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Context: Protein–protein interaction interfaces play a major role in cell signaling pathways. There is always a great interest in developing protein–protein interaction (PPI) inhibitors of kinases, as they are challenging due to their hydrophobicity, flat surface, specificity, potency, etc. 3 Phosphoinositide-dependent kinase-1 (PDK1), which is involved in the PI3K/PDK1/AKT pathway, is a cancer target that has gained insight for the past two decades. PDK1 possesses a protein interaction fragment (PIF) pocket, which is a well-known PPI that targets allosteric modulators. This work focusses on energy-based pharmacophore model development which on virtual screening could yield novel scaffolds towards the drug designing objective for the kind of PDK1 modulators. A novel pyrazolo pyridine molecule was identified as an allosteric modulator that binds to the PPI site. The metadynamics simulations with free energy profiles further revealed the conformational allosteric changes stimulated on the protein structure upon ligand binding. The cytotoxic activity (IC50-20 μM) of the identified compound against five different cancer cell lines and cell cycle analysis supported the anticancer activity of the identified compound. Methods: All the computational works were carried out by the most commonly used Schrodinger Suite software. The pharmacophore was validated by Receiver Operation Characteristics (ROC) and screening against allosteric Enamine database library. The Optimized Potential Liquid Simulations (OPLS-2005) was used to minimize the structures for molecular docking calculations, and inbuilt scoring method of ranking the compounds based on docking score and Glide energy was used. Molecular dynamics simulations were conducted by Desmond implemented in Maestro. The hit compound was purchased from Enamine and tested against different cancer cell lines by MTT assay, apoptosis by western blotting technique, and by flow cytometry analysis. [ABSTRACT FROM AUTHOR]

Phosphoinositide-dependent kinase-1 (PDK1) is a master kinase of the protein A, G, and C (AGC) family kinases that play important roles in regulating cancer cell proliferation, survival, and metabolism. Besides phosphorylating/activating AKT at the cell membrane in a PI3K-dependent manner, PDK1 also exhibits constitutive activity on many other AGC kinases for tumor-promoting activity. In the latter case, PDK1 protein levels dominate its activity. We previously reported that MAPK4, an atypical MAPK, can PI3K-independently promote AKT activation and tumor growth. Here, using triple-negative breast cancer (TNBC) cell models, we demonstrate that MAPK4 can also enhance PDK1 protein synthesis, thus phosphorylate/activate PDK1 substrates beyond AKT. This new MAPK4-PDK1 axis alone lacks vigorous tumor-promoting activity but cooperates with our previously reported MAPK4-AKT axis to promote tumor growth. Besides enhancing resistance to PI3K blockade, MAPK4 also promotes cancer cell resistance to the more stringent PI3K and PDK1 co-blockade, a recently proposed therapeutic strategy. Currently, there is no MAPK4 inhibitor to treat MAPK4-high cancers. Based on the concerted action of MAPK4-AKT and MAPK4-PDK1 axis in promoting cancer, we predict and confirm that co-targeting AKT and PDK1 effectively represses MAPK4-induced cancer cell growth, suggesting a potential therapeutic strategy to treat MAPK4-high cancers. This study shows that besides activating AKT, MAPK4 promotes PDK1 protein synthesis and thus also activates PDK1 substrates beyond AKT to promote cancer. Co-targeting AKT and PDK1 can therefore repress MAPK4-induced cancer cell growth, suggesting a potential therapeutic strategy to treat MAPK4-high cancers, including a large subset of "triple negative" breast cancer. [ABSTRACT FROM AUTHOR]

Polycystic ovarian syndrome (PCOS) is a complex multi-factorial syndrome associated with androgen excess and anovulatory infertility. In the current study, we investigated the role of dihydrotestosterone-induced exosomal miR-379-5p release in determining the destiny of the developing follicles. Our hypothesis was that androgen regulates granulosa cell miR-379-5p content by facilitating its exosomal release in a follicular-stage dependent manner, a process which determines granulosa cell fate. Compared to human non-PCOS subjects, individuals with PCOS exhibit higher follicular fluid free testosterone levels, lower exosomal miR-379-5p content and granulosa cell proliferation. Androgenized rats exhibited lower granulosa cell miR-379-5p but higher phosphoinositide-dependent kinase-1 (PDK1; a miR-379-5p target) content and proliferation. Androgen reduced granulosa cell miR-379-5p content by increasing its exosomal release in preantral follicles, but not in antral follicles in vitro. Studies with an exosomal release inhibitor confirmed that androgen-induced exosomal miR-379-5p release decreased granulosa cell miR-379-5p content and proliferation. Ovarian overexpression of miR-379-5p suppressed granulosa cell proliferation, and basal and androgen-induced preantral follicle growth in vivo. These findings suggest that increased exosomal miR-379-5p release in granulosa cells is a proliferative response to androgenic stimulation specific for the preantral stage of follicle development and that dysregulation of this response at the antral stage is associated with follicular growth arrest, as observed in human PCOS. [ABSTRACT FROM AUTHOR]

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The longevity phenomenon is entirely controlled by the insulin signaling pathway (IIS-pathway). Both vertebrates and invertebrates have IIS-pathways that are comparable to one another, though no one has previously described de novo transcriptome assembly of IIS-pathway-associated genes in termites. In this research, we analyzed the transcriptomes of both reproductive (primary kings "PK" and queens "PQ", secondary worker reproductive kings "SWRK" and queens "SWRQ") and non-reproductive (male "WM" and female "WF" workers) castes of the subterranean termite Reticulitermes chinensis. The goal was to identify the genes responsible for longevity in the reproductive and non-reproductive castes. Through transcriptome analysis, we annotated 103,589,264 sequence reads and 184,436 (7G) unigenes were assembled, GC performance was measured at 43.02%, and 64,046 sequences were reported as CDs sequences. Of which 35 IIS-pathway-associated genes were identified, among 35 genes, we focused on the phosphoinositide-dependent kinase-1 (Pdk1), protein kinase B2 (akt2-a), tuberous sclerosis-2 (Tsc2), mammalian target of rapamycin (mTOR), eukaryotic translation initiation factor 4E (EIF4E) and ribosomal protein S6 (RPS6) genes. Previously these genes (Pdk1, akt2-a, mTOR, EIF4E, and RPS6) were investigated in various organisms, that regulate physiological effects, growth factors, protein translation, cell survival, proliferation, protein synthesis, cell metabolism and survival, autophagy, fecundity rate, egg size, and follicle number, although the critical reason for longevity is still unclear in the termite castes. However, based on transcriptome profiling, the IIS-pathway-associated genes could prolong the reproductive caste lifespan and health span. Therefore, the transcriptomic shreds of evidence related to IIS-pathway genes provide new insights into the maintenance and relationships between biomolecular homeostasis and remarkable longevity. Finally, we propose a strategy for future research to decrypt the hidden costs associated with termite aging in reproductive and non-reproductive castes. [ABSTRACT FROM AUTHOR]

