mk-2206 and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Mucosal hyperplasia is common sequela of otitis media (OM), leading to the secretion of mucus and the recruitment of leukocytes. However, the pathogenic mechanisms underlying hyperplasia are not well defined. Here, we investigated the role of the AKT pathway in the development of middle mucosal hyperplasia using in vitro mucosal explants cultures and an in vivo rat model. The Akt inhibitor MK2206 treatment inhibited the growth of middle ear mucosal explants in a dose-dependent manner. In vivo, MK2206 also reduced mucosal hyperplasia. Unexpectedly, while PTEN is generally thought to act in opposition to AKT, the PTEN inhibitor BPV reduced mucosal explant growth in vitro. The results indicate that both AKT and PTEN are mediators of mucosal growth during OM, and could be potential therapeutic targets.

Topics: Animals; Disease Models, Animal; Female; Heterocyclic Compounds, 3-Ring; Humans; Hyperplasia; Male; Mice; Mice, Inbred C57BL; Mucous Membrane; Otitis Media; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley; Signal Transduction

BACKGROUND Oxidative stress and myocardial apoptosis are features of doxorubicin-induced cardiac toxicity that can result in cardiac dysfunction. Previous studies showed that microRNA-143 (miR-143) was expressed in the myocardium and had a role in cardiac function. This study aimed to investigate the effects and possible molecular mechanisms of miR-143 on oxidative stress and myocardial cell apoptosis in a mouse model of doxorubicin-induced cardiac toxicity. MATERIAL AND METHODS Mice underwent intraperitoneal injection of doxorubicin (15 mg/kg) daily for eight days to develop the mouse model of doxorubicin-induced cardiac toxicity. Four days before doxorubicin administration, a group of mice was pretreated daily with a miR-143 antagonist (25 mg/kg/day) for four consecutive days by tail vein injection. The study included the use of a miR-143 antagomir, or anti-microRNA, an oligonucleotide that silenced endogenous microRNA (miR), and an agomir to miR-143, and also the AKT inhibitor, MK2206. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblot analysis were used to measure mRNA and protein expression, respectively. RESULTS Doxorubicin treatment increased the expression of miR-143, which was reduced by the miR-143 antagomir. Overexpression of miR-143 increased doxorubicin-induced myocardial apoptosis and oxidative stress. The use of the miR-143 antagomir significantly activated protein kinase B (PKB) and AKT, which were reduced in the presence of the AKT inhibitor, MK2206. However, the use of the miR-143 antagomir further down-regulated AKT phosphorylation following doxorubicin treatment and increased AKT activation. CONCLUSIONS In a mouse model of doxorubicin-induced cardiac toxicity, miR-143 increased oxidative stress and myocardial cell apoptosis following doxorubicin treatment by inhibiting AKT.

Topics: Animals; Antagomirs; Apoptosis; Cardiotoxicity; Cell Line; Disease Models, Animal; Doxorubicin; Heart; Heterocyclic Compounds, 3-Ring; Male; Mice; MicroRNAs; Myocardium; Myocytes, Cardiac; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Up-Regulation

The cellular and molecular mechanisms via which MK2206, an AKT inhibitor, prevents the activation of AKT in toluene diisocyanate (TDI)‑induced asthma remain unclear. Thus, the present study aimed to evaluate the potential effects of MK2206 on airway AKT activation, inflammation and remodeling in a TDI‑induced mouse model of asthma. A total of 24 BALB/c mice were selected and randomly divided into untreated (AOO), asthma (TDI), MK2206 (TDI + MK2206), and dexamethasone (TDI + DEX) groups. Phosphorylated AKT (p‑AKT), total AKT, airway remodeling indices, α‑smooth muscle actin (α‑SMA) and collagen I levels in pulmonary tissue were measured using western blotting. Airway inflammation factors, including interleukin (IL)‑4, ‑5, ‑6, and ‑13 in bronchoalveolar lavage fluid (BALF) and IgE in serum, were determined using ELISA. Additionally, the airway hyperresponsiveness (AHR) and pulmonary pathology of all groups were evaluated. The results of the present study demonstrated that p‑AKT levels in lung protein lysate were upregulated, and neutrophil, eosinophil and lymphocyte counts were increased in the lungs obtained from the asthma group compared with the AOO group. Both MK2206 and DEX treatment in TDI‑induced mice resulted not only in the attenuation of AKT phosphorylation, but also reductions in neutrophil, eosinophil and lymphocyte counts in the lungs of mice in the asthma group. Consistently, increases in the levels of the inflammatory cytokines IL‑4, ‑5, ‑6 and ‑13 analyzed in BALF, and serum IgE in the TDI group were demonstrated to be attenuated in the TDI + MK2206 and TDI + DEX groups. Furthermore, α‑SMA and AHR were significantly attenuated in the TDI + MK2206 group compared with the TDI group. These results revealed that MK2206 not only inhibited AKT activation, but also served a role in downregulating airway inflammation and airway remodeling in chemical‑induced asthma. Therefore, the findings of the present study may provide important insight into further combination therapy.

