Discovery of Small Molecule COX-1 and Akt Inhibitors as Anti-NSCLC Agents Endowed with Anti-Inflammatory Action
Mehlika Dilek AltıntopGülşen Akalın ÇiftçiNalan Yılmaz Savaşİpek ErtorunBetül CanBelgin SeverHalide Edip TemelÖzkan AlataşAhmet Özdemır
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Targeted therapies have come into prominence in the ongoing battle against non-small cell lung cancer (NSCLC) because of the shortcomings of traditional chemotherapy. In this context, indole-based small molecules, which were synthesized efficiently, were subjected to an in vitro colorimetric assay to evaluate their cyclooxygenase (COX) inhibitory profiles. Compounds 3b and 4a were found to be the most selective COX-1 inhibitors in this series with IC50 values of 8.90 µM and 10.00 µM, respectively. In vitro and in vivo assays were performed to evaluate their anti-NSCLC and anti-inflammatory action, respectively. 2-(1H-Indol-3-yl)-N′-(4-morpholinobenzylidene)acetohydrazide (3b) showed selective cytotoxic activity against A549 human lung adenocarcinoma cells through apoptosis induction and Akt inhibition. The in vivo experimental data revealed that compound 3b decreased the serum myeloperoxidase and nitric oxide levels, pointing out its anti-inflammatory action. Moreover, compound 3b diminished the serum aminotransferase (particularly aspartate aminotransferase) levels. Based on the in vitro and in vivo experimental data, compound 3b stands out as a lead anti-NSCLC agent endowed with in vivo anti-inflammatory action, acting as a dual COX-1 and Akt inhibitor.Keywords:
Anti-inflammatory
Activation of the serine-threonine kinase Akt promotes the survival and proliferation of various cancers. Hypoxia promotes the resistance of tumor cells to specific therapies. We therefore explored a possible link between hypoxia and Akt activity. We found that Akt was prolyl-hydroxylated by the oxygen-dependent hydroxylase EglN1. The von Hippel-Lindau protein (pVHL) bound directly to hydroxylated Akt and inhibited Akt activity. In cells lacking oxygen or functional pVHL, Akt was activated to promote cell survival and tumorigenesis. We also identified cancer-associated Akt mutations that impair Akt hydroxylation and subsequent recognition by pVHL, thus leading to Akt hyperactivation. Our results show that microenvironmental changes, such as hypoxia, can affect tumor behaviors by altering Akt activation, which has a critical role in tumor growth and therapeutic resistance.
Hydroxylation
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Akt is commonly overexpressed and activated in cancer cells and plays a pivotal role in cell survival, protection, and chemoresistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anticancer drugs. Here we examined whether a newly developed photo-activatable Akt (PA-Akt) probe, based on a light-inducible protein interaction module of plant cryptochrome2 (CRY2) and cryptochrome-interacting basic helixloophelix (CIB1), can regulate Akt-associated cell functions. By illuminating blue light to the cells stably transfected with PA-Akt probe, CRY2-Akt (a fusion protein of CRY2 and Akt) underwent a structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1), anchoring it at the cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probe-Akt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3, one of the main downstream signals of Akt, were also phosphorylated, depending on light intensity. These facts indicate that photo-activation of probe-Akt can activate endogenous Akt and its downstream signals. The photo-activated Akt conferred protection against nutritional deprivation and H 2 O 2 stresses to the cells significantly. Using the newly developed PA-Akt probe, endogenous Akt was activated easily, transiently, and repeatedly. This probe will be a unique tool in studying Akt-associated specific cellular functions in cancer cells and developing anticancer drugs.
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Targeting redox regulatory site of protein kinase B impedes neutrophilic inflammation in lung injury
Abstract Neutrophil activation has a pathogenic effect in inflammatory diseases. Protein kinase B (PKB)/AKT regulates diverse cellular responses. However, the significance of AKT in neutrophilic inflammation is still not well understood. Here, we identified CLLV-1 as a novel AKT inhibitor. CLLV-1 inhibited respiratory burst, degranulation, chemotaxis, and AKT phosphorylation in activated human neutrophils and dHL-60 cells. Significantly, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro . The AKT 309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Additionally, CLLV-1 ameliorated lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. CLLV-1 acts as a covalent allosteric AKT inhibitor by targeting AKT Cys310 to restrain inflammatory responses in human neutrophils and LPS-induced ALI in vivo . Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.
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Carboxy-terminal modulator protein (CTMP) was identified as binding to the carboxy terminus of Akt and inhibiting the phosphorylation and activation of Akt. In contrast to a previous study, we found CTMP overexpression to significantly enhance Akt phosphorylation at both Thr(308) and Ser(473) as well as the kinase activity of Akt, while phosphatidylinositol 3-kinase (PI3-kinase) activity was unaffected. Translocation of Akt to the membrane fraction was also markedly increased in response to overexpression of CTMP, with no change in the whole cellular content of Akt. Furthermore, the phosphorylations of GSK-3beta and Foxo1, well-known substrates of Akt, were increased by CTMP overexpression. On the other hand, suppression of CTMP with small interfering RNA partially but significantly attenuated this Akt phosphorylation. The cellular activities reportedly mediated by Akt activation were also enhanced by CTMP overexpression. UV-B-induced apoptosis of HeLa cells was significantly reversed not only by overexpression of the active mutant of Akt (myr-Akt) but also by that of CTMP. Increases in glucose transport activity and glycogen synthesis were also induced by overexpression of either myr-Akt or CTMP in 3T3-L1 adipocytes. Taking these results into consideration, it can be concluded that CTMP induces translocation of Akt to the membrane and thereby increases the level of Akt phosphorylation. As a result, CTMP enhances various cellular activities that are principally mediated by the PI3-kinase/Akt pathway.
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Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.
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The Serine/Threonine protein kinase B (PKB), which is now called Akt, has well-documented oncogenic potential and pro-survival activities that can counteract apoptosis induced by anti-cancer drugs. The goal of this review is to discuss current evidence that the pro-survival function of Akt can be overridden or converted to a pro-apoptotic function. A brief description of how upstream regulators and downstream effectors of the Akt kinase participate in a network of protection against cell death is presented. This background provides a basis for understanding how specific chemotherapeutic agents and cellular conditions can overcome the Akt pro-survival signal or alter Akt signaling in a way that converts Akt kinase activity to be directly involved in the induction of apoptosis. This pro-apoptotic activity only occurs under specific cellular conditions, since Akt can function as both a survival factor and an apoptotic factor within the same cell type. In some situations, the Akt pro-survival activity was eventually overwhelmed by prolonged treatment with chemotherapeutic agents, or was converted to a pro-apoptotic function upon prolonged hyperactivation of the Akt kinase activity, or by nuclear retention or unbalanced phosphorylation of the Akt protein. Increased levels of intracellular oxidation stimulated Akt activity and were increased by oxidative metabolism resulting from chronic Akt hyperactivity. Downstream effects on mTOR, FoxO3 transcription factors and cdk-2 affected the switch between pro-survival and proapoptotic functions through complex positive- and negative-feedback interactions. Upstream, caveolin-1 stimulated the pro-apoptotic function. Implications of the opposing functions of Akt in cancer therapy are discussed.
FOXO3
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The serine/threonine kinase, Akt, also known as PKB (Protein Kinase B) is one important signal transduction pathway that mediates the delay of neutrophil apoptosis caused by inflammatory mediators. Proteins controlled by the PKB/Akt pathway have been reported to prevent or reverse apoptotic-signaling pathways and regulate cell survival. In this review we discuss the role of PKB/Akt activation in the regulation of neutrophil activation during inflammation, and the importance of resolving the inflammatory response by inhibiting PKB/Akt activation and neutrophil survival. Furthermore, we introduce the concept of a dynamic Akt signal complex that is altered when an extracellular signal is initiated such that changes in protein-protein interactions within the Akt signal complex regulates Akt activity and cell survival. Various substrates of PKB/Akt as well as positive and negative regulators of PKB/Akt activation are discussed which in turn inhibit or enhance cellular survival.
Proto-Oncogene Proteins c-akt
AKT3
AKT2
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Abstract Akt, a protein kinase hyperactivated in many tumors, plays a major role in both cell survival and resistance to tumor therapy. A recent study, 1 along with other evidences, shows interestingly, that Akt is not a single‐function kinase, but may facilitate rather than inhibit cell death under certain conditions. This hitherto undetected function of Akt is accomplished by its ability to increase reactive oxygen species and to suppress antioxidant enzymes. The ability of Akt to down‐regulate antioxidant defenses uncovers a novel Achilles' heel, which could be exploited by oxidant therapies in order to selectively eradicate tumor cells that express high levels of Akt activity.
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AIM: The present study was designed to investigate the effect of adenovirus-mediated 14-3-3σ on Rat1-Akt cell xenografts and to explore whether the effect was mediated through negative regulation of Akt.METHODS: The effect of Ad-14-3-3σ on Rat1-Akt cells xenografts was observed in nude mice ex vivo and in vivo.Western blotting was used to detect 14-3-3σ protein,phospho-Akt(Thr 308),phospho-Akt(Ser 473),and phospho-(Ser/Thr) Akt substrate in tumor tissue after transfection of 14-3-3σ gene.RESULTS: The tumor volume was dramatically decreased and its emergence was delayed,regardless of using Ad-14-3-3σ-treated Rat1-Akt cells(ex vivo) or injected Ad-14-3-3σ in vivo,of which the effect of the continuously injected group was the best.Levels of Akt protein,phosphorylated Akt and phosphorylated Akt substrates in tumors obtained from Ad-14-3-3σ-treated mice were markedly less than those in PBS or Ad-β-gal-treated mice.CONCLUSION: 14-3-3σ suppressed Akt overexpession in cells of Rat1-Akt xenografts by negatively regulating Akt.
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Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named "Akt-in" (Akt inhibitor, NH2-AVTDHPDRLWAWEKF-COOH, encompassing the βA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases. Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named "Akt-in" (Akt inhibitor, NH2-AVTDHPDRLWAWEKF-COOH, encompassing the βA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases. Akt (also known as protein kinase B), a central component of the phosphoinositide 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PH, pleckstrin homology; GSK, glycogen synthesis kinase; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PDGF, platelet-derived growth factor; GST, glutathione S-transferase; GFP, green fluorescent protein; TUNEL, terminal dUTP nick-end labeling; H & E, hematoxylin and eosin; FKHR, fork head transcription factor; HA, hemagglutinin; MAP, mitogen-activated protein; MAPK, MAP kinase; PDK, phosphoinositide-dependent protein kinase. signaling pathways, has emerged as a pivotal regulator of many cellular processes (1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (882) Google Scholar, 2Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4770) Google Scholar, 3Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar, 4Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (973) Google Scholar). Three highly homologous Akt isoforms (Akt1, Akt2, and Akt3) exist in mammals. Akt is composed of three functionally distinct regions: an N-terminal pleckstrin homology (PH) domain, a central catalytic domain, and a C-terminal hydrophobic region. The PH domain is a small 100–120-residue module found in many proteins involved in cell signaling or cytoskeletal rearrangement. The PH domain of Akt is similar to other proteins, and it consists of seven β strands forming two orthogonal antiparallel β-sheets that are closed with the C-terminal α-helix (5Auguin D. Barthe P. Auge-Senegas M.T. Stern M.H. Noguchi M. Roumestand C. J. Biomol. NMR. 2004; 28: 137-155Crossref PubMed Scopus (46) Google Scholar, 6Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 7Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (443) Google Scholar, 8Milburn C.C. Deak M. Kelly S.M. Price N.C. Alessi D.R. Van Aalten D.M. Biochem. J. 2003; 375: 531-538Crossref PubMed Scopus (234) Google Scholar, 9Thomas C.C. Deak M. Alessi D.R. van Aalten D.M. Curr. Biol. 2002; 12: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In response to growth factors and other extracellular stimuli, Akt is activated by the lipid products (PtdIns (3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2) of PI3K, which phosphorylates the 3-OH position of the inositol core of inositol phospholipids (PtdIns) (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar, 11Frech M. Andjelkovic M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 12James S.R. Downes C.P. Gigg R. Grove S.J. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (275) Google Scholar). Recent structural studies have located the binding pocket of PtdIns(1,3–5) P4 (the polar head group of PtdIns(3,4,5)P3) in variable loop 1 (VL1, the loop between the β1 and β2strands) of the Akt PH domain (5Auguin D. Barthe P. Auge-Senegas M.T. Stern M.H. Noguchi M. Roumestand C. J. Biomol. NMR. 2004; 28: 137-155Crossref PubMed Scopus (46) Google Scholar, 7Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (443) Google Scholar, 8Milburn C.C. Deak M. Kelly S.M. Price N.C. Alessi D.R. Van Aalten D.M. Biochem. J. 2003; 375: 531-538Crossref PubMed Scopus (234) Google Scholar, 13Ferguson K.M. Kavran J.M. Sankaran V.G. Fournier E. Isakoff S.J. Skolnik E.Y. Lemmon M.A. Mol. Cell. 2000; 6: 373-384Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). An association with PtdIns(3,4,5)P3 induces a conformational change of Akt, which allows phosphoinositide-dependent protein kinase 1 (PDK1) to access and phosphorylate threonine 308 (Thr-308) (14Lawlor M.A. Mora A. Ashby P.R. Williams M.R. Murray-Tait V. Malone L. Prescott A.R. Lucocq J.M. Alessi D.R. EMBO J. 2002; 21: 3728-3738Crossref PubMed Scopus (276) Google Scholar, 15Collins B.J. Deak M. Arthur J.S. Armit L.J. Alessi D.R. EMBO J. 2003; 22: 4202-4211Crossref PubMed Scopus (161) Google Scholar) in the so-called activation loop. It is also regulated by phosphorylation events within the conserved C-terminal hydrophobic motif (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar, 15Collins B.J. Deak M. Arthur J.S. Armit L.J. Alessi D.R. EMBO J. 2003; 22: 4202-4211Crossref PubMed Scopus (161) Google Scholar). Both serine 473 (Ser-473) phosphorylation and membrane anchoring are required for Thr-308 phosphorylation (16Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (348) Google Scholar) and complete activation of Akt (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar, 15Collins B.J. Deak M. Arthur J.S. Armit L.J. Alessi D.R. EMBO J. 2003; 22: 4202-4211Crossref PubMed Scopus (161) Google Scholar). Over 20 molecules have been identified as potential physiological substrates of Akt, including GSK3α (glycogen synthesis kinase3α), GSK3β, fork head transcription factor (FKHR), BAD, and endothelial nitric-oxide synthase (1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (882) Google Scholar, 17Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3754) Google Scholar). Activation of Akt promotes cell survival (18Chen W.S. Xu P.Z. Gottlob K. Chen M.L. Sokol K. Shiyanova T. Roninson I. Weng W. Suzuki R. Tobe K. Kadowaki T. Hay N. Genes Dev. 2001; 15: 2203-2208Crossref PubMed Scopus (792) Google Scholar); thus, it could be the underlying mechanism for numerous human neoplastic diseases including lung, ovarian, and prostate cancers (1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (882) Google Scholar, 19Luo J. Manning B.D. Cantley L.C. Cancer Cells. 2003; 4: 257-262Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). Activation of Akt is also induced in the mutation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene. PTEN antagonizes PI3K function by the reduction in the levels of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Mutations of PTEN are implicated in several tumor types, including glioblastoma, endometrial tumors, and Cowden's syndrome (20Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1768) Google Scholar, 21Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2146) Google Scholar). We have demonstrated that the proto-oncogene TCL1 is an Akt kinase co-activator (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 23Pekarsky Y. Koval A. Hallas C. Bichi R. Tresini M. Malstrom S. Russo G. Tsichlis P. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3028-3033Crossref PubMed Scopus (317) Google Scholar, 24Gold M.R. Trends Immunol. 