Inhibition Of PI3K-Akt Pathway Research Articles

515MO A phase I trial of durvalumab (Durv) in combination with olaparib (Ola) and capivasertib (Cap) in patients (pts) with advanced or metastatic cancers (Ca) (MEDIPAC)

Tumor-specific homologous recombination (HR) DNA defects and the phosphatidylinositol 3-kinase (PI3K)-AKT pathway are commonly implicated in tumorigenesis, and preclinical studies have shown synergistic effects of poly (ADP-ribose) polymerase (PARP) with PI3K-AKT inhibitors. Both pathways modulate the tumor immune microenvironment by regulation of tumor-infiltrating lymphocytes and reduction of Treg cells. We hypothesized that triplet combination of immunotherapy with PARP and PI3K-AKT pathway inhibition is tolerable and may demonstrate activity in pts with relevant pathway defects.

Ursodeoxycholic acid shows antineoplastic effects in bile duct cancer cells via apoptosis induction; p53 activation; and EGFR-ERK, COX-2, and PI3K-AKT pathway inhibition.

Unlike in normal cells, ursodeoxycholic acid (UDCA) causes apoptosis rather than protection in cancer cells. Aim of this study was to demonstrate whether UDCA actually inhibits proliferation and induces apoptosis in bile duct cancer cells; the effect of UDCA on the expression of COX-2, PI3K/AKT, ERK, and EGFR; how UDCA affects cancer cell invasiveness and metastasis, since these effects are not established in bile duct cancer cells. SNU-245 cells (human extrahepatic bile duct cancer cells) were cultured. MTT assays were performed to evaluate the effect of UDCA on the cell proliferation. A cell death detection enzyme-linked immunosorbent assay and a caspase-3 activity assay were used to determine apoptosis. Western blot analysis measured expression levels of various proteins. The invasiveness of the cancer cells was evaluated by invasion assay. In cultured bile duct cancer cells, UDCA suppressed cell proliferation in bile duct cancer cells by inducing apoptosis and p53 activation, blocking deoxycholic acid (DCA)-induced activated EGFR-ERK signaling and COX-2, inhibiting DCA-induced activated PI3K-AKT signaling, and suppressing the invasiveness of bile duct cancer cells. In addition, a MEK inhibitor impaired UDCA-induced apoptosis in bile duct cancer cells. UDCA has antineoplastic and apoptotic effects in bile duct cancer cells. Thus, UDCA could be a chemopreventive agent in patients with a high risk of cancer, and/or a therapeutic option that enhances other chemotherapeutics.

M2 macrophages, but not M1 macrophages, support megakaryopoiesis by upregulating PI3K-AKT pathway activity

Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in the bone marrow (BM) microenvironment. However, the specific effects of M1 MФs or M2 MФs on regulating megakaryocytes (MKs) are largely unknown. In the current study, aberrant BM-M1/M2 MФ polarization, characterized by increased M1 MФs and decreased M2 MФs and accompanied by impaired megakaryopoiesis-supporting abilities, was found in patients with PT post-allotransplant. RNA-seq and western blot analysis showed that the PI3K-AKT pathway was downregulated in the BM MФs of PT patients. Moreover, in vitro treatment with PI3K-AKT activators restored the impaired megakaryopoiesis-supporting ability of MФs from PT patients. Furthermore, we found M1 MФs suppress, whereas M2 MФs support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2 MФs, as indicated by decreased MK count, colony-forming unit number, high-ploidy distribution, and platelet count. Importantly, genetic knockdown of the PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ-specific PI3K-knockdown murine model, indicating a critical role of PI3K-AKT pathway in regulating the megakaryopoiesis-supporting ability of M2 MФs. Furthermore, our preliminary data indicated that TGF-β released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs. Taken together, our data reveal that M1 and M2 MФs have opposing effects on MKs in a PI3K-AKT pathway-dependent manner, which may lead to new insights into the pathogenesis of thrombocytopenia and provide a potential therapeutic strategy to promote megakaryopoiesis.

A pharmacophore-based classification better predicts the outcomes of HER2-negative breast cancer patients receiving the anthracycline- and/or taxane-based neoadjuvant chemotherapy.

