Effects Of LY294002 Research Articles

Abstract A05: Phosphoinositide-3-kinase pathway promotes colon cancer stem cell proliferation

Abstract Introduction. The cancer stem cell (CSC) model posits that a select cell-subpopulation exists in solid tumors that sustain tumorigenesis. Previously, we have shown that aldehyde-dehydrogenase (ALDH) enriches colon CSCs (CCSC) from sporadic colon cancer. In this study, Affymetrix microarray was used to screen for differences between ALDHhi and ALDHlo cells isolated from sporadic human colon adenocarcinoma. As a result, the phosphoinositide-3-kinase (PI3K) pathway was identified as a potential regulator of CCSC growth and differentiation. Functional assays demonstrate that inhibition of PI3K results in significant reduction in proliferation and colony formation of by CCSCs. Experimental Procedures. Unbiased Expression-Profile Analysis: We implanted human CCSCs into flanks of NOD-SCID mice. Flow assisted cell sorting discriminated resulting tumor epithelia into ALDHhi and ALDHlo groups from 6 individual sporadic human adenocarcinomas, which were analyzed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Proliferation Assay: Bromodeoxyuridine (BrdU) incorporation measured the effect of LY294002 (Calbiochem) (1-100 μM) on proliferation. Colony Assay: Methylcellulose colony assays were used to assess the effect of LY294002 on colony formation. Summary of Data. Expression Profile Analysis revealed 136 genes that were significantly up or down-regulated in the ALDHhi cell groups (>3-fold; p<0.001). The beta regulatory subunit of phosphoinositide-3 kinase demonstrated a 5.2 fold up-regulation in the ALDHhi vs. ALDHlo CCSCs. Cell Proliferation Assay: LY294002 significantly inhibits CCSC proliferation with IC50 ~ 50 μM and near complete inhibition at the dose of 100 μM (p<0.001). Colony Assay: LY294002 significantly reduces total colony area formed by CCSCs, with a similar trend as shown in BrdU proliferation assays (p<0.001). Conclusions. We have previously shown that ALDH enriches colon cancer epithelia. Here we show that the PI3K signaling pathway is differentially expressed between ALDHhi and ALDHlo CCSCs derived from primary tumor xenografts. We demonstrate in vitro functional differences in CCSCs with drug-inhibition assays. Citation Format: Sugong Chen, Robert C. Fisher, Anitha K. Shenoy, Maria Cecilia Lopez, Lyle L. Moldawer, Henry V. Baker, Emina H. Huang. Phosphoinositide-3-kinase pathway promotes colon cancer stem cell proliferation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A05.

LY294002 Enhances Expression of Proteins Encoded by Recombinant Replication-Defective Adenoviruses via mTOR- and Non-mTOR-Dependent Mechanisms

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

PI3K-like kinases restrain Pim gene expression in endothelial cells

Pim kinases contribute to tumor formation and development of lymphoma, which shows enhanced DNA replication, DNA recombination and repair. Endothelial cells^(ECs) express all the three members of Pim kinase gene family. We hypothesized that DNA repair gene would regulate Pim expression in ECs. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium. The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining. The siRNA fragments were synthesized and transfected by using Lipofectamine LTX. The total cellular RNA was extracted from the cells by using Trizol reagent. cDNAs were quantified by semi-quantity PCR. The effects of LY294002 and wortmannin on RNA stability in ECs were also examined. Our data showed that LY294002 and wortmannin, phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors, increased Pim mRNA expression in ECs without altering the mRNA stability. RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1, respectively. Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs. But etoposide, a nucleoside analogue, which could activate DNA-PKcs and ATM, increased Pim expression in ECs. Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

The Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor kappa B inhibition, in a PI3K-independent mechanism

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor κB (NFκB) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NFκB subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NFκB in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor – wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NFκB enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNFα production in the LPS tolerant state which is regulated by p50 NFκB homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NFκB homodimer activity by LY2.