Topics: Airway Remodeling; Animals; Anti-Inflammatory Agents; Asthma; Cytokines; Dexamethasone; Disease Models, Animal; Heterocyclic Compounds, 3-Ring; Immunoglobulin E; Inflammation; Male; Mice; Mice, Inbred BALB C; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Toluene 2,4-Diisocyanate; Treatment Outcome

Cardiac steroids (CSs), such as ouabain and digoxin, increase the force of contraction of heart muscle and are used for the treatment of congestive heart failure (CHF). However, their small therapeutic window limits their use. It is well established that Na

Topics: Animals; Cardiotonic Agents; Cells, Cultured; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Heterocyclic Compounds, 3-Ring; Isolated Heart Preparation; Male; Myocardial Contraction; Myocardial Infarction; Myocytes, Cardiac; Ouabain; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction; Ventricular Function, Left

Hepatitis B virus X (HBx), a viral onco-protein encoded by HBV, can promote oncogenesis of HCC. However, the mechanism of HBx in hepatocarcinogenesis is still unclear. In this study, we establish a new mouse model with normal immune system to investigate the role of HBx and its functional mechanisms under normal immune function. The animal model was established by injecting HBx-EGFP-14-19 cells into the hepatic portal vein of KM mice. To verify the mouse model, the expression of HBx in the liver tissue of mice was detected by qRT-PCR, western blotting and immunohistochemistry. The apoptosis index was calculated using the terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, and the expression levels of apoptosis-related and cell cycle-related factors were measured. Moreover, expression of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway was detected in HBx-EGFP-14-19 mice with and without use of an Akt inhibitor. The results showed the HBx was successfully overexpressed in liver of KM mice. After overexpressing HBx, the apoptosis index was downregulated in HBx-EGFP-14-19 liver tissue, and the expression levels of caspase-9 and Bad were reduced, but Bcl-xl was increased in HBx-EGFP-14-19 liver tissue. Overexpression of HBx increased the expression of the cyclin-dependent kinase 2 (CDK2), cyclinD1 and cyclinE. Moreover, compared with the low-level HBx group, p-Akt and p-mTOR were increased in the livers of mice with high levels of HBx. However, inactivation of apoptosis by overexpression of HBx was abolished by the treatment with an Akt inhibitor. These results indicate that HBx can induce anti-apoptosis mechanisms in hepatocytes in vivo, which is mediated by the Akt/mTOR signaling pathway.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line; Cell Proliferation; Dependovirus; Disease Models, Animal; Hepatitis B virus; Hepatocytes; Heterocyclic Compounds, 3-Ring; Humans; Liver; Liver Neoplasms; Mice; Neoplasms, Experimental; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Trans-Activators; Up-Regulation; Viral Regulatory and Accessory Proteins

To explore the effects and mechanism of action of micro ribonucleic acid (miR)-21 on the proliferation and migration of vascular smooth muscle (VSM) in atherosclerosis (AS).. The rats were fed with a high-fat diet, and the oil red staining was adopted to compare AS between Sprague Dawley (SD) rats and miR-21 knockdown rats. At the in-vitro level, primary rat VSM cells (VSMCs) were selected and divided into miR-NC blank control group [miR-normal control (NC) group] and miR-21 overexpression group (miR-21 group) for relevant experimental detection. Wound healing assay and transwell assay were used to detect the effects of miR-21 on the proliferation and migration of VSMCs. Westernn blotting was applied to examine the changes in the levels of Cyclin D, a cell cycle-related protein, and the key factors of the Akt/ERK signaling pathway, such as phosphorylated-Akt (p-AKT), AKT, p-ERK1/2, and ERK1/2. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell activity assay kit was applied to determine the effects of miR-21 on the proliferation of VSMCs through regulating the Akt/ERK signaling pathway after the ERK signaling pathway inhibitor PD98059 and AKT inhibitor MK-2206 were given.. Compared with that in miR-NC group, the level of AS in miR-21 knockdown rats were decreased significantly (p < 0.05). In the cell-level experiment, the overexpression of miR-21 promoted abnormal proliferation of VSMCs and activated the Akt/ERK signaling pathway (p < 0.05). MTT assay results revealed that inhibiting the Akt/ERK pathway could reverse the effects of miR-21 promoting proliferation and migration.. MiR-21 promotes the proliferation and migration of VSMCs by activating the Akt/ERK pathway and aggravates AS. Knocking down miR-21 or inhibiting the Akt/ERK pathway can suppress the activation of VSMCs.

