Upregulation Of Akt Research Articles

MUC16/CA125 specific targeting of critical signaling kinases in ovarian cancer

e22112 Background: The serum marker CA125 is known to originate from the transmembrane protein, MUC16; however, the biological importance of MUC16 is not known. Our lab previously created fluorescent-labeled transfectants, containing truncated versions of the MUC16 molecule. MUC16 expression vectors were transfected into CA125-negative ovarian cancer cell lines. These cell lines have allowed for a unique means of studying MUC16 biology. In vivo data suggest that MUC16 drives a more aggressive phenotype in ovarian cancer, including increased growth, invasion and dissemination. MUC16 transfection is also associated with the upregulation of specific signaling kinases, including EGFR, Erk, Src and Akt. To validate these findings and explore the potential of MUC16 signal targeting through inhibition of downstream kinases, we are examining the effect of a kinase-directed siRNA library on the A2780 ovarian cancer cell line transfected with a carboxy-terminus MUC16-expression vector. With the identification of kinases important to the survival of CA125+ ovarian cancer tissue, we will identify molecular targets for novel, tumor-specific therapies. Methods: Pilot plates containing candidate kinase-directed, negative control and total kill siRNA (Qiagen) were applied to MUC16+ and MUC16- cells. Cells were grown in culture in parallel 96-well plates. Immunofluorescent readings and Alamar Blue viability staining were performed at 72 hours. Results: Transfection reagents and nonspecific siRNA did not substantially change the survival of fluorescent protein expression in MUC16± cells; while hrGFP and mCherry proteins were decreased by ∼75%. None of the final transfection conditions altered Alamar Blue survival. Preliminary studies suggest that siRNA against AKT2 and SRC had specific inhibition against MUC16+ cells compared to MUC16- controls. Studies of three additional AKT2 siRNAs and a full complement of 714 human kinases are now being tested. Kinases with MUC16-specifc effects will be confirmed with three additional siRNAs. Conclusions: Dual fluorescence isogenic cell lines can be used to explore the kinase profile of MUC16+ ovarian cancer cells. Identified target kinases will enhance our knowledge about MUC16/CA125 biology and provide future leads for the treatment of ovarian cancer. No significant financial relationships to disclose.

Expression of selected tumor suppressor and oncogenes in endometrium of women with endometriosis

It is becoming increasingly evident that the eutopic endometrium of women with endometriosis shows certain genetic alterations which are not found in the endometrium of disease-free women. The aim of the study was to compare the expression level of mammalian target of rapamycin (mTOR) tumor suppressor and oncogene-related genes in the endometrium of women with and without endometriosis as well as in ovarian endometriosis. A total of 81 regularly menstruating patients were recruited in the study. We applied the micro fluidic gene array to examine the expression of 15 human tumor suppressor and oncogenes in eutopic endometrium of 40 women with endometriosis and 41 controls without endometriosis. In 14 patients with endometriosis, gene expression was also studied in matched ovarian lesions. We studied the following genes: NF1, RHEB, mTOR, PTEN, TSC1, TSC2, KRAS, S6K1, TP53, EIF4E, LKB1, PIK3CA, BECN1, 4EBP1 and AKT1. Immunohistochemical studies were subsequently performed for selected proteins. Of the 15 studied genes, we found significantly higher levels of oncogene AKT1 (P = 0.006) and tumor suppressor gene 4EBP1 (P = 0.01) mRNAs in the eutopic endometrium of women with endometriosis compared with control patients. Immunohistochemistry showed that 4EBP1 and AKT1 proteins were expressed in eutopic endometrium. Our results suggest that up-regulation of AKT1 and 4EBP1 in eutopic endometrium may be associated with the pathogenesis of endometriosis, but their precise role remains to be established.

