Activation Of Phosphatidylinositol Research Articles

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

Memory and Learning Enhancement Caused by Phosphatidylinositol Activity in Phosphatidylinositol 50 Treated Rats

Memory and Learning Enhancement Caused by Phosphatidylinositol Activity in Phosphatidylinositol 50 Treated Rats

Asynchronous embryonic germ cell development leads to a heterogeneity of postnatal ovarian follicle activation and may influence the timing of puberty onset in mice

BackgroundOvarian follicles, which are the basic units of female reproduction, are composed of oocytes and surrounding somatic (pre) granulosa cells (GCs). A recent study revealed that signaling in somatic preGCs controlled the activation (initial recruitment) of follicles in the adult ovaries, but it is also known that there are two waves of follicle with age-related heterogeneity in their developmental dynamics in mammals. Although this heterogeneity was proposed to be crucial for female reproduction, our understanding of how it arises and its significance is still elusive.ResultsIn the current study, by deleting the key secreted factor KIT ligand from preGCs and analyzing the follicle cell developmental dynamics, we revealed distinct patterns of activation and growth associated with the two waves of follicles in mouse ovary. Our results confirmed that activation of adult wave follicles is initiated by somatic preGCs and dependent on the KIT ligand. By contrast, activation of first wave follicles, which are awakened from germ cells before follicle formation, can occur in the absence of preGC-secreted KIT ligand in postnatal ovaries and appears to be oocyte-initiated. We also found that the asynchronous activity of phosphatidylinositol 3 kinases (PI3K) signaling and meiotic process in embryonic germ cells lead to the follicle heterogeneity in postnatal ovaries. In addition, we supplied evidence that the time sequence of embryonic germ cell development and its related first wave follicle growth are correlated to the time of puberty onset in females.ConclusionTaken together, our study provides evidence that asynchronous development of embryonic oocytes leads to the heterogeneity of postnatal ovarian follicle activation and development, and affects the timing of onset of puberty in females.

Dibutyl phthalate-induced oxidative stress and apoptosis in swine testis cells and therapy of naringenin via PTEN/PI3K/AKT signaling pathway.

Dibutyl phthalate (DBP) is a phthalic acid ester (PAE) that has posed a health hazard to the organisms. Naringenin (NRG) is a flavanone compound that has shown protection against several environmental chemicals through suppression of oxidative stress and activation of phosphatidylinositol 3-kinase/threonine kinase (PI3K/AKT) signaling pathway. Herein, swine testis (ST) cells were treated with 1.8μM of DBP or/and 25.39 nM of NRG for 24 h, we described the discovery path of NRG inhibition on apoptosis in DBP-exposed ST cells through targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN). We first found that the anti-apoptosis effect of NRG is dependent on mitochondrial pathway through flow cytometry and related gene/protein expression, and then we detected PI3K/AKT pathway-related gene/protein expression, and established a computational docking assay between NRG and PTEN. We found that NRG specifically binds to three basic residues (His93, Lys125, Lys128) of P loop in PTEN, as well as phosphatase domains (Asp92, His93, Cys124, Lys125, Ala126, Lys128, and Arg130) in active dephosphorylation pockets, thereby reducing PTEN level and activating PI3K/AKT signaling pathway, and further inhibiting oxidative stress and mitochondrial pathway apoptosis. Taken together, our results push forward that NRG deserves further attention as a potential antagonistic therapy against DBP through targeting PTEN to inhibit oxidative stress and activate PI3K/AKT signaling pathway.

Resveratrol-Selenium Nanoparticles Alleviate Neuroinflammation and Neurotoxicity in a Rat Model of Alzheimer's Disease by Regulating Sirt1/miRNA-134/GSK3β Expression.