AimsPrognosis of patients for human epidermal growth factor receptor 2 (HER2)‐negative breast cancer post neoadjuvant chemotherapy is not well understood. The aim of this study was to develop a novel pharmacophore‐based signature to better classify and predict the risk of HER2‐negative patients after anthracycline‐and/or taxane‐based neoadjuvant chemotherapy (NACT).Main methodsAnthracycline and taxane pharmacophore‐based genes were obtained from PharmMapper. Drug‐targeted genes (DTG) related clinical and bioinformatic analyses were undertaken in four GEO datasets.Key findingsWe used 12 genes from the pharmacophore to develop a DTG score (DTG‐S). The DTG‐S classification exhibited significant prognostic ability with respect to disease free survival (DFS) for HER2‐negative patients who receive at least one type of neoadjuvant chemotherapy that included anthracycline and/or taxane. DTG‐S associated with a high predictive ability for pathological complete response (pCR) as well as for prognosis of breast cancer. Using the DTG‐S classification in other prediction models may improve the reclassification accuracy for DFS. Combining the DTG‐S with other clinicopathological factors may further improve its predictive ability of patients’ outcomes. Gene ontology and KEGG pathway analysis showed that the biological processes of DTG‐S high group were associated with the cell cycle, cell migration, and cell signal transduction pathways. Targeted drug analysis shows that some CDK inhibitors and PI3K‐AKT pathway inhibitors may be useful for high DTG‐S patients.SignificanceThe DTG‐S classification adds prognostic and predictive information to classical parameters for HER2‐negative patients who receive anthracycline‐and/or taxane‐based NACT, which could improve the patients’ risk stratification and may help guide adjuvant treatment.

Targeting BET Proteins BRD2 and BRD3 in Combination with PI3K-AKT Inhibition as a Therapeutic Strategy for Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare, chemo-resistant subtype of ovarian cancer. To identify novel therapeutic targets and combination therapies for OCCC, we subjected a set of patient-derived ovarian cancer cell lines to arrayed high-throughput siRNA and drug screening. The results indicated OCCC cells are vulnerable to knockdown of epigenetic gene targets such as bromodomain and extra-terminal domain (BET) proteins BRD2 and BRD3. Subsequent RNA interference assays, as well as BET inhibitor treatments, validated these BET proteins as potential therapeutic targets. Because development of resistance to single targeted agents is common, we next performed sensitizer drug screens to identify potential combination therapies with the BET inhibitor CPI0610. Several PI3K or AKT inhibitors were among the top drug combinations identified and subsequent work showed CPI0610 synergized with alpelisib or MK2206 by inducing p53-independent apoptosis. We further verified synergy between CPI0610 and PI3K-AKT pathway inhibitors alpelisib, MK2206, or ipatasertib in tumor organoids obtained directly from patients with OCCC. These findings indicate further preclinical evaluation of BET inhibitors, alone or in combination with PI3K-AKT inhibitors for OCCC, is warranted.

Chemerin/ChemR23 regulates cementoblast function and tooth resorption in mice via inflammatory factors.

Periodontitis and orthodontic treatment can lead to inflammatory root resorption (IRR) through an unclear mechanism. Chemerin, a novel chemoattractant protein, is closely associated with inflammation, affects osteoblast and osteoclast differentiation, and may play a role in IRR. We aimed to explore possible roles of the chemerin/ChemR23 interaction in cementoblast function and IRR and reveal a new IRR therapeutic target. Cementoblast function-related gene and protein expression in the immortalized murine cementoblast cell line OCCM-30 after treatment with chemerin and siChemR23 was examined by qRT-PCR and Western blotting. The roles of the MAPK and PI3K-Akt signaling pathways were studied using specific inhibitors. Cementoblast cytokine production under different treatment conditions was measured by enzyme-linked immunosorbent assay and qRT-PCR. Additionally, we modeled IRR in wild-type and chemerin-overexpressing mice and injected transgenic mice with anti-ChemR23 antibody to block ChemR23. We then calculated the root resorption volume and examined periodontal tissue cathepsin K, Runx2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) expression. Chemerin suppressed cementoblast differentiation and mineralization and exerted a proinflammatory effect on cementoblasts. These effects were partially reversed by siChemR23 and reversed to different extents by p38, Erk1/2 and PI3K-Akt pathway inhibition, suggesting p38, Erk1/2 and PI3K-Akt pathways as signaling pathways downstream of chemerin/ChemR23. In vivo, chemerin overexpression worsened IRR. Moreover, chemerin expression was positively correlated with TNF-α, IL-6, and cathepsin K expression and negatively correlated with Runx2 expression. ChemR23 downregulation reversed these effects. Chemerin/ChemR23 induced TNF-α and IL-6 expression dependent on Erk1/2, p38 MAPK, and PI3K-Akt signaling pathway activation, thereby regulating cementoblast function and affecting IRR.

