Phosphatidylinositol 3-kinase P85 Research Articles

Integrin CD11b attenuates colitis by strengthening Src-Akt pathway to polarize anti-inflammatory IL-10 expression.

Interleukin-10 (IL-10) plays a central role in regulation of intestinal mucosal homeostasis and prevention of inflammatory bowel disease (IBD). We previously reported that CD11bhi regulatory dendritic cells (DCs) can produce more IL-10, and CD11b can negatively regulate Toll-like receptors (TLRs)-induced inflammatory responses in macrophages. However whether CD11b and its signaling can control autoimmunity via IL-10 production remains unclear. Here we found that CD11b deficient (Itgam−/−) mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis, with more tumor necrosis factor α (TNF-α) while less IL-10 production. CD11b inhibited nuclear factor-kappa B (NF-κB) while promoted activator protein 1 (AP-1) activation through activating sarcoma oncogene (Src), leading to decreased TNF-α while increased IL-10 production. Src interacted with and promoted c-casitas B lineage lymphoma proto-oncogene (c-Cbl)-mediated degradation of the inhibitory subunit p85 of phosphatidylinositol 3-kinase (PI3K). Importantly, Src inhibitor dasatinib aggravated DSS-induced colitis by decreasing IL-10 while increasing TNF-α in vivo. Therefore, CD11b promotes IL-10 production by activating Src-Akt signal pathway. An axis of CD11b-Src pathway is important in balancing homeostasis of TLR-induced pro-inflammatory and anti-inflammatory responses.

Rat white adipocytes activate p85/p110 PI3K and induce PM GLUT4 in response to adrenoceptor agonists or aluminum fluoride.

Adipocyte responses to adrenergic and ß-adrenoceptor(-AR) (adrenoceptor) regulation are not sufficiently understood, and information helpful for elucidating the adrenoceptor-responsive machinery is insufficient. Here we show by using immunoprecipitated kinase analysis with a phosphatidylinositol 3-kinase (PI3K) p85 antibody that PI3K activation was induced by treatment with 10 or 100 µM norepinephrine (NE) for 15 min or with 10 mM aluminum fluoride (AF, a guanosine triphosphate (GTP)-binding (G) protein activator) for 20 min in white adipocytes (rat epididymal adipocytes) and that treatment with pertussis toxin (PTX, a G-protein inactivator) inhibited PI3K activation induced by the 20-min treatment with AF in the cells. In addition, western blot analysis revealed that glucose transporter 4 (GLUT4) level in the adipocyte plasma membrane (PM) fraction was increased by treatment with 10 µM NE, 100 µM dobutamine (DOB, a ß1-AR agonist), or 0.1 µM CL316243 (CL, a ß3-AR agonist) for 30 min or with 10 mM AF for 20 min. NE or AF treatment triggered 2-deoxyglucose (2-DG) uptake into adipocytes under the above conditions. Our results advance the understanding of responses to adrenoceptor regulation in white adipocytes and provide possible clues for clarifying the machinery involved in adrenergic and ß-AR responses in the cells.

Diosgenin and 5-Methoxypsoralen Ameliorate Insulin Resistance through ER-α/PI3K/Akt-Signaling Pathways in HepG2 Cells.

To determine the effects and the underlying mechanism of diosgenin (DSG) and 5-methoxypsoralen (5-MOP), two main active components in the classical Chinese prescription Hu-Lu-Ba-Wan (HLBW), on insulin resistance, HepG2 cells were incubated in medium containing insulin. Treatments with DSG, 5-MOP, and their combination were performed, respectively. The result showed that the incubation of HepG2 cells with high concentration insulin markedly decreased glucose consumption and glycogen synthesis. However, treatment with DSG, 5-MOP, or their combination significantly reversed the condition and increased the phosphorylated expression of estrogen receptor-α (ERα), sarcoma (Src), Akt/protein kinase B, glycogen synthase kinase-3β (GSK-3β), and the p85 regulatory subunit of phosphatidylinositol 3-kinase p85 (PI3Kp85). At the transcriptional level, expression of the genes mentioned above also increased except for the negative regulation of GSK-3β mRNA. The increased expression of glucose transport-4 (GLUT-4) was meanwhile observed through immunofluorescence. Nevertheless, the synergistic effect of DSG and 5-MOP on improving glycometabolism was not obvious in the present study. These results suggested that DSG and 5-MOP may improve insulin resistance through an ER-mediated PI3K/Akt activation pathway which may be a new strategy for type 2 diabetes mellitus, especially for women in an estrogen-deficient condition.

