Phosphatidylinositol 3-kinase Activity Research Articles

Effect of combination of chlorella intake and aerobic exercise training on glycemic control in type 2 diabetic rats.

Chlorella is a type of unicellular green algae that contains various nutrients. Habitual exercise and chlorella treatment can improve insulin resistance in obese or diabetic animal models. However, the additive effects of combined chlorella intake and aerobic exercise training remain unclear. The aim of this study was to investigate whether a combination of chlorella intake and aerobic exercise training would produce greater effects on improving glycemic control in rats with type 2 diabetes. Twenty-wk-old male rats with type 2 diabetes (Otsuka Long-Evans Tokushima Fatty [OLETF] rats) were randomly divided into four groups: sedentary control, aerobic exercise training (treadmill running for 1 h, 25m/min, 5 d/wk), chlorella intake (0.5% chlorella powder in normal diet), or combination of aerobic exercise training and chlorella intake for 8 wk (n = 7 per group). Chlorella intake and aerobic exercise training significantly decreased fasting blood glucose, insulin levels, and total glucose area under the curve during the oral glucose tolerance test and increased the insulin sensitivity index concomitant with muscle phosphatidylinositol-3 kinase (PI3K) activity, protein kinase B (Akt) phosphorylation, and glucose transporter 4 (GLUT4) translocation levels. Furthermore, a combination of chlorella intake and aerobic exercise training significantly further improved these effects compared with aerobic exercise training or chlorella intake alone. These results suggested that chlorella intake combined with aerobic exercise training had more pronounced effects on the improvement of glycemic control via further activation of muscle PI3K/Akt/GLUT4 signaling in rats with type 2 diabetes.

Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro.

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses (fresh control) or cultured in α-MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 μM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 μM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL-positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α-MEM+ and 10 μM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 μM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.

Behavioral defects induced by chronic social defeat stress are protected by Momordica charantia polysaccharides via attenuation of JNK3/PI3K/AKT neuroinflammatory pathway.

The aim of this study was to evaluate the protective effects of Momordica charantia polysaccharides (MCP) on depressive-like behaviors. The chronic social defeat stress (CSDS) mice model was used to evaluate the effects of MCP and their underlying mechanisms. Social interaction test (SIT), sucrose preference test (SPT), and tail suspension test (TST) were performed for behavioral assessments. Expression levels of inflammation mediators and phosphatidylinositol 3-kinase (PI3K) activity were determined using commercial ELISA kits. The expression of key proteins in the c-Jun N-terminal protein kinase (JNK3)/PI3K/protein kinase B (AKT) pathway were measured using western blot and RT-PCR. The results showed that chronic administration of MCP (100, 200, 400 mg/kg/day) significantly prevented depressive-like behaviors in CSDS mice as assessed by SIT, TST and SPT. Elevated levels of proinflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β)], and expression of JNK3, c-Jun, P-110β proteins were observed in the hippocampus of CSDS mice. Moreover, the activity of PI3K and phosphorylation level of AKT were reduced in the hippocampus of CSDS mice. Interestingly, the administration of MCP reversed these changes. Furthermore, the protective effects of MCP on CSDS mice were partly inhibited by the PI3K inhibitor, LY294002. In conclusion, the protective effects of MCP against depressive-like behaviors in CSDS mice might be due to a reduction in neuroinflammation and the down-regulation of the JNK3/PI3K/AKT pathway in the hippocampus.

Role of Green Macroalgae Enteromorpha Prolifera Polyphenols in the Modulation of Gene Expression and Intestinal Microflora Profiles in Type 2 Diabetic Mice.

