matrix degrading enzymes involved in cell invasion.Concomitantly,HGF stimulated Akt and extracellular signal-regulated kinase(ERK) 1/2 phosphorylation but with slightly different kinetic profiles in the two cell lines.Inhibition of the phosphoinositide 3-kinase(PI3K) /Akt pathway by the PI3K inhibitor,LY294002,markedly suppressed HGFstimulated invasion of both CCA cell lines,and inhibition of the ERK pathway by U0126 suppressed HGF-induced invasion of the KKU-M213 cell line but had a moderate effect on HuCCA-1 cells. CONCLUSION:These data indicate that HGF promotes CCA cell invasiveness through dys-localization of E-cadherin and induction of cell motility by distinct signaling pathways depending on cell line type.

Chemokine ligand 20 enhances progression of hepatocellular carcinoma via epithelial-mesenchymal transition

AIM: To identify the mechanisms of chemokine ligand 20(CCL20)-induced hepatocellular carcinoma(HCC) metastasis and evaluate it as a prognostic marker. METHODS: Expression of CCL20 was evaluated by immunohistochemistry in HCC tissues from 62 patients who underwent curative resection. The relationship between CCL20 expression and clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate its predictive value for recurrence and survival of HCC patients. The expression levels ofepithelial-mesenchymal transition(EMT)-and signaling pathway-related proteins were evaluated by Western blotting and immunocytochemistry. The effects of CCL20 on HCC cell proliferation and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium bromide(MTT) and Transwell assays. RESULTS: CCL20 immunoreactivity was detected in all 62 patient specimens. CCL20 expression was associated with preoperative alpha-fetoprotein level(P = 0.043), tumor size(P = 0.000), tumor number(P = 0.008), vascular invasion(P = 0.014), and tumor differentiation(P = 0.007). Patients with high CCL20 expression had poorer recurrence-free and overall survivals compared to those with low CCL20 expression(both P < 0.001). CCL20 induced EMT-like changes in HCC cells and increased their proliferation and migration ability(P < 0.05). Western blotting and immunofluorescence staining showed that CCL20 induced an EMT-like phenotype in HCC cells, and increased expression of phosphorylated AKT, β-catenin and vimentin, and decreased E-cadherin expression(P < 0.05). The correlation analysis revealed that high CCL20 expression in HCC tissue specimens was negatively correlated with E-cadherin expression(13.33%, 4/30), and positively correlated with vimentin(90.0%, 27/30), β-catenin(96.67%, 29/30) and p-AKT(76.67%, 23/30) expression.CONCLUSION: CCL20 expression is associated with HCC recurrence and patient survival and promotes HCC cell proliferation and migration by inducing EMT-like changes via PI3K/AKT and Wnt/β-catenin pathways.

Functional interactions between steroid hormones and neurotrophin BDNF

Brain-derived neurotrophic factor(BDNF),a critical neurotrophin,regulates many neuronal aspects including cell differentiation,cell survival,neurotransmission,and synaptic plasticity in the central nervous system(CNS) .Though BDNF has two types of receptors,high affinity tropomyosin-related kinase(Trk) B and low affinity p75 receptors,BDNF positively exerts its biological effects on neurons via activation of TrkB and of resultant intracellular signaling cascades including mitogenactivated protein kinase/extracellular signal-regulated protein kinase,phospholipase Cγ,and phosphoinositide 3-kinase pathways.Notably,it is possible that alteration in the expression and/or function of BDNF in the CNS is involved in the pathophysiology of various brain diseases such as stroke,Parkinson's disease,Alzheimer's disease,and mental disorders.On the other hand,glucocorticoids,stress-induced steroid hormones,also putatively contribute to the pathophysiology of depression.Interestingly,in addition to the reduction in BDNF levels due to increased glucocorticoid exposure,current reports demonstrate possible interactions between glucocorticoids and BDNF-mediated neuronal functions. Other steroid hormones,such as estrogen,are involved in not only sexual differentiation in the brain,but also numerous neuronal events including cell survival and synaptic plasticity.Furthermore,it is well known that estrogen plays a role in the pathophysiology of Parkinson's disease,Alzheimer's disease,and mental illness,while serving to regulate BDNF expression and/or function.Here,we present a broad overview of the current knowledge concerning the association between BDNF expression/function and steroid hormones(glucocorticoids and estrogen).