Alzheimer's disease (AD) is a brain disorder associated with a gradual weakening in neurocognitive functions, neuroinflammation, and impaired signaling pathways. Resveratrol (RSV) has neuroprotective properties, but with low bioavailability, and low solubility in vivo. Selenium (Se) is an essential micronutrient for brain function. Thus, this study aimed to evaluate the role of formulated RSV-Se nanoparticles (RSV-SeNPs) on neurochemical and histopathological approaches associated with the AD model in rats induced by Aluminum chloride (AlCl3) at a dose of 100mg/kg/day for 60days. RSV-SeNPs supplementation attenuates the impaired oxidative markers and mitochondrial dysfunction. The ameliorative effect of RSV-SeNPs on cholinergic deficits was associated with clearance of amyloid β (Aβ). Furthermore, activation of phosphatidylinositol 3 kinase (PI3K) deactivates glycogen synthase kinase 3 beta (GSK-3β)-mediated tau hyperphosphorylation. Additionally, RSV-SeNPs downregulate signal transducer and activator of transcription (STAT3) expression as well as interleukin-1β (IL-1β) levels, therefore alleviating neuroinflammation in AD. Moreover, RSV-SeNPs upregulate the expression of Sirtuin-1 (SIRT1) and lower that of microRNA-134, consequently increasing neurite outgrowth. Eventually, the obtained results showed that nano-formulation of resveratrol with selenium maximized the therapeutic potential of RSV against Alzheimer's disease not only by their antioxidant but also by anti-inflammatory effect improving the neurocognitive function and modulating the signaling pathways.

Erianin Regulates PI3K/AKT Signaling Pathway and Inhibits Cell Invasion and Migration in Gastric Cancer In Vitro

Gastric carcinoma is one of the most malignant cancers associated with lifestyle and genetics. The currently used pharmacotherapeutics are associated with serious side effects necessitating a need for safer drugs. Erianin is a natural plant bioactive substance known for its anti-cancerous and a variety of other biologic activities. Using a variety of measures such as colony formation, invasion and migration assay, and flow cytometry and apoptosis, we have examined the effect of erianin on human gastric adenocarcinoma cells representing gastric carcinoma. The results show that erianin ensured (i) inhibition of proliferation, invasion, and migration capabilities, (ii) induction of apoptosis, and (iii) inhibition of the activation of phosphatidylinositol 3-kinase/Protein kinase B signaling pathway. In conclusion, these results show a potential for erianin in the management of gastric carcinoma.

Dedicator of Cytokinesis 2 (DOCK2) Deficiency Attenuates Lung Injury Associated with Chronic High-Fat and High-Fructose Diet–Induced Obesity

Obesity is a major risk factor for lung disease development. However, little is known about the impact of chronic high-fat and high-fructose (HFHF) diet-induced obesity on lung inflammation and subsequent pulmonary fibrosis. Herein we hypothesized that dedicator of cytokinesis 2 (DOCK2) promotes a proinflammatory phenotype of lung fibroblasts (LFs) to elicit lung injury and fibrosis in chronic HFHF diet-induced obesity. An HFHF diet for 20 weeks induced lung inflammation and profibrotic changes in wild-type C57BL/6 mice. CD68 and monocyte chemoattractant protein-1 (MCP-1) expression were notably increased in the lungs of wild-type mice fed an HFHF diet. An HFHF diet further increased lung DOCK2 expression that co-localized with fibroblast-specific protein 1, suggesting a role of DOCK2 in regulating proinflammatory phenotype of LFs. Importantly, DOCK2 knockout protected mice from lung inflammation and fibrosis induced by a HFHF diet. In primary human LFs, tumor necrosis factor-α (TNF-α) and IL-1β induced DOCK2 expression concurrent with MCP-1, IL-6, and matrix metallopeptidase 2. DOCK2 knockdown suppressed TNF-α-induced expression of these molecules and activation of phosphatidylinositol 3-kinase/AKT and NF-κB signaling pathways, suggesting a mechanism of DOCK2-mediated proinflammatory and profibrotic changes in human LFs. Taken together, these findings reveal a previously unrecognized role of DOCK2 in regulating proinflammatory phenotype of LFs, potentiation of lung inflammation, and pulmonary fibrosis in chronic HFHF diet-caused obesity.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for apolipoprotein A4 (APOA4) in adipose tissue