2003; 24: 104-108Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 25Auguin D. Barthe P. Royer C. Stern M.H. Noguchi M. Arold S.T. Roumestand C. J. Biol. Chem. 2004; 279: 35890-35902Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). TCL1 contains two distinct functional motifs responsible for Akt association and homodimerization. Both Akt association and homodimerization of TCL1 are required for the complete function of TCL1 to enhance Akt kinase activity. TCL1 binds to Akt and activates Akt via a transphosphorylation reaction (26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar, 27Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). TCL1 oncogene was first implicated in human T cell prolymphocytic leukemia, a chronic adulthood leukemia (28Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (83) Google Scholar). Under physiological conditions, TCL1 expression is limited to early developmental stages such as the immune system (24Gold M.R. Trends Immunol. 2003; 24: 104-108Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Pekarsky Y. Hallas C. Croce C.M. Oncogene. 2001; 20: 5638-5643Crossref PubMed Scopus (83) Google Scholar, 29Narducci M.G. Fiorenza M.T. Kang S.M. Bevilacqua A. Di Giacomo M. Remotti D. Picchio M.C. Fidanza V. Cooper M.D. Croce C.M. Mangia F. Russo G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11712-11717Crossref PubMed Scopus (81) Google Scholar). Because the PI3K-Akt pathway is involved in various human neoplastic diseases, Akt represents an attractive target for drug development (19Luo J. Manning B.D. Cantley L.C. Cancer Cells. 2003; 4: 257-262Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 30Lock R.B. Int. J. Biochem. Cell Biol. 2003; 35: 1614-1618Crossref PubMed Scopus (11) Google Scholar). A small peptide was proven to effectively modulate activity of kinases effectively (31Barr R.K. Boehm I. Attwood P.V. Watt P.M. Bogoyevitch M.A. J. Biol. Chem. 2004; 279: 36327-36338Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 32Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 33Datta K. Sundberg C. Karumanchi S.A. Mukhopadhyay D. Cancer Res. 2001; 61: 1768-1775PubMed Google Scholar). One class of Akt inhibitors under development is based on the cross-reactivity between known kinase inhibitors (e.g. cyclic AMP-dependent protein kinase (PKA) or PI3K) (34Reuveni H. Livnah N. Geiger T. Klein S. Ohne O. Cohen I. Benhar M. Gellerman G. Levitzki A. Biochemistry. 2002; 41: 10304-10314Crossref PubMed Scopus (114) Google Scholar, 35Hu Y. Qiao L. Wang S. Rong S.B. Meuillet E.J. Berggren M. Gallegos A. Powis G. Kozikowski K. J. Med. Chem. 2000; 43: 3045-3051Crossref PubMed Scopus (199) Google Scholar, 36Niv M.Y. Rubin H. Cohen J. Tsirulnikov L. Licht T. Peretzman-Shemer A. Cna'an E. Tartakovsky A. Stein I. Albeck S. Weinstein I. Goldenberg-Furmanov M. Tobi D. Cohen E. Laster M. Ben-Sasson S.A. Reuveni H. J. Biol. Chem. 2003; 279: 1242-1255Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar); however, these drugs are not specific for Akt. With the goal to develop a putative Akt kinase inhibitor, we hypothesized that a peptide, which is spanning the Akt-binding site, binds to Akt and modulates Akt kinase activity along with its downstream biological responses. Based on the binding domain of TCL1 with Akt, we identified and characterized a peptide that encompassed the βA strand of TCL1, interacted with Akt, and inhibited Akt kinase activity. Akt-in prevented PtdIns binding to Akt, and consequently it inhibited membrane translocation of Akt and its downstream biological responses. Given the pivotal role of Akt kinase as a core intracellular survival factor implicated in the molecular mechanisms of human neoplastic diseases, the results could help to design Akt kinase-specific inhibitors for therapeutic approaches. Peptide Design—For the Akt-in peptides, the amino acid positions 10–24 of human TCL1, NH2-AVTDHPDRLWAWEKF-COOH are used. For TAT-FLAG Akt-in, the sequence is YGRKKRRQRRRDYKDDDDKAVTDHPDRLWAWEKF-COOH. Control Peptides—For βC peptides, the amino acid positions 29–40 of human TCL1 are used, NH2-EKQHAWLPLTIE-COOH. For TAT-βC, the sequence is H2-YGRKKRRQRRREKQHAWLPLTIE-COOH; and for TAT-FLAG, the sequence is NH2-YGRKKRRQRRR-DYKDDDDK-COOH. For functional assays, the Akt-in peptide was fused with TAT (YGRKKRRQRRR) (37Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (891) Google Scholar). The peptides were either purchased from Sigma Genosys and Applied Biosystems (SynthAssist®) or synthesized using FMOC (N-(9-fluorenyl)methoxycarbonyl)-protected amino acids and standard 