Roles of the PI3K/Akt pathway in Epstein-Barr virusinduced cancers and therapeutic implications

Viruses have been shown to be responsible for 10%-15% of cancer cases. Epstein-Barr virus(EBV) is the first virus to be associated with human malignancies. EBV can cause many cancers, including Burkett's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma and gastric cancer. Evidence shows that phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) plays a key role in EBV-induced malignancies. The main EBV oncoproteins latent membrane proteins(LMP) 1 and LMP2 A can activate the PI3K/Akt pathway, which, in turn, affects cell survival, apoptosis, proliferation and genomic instability via its downstream target proteins to cause cancer. It has also been demonstrated that the activation of the PI3K/Akt pathway can result in drug resistance to chemotherapy. Thus, the inhibition of this pathway can increase the therapeutic efficacy of EBV-associated cancers. For example, PI3 K inhibitor Ly294002 has been shown to increase the effect of 5-fluorouracil in an EBV-associated gastric cancer cell line. At present, dual inhibitors of PI3 K and its downstream target mammalian target of rapamycin have been used in clinical trials and may be included in treatment regimens for EBV-associated cancers.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus(DM) is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin(m TOR) is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1(m TORC1) and m TOR Complex 2(m TORC2) and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase(PI 3-K),protein kinase B(Akt),AMP activated protein kinase(AMPK),silent mating type information regulation 2 homolog 1(Saccharomyces cerevisiae)(SIRT1),Wnt1 inducible signaling pathway protein 1(WISP1),and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

Neuroendocrine tumors resistant to mammalian target of rapamycin inhibitors:A difficult conversion from biology to the clinic

Deregulation of the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)- mammalian target of rapamycin(m TOR) signaling pathway is one of the most commonlyinvolved pathways in tumorigenesis. It has also been reported as altered in neuroendocrine tumors(NETs). m TOR inhibitors used in clinical practice are derived from rapamycin,an anti-cancer agent also used as an immunosuppressor after organ transplantation. Everolimus and temsirolimus are the two rapamycin-derived m TOR inhibitors used in NETs. Notably everolimus has been approved in advanced progressive well/moderatelydifferentiated pancreatic NETs(p NETs). It inhibits specifically the m TORC1 subunit of m TOR,not interacting with m TORC2. Although everolimus produced a significant prolongation of progression-free survival a number of patients with p NETs do not benefit from the drug due to early or late progression. Two supposed mechanisms of resistance to m TOR inhibitors are Akt and PI3 K activation,by means of m TORC2 and insulin growth factor(IGF)- IGF receptor signaling,respectively. BEZ235 is a multi-targeted inhibitor binding to PI3 K,m TORC1 and m TORC2,therefore potentially turning off all the supposed molecular targets of resistance to everolimus. The two clinical trials designed in p NETs were stopped early due to unmet statistical endpoint and the global clinical development of BEZ235 was also halted. Tolerability of this drug was challenging and conditioned the feasibility of therapy. The BEZ experience is an example of the huge difference between the preclinical and clinical setting and prompts us to pay more attention to the phase Ⅰ step of clinical development and the design of phase Ⅱ clinical trials.

A likely role for a novel PH-domain containing protein,PEPP2,in connecting membrane and cytoskeleton

PH domains(pleckstrin homology)are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners.PH domain-containing proteins are found to be involved in a wide range of cellular events,including signalling,cytoskeleton rearrangement and vesicular trafficking.Here we showed that a novel PH domain-containing protein,PEPP2,displayed moderate phosphoinositide binding specificity.Full length PEPP2 associated with both plasma membrane and microtubules.The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells.Overexpression of PEPP2 increased membrane microviscosity,indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

A likely role for the PH-domain containing protein, PEPP2/ PLEKHA5, at the membrane-microtubule cytoskeleton interface

PH(pleckstrin homology)domains are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular compartments with assistances of alternative binding partners.PH domain-containing proteins have been found to be involved in a wide range of cellular events,including signalling,cytoskeleton rearrangement and vesicular trafficking.Here we showed that a novel PH domain-containing protein,PEPP2(also known as PLEKHA5),displays moderate phosphoinositide binding specificity.Full length PEPP2 was observed to variably associate with both the plasma membrane and microtubules.The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells.Overexpression of PEPP2 increased membrane microviscosity,indicating a potential role for PEPP2 in regulating function of microtubule-dependent membrane functions.