Apolipoprotein A4 (APOA4) is one of the most abundant and versatile apolipoproteins facilitating lipid transport and metabolism. APOA4 is synthesized in the small intestine, packaged onto chylomicrons, secreted into intestinal lymph and transported via circulation to several tissues, including adipose. Since its discovery nearly 4 decades ago, to date, only platelet integrin αIIbβ3 has been identified as APOA4 receptor in the plasma. Using co-immunoprecipitation coupled with mass spectrometry, we probed the APOA4 interactome in mouse gonadal fat tissue, where ApoA4 gene is not transcribed but APOA4 protein is abundant. We demonstrate that lipoprotein receptor-related protein 1 (LRP1) is the cognate receptor for APOA4 in adipose tissue. LRP1 colocalized with APOA4 in adipocytes; it interacted with APOA4 under fasting condition and their interaction was enhanced during lipid feeding concomitant with increased APOA4 levels in plasma. In 3T3-L1 mature adipocytes, APOA4 promoted glucose uptake both in absence and presence of insulin in a dose-dependent manner. Knockdown of LRP1 abrogated APOA4-induced glucose uptake as well as activation of phosphatidylinositol 3 kinase (PI3K)-mediated protein kinase B (AKT). Taken together, we identified LRP1 as a novel receptor for APOA4 in promoting glucose uptake. Considering both APOA4 and LRP1 are multifunctional players in lipid and glucose metabolism, our finding opens up a door to better understand the molecular mechanisms along APOA4-LRP1 axis, whose dysregulation leads to obesity, cardiovascular disease, and diabetes.

Lipid kinases PIP5K7 and PIP5K9 are required for polyamine-triggered K+ efflux in Arabidopsis roots.

SUMMARYPolyamines, such as putrescine, spermidine and spermine (Spm), are low‐molecular‐weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5‐bisphosphate (PIP2), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32Pi‐labelling of Arabidopsis seedlings, low physiological (μm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time‐ and dose‐dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32Pi‐labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5‐phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm‐induced PIP2 response. Using non‐invasive microelectrode ion flux estimation, we discovered that the Spm‐triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine‐mediated signalling controlling K+ flux in plants.

Abstract 3048: Murine-derived colorectal cancer organoid culture high-throughput drug screening to identify novel combinations targeting PIK3CA mutant cancers

Abstract Introduction: Colorectal cancer (CRC) treatment is becoming increasingly individualized as clinical mutation profiling is a standard of care. Oncogenic mutations of the PIK3CA gene induce a constitutively active phosphatidylinositol 3-kinase with downstream effects that promote tumor initiation and progression. Effective clinical therapeutic strategies targeting this subtype of colorectal cancer have remained elusive. Here we develop a high-throughput organoid drug screening platform to examine combination strategies targeting PIK3CA mutant CRC. Methods: Murine-derived CRC organoids (MDCOs) were derived from a transgenic mouse model of PIK3CA and APC mutated colorectal cancer (F1 (FVBxB6) Apcfl/+ Pik3caH1047R), matured for 48 hours and plated in 96 well plates. A total of 96 compounds chosen from clinically available anticancer drugs and select drugs in clinical development were analyzed for their efficacy in reducing organoid growth on their own, as well as in combination with copanlisib (PI3K/MTOR inhibitor) over 48 hours. Select treatments were confirmed using our standard 24 well plate assay. Additionally, irinotecan (SN38; topoisomerase-I inhibitor), panitumumab (EGFR inhibitor), and olaparib (PARP inhibitor) were investigated alone and in combination with copanlisib based on clinical use for this population or recent promising clinical trial data. Results: SN38, olaparib, and panitumumab yielded insignificant changes in spheroid size in the combination regimen compared to copanlisib alone (median relative change in organoid diameter - copanlisib -8%, p<0.001; SN38 combo -8%, p=0.5; olaparib combo -13%, p=0.6; and panitumumab combo 1%, p=0.9). The MDCOs were successfully utilized for the high-throughput drug screen. Differential sensitivity was identified using change in organoid diameter without the need for reagents or dyes which could complicate the screening method. Of the 96 compounds investigated in the high-throughput screen, 12 resulted in enhanced treatment effect when combined with copanlisib. Three of these 12 compounds inhibit microtubule formation, and others inhibit components of the RAS pathway. Two of these drug combinations were examined further and significant treatment responses were confirmed. Conclusion: Advances in the understanding of the molecular basis of cancer allow for a more precise approach to cancer treatment. High-throughput techniques are needed to better identify novel treatment strategies for molecular subtypes. Here we describe a label free murine-derived cancer organoid method to screen drugs for combination treatment strategies. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Gioia Sha, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Murine-derived colorectal cancer organoid culture high-throughput drug screening to identify novel combinations targeting PIK3CA mutant cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3048.

