Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Roles of phosphatidylinositol-3-kinases signaling pathway in inflammation-related cancer:Impact of rs10889677 variant and buparlisib in colitis-associated cancer

BACKGROUND Phosphatidylinositol-3-kinases(PI3K)is a well-known route in inflammationrelated cancer.Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis(UC)and colorectal cancer(CRC)with colitisassociated cancer(CAC).PI3K/AKT pathway has been recommended as a potential additional therapeutic option for CRC due to its substantial role in modifying cellular processes.Buparlisib is a pan-class I PI3K inhibitor previously shown to reduce tumor growth.AIM To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis.METHODS Genomic DNA from 32 colonic samples,including CAC(n=7),UC(n=10)and CRC(n=15),was sequenced for the rs10889677 mutation.The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector.The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line.CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium,then buparlisib was administered after 14 d.The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2.RESULTS Luciferase activity decreased by 2.07-fold in the rs10889677 mutant,confirming the hypothesis that the variant disrupted miRNA binding sites,which led to an increase in IL23R expression and the activation of the PI3K signaling pathway.Furthermore,CAC-induced mice had a significantly higher disease activity index(P<0.05).Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice(P<0.05),reduced the percentage of proliferating cells by 5%,and increased the number of apoptotic cells.The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression.CONCLUSION Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway,and buparlisib had the ability to prevent PI3K-non-AKT activation in the pathophysiology of CAC.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

Tumor-related factor complement Clq/TNF-related protein 6 affects the development of digestive system tumors through the phosphatidylinositol 3-kinase pathway

In this editorial,we review the work of Razali et al published in World J Gas-troenterology,with a particular focus on the effect of rs10889677 variation in the phosphatidylinositol 3-kinase(PI3K)pathway and buparlisib on colitis-associated cancer.The role of PI3K in promoting cancer progression has been widely recognized,as it is involved in regulating the survival,differentiation,and prolif-eration of cancer cells.The complement Clq/TNF-related protein 6(CTRP6)is a newer tumor-associated factor.Recent studies have revealed the pro-tumor effect of CTRP6 in gastric cancer,hepatocellular carcinoma,colorectal cancer,and other gastrointestinal tumors through the PI3K pathway.This article attempts to reveal the mechanism through which the CTRP6 affects the development of digestive system tumors through the PI3K pathway by summarizing recent research.

Bornyl acetate extracted from Sharen(Fructus Amomi)inhibits proliferation,invasion and induces apoptosis by suppressing phosphatidylinositol-3-kinase/protein kinase B signaling in colorectal cancer

OBJECTIVE:To investigate the antitumor effects of bornyl acetate(BA)isolated from Sharen(Fructus Amomi)in colorectal cancer(CRC)and the underlying mechanisms.METHODS:SW480 and HT29 cells were treated with increasing doses of BA in order to determine its antitumor effects in vitro.Cell viability,colony formation,cell cycle,and apoptosis as well as migration and invasion were assessed using various assays.In addition,the in vivo antitumor effects of BA were assessed using a xenograft mouse model.We then assessed the mechanism of action of BA by conducting pathway activator-mediated rescue experiments and assessed the protein levels by Western blot analysis.RESULTS:BA showed anti-CRC tumor activities in vitro by suppressing cell proliferation and colony formation,inducing apoptosis,blocking cell cycle,and inhibiting migration and invasion.These effects were mediated via suppression of the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)pathway.In the tumor xenograft experiment,BA was found to repress tumor growth in vivo with low toxicity.CONCLUSIONS:The results demonstrated that BA exerts antitumor effects by suppressing the PI3K/AKT pathway,with low toxicity.Thus,BA might be a potential novel therapeutic agent for CRC.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection

Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

Quercetin-3-O-β-D-glucuronide Inhibits Mitochondria Pathway-mediated Platelet Apoptosis via the Phosphatidylinositol-3-kinase/AKT Pathway in Immunological Bone Marrow Failure