Effects of green macroalgae 55% ethanolic extract Enteromorpha prolifera through an ultrafiltration membrane of 3 kDa (EPE3k) on antidiabetic activity, gut microbiota, and regulation mechanism were investigated in high-fat/high-sucrose diet and streptozocin-induced diabetic mice. The structural characterizations of its major compounds in EPE3k were determined by ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry. Furthermore, the intestinal microflora modulation in diabetic mice was also investigated with high-throughput 16S rRNA gene sequencing. The proposed presence of polyphenols in EPE3k was confirmed. EPE3k could significantly decrease the fasting blood glucose and improve fasting glucose tolerance. The hypoglycemic effect of EPE3k was via activation of phosphatidylinositol 3-kinase and suppression of c-Jun N-terminal kinase in liver. EPE3k treatment significantly increased the relative abundance of Akkermansia and decreased the proportion of Alistipes and Turicibacter. The above results indicated that EPE3k could be provided as a new potential therapy for the treatment of type 2 diabetic mellitus.

Anti-tumoral potential of MDA19 in human osteosarcoma via suppressing PI3K/Akt/mTOR signaling pathway.

Osteosarcoma (OS) is the most common primary malignancy of skeleton with higher mortality rates amongst children and young adults worldwide, whereas effective and secure therapies have also been sought by researches with ongoing efforts. The purpose of the present study was to investigate the impact of N′-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene] benzohydrazide (MDA19) on OS and explore its potential mechanism. Cell Counting Kit-8 (CCK8) and colony formation assay were employed to evaluate the potential effect of MDA19 on U2OS and MG-63 cells proliferation. Moreover, transwell migration and invasion assay were performed to assess the influence of MDA19 on U2OS and MG-63 cells migration and invasion. In addition, Annexin V-FITC/propidium iodide (Annexin V-FITC/PI) staining and flow cytometry were used to examine apoptotic ratio of the U2OS and MG-63 cells. Meanwhile, Western blot analysis was applied to explore change of relevant mechanism proteins in OS cells treated with MDA19. Our study showed that MDA19 had anti-proliferative activity of OS cells in a dose- and time-dependent manner, simultaneously, inhibition of colony formation was also observed in U2OS and MG-63 cells after incubation of MDA19. Besides, MDA19 could significantly inhibit the number of migrated and invaded OS cells and markedly increase the OS cells apoptosis rate. Mechanistically, we detected detectable reductions in apoptosis related proteins, epithelial–mesenchymal transition (EMT)-related proteins and activity of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling in U2OS and MG-63 cells exposure to MDA19. Overall, the current study indicates in vitro anti-proliferative, anti-metastatic, and pro-apoptotic potential of MDA19 in U2OS and MG-63 cells. Our findings propose a clue for further studies with this compound in preclinical and clinical treatment for OS.

Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1.

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on "outside-in" signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function-associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling-induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

A humanized yeast-based toolkit for monitoring phosphatidylinositol 3-kinase activity at both single cell and population levels.

Phosphatidylinositol 3-kinase (PI3K) is a key regulator of phosphoinositide-dependent signaling in mammalian cells and its dysfunction is related to multiple syndromes, including cancer. By heterologous expression in Saccharomyces cerevisiae, we have developed a humanized yeast system as a tool for functional studies on higher eukaryotic PI3K. Here we restrict PI3K activity in yeast to specific plasma membrane (PM) microdomains by fusing the p110α PI3K catalytic subunit to either a septin or an eisosome component. We engineered a Dual Reporter for PI3K (DRAPIK), useful to monitor activity on cellular membranes in vivo at a single-cell level, by simultaneous PM staining of the enzyme substrate (PtdIns4,5P2) with GFP and its product (PtdIns3,4,5P3) with mCherry. We also developed a sensitive FLUorescence by PI3K Inhibition (FLUPI) assay based on a GFP transcriptional reporter that is turned off by PI3K activity. This reporter system proved useful to monitor PI3K inhibition in vivo by active compounds. Such novel tools were used to study the performance of yeast PM microdomain-directed PI3K. Our results show that tethering heterologous PI3K to discrete PM domains potentiates its activity on PtdIns4,5P2 but different locations display distinct effects on yeast growth and endocytosis.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells.

Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.

Dorsal ruffles enhance activation of Akt by growth factors.