Topics: Animals; Atherosclerosis; Cell Movement; Cell Proliferation; Cells, Cultured; Diet, High-Fat; Disease Models, Animal; Flavonoids; Gene Expression Regulation; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Male; MAP Kinase Signaling System; MicroRNAs; Muscle, Smooth, Vascular; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley

In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca. The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I. H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I

Topics: Animals; Blotting, Western; Calcium Channels, L-Type; Carrier Proteins; Cell Line; Chromones; CREB-Binding Protein; Disease Models, Animal; DNA; Enzyme Inhibitors; Estradiol; Estrogens; Gene Expression Regulation; Heterocyclic Compounds, 3-Ring; Long QT Syndrome; Morpholines; Myocytes, Cardiac; Patch-Clamp Techniques; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Up-Regulation

A high recurrence rate after medical treatment is a major clinical problem for patients with endometriosis. Here, we have evaluated the in vitro effects of combined treatment with MK2206 (an AKT inhibitor) + chloroquine on cell growth and regrowth of endometriotic stromal cells and the in vivo effects on endometriotic implants in a mouse xenograft model of endometriosis.. We evaluated the effects of autophagy inhibition by knockdown of the ATG13, Beclin-1 and ATG12 genes and pharmacological agents (chloroquine, bafilomycin A1 or 3-methyalanine) individually and in combination with MK2206 on cell growth and/or cell regrowth of endometriotic stromal cells in vitro. Furthermore, we evaluated treatment with MK2206 + chloroquine on endometriotic implants in a mouse xenograft model of endometriosis.. Combined treatment with MK2206 and chloroquine markedly reduced cell growth and regrowth after discontinuation of treatment in endometriotic stromal cells compared with cells treated with either drug alone. Autophagy inhibition by ATG13, Beclin-1 or ATG12 gene knockdown only affected regrowth of endometriotic stromal cells, but not endometrial stromal cells from the same patients, after a 72 h discontinuation of the combined treatment. Furthermore, combined treatment reduced the size of endometriotic implants, whereas no effects on endometriotic implants treated with either drug alone were observed in a mouse xenograft model of endometriosis.. The present findings suggest that a novel strategy for treatment of endometriosis may involve decreasing the number of endometriotic cells that can survive treatment and then preventing regrowth by autophagy inhibition.

Topics: Adult; Animals; Autophagy; Cell Proliferation; Cells, Cultured; Chloroquine; Disease Models, Animal; Drug Therapy, Combination; Endometriosis; Female; Heterocyclic Compounds, 3-Ring; Humans; Mice; Young Adult

Daily vs. weekly dosing with EGFR inhibitors (gefitinib and lapatinib) and an AKT inhibitor (MK2206) were compared in two rodent breast cancer models. Female Sprague-Dawley rats were administered methylnitrosourea (MNU) at 50 days of age, and gefitinib (daily/weekly dosing at 10/70 mg/kg BW) or lapatinib (daily/weekly dosing at 75/525 mg/kg BW) were administered by gavage beginning 5 days after MNU. For the prevention studies, weekly or daily dosing with gefitinib or lapatinib reduced cancer multiplicity >75%, and all treatments reduced tumor weights by >90%. For the therapeutic studies, MNU-treated rats were followed until small palpable mammary cancers developed. The rats were then treated daily or weekly as above for 6 weeks. Either daily or weekly dosing with lapatinib or gefitinib caused regression in >50% of the tumors. Immunohistochemistry biomarker studies in palpable mammary cancers following a weekly dose of gefitinib showed that 1 day (but not 7 days) after treatment, the levels of phosphorylated EGFR1 were significantly decreased. In an ER-negative (ER-) Neu-overexpressing model employing MMTV-Neu/P53KO mice, daily (100 mg/kg BW/day, 5 days each week), or weekly dosing (500 or 250 mg/kg BW) with gefitinib reduced tumor multiplicity 65, 85 and 75%, respectively. In the MNU prevention model, daily dosing (100 mg/kg BW/day) with the allosteric AKT inhibitor MK2206 was ineffective, while weekly dosing (700 mg/kg BW) reduced the final tumor weight >70%. Combining weekly MK2206 with the aromatase inhibitor vorozole (0.12 mg/kg BW/day) showed that each compound alone reduced tumor multiplicity 40-50%. The combination reduced cancer multiplicity ~70%. These studies demonstrate the efficacy of weekly dosing with various protein kinase inhibitors; raising the possibility of employing these agents in a breast cancer preventive setting.