Objective: Quercetin-3-O-β-D-glucuronide(QG) can alleviate immunological bone marrow failure(BMF) by increasing platelet counts. However, the principal mechanism is less known. This study aimed at deciphering the possible underlying mechanism of QG that is indicated in thrombocytopenic purpura. Methods: In vitro and in vivo experiments were carried out for investigating the mechanism behind QG-facilitated inhibition of mitochondrial pathway-mediated excessive apoptosis of platelets through the phosphatidylinositol-3-kinase(PI3K)/AKT pathway. Results: Our results revealed that QG, the main effective ingredient of Herba Sarcandrae, increases the number of platelets and decreases the expression of Bax, Bad, Bid, and caspase-9 in immunological BMF, indicating the inhibition of mitochondrial pathway-mediated apoptosis. Moreover, we found that the protein and m RNA expressions, as well as the phosphorylated levels of PI3K and AKT, were increased significantly by QG, suggesting the activation of the PI3K/AKT pathway. Furthermore, the inhibition of the PI3K/AKT pathway by LY294002 antagonizes the effects of QG on platelet counts and mitochondrial pathway-mediated apoptosis. Conclusion: We demonstrate that QG inhibits the mitochondria pathway-mediated platelet apoptosis via the PI3K/AKT pathway in immunological BMF. This study thus sheds light on exploring the possible regulatory mechanism of traditional Chinese medicine in the treatment of thrombocytopenia induced by BMF.

Response of Subcutaneous Xenografts of Endometrial Cancer in Nude Mice to Inhibitors of Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase (MAPK) Pathways: An Effective Therapeutic Strategy for Endometrial Cancer

Objective: This study was designed to explore whether inhibition of the extracellular-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways can inhibit the growth of xenografts of endometrial cancer cell lines with different estrogen receptors (ER) profiles in vivo and to provide preliminary laboratory basis for the probability of endometrial adenocarcinoma treatment with blockage of the two pathways, especially to endometrial cancer with low ER status. Methods: Human endometrial cancer Ishikawa bearing ER and HEC-1Awith low ER status cells were subcutaneously injected into BALB/c nude mice to establish endometrial cancer xenograft tumor models. The effects of PI3K/Akt inhibitor LY294002, MAPK/ERK1/2 inhibitor PD-98059 and their combinations on the growth of the xenograft tumors and apoptotic state of Ishikawa and HEC-1Acells were tested in vivo using the inhibitory rate, the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, H/E-stain. Western blot analysis was used to detect the alterations of activated ERK (P-ERK) and AKT (P-AKT) during this process. Results: LY294002, a PI3K/Akt pathway inhibitor, induced significant suppression in the growth of both Ishikawa and HEC-1Acell xenograft tumors, concomitant with increased apoptosis in xenografts as evidenced by TUNEL. A similar effect was also observed when the MAPK/ERK1/2 signaling pathway was inhibited by PD98059. Concurrent inhibition of the PI3K/Akt and MAPK/ERK1/2 pathways showed enhanced anti-tumor effects in vivo as indicated by increased apoptosis. At the same time, the levels of P-ERK and P-AKT in both xenograft tumors decreased, and their levels in combination group was the lowest. Conclusions: PD98059, LY294002 and their combinations showed remarkable inhibitory effects on xenograft tumors of endometrial carcinoma cell lines with different expression status of ER in vivo through blockage of PI3K/Akt and MAPK/ERK1/2 signaling pathways. This suggests that targeting these pathways may be an effective therapeutic strategy against endometrial carcinomas, especially for ER-negative cancers which show poor response to endocrinal therapy.

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.

Phosphatidylinositol 3-kinase CB association with preoperative radiotherapy response in rectal adenocarcinoma