Insulin resistance (IR) plays a key role in the pathogenesis and clinical outcome of patients with multiple diseases and diabetes. In this study, we examined the antidiabetic effects of a terpenoid-rich extract from Dillenia indica L. bark (TRDI) in palmitic acid-induced insulin resistance (PA-IR) in C2C12 myotube and a streptozotocin (STZ)-induced diabetic mice model and explored the possible underlying mechanism. TRDI showed potential DPPH- and ABTS-radical scavenging effects with a half-maximal inhibitory concentration (IC50) value of 9.76 ± 0.50 µg/mL and 17.47 ± 1.31 µg/mL, respectively. Furthermore, TRDI strongly mitigated α-glucosidase activity with an IC50 value of 3.03 ± 1.01 µg/mL, which was 92-fold higher than the positive control, acarbose (IC50 = 279.49 ± µg/mL). TRDI stimulated the insulin receptor substrarte-1 (INS-1), downregulated phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (Akt) in both normal and PA-IR C2C12 cells as well as in STZ-induced diabetic mice, enhanced glucose transporter 4 (GLUT4) translocation to the plasma membrane (PM), and increased glucose absorption. Furthermore, TRDI administration significantly reduced PA-induced reactive oxygen species (ROS) formation in C2C12 cells and increased the protein level of numerous antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase-1 (GPx-1) and thioredoxin reductase (TrxR) both in vitro and in vivo. Furthermore, TRDI facilitated nuclear factor erythroid 2 related factor 2 (Nrf2) nuclear translocation and increased HO-1 expression in PA-IR C2C12 cells and STZ-induced diabetic mice. However, for the inhibition of Nrf2, TRDI failed to resist the effects of IR. Thus, this study provides new evidence to support the use of TRDI for diabetes treatment. [ABSTRACT FROM AUTHOR]

Reference 1 Xia X, Li X, Li F. A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1. 3C Representative images showing plate colony formations from different cell lines were incorrectly selected during figure assembly; this affects circ-AKT3 IRES mut U373 (bottom left in left-hand colony formations), and circ-AKT3 shRNA control SW1783 and HS683 (top and bottom left in right-hand colony formations). [Extracted from the article]

Objectives: The epidermal growth factor receptor (EGFR) is related to the invasion and metastasis of external auditory canal (EAC) squamous cell carcinoma (SCC). The phosphoinositide‐dependent protein kinase‐1 (PDPK1) accelerates tumor cell growth through anti‐apoptotic signaling under the influence of downstream EGFR‐mediated signaling pathways. In this study, we investigated the EGFR/PDPK1 axis in the EAC under EGF stimulation. Methods: We confirmed EGFR and PDPK1 expression in human EACSCC specimens immunohistochemically. We next transfected the EGF expression vector in the mouse EAC and then conducted a PDPK1 inhibitory experiment. Immunohistochemical analysis was performed in the mouse EAC, using anti‐EGF, anti‐EGFR, anti‐PDPK1, and anti‐Ki67 antibodies. Immunohistochemical analysis of cleaved caspase‐3 and terminal deoxy(d)‐UTP nick end labeling (TUNEL) detection assays were also performed for the assessment of apoptosis in the inhibitory experiment. Results: Immunohistochemical analysis revealed overexpression and colocalization of EGFR and PDPK1 in human EACSCC specimens. The growth of a protuberant tumor was observed in the mouse EAC in which EGF expression vector was transfected, and EGF, EGFR, PDPK1, and Ki67 labeling indexes (LIs) were significantly increased. PDPK1 inhibition then induced normal epithelial appearance in the EAC. Moreover, EGF, EGFR, PDPK1, and Ki67 LIs were decreased, and cleaved caspase‐3 and TUNEL LIs were increased in the EAC. Conclusion: We demonstrated the possibility that PDPK1 plays an important role in EACSCC. Level of Evidence: NA. [ABSTRACT FROM AUTHOR]

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function. Arginine methylation has been reported to regulate AKT-associated signalling. Here, the authors show that PRMT5-mediated arginine methylation promotes AKT activity and induces tumourigenesis. [ABSTRACT FROM AUTHOR]

The role of PI3K-mTOR pathway in regulating NK cell development has been widely reported. However, it remains unclear whether NK cell development depends on the protein kinase B (PKB), which links PI3K and mTOR, perhaps due to the potential redundancy of PKB. PKB has two phosphorylation sites, threonine 308 (T308) and serine 473 (S473), which can be phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK1) and mTORC2, respectively. In this study, we established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. We found that the single deletion of PDK1 or Rictor could lead to a significant defect in NK cell development, while combined deletion of PDK1 and Rictor severely hindered NK cell development at the early stage. Notably, ectopic expression of myristoylated PKB significantly rescued this defect. In terms of mechanism, in PDK1/Rictor-deficient NK cells, E4BP4, a transcription factor for NK cell development, was less expressed, and the exogenous supply of E4BP4 could alleviate the developmental defect of NK cell in these mice. Besides, overexpression of Bcl-2 also helped the survival of PDK1/Rictor-deficient NK cells, suggesting an anti-apoptotic role of PKB in NK cells. In summary, complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development, and PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is the most common malignancy and is the second leading cause of cancer among men globally. Using a kinome‐wide lentiviral small‐hairpin RNA (shRNA) library screen, we identified phosphoinositide‐dependent kinase‐1 (PDPK1) as a potential mediator of cell survival in PCa cells. We showed that knock‐down of endogenous human PDPK1 induced significant tumour‐specific cell death in PCa cells (DU145 and PC3) but not in the normal prostate epithelial cells (RWPE‐1). Further analyses revealed that PDPK1 mediates cancer cell survival predominantly via activation of serum/glucocorticoid‐regulated kinase 3 (SGK3). Knock‐down of endogenous PDPK1 in DU145 and PC3 cells significantly reduced SGK3 phosphorylation while ectopic expression of a constitutively active SGK3 completely abrogated the apoptosis induced by PDPK1. In contrast, no such effect was observed in SGK1 and AKT phosphorylation following PDPK1 knock‐down. Importantly, PDPK1 inhibitors (GSK2334470 and BX‐795) significantly reduced tumour‐specific cell growth and synergized docetaxel sensitivity in PCa cells. In summary, our results demonstrated that PDPK1 mediates PCa cells' survival through SGK3 signalling and suggest that inactivation of this PDPK1‐SGK3 axis may potentially serve as a novel therapeutic intervention for future treatment of PCa. [ABSTRACT FROM AUTHOR]

Background: A number of JmjC domain-containing histone demethylases have been identified and biochemically characterized in mammalian models and humans. JMJD2A is a transcriptional co-factor and enzyme that catalyzes the demethylation of histone H3 lysine 9 and 36 (H3K9 and H3K36). Here in this study, we reported the role of JMJD2A in human glioma. Methods: Quantitative real-time PCR and western blot were performed to analyzed JMJD2A expression in glioma. Log-rank was performed to plot the survival curve. JMJD2A was knocked or overexpressed with lentivirus. Cell proliferation and colony formation were performed to assess the effects of JMJD2A on glioma cell growth. Xenograft experiment was performed the evaluate the growth rate of glioma cells in vivo. The signaling pathway was analyzed with western blot and mTOR was inhibited with rapamycin. Results: Quantitative real-time PCR and western blot experiments revealed higher expression of JMJD2A and lower levels of H3K9me3/H3K36me3 in glioma tissues than that in normal brain tissues. We showed that knockdown of JMJD2A expression attenuated the growth and colony formation in three lines of glioma cells (U251, T98G, and U87MG), whereas JMJD2A overexpression resulted in opposing effects. Furthermore, we performed in vivo xenograft experiments and our data demonstrated that JMJD2A knockdown reduced the growth of glioma T98G cells in vivo. Further mechanism study implicated that JMJD2A activated the Akt-mTOR pathway and promoted protein synthesis in glioma cells via promoting phosphoinositide-dependent kinase-1 (PDK1) expression. The activation of the Akt-mTOR pathway was also validated in human glioma tissues. Finally, we showed that inhibition of mTOR with rapamycin blocked the effects of JMJD2A on protein synthesis, cell proliferation and colony formation of glioma cells. Conclusions: These findings demonstrated that JMJD2A regulated glioma growth and implicated that JMJD2A might be a promising target for intervention. [ABSTRACT FROM AUTHOR]

