Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats

AIM:To determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt,also known as PKB) pathway. METHODS:Rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane.RESULTS:After Wistar rats were fed with high-fat diet for 16 wk,their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7,P < 0.01),deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%,P < 0.05),and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%,P < 0.05). In addition,the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced,as was the expression of the anti-apoptotic protein Bcl-2,while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt,instead of high-fat diet,a similar pattern of hepatocellular fat accumulation,mitochondrial impairment,and change in the levels of PI3K,Akt,Bcl-2 was observed. CONCLUSION:High-fat diet appears to inhibit the PI3K/Akt signaling pathway,which may lead to hepa-tocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

Dickkopf-related protein 1/cytoskeleton-associated protein 4 signaling activation by Helicobacter pylori-induced activator protein-1 promotes gastric tumorigenesis via the PI3K/AKT/mTOR pathway

BACKGROUND Gastric cancer(GC)is a common malignant tumor with high incidence and mortality rates globally,especially in East Asian countries.Helicobacter pylori(H.pylori)infection is a significant and independent risk factor for GC.However,its underlying mechanism of action is not fully understood.Dickkopf-related protein(DKK)1 is a Wnt signaling antagonist,and cytoskeleton-associated protein(CKAP)4 is a newly identified DKK1 receptor.Recent studies found that the binding of DKK1 to CAKP4 mediated the procancer signaling of DKK1 independent of Wnt signaling.We hypothesize that H.pylori-induced activation of DKK1/CKAP4 signaling contributes to the initiation and progression of GC.AIM To investigate the interaction of H.pylori infection,DKK1 and CAKP4 in GC,as well as the underlying molecular mechanisms.METHODS RNA sequencing was used to identify differentially expressed genes(DEGs)between H.pylori-infected and uninfected primary GC cells.Gain-and loss-offunction experiments were performed to verify the H.pylori-induced upregulation of activator protein-1(AP-1)in GC cells.A dual-luciferase reporter assay and co-immunoprecipitation were used to determine the binding of AP-1 to the DKK1 promoter and DKK1 to CKAP4.Western blotting and immunohistochemistry detected the expression of DKK1,CKAP4,and phosphatidylinositol 3-kinase(PI3K)pathway-related proteins in GC cells and tissues.Functional experiments and tumorigenicity in nude mice detected malignant behavior of GC cells in vitro and in vivo.RESULTS We identified 32 DEGs between primary GC cells with and without H.pylori infection,including JUN,fos-like antigen-1(FOSL1),and DKK1,and confirmed that the three proteins and CKAP4 were highly expressed in H.pylori-infected GC cells,H.pylori-infected gerbil gastric tissues,and human GC tissues.JUN and FOSL1 form AP-1 to transcriptionally activate DKK1 expression by binding to the DKK1 promoter.Activated DKK1 bound to CKAP4,but not the most common Wnt coreceptor low-density lipoprotein receptor-related protein 5/6,to promote GC cell growth,colony formation,migration,invasion,and xenograft tumor growth in nude mice.All these effects were driven by activation of the PI3K/AKT/mammalian target of rapamycin(mTOR)pathway.Targeting the PI3K signaling pathway by LY294002 inhibited DKK1-mediated CKAP4/PI3K signaling activity and the malignant behavior of GC cells.CONCLUSION H.pylori induces JUN and FOSL1 expression to form AP-1,which transcriptionally activates DKK1.Binding of DKK1 to KAKP4 contributes to gastric tumorigenesis via the PI3K/AKT/mTOR pathway.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Up-regulation of PIK3CA promotes metastasis in gastric carcinoma