1-benzotriazolyloxy-trisdemethylamino-phosphonium-hexafluorophosphate-N-hydroxybenzotriazole-coupling methods as reported previously (38Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Co-immunoprecipitation Assay—Co-immunoprecipitation assays were performed as described previously (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Briefly, Akt1, Akt2, or Akt3 in pCMV6 was transfected into 293 cells (ATCC). The cells were then harvested, lysed, and pre-cleaned with protein G/A-agarose mixture (50% v/v, Pro-G/A, Amersham Biosciences). FLAG-Akt-in or control peptides (βC) at 400 μm were added to the cell lysates, incubated at 4 °C for 3 h, and incubated with Pro-G/A preconjugated with anti FLAG M2 antibody (Sigma). The resultant immune precipitants were washed and run on SDS-PAGE and immunoblotted with anti-HA antibody (3F10, Roche Applied Science). GST Pull-down Assay—293T cells (ATCC) were transfected with 10 μg of FLAG-tagged wild type Akt3, PH domain, or C-terminal Akt3 (27Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The cell lysates were immunoprecipitated with anti-FLAG antibody (FLAG M2, Sigma) bound to Pro-G/A (27Laine J. Kunstle G. Obata T. Noguchi M. J. Biol. Chem. 2002; 277: 3743-3751Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Fifty ng of GST fusion proteins were incubated with 20 μl of immobilized Akt3, PH domain, or C-terminal Akt. The samples were run on SDS gels and immunoblotted with anti-GST antibody (Amersham Biosciences). The results were consistent in at least three independent experiments. GST fusion Akt-in was generated by subcloning with the corresponding nucleotide into pGEX4T-2 vectors (Amersham Biosciences). All nucleotide sequences were verified before the experiments. GST Competition Assay—Recombinant GST-Akt-in fusion protein was generated by pGEX 4T-2 Vector (Amersham Biosciences) using oligonucleotide pairs (5′-aattcgcagtcaccgaccacccggaccgcctgtgggcctgggagaagttctagg-3′). 0.1 μg of Akt (activated, Upstate Biotechnology, Inc.) was incubated with TAT-FLAG, TAT-Akt-in, or TAT-βC at the concentration of 0, 50, 100, or 250 μm in HEPES Binding Buffer (20 mm HEPES (pH 7.0), 150 mm NaCl, 0.5 μg/μl bovine serum albumin). 0.1 μg of GST-Akt-in was then added and incubated for an additional 20 min at 4 °C. Twenty μl of immobilized Akt beads (Cell Signaling) were added to the sample, washed five times with HEPES Binding Buffer in the presence of 0.1% Nonidet P-40, resolved onto an SDS gel, and immunoblotted by anti-Akt (Cell Signaling) or anti-GST (Amersham Biosciences) antibodies using ECL (Amersham Biosciences). Kinetics of Akt-in—The kinetics of Akt-in with the human Akt2-PH domain (amino acid 1–125 of human Akt2) was performed using the Applied Biosystems 8500 Affinity Chip Analyzer. Briefly, His fusion protein of human Akt2-PH domain was generated using pQE30 (Qiagen) by PCR. 1.25 pg of GST fusion proteins (Akt-in or wild type TCL1) were spotted onto the protein-A/G Affinity Chips preconjugated with anti-GST antibody (Sigma). Fifty μm of His-Akt2-PH domain was applied, and the dissociation constant was calculated by using data analysis software (Applied Biosystems). The values (mean ± S.D.) were calculated from the 80 measurements. In Vitro Akt Kinase Assay—In vitro Akt kinase assays were performed using the Akt kinase assay kit (Cell Signaling) (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Briefly, the immobilized Akt was incubated with 0, 200, or 400 μm of indicated peptides for 2 h, and then an in vitro kinase assay reaction was performed for 4 min at 30 °C. The samples were heat-denatured, separated on SDS-PAGE, and immunoblotted with anti-phospho-GSK or anti-Akt (Cell Signaling) using ECL (Amersham Biosciences). PKA Kinase Assay—In vitro PKA kinase assays were performed using Peptag (Promega) as described previously (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The indicated concentrations of peptides (Akt-in or TAT-FLAG control peptide) were incubated with 25 ng of PKA with 100 ng of bovine serum albumin for 1 h in the presence or absence of 2 μm PKA inhibitor (Calbiochem catalog number 116805), followed by the kinase reaction for 20 min at 26 °C, and then separated on 0.8% TBE-agarose gel. In Vitro PDK1 Kinase Assay—Purified baculovirus-derived recombinant His-PDK-1-WT (1.0 μg/reaction (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar)) was incubated with or without the indicated concentrations of peptides (Akt-in or TAT-FLAG) in 20 μl of reaction mixture containing 20 mm HEPES-NaOH (pH 7.4), 100 mm NaCl, 10 mm MgCl2, 0.5 mm EGTA, 1 mm dithiothreitol, 100 μm cold ATP, and 3 μCi of [γ-32P]ATP for 10 min at 30 °C. The reactions were analyzed by SDS-PAGE with Coomassie staining followed by autoradiography (shown in Fig. 3D). Phosphorylation of Akt, BAD, FKHR, or p44/42 MAP Kinase in 293 Cells—293 cells (ATCC) were cultured in a 60-mm dish and transfected (or nontransfected in Fig. 3B) with 5 μg of m-BAD (Fig. 3C, pEBGmBad, Cell Signaling) using calcium phosphate transfection as described previously (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). 24 h after transfection, the cells were serum-starved (0.2% fetal bovine serum) and treated with either control (TAT-FLAG) or Akt-in (TAT-Akt-in) at 50 μm for additional 12 h. The cells were stimulated with or without 20 ng/ml PDGF for 8 (in Fig. 3C) or 5 min (in Fig. 3B) and lysed with Brij lysis buffer in the presence of phosphatase inhibitors (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), and the resultant samples were resolved on 4–20% SDS gel (Kyoto Daiichi Kagaku Co., Ltd.). They were then immunoblotted with the indicated antibodies purchased from Cell Signaling (Akt, 9272, phospho-Ser-473 Akt 9271, phospho-Ser-308 Akt 9275, Ser-256 FKHR 9461, FKHR 9462, phospho-Ser-136 BAD 9295, BAD 9292, anti-p44/42 MAP kinase 9102, anti-phospho-p44/42 MAP kinase 9106, anti-38 MAP kinase 9212, and phospho-p38 MAP kinase antibody 9216), and detected by ECL (Amersham Biosciences). NMR Experiment—NMR spectra were recorded on 0.25–0.3-ml (Shigemi tubes pre-coated with a silicon solution (Sigma)) samples of 0.05 mm15N-labeled Akt2-PH dissolved in the conditioning buffer (10 mm Tris/H2O (pH 7.4), 300 mm NaCl, 0.1 mm benzamidine, 0.1 mm EDTA, with 5–10% 2H2O for the lock), in the presence or absence of 20 mmAkt-in. NMR experiments were carried out at 10 °C on a Bruker AVANCE 600 spectrometer equipped with 5-mm z-shielded gradient 1H-13C-15N triple resonance cryogenic probe. 1H chemical shifts were directly referenced to the resonance of 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt, and 15N chemical shifts were indirectly referenced with the absolute frequency ratios (15N/1H) = 0.101329118. In all experiments, the 1H carrier was centered on the water resonance, and a WATERGATE sequence was incorporated to suppress the solvent resonance. All NMR spectra were acquired in the phase-sensitive mode with Digital Quadrature Detection in the F2 dimension and hypercomplex States-TPPI method in F1 dimension and processed using Gifa (version 4.22) software. 1H,15N-HSQC spectra were recorded using a time domain data size of 64 t1 × 1K t2 complex points and 32 transients per complex t1 increment. PtdIns(3,4,5)P3 Lipid-Protein Pull-down Assay—A lipid-protein pull-down assay was performed using PIP Beads (PtdIns(3,4,5)P3, Echelon Bioscience Inc.). Indicated peptides (Akt-in or βC control) were incubated with 50 ng of Akt kinase (unactivated, Upstate Biotechnology Inc., catalog number 14-279) with 400 ng/ml bovine serum albumin for 2 h with gentle agitation at 4 °C. TAT-FLAG control was added to adjust the final peptide concentration to be equal throughout the samples. 25 μl of PIP Beads were then added to each sample and incubated for an additional 16 h. The reactions were then washed four times with washing buffer (10 mm HEPES (pH 7.4), 0.25% Nonidet P-40, 140 mm NaCl), resolved on SDS gel, and immunoblotted by ECL (Amersham Biosciences). Membrane Translocation Experiment—293 cells (ATCC) were grown on a poly-l-lysine-coated cover glass and were transfected with 1 μg of HA-Akt1 or Akt-PH-GFP or Btk (Bruton tyrosine kinase)-PH-GFP in a mammalian expression vector (39Varnai P. Rother K.I. Balla T. J. Biol. Chem. 1999; 274: 10983-10989Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) using FuGENE 6 (Roche Applied Science). Six hours after the transfection, 50 μmAkt-in or TAT-FLAG control was added and incubated for 16 h. The cells were then serum-starved (0.5%) and then incubated for an additional 24 h. The cells were treated with or without 50 nm wortmannin for 20 min, stimulated with 50 ng/ml PDGF-AB (Sigma, 3226) for 10 min, fixed with 4% paraformaldehyde, stained with 10 ng/ml fluorescein isothiocyanate-conjugated anti-HA antibody (12CA5, MBL) or phospho-Ser-473 antibody (587F11, Cell Signaling), and examined using a confocal microscope (Nikon). Proliferation Assay—Cell growth was assessed by a colorimetric method using WST-8 regent (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) (347-07621, Dojin) according to the manufacturer's instructions. Briefly, 2000 T4 cells were seeded into each well of 96-well plate. The peptides (TAT-βC or TAT-Akt-in) were added at the indicated concentrations. Three days later, WST-8 regent was added and incubated for an additional 4 h at 37 °C, and the absorbance was determined using a microplate reader (Bio-Rad). Cell Death Assay and Mitochondrial Permeability Transition Assay—T4 cells (human T cell leukemia cells) were treated with the indicated concentrations of peptide (TAT-FLAG control or TAT-Akt-in) for 24 h. The cells were transfected with either myr-Akt (Upstate Biotechnology, Inc., catalog number 17-253) or a control. Cell death was assessed by staining with 2 μg/ml propidium iodide. Mitochondrial permeability transition was verified by staining with rhodamine 123 (Molecular Probe) at 5 μm for 15 min at 37 °C (26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar) and analyzed using fluorescence-activated cell sorter (Cell Quest). In Vivo Tumor Growth—Fibrosarcoma cells (QRsP-11 cells, 2 × 105 cells per mouse) were subcutaneously transplanted into syngeneic C57BL/6 mice (eight mice in each group) (40Okada F. Hosokawa M. Hamada J.I. Hasegawa J. Kato M. Mizutani M. Ren J. Takeichi N. Kobayashi H. Br. J. Cancer. 1992; 66: 635-639Crossref PubMed Scopus (62) Google Scholar). Two micromoles of the indicated peptides (TAT-Akt-in, TAT-FLAG, or phosphate-buffered saline) per mouse were injected directly into the tumor on days 5, 7, 10, 12, 14, 17, and 19. In vivo cell growth was calculated based on the diameter of the tumor. On day 9 after transplantation, the tumors were resected, fixed in formalin, embedded in paraffin, stained with hematoxylin and eosin (H & E), TUNEL (terminal dUTP nick-end labeling, MK500, Takara Shuzo), or phospho-Akt (Ser-473) monoclonal antibody (587F11, Cell Signaling). Peptide Design of Akt-in and the Structure of TCL1—TCL1 forms a closed symmetrical β-barrel structure, consisting of eight antiparallel β strands (41Hoh F. Yang Y.S. Guignard L. Padilla A. Stern M.H. Lhoste J.M. van Tilbeurgh H. Structure (Lond.). 1998; 6: 147-155Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) (Fig. 1A). In our previous studies, we showed that the surface composed of βA and βE strands of TCL1 mediated the interaction with Akt (22Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar, 42French S.W. Shen R.R. Koh P.J. Malone C.S. Mallick P. Teitell M.A. Biochemistry. 2002; 41: 6376-6382Crossref PubMed Scopus (34) Google Scholar) (Fig. 1A, top surface). Both dimerization and Akt interaction are essential for the full function of TCL1 to activate Akt (26Künstle G. Laine J. Pierron G. Kagami S. Nakajima H. Hoh F. Roumenstand C. Stern M-H. Noguchi M. Mol. Cell. Biol. 2002; 22: 1513-1525Crossref PubMed Scopus (84) Google Scholar). We hypothesized that a peptide, which spans the Akt-binding sequences, can modulate Akt kinase activity and its downstream signals. We designed a peptide (named Akt-in, Akt inhibitor, positions 10–24 of human TCL1, NH2-AVTDHPDRLWAWEKF-COOH), which encompasses the βA strand of TCL1 for further study (Fig. 1B). For functional assays, the Akt-in peptide (amino acid positions 10–24 of TCL1, Fig. 1B) was fused with TAT (YGRKKRRQRRR) and/or FLAG epitope (DYKDDDDK).
Pleckstrin homology domain
LY294002
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