Sulfaphenazole Protects Heart Against Ischemia–Reperfusion Injury and Cardiac Dysfunction by Overexpression of iNOS, Leading to Enhancement of Nitric Oxide Bioavailability and Tissue Oxygenation

The objective of this study was to establish the cardioprotective effect of sulfaphenazole (SPZ), a selective inhibitor of cytochrome P450 2C9 enzyme, in an in vivo rat model of acute myocardial infarction (MI). MI was induced by 30 min ligation of left anterior descending coronary artery, followed by 24 h reperfusion (I/R). The study used 6 groups: I/R (control); SPZ; L-NAME; L-NAME + SPZ; 1400W (an inhibitor of iNOS); 1400W + SPZ. The agents were administered orally through drinking water for 3 days prior to induction of I/R. Myocardial oxygenation (pO(2)) at the I/R site was measured using EPR oximetry. The preischemic pO(2) value was 18 +/- 2 mm Hg in all groups. At 1 h of reperfusion, the SPZ group showed a significantly higher hyperoxygenation when compared to control (45 +/- 1 vs. 34 +/- 2 mm Hg). The SPZ group showed a significant improvement in the contractile functions and reduction in infarct size. Histochemical staining of SPZ-treated hearts exhibited significantly lower levels of superoxide and peroxynitrite, and markedly increased levels of iNOS activity and nitric oxide. Western blot analysis indicated upregulation of Akt and attenuation of p38MAPK activities in the reperfused myocardium. The study established that SPZ attenuated myocardial I/R injury through overexpression of iNOS, leading to enhancement of nitric oxide bioavailability and tissue oxygenation.

Dolichol‐linked oligosaccharide synthesis inhibitor reduces the survival potential of capillary endothelial cells

Proliferation and differentiation of capillary endothelial cells (i.e., angiogenesis) are essential for breast tumor growth and metastasis. We have recently shown that dolichol‐linked oligosaccharide (i.e., LLO) biosynthesis and turnover are required for angiogenesis. Its inhibition by a glycotherapeutic, tunicamycin resulted in cell cycle arrest in G1 and induction of apoptosis mediated by unfolded protein response (upr) signaling. The objective of our study was to evaluate that tunicamycin reduces the survival potential of capillary endothelial cells. We have used vascular endothelial growth factor (VEGF) and insulin‐like growth factor‐1 (IGF‐1) to answer this question. VEGF165 (10ng/ml) stimulated cellular proliferation but it could not protect the cells from tunicamycin. A similar result was also obtained when the cells were treated with IGF‐1 (10ng/ml). There was however upregulation of p85PI3K(Tyr), Akt(S473) and p53(S15) phosphorylation in tunicamycin treated cells following IGF‐1 treatment for 10 min. The phosphorylation of NFκB (p65 S536) on the other hand remained silent under the experimental condition. These results together with the activation of caspase‐9, p21 and down‐regulation of GSK3‐β following tunicamycin treatment in the absence of IGF‐1 suggest shifting of the survival potential of VEGF165/IGF‐1 to apoptosis. Supported in part by grants U54‐CA096297 and Susan G. Komen for Cure BCTR0600582 (DKB), and G12‐RR03035 (KB).

Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference.

Eukaryotic cap-dependent mRNA translation is mediated by the initiation factor eIF4E, which binds mRNAs and stimulates efficient translation initiation. eIF4E is often overexpressed in human cancers. To elucidate the molecular signature of eIF4E target mRNAs, we analyzed sequence and structural properties of two independently derived polyribosome recruited mRNA datasets. These datasets originate from studies of mRNAs that are actively being translated in response to cells over-expressing eIF4E or cells with an activated oncogenic AKT: eIF4E signaling pathway, respectively. Comparison of eIF4E target mRNAs to mRNAs insensitive to eIF4E-regulation has revealed surprising features in mRNA secondary structure, length and microRNA-binding properties. Fold-changes (the relative change in recruitment of an mRNA to actively translating polyribosomal complexes in response to eIF4E overexpression or AKT upregulation) are positively correlated with mRNA G+C content and negatively correlated with total and 3′UTR length of the mRNAs. A machine learning approach for predicting the fold change was created. Interesting tendencies of secondary structure stability are found near the start codon and at the beginning of the 3′UTR region. Highly upregulated mRNAs show negative selection (site avoidance) for binding sites of several microRNAs. These results are consistent with the emerging model of regulation of mRNA translation through a dynamic balance between translation initiation at the 5′UTR and microRNA binding at the 3′UTR.

BI‐69A11‐mediated inhibition of AKT leads to effective regression of xenograft melanoma

The AKT/PKB pathway plays a central role in tumor development and progression and is often up-regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI-69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI-69A11 was performed in melanoma cells, a tumor type that commonly exhibits up-regulation of AKT. Treatment of the UACC903 human melanoma cells, harboring the PTEN mutation, with BI-69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI-69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP-90. BI-69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI-69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra-peritoneal injection of BI-69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI-69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.

