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4.
J Biol Chem ; 285(11): 8383-94, 2010 Mar 12.
Article in English | MEDLINE | ID: mdl-20068047

ABSTRACT

The Akt pathway is frequently hyperactivated in human cancer and functions as a cardinal nodal point for transducing extracellular and intracellular oncogenic signals and, thus, presents an exciting target for molecular therapeutics. Here we report the identification of a small molecule Akt/protein kinase B inhibitor, API-1. Although API-1 is neither an ATP competitor nor substrate mimetic, it binds to pleckstrin homology domain of Akt and blocks Akt membrane translocation. Furthermore, API-1 treatment of cancer cells results in inhibition of the kinase activities and phosphorylation levels of the three members of the Akt family. In contrast, API-1 had no effects on the activities of the upstream Akt activators, phosphatidylinositol 3-kinase, phosphatidylinositol-dependent kinase-1, and mTORC2. Notably, the kinase activity and phosphorylation (e.g. Thr(P)(308) and Ser(P)(473)) levels of constitutively active Akt, including a naturally occurring mutant AKT1-E17K, were inhibited by API-1. API-1 is selective for Akt and does not inhibit the activation of protein kinase C, serum and glucocorticoid-inducible kinase, protein kinase A, STAT3, ERK1/2, or JNK. The inhibition of Akt by API-1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor constitutively activated Akt. Furthermore, API-1 inhibited tumor growth in nude mice of human cancer cells in which Akt is elevated but not of those cancer cells in which it is not. These data indicate that API-1 directly inhibits Akt through binding to the Akt pleckstrin homology domain and blocking Akt membrane translocation and that API-1 has anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express hyperactivated Akt.


Subject(s)
Antineoplastic Agents/pharmacology , Nucleosides/pharmacology , Ovarian Neoplasms/drug therapy , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Animals , Antineoplastic Agents/chemistry , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Immediate-Early Proteins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Mice , Mice, Nude , NIH 3T3 Cells , Neoplasm Transplantation , Nucleosides/chemistry , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction/physiology , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases/metabolism
5.
J Biol Chem ; 282(15): 10981-7, 2007 Apr 13.
Article in English | MEDLINE | ID: mdl-17311912

ABSTRACT

The serine protease HtrA2/Omi is released from the mitochondria into the cytosol following apoptosis stimuli, leading to the programmed cell death in caspase-dependent and -independent manners. The function of HtrA2/Omi closely relates to its protease activity, which is required for cleavage of its substrate such as the members of the X-linked inhibitor of apoptotic protein family. However, the regulation of HtrA2/Omi by signaling molecule has not been documented. Here we report that serine/threonine kinases Akt1 and Akt2 phosphorylate mitochondria-released HtrA2/Omi on serine 212 in vivo and in vitro, which results in attenuation of its serine protease activity and pro-apoptotic function. Abolishing HtrA2/Omi phosphorylation by Akt through mutation of serine 212 to alanine (HtrA2/Omi-S212A) retains its serine protease activity and induces more apoptosis as compared with wild-type HtrA2/Omi. Conversely, HtrA2/Omi-S212D, a mutant mimicking phosphorylation, lost the protease activity and failed to induce the programmed cell death. Furthermore, the phosphorylated HtrA2/Omi fails to cleave X-linked inhibitor of apoptotic protein without interfering with their complex formation. In addition, Akt inhibits the release of HtrA2/Omi from the mitochondria into the cytoplasm in response to cisplatin treatment. These data reveal for the first time that HtrA2/Omi is directly regulated by Akt and provide a mechanism by which Akt induces cell survival at post-mitochondrial level.


