Phospatidylinositol 3-kinase Research Articles

PI3K Inhibitors in Cancer: Clinical Implications and Adverse Effects.

The phospatidylinositol-3 kinase (PI3K) pathway is a crucial intracellular signaling pathway which is mutated or amplified in a wide variety of cancers including breast, gastric, ovarian, colorectal, prostate, glioblastoma and endometrial cancers. PI3K signaling plays an important role in cancer cell survival, angiogenesis and metastasis, making it a promising therapeutic target. There are several ongoing and completed clinical trials involving PI3K inhibitors (pan, isoform-specific and dual PI3K/mTOR) with the goal to find efficient PI3K inhibitors that could overcome resistance to current therapies. This review focuses on the current landscape of various PI3K inhibitors either as monotherapy or in combination therapies and the treatment outcomes involved in various phases of clinical trials in different cancer types. There is a discussion of the drug-related toxicities, challenges associated with these PI3K inhibitors and the adverse events leading to treatment failure. In addition, novel PI3K drugs that have potential to be translated in the clinic are highlighted.

Tumor suppressor RIZ1 in obesity and the PI3K/AKT/mTOR pathway.

The aim of this study was to investigate the shared molecular pathways of obesity and cancer by exploring the role of RIZ1 in obesity and the phospatidylinositol 3-kinase (PI3K)/V-Akt murine thymoma viral oncogene homolog (PKB) (AKT)/mechanistic target of rapamycin (mTOR) pathway. Male wild type (WT) and Riz1(-/-) mice (KO) were fed a standard diet (STD) or a high-fat (HF) diet for up to 8 months. These mice were studied for phenotypic and molecular changes. Riz1(-/-) mice gained more weight on a HF diet compared to WT mice, with higher free fatty acid and increased visceral fat. Metabolic cage analysis of Riz1(-/-) mice showed lower oxygen consumption but no changes in food intake and ambulatory activity. Riz1(-/-) mice showed impaired glucose regulation but no change in insulin sensitivity. RNA-seq and quantitative RT-PCR analysis found altered expression in certain glycolysis and ATP production genes such as Ubiad1, Atp5g2, and Cyp4a12. The PI3K/AKT/mTOR pathway was activated in the Riz1(-/-) mice fed a HF diet with higher Akt3 mRNA levels and increased phosphorylation of AKT (Ser473), mTOR, and S6. The results identify RIZ1 as an important regulator of both Akt3 transcription and AKT phosphorylation and suggest a role for RIZ1 in HF-induced obesity and the AKT pathway.

Genistein sensitizes hepatic insulin signaling and modulates lipid regulatory genes through p70 ribosomal S6 kinase-1 inhibition in high-fat–high-fructose diet-fed mice

Context: Genistein reduces high-calorie diet-induced insulin resistance and fat accumulation in animals, but the mechanism is unresolved.Objective: This study explores whether action of genistein is associated with p70 ribosomal S6 kinase-1 (S6K1) inhibition.Materials and methods: Adult male mice were fed either normal diet or high-fat–high-fructose diet (HFFD) for 15 days, after which animals in each dietary group were divided into two groups and administered either genistein (1 mg kg−1 day−1, p.o.) in 0.5 ml of 30% dimethylsulfoxide (DMSO) or 30% DMSO (0.5 ml) for the next 45 days. At the end of the study, their liver was analyzed for lipid content. Semi-quantitative RT-PCR and western blotting methods were used to analyze lipid regulatory genes and insulin signaling proteins, respectively.Results: Genistein significantly (p < 0.05) lowered HFFD-induced body and liver weight gain and plasma and hepatic lipid levels. Histology showed a 2.5-fold increase of lipid in HFFD compared to control. Genistein treatment to HFFD-fed animals significantly decreased lipid accumulation (by 40%) compared to HFFD. Insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrates-1 (IRS-1), IRS-1 associated phospatidylinositol-3kinase (PI3K) and Akt Ser473 phosphorylation were improved while IRS-1 serine phosphorylation was significantly (p < 0.05) decreased by genistein in HFFD. Significant (p < 0.05) increase in adenosine monophosphate-activated protein kinase (AMPK) Thr172 phosphorylation and decrease in S6K1 Thr389 phosphorylation were observed in HFFD-plus genistein compared to HFFD. Genistein downregulated lipogenic genes and upregulated fatty acid oxidative genes in HFFD-fed mice.Conclusion: Genistein improves insulin signaling and attenuates fat accumulation in liver through S6K1 inhibition.