Topics: Animals; Disease Models, Animal; Drug Administration Schedule; ErbB Receptors; Female; Gefitinib; Heterocyclic Compounds, 3-Ring; Lapatinib; Male; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Protein Kinase Inhibitors; Quinazolines; Rats; Rats, Sprague-Dawley; Triazoles

Excess fibrosis may lead to chronic pain, scarring, and infertility as endometriosis develops and progresses. The pathogenesis of endometriosis has been linked to transforming growth factor-β (TGF-β), the most potent promoter of fibrosis.. Levels of NR4A1 and P-NR4A1 protein in human endometrial and endometriotic tissue were assessed by western blotting and immunohistochemistry. The expression levels of fibrotic markers in stromal cells were evaluated by real-time PCR. The degree of fibrosis in mouse endometriotic lesions was detected by Masson trichrome and Sirius red staining.. The level of phosphorylated-NR4A1 was higher in ovarian endometriotic tissue than in normal endometrium, and long-term TGF-β1 stimulation phosphorylated NR4A1 in an AKT-dependent manner and then promoted the expression of fibrotic markers. Furthermore, inhibition of NR4A1 in stromal cells increased the TGF-β1-dependent elevated expression of fibrotic markers, and loss of NR4A1 stimulated fibrogenesis in mice with endometriosis. Additionally, Cytosporone B (Csn-B), an NR4A1 agonist, effectively decreased the TGF-β1-dependent elevated expression of fibrotic markers in vitro and significantly inhibited fibrogenesis in vivo.. NR4A1 can regulate fibrosis in endometriosis and may serve as a new target for the treatment of endometriosis.

Topics: Adult; Animals; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Disease Models, Animal; Endometriosis; Endometrium; Female; Fibronectins; Fibrosis; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Nude; Microscopy, Fluorescence; Nuclear Receptor Subfamily 4, Group A, Member 1; Phenylacetates; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Up-Regulation; Young Adult

The FDA-approved small-molecule drug ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL). Ibrutinib inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. However, the potential regulation of neuroinflammatory responses in the brain by ibrutinib has not been comprehensively examined.. BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO), followed by lipopolysaccharide (LPS; 1 μg/ml) or PBS. RT-PCR, immunocytochemistry, and subcellular fractionation were performed to examine the effects of ibrutinib on neuroinflammatory responses. In addition, wild-type mice were sequentially injected with ibrutinib (10 mg/kg, i.p.) or vehicle (10% DMSO, i.p.), followed by LPS (10 mg/kg, i.p.) or PBS, and microglial and astrocyte activations were assessed using immunohistochemistry.. Ibrutinib significantly reduced LPS-induced increases in proinflammatory cytokine levels in BV2 microglial and primary microglial cells but not in primary astrocytes. Ibrutinib regulated TLR4 signaling to alter LPS-induced proinflammatory cytokine levels. In addition, ibrutinib significantly decreased LPS-induced increases in p-AKT and p-STAT3 levels, suggesting that ibrutinib attenuates LPS-induced neuroinflammatory responses by inhibiting AKT/STAT3 signaling pathways. Interestingly, ibrutinib also reduced LPS-induced BV2 microglial cell migration by inhibiting AKT signaling. Moreover, ibrutinib-injected wild-type mice exhibited significantly reduced microglial/astrocyte activation and COX-2 and IL-1β proinflammatory cytokine levels.. Our data provide insights on the mechanisms of a potential therapeutic strategy for neuroinflammation-related diseases.

