phosphatidylinositol-3-OH Kinase Research Articles

PTEN signaling is required for the maintenance of spermatogonial stem cells in mouse, by regulating the expressions of PLZF and UTF1

BackgroundPten plays a crucial role in the stem cell maintenance in a few organs. Pten defect also causes the premature oocytes and ovary aging. We and other groups have found that the phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling regulates the proliferation and differentiation of spermatogonial stem cells (SSCs). PTEN functions as a negative regulator of the PI3K pathway. Thus, we thought that the fate of SSCs might be controlled by Pten.ResultsWe report that promyelocytic leukaemia zinc finger (PLZF) and undifferentiated embryonic cell transcription factor 1 (UTF1), both of which are germ cell-specific transcriptional factors, are regulated by Pten. Conditional deletion of Pten leads to reduction in PLZF expression but induction of UTF1, which is associated with SSCs depletion and infertility in males with age.ConclusionOur data demonstrate that Pten is required for the long-term maintenance of SSCs and precise regulation of spermatogenesis in mouse. The finding of a Pten-regulated GFRα1+/PLZF−/UTF1+ progenitor population provides a new insight into the precise mechanisms controlling SSC fate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0034-x) contains supplementary material, which is available to authorized users.

The rLrp of Mycobacterium tuberculosis inhibits proinflammatory cytokine production and downregulates APC function in mouse macrophages via a TLR2-mediated PI3K/Akt pathway activation-dependent mechanism.

We demonstrate that Mycobacterium tuberculosis recombinant leucine-responsive regulatory protein (rLrp) inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), interleukin-6, and interleukin-12 production and blocks the nuclear translocation of subunits of the nuclear-receptor transcription factor NF-κB (Nuclear factor-kappa B). Moreover, rLrp attenuated LPS-induced DNA binding and NF-κB transcriptional activity, which was accompanied by the degradation of inhibitory IκBα and a consequent decrease in the nuclear translocation of the NF-κB p65 subunit. RLrp interfered with the LPS-induced clustering of TNF receptor-associated factor 6 and with interleukin-1 receptor-associated kinase 1 binding to TAK1. Furthermore, rLrp did not attenuate proinflammatory cytokines or the expression of CD86 and major histocompatibility complex class-II induced by interferon-gamma in the macrophages of Toll-like receptor 2 deletion (TLR2-/-) mice and in protein kinase b (Akt)-depleted mouse cells, indicating that the inhibitory effects of rLrp were dependent on TLR2-mediated activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway. RLrp could also activate the PI3K/Akt pathway by stimulating the rapid phosphorylation of PI3K, Akt, and glycogen synthase kinase 3 beta in macrophages. In addition, 19 amino acid residues in the N-terminus of rLrp were determined to be important and required for the inhibitory effects mediated by TLR2. The inhibitory function of these 19 amino acids of rLrp raises the possibility that mimetic inhibitory peptides could be used to restrict innate immune responses in situations in which prolonged TLR signaling has deleterious effects. Our study offers new insight into the inhibitory mechanisms by which the TLR2-mediated PI3K/Akt pathway ensures the transient expression of potent inflammatory mediators.

Functional compensation between Myc and PI3K signaling supports self-renewal of embryonic stem cells.

c-Myc and phosphatidylinositol 3-OH kinase (PI3K) both participate in diverse cellular processes, including cell cycle control and tumorigenic transformation. They also contribute to preserving embryonic stem cell (ESC) characteristics. However, in spite of the vast knowledge, the molecular relationship between c-Myc and PI3K in ESCs is not known. Herein, we demonstrate that c-Myc and PI3K function cooperatively but independently to support ESC self-renewal when murine ESCs are cultured under conventional culture condition. Interestingly, culture of ESCs in 2i-condition including a GSK3β and MEK inhibitor renders both PI3K and Myc signaling dispensable for the maintenance of pluripotent properties. These results suggest that the requirement for an oncogenic proliferation-dependent mechanism sustained by Myc and PI3K is context dependent and that the 2i-condition liberates ESCs from the dependence of this mechanism.