Possible Role of Phosphoinositide-3-Kinase in Mx1 Protein Translation and Antiviral Activity of Interferon-Omega-Stimulated HeLa Cells

Interferon ω (IFN-ω), a cytokine released during innate immune activation, is well known for promoting direct antiviral responses; however, the possible signal pathways that are initiated by IFN-ω binding to the type I IFN receptors have not been fully studied. Here, we provide evidence that activation of phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) signaling plays a pivotal role in the generation of IFN-ω-mediated biological responses. We found that LY294002 (PI3K inhibitor)-attenuated antiviral activities are induced by IFN-ω treatment. Although such effects of LY294002 are unrelated to regulatory activities on IFN-ω-dependent Mx1 (myxovirus resistance 1) or Mx2 gene transcriptional regulation, translation of Mx1 protein, which was known as a key mediator of cell-autonomous antiviral resistance, was significantly reduced by PI3K inhibition. Further studies showed that PI3K inhibition using LY294002 leads to a decrease in PI3K substrate Akt and mitogen-activated protein kinase extracellular signal-regulated kinase and p38 phosphorylation/activation. In addition, although LY294002 was not able to reduce STAT1 activation, we found that the mammalian target of rapamycin (mTOR)/p70 S6 kinase pathway was significantly attenuated by inhibition of the PI3K/Akt signaling pathway. These results indicate that the PI3K/Akt pathway is a common and central integrator for antiviral responses in IFN-ω signaling via its regulatory effects on mTOR that are required for initiation of mRNA translation of Mx genes.

Insulin receptor signaling in rat hippocampus: A study in STZ (ICV) induced memory deficit model

Brain insulin receptors (IRs) have been suggested as an important regulatory factor for cognitive functions but the involvement of IR signaling in memory deficit associated with neurodegenerative conditions is not yet explored. In the present study, IR gene expression was studied by RT-PCR and signaling pathways by immunoblotting in CA1, DG and CA3 subregions of hippocampus in intracerebroventricular (ICV) administered streptozotocin (STZ, 3 mg/kg twice) induced memory deficit model in rat. The effect of pre- and post-treatment of donepezil (5 mg/kg po) and melatonin (20 mg/kg po) on signaling pathways were studied. Effect of LY294002 (ICV), a PI3 Kinase inhibitor, was also investigated on memory functions and Akt phosphorylation. An increased IR expression (both gene and protein), phosphorylation of Shc, Erk1/2, IRS-1 and Akt in CA1 and CA3 region of P2M fraction was observed after training as compared to control. STZ treated rats showed memory deficit and significant decrease in IR expression, phosphorylation of IRS-1 and Akt only in CA3 region as compared to trained group which were reversed by pre and post-treatment of melatonin but donepezil was effective only against memory deficit. LY294002 (3 mM) treatment showed delayed learning and decrease in Akt phosphorylation. This study suggests that IR expression and its signaling pathways in hippocampal CA1 and CA3 regions are involved in memory functions and STZ (ICV) induced memory deficit. Hippocampal IR system might be playing an important role in regulation of memory functions, however only IR/IRS-1/Akt pathway in CA3 region is associated with STZ induced memory deficit.

Phosphoinositide 3-kinase regulates myogenin expression at both the transcriptional and post-transcriptional level during myogenesis

It is well-established that phosphoinositide 3-kinase (PI3-kinase) regulates myogenesis by inducing transcription of myogenin, a key muscle regulatory factor, at the initiation of myoblast differentiation. In this study, we investigated the role of PI3-kinase in cells that have committed to differentiation. PI3-kinase activity increases during myogenesis, and this increase is sustained during the myogenic process; however, its function after the induction of differentiation has not been investigated. We show that LY294002, a PI3-kinase inhibitor, blocked myoblast fusion even after myogenin expression initially increased. In contrast to the inhibitory effects of LY294002 on myogenin mRNA levels during the initiation of differentiation, LY294002 blocked the accumulation of myogenin protein without affecting its mRNA level after differentiation was induced. Treatment with cycloheximide, a translation inhibitor, or actinomycin D, a transcription inhibitor, indicated that the stability of myogenin protein is lower than that of its mRNA. LY294002 inhibited the activities of several important translation factors, including eukaryotic elongation factor-2 (eEF2), by altering their phosphorylation status. In addition, LY294002 blocked the incorporation of [35S]methionine into newly synthesized proteins. Since myogenin has a relatively short half-life, LY294002-mediated inhibition of post-transcriptional processes resulted in a rapid depletion of myogenin protein. In summary, these results suggest that PI3-kinase plays an important role in regulating the expression of myogenin through post-transcriptional mechanisms after differentiation has been induced.

Bone marrow mesenchymal stem cells from leukemia patients inhibit growth and apoptosis in serum-deprived K562 cells

BackgroundThe regulation of growth and apoptosis in K562 cells by human bone marrow mesenchymal stem cells (MSCs) from leukemia patients was investigated.MethodsK562 cells were cocultured with leukemic MSCs under serum deprivation. Cell Counting Kit-8 (CCK-8), PI staining, Annexin V/PI binding and FACS assays were used to investigate cell proliferation, cell cycle status, and apoptosis of K562 cells cultures in the presence or absence of 10% serum. Western blotting was used to determine the levels of Akt, phosphorylated Akt (p-Akt), the BCL-2 family member Bad, and phosphorylated Bad (p-Bad) proteins in K562 cells after coculturing with MSCs. The effects of LY294002 (a specific inhibitor of PI3K) on protein expression were also determined.ResultsK562 cell proliferation was inhibited by coculture with MSCs and the dominant cell cycle was the G0-G1 phase. The proportion of apoptotic K562 cells was decreased and the levels of p-Akt and p-Bad were upregulated after exposing K562 cells to MSCs. However, when LY294002 was used, p-Akt and p-Bad proteins inK562 cells showed a significant reduction, while no distinct variation was seen in the nonphosphorylated Akt and Bad protein levels.ConclusionLeukemic MSCs can inhibit K562 cell expansion and modulate the cell cycle to a state of relative quiescence. This allows the K562 cells to endure adverse conditions such as serum starvation. The PI3K-Akt-Bad signaling pathway may be involved in this antiapoptotic process via phosphorylation of the Akt and Bad proteins. Blocking MSC-induced transduction of the PI3K-Akt-Bad pathway may be a potential strategy for a targeted therapy to combat leukemia.

PI-3 kinase pathway can mediate the effect of TGF-β1 in inducing the expression of SHARP-2 in LLC-PK1 cells

We aim to investigate the effect of transforming growth factor (TGF)-beta1 on the expression of enhancer of split- and hairy-related protein-2 (SHARP-2) messenger RNA (mRNA) and its signaling pathway. In this study, several cell lines including LLC-PK1 (a porcine kidney tubular epithelial cell line), MDCK (Madin-Darby canine kidney) and CTLL-2 (cytotoxic T-lymphocyte line) were treated with recombinant human TGF-beta1, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-beta1 treatment to probe the signaling pathway. The results showed that TGF-beta1 can significantly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-beta1 stimulation. While one phosphoinositide 3-kinases (PI-3) kinase inhibitor, LY294002, completely blocked the effect of TGF-beta1 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-beta1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect.

Inhibition of Phosphatidylinositol 3-Kinase/Akt Signaling Suppresses Tumor Cell Proliferation and Neuroendocrine Marker Expression in GI Carcinoid Tumors

Overactivation of PI3K/Akt signaling facilitates tumor proliferation in several cancers. We have shown that various signal transduction pathways promote tumorigenesis in carcinoid tumors, which exhibit endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that inhibition of the PI3K/Akt pathway would suppress carcinoid tumor cell growth and neuroendocrine (NE) marker production. Human carcinoid BON cells were treated in vitro with LY294002, a PI3-kinase inhibitor, or transfected with Akt1 siRNA. Tumor cell proliferation was measured by MTT for 6 days. The effect of LY294002 or Akt1 siRNA treatment was assessed by Western analysis. We examined the levels of phosphorylated Akt, total Akt, Akt1, and the NE markers human achaete-scute homolog1 (ASCL1) and chromogranin A (CgA). Treatment of BON cells with LY294002 reduced tumor cell proliferation (76%) in a dose-dependent manner. Growth also decreased in Akt1 siRNA transfected cells (26%). Levels of active, phosphorylated Akt and the NE tumor markers, ASCL1 and CgA, were diminished with both LY294002 and Akt1 siRNA treatments proportional to the degree of Akt inhibition. Total Akt, Akt2, and Akt3 levels were unaffected by these experiments. These data indicate that PI3K/Akt signaling performs a critical role in human carcinoid tumor cell survival and NE hormone generation. Furthermore, the development of novel therapeutics targeting Akt1 or components of the PI3K/Akt pathway may enhance the management of carcinoid disease.

Inhibitory actions of the phosphatidylinositol 3‐kinase inhibitor LY294002 on the human Kv1.5 channel

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I(Kur)), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K). hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method. LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC(50) of 7.9 micromol.L(-1)). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k(+1)) and unbinding (k(-1)) rate constants were calculated to be 1.6 micromol.L(-1) (-1).s(-1) and 5.7 s(-1) respectively. Inhibition by LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A. LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for LY294002.

Effects of LY294002 on the invasiveness of human gastric cancer in vivo in nude mice

To investigate the effects of class I phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 on the invasiveness and related mechanisms of implanted tumors of SGC7901 human gastric carcinoma cells in nude mice. Nude mice were randomly divided into model control groups and LY294002 treatment groups. On days 5, 10 and 15 after treatment, the inhibitory rate of tumor growth, pathological changes in tumor specimens, expression levels of matrix metalloproteinase (MMP)-2, MMP-9, CD34 [representing microvessel density (MVD)] and vascular endothelial growth factor (VEGF), as well as apoptosis indexes in tumor samples were observed. In this study, we showed that treating the tumors with LY294002 could significantly inhibit carcinoma growth by 11.3%, 29.4% and 36.7%, after 5, 10 and 15 d, respectively, compared to the control group. Hematoxylin & eosin staining indicated that the rate of inhibition increased progressively (23.51% +/- 3.11%, 43.20% +/- 3.27% and 63.28% +/- 2.10% at 5, 10 and 15 d, respectively) along with apoptosis. The expression of MMP-2 was also downregulated (from 71.4% +/- 1.6% to 47.9% +/- 0.7%, 31.9% +/- 0.9% and 7.9% +/- 0.7%). The same effects were observed in MMP-9 protein expression (from 49.4% +/- 1.5% to 36.9% +/- 0.4%, 23.5% +/- 0.9% and 7.7% +/- 0.6%), the mean MVD (from 51.2% +/- 3.1% to 41.9% +/- 1.5%, 30.9% +/- 1.7% and 14.9% +/- 0.8%), and the expression of VEGF (from 47.2% +/- 3.1% to 25.9% +/- 0.5%, 18.6% +/- 1.2% and 5.1% +/- 0.9%) by immunohistochemical staining. The class I PI3K inhibitor LY294002 could inhibit the invasiveness of gastric cancer cells by downregulating the expression of MMP-2, MMP-9, and VEGF, and reducing MVD.

Abstract 3944: Converged Signaling of β-Catenin and Notch Induces Differentiation of Arterial Endothelial Cells from VEGFR2+ Vascular Progenitors

We have been investigating molecular mechanisms of vascular development and diversification using embryonic stem (ES) cells. Previously, we established an ES cell differentiation system that reproduces early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors (Nature, 2000). Endothelial cells (ECs) were induced from VEGFR2 + progenitor cells with VEGF. VEGF alone mainly induced venous ECs, which are negative for arterial EC markers, ephrinB2 and CXCR4. Interestingly, addition of 8bromo-cAMP with VEGF activated Notch signaling in ECs and substantially induced arterial ECs. Nevertheless, activation of Notch using notch intracellular domain (NICD)-estrogen receptor fusion protein failed to induce arterial ECs (Arterioscler Thromb Vasc Biol, 2006). These results indicate that Notch activation was not sufficient to induce arterial ECs from vascular progenitors. Then we further investigated the downstream of cAMP, which would suffice signals for constructive induction of arterial ECs. We found that phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, completely inhibited cAMP-induced arterial EC differentiation as well as Notch activation. GSK3β inhibitor, Bio, partially reversed the inhibitory effects of LY294002. Whereas activation of β-catenin signaling alone using Doxycycline-induced constitutive active form of β-catenin expression showed only a weak effect on arterial EC induction, simultaneous activation of β-catenin and Notch signaling completely restored arterial EC induction even in the absence of cAMP. Thus, cAMP-PI3K-GSK3β-β-catenin pathway plays a critical role in arterial EC differentiation. Furthermore, chromatin immunoprecipitation assay on the two RBP-J binding sites of mouse EphrinB2 revealed that RBP-J, NICD, and β-catenin formed a protein complex on these two sites only in arterial ECs, but not in venous ECs. These findings suggest that Notch and β-catenin signaling converged to form a novel arterial EC-specific protein complex to induce arterial ECs. This study would provide novel understandings of cellular and molecular mechanisms for arterial-venous specification and novel molecular targets for drug discovery.

Effects of the Phosphatidylinositol 3-Kinase Inhibitor in a Mouse Model of Retinal Neovascularization

Purpose: To determine the effectsof LY294002, a phosphatidylinositol 3-kinase inhibitor, on suppressing experimental retinal neovascularization in an animal model of ischemic retinopathy. Methods: The effect of LY294002 on the survival of RF/6A cells stimulated by vascular endothelial growth factor (VEGF) was investigated colorimetrically. The inhibitory activity of LY294002 on the migration of cells stimulated with VEGF was measured by cell counting. C57BL/6N mice at postnatal day (P) 7 were exposed to 75 ± 2% oxygen for 5 days (P7–P11) and then returned to room air for 5 days (P12–P17) to induce retinal neovascularization. Beginning on P12, mice received daily intraperitoneal injections of LY294002 or dimethyl sulfoxide and phosphate-buffered saline (control) through P17. Retinal neovascularization was examined by adenosine diphosphatase staining after 5 days in room air and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to the inner limiting membrane. Results: LY294002 significantly inhibited VEGF-induced survival and migration. LY294002-treated and control animals demonstrated no perfusion regions in the posterior retina. Retinas from control mice at P17 contained neovascular tufts at the junction between the perfused and nonperfused retina. The tufts contained numerous neovascular nuclei. Retinas from mice treated with LY294002 demonstrated a significant reduction in neovascular cell nuclei compared with control mice. Conclusions: LY294002 significantly inhibits retinal neovascularization in a mouse model of retinal neovascularization.

Oxidative Stress Modulates Complement Factor H Expression in Retinal Pigmented Epithelial Cells by Acetylation of FOXO3

Age-related macular degeneration (AMD), the leading cause of severe vision loss in the elderly, is a complex disease that results from genetic modifications that increase susceptibility to environmental exposures. Smoking, a major source of oxidative stress, increases the incidence and severity of AMD, and antioxidants slow progression, suggesting that oxidative stress plays a major role. Polymorphisms in the complement factor H (CFH) gene that reduce activity of CFH increase the risk of AMD. In this study we demonstrate an interaction between these two risk factors, because oxidative stress reduces the ability of an inflammatory cytokine, interferon-gamma, to increase CFH expression in retinal pigmented epithelial cells. The interferon-gamma-induced increase in CFH is mediated by transcriptional activation by STAT1, and its suppression by oxidative stress is mediated by acetylation of FOXO3, which enhances FOXO3 binding to the CFH promoter, reduces its binding to STAT1, inhibits STAT1 interaction with the CFH promoter, and reduces expression of CFH. Expression of SIRT1, a mammalian homolog of NAD-dependent protein deacetylase sir2, attenuated FOXO3 recruitment to the CFH regulatory region and reversed the H(2)O(2)-induced repression of CFH gene expression. These data suggest an important interaction between environmental exposure and genetic susceptibility in the pathogenesis of AMD and, by elucidating molecular signaling involved in the interaction, provide potential targets for therapeutic intervention.

CpG DNA Activates Survival in Murine Macrophages through TLR9 and the Phosphatidylinositol 3-Kinase-Akt Pathway

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

Opposite Effects of Wortmannin and 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one Hydrochloride on Toll-Like Receptor-Mediated Nitric Oxide Production: Negative Regulation of Nuclear Factor-κB by Phosphoinositide 3-Kinase

A number of previous studies have suggested the involvement of phosphoinositide 3-kinase (PI3K) in Toll-like receptor (TLR) signaling. However, there have also been a number of conflicting reports. The PI3K inhibitor wortmannin greatly enhanced TLR-mediated inducible nitric-oxide synthase (iNOS) expression and cytokine production in the mouse macrophage cell line Raw264.7. The effect of wortmannin was common to TLR2, -3, -4, and -9 and was accompanied by activation of nuclear factor-kappaB and up-regulation of cytokine mRNA production. We were surprised to find that another PI3K inhibitor, LY294002, strongly suppressed the production of iNOS and cytokines. This effect of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) was based on its inhibitory effect on mRNA synthesis. Expression of dominant-negative mutants of PI3K in macrophages augmented the lipopolysaccharideinduced expression of iNOS. Introduction of a pH1 vector producing short hairpin RNA that targets a catalytic subunit of PI3K (p110beta) also enhanced the TLR-mediated responses. Thus, the augmentation of TLR signals by wortmannin was mediated through the inhibition of PI3K, whereas the effect of LY294002 was not explained by its effect on PI3K. These discrepancies in the effects of pharmacological inhibitors in TLR-signaling may have caused confusion regarding the role of PI3K in innate immunity.