Genistein promotes insulin action through adenosine monophosphate–activated protein kinase activation and p70 ribosomal protein S6 kinase 1 inhibition in the skeletal muscle of mice fed a high energy diet

Genistein (GEN), a soy isoflavone, exerts insulin-sensitizing actions in animals; however, the underlying mechanisms have not been determined. Because GEN is a known activator of adenosine monophosphate-activated protein kinase (AMPK), we hypothesize that GEN activates insulin signaling through AMPK activation. To test this hypothesis, a high fat-high fructose diet (HFFD)-fed mice model of insulin resistance was administered GEN, and the insulin signaling pathway proteins in the skeletal muscle were examined. Hyperglycemia and hyperinsulinemia observed in HFFD-fed mice were significantly lowered by GEN. GEN increased insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrate (IRS) 1 but down-regulated IRS-1 serine phosphorylation in the skeletal muscle of HFFD-fed mice. Furthermore, GEN treatment improved muscle IRS-1-associated phospatidylinositol-3 kinase expression, phosphorylation of Akt at Ser(473), and translocation of glucose transporter subtype 4. Phosphorylation of AMPK at Thr(172) and acetyl coenzyme A carboxylase (ACC) at Ser(79) was augmented, whereas phosphorylation of p70 ribosomal protein S6 kinase 1 at Thr(389) was significantly decreased after GEN treatment in the skeletal muscle of HFFD-fed mice. These results suggest that GEN might improve insulin action in the skeletal muscle by targeting AMPK.

Growth of the pancreatic cancer cell line PANC-1 is inhibited by protein phosphatase 2A inhibitors through overactivation of the c-Jun N-terminal kinase pathway

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that can dephosphorylate multiple kinases. It is generally considered to be a cancer suppressor as its inhibition can induce phosphorylation and activation of substrate kinases that mainly accelerate growth. We previously reported that cantharidin, an active constituent of a traditional Chinese medicine, potently and selectively inhibited PP2A, yet efficiently repressed the growth of pancreatic cancer cells through activation of the c-Jun N-terminal kinase (JNK) pathway. This suggested that activation of kinase pathways might also be a potential strategy for cancer therapy. In this study, we have confirmed that the basal activity of the phospatidylinositol 3-kinase (PI3K)/JNK/activator protein 1 (AP-1) pathway promoted pancreatic cancer cell growth when stimulated by growth factors. Interestingly, although treatment with the PP2A inhibitors, cantharidin or okadaic acid (OA), amplified the PI3K-dependent activation of JNK, cell growth was repressed. We therefore hypothesised that a specific level of activity of the JNK pathway might be required to maintain the promitogenic function, as both repression and overactivation of JNK could inhibit cell proliferation. It was found that the JNK-dependent growth inhibition was independent of the activation of AP-1, but dependent on the repression of Akt. Although the PP2A inhibitors triggered overactivation of JNK and inhibited cell growth, excessively activated protein kinase C (PKC) improved cell survival. Combined treatment with a PP2A inhibitor and a PKC inhibitor produced a synergistic effect, which indicates a potentially promising therapeutic approach to pancreatic cancer treatment.

High concentrations of vascular endothelial growth factor reduce stretch‐induced apoptosis of alveolar type II cells

Vascular endothelial growth factor (VEGF) has protective as well as injurious effects in ARDS/acute lung injury. The influence of VEGF was investigated in a model of stretch-induced apoptosis. High-amplitude mechanical stretch induced the secretion of VEGF. High VEGF concentrations may prevent stretch-induced apoptosis by restoring stretch-impaired phospatidylinositol-3 kinase signalling. Vascular endothelial growth factor (VEGF) is strongly expressed in the alveolar epithelium. VEGF has been shown to exhibit protective as well as injurious effects in ARDS/acute lung injury. We therefore investigated the influence of VEGF in a model of stretch-induced apoptosis. Isolated rat alveolar type II (ATII) cells were subjected to high-amplitude cyclic mechanical stretch (40 per minute, 30% change in surface area) for 24 h. VEGF gene expression was investigated by real-time reverse transcription-PCR. Concentrations of VEGF in culture supernatants of stretched cells were determined by ELISA. Apoptosis of cells following stretching was assessed by flow cytometry. Vascular endothelial growth factor gene expression increased during the first 4 h of stretching and then declined to a similar level to that of static control cells. VEGF concentrations in cell supernatants increased in response to mechanical stretch, as compared with those in supernatants of static control cells. Incubation of ATII cells with higher concentrations of VEGF (50 ng/mL) during stretching inhibited apoptosis, presumably by restoring stretch-impaired phosphatidylinositol-3 kinase signalling. However, blocking free VEGF in the supernatant with an anti-VEGF antibody did not influence stretch-induced apoptosis. These findings suggest that high-amplitude mechanical stretch induced secretion of VEGF, which in high concentrations, may prevent stretch-induced apoptosis. In this model, however, the protective influence of VEGF was not essential for survival of ATII cells subjected to high-amplitude mechanical stretch.