In fibroblasts, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulate the formation of actin-rich, circular dorsal ruffles (CDRs) and phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of Akt. To test the hypothesis that CDRs increase synthesis of phosphorylated Akt1 (pAkt), we analyzed the contributions of CDRs to Akt phosphorylation in response to PDGF and EGF. CDRs appeared within several minutes of growth factor addition, coincident with a peak of pAkt. Microtubule depolymerization with nocodazole blocked CDR formation and inhibited phosphorylation of Akt in response to EGF but not PDGF. Quantitative immunofluorescence showed increased concentrations of Akt, pAkt and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), the phosphoinositide product of PI3K that activates Akt, concentrated in CDRs and ruffles. EGF stimulated lower maximal levels of pAkt than did PDGF, which suggests that Akt phosphorylation requires amplification in CDRs only when PI3K activities are low. Accordingly, stimulation with low concentrations of PDGF elicited lower levels of Akt phosphorylation, which, like responses to EGF, were inhibited by nocodazole. These results indicate that when receptor signaling generates low levels of PI3K activity, CDRs facilitate local amplification of PI3K and phosphorylation of Akt.This article has an associated First Person interview with the first author of the paper.

212 - The role of Nrf2 in redox-dependent cobalamin processing

Objective Define the role of Nrf2 in regulating cellular cobalamin content. Background Vitamin B12 (cobalamin) serves as a cofactor for methionine synthase, which catalyzes regeneration of methionine from homocysteine. Cellular cobalamin processing requires the availability of cytoplasmic glutathione and nicotinamide adenine dinucleotide phosphate (NADPH). Neuregulin-1 is a neurotrophic factor that stimulates phosphatidylinositol 3 kinase (PI3K) signaling. PI3K activation downstream of neuregulin-1 has been demonstrated to promote cobalamin processing to active species, by enhancing cysteine uptake and subsequent glutathione production. PI3K signaling and lithium can upregulate Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activity by inhibiting glycogen synthase kinase 3 beta. Whether Nrf2 promotes cobalamin processing by modulating redox conditions through its role as a transcription factor is unclear. Methods SH-SY5Y cells grown in AMEM with 10% FBS were treated with 10 mM lithium chloride (LiCl). Cobalamin and thiol analysis were done via HPLC. SH-SY5Y cells grown in AMEM with 1% FBS were pre-treated with PI3K inhibitors pictilisib (pict) (100 nM) or wortmannin (wort) (100 nM), prior to 1h neuregulin-1 (1 nM) exposure. RT-qPCR was performed to measure gene expression of enzymes involved in redox-related cobalamin processing. Results 1 or 4h LiCl exposure increased glutathione. Cyanocobalamin (CNCbl) was decreased at both time points. Adenosylcobalamin (AdoCbl) was increased at 1h, but decreased after 4h. Neuregulin-1 treatment following pre-treatment with pict or wort modulated expression of Methylmalonic aciduria and homocystinuria type C protein (MMACHC) and Methionine synthase reductase (MTRR). MMACHC was increased by neuregulin-1 alone and decreased by neuregulin-1 with wort. MTRR was increased by wort alone or combination with neuregulin-1. Conclusion Our research strengthens the role of PI3K in regulating cobalamin processing and suggests Nrf2 may positively regulate cobalamin processing. Future research may directly implicate Nrf2.

Long Non-Coding RNA (lncRNA) Growth Arrest Specific 5 (GAS5) Suppresses Esophageal Squamous Cell Carcinoma Cell Proliferation and Migration by Inactivating Phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian Target of Rapamycin (mTOR) Signaling Pathway.