RXFP1-inactive relaxin activates human glucocorticoid receptor: Further investigations into the relaxin–GR pathway

The relaxin peptide family regulates diverse biological functions (central nervous processes, reproduction, cardiovascular and kidney function, and connective tissue composition) through different G protein-coupled receptors. We reported earlier that human relaxin-2 and porcine relaxin additionally interact with the human glucocorticoid receptor (GR) in an agonistic manner. Here we investigated whether the membrane receptor RXFP1 is critically involved in this pathway. We used chemically modified porcine relaxin which was biologically inactive at RXFP1. Native porcine relaxin, but not the modified peptide affected RXFP1-dependent and GR-independent readouts: ERK-1/2 and Akt phosphorylation as well as up-regulation of Akt and endothelin type-B receptor. In contrast, relaxin and modified relaxin inhibited endotoxin-stimulated secretion of TNF-α and IL-6 by human macrophages, an effect sensitive to the glucocorticoid receptor antagonists RU-486 and D-06. Both relaxins caused Ser 211 phosphorylation of GR, a biomarker of agonist-related receptor activation. Relaxin-induced accumulation of Ser 211-phosphorylated GR was found in the cytoplasm and nucleus of HeLa cells, in endothelial cells, and in transfected HT-29 cells. In AP-1-luciferase assays, relaxin and modified relaxin inhibited endotoxin-induced activation of AP-1, a transcription factor essentially involved in endotoxin signaling. This study suggests that the inability of relaxin to interact with its membrane receptor does not interfere with its ability to activate GR.

Apoptogenic effect of 7,8-diacetoxy-4-methylcoumarin and 7,8-diacetoxy-4-methylthiocoumarin in human lung adenocarcinoma cell line: Role of NF-κB, Akt, ROS and MAP kinase pathway

Coumarin (1,2-benzopyrone) is a naturally occurring fragrant compound found in a variety of plants and spices. Coumarins have attracted intense interest in recent years because of their diverse pharmacological activities. This study examines the antioxidant coumarin 7,8-diacetoxy-4-methylcoumarin (DAMC) and its thiocoumarin derivative 7,8-diacetoxy-4-methylthiocoumarin (DAMTC) for their effect on human non-small cell lung cancer A549 cells. Here we show that both DAMC and DAMTC not only inhibited cell proliferation, but also induced apoptosis with an IC 50 of 160 μg/ml as confirmed by morphological examination, annexin-V assay and flow cytometric analysis. Interestingly, it was observed that these two coumarin compounds exhibited little cytotoxicity towards peripheral blood mononuclear cells but induced apoptosis in malignant cells. DAMC/DAMTC treatment also resulted in pronounced release of apoptogenic cytochrome c from mitochondria to cytosol, alteration of mitochondrial membrane potential (Δ Ψ m), and activation of caspase-9 and caspase-3. Although an increase in the levels of reactive oxygen species (ROS) was observed, pre-treatment with antioxidant showed no protective effect against DAMC/DAMTC-induced apoptosis. Results of present study suggest that downregulation of Bcl-xl, Cox-2 and mitogen activated protein kinase pathway and upregulation of p53, Akt and NF-κB pathway are involved in the underlying molecular mechanism of apoptosis induction by DAMC and DAMTC in A549 cells.

Epidermal Growth Factor Receptor Pathway Substrate 8 Is Overexpressed in Human Pituitary Tumors: Role in Proliferation and Survival

Based on prior work showing that human pituitary tumors overexpress epidermal and fibroblast growth factor receptors, we hypothesized that downstream components of growth factor signaling pathways may also be dysregulated. Epidermal growth factor pathway substrate number 8 (Eps8) was identified as a transcript overexpressed (5.9-fold) in human pituitary tumors compared with normal pituitary by DNA microarrays. Eps8 mRNA up-regulation was confirmed by semiquantitative RT-PCR. Immunoblot analysis showed that Eps8 protein levels and its downstream target phosphorylated ERK were also up-regulated in human pituitary tumors. Stable overexpression of Eps8 in LbetaT2 gonadotrope pituitary cells augmented colony formation in soft agar at d 21. Eps8 cells proliferated more robustly compared with controls in growth factor replete as well as growth-restricted conditions. In addition, the Eps8 overexpressing cells were protected from serum withdrawal-induced apoptosis compared with controls as assessed by caspase-3 cleavage. Epidermal growth factor activated a robust amplification of ERK and modest up-regulation of Akt in Eps8-overexpressing pituitary cells compared with vector controls. MAPK kinase inhibition or silencing of Eps8 blunted the proliferation of the cells in response to growth factor stimulation. Blockade of the phosphatidylinositol 3-kinase pathway or silencing of Eps8 resulted in a loss of the Eps8 protection from growth factor withdrawal-induced apoptosis. Together these data support a role of Eps8 in amplifying growth factor receptor signaling in human pituitary tumors to promote proliferation and cell survival.