A large number of heterogeneous macrophages can be observed in solid tumor lesions. Classically activated M1 macrophages are a powerful killer of cancer cells. In contrast, tumor-associated macrophages (TAMs) are often referred to as M2 phenotype and usually impair tumor immunity mediated by cytotoxic lymphocytes, natural killer (NK) cells and CD8+ T cells. Therefore, orchestrating M2 to M1 reprogramming will provide a promising approach to tumor immunotherapy. Here we used a PyMT-induced spontaneous breast cancer model in which M2-polarized macrophages were abundant. This M2 phenotype was closely related to tumor progression and immune dysfunction of NK cells and CD8+ T cells. We then found that these TAMs showed increased energy expenditure and over-activation of two kinases, Akt and mammalian target of rapamycin (mTOR). Myeloid inactivation of phosphoinositide-dependent kinase-1 (PDK1), the upstream regulator for Akt and mTOR signaling, significantly reduced excessive metabolic activation of macrophages. Notably, the loss of PDK1 significantly led to regression of breast cancer and prevented lung metastasis. Mechanistically, PDK1 deficiency mainly inhibited the activation of mTOR complex 1 (mTORC1), transforming TAMs into M1 phenotype, thereby reversing tumor-related dysfunction of T cells and NK cells. Therefore, targeting PDK1 may be a new approach for M2 macrophage-enriched solid tumor immunotherapy. [ABSTRACT FROM AUTHOR]

Phosphoinositide-dependent protein kinase-1 (PDK1) is an important protein in mediating the PI3K-AKT pathway and is thus identified as a promising target. The catalytic activity of PDK1 is tightly regulated by allosteric modulators, which bind to the PDK1 Interacting Fragment (PIF) pocket of the kinase domain that is topographically distinct from the orthosteric, ATP binding site. Allosteric modulators by attaching to the less conserved PIF-pocket have remarkable advantages such as higher selectivity, less side effect, and lower toxicity. Targeting allosteric PIF-pocket of PDK1 has become the focus of recent attention. In this review, we summarise the current advances in the structure-based discovery of PDK1 allosteric modulators. We will first present the three-dimensional structure of PDK1 and illustrate the allosteric regulatory mechanism of PDK1 through the modulation of the PIF-pocket. Then, the recent advances of PDK1 allosteric modulators targeting the PIF-pocket will be recapitulated detailly according to the structural similarity of allosteric modulators. [ABSTRACT FROM AUTHOR]

The patent describes novel useful compounds, such as PI3K protein kinase inhibitors, in particular as PI3K delta (δ) and/or gamma (γ) protein kinase modulators. The present disclosure also provides methods for preparing PI3K protein kinase inhibitors, pharmaceutical compositions containing them, and methods of treatment, prevention, and amelioration of PI3K kinase-mediated diseases, and disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Metabolic remodeling plays an essential role in the pathophysiology of heart failure (HF). Many studies have shown that the disruption of phosphoinositide-dependent protein kinase-1 (PDK1) caused severe and lethal HF; however, the metabolic pattern of PDK1 deletion remains ambiguous. 1H nuclear magnetic resonance-based metabolomics was applied to explore the altered metabolic pattern in Pdk1-deficient mice. Principle component analysis showed significant separation as early as 4 weeks of age, and dysfunction of metabolism precedes a morphological change in Pdk1-deficient mice. A time trajectory plot indicated that disturbed metabolic patterns were related to the pathological process of the HF in Pdk1-deficient mice, rather than the age of mice. Metabolic profiles demonstrated significantly increased levels of acetate, glutamate, glutamine, and O-phosphocholine in Pdk1 deletion mice. Levels of lactate, alanine, glycine, taurine, choline, fumarate, IMP, AMP, and ATP were significantly decreased compared with controls. Furthermore, PDK1 knockdown decreased the oxygen consumption rate in H9C2 cells as determined using a Seahorse XF96 Analyzer. These findings imply that the disruption of metabolism and impaired mitochondrial activity might be involved in the pathogenesis of HF with PDK1 deletion. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effect of phosphoinositide-dependent protein kinase-1 (PDK1) on hypoxia-induced pulmonary arterial hypertension (PAH). A mouse model of hypoxia-induced PAH was generated using normal or PDK1-knockout mice. Histological analysis and hemodynamic evaluations were performed to identify the progression of PAH. The expression and phosphorylation of PDK1/protein kinase B (Akt) signaling pathway associated proteins were detected by western blot analysis. Increased lung vessel thickness, right ventricular (RV) systolic pressure (RVSP), RV hypertrophy index (RVHI) values [the RV weight-to-left ventricular (LV) plus septum (S) weight ratio] and PDK1 expression were observed in the hypoxia-induced PAH model compared with the normal control. The phosphorylation of AktT308, proline-rich Akt1 substrate 1 (PRAS40) and S6KT229 was also notably increased in the PAH model compared with the control. The changes of proteins were not observed in the hypoxia treated PDK1flox/+: Tie2-Cre mice. Similarly, the RVSP and RVHI values, and PDK1 expression were reduced in the hypoxia treated PDK1flox/+: Tie2-Cre mice to a level comparable with those in the control, suggesting that PDK1 partial knockout significantly limited hypoxia-induced PAH. The results of the present study indicate that PDK1 is essential for hypoxia-induced PAH through the PDK1/Akt/S6K signaling cascades. [ABSTRACT FROM AUTHOR]

A correction to the article "The AGC Protein Kinase UNICORN Controls Planar Growth by Attenuating PDK1 in Arabidopsis thaliana" by Sebastian Scholz and colleagues is presented.