BackgroundlncRNA GAS5 acts as a tumor-suppressor gene in various types of malignancies, but its involvement in esophageal cancer has not been well studied.Material/MethodsA total of 112 patients with esophageal cancer and 55 volunteers with normal physiological conditions were included in this study. Tumor tissues and adjacent healthy tissues were collected from esophageal cancer patients and blood was extracted from patients and controls. Expression of GAS5 in those tissues was detected by qRT-PCR. All patients were followed up for 5 years and diagnostic and prognostic values of serum GAS5 for esophageal cancer were investigated by ROC curve analysis and survival curve analysis, respectively. Effects of GAS5 expression on cell proliferation and migration were investigated by CCK-8 assay and Transwell cell migration assay, respectively. Effects of GAS5 overexpression on expression of PI3K/AKT/mTOR-related proteins were explored by Western blot analysis.ResultsGAS5 expression level was lower in tumor tissues than in adjacent healthy tissues. Serum level of GAS5 was lower in cancer patients than in healthy controls, and serum level of GAS5 was decreased with increase in stage of primary tumor (T stage). GAS5 overexpression inhibited tumor cell proliferation and migration, while treatment with PI3K activator reduced the inhibitory effects. GAS5 overexpression decreased the expression level of PI3K and phosphorylation levels of Akt and mTOR in esophageal cancer cells, while PI3K activator treatment showed no significant effects on GAS5 expression.ConclusionsGAS5 was downregulated in esophageal cancer patients compared to healthy controls, and GAS5 overexpression suppressed proliferation and migration of esophageal cancer cells by inactivating the PI3K/AKT/mTOR pathway.

Protective effect of irbesartan against doxorubicin-induced nephrotoxicity in rats: implication of AMPK, PI3K/Akt, and mTOR signaling pathways.

Nephrotoxicity is one of the serious undesirable effects related to doxorubicin (DOX). Herein, we have investigated the potential protective effect of irbesartan (IRB) against chronic nephrotoxicity induced by DOX, and the implication of different mechanistic pathways underlying these effects. Rats were treated with either DOX (2.5 mg/kg i.p., 3 times/week) for 2 weeks, and (or) IRB (40 mg/kg, daily) for 3 weeks. IRB prohibited nephrotoxicity induced by DOX, which was evident by the increase in blood urea nitrogen and creatinine levels and histopathological changes. IRB improved DOX-induced alterations in oxidative status by diminishing lipid peroxidation and upregulating the antioxidant enzymes. Also, upon DOX treatment, the renal expression of tumor necrosis factor-α, interleukin-6, and caspase-3 were significantly increased; IRB diminished DOX-induced alterations in these parameters. Moreover, DOX significantly decreased the expression level of AMP-activated protein kinase (AMPK). Meanwhile, DOX induced activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt/PKB) and mammalian target of rapamycin (mTOR) pathways that cross talked with AMPK. On the contrary, IRB successfully counterbalanced all these effects. Collectively, these outcomes suggest that the modulation of AMPK, PI3K, Akt, and mTOR pathways plays a critical role in conferring the protective effects of IRB against DOX nephrotoxicity.

Anti-Inflammatory Activities of Compounds Isolated from the Rhizome of Anemarrhena asphodeloides.

Fifteen unreported compounds in Anemarrhena asphodeloides, iriflophene (3), hostaplantagineoside C (7), tuberoside G (8), spicatoside B (9), platycodin D (14), platycoside A (15), platycodin D2 (16), polygalacin D2 (17), platycodin D3 (18), isovitexin (20), vitexin (21), 3,4-dihydroxyallylbenzene-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (22), iryptophan (24), adenosine (25), α-d-Glucose monoallyl ether (26), together with eleven known compounds (1, 2, 4–6, 10–13, 19 and 23), were isolated from the rhizomes of Anemarrhena asphodeloides. The chemical structures of these compounds were characterized using HRMS and NMR. The anti-inflammatory activities of the compounds were evaluated by investigating their ability to inhibit LPS-induced NO production in N9 microglial cells. Timosaponin BIII (TBIII) and trans-hinokiresinol (t-HL) exhibited significant inhibitory effects on the NO production in a dose-dependent manner with IC50 values of 11.91 and 39.08 μM, respectively. Immunoblotting demonstrated that TBIII and t-HL suppressed NO production by inhibiting the expressions of iNOS in LPS-stimulated N9 microglial cells. Further results revealed that pretreatment of N9 microglial cells with TBIII and t-HL attenuated the LPS-induced expression tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) at mRNAs and protein levels. Moreover, the activation of nuclear factor-κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were inhibited by TBIII and t-HL, respectively. Our findings indicate that the therapeutic implication of TBIII and t-HL for neurogenerative disease associated with neuroinflammation.