Abstract 1845: Adult Cardiac Progenitor Cells Promote Angiogenesis and Cardioprotection through Their Secreted Soluble Vcam-1

Although cardiac progenitor cells have been thought to be the promising source of cell therapy, the precise mechanisms of their paracrine action have not been fully elucidated. Since we observed that the transplantation of clonal expanded Sca-1 positive cardiac progenitor cells (cSca-1 cells) derived from adult murine heart by using cell sheet technique improved cardiac function of infarcted heart compared to adipose tissue derived mesenchymal cells (ATMC), we explored the secreted factors highly expressed in cSca-1 cells and identified that soluble VCAM-1 (sVCAM-1) was much abundant in cSca-1 cells compared to ATMC by using cytokine antibody array. cSca-1 cells-derived conditioned medium (CM) significantly enhanced endothelial migration and matrigel tube formation and these effects were abolished by knock down of VCAM-1 (Fold increase: control, 1.0; CM, 2.97 ± 0.21; siVCAM-1, 1.98 ± 0.09; siControl, 2.76 ± 0.05, p<0.01), suggesting that cSca-1 cells promote angiogenesis via their secreted sVCAM-1. We next examined whether sVCAM-1 conferred direct protective effects on cardiomyocytes. We exposed cardiomyocytes to 0.2 mM H 2 O 2 in the absence or presence of sVCAM-1 or CM and examined cardiomyocyte viability by MTT assay. The exposure of cardiomyocytes to H 2 O 2 significantly induced the cell injury. Interestingly when pretreated with sVCAM-1 or CM, the cell damages of cardiomyocytes by H 2 O 2 were significantly reduced. However when pretreated with anti-VLA4 antibody, a principal coreceptor of sVCAM-1, CM mediated cell protected effect was completely inhibited (Fold increase: control, 1.0; anti-VLA4, 0.89 ± 0.33; sVCAM-1, 1.69 ± 0.27; CM, 2.08 ± 0.28; CM+anti-VLA4, 1.07 ± 0.07, p<0.01), suggesting that a crucial role of the VLA4 in inducing survival of cardiomyocytes by CM. sVCAM-1 and CM induced phosphorylation of FAK, Akt, Erk and p38 MAPK in neonatal rat cardiomyocytes. When pretreated with wortmannin, SB203580 and PD98059, the cardioprotective effects of sVCAM-1 and CM significantly inhibited, suggesting that sVCAM-1 might protect cardiomyocytes from oxidative stress via integrated upregulation of Akt, Erk and p38MAPK. These findings suggest cardiac progenitor cells promote angiogenesis and cardioprotection through their secreted sVCAM-1.

Polymerized Collagen Inhibits Fibroblast Proliferation via a Mechanism Involving the Formation of a β1 Integrin-Protein Phosphatase 2A-Tuberous Sclerosis Complex 2 Complex That Suppresses S6K1 Activity

Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G(1)/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a beta1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, beta1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G(1)/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a beta1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G(1)/S phase transition.

Follistatin-Like 1 Is an Akt-Regulated Cardioprotective Factor That Is Secreted by the Heart

The Akt protein kinase is an important mediator of cardiac myocyte growth and survival. To identify factors with novel therapeutic applications in cardiac diseases, we focused on the identification of factors secreted from Akt1-activated cells that have cardioprotective effects through autocrine/paracrine mechanisms. Using an inducible Akt1 transgenic mouse model, we have found that follistatin-like 1 (Fstl1) protein and transcript expression are increased 4.0- and 2.0-fold, respectively, by Akt activation in the heart (P<0.05). Fstl1 transcript was also upregulated in response to myocardial stresses including transverse aortic constriction, ischemia/reperfusion injury, and myocardial infarction. Adenovirus-mediated overexpression of Fstl1 protected cultured neonatal rat ventricular myocytes from hypoxia/reoxygenation-induced apoptosis (P<0.01), and this protective effect was dependent on the upregulation of both Akt and ERK activities. Conversely, knockdown of Fstl1 in cardiac myocytes decreased basal Akt signaling and increased the frequency of apoptotic death in vitro (P<0.01). The intravenous administration of an adenoviral encoding Fstl1 to mice resulted in a 66.0% reduction in myocardial infarct size after ischemia/reperfusion injury that was accompanied by a 70.9% reduction in apoptosis in the heart (P<0.01). These results indicate that Fstl1 is a cardiac-secreted factor that functions as an antiapoptotic protein. Fstl1 could play a role in myocardial maintenance and repair in response to harmful stimuli.

Preclinical rationale for combination targeted therapy in advanced clear cell renal cell carcinoma (RCC): Abrogation of rapamycin-mediated induction of AKT phosphorylation by perifosine

16083 Background: RCCs are highly vascularized tumors characterized by VHL abnormalities resulting in the upregulation of hypoxia inducible factor (HIF). Inhibition of mTOR, an upstream modulator of HIF, is an established therapeutic strategy in advanced RCC. One proposed resistance mechanism for mTOR inhibition is the compensatory upregulation of AKT, a promoter of cell growth and proliferation. Perifosine, an orally bioavailable AKT inhibitor, has clinical activity against RCC. We sought to determine whether treatment with perifosine and an mTOR-inhibitor (rapamycin) would synergistically inhibit RCC in preclinical models. Methods: Four clear cell RCC cell lines were studied: A498, CAKI-1, 769-P, and 786-O. Dose curves of single-agent and combination perifosine + rapamycin were performed by MTT. Combination studies were designed for median-effect. Status of pAKT (S473), pmTOR (S2481) and HIF-2alpha was assessed by Western blot. Cell lines had low basal levels of pAKT and VHL, moderate levels of pmTOR and HIF-2alpha, and no detectable HIF-1alpha. Results: CAKI-1, 769-P, and 786-O were sensitive to perifosine with an IC50 between 5–10 uM, while the IC50 of A498 was > 20 uM. Growth inhibition by rapamycin plateaued by 0.5–1 nM with none of the lines achieving an IC50. Combination treatments were essentially additive at lower doses of perifosine, becoming sub-additive or antagonistic at higher doses. Perifosine blocked phosphorylation of AKT induced by rapamycin. As a single agent and in combination, perifosine inhibited HIF-2alpha expression in the 769-P and CAKI-1 cell lines. Conclusions: Perifosine has high single-agent activity in select RCC lines and can abrogate the induction of AKT phosphorylation mediated by mTOR inhibition. Perifosine + rapamycin was at best additive, suggesting that pAKT is not the sole mechanism of resistance. Studies to explore the roles of VHL and HIF in response to this combination are ongoing. A Southwest Oncology Group trial in RCC patients testing the rational combination of perifosine and the mTOR inhibitor temsirolimus is now under development. No significant financial relationships to disclose.

Dexrazoxane protects against doxorubicin-induced cardiomyopathy: upregulation of Akt and Erk phosphorylation in a rat model

Dexrazoxane (DZR), a clinically approved cation chelator, is effective in reducing doxorubicin (DOX)-induced heart damage, yet its cardioprotective mechanism is not fully understood. We aimed to investigate the effects of DZR on the activation of Akt and Erk 1/2 signals in a rat model of DOX-induced cardiomyopathy. Male Sprague-Dawley rats received weekly DOX injection (2.5 mg/kg) for 6 weeks, with or without DZR pretreatment at a dose ratio of 20:1. The ventricular functions of these animals were monitored at week 6, 9 and 11 by echocardiography. At week 11, their heart morphology was studied by light and electron microscopy. Phosphorylation of Akt and Erk in heart tissues was measured by Western blot analysis. DOX caused myocardial damage with compromised left ventricular function, increased myocardium injury and reduced phosphorylation of Akt and Erk. DZR exerted a significant cardioprotective effect in terms of improved fractional shortening, cardiac output and cardiomyopathy score at one or more time points. We also provided the first evidence that dexarazoxane-treated animals had increased levels of Akt and Erk activation, whilst total Akt and Erk remained unchanged. Our results showed that the cardioprotective effect of dexarazoxane has been sustained beyond the treatment period. The data also suggested that activation of the Akt and Erk signaling pathways was regulated in the course of DOX-induced cardiomyopathy and protection by DZR.

Bone Marrow Mesenchymal Stem Cells Induce Angiogenesis and Attenuate the Remodeling of Diabetic Cardiomyopathy

Independent of the severity of coronary artery disease, diabetic patients have an increased risk of developing heart failure. Diabetic cardiomyopathy (DCM) is characterized by microvascular pathologies and interstitial fibrosis. Mesenchymal stem cells (MSCs) are pluripotent and are able to differentiate into cardiomyocytes and vascular endothelial cells. Studies have demonstrated MSCs transplantation can prevent apoptosis of ischemic heart via upregulation of Akt and eNOS and inhibit myocardial fibrosis of dilated cardiomyopathy by decreasing the expression of matrix metalloproteinase (MMP) in rat models. In order to find out whether transplantation of MSCs is a promising treatment in DCM, we used streptozotocin (STZ) -induced diabetic rats as the model. Exogenous MSCs were injected into the femoral vein 8 weeks after STZ injection. Using independent experimental approaches, we showed that MSCs presented in the myocardium 4 weeks after transplantation and some of them were positive for the cardiac markers Troponin T and myosin heavy chain. MSCs transplantation significantly increased myocardial arteriolar density and decreased the collagen volume in diabetic myocardium resulting in improved cardiac function. Furthermore, MSCs transplantation increased MMP-2 activity and decreased transcriptional level of MMP-9. These results show that MSCs transplantation improved cardiac function in the rat DCM model, possibly through angiogenesis and attenuation of cardiac remodeling.

Mammalian target of rapamycin (mTOR) inhibition activates phosphatidylinositol 3-kinase/Akt by up-regulating insulin-like growth factor-1 receptor signaling in acute myeloid leukemia: rationale for therapeutic inhibition of both pathways

The phosphatidylinositol 3-kinase (PI3K)/Akt and mTORC1 pathways are frequently activated, representing potential therapeutic targets in acute myeloid leukemia (AML). In 19 AML samples with constitutive PI3K/Akt activation, the rapamycin derivative inhibitor everolimus (RAD001) increased Akt phosphorylation. This mTOR C1-mediated Akt up-regulation was explained by an insulin-like growth factor-1 (IGF-1)/IGF-1 receptor autocrine loop: (1) blast cells expressed functional IGF-1 receptors, and IGF-1-induced Akt activation was increased by RAD001, (2) a neutralizing anti-IGF-1R alpha-IR3 monoclonal antibody reversed the RAD001-induced Akt phosphorylation, and (3) autocrine production of IGF-1 was detected in purified blast cells by quantitative reverse transcription-polymerase chain reaction and immunofluorescence. This RAD001-induced PI3K/Akt up-regulation was due to an up-regulated expression of the IRS2 adaptor. Finally, we observed that concomitant inhibition of mTORC1 and PI3K/Akt by RAD001 and IC87114 induced additive antiproliferative effects. Our results suggest that dual inhibition of the mTORC1 complex and the IGF-1/IGF-1R/PI3K/Akt pathway in AML may enhance the efficacy of mTOR inhibitors in treatment of this disease.

Maternal protein restriction leads to early life alterations in the expression of key molecules involved in the aging process in rat offspring

Recent findings demonstrate that nutrition during the fetal and neonatal periods can affect the life span of an organism. Our previous studies in rodents using a maternal low protein diet have shown that limiting protein and growth during lactation [postnatal low protein (PLP group)] increases longevity, while in utero growth restriction (IUGR) followed by "catch up growth" (recuperated group) shortens life span. The aim of this study was to investigate mechanisms in early postnatal life that could underlie these substantial differences in longevity. At weaning, PLP animals had improved insulin sensitivity as suggested by lower concentrations of insulin required to maintain concentrations of glucose similar to those of the control group and significant upregulation of insulin receptor-beta, IGF-1 receptor, Akt1, Akt2, and Akt phosphorylated at Ser 473 in the kidney. These animals also had significantly increased SIRT1 (mammalian sirtuin) expression. Expression of the antioxidant enzymes catalase, CuZnSOD, and glutathione peroxidase-1 was elevated in these animals. In contrast, recuperated animals had a significantly increased fasting glucose concentration, while insulin levels remained comparable to those of the control group suggesting relative insulin resistance. MnSOD expression was increased in these animals. These data suggest that early nutrition can lead to alterations in insulin sensitivity and antioxidant capacity very early in life, which may influence life span.