Fragile histidine triad (FHIT) suppresses proliferation and promotes apoptosis in cholangiocarcinoma cells by blocking PI3K-Akt pathway.

Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3.

Lipopolysaccharide Enhances Wnt5a Expression through Toll-like Receptor 4, Myeloid Differentiating Factor 88, Phosphatidylinositol 3-OH Kinase/AKT and Nuclear Factor Kappa B Pathways in Human Dental Pulp Stem Cells

IntroductionLipopolysaccharide (LPS) has been implicated in mesenchymal stem cell differentiation processes. Wnt5a, one of the “non-canonical” Wnt family members, is important in signaling stem cell differentiation and in the inflammatory responses of immune cells. Here we studied whether LPS can regulate the expression of Wnt5a in human dental pulp stem cells (hDPSCs) and investigated the intracellular signaling pathways activated by LPS. MethodsWnt5a mRNA and protein expression changes in hDPSCs were investigated by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay. In addition, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase activity assays were used to determine whether toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), nuclear factor kappa B (NF-kB), or the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathways are involved in LPS-induced Wnt5a expression. The activation of PI3K and AKT in hDPSCs was measured by Western blot analysis. ResultsWnt5a mRNA and protein expression was rapidly increased in response to LPS in a time- and dose-dependent manner. LPS-induced Wnt5a expression was effectively attenuated by administration of a TLR4 neutralizing antibody, MyD88 inhibitory peptide, PI3-kinase inhibitors (LY294002 and wortmannin), an AKT inhibitor, or NF-κB inhibitor (pyrrolidine dithiocarbamate), IκBa phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). Treatment of hDPSCs with LPS activated PI3-kinase (p85) and AKT signaling in a time-dependent manner. Moreover, LPS-mediated increases in κB-luciferase activity were diminished by the overexpression of dominant negative mutants of TLR4, MyD88, p85, AKT, and IκBa. ConclusionsThese results demonstrated that LPS-induced Wnt5a expression was mediated through the TLR4/MyD88/PI3-kinase/AKT pathway, which then initiated NF-κB activation in hDPSCs.

Erlotinib resistance in EGFR-amplified glioblastoma cells is associated with upregulation of EGFRvIII and PI3Kp110δ.

The treatment efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors like erlotinib has not met expectations for glioblastoma therapy, even for EGFR-overexpressing tumors. We determined possible mechanisms of therapy resistance using the unique BS153 glioblastoma cell line, which has retained amplification of the egfr gene and expression of EGFR variant (v)III. Functional effects of erlotinib, gefitinib, and cetuximab on BS153 proliferation, migration, and EGFR-dependent signal transduction were systematically compared in vitro. The tumor-initiating capacity of parental and treatment-resistant BS153 was studied in Naval Medical Research Institute/Foxn1(nu) mice. Potential mediators of resistance were knocked down using small interfering (si)RNA. Erlotinib and gefitinib inhibited proliferation and migration of BS153 in a dose-dependent manner, whereas cetuximab had no effect. BS153 developed resistance to erlotinib (BS153(resE)) but not to gefitinib. Resistance was associated with strong upregulation of EGFRvIII and subsequent activation of the phosphatidylinositol-3-OH kinase (PI3K) pathway in BS153(resE) and an increased expression of the regulatory 110-kDa delta subunit of PI3K (p110δ). Knockdown of EGFRvIII in BS153(resE) largely restored sensitivity to erlotinib. Targeting PI3K pharmacologically caused a significant decrease in cell viability, and specifically targeting p110δ by siRNA partially restored erlotinib sensitivity in BS153(resE). In vivo, BS153 formed highly invasive tumors with an unusual growth pattern, displaying numerous satellites distant from the initial injection site. Erlotinib resistance led to delayed onset of tumor growth as well as prolonged overall survival of mice without changing tumor morphology. EGFRvIII can mediate resistance to erlotinib in EGFR-amplified glioblastoma via an increase in PI3Kp110δ. Interfering with PI3Kp110δ can restore sensitivity toward the tyrosine kinase inhibitor.

Phase II study of PX-866 in recurrent glioblastoma.

2053 Background: Glioblastoma (GBM) is the most aggressive malignancy of the central nervous system. The majority have genetic changes that increase the activity of the phosphatidylinositol-3-OH kinase (PI3K) signal transduction pathway, critical for cell motility, proliferation, and survival. We present the results of PX-866, an oral PI3K inhibitor, in patients (pts) with recurrent GBM. Methods: A multinomial design of response and early progression (< 8 weeks on study) was used. In stage 1 (15 pts), 0 responses and ≥ 10 early progressions would stop accrual; after full accrual, ≥ 4 responses OR ≤ 13 early progressions was prespecified as of interest. Pts with histologically confirmed GBM, at first recurrence after chemoradiation and adjuvant temozolomide were given PX-866 8 mg daily on this single-arm phase II study. MRI and clinical exam were done every cycle (8 weeks). Tumour tissue was collected for analysis of potential markers of PI3K inhibitory activity (PTEN, EGFRviii, PIK3CA mutations). Results: A total of 33 pts were enrolled, eligible and evaluable. Median age was 56 (range 35-78), 12 were female; 29 had performance status (PS) 0-1 and 4 had PS 2. Median time from initial diagnosis to enrolment was 308 days (range 141-1256). Median number of cycles was 1 (range 1-7). Thirty-two pts have discontinued therapy, 26 due to disease/symptomatic progression and 6 due to toxicity (5 LFT elevation and 1 allergic reaction). Other adverse effects (AE): fatigue (16 pts/2 grade 3), diarrhea (11 pts/5 grade 3), nausea (19 pts/1 grade 3), vomiting (11 pts/1 grade 3) and lymphopenia (29 pts/7 grade 3/4). Five pts had related serious AEs (1 LFTs, 1 GI and 3 venous thromboembolism) All pts were evaluable for response; 25 had a best response of progression, 1 had partial response (overall response rate 3%) and seven (21%) had stable disease (SD, median 7.3 months; range 3.1-13.6). Six month PFS was 17%. In preliminary analyses, no statistical association was found between SD and PTEN or EGFRviii status (results pending in 16 pts). Conclusions: PX-866 was relatively well tolerated. Overall response rate was low, and the study did not meet its primary endpoint; however, 21% of pts obtained durable stable disease. Further correlative work is required to identify the predictor of this effect. Clinical trial information: NCT01259869.

Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy

Autophagy is a stress-induced catabolic process in which cytoplasmic components, sequestered in double-membrane autophagic vesicles (AVs) or autophagosomes, are delivered to lysosomes for degradation and recycling [Kroemer G, Mariño G, Levine B (2010) Mol Cell 40(2):280-293]. Activity of the class III phosphatidylinositol-3-OH-kinase (PI3K) vacuolar protein-sorting (Vps) 34, bound to coiled-coil moesin-like B-cell lymphoma 2 (Bcl-2)-interacting protein Beclin-1, is required for phosphoinositide generation, essential for AV formation in autophagy [Cuervo AM (2010) Nat Cell Biol 12(8):735-737]. However, how autophagy-inducing stress regulates Vps34 activity has not been fully elucidated. Our findings demonstrate that autophagy-inducing stress increases intracellular levels of acetylated inducible heat shock protein (hsp) 70, which binds to the Beclin-1-Vps34 complex. Acetylated hsp70 also recruits E3 ligase for SUMOylation, KRAB-ZFP-associated protein 1 (KAP1), inducing Lys840 SUMOylation and increasing Vps34 activity bound to Beclin 1. Knockdown of hsp70 abolished the Beclin-1-Vps34 complex formation, as well as inhibited KAP1 binding to Vps34 and AV formation. Notably, autophagy-inducing stress due to histone deacetylase inhibitor treatment induced AV formation in the wild-type but not hsp70.1/3 knockout mouse embryonic fibroblasts MEFs. These findings highlight a regulatory mechanism of Vps34 activity, which involves acetylated hsp70 and KAP1-dependent SUMOylation of Vps34 bound to Beclin 1.