Effect of Methylene Blue and PI3K-Akt Pathway Inhibitors on the Neurovascular System after Chronic Cerebral Hypoperfusion in Rats.

Methylene blue (MB) has a protective effect on cognitive decline caused by chronic hypoperfusion, but the specific mechanism is not clear. This article aims to determine whether MB protects vascular neurons through PI3K/Akt and plays a role in improving cognitive impairment. Molecular biological methods, the hippocampal neuronal density test, the hippocampal vascular network density test, and dynamic enhanced magnetic resonance imaging (MRI) were used to detect the blood-brain barrier permeability and Evans blue leakage rate in the hippocampus. We also observed and evaluated the changes in the above results after administration of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway protein inhibitor LY294002. There were significant differences for cerebral blood flow (CBF) between the chronic cerebral hypoperfusion (CCH) + MB group (100ml/100g/min) and the CCH group (60ml/100g/min, P < 0.05). After using LY294002, the CBF of the CCH + MB + LY294002 group dropped to 82ml/100g/min. The vascular density in the CCH + MB group was 23%, which is significantly higher than that in the CCH group (15.1%) (P < 0.05). The vascular density (17.5%) in the CCH + MB + LY294002 group was significantly higher than that in the CCH group but lower than that in the CCH + MB group. Western blotting results showed that one week after intraperitoneal injection of MB, the expression of t-Akt and p-Akt in the CCH + MB group was increased after CCH, and LY294002 partially blocked this up-regulation effect (CCH + MB + LY294002 group). MB is a potential therapy for the relief of mild cognitive impairment associated with CCH, vascular dementia, and Alzheimer's disease.

Akt/survivin pathway inhibition enhances the apoptotic cell death-induced by alpha-santalol in human prostate cancer cells

We have shown previously that alpha-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis, but the mechanism of cell death is not fully elucidated. The present study was undertaken to investigate the role of PI3K/Akt/survivin pathway in alpha-santalol-induced apoptosis employing cultured LNCaP and PC-3 human prostate cancer cells. Treatment of prostate cancer cells with alpha-santalol (20, 40 μM) resulted in the down regulation of survivin and p-AKT (s-473) expression and statistically significant reduction in total survivin levels as evidenced by survivin ELISA assay. Furthermore, inhibition of PI3K-Akt pathway by pharmacological inhibitor, LY294002 enhanced the apoptotic cell death induced by alpha-santalol as determined by cell viability, cellular morphology, active caspase-3 activity and expression of cleaved PARP, cleaved caspase-3 levels. In conclusion, the present study provides novel insight into the molecular circuitry of alpha-santalol-induced cell death and reveals that alpha-santalol targets Akt/Survivin pathway to induce cell death and that the cell death is increased in the presence of a known inhibitor of the pathway.

Triterpenoids from Liquidambar Fructus induced cell apoptosis via a PI3K-AKT related signal pathway in SMMC7721 cancer cells

A previously undescribed taraxerene-type triterpenoid possessing a class of rare natural taraxerene triterpenoid possessing skeleton with 14, 28-lactone, two undescribed oleane-type triterpenoids, and twenty-five known triterpenoids were isolated from Liquidambar formosana (Hamamelidaceae). The structures of undescribed compounds were determined on the basis of 1D and 2D NMR spectroscopic, HR-ESI-MS, and X-ray crystallographic data analysis. Among the isolates, ursolic acid, 3,6-dion-20(29)-lupen-28-oic acid, and 3-oxo-12α-hydroxyoleanan-28,13β-olide induced a significant apoptosis in SMMC7721 cells in the flow cytometer experiment with apoptosis rates of 94.5%, 57.3% and 89.9% at 8.0 μM, respectively, exhibiting near equivalent apoptosis-inducing abilities to that positive drug taxol (apoptotic rate of 71.2% at 1.4 μM). Mechanism studies suggested that these three compounds could regulate the mitochondrial pathway by up-regulating the expressions of pro-apoptotic factors (Bad and Bax) and activating caspase-3 and caspase-9 to induce apoptosis. Further studies indicated that the pro-apoptotic effects of these three compounds were associated with PI3K-AKT pathway inhibition. Taken together, these studies provide evidence that triterpenoids from L. Fructus are promising candidates for the hepatocellular carcinoma therapy.