The role of ginsenoside in the prevention of cancer has been well established. Ginsenoside Rg5 is one of the main components isolated from red ginseng, which has been demonstrated to have anti-tumor effects by inhibiting cell proliferation and causing DNA damage. However, the role of ginsenoside Rg5 and its molecular mechanisms remain unclear in human esophageal cancer. In the present study, Rg5 was investigated as a novel drug for the chemotherapy of esophageal cancer in in vitro experiments. Esophageal cancer Eca109 cells were exposed to various concentrations of ginsenoside Rg5 (0–32 µΜ) for 24 h. Subsequent cell proliferation assays demonstrated that treatment with ginsenoside Rg5 resulted in the dose-dependent inhibition of proliferation, while a significant increase in apoptotic rate and increased activities of caspase-3, −8 and −9 were observed. In addition, the mitochondrial membrane potential was decreased and the cytoplasmic free calcium level increased following treatment with ginsenoside Rg5. Furthermore, the expression of B-cell lymphoma 2 and phosphorylated-protein kinase B (p-Akt) decreased. The specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002 promoted this effect, while insulin-like growth factor-1, a specific PI3K activator, inhibited this action. Taken together, the results suggested that ginsenoside Rg5 may have a tumor-suppressive effect on esophageal cancer by promoting apoptosis and may be associated with the downregulation of the PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract As the most commonly occurring form of primary renal tumor, renal cell carcinoma (RCC) is a malignancy accompanied by a high mortality rate. 3-phosphoinositide-dependent protein kinase 1 (PDK1) has been established as a protein target and generated considerable interest in both the pharmaceutical and academia industry. The aim of the current study was to investigate the effect of si-PDK1 on the RCC cell apoptosis, proliferation, migration, invasion and epithelial mesenchymal transition (EMT) in connection with the PI3K-PDK1-Akt pathway. Microarray analysis from the GEO database was adopted to identify differentially expressed genes (DEGs) related to RCC, after which the positive expression of the PDK1 protein in tissue was determined accordingly. The optimal silencing si-RNA was subsequently selected and RCC cell lines 786-O and A498 were selected and transfected with either a si-PDK1 or activator of the PI3K-PDK1-Akt pathway for grouping purposes. The mRNA and protein expressions of PDK1, the PI3K-PDK1-Akt pathway-, EMT- and apoptosis-related genes were then evaluated. The effect of si-PDK1 on cell proliferation, apoptosis, invasion and migration was then analyzed. Through microarray analysis of GSE6344, GSE53757, GSE14762 and GSE781, PDK1 was examined. PDK1 was determined to be highly expressed in RCC tissues. Si-PDK1 exhibited marked reductions in relation to the mRNA and protein expression of PDK1, PI3K, AKT as well as Vimentin while elevated mRNA and protein expressions of E-cadherin were detected, which ultimately suggested that cell migration, proliferation and invasion had been inhibited coupled with enhanced levels of cell apoptosis. While a notable observation was made highlighting that the PI3K-PDK1-Akt pathway antagonized the effect of PDK1 silencing. Taken together, the key observations of this study provide evidence suggesting that high expressions of PDK1 are found in RCC, while highlighting that silencing PDK1 could inhibit RCC cell proliferation, migration, invasion and EMT by repressing the PI3K-PDK1-Akt pathway. Highlights • Si-PDK1 regulates RCC cells through PI3K-PDK1-Akt pathway. • PDK1 is high expressed in RCC cells, and PI3K-PDK1-Akt pathway was activated. • Si-PDK1 inhibits PI3K-PDK1-Akt pathway by suppressing p PI3K and p-AKT. • Si-PDK1 suppresses EMT by down-regulating E-cadherin and up-regulating vimentin. • PDK1 can be a biomarker for clinical diagnosis of RCC. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR) signaling strength is a dominant factor regulating T-cell differentiation, thymic development, and cytokine signaling. The molecular mechanisms by which TCR signal strength is transduced to downstream signaling networks remains ill-defined. Using computational modeling, biochemical assays, and imaging flow cytometry, we found here that TCR signal strength differentially generates phosphatidylinositol species. Weak TCR signals generated elevated phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and reduced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, whereas strong TCR signals reduced PI(4,5)P2 and elevated PIP3 levels. A proteomics screen revealed that focal adhesion kinase bound PI(4,5)P2, biochemical assays disclosed that focal adhesion kinase is preferentially activated by weak TCR signals and is required for optimal Treg induction, and further biochemical experiments revealed how TCR signaling strength regulates AKT activation. Low PIP3 levels generated by weak TCR signals were sufficient to activate phosphoinositide-dependent kinase-1 to phosphorylate AKT on Thr-308 but insufficient to activate mTOR complex 2 (mTORC2), whereas elevated PIP3 levels generated by a strong TCR signal were required to activate mTORC2 to phosphorylate Ser-473 on AKT. Our results provide support for a model that links TCR signaling to mTORC2 activation via phosphoinositide 3-kinase signaling. Together, the findings in this work establish that T cells measure TCR signal strength by generating different levels of phosphatidylinositol species that engage alternate signaling networks to control cell fate decisions. [ABSTRACT FROM AUTHOR]

Summary: Diffuse large B‐cell lymphoma (DLBCL) is the most common subtype of non‐Hodgkin lymphoma. Because the prognosis of DLBCL patients varies considerably, there is an urgent need to identify novel prognostic factors. In this study, we investigated the expression levels of the signalling enzyme 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1), the cell cycle regulatory enzyme Polo‐like kinase 1 (PLK1) and the transcription factor (c‐Myc) in DLBCL tissues and evaluated their clinical and prognostic significance. PDK1, PLK1 and c‐Myc were detected by immunohistochemical staining of paraffin‐embedded specimens from 152 DLBCL and 48 lymphadenitis patients. Expression levels were correlated with clinicopathological factors. PDK1, PLK1 and c‐Myc were more commonly expressed in DLBCL specimens than in lymphadenitis specimens, and the expression of each protein correlated positively with that of the other two molecules. High PDK1, PLK1 and c‐Myc expression, high international prognostic index score, high lactate dehydrogenase levels and late Ann Arbor stage were shown to correlate with shorter overall survival time. A multivariate Cox regression model showed that high expression levels of PLK1 and c‐Myc were independent prognostic factors for DLBCL. Our findings indicate that PLK1 and c‐Myc expression might be promising predictive biomarkers for DLBCL patients. [ABSTRACT FROM AUTHOR]

Graphical abstract The virtual screening, molecular docking, ADMET prediction and molecular dynamics simulations were performed on Asinex database to find the novel compounds against PI3Kδ, which were demonstrated as the following flow chart: Highlights • Phosphoinositide-3-kinase Delta (PI3Kδ) is a well-validated target for anticancer drug design and development. • Structure-based virtual screening of ∼300,000 druglike compounds retrieved from Asinex Gold-Platinum database. • A total of 3 potential PI3Kδ-selective inhibitors were identified. • Molecular dynamics simulation of their complexes with PI3Kδ suggested their stable binding mode over time. Abstract Background Phosphoinositide-3-kinase Delta (PI3Kδ) plays a key role in B-cell signal transduction and inhibition of PI3Kδ is confirmed to have clinical benefit in certain types of activation of B-cell malignancies. Virtual screening techniques have been used to discover new molecules for developing novel PI3Kδ inhibitors with little side effects. Method Computer aided drug design method were used to rapidly screen optimal PI3Kδ inhibitors from the Asinex database. Virtual screening based molecular docking was performed to find novel and potential lead compound targeting PI3Kδ, at first. Subsequently, drug likeness studies were carried out on the retrieved hits to evaluate and analyze their drug like properties such as absorption, distribution, metabolism, excretion, and toxicity (ADMET) for toxicity prediction. Three least toxic compounds were selected for the molecular dynamics (MD) simulations for 30 ns in order to validate its stability inside the active site of PI3Kδ receptor. Results Based on the present in silico analysis, two molecules have been identified which occupied the same binding pocket confirming the selection of active site. ASN 16296138 (Glide score: −12.175 kcal/mol, cdocker binding energy: −42.975 kcal/mol and ΔG bind value: −90.457 kcal/mol) and BAS 00227397 (Glide score: −10.988 kcal/mol, cdocker binding energy: −39.3376 kcal/mol and ΔG bind value: −81.953 kcal/mol) showed docking affinities comparatively much stronger than those of already reported known inhibitors against PI3Kδ. These two ligand's behaviors also showed consistency during the simulation of protein-ligand complexes for 30000 ps respectively, which is indicative of its stability in the receptor pocket. Conclusion Compound ASN 16296138 and BAS 00227397 are potential candidates for experimental validation of biological activity against PI3Kδ in future drug discovery studies. This study smoothes the path for the development of novel leads with improved binding properties, high drug likeness, and low toxicity to humans for the treatment of cancer. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LAD) is one of the most common malignancies that threats human health worldwide. Long non-coding RNAs (lncRNAs) have been reported to play significant roles in tumorigenesis and might be novel biomarkers and targets for diagnosis and treatment of cancers. TP73-AS1 is a newly discovered lncRNA involved in the tumorigenesis and development of several cancers. However, its role in LAD has not been investigated yet. In the present study, we first found that TP73-AS1 expression was markedly increased in LAD tissues and cell lines and its overexpression was strongly associated with poor clinical outcomes. Then the loss/gain-of-function assays elucidated that TP73-AS1 contributed to cell proliferation, migration, and invasion in vitro, and the in vivo experiments illustrated that its knockdown inhibited tumor growth and metastasis. What was more, we discovered that phosphoinositide 3-kinase and AKT (PI3K/AKT) pathway was activated both in LAD tissues and cell lines but inactivated under TP73-AS1 silence. Moreover, the activation of this pathway could rescue the inhibitory effects of TP73-AS1 suppression on LAD cellular processes partially. These data suggested that TP73-AS1 served as an oncogene in LAD partially through activating PI3K/AKT pathway and it could be a potential target for diagnosis and treatment of LAD. [ABSTRACT FROM AUTHOR]

Abstract Phosphoinositide 3-kinase (PI3K)/Akt plays a pivotal role in the vascular response. The present study is to determine whether PI3K/Akt pathway in vascular smooth muscle cells is involved in nitroglycerin (NTG) tolerance and the underlying mechanism. Nitrate tolerance of porcine coronary arteries in vitro was induced by incubation of NTG (10−5 M) for 24 h. Nitrate tolerance in vivo was obtained by subcutaneous injection of mice with NTG (20 mg kg−1, tid, 3 days) and the aortas were used. Protein levels of total and phosphorylated Akt, forkhead box protein O1 (FoxO1), and cGMP-dependent protein kinase (PKG) were determined by western blot analysis. Isometric vessel tension was recorded by organ chamber technique. PKG mRNA was determined by real-time PCR. The cellular translocation of FoxO1 was observed by immunofluorescence. Reactive oxygen species (ROS) level was measured by DHE staining. The vascular relaxation to NTG was significantly inhibited in in vivo and in vitro NTG tolerant arteries. Meanwhile, the protein level of phosphorylated Akt at Ser473 was increased in the tolerant arteries. The attenuated relaxation and the augmented Akt-p were ameliorated by LY294002, a specific inhibitor of PI3K. The protein and mRNA expression of PKG were significantly down-regulated in NTG tolerant arteries, which were reversed by LY294002. The level of phosphorylated FoxO1 at Ser256 and its translocation from the nucleus to the cytosol were both increased in NTG tolerance and were also inhibited by LY294002. ROS production was significantly increased in NTG tolerant arteries, which was not be affected by LY294002 but inhibited by N-acetyl-L-cysteine. In conclusion, the present study suggests that PI3K/Akt in vascular smooth muscle is involved in the development of NTG tolerance via inhibiting PKG transcription and the effect is mediated by FoxO1. Highlights • Both in vitro and in vivo conditions, the activation of PI3K/Akt contributes to the development of NTG tolerance. • PI3K/Akt is involved in the NTG tolerance mainly by inhibiting PKG transcription via suppressing FoxO1 transcriptional activity. • The role of PI3K/Akt in the NTG tolerance is unlikely related to ROS production. [ABSTRACT FROM AUTHOR]

Abstract Phosphoinositide-3-kinase-δ (PI3Kδ) is a key regulator in the process of IgE mediated mast cell degranulation, which directly induces allergic diseases, such as asthma. This study is aimed at discovery of natural PI3Kδ inhibitors from Chinese medicine and evaluating their anti-mast cell degranulation activity. A combined virtual screening based on 3D pharmacophore model and molecular docking was used to screen for bioactive ingredients directly targeting PI3Kδ. Then, an in vitro kinase inhibition assay was conducted to evaluate the PI3Kδ inhibitory activity of the virtual screening hits. Subsequently, a β-hexosaminidase release assay was performed to verify the anti-mast cell degranulation activity of the active compounds. Finally, ginkgoneolic acid was identified as a PI3Kδ inhibitor (IC 50 = 2.49 μM) and exhibited anti-mast cell degranulation activity in vitro (IC 50 = 2.40 μM). Docking studies showed that Glu826, Val827 and Val828 were key amino acid residues for PI3Kδ inhibitory activity. Ginkgoneolic acid may be a potential lead compound for developing effective and safe PI3Kδ-inhibiting drugs. Graphical abstract Image 1 Highlights • Ginkgoneolic acid derived from TCM was identified as a PI3Kδ inhibitor with IC 50 of 2.49μM. • Ginkgoneolic acid significantly inhibited degranulation of mast cellswith an IC 50 of 2.40μM. • The inhibition of ginkgoneolic acid on PI3Kδ was revealed by interacting with residues such as Glu826, Val827 and Val828. [ABSTRACT FROM AUTHOR]

Background/Aims: Increasing wall stress or biomechanical stretch experienced by arteries influences the initiation of atherosclerotic lesions. This initiation is mediated by Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which are both effectors of the Hippo pathway. In this study, the functional roles of YAP/TAZ proteins in the regulation of the stretch-mediated programing of human umbilical arterial smooth muscle cells (HUASMCs) to a proliferative phenotype were examined. Methods: HUASMCs were seeded on a Matrigel-coated silicone chamber and subjected to biomechanical stretch for 24 h after 48 h of growth. YAP/TAZ small interfering RNA was used to specifically knockdown YAP/ TAZ expression in HUASMCs. Results: We observed that YAP/TAZ activation via biomechanical stretching is involved in the regulation of critical aspects of the HUASMC phenotypic switch. YAP/TAZ knockdown significantly attenuated the stretch-induced proliferative and pro-inflammatory phenotypes in HUASMCs. Furthermore, treatment with atorvastatin, an anti-atherosclerotic drug, attenuated the stretch-induced phenotypic switch of HUASMCs from the contractile to synthetic state by suppressing YAP/TAZ expression. Additional investigations demonstrated the role of stretch in inhibiting the Hippo pathway, leading to the activation of PI3-kinase (PI3K) and phosphoinositide dependent kinase (PDK1); the key molecule for the regulation of the PDK1 and Hippo complex interaction was Sav1. These results showed the importance of YAP/TAZ activation, induced by biomechanical stretch, in promoting atheroprone phenotypes in HUASMCs. Conclusion: Taken together, our findings revealed a mechanism by which YAP/TAZ activation contributes to the pathogenesis of atherosclerosis. [ABSTRACT FROM AUTHOR]

Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, thePDK1knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation. [ABSTRACT FROM AUTHOR]

Summary: Although low‐molecular‐mass hyaluronan (LMMHA) has been implicated in pulmonary inflammatory diseases, the signalling pathway of LMMHA (200 000 molecular weight) that initiates the inflammatory response in lung is still unknown. In this study, we evaluate the role of phosphoinositide 3‐kinase (PI3K) and its downstream signalling pathway in LMMHA‐induced lung inflammatory responses. Our results indicate that pharmacological inhibition of PI3K or genetic deletion of Akt1 enhances neutrophil apoptosis, attenuates neutrophil influx into the lungs of mice and diminishes the expression of pro‐inflammatory factors such as interleukin‐6, keratinocyte cell‐derived chemokine and pro‐matrix metalloproteinase‐9 in bronchoalveolar lavage fluid after intratracheal administration of LMMHA. More importantly, we found that PI3K/Akt1 participates in LMMHA‐induced inflammatory responses, which are mainly mediated by the myeloid leukaemia cell differentiation protein (Mcl‐1). Our study suggests that LMMHA induced significantly increased levels of inflammatory factors in bronchoalveolar lavage fluid and activation of the PI3K/Akt1 pathway, which up‐regulates the expression of the anti‐apoptotic protein Mcl‐1 and inhibits the activation of caspase‐3, thereby suppressing neutrophil apoptosis to trigger lung inflammation. These findings reveal a novel molecular mechanism underlying sterile inflammation and provides a new potential target for the treatment of pulmonary disease. Low‐molecular‐mass hyaluronan (LMMHA; 200 000 MW) is a critical signal in neutrophil activation and recruitment, leading to lung inflammation. Our study indicates that LMMHA induced activation of the PI3K/Akt1 pathway, which up‐regulates the expression of the anti‐apoptotic protein Mcl‐1 to delay lung neutrophil apoptosis. Hence, inhibition of the PI3K/Akt1 pathway exerts a protective role in LMMHA‐induced lung inflammation. [ABSTRACT FROM AUTHOR]