AIM:To explore expressions of PIK3CA in the progression of gastric cancer from primary to metastasis and its effects on activation of phosphatidylinositol 3-kinase(PI3K)/Akt pathway.METHODS:mRNA and protein levels of PIK3CA were assessed,respectively,by real-time quantitative polymerase chain reaction and immunohistochemistry in specimens of normal gastric mucosa,primary foci and lymph node and distant metastasis of gastric cancer.Akt and phosphorylated Akt protein were also examined by Western blotting in these tissues,in order to analyze the effect of PIK3CA expression level changes on the activation of PI3K/Akt signaling pathway.RESULTS:PIK3CA mRNA in lymph node metastasis were approximately 5 and 2 folds higher,respectively,than that in the corresponding normal gastric mucosa and primary gastric cancer tissues(P<0.05),while no statistical significance was found compared with distant metastasis.Immunohistochemically,PIK3CA protein expression was discovered in 7(35%)specimens of 20 primary foci vs 10(67%)of 15 of lymph node metastasis or 11(61%)of 18 of distant metastasis(35%vs 67%,P=0.015;35%vs 61%,P=0.044).With the increased level of PIK3CA expression,the total Akt protein expression remained almost unchanged,but p-Akt protein was upregulated markedly.CONCLUSION:Increased expression of PIK3CA is expected to be a promising indicator of metastasis in gastric cancer.Up-regulation of PIK3CA may promote the metastasis of gastric cancer through aberrant activation of PI3K/Akt signaling.

LY294002 potentiates the anti-cancer effect of oxaliplatin for gastric cancer via death receptor pathway

AIM:To examine the effects of combined treatment of oxaliplatin and phosphatidylinositol 3'-kinase inhibitor,2-(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one(LY294002) for gastric cancer. METHODS:Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay.Apoptotic cells were detected by flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.Western blotting and immuno-precipitation were used to examine protein expression and recruitment,respectively.Nuclear factorκB(NFκB) binding activities were investigated using electrophoretic mobility shift assay.Nude mice were used to investigate tumor growth. RESULTS:Treatment with combined oxaliplatin and LY294002 resulted in increased cell growth inhibi-tion and cell apoptosis in vitro,and increased tumor growth inhibition and cell death in the tumor mass in vivo.In MKN45 and AGS cells,oxaliplatin treatment promoted both protein kinase B(Akt) and NFκB activation,while pretreatment with LY294002 significantly attenuated oxaliplatin-induced Akt activity and NFκB binding.LY294002 promoted oxaliplatin-induced Fas ligand(FasL) expression,Fas-associated death domain protein recruitment,caspase-8,Bid,and caspase-3 activation,and the short form of cellular caspase-8/FLICEinhibitory protein(c-FLIPS) inhibition.In vivo,LY294002 inhibited oxaliplatin-induced activation of Akt and NFκB,and increased oxaliplatin-induced expression of FasL,inhibition of c-FLIPS,and activation of caspase-8,Bid,and caspase-3. CONCLUSION:Combination of oxaliplatin and LY294002 was therapeutically promising for gastric cancer treatment.The enhanced sensitivity of the combined treatment was associated with the activation of the death receptor pathway.

Quercetin Increased Protein Utilization and Decreased Nitrogen Excretion in Broilers by Activating TOR Signaling Pathway

The study was conducted to investigate the effect and mechanism of dietary quercetin supplementation on protein utilization of Arbor Acres(AA)broilers.A total of 2401-day-old AA broilers were randomly allocated to four treatments with six replicates,comprising 10 broilers each replicate(60 broilers per treatment).Birds were fed either a corn-soybean meal basal diet without quercetin(control)or a basal diet supplemented with 0.2,0.4 or 0.6 g of quercetin per kg feed,and the trial lasted 42 days.Dietary quercetin supplementation tended to increase the apparent metabolic rate of protein(p=0.076)and the content of serum albumin(p=0.062)in AA broilers.Compared with the control,dietary quercetin supplementation increased the contents of protein in breast muscle(p<0.05)and in thigh muscle(p=0.053).In addition,quercetin up-regulated mRNA expression of insulin-like growth factor 1(IGF-1),phosphatidylinositol 3-kinase(PI3K),target of rapamycin(TOR),ribosomal protein S6 kinase 1(S6K1),eukaryotic translation initiation factor 4E(eIF4E),eukaryotic translation initiation factor 4G(eIF4G),eukaryotic elongation factor 2(eEF2)and eukaryotic translation initiation factor 4B(eIF4B)genes and down-regulated mRNA expression of eukaryotic elongation factor 2 kinase(eEF2K)and eukaryotic initiation factor 4E binding protein1(4E-BP1)genes in breast muscle,thigh muscle and liver of AA broilers(p<0.05).The present results suggested that dietary quercetin supplementation enhanced protein utilization in broilers by activating TOR signaling pathway.

Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment

BACKGROUND Mesenchymal stem cells(MSCs)are a type of stem cells that possess relevant regenerative abilities and can be used to treat many chronic diseases.Diabetes mellitus(DM)is a frequently diagnosed chronic disease characterized by hyperglycemia which initiates many multisystem complications in the long-run.DM patients can benefit from MSCs transplantation to curb down the pathological consequences associated with hyperglycemia persistence and restore the function of damaged tissues.MSCs therapeutic outcomes are found to last for short period of time and ultimately these regenerative cells are eradicated and died in DM disease model.AIM To investigate the impact of high glucose or hyperglycemia on the cellular and molecular characteristics of MSCs.METHODS Human adipose tissue-derived MSCs(hAD-MSCs)were seeded in low(5.6 mmol/L of glucose)and high glucose(25 mmol/L of glucose)for 7 d.Cytotoxicity,viability,mitochondrial dynamics,and apoptosis were deplored using specific kits.Western blotting was performed to measure the protein expression of phosphatidylinositol 3-kinase(PI3K),TSC1,and mammalian target of rapamycin(mTOR)in these cells.RESULTS hAD-MSCs cultured in high glucose for 7 d demonstrated marked decrease in their viability,as shown by a significant increase in lactate dehydrogenase(P<0.01)and a significant decrease in Trypan blue(P<0.05)in these cells compared to low glucose control.Mitochondrial membrane potential,indicated by tetramethylrhodamine ethyl ester(TMRE)fluorescence intensity,and nicotinamide adenine dinucleotide(NAD+)/NADH ratio were significantly dropped(P<0.05 for TMRE and P<0.01 for NAD+/NADH)in high glucose exposed hAD-MSCs,indicating disturbed mitochondrial function.PI3K protein expression significantly decreased in high glucose culture MSCs(P<0.05 compared to low glucose)and it was coupled with significant upregulation in TSC1(P<0.05)and downregulation in mTOR protein expression(P<0.05).Mitochondrial complexes I,IV,and V were downregulated profoundly in high glucose(P<0.05 compared to low glucose).Apoptosis was induced as a result of mitochondrial impairment and explained the poor survival of MSCs in high glucose.CONCLUSION High glucose impaired the mitochondrial dynamics and regulatory proteins in hAD-MSCs ensuing their poor survival and high apoptosis rate in hyperglycemic microenvironment.

Protective effects of curcumin in APPswe transfected SH-SY5Y cells

The APPswe plasmid was transfected into the neuroblastoma cell line SH-SY5Y to establish a cell model of Alzheimer＇s disease. Graded concentration and time course experiments demonstrate that curcumin significantly upregulates phosphatidylinositol 3-kinase （PI3K）, Akt, nuclear factor E2-related factor-2 （Nrf2）, heme oxygenase 1 and ferritin expression, and that it significantly downregulates heme oxygenase 2, reactive oxygen species and amyloid-beta 40/42 expression. These effects of curcumin on PI3K, Akt and Nrf2 were blocked by LY294002 （PI3k inhibitor） and NF-E2-related factor-2 siRNA. The results indicate that the cytoprotection conferred by curcumin on APPswe transfected SH-SY5Y cells is mediated by its ability to regulate the balance between heme oxygenase 1 and 2 via the PI3K/Akt/Nrf2 intracellular signaling pathway.