Elucidation of the hepatoprotective effect and mechanism of Melastoma dodecandrum Lour. based on network pharmacology and experimental validation

Objective:To systematically explore the effect and mechanism of melastomatis dodecandri herba(Melastoma dodecandrum Lour.)in the treatment of hepatitis based on network pharmacology.Method:We evaluated the hepatoprotective effects of M.dodecandrum in concanavalin A(Con A)-induced hepatitis in mice by assessing survival rate,histological analysis,serum transaminases,and related cytokines.Then the mechanism of action was predicted by a network pharmacology-based strategy.Based on the results,we measured the hepatic expression of related genes at mRNA level and proteins related to the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)and nuclear factorkappa B(NF-кB)pathways.Results:Our study results clearly demonstrated that M.dodecandrum pretreatment significantly alleviated liver injury.This was demonstrated by an increase in survival rate,decreased severity of liver damage,and reduced serum transaminase levels compared with those in the Con A group.Moreover,M.dodecandrum significantly reduced the serum levels of tumor necrosis factor-a,interleukin-6,and interferon-g and increased the liver levels of superoxide dismutase,which indicated that M.dodecandrum exhibits anti-inflammatory and antioxidant activities.On the basis of network pharmacology,50 nodes were selected as major hubs based on their topological importance.Pathway enrichment analyses indicated that the putative targets of M.dodecandrum mostly participate in various pathways associated with the anti-inflammation response,which implies the underlying mechanism by which M.dodecandrum acts on hepatitis.Real-time fluorescent quantitative PCR analysis showed that M.dodecandrum downregulates the mRNA expression of interleukin-6,Toll-like receptor 7,interleukin-1 receptor-associated kinase-4,NF-кB and tumor necrosis factor-a in liver tissues.Western blotting showed that M.dodecandrum pretreatment protected against inflammation through activating the PI3K-Akt pathway by upregulating phosphorylated Akt(p-Akt)expression and suppressing NF-кB activation by inhibiting the phosphorylation of IKK,IkBa,and p65.Conclusion:The present work demonstrated the hepatoprotective effects of M.dodecandrum by regulating the PI3K/Akt and NF-кB pathways in Con A-induced mice,which provide insights into the treatment of hepatitis using M.dodecandrum.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair

Objective:To investigate the effects of Danmu Extract Syrup(DMS) on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice and explore the mechanism.Methods:Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table(n=12),including control(normal saline),LPS(5 mg/kg),LPS+DMS 2.5 mL/kg,LPS+DMS 5 mL/kg,LPS+DMS 10 mL/kg,and LPS+Dexamethasone(DXM,5 mg/kg) groups.After pretreatment with DMS and DXM,the ALI mice model was induced by LPS,and the bronchoalveolar lavage fluid(BALF) were collected to determine protein concentration,cell counts and inflammatory cytokines.The lung tissues of mice were stained with hematoxylin-eosin,and the wet/dry weight ratio(W/D) of lung tissue was calculated.The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1βin BALF of mice were detected by enzyme linked immunosorbent assay.The expression levels of Claudin-5,vascular endothelial(VE)-cadherin,vascular endothelial growth factor(VEGF),phospho-protein kinase B(p-Akt) and Akt were detected by Western blot analysis.Results:DMS pre-treatment significantly ameliorated lung histopathological changes.Compared with the LPS group,the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment(P<0.05 or P<0.01).The number of cells in BALF and myeloperoxidase(MPO) activity decreased significantly after DMS pretreatment(P<0.05 or P<0.01).DMS pre-treatment decreased the levels of TNF-α,IL-6 and IL-1β(P<0.01).Meanwhile,DMS activated the phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) pathway and reversed the expressions of Claudin-5,VE-cadherin and VEGF(P<0.01).Conclusions:DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier.It might be a potential therapeutic drug for LPS-induced lung injury.