Calycosin‑7‑O‑β‑D‑glucoside attenuates ischemia‑reperfusion injury in vivo via activation of the PI3K/Akt pathway.

The aim of the present study was to investigate the effects and mechanisms of calycosin-7-O-β-d-glucoside (CG) on ischemia-reperfusion (I/R) injury in vivo. Hemodynamic parameters, including ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic pressure (LVESP) and left ventricular end-diastolic pressure (LVEDP) were monitored using an ultrasound system, and infarct size was measured using Evans blue/tetrazolium chloride double staining. The activities of serum creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA) were determined to assess the degree of myocardial injury and oxidative stress-induced damage. The protein expression levels of cleaved-caspase-3, cleaved-caspase-9, phosphorylated (p)-phosphatidylinositol 3-kinase (PI3K) p85, PI3K p85, p-Akt and Akt were determined using western blotting. The results demonstrated that pretreatment with high dose (H)-CG markedly improved cardiac function, as evidenced by upregulated EF, FS and LVESP, and downregulated LVEDP. In addition, administration of CG resulted in significant decreases in infarct size in the I/R+low dose-CG and I/R+H-CG groups, compared with the I/R group. The activities of CK and LDH, and the levels of MDA in the I/R+H-CG group were reduced, compared with those in the I/R group, whereas SOD activity was elevated. Treatment with CG inhibited the cleavage and activity of caspase-3 and caspase-9, and enhanced the phosphorylation of PI3K p85 and Akt. Notably, administration of the PI3K inhibitor, LY294002, markedly lowered the levels of p-PI3K p85/p-Akt, and eradicated the inhibitory effects of H-CG on infarct size, myocardial injury and oxidative stress-induced damage. Taken together, the results suggested that CG may alleviate I/R injury by activating the PI3K/Akt signaling pathway.

TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

Impaired insulin signaling pathway in ovarian follicles of cows with cystic ovarian disease

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence.

GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway.

Angiopoietin-like protein 2 (ANGPTL2) plays an important role in inflammatory carcinogenesis and tumor metastasis. The compound GDC-0152 is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. However, the interaction between ANGPTL2 and GDC-0152 has not been studied. It has been proven that ANGPTL2 promotes metastasis of osteosarcoma. Therefore, in the present study, the effect of GDC-0152 on the malignant progression of osteosarcoma promoted by ANGPTL2 was investigated. Human osteosarcoma cell line SaOS2 cells were pre-treated or non-treated with GDC-0152 and then exposed to recombinant human ANGPTL2. The viability of SaOS2 cells was determined by MTT assay, the migration of SaOS2 cells was analyzed by chamber migration assay kit, and the SaOS2 cell apoptosis was determined by fluorescence-activated cell sorting (FACS) and nuclear staining. Treatment with ANGPTL2 increased SaOS2 cell growth and migration and decreased cell apoptosis. The increased cell growth and decreased cell apoptosis were significantly attenuated in SaOS2 cells receiving GDC-0152. However, the ANGPTL2-increased SaOS2 cell migration was not inhibited by GDC-0152 treatment. Furthermore, western blot analysis showed that the activation of phosphatidyl inositol 3-kinase (PI3K) (p85), PI3K (p110), protein kinase B (Akt) (Ser473), Akt (Thr308) and p38 mitogen-activated protein kinase (p38MAPK) were upregulated by ANGPTL2. Quantitative real-time polymerase chain reaction (qTR-PCR) and gelatin zymography showed that the mRNA expression and activity of matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-2 (MMP-2) were also increased by ANGPTL2. The upregulated activation of PI3K and Akt were significantly suppressed by the treatment of GDC-0152. In contrast, GDC-0152 did not suppress ANGPTL2-induced p38MAPK phosphorylation, MMP-9/MMP-2 mRNA expression or MMP-9/MMP-2 activity. Taken together, these data indicate that GDC-0152 attenuates the malignant progression of osteosarcoma promoted by ANGPTL2 via PI3K/AKT but not p38MAPK signaling pathway. The present study indicated a novel therapeutic strategy to inhibit tumor growth by indirectly preventing ANGPTL2 signaling.