Therapeutic effect of magnesium lithospermate B on neointimal formation after balloon-induced vascular injury

Vascular smooth muscle cell (VSMC) proliferation and migration in response to platelet-derived growth factor (PDGF) play an important role in the development of atherosclerosis and restenosis. Recent evidence indicates that PDGF increases intracellular levels of reactive oxygen species in VSMCs and that both PDGF-induced VSMC proliferation and migration are reactive oxygen species-dependent. Danshen is a representative oriental medicine used for the treatment of vascular disease. Previously, we reported that magnesium lithospermate B, an active component of Danshen, is a potent antioxidant. Thus we investigated the therapeutic potential of magnesium lithospermate B in neointimal formation after carotid artery injury in rats along with its effects on the PDGF signaling pathway for stimulating VSMC proliferation and migration in vitro. PDGF is dimeric glycoprotein composed of two A or two B chains. In this study, we used PDGF-BB, which is one of the isoforms of PDGF (i.e., PDGF-AA, PDGF-BB, and PDGF-AB). Our results demonstrated that magnesium lithospermate B directly scavenged reactive oxygen species in a xanthine/xanthine oxidase system and reduced PDGF-BB-induced intracellular reactive oxygen species generation in VSMCs. In a rat carotid artery balloon injury model, magnesium lithospermate B treatment (10 mg/kg/day, i.p) showed a significant effect on the prevention of neointimal formation compared with vehicle treatment. In cultured VSMCs, magnesium lithospermate B significantly attenuated PDGF-BB-induced cell proliferation and migration as measured by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and transwell migration assays, respectively. Further, magnesium lithospermate B inhibited PDGF-BB-induced phosphorylation of phospatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways by scavenging reactive oxygen species. Together, these data indicated that magnesium lithospermate B, a potent reactive oxygen species scavenger, prevented both injury-induced neointimal formation in vivo and PDGF-BB-induced VSMC proliferation and migration in vitro, suggesting that magnesium lithospermate B may be a promising agent to prevent atherosclerosis and restenosis following angioplasty.

Hyperinsulinemia: effect on cardiac mass/function, angiotensin II receptor expression, and insulin signaling pathways

To investigate the association between hyperinsulinemia and cardiac hypertrophy, we treated rats with insulin for 7 wk and assessed effects on myocardial growth, vascularization, and fibrosis in relation to the expression of angiotensin II receptors (AT-R). We also characterized insulin signaling pathways believed to promote myocyte growth and interact with proliferative responses mediated by G protein-coupled receptors, and we assessed myocardial insulin receptor substrate-1 (IRS-1) and p110 alpha catalytic and p85 regulatory subunits of phospatidylinositol 3 kinase (PI3K), Akt, MEK, ERK1/2, and S6 kinase-1 (S6K1). Left ventricular (LV) geometry and performance were evaluated echocardiographically. Insulin decreased AT1a-R mRNA expression but increased protein levels and increased AT2-R mRNA and protein levels and phosphorylation of IRS-1 (Ser374/Tyr989), MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), S6K1 (Thr421/Ser424/Thr389), Akt (Thr308/Thr308), and PI3K p110 alpha but not of p85 (Tyr508). Insulin increased LV mass and relative wall thickness and reduced stroke volume and cardiac output. Histochemical examination demonstrated myocyte hypertrophy and increases in interstitial fibrosis. Metoprolol plus insulin prevented the increase in relative wall thickness, decreased fibrosis, increased LV mass, and improved function seen with insulin alone. Thus our data demonstrate that chronic hyperinsulinemia decreases AT1a-to-AT2 ratio and increases MEK-ERK1/2 and S6K1 pathway activity related to hypertrophy. These changes might be crucial for increased cardiovascular growth and fibrosis and signs of impaired LV function.