Klebsiella pneumoniaetargets an EGF receptor-dependent pathway to subvert inflammation

The NF-κB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identified 17 kinases that when targeted by siRNA restored IL-1β-dependent NF-κB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)-phosphatidylinositol 3-OH kinase (PI3K)-AKT-PAK4-ERK-GSK3β signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-κB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR-PI3K-AKT-PAK4-ERK-GSK3β signalling pathway. Our efforts to identify the bacterial factor(s)responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence suggesting that CPS could mediate the activation of EGFR. Supporting this notion, purified CPS did activate EGFR as well as the EGFR-dependent PI3K-AKT-PAK4-ERK-GSK3β signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4-MyD88-c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Red wine consumption may affect sperm biology: The effects of different concentrations of the phytoestrogen Myricetin on human male gamete function

Myricetin is a natural flavonoid, particularly enriched in red wines, whose occurrence is widespread among plants. Despite extensive research, the beneficial effects of Myricetin on human health are still controversial. Here, we tested the estrogen-like effect of the phytoestrogen Myricetin on human ejaculated sperm biology. To this aim, human normozoospermic samples were exposed to increasing concentrations (10 nM, 100 nM, and 1 µM) of Myricetin. Motility, viability, capacitation-associated biochemical changes (i.e., cholesterol efflux and tyrosine phosphorylation), acrosin activity, as well as glucose utilization and fatty-acid oxidation (i.e., glucose and lipid metabolism) were all significantly increased by low doses of Myricetin. Importantly, both estrogen receptors α and β (ERs) and phosphatidylinositol-3-OH kinase (PI3K)/AKT signaling are activated in the presence of Myricetin since these were both abrogated by specific inhibitors of each pathway. Our results show how Myricetin, through ERs and PI3K/AKT signalings, potentiates sperm function. This effect is dose-dependent at low concentrations of Myricetin (up to 100 nM), whereas higher amounts do not seem to improve any further sperm motility, viability, or other tested features, and, in some cases, they reduced or even abrogated the efficacy exerted by lower doses. Further studies are needed to elucidate if high levels of Myricetin, which could be attained even with moderate wine consumption, could synergize with endogenous estrogens in the female reproductive tract, interfering with the physiological sperm fertilization process.

Synthesis and SAR study of modulators inhibiting tRXRα-dependent AKT activation

RXRα represents an intriguing and unique target for pharmacologic interventions. We recently showed that Sulindac and a designed analog could bind to RXRα and modulate its biological activity, including inhibition of the interaction of an N-terminally truncated RXRα (tRXRα) with the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K). Here we report the synthesis, testing and SAR of a series of novel analogs of Sulindac as potential modulators for inhibiting tRXRα-dependent AKT activation. A new compound 30 was identified to have improved biological activity.

Hydrogen Improves Glycemic Control in Type1 Diabetic Animal Model by Promoting Glucose Uptake into Skeletal Muscle

Hydrogen (H2) acts as a therapeutic antioxidant. However, there are few reports on H2 function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H2 in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H2 promoted 2-[14C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H2 significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H2 had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H2 exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