Acute Hepatic Insulin Resistance Contributes to Hyperglycemia in Rats Following Myocardial Infarction.

Although hyperglycemia is common in patients with acute myocardial infarction (MI), the underlying mechanisms are largely unknown. Insulin signaling plays a key role in the regulation of glucose homeostasis. In this study, we test the hypothesis that rapid alteration of insulin signaling pathways could be a potential contributor to acute hyperglycemia after MI. Male rats were used to produce MI by ligation of the left anterior descending coronary artery. Plasma glucose and insulin levels were significantly higher in MI rats than those in controls. Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) was reduced significantly in the liver tissue of MI rats compared with controls, followed by decreased attachment of phosphatidylinositol 3-kinase (PI3K) p85 subunit with IRS1 and Akt phosphorylation. However, insulin-stimulated signaling was not altered significantly in skeletal muscle after MI. The relative mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase were slightly higher in the liver tissue of MI rats than those in controls. Rosiglitazone (ROSI) markedly restored hepatic insulin signaling, inhibited gluconeogenesis and reduced plasma glucose levels in MI rats. Insulin resistance develops rapidly in liver but not skeletal muscle after MI, which contributes to acute hyperglycemia. Therapy aimed at potentiating hepatic insulin signaling may be beneficial for MI-induced hyperglycemia.

Abstract 4109: Cucurbitacin B: A novel natural agent for the management of non-small cell lung cancer

Abstract Lung cancer is the primary cause of cancer death in both men and women in the United States and worldwide. The general prognosis is still very low despite of developments in the treatment due to improved surgical techniques, increased application of combined modality treatments and the use of new drugs. Since the past decade, researchers have been investigating a range of purified compounds from dietary sources as possible anticancer drugs. The main issue with the use of natural products is their effectiveness at high doses which generally could not be delivered to humans through dietary consumption. Here, we observed that cucurbitacin B exhibits antiproliferative effects on human non-small cell lung cancer (NSCLC) cells at very low concentrations. The cucurbitacins are highly diverse and oxygenated tetracyclic triterpenoids isolated from plants of Cucurbitaceae family which are well-known for their bitterness of edible products like pumpkins, gourds and squashes. Cucurbitacin B is one of the most abundant and has been most widely used. In this study, we determined the effect of cucurbitacin B on the inhibition of cell-growth and suppression of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling in human NSCLC cells. Treatment of cucurbitacin B (0.2-0.6 μM; 24 h) was found to result in 34-61% and 45-65% decrease in the viability of human NSCLC H1792 and A549 cells respectively, but had only minimal effect on normal human bronchial epithelial cells. The IC50 of cucurbitacin B in these cells ranged from 0.43-0.49 μM. The A549 cell colonies were also reduced by treatment with cucurbitacin B in a dose-dependent manner. It has been well documented that mTOR functions downstream of the PI3K/Akt pathway and is phosphorylated in response to stimuli that activate the PI3K/Akt pathway. Treatment of A549 cells with cucurbitacin B (0.2-0.6 μM; 24 h) caused decrease in the protein expression of PI3K (p85 and p110) and inhibition of the phosphorylation of Akt and mTOR. PI3K/Akt-dependent phosphorylation signals through tuberin, the protein product of tuberous sclerosis complex (TSC)1/TSC2 complex, which leads to activation of mTOR. The mTOR also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) which disassociates from the eukaryotic translation initiation factor (eIF) 4E, allowing activation of protein translation. Treatment of A549 cells with cucurbitacin B also caused inhibition of the phosphorylation of p70S6K1, eIF-4E and 4E-BP1 and downstream targets of mTOR. In cucurbitacin B treated cells, there was also inhibition of the constituents of mTOR signaling complex such as Rictor, Raptor, GβL and PRAS40 and activation of AMP-activated protein kinase (AMPKα) and tuberous sclerosis complex (TSC)2. We suggest that cucurbitacin B could be developed as an agent for the management of lung cancer. Citation Format: Naghma Khan, Farah Jajeh, Sameh M. Shabana, Hasan Mukhtar. Cucurbitacin B: A novel natural agent for the management of non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4109. doi:10.1158/1538-7445.AM2014-4109

Role of c-Met/Phosphatidylinositol 3-Kinase (PI3k)/Akt Signaling in Hepatocyte Growth Factor (HGF)-mediated Lamellipodia Formation, Reactive Oxygen Species (ROS) Generation, and Motility of Lung Endothelial Cells

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

The Chinese herbal medicine FTZ attenuates insulin resistance via IRS1 and PI3K in vitro and in rats with metabolic syndrome

BackgroundInsulin resistance plays an important role in the development of metabolic syndrome (MS). Fu Fang Zhen Zhu Tiao Zhi formula (FTZ), a Chinese medicinal decoction, has been used to relieve hyperlipidemia, atherosclerosis and other symptoms associated with metabolic disorders in the clinic.MethodsTo evaluate the effect of FTZ on insulin resistance, HepG2 cells were induced with high insulin as a model of insulin resistance and treated with FTZ at one of three dosages. Next, the levels of glucose content, insulin receptor substrate1 (IRS1) protein expression and phosphatidylinositol 3-kinase (PI3K) subunit p85 mRNA expression were measured. Alternatively, MS was induced in rats via gavage feeding of a high-fat diet for four consecutive weeks followed by administration of FTZ for eight consecutive weeks. Body weight and the plasma levels of lipids, insulin and glucose were evaluated. Finally, the expression of PI3K p85 mRNA in adipose tissue of rats was measured.ResultsOur results revealed that FTZ attenuated glucose content and up-regulated the expression of PI3K p85 mRNA and IRS1 protein in insulin-resistant HepG2 cells in vitro. Moreover, FTZ reduced body weight and the plasma concentrations of triacylglycerol, cholesterol, fasting glucose and insulin in insulin resistant MS rats. FTZ also elevated the expression of PI3K p85 mRNA in the adipose tissues of MS rats.ConclusionFTZ attenuated MS symptoms by decreasing the plasma levels of glucose and lipids. The underlying mechanism was attenuation of the reduced expression of PI3K p85 mRNA and IRS1 protein in both insulin-resistant HepG2 cells and MS rats.

PIK3CA Mutations Frequently Coexist with EGFR/KRAS Mutations in Non-Small Cell Lung Cancer and Suggest Poor Prognosis in EGFR/KRAS Wildtype Subgroup

Purpose PIK3CA gene encoding a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) is mutated and/or amplified in various neoplasia, including lung cancer. Here we investigated PIK3CA gene alterations, the expression of core components of PI3K pathway, and evaluated their clinical importance in non-small cell lung cancer (NSCLC).Materials and methodsOncogenic mutations/rearrangements in PIK3CA, EGFR, KRAS, HER2, BRAF, AKT1 and ALK genes were detected in tumors from 1117 patients with NSCLC. PIK3CA gene copy number was examined by fluorescent in situ hybridization and the expression of PI3K p110 subunit alpha (PI3K p110α), p-Akt, mTOR, PTEN was determined by immunohistochemistry in PIK3CA mutant cases and 108 patients without PIK3CA mutation.Results PIK3CA mutation was found in 3.9% of squamous cell carcinoma and 2.7% of adenocarcinoma. Among 34 PIK3CA mutant cases, 17 tumors harbored concurrent EGFR mutations and 4 had KRAS mutations. PIK3CA mutation was significantly associated with high expression of PI3K p110α (p<0.0001), p-Akt (p = 0.024) and mTOR (p = 0.001), but not correlated with PIK3CA amplification (p = 0.463). Patients with single PIK3CA mutation had shorter overall survival than those with PIK3CA-EGFR/KRAS co-mutation or wildtype PIK3CA (p = 0.004). A significantly worse survival was also found in patients with PIK3CA mutations than those without PIK3CA mutations in the EGFR/KRAS wildtype subgroup (p = 0.043)Conclusions PIK3CA mutations frequently coexist with EGFR/KRAS mutations. The poor prognosis of patients with single PIK3CA mutation in NSCLC and the prognostic value of PIK3CA mutation in EGFR/KRAS wildtype subgroup suggest the distinct mutation status of PIK3CA gene should be determined for individual therapeutic strategies in NSCLC.

Expression of PI3-K, PKB and GSK-3 β in the skeletal muscle tissue of gestational diabetes mellitus

ObjectiveTo analyze the expression of phosphatidylinositol 3 kinase (PI3-K), protein kinase B (PKB) and glycogen synthase kinase 3 beta (GSK-3 β) in skeletal muscle tissue of gestational diabetes mellitus (GDM). MethodsA total of 90 cases of pregnant women were divided into observation group and control group according to the occurrence of GDM with 45 cases in either, and the expression of PI3-K, PKB, GSK-3 β mRNA expression in skeletal muscle tissue was compared between two groups. ResultsThe total PI3-K p85 protein was significantly higher in the observation group compared with the control group, the activity of PI3-K was lower than that of the latter; The total PKB, GSK-3 β protein in skeletal tissue had no significant difference between two groups, while the serine phosphorylation levels of PKB and GSK-3βwere significantly lower in observation group compared with the control group. ConclusionsThe downregulation of PI3-K, PKB and GSK-3βin skeletal tissue of GDM caused by phosphorylation dysfunction of signaling molecules is the reason for insulin resistance and transporter function decline which lead to GDM.

Regulation of matrix metalloproteinase‐1, ‐3, and ‐9 inMycobacterium tuberculosis‐dependentrespiratory networks by the rapamycin‐sensitive PI3K/p70S6Kcascade

This study was designed to investigate the role of the phosphatidyl inositol 3-kinase (PI3K)/AKT/p70(S6K) signaling path on regulation of primary normal human bronchial epithelial cell-derived matrix metalloproteinase (MMP)-1, -3, and -9 expression in tuberculosis (TB). These MMPs are key in pathological extracellular matrix degradation in TB. Normal human bronchial epithelials were stimulated with conditioned medium from monocytes infected with virulent TB (CoMTb) and components of the PI3K/AKT signaling pathway blocked using specific chemical inhibitors and siRNA. MMP gene expression was measured by RT-PCR and secretion by ELISA, luminex, or zymography. Phospho-p70 S6K was detected by Western blot analysis and activity blocked by rapamycin. Chemical blockade of the proximal catalytic PI3K p110 subunit augmented MMP-1 and MMP-9 in a dose-dependent manner (all P<0.001) but suppressed MMP-3 (P<0.01). Targeted siRNA studies identified the p110α isoform as key causing 5-fold increase in TB network-dependent MMP-1 secretion to 4900 ± 1100 pg/ml. Specific inhibition of the AKT node suppressed all 3 MMPs. Phospho-p70(S6K) was identified in the cellular model, and rapamycin, a p70(S6K) inhibitor, inhibited MMP-1 (P<0.001) and MMP-3 (P<0.01) but not MMP-9. Controls were epithelial cells that were unstimulated or exposed to conditioned medium from monocytes not exposed to TB. In summary, blockade of the proximal PI3K catalytic subunit increases MMP-1 and MMP-9, whereas rapamycin decreased both MMP-1 and MMP-3. The regulation of the PI3K path in TB is complex, MMP specific, and a potential immunotherapeutic target in diseases characterized by tissue destruction.

Jiaotai pill (交泰丸) enhances insulin signaling through phosphatidylinositol 3-kinase pathway in skeletal muscle of diabetic rats

To investigate the effect of Jiaotai Pill (, JTP) at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase (PI3K) pathway in the skeletal muscle of diabetic rats. 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. All the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β (InsRβ) 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. 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.