Identification of novel chemoattractant peptides for human leukocytes

AbstractSuperoxide is the most important armory on the primary defense line of monocytes against invading pathogens, and the identification of new stimuli and the characterization of the regulatory mechanism of superoxide generation are of paramount importance. In this study, we identified 3 novel peptides by screening a synthetic hexapeptide combinatorial library and modification of 1 of the peptides. The isolated peptides that can induce superoxide generation in human monocytes are His-Phe-Tyr-Leu-Pro-Met-CONH2 (HFYLPM), Met-Phe-Tyr-Leu-Pro-Met-CONH2 (MFYLPM), and His-Phe-Tyr-Leu-Pro-D-Met-CONH2 (HFYLPm). All 3 peptides also caused intracellular calcium ([Ca++]i) rise. We tested the specificities of the peptides on cells of different origin by looking at [Ca++]i rise. All 3 peptides acted specifically on leukocytes and not on nonimmune cells. Among leukocytes, HL60 and Jurkat T cells were stimulated specifically by MFYLPM or HFYLPM, respectively. As a physiologic characteristic of the peptides, we observed that all 3 peptides induced chemotactic migration of monocytes. Studying receptor specificity, we concluded that the 3 peptides might act on some shared and some distinct receptor(s) on leukocytes. Studying intracellular signaling set in motion by the peptides revealed that HFYLPM, but not MFYLPM or HFYLPm, induced chemotaxis via phospatidylinositol-3 kinase and protein kinase C. Because HFYLPM, MFYLPM, and HFYLPm not only exhibit different specificities depending on cell type and status of differentiation but also stimulate cells via distinct receptors and signaling, the 3 novel peptides might be useful tools to study leukocyte activation.

Phospatidylinositol 3-kinase and P42/44 map kinases are involved in a novel effect of glycine-extended epithelial cell migration

Phospatidylinositol 3-kinase and P42/44 map kinases are involved in a novel effect of glycine-extended epithelial cell migration

Defective radiation signal transduction in ataxia-telangiectasia cells

Purpose : The product of the gene ATM mutated in the human genetic disorder ataxia-telangiectasia (A-T) is predominantly present in the nucleus compatible with a role in DNA-damage recognition and cell-cycle control. However, ATM is also present outside the nucleus in cytoplasmic and membrane associated vesicles, which may explain the more general signalling defect in A-T. This led us to investigate signalling events initiated by ionizing radiation, remote from the nucleus. Materials and Methods : A-T and control lymphoblastoid cells were employed to study radiation-induced signalling at the level of protein activation using immunoprecipitation and immunoblotting. Flow cytometry was used to determine mobilization of intracellular Ca 2. Results : Lymphoblastoid cells from A-T patients were found to be defective in the radiation-induced activation of protein tyrosine kinase p53/p56lyn. In control cells Ca 2 was mobilized in response to gamma-radiation largely from internal stores, and increased significantly over a 20 min period. This mobilization of Ca2 was either absent or increased very slowly in A-T cells post-irradiation. The same pattern of release was observed after treatment with the radiometric agent, streptonigrin. In addition the phospatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin suppressed the release of Ca 2. Conclusion : These data demonstrate that ionizing radiation activates lyn kinase and leads to the release of Ca 2, and-for the first time-that these steps are ATM-dependent.

A Thrombopoietin Receptor Mutant Deficient in Jak-STAT Activation Mediates Proliferation But Not Differentiation in UT-7 Cells