MiR-221 affects the proliferation and apoptosis of laryngeal cancer cells through the PI3K/AKT signaling pathway

To investigate the effect of MiR-221 on proliferation and apoptosis of laryngeal carcinoma cells through the PI3K/AKT signaling pathway. LipofectamineTM 2000 liposomes were used to transfer MiR-221 analogue, MiR-221 NC into Hep-2 cells of laryngeal carcinoma. Real-time fluorescence quantitative polymerase chain reaction (PCR) method was used to detect the expression of MiR-221, MTT method was used to detect the proliferation of cells, flow cytometry was used to detect cell cycle, Western blotting was used to detect the expression of apoptosis proteinase-1 (Apaf-1) and cyclin-dependent kinase (CDK1, CDK2) protein and the activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT). Compared with MiR-221 NC group, the expression of MiR-221 in MiR-221 analogue group was up-regulated (p<0.01), the cell proliferation rate was decreased (p<0.01), the cell cycle was stagnated in the G1 phase (p<0.01), the expression levels of Cyclin A, CDK1, CDK2, PI3K, and p-AKT were significantly down-regulated (p<0.01), and the expression levels of Bax and Apaf-1 were significantly up-regulated (p<0.01). MiR-221 analogues can significantly inhibit the proliferation and induce apoptosis of Hep-2 cells in laryngeal cancer, and this is achieved by blocking the PI3K/AKT signaling pathway, which also indicates that MiR-221 affects the proliferation and apoptosis of laryngeal cancer cells through the PI3K/AKT signaling pathway.

Neutrophil Extracellular Traps in Tissue and Periphery in Juvenile Dermatomyositis.

Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). This study was undertaken to investigate the role of neutrophils in juvenile dermatomyositis (JDM). Electron microscopy was used to identify neutrophils in tissue. NETs were also imaged using fluorescence microscopy and quantified using a myeloperoxidase-DNA enzyme-linked immunosorbent assay (ELISA) in plasma obtained from healthy children (n = 20), disease controls (n = 29), JDM patients (n = 66), and JDM patients with history of calcifications (n = 20). Clinical data included disease activity scores and complement C4 levels. Levels of immune complexes (ICs) and calprotectin were analyzed using ELISA. Using electron microscopy, neutrophils were found to infiltrate affected muscle tissue, engulfing deposited calcium crystals. Uptake of the crystals led to neutrophil activation (P < 0.01) and subsequent phosphatidylinositol 3-kinase- and NADPH oxidase-dependent but peptidylarginine deiminase 4-independent formation of NETs, which contained mitochondrial DNA (P < 0.05), as confirmed in vivo (P < 0.001) and in vitro (P < 0.01). Peripheral NET levels were associated with calcinosis (P = 0.01), ICs (P = 0.008), and interleukin-8 levels (P = 0.004). Children with JDM had impaired NET clearance (P = 0.01), associated with autoantibody profiles including melanoma differentiation-associated protein 5 (P = 0.005), and depressed complement C4 levels (r = -0.72, P = 0.002). Furthermore, children with JDM showed evidence of neutrophil activation, with elevated levels of peroxidase activity (P = 0.02) and calprotectin (P < 0.01), which were associated with disease activity (P = 0.007), and dyslipidemia (odds ratio 4.7, P < 0.05). We found novel mechanisms of both calcium crystal-mediated neutrophil activation and cell death in JDM pathophysiology. Targeting this pathway may reduce the frequency and extent of calcinosis, as well as prevent long-term development of comorbidities, including atherosclerosis.

Perilipin 5 deficiency promotes atherosclerosis progression through accelerating inflammation, apoptosis, and oxidative stress.

Excessive plasma triglyceride(TG) and cholesterol levels promote the progression of several prevalent cardiovascular risk factors, including atherosclerosis, which is a leading death cause. Perilipin 5 (Plin5), an important perilipin protein, is abundant in tissues with very active lipid catabolism and is involved in the regulation of oxidative stress. Although inflammation and oxidative stress play a critical role in atherosclerosis development, the underlying mechanisms are complex and not completely understood. In the present study, we demonstrated the role of Plin5 in high-fat-diet-induced atherosclerosis in apolipoprotein E null (ApoE-/- ) mice. Our results suggested that Plin5 expressions increased in the artery tissues of ApoE-/- mice. ApoE/Plin5 double knockout (ApoE-/- Plin5-/- ) exacerbated severer atherogenesis, accompanied with significantly disturbed plasma metabolic profiles, such as elevated TG, total cholesterol, and low-density lipoprotein cholesterol levels and reduced high-density lipoprotein cholesterol contents. ApoE-/- Plin5-/- exhibited a higher number of inflammatory monocytes and neutrophils, as well as overexpression of cytokines and chemokines linked with an inflammatory response. Consistently, the IκBα/nuclear factor kappa B pathway was strongly activated in ApoE-/- Plin5-/- . Notably, apoptosis was dramatically induced by ApoE-/- Plin5-/- , as evidenced by increased cleavage of Caspase-3 and Poly (ADP-ribose) polymerase-2. In addition, ApoE-/- Plin5-/- contributed to oxidative stress generation in the aortic tissues, which was linked with the activation of phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinases pathways. In vitro, oxidized low-density lipoprotein (ox-LDL) increased Plin5 expression in RAW264.7 cells. Its knockdown enhanced inflammation, apoptosis, oxidative stress, and lipid accumulation, while promotion of Plin5 markedly reduced all the effects induced by ox-LDL in cells. These studies strongly supported that Plin5 could be a new regulator against atherosclerosis, providing new insights on therapeutic solutions.

Abstract 1606: The ethyl ester forms of the n-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid decrease the proliferation, invasion, and matrixmetalloproteinase activity of colorectal cancer cells