Background/Aims: We previously showed that the major bioactive compound of Atractylodes macrocephula Koidz atractylenolide 1 (ATL-1) inhibited human lung cancer cell growth by suppressing the gene expression of 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1). However, the potentially associated molecules and downstream effectors of PDK1 underlying this inhibition, particularly the mechanism for enhancing the anti-tumor effects of epidermal growth factor receptor-tyrosine-kinase inhibitors (EGFR-TKIs), remain unknown. Methods: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Western blot analyses were performed to examine the protein expressions of PDK1 and of zeste homolog 2 (EZH2). The levels of long non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) were examined via qRT-PCR. RNA-binding protein immunoprecipitation assays were used to analyze HOTAIR interaction with EZH2. The promoter activity of the EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. Exogenous expressions of PDK1, HOTAIR, and EZH2 were conducted via transient transfection assays. A xenografted tumor model was used to further evaluate the effect of ATL-1 in the presence or absence of erlotinib in vivo. Results: We showed that the combination of ATL-1 and EGFR-TKI erlotinib further inhibited growth and induced cell arrest of the human lung cancer cells, determined by both MTT and flow cytometry assays. ATL-1 inhibited the protein expression and the promoter activity of EZH2, which was reversed in cells with PDK1 overexpression. Interestingly, ATL-1 inhibited the expression levels of HOTAIR. While silencing HOTAIR inhibited the expressions of PDK1 and EZH2, overexpression of HOTAIR reduced the ATL-1-reduced PDK1 and EZH2 protein expressions and EZH2 promoter activity. In addition, ATL-1 reduced the HOTAIR binding to the EZH2 protein. Moreover, we found that exogenously expressed EZH2 antagonized the effect of ATL-1 on cell growth inhibition. Consistent with the in vitro results, ATL-1 inhibited tumor growth and the expression levels of HOTAIR, protein expressions of EZH2 and PDK1 in vivo. Importantly, there was synergy of the combination of ATL-1 and erlotinib in this process. Conclusion: Here, we provide the first evidence that ATL-1 inhibits lung cancer cell growth through inhibiting not only the PDK1 but also the lncRNA HOTAIR, which results in the reduction of one downstream effector EZH2 expression. The novel interplay between the HOTAIR and EZH2, as well as repressions of the PDK1 and HOTAIR coordinate the overall effects of ATL-1. Importantly, the combination of ATL-1 and EGFR-TKI erlotinib exhibits synergy. Thus, targeting the PDK1- and HOTAIR-mediated downstream molecule EZH2 by the combination of ATL-1 and erlotinib potentially facilitates the development of an additional novel strategy to combat lung cancer. [ABSTRACT FROM AUTHOR]

As professional antigen‐presenting cells, dendritic cells (DCs) initiate and regulate immune responses against inflammation. The invasion of pathogens into the body, however, can in turn cause the change of DCs in both activity and viability, which ultimately affect immune homeostasis. The exact mechanisms that the bacteria utilize to alter the lifespan of DCs, however, are far from clear. In this study, we found that the bacterial wall compound lipopolysaccharide (LPS) can promote the survival of GM‐CSF‐differentiated DCs (GM‐DCs). At molecular levels, we demonstrated that GM‐DCs had distinct pattern of mRNA expression for anti‐apoptotic BCL‐2 family members, of which, Bcl‐x increased significantly following LPS stimulation. Interestingly, specific inhibition of BCL‐XL protein alone was sufficient to remove the anti‐apoptotic effects of LPS on BM‐DCs. Further study of the signaling mechanisms revealed that although LPS can activate both Erk MAP kinase and PI3 kinase pathways, only blocking of PI3K abolished both Bcl‐x upregulation and the enhanced survival phenotype, suggesting that the PI3K signaling mediated the upregulation of Bcl‐x for the LPS‐induced pro‐survival in GM‐DCs. Collectively, this study unveils a molecular mechanism that DCs adapt to maintain innate immunity against the invasion of pathogens. PI3K, rather than MAP kinase pathway, controls Bcl‐x upregulation in LPS promoted DC survival. [ABSTRACT FROM AUTHOR]

Antibody production and function represent an essential part of the immune response, particularly in fighting bacterial and viral infections. Multiple immunological phenotypes can result in dysregulation of the immune system humoral compartment, including class-switch recombination (CSR) defects associated with hyper-IgM (HIGM) syndromes. The CSR/HIGM syndromes are defined by the presence of normal or elevated plasma IgM levels in the context of low levels of switched IgG, IgA, and IgE isotypes. Recently described autosomal dominant gain-of-function (GOF) mutations in PIK3CD and PIK3R1 cause combined immunodeficiencies that can also present as CSR/HIGM defects. These defects, their pathophysiology and derived clinical manifestations are described in depth. Previously reported forms of CSR/HIGM syndromes are briefly reviewed and compared to the phosphoinositide 3-kinase (PI3K) pathway defects. Diseases involving the PI3K pathway represent a distinctive subset of CSR/HIGM syndromes, presenting with their own characteristic clinical and laboratory attributes as well as individual therapeutic approaches. [ABSTRACT FROM AUTHOR]

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. [ABSTRACT FROM AUTHOR]

Leonurine is a compound derived from Herba leonuri, which has been reported to protect cardiac tissue against ischemic injury via antioxidant and anti‑apoptosis effects. The present study investigated whether these effects may be applied to acute myocardial infarction (MI) and examined the underlying mechanisms of leonurine treatment. A rat model of MI was induced by coronary artery ligation. Leonurine was administered at 15 mg/kg/day by oral gavage following the onset of MI. Rats in the sham group and the saline group were administered with an equal volume of saline. Echocardiography, Masson's trichrome staining, and terminal‑deoxynucleotidyl transferase‑mediated dUTP nick end labeling assays were performed 28 days post MI. The expression of B‑cell lymphoma‑2 and Bax were assessed by western blot analysis and reverse transcription‑quantitative polymerase chain reaction. Phosphoinositide 3‑kinase (PI3K), protein kinase B and glycogen synthase kinase‑3β (GSK3β) protein expression were investigated by western blot analysis. Leonurine significantly alleviated collagen deposition and MI size, inhibited cell apoptosis and improved myocardial function. This was accompanied by significantly increased levels of phosphorylated (p)‑PI3K, p‑AKT, p‑GSK3β and Bcl‑2, as well as significantly decreased levels of caspase3, cleaved‑caspase3 and Bax following MI. The results demonstrated that leonurine exerts potent cardio‑protective effects in a rat model of MI by inducing anti‑apoptotic effects by activating the PI3K/AKT/GSK3β signaling pathway. [ABSTRACT FROM AUTHOR]

In the present study, the effects and underlying mechanism of RbAp48 on the radiosensitivity of AGS gastric cancer cells was investigated. Cell proliferation was determined with an MTT assay. Flow cytometry was performed to evaluate the cell cycle and apoptosis. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were performed to detect mRNA and protein expression, respectively, including RbAp48, phosphoinositide 3‑kinase (PI3K) and protein kinase B (Akt). The results revealed that radiation enhanced the expression level of RbAp48 in AGS cells, and that RbAp48 combined with radiation reduced AGS cell proliferation. In addition, RbAp48 combined with radiation resulted in G2 phase arrest and induced apoptosis via regulation of the PI3K/Akt pathway. In conclusion, it was demonstrated that overexpression of RbAp48 enhanced the radiosensitivity of AGS gastric cancer cells via suppression of PI3K/Akt pathway activity, suggesting that RbAp48 may hold potential as a gene therapeutic strategy in the future, aiding in the treatment of gastric cancer. [ABSTRACT FROM AUTHOR]