Thrombopoietin (TPO) stimulates proliferation and differentiation of cells of the megakaryocytic lineage. It exerts its function by binding and activating c-mpl, a member of the hematopoietic receptor superfamily. Upon binding of TPO to its receptor, numerous signaling events are triggered. These include activation of the Jak-STAT (signal transducers and activators of transcription) pathway, mitogen-activated protein kinase (MAPK), Tec, and phospatidylinositol (PI) 3-kinase and phosphorylation of Shc and Vav. The contribution of different signaling pathways to the induction of specific cellular processes such as proliferation and differentiation is incompletely understood. We have previously described a mutant of c-mpl that fails to activate the Jak-STAT pathway but nevertheless retains its ability to mediate proliferation and activation of most signaling events in the murine hematopoietic precursor cell lines BAF/3 and 32D. We confirm here the ability of this mutant to mediate proliferation in the absence of Jak-STAT activation in the human cell line UT-7 and further show that this mutant fails to mediate TPO-induced megakaryocytic differentiation. Comparison of the signaling capacity of this mutant in UT-7 and BAF/3 cells shows considerable cell-type-specific differences. Whereas in BAF/3 cells the mutant still mediates activation of Shc, MAPK, Vav, and PI 3-kinase at levels comparable to the wild-type receptor, these events are strongly diminished in UT-7 cells expressing the mutant. Furthermore, we show that the C-terminal 25 amino acid residues of the receptor mutant are crucial for the mitogenic response in UT-7 cells.

Phospatidylinositol 3-kinase expression in human breast cancer

Phospatidylinositol 3-kinase (PI 3-kinase) expression was analysed by Western blotting with monoclonal antibodies to the p85 subunit in a series of tumour and adjacent mammary gland samples collected at surgery from 33 breast cancer patients. Seventy-nine percent of the investigated pairs of the samples were characterised by an increased level of PI 3-kinase in the tumour in comparison with the adjacent mammary gland. PI 3-kinase activation was not associated with tumour steroid receptor status, histologic grade and other clinico-morphological characteristics. Furthermore, immunoblotting of epidermal growth factor receptor (EGFR) in the tumours with increased PI 3-kinase and corresponding adjacent tissues revealed no association between EGFR and PI 3-kinase activation. Thus, increased PI 3-kinase expression appears to be a widespread feature of breast cancer not associated with the main biological markers of its prognosis and hormone sensitivity.

Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart.

Because tyrosine kinase blockade prevents protection by ischemic preconditioning (p.c.) in several species, activation of tyrosine kinase appears to be critical for cardioprotection. The tyrosine kinase's identity, however, is unknown. The present study tested whether activation of a receptor tyrosine kinase, the insulin receptor, could mimic p.c. and if the mechanism of protection was similar to that of p.c. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarct size in control hearts was 32.6 +/- 2.3%. A 5-min infusion of insulin (5 mU/ml) followed by a 10-min washout period prior to ischemia significantly reduced infarction to 14.7 +/- 2.1% (P < 0.05). The tyrosine kinase inhibitor genistein (50 microM) given around the insulin infusion blocked protection (28.9 +/- 2.8%). However, when present during the onset of ischemia, genistein had no effect on protection triggered by insulin (14.0 +/- 2.4%; P < 0.05). Inhibition of either PKC by polymyxin B (50 microM) or KATP channels by 5-hydroxydecanoate (100 microM) also failed to prevent protection by insulin (17.5 +/- 3.2% and 17.6 +/- 3.0%, respectively). However, the reduction in infarct size by insulin was significantly attenuated by wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (PI3K, 28.3 +/- 2.2%). Insulin was still able to protect the heart when given only during the reperfusion period (13.2 +/- 3.4%). P.c. reduced infarction to 12.8 +/- 2.0% (P < 0.05) and still offered significant protection in the presence of wortmannin (22.1 +/- 2.4%; P < 0.05). In conclusion, activation of the insulin receptor reduces infarct size in the rabbit heart even when instituted upon reperfusion. However, the mechanism of protection is quite different from that of p.c. and involves activation of PI3K but not PKC or KATP channels.

Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis.

W/Wv mice are deficient in tissue mast cells, and mast cells cultured from these mice do not proliferate in response to the c-kit ligand, stem cell factor (SCF). In this paper, we report that mouse bone marrow cultured mast cells derived from W/Wv mice do adhere to fibronectin in the presence of SCF and exhibit chemotaxis to SCF, and we explore this model for the understanding of c-kit-mediated signaling pathways. Both in vitro and in vivo (in intact cells) phosphorylation experiments demonstrated a low residual level of W/Wv c-kit protein phosphorylation. SCF-induced responses in W/Wv mast cells were abolished by the tyrosine kinase inhibitor herbimycin A and by the phospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin but were not affected by protein kinase C inhibitors. These observations are consistent with the conclusions that Wv c-kit initiates a signaling process that is PI 3-kinase dependent and that mutated Wv c-kit retains the ability to initiate mast cell adhesion and migration.