Abstract LB-302: Whole genome sequencing reveals a significant role of ZNFs-PHLPP2-AKT axis in familial breast cancer of chaoshan area

Abstract Introduction Up to 15% cases of breast cancer are familial. However, the two most prevalent susceptibility genes, BRCA1 and BRCA2, only account for 25% of the familial cases. To this regard, we aimed to identify recurrent variants associated with familial breast cancer in Chaoshan Area of China by whole genome sequencing. Materials &amp; Methods In the present study, Whole Genome Sequencing was performed in 8 familial breast cancer families with 13 familial breast cancer cases and 25 healthy relatives from Chaoshan Area. All the variants were filtered and prioritized by co-segregation analysis and bioinformatic tools. Results A total of 714 variants in 609 genes were selected as rare, probably damaging pathogenic mutations after co-segregation and functional prediction analysis in each family. In all the 609 genes, the zinc finger protein family was overlapped in all the 8 families, among which the c.120-130del of ZNF77 was overlapped in 3 families, c.279_791de of ZNF544 and c.T895A of ZNF479 in 2 families respectively, which emphasized the great significance of ZNF family in the mutational spectrum of Chaoshan Area. After that, the KEGG Pathway analysis in KOBAS 3.0 implied the significance of PI3K-Akt pathway in all the mutated 609 genes. At the same time, the PPI analysis by Cytoscape 3.6.1 indicated that all ZNF genes were interacted with one specific gene, PHLPP2, which inhibits the PI3K-Akt pathway similar to PTEN. Furthermore, two website-based bioinformatic tools cBioportal and Oncomine have predicted the positive correlations between ZNF77 and PHLPP2. All the analysis above was demonstrated the great significance of ZNFs-PHLPP2-AKT axis in the mutational spectrum of familial breast cancer of Chaoshan Area, in China. Conclusions Zinc finger protein family, especially ZNF77, are frequently mutated in the familial breast cancer of Chaoshan Area, which results in the down regulation of PHLPP2 and subsequently reverse the inhibition of PI3K-Akt pathway, and finally lead to tumorigenesis. Citation Format: Can-Bin Fang, Qian-Qian Ye, Jing Liu, Guo-Jun Zhang. Whole genome sequencing reveals a significant role of ZNFs-PHLPP2-AKT axis in familial breast cancer of chaoshan area [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 LB-302.

Jarid1b promotes epidermal differentiation by mediating the repression of Ship1 and activation of the AKT/Ovol1 pathway.

ObjectivesTerminally differentiated stratified squamous epithelial cells play an important role in barrier protection of the skin. The integrity of epidermal cells is maintained by tight regulation of proliferation and differentiation. The aim of this study was to investigate the role of epigenetic regulator H3K4me3 and its demethylase Jarid1b in the control of epithelial cell differentiation.Materials and methodsRT‐qPCR, Western blotting and IHC were used to detect mRNA and protein levels. We analysed cell proliferation by CCK8 assay and cell migration by wound healing assay. ChIP was used to measure H3K4me3 enrichment. A chamber graft model was established for epidermal development.ResultsOur studies showed that H3K4me3 was decreased during epidermal differentiation. The H3K4me3 demethylase Jarid1b positively controlled epidermal cell differentiation in vitro and in vivo. Mechanistically, we found that Jarid1b substantially increased the expression of mesenchymal‐epithelial transition (MET)‐related genes, among which Ovol1 positively regulated differentiation gene expression. In addition, Ovol1 expression was repressed by PI3K‐AKT pathway inhibitors and overexpression (O/E) of the PI3K‐AKT pathway suppressor Ship1. Knockdown (KD) of Ship1 activated downstream PI3K‐AKT pathway and enhanced Ovol1 expression in HaCaT. Importantly, we found that Jarid1b negatively regulated Ship1 expression, but not that of Pten, by directly binding to its promoter to modulate H3K4me3 enrichment.ConclusionOur results identify an essential role of Jarid1b in the regulation of the Ship1/AKT/Ovol1 pathway to promote epithelial cell differentiation.

Cubeben induces autophagy via PI3K-AKT-mTOR pathway to protect primary neurons against amyloid beta in Alzheimer's disease.

Autophagy is a lysosomal degradative process by which it recycles cytosolic components and degrades protein aggregates inside cells. Here, we identified cubeben as an inducer of autophagy in primary neuronal cells. Autophagy induction was accompanied the upregulation of autophagic proteins like Beclin, ATG5, ATG12 and lipidation of LC3-II in primary neuronal cells. Cubeben induces autophagy by the inhibition of PI3K-AKT pathway in a dose dependent manner. Constitutive active P110α abrogates cubeben induced autophagic induction in primary neuronal cells. Furthermore, cubeben inhibits amyloid beta induced toxicity in primary neuronal cells. Thus our data suggests that cubeben as a potential anti-Alzheimer therapeutic lead.

Jarid1b Promotes Epidermal Differentiation by Mediating Repression of Ship1 and Activation of AKT/Ovol1 Pathway

The role of epigenetic regulators in the control of epithelial cell differentiation is largely undefined. Our studies showed that H3K4me3 was decreased during epidermal differentiation. Unexpectedly, both expression of histone methyltransferases and demethylases over H3K4me3 were elevated upon differentiation, suggesting the crucial role of demethylases in controlling of differentiation. Next we found that the demethylase Jarid1b positively regulated epidermal differentiation. ShRNA-mediated depletion of Jarid1b delayed the calcium induced HaCaT cell differentiation in vitro and impaired differentiation layer formation in reconstituted epidermis in vivo. Conversely, overexpression of Jarid1b increased epidermal differentiation gene expression and inhibited cell proliferation and migration in culture. Mechanistically, we found that Jarid1b substantially increased mesenchymal-epithelial transition (MET) gene expression, among which Ovol1 positively regulated differentiation gene expression. In addition，Ovol1 expression was repressed by PI3K-AKT pathway inhibitors and suppressor Ship1 overexpression. Knockdown of Ship1 activated downstream PI3K-AKT pathway and enhanced Ovol1 expression in HaCaT cells. Importantly, we found that Jarid1b negatively regulated Ship1 expression, but not of Pten, via directly binding to its promoter to modulate H3K4me3 enrichment. Taken together, our results indentify an essential role for Jarid1b in regulation of Ship1/AKT/Ovol1 pathway to promote epithelial cell differentiation. Funding Statement: This work was supported by National Natural Science Foundation of China (81672662, 81701381, 81770978 and 81774248), Key Research and Development Program of Shandong Province (2017GSF18193, 2018GSF118012), Natural Science Foundation of Shandong Province (ZR2014CM040) and Shandong Provincial Education Department Program (J14LK02). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All experimental protocols were approved by the ethics committee of animal care of Affiliated Hospital of Qingdao University. All animal studies were performed in strict accordance with the approved protocol.

PI3K-Akt signaling controls PFKFB3 expression during human T-lymphocyte activation.

Lymphocyte activation is associated with rapid increase of both the glycolytic activator fructose 2,6-bisphosphate (Fru-2,6-P2) and the enzyme responsible for its synthesis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFKFB3 gene, which encodes for the most abundant PFK-2 isoenzyme in proliferating tissues, has been found overexpressed during cell activation in several models, including immune cells. However, there is limited knowledge on the pathways underlying PFKFB3 regulation in human T-lymphocytes, and the role of this gene in human immune response. The aim of this work is to elucidate the molecular mechanisms of PFKFB3 induction during human T-lymphocyte activation by mitotic agents. The results obtained showed PFKFB3 induction during human T-lymphocyte activation by mitogens such as phytohemagglutinin (PHA). PFKFB3 increase occurred concomitantly with GLUT-1, HK-II, and PCNA upregulation, showing that mitotic agents induce a metabolic reprograming process that is required for T-cell proliferation. PI3K-Akt pathway inhibitors, Akti-1/2 and LY294002, reduced PFKFB3 gene induction by PHA, as well as Fru-2,6-P2 and lactate production. Moreover, both inhibitors blocked activation and proliferation in response to PHA, showing the importance of PI3K/Akt signaling pathway in the antigen response of T-lymphocytes. These results provide a link between metabolism and T-cell antigen receptor signaling in human lymphocyte biology that can help to better understand the importance of modulating both pathways to target complex diseases involving the activation of the immune system.

Identification of a novel angiogenic peptide from periostin.