Xanthoceraside attenuates tau hyperphosphorylation and cognitive deficits in intracerebroventricular-streptozotocin injected rats

Xanthoceraside, a novel triterpenoid saponin extracted from the fruit husks of Xanthoceras sorbifolia Bunge, reverses cognitive deficits in intracerebroventricular injection of Aβ25-35 or Aβ1-42 mice. However, whether xanthoceraside has a positive effect on hyperphosphorylated tau protein remains unclear. We investigated the effects of xanthoceraside on behavioural impairments induced by intracerebroventricular injection of streptozotocin (STZ) in rats and its potential mechanisms. The rats were administered with xanthoceraside (0.06, 0.12 or 0.24 mg/kg) or vehicle once daily after STZ intracerebroventricular injections. The Y-maze test and novel object recognition test were performed 21 and 22 days after the second STZ injection, respectively. The levels of hyperphosphorylated tau, phosphatidylinositol-3-kinase (PI3K)/serine/threonine protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A) were also tested by Western blot. Xanthoceraside treatment significantly attenuated learning and memory impairments and reduced the level of STZ-induced hyperphosphorylated tau protein. Xanthoceraside also enhanced PP-2A and PP-1 expressions, increased PI3K (p85) and Akt (Ser473) phosphorylation and decreased GSK-3β (tyr216) phosphorylation. Xanthoceraside has protective effect against learning and memory impairments and inhibits tau hyperphosphorylation in the hippocampus, possibly through the inhibition of the PI3K/Akt-dependent GSK-3β signalling pathway and an enhancement of phosphatases activity.

Carbamylated erythropoietin attenuates cardiomyopathy via PI3K/Akt activation in rats with diabetic cardiomyopathy

The aim of the present study was to investigate the protective effect of carbamylated erythropoietin (CEPO) against cardiomyopathy in high-fat, high-carbohydrate diet-fed rats with streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM). Healthy male Wistar rats were fed a high-fat, high-carbohydrate diet for four weeks, and then were injected with STZ twice (50 mg/kg, intraperitoneally). Once DCM was confirmed, the rats were divided randomly into the following groups: DCM without treatment, CEPO treatment at different dosages (500, 1,000 or 2,000 IU/kg) or recombinant human erythropoietin (rhEPO) treatment (1,000 IU/kg), for a four-week short intervention or an eight-week long intervention protocol. Healthy rats were used as normal controls. Venous blood samples were drawn for routine hematological examinations, and heart tissues were collected for histological analysis, as well as the determination of myocardial apoptosis and phosphatidylinositol-3-kinase (PI3K)/Akt signaling. CEPO treatment had no significant effect on the erythrocyte or hemoglobin levels in the rats with DCM; however, it reduced myocardial cell apoptosis in the rats and protected the cellular ultrastructure. In addition, CEPO treatment inhibited caspase-3 and increased Bcl-xl protein expression (P<0.05). It also increased PI3K (p85) and Akt1 expression at the mRNA and protein levels in the hearts of the rats with DCM, with a dose-response relationship. An eight-week treatment using CEPO, in comparison with a four-week protocol, marginally increased PI3K (p85) and Akt1 expression, and did not demonstrate significant benefit. The study indicated that CEPO protects against DCM, without markedly affecting erythropoiesis, and that the activation of PI3K/Akt may be a key mechanism in the protection conferred by CEPO.

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.

Abstract 551: The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis.