Thyroid hormones stimulate L-arginine transport in human endothelial cells

Thyroid hormone activity is associated with L-arginine metabolism and nitric oxide (NO) production, which participate in the cardiovascular manifestations of thyroid disorders. L-arginine transporters play an important role in activating L-arginine uptake and NO production. However, the effects of thyroid hormones on L-arginine transporters in endothelial cells have not yet been evaluated. The following methods were used. We measured L-arginine uptake, mRNA expression of L-arginine transporters, endothelial nitric oxide synthase (eNOS) mRNA and NO generation after the administration of T3, T4 and the T3 analog, 3,3′,5-triiodothyroacetic acid TRIAC in human umbilical vein endothelial cells (HUVECs). We also analyzed the role of αvβ3 integrin and of phosphatidyl-inositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPKs: ERK1/2, p38 and SAPK-JNK) and intracellular calcium signaling pathways as underlying mechanisms. To this end, αvβ3 integrin was pharmacologically inhibited by tetraiodothyroacetic acid (TETRAC) or genetically blocked by silencing αv mRNA and PI3K, MAPKs and intracellular calcium by selective inhibitors. The following results were obtained. Thyroid hormones and the T3 analog TRIAC increased L-arginine uptake in HUVECs, the sodium-independent y+/CAT isoforms, except CAT2b, sodium-dependent y+L system and sodium-independent system b0,+L-arginine transporters, eNOS mRNA and NO production. These effects were suppressed by αvβ3 integrin inhibition with TETRAC or αv integrin downregulation or by PI3K, MAPK or intracellular Ca2+ signaling inhibitors. In conclusion, we report for the first time that activation of L-arginine uptake by thyroid hormones is related to an upregulation of L-arginine transporters. This effect seems to be mediated by activation of αvβ3 integrin receptor and subsequent PI3K, MAPK and intracellular Ca2+ signaling pathways.

Integrin β1 promotes gemcitabine resistance in pancreatic cancer through Cdc42 activation of PI3K p110β signaling

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignancies with very poor prognosis due to its broad resistance to chemotherapy. Our previous study showed that integrin β1 expression is upregulated in PDAC and confers gemcitabine resistance in PDAC cells via the signaling pathway including Cdc42 and AKT activation. But the accurate signal transductions are not clear. Here, we aimed to illuminate the signal transductions of integrin β1 in the acquisition of gemcitabine resistance in PDAC. Drug-resistance (DR) cells from AsPC-1 parent cell line (PCL) were selected. Integrin β1 expression was determined using western blot assay. Changes in drug response and the activity of phosphatidylinositol 3-kinase (PI3K) signaling after knockdown of integrin β1, Cdc42 or p110β were evaluated using MTT, cleaved caspase-3 immunofluorescence and western blot assay. Western blot assays also detected the variations in Cdc42 activity and p110β expression after integrin β1 knockdown. The interaction between Cdc42 and p110β was determined by Glutathione S-transferase (GST) pull-down assay. The results showed that integrin β1 expression was upregulated in DR-AsPC-1 cells, and integrin β1 knockdown significantly decreased the activity of Cdc42, a target molecule of integrin β1, and p110β expression. Knockdown of anyone of integrin β1, Cdc42 and p110β inhibited the activity of PI3K signaling, and sensitized DR-AsPC-1 cells to gemcitabine. GST pull-down assay showed that GTP-Cdc42 interacted with p110β. Collectively, these data indicated that integrin β1 promoted gemcitabine resistance in PDAC through Cdc42 activation of PI3K p110β signaling. In vivo experiments also confirmed this conclusion. These findings contribute to a better understanding the molecular mechanism of chemoresistance and facilitate the development of more targeted and effective treatment strategy for PDAC.

Propranolol inhibits the activity of PI3K, AKT, and HIF-1α in infantile hemangiomas.