Angiogenic peptides have therapeutic potential for the treatment of chronic ischemic diseases. Periostin, an extracellular matrix protein expressed in injured tissues, promotes angiogenesis and tissue repair. We previously reported that in vivo administration of both recombinant full-length protein and the first FAS I domain of periostin alleviated peripheral artery occlusive disease by stimulating the migration of humane endothelial colony forming cells (ECFCs) and subsequent angiogenesis. In the present study, we ascertained the peptide sequence responsible for the periostin-induced angiogenesis. By serial deletion mapping of the first FAS I domain, we identified a peptide sequence (amino acids 142–151) of periostin for stimulation of chemotactic migration, adhesion, proliferation and endothelial tube formation of human ECFCs in vitro. Chemotactic migration of ECFCs induced by the periostin peptide was blocked by pre-incubation with an anti-β5 integrin neutralizing antibody. Treatment of ECFCs with the periostin peptide led to phosphorylation of both AKT and ERK, and pretreatment of ECFCs with the MEK-ERK pathway inhibitor U0126 or the PI3K-AKT pathway inhibitors, LY294002 or Wortmannin, blocked the periostin peptide-stimulated migration of ECFCs. These results suggest that the synthetic periostin peptide can be applied for stimulating angiogenic and therapeutic potentials of ECFCs.

Effects of ginkgobalide B on neurocyte apoptosis and the expression of protein kinase B after experimental hypoxic-ischemic brain injury

Objective To observe the effect of ginkgobalide B (GB) on neurocyte apoptosis and protein kinase B expression in neonatal rats after hypoxic-ischemic brain damage (HIBD). Methods Ninety seven-day-old Sprague-Dawley rats were randomly divided into a sham group, an HIBD group and a GB group, each of 30. HIBD was induced in the HIBD and GB groups using the classical Rice method, while the sham group was given a sham operation. GB (10 mg/kg) was injected intraperitoneally to the rats in the GB group at 0 h and 24 h after the modeling. Then 6 rats were killed 6 h, 12 h, 24 h and 48 h after the modeling, and the expression of caspase-3 mRNA was detected using a real-time PCR to find the time point of maximum effectiveness. Then to further explore the role of the PI3K-AKT pathway in the anti-apoptosis effect of ginkgolide B, a a GB+ LY294002 group of 6 rats, which was injected with PI3K-AKT pathway inhibitor LY294002 (1.8 mg/kg) intraperitoneally at 30 min before the modeling and with GB(10 mg/kg) at 0 h and 24 h after the modeling, was added to the experiment. Hematoxylin-eosin staining, terminal-deoxynucleotidyl transferase-mediated nick end labeling and immunohistochemical staining were then used to observe any morphological changes in the cortex, to detect neuronal apoptosis and to quantify the expression of P-AKT protein. Results The expression of caspase-3 in the HI and GB groups began to increase 6 hours after the HIBD and reached a peak after 24 hours, followed by a gradual decline. The expression of caspase-3 in the GB group was significantly lower than in the HI group throughout, while that of both of those groups was significantly higher than in the sham group. Apoptosis-positive cells and the expression of caspase-3increased had significantly in the HI, GB and GB+ LY294002 groups 24 hours after the HIBD compared with the sham group, while the expression of P-AKT protein had decreased significantly. Moreover, the apoptosis-positive cells and the expression of caspase-3 of the HI and GB+ LY294002 groups were significantly higher than those of the GB group, while their expression of P-AKT protein was significantly lower after 24 hours. Conclusion Ginkgobalide B can decrease neurocyte apoptosis caused by hypoxic-ischemic brain damage, especially at 24 h after the damage. The PI3K-AKT signaling pathway plays an important role in this effect. Key words: Ginkgobalide B; Hypoxic-ischemic brain injury; Apoptosis

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly.

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.