Abstract Contribution to Special Issue on Fast effects of steroids. Estrogens affect learning and memory through rapid and delayed mechanisms. Here we review studies on rapid effects on short-term memory. Estradiol rapidly improves social and object recognition memory, spatial memory, and social learning when administered systemically. The dorsal hippocampus mediates estrogen rapid facilitation of object, social and spatial short-term memory. The medial amygdala mediates rapid facilitation of social recognition. The three estrogen receptors, α (ERα), β (ERβ) and the G-protein coupled estrogen receptor (GPER) appear to play different roles depending on the task and brain region. Both ERα and GPER agonists rapidly facilitate short-term social and object recognition and spatial memory when administered systemically or into the dorsal hippocampus and facilitate social recognition in the medial amygdala. Conversely, only GPER can facilitate social learning after systemic treatment and an ERβ agonist only rapidly improved short-term spatial memory when given systemically or into the hippocampus, but also facilitates social recognition in the medial amygdala. Investigations into the mechanisms behind estrogens' rapid effects on short term memory showed an involvement of the extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K) kinase pathways. Recent evidence also showed that estrogens interact with the neuropeptide oxytocin in rapidly facilitating social recognition. Estrogens can increase the production and/or release of oxytocin and other neurotransmitters, such as dopamine and acetylcholine. Therefore, it is possible that estrogens' rapid effects on short-term memory may occur through the regulation of various neurotransmitters, although more research is need on these interactions as well as the mechanisms of estrogens' actions on short-term memory. Highlights • Estrogens rapidly facilitate social learning, recognition, spatial short-term memory. • Dorsal hippocampus and medial amygdala mediate rapid estrogen short-term memory effects. • ERK/PI3K kinase pathways mediate rapid estrogenic effects on short-term memory. • Estrogens interact with oxytocin to rapidly facilitate social recognition. • Estrogens might also interact with dopamine or acetylcholine to exert rapid effects. [ABSTRACT FROM AUTHOR]

Background: Label-retaining cancer cells (LRCC) have been proposed as a model of slowly cycling cancer stem cells (CSC) which mediate resistance to chemotherapy, tumor recurrence, and metastasis. The molecular mechanisms of chemoresistance in LRCC remain to-date incompletely understood. This study aims to identify molecular targets in LRCC that can be exploited to overcome resistance to gemcitabine, a standard chemotherapy agent for the treatment of pancreas cancer.Methods: LRCC were isolated following Cy5-dUTP staining by flow cytometry from pancreatic cancer cell lines. Gene expression profiles obtained from LRCC, non-LRCC (NLRCC), and bulk tumor cells were used to generate differentially regulated pathway networks. Loss of upregulated targets in LRCC on gemcitabine sensitivity was assessed via RNAi experiments and pharmacological inhibition. Expression patterns of PDPK1, one of the upregulated targets in LRCC, was studied in patients' tumor samples and correlated with pathological variables and clinical outcome.Results: LRCC are significantly more resistant to gemcitabine than the bulk tumor cell population. Non-canonical EGF (epidermal growth factor)-mediated signal transduction emerged as the top upregulated network in LRCC compared to non-LRCC, and knock down of EGF signaling effectors PDPK1 (3-phosphoinositide dependent protein kinase-1), BMX (BMX non-receptor tyrosine kinase), and NTRK2 (neurotrophic receptor tyrosine kinase 2) or treatment with PDPK1 inhibitors increased growth inhibition and induction of apoptosis in response to gemcitabine. Knockdown of PDPK1 preferentially increased growth inhibition and reduced resistance to induction of apoptosis upon gemcitabine treatment in the LRCC vs non-LRCC population. These findings are accompanied by lower expression levels of PDPK1 in tumors compared to matched uninvolved pancreas in surgical resection specimens and a negative association of membranous localization on IHC with high nuclear grade (p < 0.01).Conclusion: Pancreatic cancer cell-derived LRCC are relatively resistant to gemcitabine and harbor a unique transcriptomic profile compared to bulk tumor cells. PDPK1, one of the members of an upregulated EGF-signaling network in LRCC, mediates resistance to gemcitabine, is found to be dysregulated in pancreas cancer specimens, and might be an attractive molecular target for combination therapy studies. [ABSTRACT FROM AUTHOR]

Sphingolipids such as ceramide are important constituents of cell membranes. The ceramide transfer protein (CERT) moves ceramide from the endoplasmic reticulum to the Golgi apparatus in a nonvesicular manner. Hyperphosphorylation of the serine-repeat motif (SRM) adjacent to the pleckstrin homology (PH) domain of CERT down-regulates the inter-organelle ceramide transport function of CERT. However, the mechanistic details of this down-regulation remain elusive. Using solution NMR and binding assays, we herein show that a hyperphosphorylation-mimetic CERT variant in which 10 serine/threonine residues of SRM had been replaced with glutamate residues (the 10E variant) displays an intramolecular interaction between SRM and positively charged regions of the PH domain, which are involved in the binding of this domain to phosphati-dylinositol 4-monophosphate (PI4P). Of note, the binding of the PH domain to PI4P-embedded membranes was attenuated by the SRM 10E substitutions in cell-free assays. Moreover, the 10E substitutions reduced the Golgi-targeting activity of the PH-SRM construct in living cells. These results indicate that hyperphosphorylated SRM directly interacts with the surface of the PH domain in an intramolecular manner, thereby decreasing the PI4P-binding activity of the PH domain. In light of these findings, we propose that the hyperphosphorylation of SRM may trigger the dissociation of CERT from the Golgi apparatus, resulting in a functionally less active conformation of CERT. [ABSTRACT FROM AUTHOR]

In the hypothalamic arcuate nucleus (ARC), orexigenic agouti-related peptide (AgRP) neurons regulate feeding behavior and energy homeostasis. The 3-phosphoinositide-dependent protein kinase-1 (PDK1) in AgRP neurons serves as a major signaling molecule for leptin and insulin, the hormones regulating feeding behavior, energy homeostasis and circulation. However, it is unclear whether PDK1 in AGRP neurons is also involved in regulation of blood pressure. This study explored it by generating and analyzing AgRP neuron-specific PDK1 knockout ( Agrp-Pdk1 flox/flox ) mice and effect of high salt diet on blood pressure in KO and WT mice was analyzed. Under high salt diet feeding, systolic blood pressure (SBP) of Agrp-Pdk1 flox/flox mice was significantly elevated compared to Agrp-Cre mice. When the high salt diet was switched to control low salt diet, SBP of Agrp-Pdk1 flox/flox mice returned to the basal level observed in Agrp-Cre mice within 1 week. In Agrp-Pdk1 flox/flox mice, urinary noradrenalin excretion and NUCB2 mRNA expression in hypothalamic paraventricular nucleus (PVN) were markedly upregulated. Moreover, silencing of NUCB2 in the PVN counteracted the rises in urinary noradrenalin excretions and SBP. These results demonstrate a novel role of PDK1 in AgRP neurons to counteract the high salt diet-induced hypertension by preventing hyperactivation of PVN nesfatin-1 neurons. [ABSTRACT FROM AUTHOR]

In this study, a novel series of 6-aryl substituted 4-pyrrolidineaminoquinazoline derivatives were designed and evaluated as potent PI3Kδ inhibitors. The preliminary SAR was established, and compounds 12d , 20a and 20c displayed leading potent PI3Kδ inhibition, with IC 50 values of 4.5, 2.7 and 3.1 nM, respectively, that were comparable to idelalisib (IC 50  = 2.7 nM). Moreover, these three compounds showed favorable PI3Kδ isoform selectivity over PI3Kα, PI3Kβ, and PI3Kγ, and showed distinct anti-proliferation profiles against four human B cell lines of Ramos, Raji, RPMI-8226 and SU-DHL-6. In addition, molecular docking simulation showed that several key hydrogen bonding interactions were formed for compounds 12d , 20a and 20c in the PI3Kδ pocket, which might explain their potent PI3Kδ inhibition. These results indicate the 6-aryl substituted 4-pyrrolidineaminoquinazolines were potent PI3Kδ inhibitors. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a fatal form of musculoskeletal tumor that commonly leads to pulmonary metastatic disease. Traditional therapies such as surgery and chemotherapy are not effective treatment modalities in certain patients with OS; therefore, identifying the molecular mechanism of OS is imperative for the development of novel therapeutics. Previous studies have reported that focal adhesion kinase (FAK) is associated with numerous types of human malignancies. Therefore, in order to investigate the biological function of FAK in OS, the present study examined the expression levels of FAK in OS cell lines, OS tissues and paired normal tissue specimens by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). FAK expression in vitro was blocked using small interfering RNA (siRNA) to observe the invasion, proliferation and apoptosis trends of OS cells. Phosphoinositide-dependent kinase-1 (PDK1), AKT and BRAF protein levels were also evaluated by western blotting to analyze the effects of FAK depletion on the AKT and mitogen-activated protein kinase (MAPK) signaling pathways. A significantly reduced level of FAK mRNA was identified in paired normal tissues compared with OS tissues and cell lines. The invasive capability and proliferative potential of OS cells were suppressed due to the transient in vitro transfection of FAK siRNA. It was also demonstrated that decreased FAK expression facilitated the apoptosis of OS cells, as demonstrated by flow cytometric and western blotting analyses. Decreased FAK expression resulted in the downregulation of phosphorylated (p)-AKT, p-PDK1 and p-BRAF protein levels. Higher FAK expression levels are positively associated with clinicopathological characteristics of advanced Enneking stages (P<0.001) and recurrence (P=0.041) in patients with OS. Collectively, these data demonstrated that FAK is an important diagnostic biomarker for OS, and FAK siRNA therapy may be a potentially promising approach for the treatment of OS. [ABSTRACT FROM AUTHOR]

Abstract: Cervical cancer continues to be among the most frequent gynaecologic cancers worldwide. The phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) pathway is constitutively activated in cervical cancer. Inositol polyphosphate 4‐phosphatase type II (INPP4B) is a phosphoinositide phosphatase and considered a negative regulatory factor of the PI3K/AKT pathway. INPP4B has diverse roles in various tumours, but its role in cervical cancer is largely unknown. In this study, we investigated the role of INPP4B in cervical cancer. Overexpression of INPP4B in HeLa, SiHa and C33a cells inhibited cell proliferation, metastasis and invasiveness in CCK‐8, colony formation, anchorage‐independent growth in soft agar and Transwell assay. INPP4B reduced the expression of some essential proteins in the PI3K/AKT/SGK3 pathway including p‐AKT, p‐SGK3, p‐mTOR, phospho‐p70S6K and PDK1. In addition, overexpression of INPP4B decreased xenograft tumour growth in nude mice. Loss of INPP4B protein expression was found in more than 60% of human cervical carcinoma samples. In conclusion, INPP4B impedes the proliferation and invasiveness of cervical cancer cells by inhibiting the activation of two downstream molecules of the PI3K pathway, AKT and SGK3. INPP4B acts as a tumour suppressor in cervical cancer cells. [ABSTRACT FROM AUTHOR]

Long non‑coding RNAs (lncRNAs) are defined as non‑coding transcripts (>200 nucleotides) that serve important roles in the proliferation and differentiation of stem cells. Hair follicle stem cells (HFTs) have multidirectional differentiation potential and are able to differentiate into skin, hair follicles and sebaceous glands, serving a role in skin wound healing. The aim of the present study was to analyze the regulatory role of lncRNA AK015322 (IncRNA5322) in HFTs and the potential mechanism of IncRNA5322‑mediated differentiation of HFTs. The results demonstrated that lncRNA5322 transfection promoted proliferation and differentiation in HFTs. It was identified that lncRNA5322 transfection upregulated the expression and phosphorylation of phosphoinositide 3‑kinase (PI3K) and protein kinase B (AKT) in HFTs. It was also observed that lncRNA5322 transfection upregulated microRNA (miR)‑21 and miR‑21 agonist (agomir‑21) eliminated lncRNA5322‑induced expression and phosphorylation of PI3K and AKT. The present study also demonstrated that agomir‑21 blocked IncRNA5322‑induced expression and phosphorylation of PI3K and AKT in HFTs. The results indicated that agomir‑21 transfection also suppressed the IncRNA5322‑induced proliferation and differentiation of HFTs. In conclusion, the results of the present study suggest that lncRNA5322 is able to promote the proliferation and differentiation of HFTs by targeting the miR‑21‑mediated PI3K‑AKT signaling pathway in HFTs. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV)-encoded X antigen (HBx) contributes to the development of hepatocellular carcinoma (HCC). Although HBx has been implicated in the progression of HCC, its precise function in HBV-associated HCC remains unclear. In the present study, HBx affected 3-phosphoinositide-dependent protein kinase-1 (PDK1) and with-no-lysine (K) kinase (WNK1) signaling, which was identified to be involved in the viability and metastasis of hepatic cells. The phosphorylation of WNK1 was decreased when the hepatic cells were treated with a PDK1 inhibitor. The inhibition of PDK1 activity inhibited the viability and migration of hepatic cells. To the best of our knowledge, the present study is the first to identify the activation of PDK1 in HCC tissues, confirmed using western blot analysis. PDK1-WNK1 signaling may be a potential therapeutic target in HBV-associated liver cancer. [ABSTRACT FROM AUTHOR]

The PI3K-AKT kinase signaling pathway is frequently deregulated in human cancers, particularly breast cancer, where amplification and somatic mutations of PIK3CA occur with high frequency in patients. Numerous small-molecule inhibitors targeting both PI3K and AKT are under clinical evaluation, but dose-limiting toxicities and the emergence of resistance limit therapeutic efficacy. Various resistance mechanisms to PI3K inhibitors have been identified, including de novo mutations, feedback activation of AKT, or cross-talk pathways. We found a previously unknown resistance mechanism to PI3K pathway inhibition that results in AKT rebound activation. In a subset of triple-negative breast cancer cell lines, treatment with a PI3K inhibitor or depletion of PIK3CA expression ultimately promoted AKT reactivation in a manner dependent on the E3 ubiquitin ligase Skp2, the kinases IGF-1R (insulin-like growth factor 1 receptor) and PDK-1 (phosphoinositide-dependent kinase-1), and the cell growth and metabolism-regulating complex mTORC2 (mechanistic target of rapamycin complex 2), but was independent of PI3K activity or PIP3 production. Resistance to PI3K inhibitors correlated with the increased abundance of Skp2, ubiquitylation of AKT, cell proliferation in culture, and xenograft tumor growth in mice. These findings reveal a ubiquitin signaling feedback mechanism by which PI3K inhibitor resistance may emerge in aggressive breast cancer cells. [ABSTRACT FROM AUTHOR]