Abstract More than half of individuals diagnosed with colorectal cancer (CRC) will die from their cancer. The 5-year survival rate for patients diagnosed metastatic CRC is only 14%. Effective and well-tolerated therapies, such as dietary supplements, that prevent metastasis may increase the survival of these patients. The ability of the n-3 fatty acids (n-3FAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to decrease the proliferation of a variety of cancers in vitro, including CRC, is well established; however, the efficacy of n-3FAs with respect to inhibition of CRC metastasis has received little attention. EPA- and DHA ethyl esters (EE) are alternative forms of n-3FAs that may be more efficacious than free, non-esterified n-3FAs due to their ability to resist degradation allowing them to exert their effects for longer durations. Our objectives were to (1) compare the efficacy of free vs EE-n-3FAs with respect to inhibition of CRC cell proliferation, (2) to determine if EE-n-3FAs decreased CRC cell invasion, and (3) to begin to elucidate the mechanism through which EE-n-3FAs decrease cell invasion. We used two human invasive CRC cell lines, HCT-116 and SW620. To examine the effects of each n-3FA on cellular proliferation, cells were treated with 12.5, 25, 50 and 100 µM free or EE- EPA or DHA for 72 h [CAS numbers: free EPA (10417-94-4), free DHA (6217-54-5), EE-EPA (86227-47-6), EE-DHA (81926-94-5)]. All cells received equal volumes of ethanol vehicle. Data shown are mean +/- n=3 unless indicated. As n-3FA concentration increased, cellular proliferation decreased, regardless of n-3 FA type. Importantly, the IC50 (inhibitory concentration) for EE-EPA (7.7 ± 1.6 µM) was lower than that of free EPA (24.5 ±4.2 µM). The ability of EE-EPA and -DHA to decrease the invasion of CRC cells through Matrigel coated Boyden chambers was assessed following treatment with 12.5 µM EE-EPA or -DHA for 24 h. EE-EPA and EE-DHA inhibited the invasion of HCT-116 cells to 37.1 ± 13.5% and 50.6 ± 17.5% of control, respectively (n=4). Similarly, SW620 cell invasion was decreased to 27.5 ± 9.7% of control by EE-EPA and 24.2 ± 6.7% of control by EE–DHA. The decrease in metalloproteinase-2 (MMP-2) activity, assessed via zymography following EE-n-3FA treatment, reflected the decrease in CRC cell invasion. Finally, EE-n-3FAs failed to inhibit the invasion of HCT-116 cells expressing two alleles of constitutively active phosphatidylinositol 3-kinase, suggesting their ability to decrease cell invasion is conferred by inhibition of PI3K activity. In conclusion, EE-n-3FAs are likely more effective inhibitors of CRC proliferation than free n-3FAs, potentially because they are more stable. Additionally, EE-n-3FAs also inhibit CRC invasion and decrease MMP-2 activity through a mechanism that may involve PI3K. Citation Format: Eric Pfister, Michelle A. Lane. The ethyl ester forms of the n-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid decrease the proliferation, invasion, and matrixmetalloproteinase activity of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1606.

Ascl1 Regulates Electric Field-Induced Neuronal Differentiation Through PI3K/Akt Pathway

Directing differentiation of neural stem/progenitor cells (NSCs/NPCs) to produce functional neurons is one of the greatest challenges in regenerative medicine. Our previous paper has confirmed that electrical stimulation has a high efficiency of triggering neuronal differentiation by using isolated filum terminale (FT)-derived NPCs. To further clarify the intrinsic molecular mechanisms, protein–protein interaction (PPI) network analysis was applied to pinpoints novel hubs in electric field (EF)-induced neuronal differentiation. In this study, siRNA transfection of Achaete-scute homolog 1 (Ascl1) in NPCs or NPCs was followed by direct current stimulation at 150 mV/mm. Neuronal differentiation rate and protein expression level were analyzed after 7 or 14 days of electrical stimulation. The data showed that the expression level of Ascl1 was enhanced by electrical stimulation and positively correlated to EF strength. Moreover, we identified that the expression of Ascl1 positively regulated neuronal differentiation of NPCs and can be up-regulated by EF-stimulation through the activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway. Therefore, this study provides new insights into the role of Ascl1 and its relevant PI3K/Akt pathway in regulating of EF-induced neuronal differentiation and pointed out that continuous expression of Ascl1 in NPCs is required for EF-induced neuronal differentiation.

Mediatory role of BLT2 in the proliferation of KRAS mutant colorectal cancer cells

Inflammatory lipid mediators play various roles in colorectal cancer progression through complex pathways. However, the mechanism by which lipoxygenase-derived inflammatory lipid mediators contribute to colorectal cancer progression remains elusive. In this study, we found that BLT2, a cell surface GPCR for leukotriene B4 and 12‑hydroxyeicosatetraenoic acid, is highly upregulated in KRAS mutant LOVO and SW480 colorectal cancer cells and plays critical roles in mediating proliferation through activation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) and subsequent upregulation of cyclin D1. Exposure to BLT2 siRNA or LY255283, a specific BLT2 inhibitor, clearly suppressed the proliferation of KRAS mutant colorectal cancer cells and markedly increased cell cycle arrest by downregulating the PI3K/Akt-cyclin D1 cascade. Xenograft tumor formation by LOVO and SW480 cells in athymic mice was also substantially reduced by treatment with the BLT2 inhibitor in vivo. Together, our study demonstrates that BLT2 is necessary for the proliferation of LOVO and SW480 cells and thus may be a potential therapeutic target for the treatment of KRAS mutant colorectal cancer.