Topics: Adenine; Animals; Animals, Newborn; Anti-Inflammatory Agents; Cell Line, Transformed; Cells, Cultured; Culture Media, Serum-Free; Cyclooxygenase 2; Cytokines; Disease Models, Animal; Heterocyclic Compounds, 3-Ring; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Microglia; Piperidines; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Rats; Signal Transduction; Wound Healing

Gastrointestinal stromal tumors (GIST) generally harbor activating mutations in the receptor tyrosine kinase KIT or in the related platelet-derived growth factor receptor alpha (PDGFRA). GIST treated with imatinib mesylate or second-line therapies that target mutant forms of these receptors generally escape disease control and progress over time. Inhibiting additional molecular targets may provide more substantial disease control. Recent studies have implicated the PI3K/AKT pathway in the survival of imatinib mesylate-resistant GIST cell lines and tumors.. Here, we performed in vitro and in vivo studies evaluating the novel combination of imatinib mesylate with the AKT inhibitor MK-2206 in GIST. Whole-transcriptome sequencing (WTS) of xenografts was performed to explore the molecular aspects of tumor response to this novel combination and to potentially identify additional therapeutic targets in GIST.. This drug combination demonstrated significant synergistic effects in a panel of imatinib mesylate-sensitive and -resistant GIST cell lines. Furthermore, combination therapy provided significantly greater efficacy, as measured by tumor response and animal survival, in imatinib mesylate-sensitive GIST xenografts as compared with treatment with imatinib mesylate or MK-2206 alone. WTS implicated two neural genes, brain expressed X-linked 1 and neuronal pentraxin I, whose expression was significantly upregulated in combination-treated tumors compared with tumors treated with the two monotherapies.. These studies provide strong preclinical justification for combining imatinib mesylate with an AKT inhibitor as a front-line therapy in GIST. In addition, the WTS implicated the BCL-2/BAX/BAD apoptotic pathway as a potential mechanism for this enhanced combination effect. Clin Cancer Res; 23(1); 171-80. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Synergism; Exome Sequencing; Gastrointestinal Stromal Tumors; Gene Expression Profiling; Heterocyclic Compounds, 3-Ring; Humans; Imatinib Mesylate; Mice; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-kit; Signal Transduction; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

Topics: Animals; Astrocytes; Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Brain; Cell Nucleus; Disease Models, Animal; Fibroblasts; Gene Knockdown Techniques; HeLa Cells; Heterocyclic Compounds, 3-Ring; Humans; Male; Mechanistic Target of Rapamycin Complex 1; Membrane Glycoproteins; Mice; Mice, Transgenic; Molecular Chaperones; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Phosphorylation; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Signal Transduction; Trehalose

There is extensive evidence for the role of aberrant cell survival signaling mechanisms in cancer progression and metastasis. Akt is a major component of cell survival-signaling mechanisms in several types of cancer. It has been shown that activated Akt stabilizes XIAP by S87 phosphorylation leading to survivin/XIAP complex formation, caspase inhibition and cytoprotection of cancer cells. We have reported that TGFβ/PKA/PP2A-mediated tumor suppressor signaling regulates Akt phosphorylation in association with the dissociation of survivin/XIAP complexes leading to inhibition of stress-dependent induction of cell survival.. IGF1R-dependent colon cancer cells (GEO and CBS) were used for the study. Effects on cell proliferation and cell death were determined in the presence of MK-2206. Xenograft studies were performed to determine the effect of MK-2206 on tumor volume. The effect on various cell death markers such as XIAP, survivin, AIF, Ezrin, pEzrin was determined by western blot analysis. Graph pad 5.0 was used for statistical analysis. P < 0.05 was considered significant.. We characterized the mechanisms by which a novel Akt kinase inhibitor MK-2206 induced cell death in IGF1R-dependent colorectal cancer (CRC) cells with upregulated PI3K/Akt signaling in response to IGF1R activation. MK-2206 treatment generated a significant reduction in tumor growth in vivo and promoted cell death through two mechanisms. This is the first report demonstrating that Akt inactivation by MK-2206 leads to induction of and mitochondria-to-nuclear localization of the Apoptosis Inducing Factor (AIF), which is involved in caspase-independent cell death. We also observed that exposure to MK-2206 dephosphorylated Ezrin at the T567 site leading to the disruption of Akt-pEzrin-XIAP cell survival signaling. Ezrin phosphorylation at this site has been associated with malignant progression in solid tumors.. The identification of these 2 novel mechanisms leading to induction of cell death indicates MK-2206 might be a potential clinical candidate for therapeutic targeting of the subset of IGF1R-dependent cancers in CRC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Cell Death; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cytoskeletal Proteins; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Proto-Oncogene Proteins c-akt; RNA Interference; Tumor Burden; Xenograft Model Antitumor Assays

The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PR(cre/+)Pten(f/+) heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Pten(f/+) mouse and 7.5 ± 0.43 lesions in the PR(cre/+)Pten(f/+) mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Pten(f/+) and PR(cre/+)Pten(f/+) mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206-treated mice compared with vehicle-treated mice. Furthermore, levels of FOXO1 and progesterone receptor increased in lesions of mice receiving MK-2206. These results demonstrate that heightened AKT activity plays an active role in the establishment of ectopic endometrial tissues.