The Novel Akt Inhibitor Perifosine Induces Apoptosis, Cell Cycle Arrest and Synergizes with Chemotherapeutic Drugs in Acute Myelogenous Leukemia Cells by a JNK Dependent Mechanism - A Novel Therapeutic Approach for Leukemia Displaying Elevated Akt Signaling.

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). In the following study, we have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents which target the plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV4-11 cell lines, as well as primary leukemia cells. The drug blocked AML cells in G2/M phase of the cell cycle and, at longer incubation times (24 hr), decreased survival and induced cell death by apoptosis, as demonstrated by MTT assays and flow cytometric analysis of annexin V-FITC/propidium iodide stained samples. The IC50 for perifosine with THP-1 cells was 15.8 microM, while MV4-11 cells were more sensitive to the drug, as the IC50 was 2.7 microM. In THP-1 cells, perifosine caused complete Akt dephosphorylation on Ser 473 and Thr 308, without affecting total Akt expression levels. Perifosine treatment also resulted in dephosphorylation of the Akt downstream target p70S6 kinase. Perifosine did not affect phosphorylation and activity of the Akt upstream kinase, PDK1 nor altered formation or equilibrium of mTORC1 and mTORC2 complexes. Perifosine induced activation of apical caspases-2, -8, -9, and of executioner caspases-3, -6-, and -7, as well as cleavage of PARP and Bid. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on Fas/FasL interactions, as blocking antibodies to either Fas or FasL significantly reduced perifosine mediated cytotoxicity. Perifosine also activated JNK, and a JNK selective inhibitor (SP600125) markedly reduced perifosine-dependent apoptosis. In MV4-11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine was cytotoxic for AML blasts with activated Akt (IC50 range: 5.6–7.8 microM). Perifosine also reduced the clonogenic activity of CD34+ cells from AML patients displaying constitutive Akt upregulation, but not the clonogenic activity of CD34+ cells from AML patients without Akt activation, or from healthy donors. The inhibitor markedly increased blast cell sensitivity to etoposide. Taken together, our findings indicate that perifosine, either alone or in combination with existing drugs, is a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K/Akt survival pathway. Furthermore this novel Akt inhibitor perifosine appeared not to affect normal human hematopoietic stem cells, further documenting its therapeutic potential.

Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors

Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous beta HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activity can be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC.

Swim training sensitizes myocardial response to insulin: Role of Akt-dependent eNOS activation

Physical activity has been well known to benefit heart function. The improved autonomic nervous activity is considered to be mainly responsible for this beneficial effect. However, the precise mechanism behind the intrinsic myocardial responsiveness to exercise is still unclear. This study was designed to examine the effect of swim training on myocardial response to insulin with a special focus on the endogenous endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) cascade. Adult male Sprague-Dawley (SD) rats were subjected to a 10-week free-loading swim training (3 h/day, 5 days/week). Contractile response to insulin at the levels of cardiomyocytes and isolated perfused heart, myocardial glucose uptake and post-insulin receptor signaling cascades were evaluated. Swim training enhanced cardiac contractile response to insulin in cardiomyocytes and isolated perfused heart, respectively. The improved cardiac response was accompanied by facilitated insulin-stimulated glucose uptake, GLUT4 translocation and upregulation of Akt and eNOS expression (p<0.01). Treatment with insulin resulted in a 3.6- and 2.2-fold increase of eNOS phosphorylation (p<0.01), as well as a 3.0- and 1.9-fold increase of Akt phosphorylation in exercise and sedentary groups, respectively (p<0.01). In addition, exercise significantly facilitated insulin-induced myocardial NO production (p<0.01 vs. sedentary). Moreover, pretreatment with either LY294002, a phosphatidylinositol-3 kinase (PI-3K) inhibitor or L-NAME, a NOS inhibitor, abolished the exercise-induced sensitization of myocardial contractile response to insulin, insulin-induced NO production and phosphorylation of Akt and eNOS. These results demonstrate that swim training is capable of sensitizing myocardial contractile response to insulin via upregulation of Akt- and eNOS signaling cascades.