We sought to evaluate effect of propranolol in the treatment of infantile hemangiomas by quantifying the amount of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and hypoxia-inducible factor-1α (HIF-1α). Hemangioma tissue was isolated from an infant patient and implanted into nude mice to establish a hemangioma model. Twenty-four hemangioma-model nude mice were divided into two groups including a control group (saline, by gastrogavage) and an experimental group (propranolol, by gastrogavage). The hemangioma-model nude mice were euthanized and tumors were removed at 30 and 50days (before and after treatment). HE staining was used to observe the histopathological changes, and western blot and quantitative real-time PCR were used to describe levels of protein and mRNA expression of PI3K, AKT, and HIF-1α. Propranolol treatment decreased tumor size as compared to the control group. Protein and mRNA expression levels of PI3K, AKT, and HIF-1α were lower in the experimental group at day 50 compared to the control group at day 50 and the experimental group at day 30 (p < 0.05). Propranolol can promote regression of infantile hemangiomas, which may be related to the inhibition of PI3K, AKT, and HIF-1α activity.

Immunosuppressive Effect of Ticagrelor on Dendritic Cell Function: A New Therapeutic Target of Antiplatelet Agents in Cardiovascular Disease

Atherosclerosis is being thought of as an autoimmune disease. As the most potent antigen-presenting cells, dendritic cells (DCs) have been regarded as a major target for the control of this harmful immune response. In this study, we investigated the effect of ticagrelor, a new antiplatelet drug antagonizing the P2Y12 receptor, on the function of mouse bone marrow-derived DCs. RT-PCR revealed relatively high P2Y12 mRNA levels in DCs, and expression of the P2Y12 protein was documented by western blot analysis. Moreover, antigen (Ag) uptake by DCs was markedly increased following activation of the P2Y12 receptor by adenosine-5'-O-(2-thiodiphosphate) (ADPβS). Ticagrelor reduced the ADPβS-stimulated uptake of fluorescein-labeled dextran by DCs while exerting no significant effect on spontaneous endocytosis. In addition, ticagrelor suppressed the capacity of ADPβS-stimulated DCs to induce activation of T lymphocytes. Ticagrelor blocked the activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK1/2) in ADPβS-treated DCs. Preventing the activation of PI3K reduced significantly ADPβS-induced endocytosis by DCs. Thus, ticagrelor decreases Ag uptake by DCs via the inhibition of P2Y12 receptor-mediated PI3K activity, attenuating the stimulation of Ag-specific T cells. Our findings indicate that ticagrelor may directly target DCs and inhibit their function, suggesting a possible explanation for the immunoregulatory activity of this drug.

Survivin Ser81 Plays An Important Role in PI3K/Akt/mTOR Signaling Pathway

Background: Survivin, a member of the inhibitor of apoptosis protein family, has been associated with protection from cell apoptosis and regulation of mitosis. Phosphorylated-Survivin at Ser81 was reported to provide cytoprotection against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in L929 cells by inducing a backloop activation of phosphatidylinositol 3-kinase (PI3K). Therefore Akt as a possible substrate of PI3K was investigated.Methods: L929 cells were pretreated with/without 50 μM LY294002 or 10 μM Perifosine, and infected with viral particle of Survivin, anti sense of Survivin, Ser81Ala mutated Survivin or vector only. Cells were then harvested, lysed and subjected to immunoblot assay to detect Akt, phosphorylated Akt (Ser473), mammalian target of rapamycin (mTOR), phosphorylated-mTOR (Ser2448).Results: Survivin induced Akt and mTOR phosphorylations in a viral particle concentration dependent manner. Pretreatment of LY294002 or Perifosine prior to Survivin infection, attenuated Akt or mTOR phosphorylations, respectively. Low Akt or mTOR phosphorylations were observed when L929 cells were infected with Ser81Ala mutated Survivin.Conclusion: Ser81 phosphorylation site of Survivin played an important role in activating Survivin/PKA/PI3K/Akt/mTOR signaling pathway.Keywords: survivin, Ser81, Akt, mTOR, LY294002, perifosine

Preconditioned neurons with NaB and nicorandil, a favorable source for stroke cell therapy.