HSP70 Inhibitor Suppresses IGF-I-Stimulated Migration of Osteoblasts through p44/p42 MAP Kinase.

Heat shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone in a variety of cells including osteoblasts. We previously showed that insulin-like growth factor-I (IGF-I) elicits migration of osteoblast-like MC3T3-E1 cells through the activation of phosphatidylinositol 3-kinase/Akt and p44/p42 mitogen-activated protein (MAP) kinase. In the present study, we investigated the effects of HSP70 inhibitors on the IGF-I-elicited migration of these cells and the mechanism involved. The IGF-I-stimulated osteoblast migration evaluated by a wound-healing assay and by a transwell cell migration was significantly reduced by VER-155008 and YM-08, which are both HSP70 inhibitors. VER-155008 markedly suppressed the IGF-I-induced phosphorylation of p44/p42 MAP kinase without affecting that of Akt. In conclusion, our results strongly suggest that the HSP70 inhibitor reduces the IGF-I-elicited migration of osteoblasts via the p44/p42 MAP kinase.

Baicalin prevents tumor necrosis factor-α-induced apoptosis and dysfunction of pancreatic β-cell line Min6 via upregulation of miR-205.

Baicalin (BAI), one major flavonoid from Scutellaria baicalensis, possesses anticancer and anti-inflammatory properties. However, the effect of BAI on diabetes mellitus has not been investigated. This study explored the antidiabetic effect of BAI on pancreatic β-cell line Min6. Min6 cells were treated with tumor necrosis factor-α (TNF-α) to mimic β-cell destruction in type 1 diabetes mellitus. The effects of BAI on viability and apoptosis of Min6 cells were analyzed by the cell counting kit-8 assay and Annexin V-fluoresceine isothiocyanate/propidium iodide staining method. The insulin secretion of Min6 cells was determined using radioimmunoassay. Expression of apoptosis-associated proteins and inducible nitric oxide synthase (iNOS), and activation of phosphatidylinositol 3'-kinase/protein kinase B (PI3K/AKT) and nuclear factor ΚB (NF-κB) pathways were analyzed by Western blot analysis. Relative microRNA-205 (miR-205) expression was determined by quantitative real time polymerase chain reaction. TNF-α treatment inhibited cell growth and insulin secretion, but promoted iNOS expression. All of these effects were reversed by BAI treatment. BAI promoted viability; suppressed apoptosis; regulated caspase-3, B-cell lymphoma 2 and Bcl-2-associated X protein; decreased iNOS level; and increased insulin production. BAI protected Min6 cells by upregulating miR-205. Besides, the Min6 cell-protective effect of BAI was PI3K/AKT pathway and NF-κB pathway dependent. BAI activated the PI3K/AKT pathway and inhibited the NF-κB pathway by regulating miR-205. In conclusion, BAI protected Min6 cells from TNF-α-induced injury by upregulating miR-205, which acts, at least in part, via activation of the PI3K/AKT pathway and inactivation of the NF-κB pathway.

Abstract 3939: Activation of phosphatidylinositol-3′-kinase/Akt signaling in myxoid liposarcoma