AIM:To examine the correlation of phosphatidylinositol3-kinase(PIK3)CB expression with preoperative radiotherapy response in patients with stageⅡ/Ⅲrectal adenocarcinoma.METHODS:PIK3CB immunoexpression was retrospectively assessed in pretreatment biopsies from 208 patients with clinical stageⅡ/Ⅲrectal adenocarcinoma,who underwent radical surgery after 30-Gy/10-fractionpreoperative radiotherapy.The relation between PIK3CB expression and tumor regression grade,clinicopathological characteristics,and survival time was statistically analyzed.Western blotting and in vitro clonogenic formation assay were used to detect PIK3CB expression in four colorectal cancer cell lines(HCT116,HT29,Lo Vo,and LS174T)treated with 6-Gy ionizing radiation.Pharmacological assays were used to evaluate the therapeutic relevance of TGX-221(a PIK3CB-specific inhibitor)in the four colorectal cancer cell lines.RESULTS:Immunohistochemical staining indicated that PIK3CB was more abundant in rectal adenocarcinoma tissues with poor response to preoperative radiotherapy.High expression of PIK3CB was closely correlated with tumor height(P<0.05),yp T stage(P<0.05),and high-degree tumor regression grade(P<0.001).High expression of PIK3CB was a potential prognostic factor for local recurrence-free survival(P<0.05)and metastasis-free survival(P<0.05).High expression of PIK3CB was also associated with poor therapeutic response and adverse outcomes in rectal adenocarcinoma patients treated with 30-Gy/10-fraction preoperative radiotherapy.In vitro,PIK3CB expression was upregulated in all four colorectal cancer cell lines concurrently treated with 6-Gy ionizing radiation,and the PIK3CB-specific inhibitor TGX-221 effectively inhibited the clonogenic formation of these four colorectal cancer cell lines.CONCLUSION:PIK3CB is critically involved in response to preoperative radiotherapy and may serve as a novel target for therapeutic intervention.

Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn’s disease via signaling involving distinct classes of phosphatidylinositol 3-kinases

BACKGROUND Autophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components.Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn’s disease(CD).Herbal cake-partitioned moxibustion(HM)has been historically practiced to treat CD.However,the mechanism by which HM regulates colonic autophagy in CD remains unclear.AIM To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism.METHODS Rats were randomly divided into a normal control(NC)group,a CD group,an HM group,an insulin+CD(I+CD)group,an insulin+HM(I+HM)group,a rapamycin+CD(RA+CD)group,and a rapamycin+HM(RA+HM)group.2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model.The morphology of the colonic mucosa was observed by hematoxylin-eosin staining,and the formation of autophagosomes was observed by electron microscopy.The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta(LC3B)was observed by immunofluorescence staining.Insulin and rapamycin were used to inhibit and activate colonic autophagy,respectively.The mRNA expression levels of phosphatidylinositol 3-kinase class I(PI3KC1),Akt1,LC3B,sequestosome 1(p62),and mammalian target of rapamycin(mTOR)were evaluated by RT-qPCR.The protein expression levels of interleukin 18(IL-18),tumor necrosis factor-α(TNF-α),nuclear factorκB/p65(NF-κB p65),LC3B,p62,coiled-coil myosin-like BCL2-interacting protein(Beclin-1),p-mTOR,PI3KC1,class III phosphatidylinositol 3-kinase(PI3KC3/Vps34),and p-Akt were evaluated by Western blot analysis.RESULTS Compared with the NC group,the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues(P<0.01 for both).Compared with the CD group,the HM group showed significantly lower levels of these proteins(PIL-18<0.01 and Pp65<0.05).There were no significant differences in the expression of TNF-αprotein in colon tissue among the rat groups.Typical autophagic vesicles were found in both the CD and HM groups.The expression of the autophagy proteins LC3B and Beclin-1 was upregulated(P<0.01 for both)in the colon tissues of rats in the CD group compared with the NC group,while the protein expression of p62 and p-mTOR was downregulated(P<0.01 for both).However,these expression trends were significantly reversed in the HM group compared with the CD group(PLC3B<0.01,PBeclin-1<0.05,Pp62<0.05,and Pm-TOR<0.05).Compared with those in the RA+CD group,the mRNA expression levels of PI3KC1,Akt1,mTOR,and p62 in the RA+HM group were significantly higher(PPI3KC1<0.01 and PAkt1,mTOR,and p62<0.05),while those of LC3B were significantly lower(P<0.05).Compared with the RA+CD group,the RA+HM group exhibited significantly higher PI3KC1,p-Akt1,and pmTOR protein levels(PPI3KC1<0.01,Pp-Akt1<0.05,and Pp-mTOR<0.01),a higher p62 protein level(P=0.057),and significantly lower LC3B and Vps34 protein levels(P<0.01 for both)in colon tissue.CONCLUSION HM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3(Vps34)-Beclin-1 protein complex in the colon tissues of CD rats,thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.

Analysis of Phosphatidylinositol 3-kinase Activation in the Adipose Tissue of Gestational Diabetes Mellitus Patients and Insulin Resistance

The P85 regulatory subunit protein and gene expression and P110 catalylic subunit activity of phosphatidylinositol 3-kinase (PI-3K) were investigated in adipose tissue of patients with gestational diabetes mellitus (GDM) in order to explore the molecular mechanisms of insulin resistance (IR) of GDM. Samples from patients with GDM (n=50), and controls (n=50) were collected. Fasting insulin (FIN) was determined by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Western blot technique was used to detect the levels of PI-3K P85 subunit in adipose tissues of patients with GDM. The mRNA expression of PI-3K P85 subunit was detected by reverse transcription polymerase chain reaction (RT-PCR) method in the adipose tissue. PI-3K activity was examined by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed statistically. It was found that the levels of FPG, FIN and HOMA-IR in GDM group were significantly higher than those in control group (all P0.05). PI-3K activity was significantly decreased to 82.89% in GDM group as compared with control group (P<0.01) and negatively correlated with HOMA-IR (r=-0.75, P<0.01). It was concluded that PI-3K in GDM patients may be involved in the insulin signaling pathway, resulting in IR of GDM.

Micro RNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of micro RNA-21(mi R-21), phosphatase gene(p TEN), and its downstream proteins, protein kinase B(AKT) and phosphatidylinositol 3-kinase(p I3K), in colorectal cancer(CRC) cells. METHODS: Quantitative real-time p CR(q RT-p CR) and Western blot were used to detect the expression levels of mi R-21 and p TEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of p TEN m RNA and its downstream proteins AKT and p I3 K in HCT116 cells after downregulating mi R-21 were investigated. RESULTS: Comparing the mi R-21 expression in CRC cells, the expression levels of mi R-21 were highest in HCT116 cells, and the expression levels of mi R-21 were lowest in SW480 cells. In comparing mi R-21 and p TEN expression in CRC cells, we found that the protein expression levels of mi R-21 and p TEN were inversely correlated(p < 0.05); when mi R-21 expression was reduced, m RNA expression levels of p TEN did not significantly change(p > 0.05), but the expression levels of its protein significantly increased(p < 0.05). In comparing the levels of p TEN protein and downstream AKT and p I3 K in HCT116 cells after downregulation of mi R-21 expression, the levels of AKT and p I3 K protein expression significantly decreased(p < 0.05). CONCLUSION: p TEN is one of the direct target genesof mi R-21. Thus, phosphatase gene and its downstream AKT and p I3 K expression levels can be regulated by regulating the expression levels of mi R-21, which in turn regulates the development of CRC.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition

BACKGROUND Intestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIM To explore the protective effects of panax notoginseng saponin(PNS) against dextran sulfate sodium(DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B(PI3K/AKT) signaling pathway inhibition in rats.METHODS Colitis rat models were generated via DSS induction, and rats were divided into control(no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3 K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTS Compared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers,and showed significantly increased M1 macrophages in spleen and colon tissues.They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway(all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency(all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group(P < 0.05).CONCLUSION PNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

Promoting axon regeneration in the central nervous system by increasing PI3-kinase signaling

Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system.Axons in the central nervous system fail to regenerate,meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences.In 2008,genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve.PTEN is a phosphatase that opposes the actions of PI3-kinase,a family of enzymes that function to generate the membrane phospholipid PIP_(3) from PIP_(2)(phosphatidylinositol(3,4,5)-trisphosphate from phosphatidylinositol(4,5)-bisphosphate).Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase,and was initially demonstrated to promote axon regeneration by signaling through mTOR.More recently,additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability.This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3,and considers them in relation to both developmental and regenerative axon growth.We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability,and describe how these are affected by signaling through PI3-kinase.We highlight the recent finding of a developmental decline in the generation of PIP_(3) as a key reason for regenerative failure,and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system.Finally,we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.

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.

Xuebijing improves intestinal microcirculation dysfunction in septic rats by regulating the VEGF-A/PI3K/Akt signaling pathway

BACKGROUND:This study aims to explore whether Xuebijing(XBJ) can improve intestinal microcirculation dysfunction in sepsis and its mechanism.METHODS:A rat model of sepsis was established by cecal ligation and puncture(CLP).A total of 30 male SD rats were divided into four groups:sham group,CLP group,XBJ + axitinib group,and XBJ group.XBJ was intraperitoneally injected 2 h before CLP.Hemodynamic data(blood pressure and heart rate) were recorded.The intestinal microcirculation data of the rats were analyzed via microcirculation imaging.Enzyme-linked immunosorbent assay(ELISA) kits were used to detect the serum levels of interleukin-6(IL-6),C-reactive protein(CRP),and tumor necrosis factor-α(TNF-α) in the rats.Histological analysis and transmission electron microscopy were used to analyze the injury of small intestinal microvascular endothelial cells and small intestinal mucosa in rats.The expression of vascular endothelial growth factor A(VEGF-A),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt) in the small intestine was analyzed via Western blotting.RESULTS:XBJ improved intestinal microcirculation dysfunction in septic rats,alleviated the injury of small intestinal microvascular endothelial cells and small intestinal mucosa,and reduced the systemic inflammatory response.Moreover,XBJ upregulated the expression of VEGF-A,p-PI3K/total PI3K,and p-Akt/total Akt in the rat small intestine.CONCLUSION:XBJ may improve intestinal microcirculation dysfunction in septic rats possibly through the VEGF-A/PI3K/Akt signaling pathway.

Impacts of PI3K/protein kinase B pathway activation in reactive astrocytes: from detrimental effects to protective functions

Astrocytes are the most abundant type of glial cell in the central nervous system.Upon injury and inflammation,astrocytes become reactive and undergo morphological and functional changes.Depending on their phenotypic classification as A1 or A2,reactive astrocytes contribute to both neurotoxic and neuroprotective responses,respectively.However,this binary classification does not fully capture the diversity of astrocyte responses observed across different diseases and injuries.Transcriptomic analysis has revealed that reactive astrocytes have a complex landscape of gene expression profiles,which emphasizes the heterogeneous nature of their reactivity.Astrocytes actively participate in regulating central nervous system inflammation by interacting with microglia and other cell types,releasing cytokines,and influencing the immune response.The phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway is a central player in astrocyte reactivity and impacts various aspects of astrocyte behavior,as evidenced by in silico,in vitro,and in vivo results.In astrocytes,inflammatory cues trigger a cascade of molecular events,where nuclear factor-κB serves as a central mediator of the pro-inflammatory responses.Here,we review the heterogeneity of reactive astrocytes and the molecular mechanisms underlying their activation.We highlight the involvement of various signaling pathways that regulate astrocyte reactivity,including the PI3K/AKT/mammalian target of rapamycin(mTOR),αvβ3 integrin/PI3K/AKT/connexin 43,and Notch/PI3K/AKT pathways.While targeting the inactivation of the PI3K/AKT cellular signaling pathway to control reactive astrocytes and prevent central nervous system damage,evidence suggests that activating this pathway could also yield beneficial outcomes.This dual function of the PI3K/AKT pathway underscores its complexity in astrocyte reactivity and brain function modulation.The review emphasizes the importance of employing astrocyte-exclusive models to understand their functions accurately and these models are essential for clarifying astrocyte behavior.The findings should then be validated using in vivo models to ensure real-life relevance.The review also highlights the significance of PI3K/AKT pathway modulation in preventing central nervous system damage,although further studies are required to fully comprehend its role due to varying factors such as different cell types,astrocyte responses to inflammation,and disease contexts.Specific strategies are clearly necessary to address these variables effectively.