GSK-3β reactivation with LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis

Constitutive activation of phosphatidylinositol 3-kinase (PI3K) confers resistance to apoptotic stimuli induced by chemotherapeutic agents in a variety of cancer cells. Therefore, the comprehension of mechanisms whereby PI3K downregulation interferes with chemotherapy is of major clinical interest for the elaboration of combined anticancer treatment modalities. Here, we examined the molecular mechanisms whereby the PI3K inhibitor LY294002 sensitized p53- and Fas-deficient hepatoma cells to etoposide and camptothecin. LY294002 increased Hep3B cell susceptibility to chemotherapy-induced apoptosis by enhancing the expression of DR4 and DR5 and the activation of caspase-8 and -3. Moreover, LY294002-mediated sensitization to chemotherapy involved mitochondrial Bax translocation and cytosolic cytochrome c accumulation. In Hep3B cells, LY294002 led to the reactivation of glycogen synthase kinase-3beta (GSK-3beta) by promoting its dephosphorylation on the serine 9 residue independently from Akt inhibition. The transient transfection of a constitutively active and non-phosphorylable S9AGSK-3beta mutant sensitized cells to etoposide cytotoxic effects while cell treatment with the small GSK-3beta inhibitor SB-415286 repressed the sensitizing effect of LY294002 on chemotherapy-induced apoptosis and caspase-8 activation. Altogether, our results show that LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis via death receptor and mitochondria signalling pathways and that GSK-3beta reactivation is involved in this process. Therefore, PI3K-mediated GSK-3beta inhibition could be a mechanism by which cancer cells escape from chemotherapy-induced apoptosis.

Glimepiride induces nitric oxide production in human coronary artery endothelial cells via a PI3-kinase-Akt dependent pathway

Diabetes mellitus is one of the major risk factors for coronary artery disease (CAD). A recent study reported that glimepiride, a new third-generation sulfonylurea, inhibited the formation of atheromatous plaques in high-cholesterol fed rabbits. However, the mechanism by which glimepiride induces atheroprotection remains unknown. In the present study, we tested the hypothesis that glimepiride may stimulate NO production in vascular endothelial cells. Human coronary artery endothelial cells (HCAECs) were treated with glimepiride, glibenclamide or vehicle, and NO release was measured. Akt phosphorylation was evaluated by Western blot. The effects of LY294002, a specific PI3-kinase inhibitor, and antisense oligonucleotides directed to Akt, on glimepiride-induced NO production were examined. Glimepiride (0.1–10 μM), but not glibenclamide, induced NO production, significantly increasing it by 1.8-fold ( n = 6, p < 0.05). LY294002 inhibited glimepiride-induced NO production by 68%. Akt was rapidly phosphorylated by glimepiride and antisense oligonucleotides directed to Akt completely inhibited glimepiride-induced NO production. These data demonstrate that glimepiride induces NO production in HCAECs by activating PI3-kinase and Akt, and also suggest that use of glimepiride in type 2 diabetes may show promise for preventing CAD in addition to lowering glucose levels.

PKB/Akt mediates radiosensitization by the signaling inhibitor LY294002 in human malignant gliomas

The phosphoinositide 3-kinase (PI3-kinase) signaling pathway is frequently aberrantly activated in glioblastoma multiforme (GM) by mutation or loss of the 3' phospholipid phosphatase PTEN. PTEN abnormalities result in inappropriate signaling to downstream molecules including protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR). PI3-kinase activation increases resistance to radiation-induced cell death; conversely, PI3-kinase inhibition enhances the sensitivity of tumors to radiation. The effects of LY294002, a biochemical inhibitor of PI3-kinase, on the response to radiation were examined in the PTEN mutant glioma cell line U251 MG. Low doses of LY294002 sensitized U251 MG to clinically relevant doses of radiation. In contrast to LY294002, rapamycin, an inhibitor of mTOR, did not result in radiosensitization. We demonstrate that among multiple known targets of LY294002, PI3-kinase is the most likely molecule responsible for LY294002-induced radiosensitization. Furthermore, using a myristoylated PKB/Akt construct, we identified PKB/Akt as the downstream molecule that mediates the synergistic cytotoxicity between LY294002 and radiation. Thus PI3-kinase dysregulation may contribute to the notable radioresistance of GM tumors and inhibition of PKB/Akt offers an excellent target to enhance radiosensitivity.