Abstract Introduction: Myxoid liposarcoma (MLS) is the second most common type of liposarcoma and characterized by a high tendency to develop metastases. The molecular hallmark of MLS (≈90%) is a pathognomonic reciprocal t(12;16) (q13;p11) translocation, leading to the specific gene fusion of FUS and DDIT3. The resulting chimeric FUS-DDIT3 fusion protein is suggested to play a crucial role in MLS tumorigenesis, although its specific biological function and mechanism of action remain to be substantiated. While radiotherapy and chemotherapy with high-dose ifosfamide and doxorubicin represent established therapeutic options, prognosis in the metastasized situation is poor. Molecularly targeted therapeutic approaches are currently not available. Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional relevance of phosphatidylinositol-3'-kinase (PI3-kinase)/Akt signaling in MLS. Experimental procedures: Immunohistochemical and FISH analyses of PI3-kinase/Akt signaling effectors were performed in a large cohort of clinical MLS tumor specimens. Mutational burden was studied by targeted next-generation sequencing (NGS; Illumina MiSeq). PI3-kinase/Akt-mediated signaling transduction was modulated by specific RNAi knockdown and a pharmacological approach applying the small molecule inhibitor BKM120 (Buparlisib; NVP-BKM120). Cell proliferation and FACS assays were performed in different MLS cell lines. An established MLS chorioallantoic membrane model (CAM) was employed for in vivo confirmation of the preclinical in vitro data. Results: In a significant subset of MLS tumor specimens, immunohistochemical staining revealed elevated phosphorylation levels of various signaling components, indicating that activation of PI3-kinase/Akt signaling is a frequent feature in MLS. Activating PIK3CA mutations and loss of PTEN as mechanism for PI3-kinase/Akt activation were detected in ≈15%. PI3-kinase inhibition significantly suppressed the signaling cascade, associated with reduction of MLS cell viability and induction of apoptosis in vitro and in vivo. Conclusions: Our preclinical study emphasizes the pivotal role of the PI3-kinase/Akt signaling cascade in MLS pathogenesis and indicates the occurrence of specific mutational aberrations apart from the pathognomonic FUS-DDIT3 gene fusion. Our in vitro and in vivo results suggest that targeting the PI3-kinase/Akt signaling pathway provides a rational, molecularly founded therapeutic strategy in the treatment of MLS. Citation Format: Marcel Trautmann, Magdalene Cyra, Christian Bertling, Ilka Isfort, Bianca Altvater, Claudia Rossig, Susanne Hafner, Thomas Simmet, Jessica Becker, Inga Grünewald, Pierre Åman, Reinhard Büttner, Eva Wardelmann, Sebastian Huss, Wolfgang Hartmann. Activation of phosphatidylinositol-3′-kinase/Akt signaling in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3939.

Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity.

Background: Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of insulin signaling. The p110 catalytic and p85 regulatory subunits of PI3K are the products of separate genes, and while they come together to make the active heterodimer, they have opposing roles in insulin signaling and action. Deletion of hepatic p110α results in an impaired insulin signal and severe insulin resistance, whereas deletion of hepatic p85α results in improved insulin sensitivity due to sustained levels of phosphatidylinositol (3,4,5)-trisphosphate. Here, we created mice with combined hepatic deletion of p110α and p85α(L-DKO) to study the impact on insulin signaling and whole body glucose homeostasis. Methods: Six-week old male flox control and L-DKO mice were studied over a period of 18 weeks, during which weight and glucose levels were monitored, and glucose tolerance tests, insulin tolerance test and pyruvate tolerance test were performed. Fasting insulin, insulin signaling mediators, PI3K activity and insulin receptor substrate (IRS)1-associated phosphatidylinositol kinase activity were examined at 10 weeks. Liver, muscle and white adipose tissue weight was recorded at 10 weeks and 25 weeks. Results: The L-DKO mice showed a blunted insulin signal downstream of PI3K, developed markedly impaired glucose tolerance, hyperinsulinemia and had decreased liver and adipose tissue weights. Surprisingly, however, these mice displayed normal hepatic glucose production, normal insulin tolerance, and intact IRS1-associated phosphatidylinositol kinase activity without compensatory upregulated signaling of other classes of PI3K. Conclusions: The data demonstrate an unexpectedly overall mild metabolic phenotype of the L-DKO mice, suggesting that lipid kinases other than PI3Ks might partially compensate for the loss of p110α/p85α by signaling through other nodes than Akt/Protein Kinase B.