Selenium deficiency-induced thioredoxin suppression and thioredoxin knock down disbalanced insulin responsiveness in chicken cardiomyocytes through PI3K/Akt pathway inhibition

Thioredoxin (Txn) system is the most crucial antioxidant defense mechanism in cell consisting of Txn, thioredoxin reductase (TR) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH). Perturbations in Txn system may compromise cell survival through oxidative stress induction. Metabolic activity of insulin plays important roles in fulfilling the stable and persistent demands of heart through glucose metabolism. However, the roles of Txn and Txn system in insulin modulated cardiac energy metabolism have been less reported. Therefore, to investigate the role of Txn in myocardial metabolism, we developed a Se-deficient chicken model (0.033mg/kg) for in-vivo and Txn knock down cardiomyocytes culture model (siRNA) for in-vitro studies. Quantitative real time PCR and western blotting was performed. Se deficiency suppressed Txn and TR in cardiac tissues. Significant increases in ROS (P<0.05) levels signify the onset of oxidative stress and in both models. Se deficiency-induced Txn suppression model and Txn knock down cardiomyocytes models significantly decreased (P<0.05), the mRNA and protein levels of insulin-like growth factors (IGF1, IGF2), IGF-binding proteins (IGFBP2, IGFBP4), insulin receptor (IR), insulin receptor substrates (IRS1, IRS2), and glucose transporters (GLUT1, GLUT3, GLUT8), however, IGFBP3 expression increased in Txn knock down cardiomyocytes. In addition, in contrast to their respective controls, Se deficiency-induced Txn depleted tissues and Txn deleted cardiomyocytes showed suppression in mRNA and protein levels of PI3K, AKT, P-PI3K, and repression in FOX, P-FOX JNK genes. Combing the in vitro and in vivo experiments, we demonstrate that Txn gene suppression can cause dysfunction of insulin-modulated cardiac energy metabolism and increase insulin resistance through PI3K-Akt pathway inhibition. Herein, we conclude that inactivation of Txn system can alter cellular insulin response through IRS/PI3K/Akt pathway repression and JNK and FOX expression. These findings point out that Txn system can redox regulate the insulin dependent glucose metabolism in heart and is essential for cell vitality. Moreover, the increased expression of IGFBP3 indicates that it can be a potential negative modulator of metabolic activity of insulin in Txn deficient cells.

Abstract 1231: Restoration of tumor suppression in vivo by systemic delivery of PTEN mRNA nanoparticles

Abstract PTEN (phosphatase and tensin homolog on chromosome ten) is one of the most common lost or mutated tumor suppressor genes in human cancers, including ~50% of metastatic castration-resistant prostate cancer (mCRPC). By catalyzing PIP3 dephosphorylation, PTEN negatively regulates the PI3K-AKT-mTOR pathway, which is frequently altered in mCRPC. Reintroduction of functional PTEN for mCRPC treatment has proven difficult. By employing self-assembled lipid-polymer hybrid nanoparticle platforms, we successfully reintroduced PTEN mRNA to PTEN-null prostate cancer cells both in vitro and in vivo. These mRNA-loaded nanoparticles demonstrate high protein expression efficiency, low toxicity and good stability in serum and tumor accumulation. We confirmed that restoration of PTEN in PTEN-null prostate cancer cells inhibits the PI3k-AKT pathway, reduces cell viability and enhances apoptosis in vitro. Systemic delivery of PTEN mRNA-loaded nanoparticles in prostate xenograft tumors results in ~85% inhibition of tumor growth and leads to tumor cell death without toxic side effects in vivo. In summary, this work provides proof of concept of mRNA-based gene therapy for systemic restoration of functional PTEN for tumor suppression in vivo. It represents a novel approach to PI3K-AKT pathway inhibition, with the potential to specifically target cancers with loss of PTEN function. Citation Format: Yingjie Xu, Mohammad Ariful Islam, Harshal Zope, Morteza Mahmoudi, Robert S. Langer, Jinjun Shi, Bruce R. Zetter, Omid C. Farokhzad. Restoration of tumor suppression in vivo by systemic delivery of PTEN mRNA nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2017-1231

RETRACTED: Activation of the PI3K-Akt pathway promotes neuroprotection of the δ-opioid receptor agonist against cerebral ischemia-reperfusion injury in rat models

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. The authors contacted the journal to request a retraction: "Recently, we found that some experimental animal samples from another research were mistakenly used in this paper, resulting in the results and conclusions unreliable". Concern was also raised about the reliability of the brain section images in Figure 1, which seem to appear in other publications, as detailed here: https://pubpeer.com/publications/8AF402957928F3F27A1F46B6D556AD; and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. In addition, suspected image duplications were detected in Figures 2A, 3B and 4B. The journal requested the corresponding author comment on these concerns and provide the raw data. The author did not fulfil this request and the Editor-in-Chief decided to retract the article.