Effects and Mechanism of Organ Protection by Cardiotrophin-1

Cardiotrophin-1 (CT-1), a member of the interleukin (IL)-6 family, is reported to exhibit a plethora of pleiotropic effects in the heart such as cytoprotective, pro-proliferative and pro-fibrotic ones. An extensive research has been devoted on proliferative and profibrotic effects of CT-1 on the heart. Thus the present review has been aimed to critically define the cytoprotective effects of CT-1 and the cellular and molecular mechanisms involved in them. Although many effects of CT-1 have been described on the heart, CT-1 has now also been reported to exhibit important protective effects in other organs such as liver, kidney or nervous system. CT-1 produces its effects through a unique receptor system comprising LIF receptor (LIFRβ) and a common signal transducer, the glycoprotein 130 (gp130). The signaling pathway downstream from gp130 is based on at least, three distinct pathways: 1) the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, 2) the p42/44 mitogen-activated protein kinase (p42/44 MAPK) pathway, also known as the extracellular receptor kinase-1/2 (ERK1/2) pathway, and 3) the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway. Since CT-1 easily achieves its cytoprotective effects via a combination of the above three signaling pathways, it becomes quite necessary to determine which pathway(s) is involved in each particular effect of CT-1. In each of its target organs, CT-1 may also display differential mechanisms of cytoprotection, and thus it is relevant to understand how these mechanisms are locally regulated.

Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H2O2)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H2O2 to establish a model of H2O2 injury to cells. H2O2 was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H2O2-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H2O2-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins' expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H2O2 treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H2O2-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H2O2-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H2O2-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H2O2-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H2O2 injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H2O2-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.

The Role of the Insulin-like Growth Factor-1 Receptor (IGF-1R), Phosphatase and Tensin Homolog (PTEN), c-Met, and the PI3-Kinase Pathway in Colorectal Cancer

Colorectal cancer (CRC) is the second-leading cause of cancer deaths in the US and the third most common cancer malignancy annually. Overall prognosis of metastatic CRC is still poor, with five-year survival within 5–8 %. Previous molecular studies of CRC established several key events in CRC disease progression, including APC inactivation, β-catenin activation, and activating KRAS and BRAF mutations. More recent development of molecular therapeutics has led to the current use of anti-EGFR monoclonal antibody in targeted therapy of advanced CRC. Also, high-throughput cancer genome mutational analysis and newer second-generation cancer genome sequencing have, in recent years, resulted in a deeper understanding of the genome and of regulation of CRC. Most importantly, improved understanding of the involvement of many of the novel molecular targets in CRC tumorigenesis and tumor progression would engender the development of novel targeted therapeutics to treat the disease. Moreover, studies in molecular determinants of predictive and prognostic biomarkers of targeted therapy would further enable better strategies for the use of targeted therapeutics in preventing or overcoming treatment resistance. In this review, we focus on recent findings and their clinical relevance to actionable molecular targets—insulin-like growth factor-1 receptor (IGF-1R), phosphatase and tensin homolog (PTEN), c-Met and phosphatidylinositol 3-OH kinase (PI3-K)—in CRC personalized cancer therapeutics.

Mild Electrical Stimulation and Heat Shock Ameliorates Progressive Proteinuria and Renal Inflammation in Mouse Model of Alport Syndrome

Alport syndrome is a hereditary glomerulopathy with proteinuria and nephritis caused by defects in genes encoding type IV collagen in the glomerular basement membrane. All male and most female patients develop end-stage renal disease. Effective treatment to stop or decelerate the progression of proteinuria and nephritis is still under investigation. Here we showed that combination treatment of mild electrical stress (MES) and heat stress (HS) ameliorated progressive proteinuria and renal injury in mouse model of Alport syndrome. The expressions of kidney injury marker neutrophil gelatinase-associated lipocalin and pro-inflammatory cytokines interleukin-6, tumor necrosis factor-α and interleukin-1β were suppressed by MES+HS treatment. The anti-proteinuric effect of MES+HS treatment is mediated by podocytic activation of phosphatidylinositol 3-OH kinase (PI3K)-Akt and heat shock protein 72 (Hsp72)-dependent pathways in vitro and in vivo. The anti-inflammatory effect of MES+HS was mediated by glomerular activation of c-jun NH2-terminal kinase 1/2 (JNK1/2) and p38-dependent pathways ex vivo. Collectively, our studies show that combination treatment of MES and HS confers anti-proteinuric and anti-inflammatory effects on Alport mice likely through the activation of multiple signaling pathways including PI3K-Akt, Hsp72, JNK1/2, and p38 pathways, providing a novel candidate therapeutic strategy to decelerate the progression of patho-phenotypes in Alport syndrome.

Phase II study of PX-866 in recurrent glioblastoma.

2051 Background: Glioblastoma (GBM) is the most aggressive malignancy of the central nervous system. The majority of GBM have genetic changes that increase the activity of the phosphatidylinositol-3-OH kinase (PI3K) signal transduction pathway, critical for cell motility, proliferation, and survival. We present the interim results of PX-866, an oral PI3K inhibitor, in patients (pts) with recurrent GBM. Methods: Pts with histologically confirmed GBM at first recurrence after treatment with chemoradiation and adjuvant temozolomide are given PX-866 8 mg daily on this single-arm phase II study. MRI and clinical exam are done every 8 weeks to determine treatment response. The trial has a 2-stage design with dual endpoints of objective response and early progression (within 8 weeks). In Stage I, 15 pts are evaluated and if 0 responses and 10 or more early progressions are seen, enrolment will stop. Otherwise, Stage II will enrol another 15 pts for efficacy analysis. Tumour tissue is collected for analysis of potential markers of PI3K inhibitory activity (PTEN, EGFRvIII, PIK3CA mutations). Results: Seventeen pts have been enroled to date: 14 evaluable for response and 15 for toxicity. Median age was 54 years (range 35-70), with 7 females and 10 males. No pts had received treatment for recurrent GBM, and median time between initial diagnosis and study enrolment was 300 days (range: 113-447 days). Pts have received a median of one 8-week cycle of PX-866 (range: 1-4). Twelve pts have discontinued therapy, 9 due to disease progression and 3 due to grade 3/4 liver enzyme abnormalities. Other adverse effects have included fatigue (10 pts/1 grade 3), diarrhea (6 pts/3 grade 3), nausea (7 pts/0 grade 3), vomiting (6 pts/0 grade 3), lymphopenia (14 pts/3 grade 3). Stage I response data are premature; it is not yet known if the trial will continue to Stage II. Archival tissue is available on all patients and is undergoing analysis. Conclusions: This is one of the first trials of a PI3K inhibitor in pts with recurrent GBM. PX-866 has been relatively well tolerated. Stage I response data are premature; while it is not yet known if the criteria will be met to continue to Stage II, prolonged SD has been observed in some pts. The correlative biomarker assays underway will be important to understand this observation.

PI3K‐independent AKT activation in cancers: A treasure trove for novel therapeutics

AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors.

Antagonist Effect of Triptolide on AKT Activation by Truncated Retinoid X Receptor-alpha

BackgroundRetinoid X receptor-alpha (RXRα) is a key member of the nuclear receptor superfamily. We recently demonstrated that proteolytic cleavage of RXRα resulted in production of a truncated product, tRXRα, which promotes cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K)/AKT pathway. However, how the tRXRα-mediated signaling pathway in cancer cells is regulated remains elusive.Methodology/Principal FindingsWe screened a natural product library for tRXRα targeting leads and identified that triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRα-mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell apoptosis dependent on intracellular tRXRα expression levels, demonstrating that tRXRα serves as an important intracellular target of triptolide. We show that triptolide selectively induces tRXRα degradation and inhibits tRXRα-dependent AKT activity without affecting the full-length RXRα. Interestingly, such effects of triptolide are due to its activation of p38. Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRα degradation and AKT activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRα interaction with p85α leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and AKT through tRXRα mediation. We finally show that targeting tRXRα by triptolide strongly activates TNFα death signaling and enhances the anticancer activity of other chemotherapiesConclusions/SignificanceOur results identify triptolide as a new xenobiotic regulator of the tRXRα-dependent survival pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide represents one of the most promising therapeutic leads of natural products of traditional Chinese medicine with unfortunate side-effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.