Subject(s)
Mitochondrial Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Serine Endopeptidases/metabolism , Serine/metabolism , Animals , Apoptosis , Cell Line , Cytoplasm/enzymology , Down-Regulation , Gene Expression Regulation, Enzymologic , High-Temperature Requirement A Serine Peptidase 2 , Humans , Mice , Mitochondria/enzymology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Rats , Serine/genetics , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics
6.
Cancer Res ; 64(13): 4394-9, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15231645

ABSTRACT

Accumulated studies have shown that activation of the Akt pathway plays a pivotal role in malignant transformation and chemoresistance by inducing cell survival, growth, migration, and angiogenesis. Therefore, Akt is believed to be a critical target for cancer intervention. Here, we report the discovery of a small molecule Akt pathway inhibitor, Akt/protein kinase B signaling inhibitor-2 (API-2), by screening the National Cancer Institute Diversity Set. API-2 suppressed the kinase activity and phosphorylation level of Akt. The inhibition of Akt kinase resulted in suppression of cell growth and induction of apoptosis in human cancer cells that harbor constitutively activated Akt due to overexpression of Akt or other genetic alterations such as PTEN mutation. API-2 is highly selective for Akt and does not inhibit the activation of phosphatidylinositol 3'-kinase, phosphoinositide-dependent kinase-1, protein kinase C, serum- and glucocorticoid-inducible kinase, protein kinase A, signal transducer and activators of transcription 3, extracellular signal-regulated kinase-1/2, or c-Jun NH(2)-terminal kinase. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells in which Akt is aberrantly expressed/activated but not of those cancer cells in which it is not. These findings provide strong evidence for pharmacologically targeting Akt for anticancer drug discovery.


Subject(s)
Enzyme Inhibitors/pharmacology , Nuclear Proteins , Nucleosides/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridazines/pharmacology , Animals , Apoptosis/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Female , Humans , Immediate-Early Proteins , JNK Mitogen-Activated Protein Kinases , MAP Kinase Signaling System/drug effects , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NIH 3T3 Cells , Protein Kinase C/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-akt , Signal Transduction/drug effects , Signal Transduction/physiology , Substrate Specificity , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases
7.
J Biol Chem ; 278(44): 42992-3000, 2003 Oct 31.
Article in English | MEDLINE | ID: mdl-12933816

ABSTRACT

Recent studies have demonstrated that the cell growth and antiapoptotic actions of androgen could be dissociated from the transcriptional activity of the receptor and were, instead, mediated by activation of a mitogen-activated protein kinase pathway. This finding suggests an important cellular function of androgen receptor (AR) outside the nucleus. In this report, we demonstrate that androgen activates phosphatidylinositol 3-kinase (PI3K) and Akt, including AKT1 and AKT2, in AR-positive cells. Androgen-induced cell growth and survival were inhibited by PI3K inhibitor and dominant-negative Akt. AR interacts with the p85alpha regulatory subunit of PI3K, and its binding affinity is increased after androgen stimulation. The sites of interaction on the two proteins were mapped to the C-terminal Src-homology 2 domain of p85alpha and N terminus of AR. Activation of PI3K/Akt by androgen was inhibited by dominant-negative Src. Neither N-terminal truncated nor proline-rich region-deleted AR mutants, which are unable to bind to p85alpha and Src, respectively, was able to mediate androgen-induced PI3K/Akt activation. AR with deletion of C-terminal region including ligand binding domain, however, retains the ability to activate PI3K/Akt upon androgen stimulation, which supports the notion that nongenomic function of androgen is mediated by its interaction with membrane receptors (1, 3, 4). These findings indicate that a triple complex between AR, p85alpha, and Src is required for androgen-stimulated PI3K/Akt activation, and that the PI3K/Akt pathway, in addition to mitogen-activated protein kinase, mediates androgen-induced cell growth and cell survival.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Receptors, Androgen/metabolism , src-Family Kinases/metabolism , Apoptosis , Blotting, Western , Cell Division , Cell Line , Cell Line, Tumor , Cell Survival , Enzyme Activation , Glutathione Transferase/metabolism , Humans , Ligands , MAP Kinase Signaling System , Models, Biological , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary
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