Phosphotidylinositol (PI) can be hydrolyzed into inositol 1,4,5_ trisphosphate/diacylglycerol (IP 3/DAG), the second signalling messengers which can be further phosphorylated by phospholipases to other signalling mole...Phosphotidylinositol (PI) can be hydrolyzed into inositol 1,4,5_ trisphosphate/diacylglycerol (IP 3/DAG), the second signalling messengers which can be further phosphorylated by phospholipases to other signalling molecules, and phosphatidic acid (PA) and lysophosphoglyceride (lysoPI) involved in many signalling cascades. It has been shown that PI signalling pathway and the involved enzymes are critical for plant development and response to environmental factors. Three classes of phospholipases, named PLA 2, PLC and PLD, were classified based on their cleavage sites, and the presence of variants of isoforms indicating their different functions in plant growth/development. The update information of phospholipases with focus on the interaction of plant hormone and environmental factors has been provided.展开更多
Free fatty acids are known to play a key role in promoting loss of insulin sensitivity in type 2 diabetes mellitus but the underlying mechanism is still unclear.It has been postulated that an increase in the intracell...Free fatty acids are known to play a key role in promoting loss of insulin sensitivity in type 2 diabetes mellitus but the underlying mechanism is still unclear.It has been postulated that an increase in the intracellular concentration of fatty acid metabolites activates a serine kinase cascade,which leads to defects in insu-lin signaling downstream to the insulin receptor.In addition,the complex network of adipokines released from adipose tissue modulates the response of tissues to insulin.Among the many molecules involved in the intracellular processing of the signal provided by insulin,the insulin receptor substrate-2,the protein kinase B and the forkhead transcription factor Foxo 1a are of particular interest,as recent data has provided strong evidence that dysfunction of these proteins results in insulin resistance in vivo.Recently,studies have revealed that phosphoinositidedependent kinase 1-independent phosphorylation of protein kinase Cε causes a reduction in insulin receptor gene expression.Additionally,it has been suggested that mitochondrial dysfunction triggers activation of several serine kinases,and weakens insulin signal transduction.Thus,in this review,the current developments in understanding the pathophysiological processes of insulin resistance in type 2 diabetes have been summarized.In addition,this study provides potential new targets for the treatment and prevention of type 2 diabetes.展开更多
AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC). METHODS: An activated HSC cell line was ...AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC). METHODS: An activated HSC cell line was used in this study. LY 294002, the PI 3-K/Akt signal pathway block-er was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this path-way in HSC apoptosis. Immunocytochemistry, Western blot and reverse transcription polymerase chain reac-tion (RT-PCR) analysis were applied to detect the ex-pression of PI 3-K, and simultaneously phosphorylated-Akt (p-Akt) and total-Akt were determined by Western blot. The HSC apoptosis was examined by annexin-V/ propidium iodide double-labelled flow cytometry and transmission electron microscopy. RESULTS: The apoptosis rates in LY 294002 (30.82% ± 2.90%) and LY 294002 + PDGF-BB (28.16% ± 2.58%) groups were signif icantly increased compared with those of control (9.02% ± 1.81%) and PDGF-BB (4.35% ± 1.18%). PDGF-BB augmented PI 3-K and p-Akt expres-sion. LY 294002 signif icantly reduced the contents of PI 3-K and p-Akt. mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression. CONCLUSION: Inhibition of PI 3-K/Akt signaling path-way induces apoptosis in HSC.展开更多
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...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.展开更多
The phosphatidylinositol 3-kinase(PI3K) pathway is frequently activated in human cancers.Class I PI3 Ks are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate(PIP2) at the 3-OH of the inositol ring...The phosphatidylinositol 3-kinase(PI3K) pathway is frequently activated in human cancers.Class I PI3 Ks are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate(PIP2) at the 3-OH of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate(PIP3), which in turn activates Akt and the downstream effectors like mammalian target of rapamycin(m TOR) to play key roles in carcinogenesis. Therefore, PI3 K has become an important anticancer drug target, and currently there is very high interest in the pharmaceutical development of PI3 K inhibitors. Idelalisib has been approved in USA and Europe as the first-in-class PI3 K inhibitor for cancer therapy. Dozens of other PI3 K inhibitors including BKM120 and ZSTK474 are being evaluated in clinical trials. Multifaceted studies on these PI3 K inhibitors are being performed, such as single and combinational efficacy, resistance, biomarkers,etc. This review provides an introduction to PI3 K and summarizes key advances in the development of PI3 K inhibitors.展开更多
Background Apoptosis is a major cause of ischemic heart dysfunction. Apelin, the endogenous ligand for the G-protein-coupled APJ receptor, has been reported to exert cardioprotective effects during myocardial injury. ...Background Apoptosis is a major cause of ischemic heart dysfunction. Apelin, the endogenous ligand for the G-protein-coupled APJ receptor, has been reported to exert cardioprotective effects during myocardial injury. The aim of this study was to investigate the effects of apelin on apoptosis of rat cardiomyocytes induced by glucose deprivation （GD） and study the related signaling pathway. Methods Apelin and APJ mRNA expression were determined by RT-PCR in neonatal rat cardiomyocytes during different durations of GD. Cardiomyocyte apoptosis was detected by annexin V-FITC/propidium iodide （PI） staining after GD for 12 hours with or without apelin-13 （10 and 100 nmol/L） pretreatment. Protein levels of Akt and the mammalian target of rapamycin （mTOR） as well as cell apoptosis were detected in the presence or absence of LY294002 （a phosphatidylinositol 3-kinases （PI3K） inhibitor） or rapamycin （a mTOR inhibitor）. Results Apelin mRNA expression was up-regulated when cardiomyocytes were exposed to GD for 6, 12, 18, and 24 hours compared with the base level （P 〉0.05, P 〈0.01, P 〈0.01, P 〈0.01）. However, when cardiomyocytes were exposed to GD for up to 36 hours, apelin mRNA expression was 17% lower than the base level （P〈0.05）. APJ mRNA expression paralleled that of apelin. Apelin-13 pretreatment at 100 nmol/L significantly inhibited GD-induced cardiomyocyte apoptosis （P 〈0.05） and increased Akt and mTOR phosphorylation （P 〈0.01, P 〈0.01）. At the same time apelin-13 （100 nmol/L） up-regulated Bcl-2 protein expression and down-regulated Bax and cleaved caspase-3 expression （P 〈0.01, P 〈0.05, P 〈0.05）. The anti-apoptotic effect of apelin-13 was blocked by LY294002 （P 〈0.01） but not by rapamycin. Conclusions The endogenous apelin-APJ system is compensatorily up-regulated and ultimately down-regulated following sustained myocardial ischemia. Apelin protects against ischemic cardiomyocyte apoptosis via activation of the PI3K/Akt pathway.展开更多
Objective Our previous study showed that tumor tissue-derived formaldehyde at low concentrations plays an important role in bone cancer pain through activating transient receptor potential vanilloid subfamily member 1...Objective Our previous study showed that tumor tissue-derived formaldehyde at low concentrations plays an important role in bone cancer pain through activating transient receptor potential vanilloid subfamily member 1 （TRPV 1）. The present study further explored whether this tumor tissue-derived endogenous formaldehyde regulates TRPV1 expres- sion in a rat model of bone cancer pain, and if so, what the possible signal pathways are during the development of this type of pain. Methods A rat model of bone cancer pain was established by injecting living MRMT-1 tumor cells into the tibia. The formaldehyde levels were determined by high performance liquid chromatography, and the expression of TRPV1 was examined with Western blot and RT-PCR. In primary cultured dorsal root ganglion （DRG） neurons, the ex- pression of TRPV1 was assessed after treatment with 100 ~tmol/L formaldehyde with or without pre-addition of PD98059 [an inhibitor for extracellular signal-regulated kinase], SB203580 （a p38 inhibitor）, SP600125 [an inhibitor for c-Jun N- terminal kinase], BIM [a protein kinase C （PKC） inhibitor] or LY294002 [a phosphatidylinositol 3-kinase （PI3K） inhibi- tor]. Results In the rat model of bone cancer pain, formaldehyde concentration increased in blood plasma, bone marrow and the spinal cord. TRPV1 protein expression was also increased in the DRG. In primary cultured DRG neurons, 100 p^mol/L formaldehyde significantly increased the TRPV1 expression level. Pre-incubation with PD98059, SB203580, SP600125 or LY294002, but not BIM, inhibited the formaldehyde-induced increase of TRPV1 expression. Conclusion Formaldehyde at a very low concentration up-regulates TRPV1 expression through mitogen-activated protein kinase and PI3K, but not PKC, signaling pathways. These results further support our previous finding that TRPV1 in peripheral after- ents plays a role in bone cancer pain.展开更多
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 ...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.展开更多
Background The vascular endothelial growth factor （VEGF） is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 （ICAM-1） is the key mediator o...Background The vascular endothelial growth factor （VEGF） is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 （ICAM-1） is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells （ECs） is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs. Methods Bovine retinal ECs （BRECs） were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF （100 ng/ml） and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase （eNOS） were detected using Western blotting. Griess reaction was used to detect nitric oxide （NO）. Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C （PKC） altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase （PI3K） or reactive oxygen species （ROS） significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased. Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.展开更多
AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedde...AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment.The patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical records.Expression of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM kit.Standard criteria for HER2 positivity(0,1+,2+,and 3+) were used.Tumors that scored 3+ were considered HER2-positive.Expression of phospho Akt(pAkt) was also analyzed by IHC.Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more.PI3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by pyrosequencing.Epstein-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA probe.Microsatellite instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.RESULTS:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,respectively.HER2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 cancers.Mutations in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,respectively.Two new types of PIK3CA mutation,R88Q and R108H,were found in exon1.All PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in exon20.Eighteen cancers(8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type(13/18 vs 93/198,P = 0.04).T展开更多
AIM: To examine the potential anti-tumor activity of paeoniflorin in the human gastric carcinoma cell line MGC-803.METHODS: Cell viability and cytotoxic effects in MGC-803 cells were analyzed using a 3-(4,5-dimethylth...AIM: To examine the potential anti-tumor activity of paeoniflorin in the human gastric carcinoma cell line MGC-803.METHODS: Cell viability and cytotoxic effects in MGC-803 cells were analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay, respectively. Cell apoptosis of MGC-803 cells was measured using flow cytometry,DAPI staining assay and caspase-3 activity assay.Quantitative reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the expression of microRNA-124(miR-124) in response to paeoniflorin.The expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt), phospho-Akt(p-Akt) and phospho-signal transducer and activator of transcription3(p-STAT3) were also measured by quantitative RTPCR and Western blot analysis in normal, miR-124 and anti-miR-124 over-expressing MGC-803 cells, treated with paeoniflorin.RESULTS: Paeoniflorin was found to inhibit MGC-803 cell viability in a dose-dependent manner. Paeoniflorin treatment was associated with the induction of apoptosis and caspase-3 activity in MGC-803 cells. Paeoniflorin treatment significantly increased miR-124 levels and inhibited the expression of PI3 K, Akt, p-Akt and p-STAT3 in MGC-803 cells. Interestingly, the over-expression of miR-124 inhibits PI3K/Akt and phospho-STAT3 expressions in MGC-803 cells. PI3 K agonist(IGF-1, 1μg/10 μL) or over-expression of STAT3 reversed the effect of paeoniflorin on the proliferation of MGC-803 cells. Over-expression of anti-miR-124 in MGC-803 cells reversed paeoniflorin-induced up-regulation.CONCLUSION: In summary, the in vitro data suggest that paeoniflorin is a potential novel therapeutic agent against gastric carcinoma, which inhibits cell viability and induces apoptosis through the up-regulation of miR-124 and suppression of PI3K/Akt and STAT3 signaling.展开更多
objective： To investigate the effect of Jiaotai Pill （交泰丸, JTP） at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase （PI3K） pathway in the skeletal muscle of diabe...objective： To investigate the effect of Jiaotai Pill （交泰丸, JTP） at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase （PI3K） pathway in the skeletal muscle of diabetic rats. Methods： The rat model of type 2 diabetes mellitus （T2DM） was established by intravenous injection of a small dose of streptozotoein plus high fat diet feeding. JTP at the same dosage of cinnamon and the increasing dosage of Coptis chinensis was administered to diabetic rats for nine weeks respectively. Plasma glucose and insulin levels were assayed. The expressions of proteins were determined by Western blot method. Results： All the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β （InsR β） subunit, insulin receptor substrate-1 （IRS-1）, PI3K p85 subunit and glucose transporter 4 （GLUT4） in skeletal muscle. Meanwhile, JTP increased the tyrosine phosphorylation of InsRβ subunit and IRS-1, and reduced the serine phosphorylation of IRS-1 in skeletal muscle. Interestingly, the effect of JTP on improving insulin sensitivity was not dose-dependent. In contrast, JTP containing the least amount of Coptis chinensis exhibited the best effect. Conclusion： JTP at different constitutional proportions attenuates the development of diabetes in a rat model of T2DM. The mechanism might be associated with enhancing insulin signaling through PI3K pathway in the skeletal muscle.展开更多
The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt ac...The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt activity is regarded as an attractive strategy to enhance the efficacy of cancer therapy and irradiation. This pathway consists of phosphatidylinositol 3 kinase(PI3K), mammalian target of rapamycin, and the transforming serine-threonine kinase Akt protein isoforms, also known as protein kinase B. DNA-targeted agents, such as platinum agents, taxanes, and antimetabolites, as well as radiation have had a significant impact on cancer treatment by affecting DNA replication, which is aberrantly activated in malignancies. However, the caveat is that they may also trigger the activation of repairing mechanisms, such as upstream and downstream cascade of Akt survival pathway. Thus, each target can theoretically be inhibited in view of improving the potency of conventional treatment. Akt inhibitors, e.g., MK-2206 and perifosine, or PI3K modulators, e.g., LY294002 and Wortmannin, have shown some promising results in favor of sensitizing the cancer cells to the therapy in vitro and in vivo, which have provided the rationale for incorporation of these novel agents into multimodality treatment of different malignancies. Nevertheless, despite the acceptable safety profile of some of these agents in the clinical studies, with regard to the efficacy, the results are still too preliminary. Hence, we need to wait for the upcoming data from the ongoing trials before utilizing them into the standard care of cancer patients.展开更多
AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 a...AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor. RESULTS: All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% ± 2.4% (mean ± SD), 30.9% ± 5.5%, 23.8% ± 3.9%, 37.1% ± 5.3%, 10.4% ± 4.5%, 52.5% ± 4.5% and 22.6% ± 0.4%, at 2 μmol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% ± 7.6%, 32.9% ± 8.2%, 53.9% ± 8.0%, 52.8% ± 4.0%, 32.3% ± 4.2%, 51.8% ± 4.5%, and 30.6% ± 9.4%, at 50 μmol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% ± 0.2%, 0%, 0%, 2.0% ± 0.1%, 5.0% ± 0.2%, 3.0% ± 0.1%, and 5.0% ± 0.2%, respectively (P < 0.05). LY294002 at 50 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% ± 0.2%, 0%, 3.0% ± 0.2%, 0%, 0%, 0% and 3% ± 0.1%, respectively (P < 0.05). PD980509 at 20 μmol/L did not suppress motility. Cells wer展开更多
The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors,and previous studies have indicated the involvement of multiple PI-related gene families during c...The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors,and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome,82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS),PI-phosphate kinases (PIPK),phospholipases (PL),inositol polyphosphate phosphatases (IPPase),inositol polyphosphate kinases (IPK),PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK,PLC,PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly,DNA chip technology was employed to study the expression patterns of various isoforms.In total,79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf,stem and flower tissues,and leaves from plants treated with various hormones (auxin,cytokinin,gibberellin,abscisic acid and brassinosteroid) or environmental factors (temperature,calcium,sodium,drought,salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular,the different isoforms of each family were specifically expressed in many cases,suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.展开更多
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 w...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.展开更多
OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carci...OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carcinoma (HCC) nude mice. METHODS: Forty male nude mice were randomly divided into 4 groups. Human HCC tissue was implanted in the livers of three groups. After 24 h, the three groups were treated respectively with JPJDF (37.5 g/kg), saline (20 mL/kg) and Tegafur (FT-207, 160 mg/kg) once a day for 10 weeks. The control group without implanting the tissue was concurrently treated with saline (20 mL/kg). The survival data and body weight of all mice were recorded, and expression levels of PTEN, PI3K and FAK in normal tissue and cancer tissue of the livers were eval-uated with immunohistochemical method. RESULTS: The cumulative survival rate of the mice in the JPJDF group was higher than those of the other groups. The rate of weight loss was the lowest in JPJDF group. The survivability and weight loss rate in FT-207 group were the poorest in all groups. The expression intensity of PTEN was higher in normal tissues than in cancer tissues, and lower in the normal tissues of HCC models than in that of mice without HCC. The PTEN expression intensity in normal tissue and cancer tissue from mice treated with FT-207 were lower than that from the mice treated with JPJDF or saline.The expression intensity of PI3K was higher in cancer tissue than in normal tissue. The PI3K expression intensity was the lowest in normal tissue and cancer tissue from mice treated with JPJDF, and the intensity from mice treated with FT-207 was the highest. In mice treated with JPJDF, the expression intensity of FAK was higher in the normal tissue and lower in the cancer tissue than those of the other treatment groups. CONCLUSION: The mechanism accounting for the prolonged survival of HCC-bearing mice treated with JPJDF might be related to the reduction in weight loss and the benign regulation of PTEN, PI3K, and FAK.展开更多
RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in pl...RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.展开更多
文摘Phosphotidylinositol (PI) can be hydrolyzed into inositol 1,4,5_ trisphosphate/diacylglycerol (IP 3/DAG), the second signalling messengers which can be further phosphorylated by phospholipases to other signalling molecules, and phosphatidic acid (PA) and lysophosphoglyceride (lysoPI) involved in many signalling cascades. It has been shown that PI signalling pathway and the involved enzymes are critical for plant development and response to environmental factors. Three classes of phospholipases, named PLA 2, PLC and PLD, were classified based on their cleavage sites, and the presence of variants of isoforms indicating their different functions in plant growth/development. The update information of phospholipases with focus on the interaction of plant hormone and environmental factors has been provided.
文摘Free fatty acids are known to play a key role in promoting loss of insulin sensitivity in type 2 diabetes mellitus but the underlying mechanism is still unclear.It has been postulated that an increase in the intracellular concentration of fatty acid metabolites activates a serine kinase cascade,which leads to defects in insu-lin signaling downstream to the insulin receptor.In addition,the complex network of adipokines released from adipose tissue modulates the response of tissues to insulin.Among the many molecules involved in the intracellular processing of the signal provided by insulin,the insulin receptor substrate-2,the protein kinase B and the forkhead transcription factor Foxo 1a are of particular interest,as recent data has provided strong evidence that dysfunction of these proteins results in insulin resistance in vivo.Recently,studies have revealed that phosphoinositidedependent kinase 1-independent phosphorylation of protein kinase Cε causes a reduction in insulin receptor gene expression.Additionally,it has been suggested that mitochondrial dysfunction triggers activation of several serine kinases,and weakens insulin signal transduction.Thus,in this review,the current developments in understanding the pathophysiological processes of insulin resistance in type 2 diabetes have been summarized.In addition,this study provides potential new targets for the treatment and prevention of type 2 diabetes.
基金The Natural Science Foundation of Hebei Province, China, No.C2007000843
文摘AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC). METHODS: An activated HSC cell line was used in this study. LY 294002, the PI 3-K/Akt signal pathway block-er was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this path-way in HSC apoptosis. Immunocytochemistry, Western blot and reverse transcription polymerase chain reac-tion (RT-PCR) analysis were applied to detect the ex-pression of PI 3-K, and simultaneously phosphorylated-Akt (p-Akt) and total-Akt were determined by Western blot. The HSC apoptosis was examined by annexin-V/ propidium iodide double-labelled flow cytometry and transmission electron microscopy. RESULTS: The apoptosis rates in LY 294002 (30.82% ± 2.90%) and LY 294002 + PDGF-BB (28.16% ± 2.58%) groups were signif icantly increased compared with those of control (9.02% ± 1.81%) and PDGF-BB (4.35% ± 1.18%). PDGF-BB augmented PI 3-K and p-Akt expres-sion. LY 294002 signif icantly reduced the contents of PI 3-K and p-Akt. mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression. CONCLUSION: Inhibition of PI 3-K/Akt signaling path-way induces apoptosis in HSC.
基金Supported by The Natural Science Foundation of Heilongjiang Province, No. 2005-13
文摘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.
文摘The phosphatidylinositol 3-kinase(PI3K) pathway is frequently activated in human cancers.Class I PI3 Ks are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate(PIP2) at the 3-OH of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate(PIP3), which in turn activates Akt and the downstream effectors like mammalian target of rapamycin(m TOR) to play key roles in carcinogenesis. Therefore, PI3 K has become an important anticancer drug target, and currently there is very high interest in the pharmaceutical development of PI3 K inhibitors. Idelalisib has been approved in USA and Europe as the first-in-class PI3 K inhibitor for cancer therapy. Dozens of other PI3 K inhibitors including BKM120 and ZSTK474 are being evaluated in clinical trials. Multifaceted studies on these PI3 K inhibitors are being performed, such as single and combinational efficacy, resistance, biomarkers,etc. This review provides an introduction to PI3 K and summarizes key advances in the development of PI3 K inhibitors.
文摘Background Apoptosis is a major cause of ischemic heart dysfunction. Apelin, the endogenous ligand for the G-protein-coupled APJ receptor, has been reported to exert cardioprotective effects during myocardial injury. The aim of this study was to investigate the effects of apelin on apoptosis of rat cardiomyocytes induced by glucose deprivation （GD） and study the related signaling pathway. Methods Apelin and APJ mRNA expression were determined by RT-PCR in neonatal rat cardiomyocytes during different durations of GD. Cardiomyocyte apoptosis was detected by annexin V-FITC/propidium iodide （PI） staining after GD for 12 hours with or without apelin-13 （10 and 100 nmol/L） pretreatment. Protein levels of Akt and the mammalian target of rapamycin （mTOR） as well as cell apoptosis were detected in the presence or absence of LY294002 （a phosphatidylinositol 3-kinases （PI3K） inhibitor） or rapamycin （a mTOR inhibitor）. Results Apelin mRNA expression was up-regulated when cardiomyocytes were exposed to GD for 6, 12, 18, and 24 hours compared with the base level （P 〉0.05, P 〈0.01, P 〈0.01, P 〈0.01）. However, when cardiomyocytes were exposed to GD for up to 36 hours, apelin mRNA expression was 17% lower than the base level （P〈0.05）. APJ mRNA expression paralleled that of apelin. Apelin-13 pretreatment at 100 nmol/L significantly inhibited GD-induced cardiomyocyte apoptosis （P 〈0.05） and increased Akt and mTOR phosphorylation （P 〈0.01, P 〈0.01）. At the same time apelin-13 （100 nmol/L） up-regulated Bcl-2 protein expression and down-regulated Bax and cleaved caspase-3 expression （P 〈0.01, P 〈0.05, P 〈0.05）. The anti-apoptotic effect of apelin-13 was blocked by LY294002 （P 〈0.01） but not by rapamycin. Conclusions The endogenous apelin-APJ system is compensatorily up-regulated and ultimately down-regulated following sustained myocardial ischemia. Apelin protects against ischemic cardiomyocyte apoptosis via activation of the PI3K/Akt pathway.
基金supported by grants from the National Natural Science Foundation of China (81070893, 81171042 and 31171063)Beijing Municipal Commission of Education "Grants for Outstanding Ph.D. Program Tutors"+2 种基金the "111" Project of the Ministry of Education of ChinaThe Ministry of Education(BMU20100014)the China Postdoctoral Science Foundation (20090450266)
文摘Objective Our previous study showed that tumor tissue-derived formaldehyde at low concentrations plays an important role in bone cancer pain through activating transient receptor potential vanilloid subfamily member 1 （TRPV 1）. The present study further explored whether this tumor tissue-derived endogenous formaldehyde regulates TRPV1 expres- sion in a rat model of bone cancer pain, and if so, what the possible signal pathways are during the development of this type of pain. Methods A rat model of bone cancer pain was established by injecting living MRMT-1 tumor cells into the tibia. The formaldehyde levels were determined by high performance liquid chromatography, and the expression of TRPV1 was examined with Western blot and RT-PCR. In primary cultured dorsal root ganglion （DRG） neurons, the ex- pression of TRPV1 was assessed after treatment with 100 ~tmol/L formaldehyde with or without pre-addition of PD98059 [an inhibitor for extracellular signal-regulated kinase], SB203580 （a p38 inhibitor）, SP600125 [an inhibitor for c-Jun N- terminal kinase], BIM [a protein kinase C （PKC） inhibitor] or LY294002 [a phosphatidylinositol 3-kinase （PI3K） inhibi- tor]. Results In the rat model of bone cancer pain, formaldehyde concentration increased in blood plasma, bone marrow and the spinal cord. TRPV1 protein expression was also increased in the DRG. In primary cultured DRG neurons, 100 p^mol/L formaldehyde significantly increased the TRPV1 expression level. Pre-incubation with PD98059, SB203580, SP600125 or LY294002, but not BIM, inhibited the formaldehyde-induced increase of TRPV1 expression. Conclusion Formaldehyde at a very low concentration up-regulates TRPV1 expression through mitogen-activated protein kinase and PI3K, but not PKC, signaling pathways. These results further support our previous finding that TRPV1 in peripheral after- ents plays a role in bone cancer pain.
基金Supported by Medical Research Fund of Guangdong Province,No.A2007284Health Bureau Fund of Guangzhou,No.2009-YB-169
文摘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.
基金This study was supported in part by grants from the National Natural Science Foundation of China （No. 30571994, No. 30570713 and No. 30630032）.
文摘Background The vascular endothelial growth factor （VEGF） is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 （ICAM-1） is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells （ECs） is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs. Methods Bovine retinal ECs （BRECs） were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF （100 ng/ml） and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase （eNOS） were detected using Western blotting. Griess reaction was used to detect nitric oxide （NO）. Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C （PKC） altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase （PI3K） or reactive oxygen species （ROS） significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased. Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.
基金Supported by Grants-in-Aid for Scientific Research from the Ministry of Education,Culture,Sports,Science and Technology of Japan,to Yamamoto H and Shinomura Y
文摘AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment.The patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical records.Expression of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM kit.Standard criteria for HER2 positivity(0,1+,2+,and 3+) were used.Tumors that scored 3+ were considered HER2-positive.Expression of phospho Akt(pAkt) was also analyzed by IHC.Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more.PI3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by pyrosequencing.Epstein-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA probe.Microsatellite instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.RESULTS:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,respectively.HER2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 cancers.Mutations in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,respectively.Two new types of PIK3CA mutation,R88Q and R108H,were found in exon1.All PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in exon20.Eighteen cancers(8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type(13/18 vs 93/198,P = 0.04).T
基金Supported by National Natural Science Foundation of China,No.81372553
文摘AIM: To examine the potential anti-tumor activity of paeoniflorin in the human gastric carcinoma cell line MGC-803.METHODS: Cell viability and cytotoxic effects in MGC-803 cells were analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay, respectively. Cell apoptosis of MGC-803 cells was measured using flow cytometry,DAPI staining assay and caspase-3 activity assay.Quantitative reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the expression of microRNA-124(miR-124) in response to paeoniflorin.The expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt), phospho-Akt(p-Akt) and phospho-signal transducer and activator of transcription3(p-STAT3) were also measured by quantitative RTPCR and Western blot analysis in normal, miR-124 and anti-miR-124 over-expressing MGC-803 cells, treated with paeoniflorin.RESULTS: Paeoniflorin was found to inhibit MGC-803 cell viability in a dose-dependent manner. Paeoniflorin treatment was associated with the induction of apoptosis and caspase-3 activity in MGC-803 cells. Paeoniflorin treatment significantly increased miR-124 levels and inhibited the expression of PI3 K, Akt, p-Akt and p-STAT3 in MGC-803 cells. Interestingly, the over-expression of miR-124 inhibits PI3K/Akt and phospho-STAT3 expressions in MGC-803 cells. PI3 K agonist(IGF-1, 1μg/10 μL) or over-expression of STAT3 reversed the effect of paeoniflorin on the proliferation of MGC-803 cells. Over-expression of anti-miR-124 in MGC-803 cells reversed paeoniflorin-induced up-regulation.CONCLUSION: In summary, the in vitro data suggest that paeoniflorin is a potential novel therapeutic agent against gastric carcinoma, which inhibits cell viability and induces apoptosis through the up-regulation of miR-124 and suppression of PI3K/Akt and STAT3 signaling.
基金Supported by grants from National Natural Science Foundation of China(No.30801492)
文摘objective： To investigate the effect of Jiaotai Pill （交泰丸, JTP） at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase （PI3K） pathway in the skeletal muscle of diabetic rats. Methods： The rat model of type 2 diabetes mellitus （T2DM） was established by intravenous injection of a small dose of streptozotoein plus high fat diet feeding. JTP at the same dosage of cinnamon and the increasing dosage of Coptis chinensis was administered to diabetic rats for nine weeks respectively. Plasma glucose and insulin levels were assayed. The expressions of proteins were determined by Western blot method. Results： All the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β （InsR β） subunit, insulin receptor substrate-1 （IRS-1）, PI3K p85 subunit and glucose transporter 4 （GLUT4） in skeletal muscle. Meanwhile, JTP increased the tyrosine phosphorylation of InsRβ subunit and IRS-1, and reduced the serine phosphorylation of IRS-1 in skeletal muscle. Interestingly, the effect of JTP on improving insulin sensitivity was not dose-dependent. In contrast, JTP containing the least amount of Coptis chinensis exhibited the best effect. Conclusion： JTP at different constitutional proportions attenuates the development of diabetes in a rat model of T2DM. The mechanism might be associated with enhancing insulin signaling through PI3K pathway in the skeletal muscle.
文摘The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt activity is regarded as an attractive strategy to enhance the efficacy of cancer therapy and irradiation. This pathway consists of phosphatidylinositol 3 kinase(PI3K), mammalian target of rapamycin, and the transforming serine-threonine kinase Akt protein isoforms, also known as protein kinase B. DNA-targeted agents, such as platinum agents, taxanes, and antimetabolites, as well as radiation have had a significant impact on cancer treatment by affecting DNA replication, which is aberrantly activated in malignancies. However, the caveat is that they may also trigger the activation of repairing mechanisms, such as upstream and downstream cascade of Akt survival pathway. Thus, each target can theoretically be inhibited in view of improving the potency of conventional treatment. Akt inhibitors, e.g., MK-2206 and perifosine, or PI3K modulators, e.g., LY294002 and Wortmannin, have shown some promising results in favor of sensitizing the cancer cells to the therapy in vitro and in vivo, which have provided the rationale for incorporation of these novel agents into multimodality treatment of different malignancies. Nevertheless, despite the acceptable safety profile of some of these agents in the clinical studies, with regard to the efficacy, the results are still too preliminary. Hence, we need to wait for the upcoming data from the ongoing trials before utilizing them into the standard care of cancer patients.
文摘AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor. RESULTS: All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% ± 2.4% (mean ± SD), 30.9% ± 5.5%, 23.8% ± 3.9%, 37.1% ± 5.3%, 10.4% ± 4.5%, 52.5% ± 4.5% and 22.6% ± 0.4%, at 2 μmol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% ± 7.6%, 32.9% ± 8.2%, 53.9% ± 8.0%, 52.8% ± 4.0%, 32.3% ± 4.2%, 51.8% ± 4.5%, and 30.6% ± 9.4%, at 50 μmol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% ± 0.2%, 0%, 0%, 2.0% ± 0.1%, 5.0% ± 0.2%, 3.0% ± 0.1%, and 5.0% ± 0.2%, respectively (P < 0.05). LY294002 at 50 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% ± 0.2%, 0%, 3.0% ± 0.2%, 0%, 0%, 0% and 3% ± 0.1%, respectively (P < 0.05). PD980509 at 20 μmol/L did not suppress motility. Cells wer
基金supported by grants from the National Natural Science Foundation of China(No.30100101)the State Key Project of Basic Research(No.G19990-11604)
文摘The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors,and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome,82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS),PI-phosphate kinases (PIPK),phospholipases (PL),inositol polyphosphate phosphatases (IPPase),inositol polyphosphate kinases (IPK),PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK,PLC,PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly,DNA chip technology was employed to study the expression patterns of various isoforms.In total,79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf,stem and flower tissues,and leaves from plants treated with various hormones (auxin,cytokinin,gibberellin,abscisic acid and brassinosteroid) or environmental factors (temperature,calcium,sodium,drought,salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular,the different isoforms of each family were specifically expressed in many cases,suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.
基金Supported by The National Natural Science Foundation of China,No.30470782
文摘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.
基金Supported by The National Natural Science Foundation of China(No. 81072806)The National Natural Science Foundation of China for Young Scholars(No. 811102581)The Chinese Medicines Agency Project of Guangdong Province(No.2010098)
文摘OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carcinoma (HCC) nude mice. METHODS: Forty male nude mice were randomly divided into 4 groups. Human HCC tissue was implanted in the livers of three groups. After 24 h, the three groups were treated respectively with JPJDF (37.5 g/kg), saline (20 mL/kg) and Tegafur (FT-207, 160 mg/kg) once a day for 10 weeks. The control group without implanting the tissue was concurrently treated with saline (20 mL/kg). The survival data and body weight of all mice were recorded, and expression levels of PTEN, PI3K and FAK in normal tissue and cancer tissue of the livers were eval-uated with immunohistochemical method. RESULTS: The cumulative survival rate of the mice in the JPJDF group was higher than those of the other groups. The rate of weight loss was the lowest in JPJDF group. The survivability and weight loss rate in FT-207 group were the poorest in all groups. The expression intensity of PTEN was higher in normal tissues than in cancer tissues, and lower in the normal tissues of HCC models than in that of mice without HCC. The PTEN expression intensity in normal tissue and cancer tissue from mice treated with FT-207 were lower than that from the mice treated with JPJDF or saline.The expression intensity of PI3K was higher in cancer tissue than in normal tissue. The PI3K expression intensity was the lowest in normal tissue and cancer tissue from mice treated with JPJDF, and the intensity from mice treated with FT-207 was the highest. In mice treated with JPJDF, the expression intensity of FAK was higher in the normal tissue and lower in the cancer tissue than those of the other treatment groups. CONCLUSION: The mechanism accounting for the prolonged survival of HCC-bearing mice treated with JPJDF might be related to the reduction in weight loss and the benign regulation of PTEN, PI3K, and FAK.
基金This work was supported by grants from the National Natural Science Foundation of China （No. 30971889）, National Science and Technology Major Projects （No. 2009ZX08005-003B）, and the Natural Science Foundation of Jiangsu Province （No. BK2012027）.We appreciate Prof. Brett Tyler （Oregon State） for manuscript preparation, and Yuanming Zhang （Nanjing Agricultural University） for suggestions in data analysis. No conflict of interest declared.
文摘RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.