Abstract Breast cancer is not only most commonly diagnosed in women worldwide but its metastasis shows serious life threatening consequences. The cell survival and mitogenesis via phosphatidyl inositol 3’ kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling pathways are critical for breast cancer cell survival, proliferation, metabolism and metastasis. A de-regulation of osteogenic Runt-related transcription factor-2 (Runx2) is recently implicated in mammary carcinogenesis. We found that Runx2 differentially regulates the serine/threonine kinase Akt phosphorylation (pAkt), a downstream effecter of PI3K signaling, in non-tumorigenic and invasive mammary epithelial cell lines. The decline in pAkt upon Runx2 suppression in invasive MDA-MB-231 cells was completely abrogated by PI3K inhibitor LY294002. In contrast, the MAPK inhibitor, U0126, showed partial restoration of pAkt upon Runx2 depletion suggesting that Runx2-mediated crosstalk stimulates feedback mechanism within the PI3K and MAPK signaling pathways. The expression analysis of kinases and phosphatases functional in pAkt regulation revealed that the decline in pAkt upon Runx2 knockdown in MDA-MB-231 cells is due to the reduction in PI3K p85 subunit and mammalian target of rapamycin complex-2 (mTORC2). Additionally, we find altered expression of FOXO1, a downstream molecule of PI3K/Akt signaling, due to Runx2-mediated regulation of pAkt levels in MCF-10A and MDA-MB-231 cell lines. To examine the phenotypic effects of Runx2 and PI3K/Akt pathway crosstalk, we evaluated the Runx2-depleted MDA-MB-231 cells for survival under stress conditions. These studies revealed an altered morphology coupled with increased rounding and sub-G1 population in response to glucose- and serum-deprivation upon Runx2 knockdown. The increase in sub-G1 population was restored by glucose and serum re-constitution in the culture media. An increase in Annexin V staining, decline in pAkt levels and cleavage of caspase-3 confirmed the induction of apoptotic cell death. To understand the Runx2-mediated regulation of PI3K/Akt signaling pathway in breast cancer metastasis, the control or Runx2-depleted MDA-MB-231 cells were inoculated intra-cardiacally into immunodeficient mice. The tumors at lung and bone metastatic sites were harvested, FACS-sorted and examined for pAkt levels. Interestingly, to this end, the in-vivo metastatic tumor-derived Runx2 knockdown MDA-MB-231 cells showed an increase in pAkt levels in contrast to in-vitro cultured Runx2 knockdown MDA-MB-231 cells. The data suggests that the mechanism of pAkt activation might involve a dynamic requirement of transcriptional control and feedback regulation within PI3K/Akt and MAPK signaling pathways during different phases of tumor growth in primary and metastatic microenvironment. Citation Format: Manish Tandon, Zujian Chen, Jitesh Pratap. The requirement of Runx2-dependent Akt phosphorylation in breast cancer cell survival and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 551. doi:10.1158/1538-7445.AM2013-551

Targeted inhibition of phosphatidyl inositol-3-kinase p110β, but not p110α, enhances apoptosis and sensitivity to paclitaxel in chemoresistant ovarian cancers

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the critical signaling cascades playing important roles in the chemoresistance of human cancer cells, including ovarian cancer. In this study, we investigated the potential of targeting the PI3K p110β-isoform as a novel approach to overcome the chemoresistance in ovarian cancer. The effects on apoptosis, cell viability, proliferation and migration in chemoresistant ovarian cancer cell were determined following targeted p110β inhibition by small interfering RNA (siRNA). Seven paclitaxel (PTX)-resistant sublines (SKpacs and A2780pac) were produced from SKOV3 and A2780 ovarian cancer cell lines. We, first, evaluated the expression of PI3K p110 isoforms in chemosensitive and chemoresistant ovarian cancer cell lines and patient specimens, and found that p110β-isoform was significantly overexpressed both in a panel of ovarian cancer samples, and in PTX-resistant sublines compared with their parent cell lines. RNA interference-mediated p110β silencing augmented PTX-mediated apoptosis (31.15 ± 13.88 %) and reduced cell viability (67 %) in PTX-resistant cells, whereas targeting p110α did not show a significant change in cell viability and apoptosis. In addition, p110β silencing impaired cell proliferation (60 %) in PTX-resistant SKpac cells. We also found the combined treatment group with p110β siRNA and PTX showed a significant inhibition of tumor growth of SKpac cells compared to the PTX-only treated group in a xenograft nude mouse model. Thus, the siRNA-mediated silencing of PI3K p110β resensitizes PTX-resistant ovarian cancer cells, and may be a useful therapeutic strategy for PTX-resistant ovarian cancers.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-013-0807-9) contains supplementary material, which is available to authorized users.