Poor survival of stem cells in the harsh microenvironment at the site of stroke, especially during acute phase of injury, remains a serious obstacle to achieve the desired prognosis. We hypothesized that combined treatment of neural stem cells (NSCs) with small molecules would precondition them to become robust and survive better as compared with the native nonpreconditioned cells. Mouse ganglionic NSCs were isolated, cultured, and characterized. The cells were preconditioned by treatment with sodium butyrate (NaB) and nicorandil (Nico) and transplanted in an experimentally induced stroke model. Sham-operated animals without treatment or animals with experimental stroke treated with basal medium, native NSCs, NSCs preconditioned with NaB or Nico alone were used as controls. The tissue samples and cells with different treatments were used to measure brain-tissue-derived neurotrophic factor (BDNF) level and the activity of phosphatidylinositol-3 kinase (PI3K), apurinic/apyrimidinic endonuclease 1 (APE1), and nuclear factor-κB (NF-κB) p50 both in vitro and in vivo, respectively. Additionally, survival of the cells and recovery indices for stroke were studied. The combined treatment with NaB + Nico resulted in increased BDNF level and higher PI3K, APE1, and the downstream NF-κB activation, which were blocked by pretreatment with their respective inhibitors. Donor cell survival increased postengraftment as assessed by 5-bromo-2'-deoxyuridineimmunostaining and reduced Terminal deoxynucleotide transferase dUTP Nick End Labeling positivity at the site of engraftment. There was reduction in proinflammatory cytokines and infiltration of both GFAP + and CD68 + at the injury site. There was reduction in the infarct size and neurological function was preserved in the preconditioned cell treatment group. Our preconditioning approach with small molecules effectively improved the survival as well as functionality of NSCs.

Sitagliptin improves renal function in diabetic nephropathy in male Sprague Dawley rats through upregulating heme oxygenase-1 expression.

Oxidative stress is an important mechanism for diabetic nephropathy. Studies showed that hemo oxygenase-1 (HO-1) expression in renal tissue of patients with diabetic nephropathy has upregulated, while the HO-1 can protect the body through anti-oxidative stress. The study aimed to preliminarily explore the molecular mechanism by observing the effect of Sitagliptin on HO-1 expression in renal tissue of rats with diabetic nephropathy. The diabetic nephropathy rat model was established by STZ injection followed by intraperitoneal injection of sitagliptin with different concentrations. The mRNA expressions of HO-1 were detected by real-time PCR and Western blot and HO-1 enzyme activity change was detected by colorimetry. Human renal mesangial cell (HRMC) were cultured in vitro with high glucose concentration (30 μmol/L), phosphatidylinositol-3-kinase (PI3K) level and nuclear factor erythroid-2-related factor (Nrf2) content in cytoplasm and cell nucleus were observed before and after treatment with sitagliptin, as well as the action of in meditating HO-1 expression. HO-1 mRNA, protein level, and HO-1 enzyme activity in renal tissue of rats with diabetic nephropathy were significantly increased after treatment with sitagliptin (P < 0.05). As comparison, the 24 h urinary microalbumin, creatinine, and boold urea nitrogen were all decreased after treatment of sitagliptin (P < 0.05). Similar results were observed after CoPP (an agonist of HO-1) treatment (P < 0.05). In contrast, ZnPP, an inhibitor of HO-1, significantly abrogated the inhibitory effect of sitagliptin (P < 0.05). Phosphorylation of PI3K and Nrf2 nuclear translocation under high-glucose concentration condition was induced by sitagliptin in HRMC. HO-1 expression was suppressed by pretreating HRMC with PI3K inhibitor or RNA interference. Sitagliptin may induce HO-1 expression via activation of PI3K and Nrf2 in rats with diabetic nephropathy; HO-1 can improve the oxidative stress of diabetic nephropathy, eventually protect from diabetic nephropathy.