Topics: Animals; Cell Proliferation; Cell Survival; Cells, Cultured; Crosses, Genetic; Disease Models, Animal; Endometriosis; Endometrium; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Transgenic; Ovarian Diseases; Ovary; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Signal Transduction; Stromal Cells

Basal-like breast cancer is an aggressive disease for which targeted therapies are lacking. Recent studies showed that basal-like breast cancer is frequently associated with an increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway, which is critical for cell growth, survival, and angiogenesis. To investigate the therapeutic potential of PI3K pathway inhibition in the treatment of basal-like breast cancer, we evaluated the antitumor effect of the mTOR inhibitor MK-8669 and AKT inhibitor MK-2206 in WU-BC4 and WU-BC5, two patient-derived xenograft models of basal-like breast cancer. Both models showed high levels of AKT phosphorylation and loss of PTEN expression. We observed a synergistic effect of MK-8669 and MK-2206 on tumor growth and cell proliferation in vivo. In addition, MK-8669 and MK-2206 inhibited angiogenesis as determined by CD31 immunohistochemistry. Biomarker studies indicated that treatment with MK-2206 inhibited AKT activation induced by MK-8669. To evaluate the effect of loss of PTEN on tumor cell sensitivity to PI3K pathway inhibition, we knocked down PTEN in WU-BC3, a basal-like breast cancer cell line with intact PTEN. Compared with control (GFP) knockdown, PTEN knockdown led to a more dramatic reduction in cell proliferation and tumor growth inhibition in response to MK-8669 and MK-2206 both in vitro and in vivo. Furthermore, a synergistic effect of these two agents on tumor volume was observed in WU-BC3 with PTEN knockdown. Our results provide a preclinical rationale for future clinical investigation of this combination in basal-like breast cancer with loss of PTEN.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Female; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Humans; Inhibitory Concentration 50; Neoplasms, Basal Cell; Neovascularization, Pathologic; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

Topics: Animals; Apoptosis; Bone Marrow; Cell Cycle Checkpoints; Cell Line; Cell Proliferation; Disease Models, Animal; Female; Fusion Proteins, bcr-abl; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinase 2; Liver; Megakaryocytes; Mice; Mutation; Myeloproliferative Disorders; Neoplastic Stem Cells; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Spleen

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression; Heterocyclic Compounds, 3-Ring; Humans; Lung Neoplasms; Mice; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Tumor Burden; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Although the prognosis for clinically localized prostate cancer is now favorable, there are still no curative treatments for castration-resistant prostate cancer (CRPC) and, therefore, it remains fatal. In this study, we investigate a new therapeutic approach for treatment of CRPC, which involves dual targeting of a major signaling pathway that is frequently deregulated in the disease. We found that dual targeting of the Akt and mTOR signaling pathways with their respective inhibitors, MK-2206 and ridaforolimus (MK-8669), is highly effective for inhibiting CRPC in preclinical studies in vivo using a refined genetically engineered mouse model of the disease. The efficacy of the combination treatment contrasts with their limited efficacy as single agents, since delivery of MK-2206 or MK-8669 individually had a modest impact in vivo on the overall tumor phenotype. In human prostate cancer cell lines, although not in the mouse model, the synergistic actions of MK-2206 and ridaforolimus (MK-8669) are due in part to limiting the mTORC2 feedback activation of Akt. Moreover, the effects of these drugs are mediated by inhibition of cellular proliferation via the retinoblastoma (Rb) pathway. Our findings suggest that dual targeting of the Akt and mTOR signaling pathways using MK-2206 and ridaforolimus (MK-8669) may be effective for treatment of CRPC, particularly for patients with deregulated Rb pathway activity.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cluster Analysis; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Male; Mice; Mice, Transgenic; Orchiectomy; Phenotype; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays