Transient Activation Of Akt Research Articles

Regulation of Stress-Activated Kinases in Response to Tacaribe Virus Infection and Its Implications for Viral Replication.

Arenaviruses include important zoonotic pathogens that cause hemorrhagic fever (e.g., Junín virus; JUNV) as well as other viruses that are closely related but apathogenic (e.g., Tacaribe virus; TCRV). We have found that, while TCRV and JUNV differ in their ability to induce apoptosis in infected cells, due to active inhibition of caspase activation by the JUNV nucleoprotein, both viruses trigger similar upstream pro-apoptotic signaling events, including the activation/phosphorylation of p53. In the case of TCRV, the pro-apoptotic factor Bad is also phosphorylated (leading to its inactivation). These events clearly implicate upstream kinases in regulating the induction of apoptosis. Consistent with this, here we show activation in TCRV-infected cells of the stress-activated protein kinases p38 and JNK, which are known to regulate p53 activation, as well as the downstream kinase MK2 and transcription factor c-Jun. We also observed the early transient activation of Akt, but not Erk. Importantly, the chemical inhibition of Akt, p38, JNK and c-Jun all dramatically reduced viral growth, even though we have shown that inhibition of apoptosis itself does not. This indicates that kinase activation is crucial for viral infection, independent of its downstream role in apoptosis regulation, a finding that has the potential to shed further light on the determinants of arenavirus pathogenesis, as well as to inform future therapeutic approaches.

AKT/PKB‐Megalin Positive Feedback Loop Expands Albumin Endocytosis in Proximal Tubule Cells

Albuminuria is an independent risk factor for mortality in cardiovascular diseases and is a marker of progression to end-stage renal disease (ESRD). It is accepted that proximal tubule epithelial cells (PTECs) play a central role in the genesis of albuminuria. PTECs reabsorb 3-7g of filtered albumin daily through a receptor-mediated endocytosis being megalin one of the main receptors. Nowadays, one of the main challenges is to understand the regulatory mechanisms involved in this process. In this context, PI3K and AKT have been pointed out as a central mechanism to control albumin endocytosis in PTECs but the mechanism involved is poorly known. Here, we investigated how PI3K/AKT pathway and receptor-mediated albumin endocytosis are interconnected. The incubation of LLC-PK1 cells, a model of PTECs, with albumin 0.01 mg/mL promoted a transient AKT activation measured by its phosphorylation (pAKT) at Ser473 (S473) and by phosphorylation of GSK-3β at Ser9 (S9). Pre-incubation with Pit-Stop-2, an endocytosis inhibitor, prevented AKT phosphorylation (S473) induced by albumin. Using confocal microscopy, the increase of pAKT S473 in subapical vesicles was observed indicating that albumin endocytosis promotes endosomal AKT activation. Acute inhibition of PI3K and AKT by wortmannin (10-6M) and MK-2206 (10-5M) reduced albumin endocytosis capacity and lysosomal delivery by 50%, accessed by BSA-FITC and DQ-BSA uptake, respectively. To study megalin trafficking, LLC-PK1 cells were transfected with a truncated megalin tagged with HA, named MegT0. The inhibition of PI3K/AKT pathway reduced MegT0 surface expression what was followed by an increase in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to the reduction of megalin recycling. Conversely, we studied the possible feedback between PI3K/AKT pathway and receptor-mediated albumin endocytosis. LLC-PK1 cells were pre-incubated with albumin 0.01 mg/mL, washed, and BSA-FITC endocytosis was accessed. Albumin pre-incubation induced receptor-mediated albumin endocytosis what was abolished when PI3K/AKT pathway was inhibited. This result shows that albumin expands albumin endocytosis capacity at physiological concentration. To test this mechanism in vivo, BALB/c micewere i.v. injected with L-lysine, a known inhibitor of megalin-mediated albumin endocytosis in PTECs. L-lysine reduced pAKT S473 and albumin endocytosis, promoting albuminuria. Additionally, wortmannin and MK-2206 incubation reduced albumin endocytosis ex vivo in renal cortex obtained from BALB/c mice. In conclusion, our results suggest a positive feedback between megalin and PI3K/AKT pathway what could be correlated with the capacity of albumin reabsorption in PTECs and the genesis of albuminuria.

Left ventricular phosphorylation patterns of Akt and ERK1/2 after triiodothyronine intracoronary perfusion in isolated hearts and short-term in vivo treatment in Wistar rats.

Objective(s):To determine the effects of triiodothyronine (T3) intracoronary perfusion in isolated hearts and short-term administration in rats on the left ventricular (LV) phosphorylation patterns of Akt and ERK1/2. Materials and Methods:Cardiodynamic and hemodynamic parameters were evaluated in Langendorff–perfused hearts. Left ventricles were used for histomorphometric and Western blot analyses. Short-term hyperthyroidism was established by T3 (500 μg.kg-1.d-1; subcutaneous injection) for 1 (T31d), 3 (T33d), and 10 (T310d) days. Results:Isolated hearts receiving T3 perfusion did not modify LV developed pressure, +dP/dtmax, -dP/dtmin, heart rate, and coronary perfusion pressure compared with vehicle-perfused hearts. P-ERK1/2 and p-Akt levels in LV tissues after 5, 15, or 60 min of T3 or vehicle perfusion were similar. Compared with their time-matched controls, isolated hearts of T33d and T310d rats exhibited LV hypertrophy and increased absolute values of +dP/dtmax and -dP/dtmin (i.e., positive inotropic and lusitropic effects). P-ERK1/2 decreased in LV tissues of T31d and T310d but not in those of T33d rats, and p-Akt levels augmented in left ventricles of T33d and stayed unaltered in those of T31d and T310d rats.Conclusion:T3 intracoronary perfusion did not alter cardiodynamics and hemodynamics nor influence the activation of Akt and ERK of normal hearts. Accordingly, the rapid non-genomic effects of T3 were not evident. Short-term T3 treatment provoked cardiac hypertrophy coincidental with increased LV function and associated with transient Akt activation and cyclic ERK1/2 inhibition; which implies activation of physiological hypertrophy signaling and deactivation of pathological hypertrophy signaling, respectively.

MENS-associated increase of muscular protein content via modulation of caveolin-3 and TRIM72.

Microcurrent electrical neuromuscular stimulation (MENS) is known as an extracellular stimulus for the regeneration of injured skeletal muscle in sports medicine. However, the effects of MENS-associated increase in muscle protein content are not fully clarified. The purpose of this study was to investigate the effects of MENS on the muscular protein content, intracellular signals, and the expression level of caveolin-3 (Cav-3), tripartite motif-containing 72 (TRIM72) and MM isoenzyme of creatine kinase (CK-MM) in skeletal muscle using cell culture system. C2C12 myotubes on the 7th day of differentiation phase were treated with MENS (intensity: 10-20 microA, frequency: 0.3 Hz, pulse width: 250 ms, stimulation time: 15-120 min). MENS-associated increase in the protein content of myotubes was observed, compared to the untreated control level. MENS upregulated the expression of Cav-3, TRIM72, and CK-MM in myotubes. A transient increase in phosphorylation level of Akt was also observed. However, MENS had no effect on the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 and 5'AMP-activated protein kinase. MENS may increase muscle protein content accompanied with a transient activation of Akt and the upregulation of Cav-3 and TRIM72.

Signaling of IL13 through IL4Ra Promotes Cardiac Regeneration

IntroductionDuring the first week of life, neonatal mice are able to regenerate their hearts after injury with minimal scarring. Our lab has previously published that the global knockout of anti‐inflammatory cytokine Interleukin 13 (IL13) inhibits neonatal cardiac regeneration in mice. IL13 primarily acts through the IL4Ra/IL13Ra1 heterodimer receptor to promote cell cycle activity. However, how IL13 specifically signals in the heart to promote this phenotype remains unexplored. We hypothesize that the deletion of the IL4Ra component of IL13's receptor complex will lead to a similar lack of regeneration in the mouse heart.MethodsTo delineate IL13 signaling mechanisms in the mouse, we utilized various knockouts of IL13 signaling—IL13 global knockout mice, IL4Ra global knockout mice, and C57/BL6 wildtype mice. With cardiomyocytes (CMs) isolated from these models, we analyzed the dynamics of downstream factors of recombinant IL13 (rIL13) in vitro. Additionally, at postnatal day 1 (P1), our mouse models were given apical resections to remove up to 10% of the apex of the heart. After resection, we investigated mRNA and protein expression of downstream markers, as well as CM proliferation at different time points. We also analyzed overall regenerative potential through echocardiography and Gömöri trichrome staining.ResultsIn vitro administration of rIL13 to wildtype neonatal mouse CMs resulted in strong, but transient activation of AKT and ERK1/2, which have been shown to be pro‐survival and pro‐proliferative respectively. In our IL4Ra KO mice, we see an attenuation of this signaling. At 7 days post resection (dpr), we see a significant decrease in proliferating CMs from IL13 KO mice when compared to wildtype. Additionally, at 21dpr in vivo, we observe significant fibrotic apical scars in IL13 KO mice, indicating lack of cardiac regeneration.ConclusionFrom these results, our data suggests that depletion of IL13 and its receptor, IL4Ra, inhibits regenerative pathways in the heart.Support or Funding InformationAmerican Heart AssociationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lopinavir-NO, a nitric oxide-releasing HIV protease inhibitor, suppresses the growth of melanoma cells in vitro and in vivo.

We generated a nitric oxide (NO)-releasing derivative of the anti-HIV protease inhibitor lopinavir by linking the NO moiety to the parental drug. We investigated the effects of lopinavir and its derivative lopinavir-NO on melanoma cell lines in vitro and in vivo. Lopinavir-NO exhibited a twofold stronger anticancer action than lopinavir in vitro. These results were successfully translated into syngeneic models of melanoma in vivo, where a significant reduction in tumour volume was observed only in animals treated with lopinavir-NO. Both lopinavir and lopinavir-NO inhibited cell proliferation and induced the trans-differentiation of melanoma cells to Schwann-like cells. In melanoma cancer cell lines, both lopinavir and lopinavir-NO induced morphological changes, minor apoptosis and reactive oxygen species (ROS) production. However, caspase activation and autophagy were detected only in B16 cells, indicating a cell line-specific treatment response. Lopinavir-NO released NO intracellularly, and NO neutralization restored cell viability. Treatment with lopinavir-NO induced only a transient activation of Akt and inhibition of P70S6 kinase. The results of this study identify lopinavir-NO as a promising candidate for further clinical trials in melanoma and possibly other solid tumours.

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling.

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling.

Systems biology approach to determine critical T cell receptor signaling events for the induction of regulatory T cells (LYM8P.639)

Abstract Antigen stimulation of the T cell receptor (TCR) in a naïve T cell triggers a complex network of signaling pathways that determine whether the cell becomes a tolerogenic T regulatory (Treg) or immunogenic T helper (TH) cell. We previously developed a Boolean model of T cell differentiation that predicted variation of TCR signal duration could be used to optimize the production of Tregs, a strategy that we confirmed in vitro. This model suggested that dynamics of PTEN, a lipid phosphatase that opposes the activation of Akt, might be predictive of cell fate. Subsequent experiments have demonstrated that several mechanisms for regulating PTEN, including posttranslational modification, proteasomal degradation, and transcriptional inhibition, play an important role in T cell fate determination. A more fine-grained computational model of T cell signaling, which considers site-specific phosphorylation of Akt and PTEN, negative regulation of mTORC2 by kinases downstream of Akt, and negative regulation of PTEN through several Akt-dependent and independent mechanisms, recapitulates the dose-dependent dynamics of PTEN and Akt activity. The net effect of these positive and negative feedback interactions is to create a strength- and time-dependent threshold for TCR stimulation to drive the sustained Akt activity required for the differentiation and proliferation of TH cells. The model also shows that sub-threshold TCR signals lead to transient Akt activation leading to a Treg phenotype.

Role of Akt and Ca2 + on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition

Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion.

Abstract 1804: New insights in the induction of cell death by the imidazolium based compound YM155

Abstract The inhibitor of apoptosis protein (IAP) Survivin is robustly over-expressed in a variety of cancers including prostate carcinomas, and has been shown to play pivotal roles in cancer progression and resistance to chemotherapy. The imidazolium based small molecule YM155, which was first identified as a robust suppressor of the Survivin promoter of PC-3 prostate carcinoma and under clinical investigation for the treatment of multiple cancers, is a potent apoptosis inducer of castration-resistant prostate cancer (CRPC) cells. However, the mechanisms by which it inhibits the expression of Survivin and kills cancer cells remain unclear. Here we studied early signaling responses to YM155 in a panel of prostate and kidney cancer cell lines in standard cell culture conditions and showed that YM155 rapidly decreases the expression of cyclin Ds 1, 2 and 3, and activates the retinoblastoma protein (Rb), leading to the downregulation of the Survivin promoter. However, despite the robust lost of cyclin Ds, YM155 promotes an S-phase arrest rather than arrests cells at either G1 or G2/M. This suggests cell killing by YM155 involves S-phase arrest, consistent with a DNA damage response. In addition, we show that YM155 also downregulates the expression of the anti-apoptotic protein Mcl-1 and induces the expression of proapoptotic proteins Bad and Bim followed by the activation of caspases -3 and -8. Moreover, early time course experiments show that YM155 induces rapid (10 - 60 min) but transient activation of Akt and Erk1/2 followed by a marked suppression by 4 to 8 h likely via the induced expression of phosphatases 1 and 5 (DUSP1 and 5). Taken together we suggest YM155 induces apoptosis of prostate epithelial cells through a complex mechanism involving Akt, Erk1/2, DUSPs, cyclin Ds, Rb, S-phase arrest, Bim, Bad, Mcl-1,Survivin, and activation of caspases -3 and -8. Citation Format: David Danielpour, Eswar Shankar, Sarah L. Corum. New insights in the induction of cell death by the imidazolium based compound YM155. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1804. doi:10.1158/1538-7445.AM2014-1804

Effect of thrombopoietin receptor agonists on the apoptotic profile of platelets in patients with chronic immune thrombocytopenia.

Platelet survival depends upon mediators of apoptosis e.g., Bcl-xL, Bax, and Bak, which are regulated by thrombopoietin (TPO)-mediated AKT signaling. Thrombopoietin receptor (TPO-R) signaling might decrease platelet and/or megakaryocyte apoptosis and increase the platelet count. This study therefore explored anti-apoptotic effects of TPO-R-agonists in vivo on platelets of patients with immune thrombocytopenia. Patients received eltrombopag or romiplostim for two weeks. Total, immature, and large platelet counts were assessed as were Bcl-xL inhibitor assay; Bcl-xL Western blot; and flow cytometric (FACS) analysis of the AKT-signaling pathway. Eight/ten patients had platelet responses to eltrombopag and all three to romiplostim. Platelet sensitivity to apoptosis by Bcl-xL inhibition was greater in pretreatment patients than controls. This sensitivity normalized after one week of therapy, but surprisingly returned to pretreatment levels at week two. FACS analysis revealed increased AKT-pathway signaling after one week, followed by a decrease at week two. Platelet counts correlated with the Bcl-xL /Bak ratio. Platelet survival may be enhanced by TPO-R-agonists as a transient decrease in platelet sensitivity to apoptosis was accompanied by transient activation of AKT. However, this mechanism has only a short-lived effect. Megakaryocytes and platelets already present at the start of TPO-R-agonist treatment appear to respond differently than those generated de novo.

Phosphorylation accelerates geldanamycin-induced Akt degradation

Hsp90 (Heat shock protein-90) is a cellular buffer against erroneous gene products and also plays an essential role in facilitating proper folding, maturation, and activity of its client proteins. The phosphatidylinositol-3 kinase (PI-3K)–Akt pathway transduces a survival signal involved in tumor development. The kinase activity of Akt depends on its association with Hsp90. Hsp90 inhibition causes Akt degradation, but the mechanism remains unclear. Several reports showed that the Hsp90 inhibitor geldanamycin (GA) induces Thr308 and Ser473 phosphorylations of Akt, however, it is still unknown about the significance of GA-induced Akt activation in degradation of the kinase. We treated Hela cells with GA to observe Akt degradation and found that LY294002 delayed Akt degradation. Mutation of Thr308 or Ser473 also caused delayed Akt ubiquitination and degradation. Inhibition of Akt dephosphorylation enhanced GA-mediated Akt degradation. In this report, we show that GA-mediated transient activation of Akt accelerates its association with the E3 ligase CHIP (C-terminal Hsp70-interacting protein)-mediated ubiquitination and subsequent proteasome degradation.

Abstract 603: CAL-101 acts synergistic with arsenic trioxide in chronic lymphocytic leukemia by preventing p21 phosphorylation through AKT1.

Abstract Chronic lymphocytic leukemia (CLL) is the most abundant leukemia of the elderly in the Western world and characterized by the accumulation of CD5/CD19/CD23 positive cells in the peripheral blood, lymph nodes and spleen. It has a very heterogeneous course of disease with patients living longer than 10 years after diagnosis and others needing immediate treatment. CLL cells of the latter often bear aberrations in the p53 tumor suppressor gene. We have shown before that arsenic trioxide preferentially induces apoptosis in CLL cells deficient for p53 (n = 51, P = 0.029). We now reproduced this finding in mouse embryo fibroblasts (MEF) that are p53 deficient. Next we wanted to clarify the role of p53 downstream targets p21 and PUMA and found that p21 but not PUMA knockout led to increased arsenic trioxide susceptibility. When localized in the cytosol p21 has been described to have and anti-apoptotic function and accordingly is associated with negative prognosis in breast, ovarian and prostate cancer. To clarify p21’s localization in CLL cells we used differential detergent cell fractionation and demonstrate that upon arsenic trioxide treatment p21 is induced, but accumulates in the cytoplasm. This is in contrast to fludarabine treated cells, where p21 is strongly induced and shuttles to the nucleus. Moreover, we show here that arsenic trioxide leads to transient AKT activation and AKT phosphorylation of p21 has been shown to promote cytosolic localization. We next hypothesized that by combining the PI3Kdelta/AKT inhibitor CAL-101 with arsenic trioxide we might prevent cytosolic accumulation of p21. Recently, the idea of therapeutic inhibition of tyrosinse kinases in the B-cell receptor pathway has gained new momentum through pivotal phase-2 clincal studies with CAL-101 and ibrutinib targeting the PI3Kdelta- and the Bruton's tyrosine-kinase, respectively. We show here, that the PI3K inhibitor CAL-101 abrogates AKT activation through arsenic trioxide. This leads to strongly, synergistic apoptosis induction when CAL-101 and arsenic trioxide are combined irrespective of p53 status of CLL cells. Thus, we think that these findings not only shed light on the role of p21 in apoptosis induction in CLL, but open the path to novel therapeutic combination regimens. Citation Format: Olaf Merkel, Eveline Sifft, Simone Schweiggl, Daniela Asslaber, Nathalie Wacht, Richard Greil. CAL-101 acts synergistic with arsenic trioxide in chronic lymphocytic leukemia by preventing p21 phosphorylation through AKT1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2013-603

Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model

BackgroundRegardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model.MethodsBoswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0–2 h, Fraction II at 8–10 h, and Fraction III at 11–12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11–12 h (Fraction IV). Chemical compositions were identified by gas chromatography–mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry.ResultsLonger duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic acids, in frankincense essential oil fraactions. Human pancreatic cancer cells were sensitive to Fractions III and IV (containing higher molecular weight compounds) treatment with suppressed cell viability and increased cell death. Essential oil activated the caspase-dependent apoptotic pathway, induced a rapid and transient activation of Akt and Erk1/2, and suppressed levels of cyclin D1 cdk4 expression in cultured pancreatic cancer cells. In addition, Boswellia sacra essential oil Fraction IV exhibited anti-proliferative and pro-apoptotic activities against pancreatic tumors in the heterotopic xenograft mouse model.ConclusionAll fractions of frankincense essential oil from Boswellia sacra are capable of suppressing viability and inducing apoptosis of a panel of human pancreatic cancer cell lines. Potency of essential oil-suppressed tumor cell viability may be associated with the greater abundance of high molecular weight compounds in Fractions III and IV. Although chemical component(s) responsible for tumor cell cytotoxicity remains undefined, crude essential oil prepared from hydrodistillation of Boswellia sacra gum resins might be a useful alternative therapeutic agent for treating patients with pancreatic adenocarcinoma, an aggressive cancer with poor prognosis.

Transforming Growth Factor Alpha (TGFα) Regulates Granulosa Cell Tumor (GCT) Cell Proliferation and Migration through Activation of Multiple Pathways

Granulosa cell tumors (GCTs) are the most common ovarian estrogen producing tumors, leading to symptoms of excessive estrogen such as endometrial hyperplasia and endometrial adenocarcinoma. These tumors have malignant potential and often recur. The etiology of GCT is unknown. TGFα is a potent mitogen for many different cells. However, its function in GCT initiation, progression and metastasis has not been determined. The present study aims to determine whether TGFα plays a role in the growth of GCT cells. KGN cells, which are derived from an invasive GCT and have many features of normal granulosa cells, were used as the cellular model. Immunohistochemistry, Western blot and RT-PCR results showed that the ErbB family of receptors is expressed in human GCT tissues and GCT cell lines. RT-PCR results also indicated that TGFα and EGF are expressed in the human granulosa cells and the GCT cell lines, suggesting that TGFα might regulate GCT cell function in an autocrine/paracrine manner. TGFα stimulated KGN cell DNA synthesis, cell proliferation, cell viability, cell cycle progression, and cell migration. TGFα rapidly activated EGFR/PI3K/Akt and mTOR pathways, as indicated by rapid phosphorylation of Akt, TSC2, Rictor, mTOR, P70S6K and S6 proteins following TGFα treatment. TGFα also rapidly activated the EGFR/MEK/ERK pathway, and P38 MAPK pathways, as indicated by the rapid phosphorylation of EGFR, MEK, ERK1/2, P38, and CREB after TGFα treatment. Whereas TGFα triggered a transient activation of Akt, it induced a sustained activation of ERK1/2 in KGN cells. Long-term treatment of KGN cells with TGFα resulted in a significant increase in cyclin D2 and a decrease in p27/Kip1, two critical regulators of granulosa cell proliferation and granulosa cell tumorigenesis. In conclusion, TGFα, via multiple signaling pathways, regulates KGN cell proliferation and migration and may play an important role in the growth and metastasis of GCTs.

CXCL10 contributes to p38-mediated apoptosis in primary T lymphocytes in vitro

CXCL10 is part of the group of interferon-stimulated genes and it plays an important role during different viral infections by inducing cell activation, chemotaxis and lymphocyte priming toward the Th1 phenotype. In this study, we investigated in vitro the effects of CXCL10 in activated human primary T lymphocytes in terms of apoptosis or survival, and delineated the signaling pathways that are involved. CXCL10, in combination with IL-2 and/or IFNα, induces apoptosis in T lymphocytes. Moreover, CXCL10-induced activation of CXCR3 also triggers pro-survival signals that can be blocked by pertussis toxin. The analysis of the downstream signaling kinases shows that apoptosis is p38 MAPK-dependent and the pro-survival signals rely on the sustained activation of PI3K and the transient activation of Akt. On the other hand, the transient activation of p44/p42 ERK did not have an impact on T lymphocyte survival. We propose an immunological model in which CXCL10, together with other co-stimulating cytokines, participates in the activation of T lymphocytes, promotes survival and expansion of certain lymphocyte subsets, and induces chemotaxis toward the infected tissues. On the other hand, CXCL10 might contribute to the triggering of apoptosis in other subsets of T lymphocytes, including those lymphocytes that were transiently activated but later lacked the appropriate sets of specific co-stimulating signals to ensure their survival.

The Effect of Eltrombopag on Platelet Resistance to Apoptosis: The Role of the Bcl-Xl Pathway

Abstract 1158 INTRODUCTION:Immune thrombocytopenia (ITP) is typically characterized by increased platelet destruction and reduced platelet production. Eltrombopag and Romiplostim are thrombopoietin receptor (TPO-R) agonists that are known to increase platelet counts in patients with ITP by stimulating thrombopoiesis. Platelets also express TPO-R on their surface, but it is unknown whether the thrombopoietin mimetics (TPO-M) have a direct effect on the circulating platelets. Although controversial, in a very small number of ITP patients, TPO-M agents may increase platelet counts in 2–5 days, earlier than would be expected from de novo megakaryocytopoiesis. Platelet survival is hypothesized to be mediated by two molecular intermediates in an apoptotic pathway, Bcl-xL and Bak. Bcl-xL/Bak protein expression in megakaryocytes is regulated in part by TPO-mediated activation of Akt pathways through Jak2 and Stat5. We hypothesized that an increase in platelet count in the first week of treatment might be mediated by TPO-R signaling, resulting in decreased platelet apoptosis. This study explored whether Eltrombopag or Romiplostim treatment has anti-apoptotic effects on platelets of patients with ITP. METHODS:Following a treatment wash out period, 75 mg of Eltrombopag once daily or 10 mcg/kg weekly of Romiplostim was initiated for 2 weeks. Blood counts were measured on days 1, 3, 5, 8, 10, 12, and 15. Platelet function and survival was assessed on days 1, 8, and 15 by: immature platelet fraction (IPF), glycocalicin index, Bcl-xL inhibitor (ABT-737) assay, measurement of Bcl-xL by western blot, measurement of several members of the Bcl-xL Akt mediated, apoptotic pathway by flow cytometry (FACS), bleeding score, measurement of thrombin-anti-thrombin complexes (TATs), and quantification of microparticles. RESULTS:Eight of 10 patients responded to treatment with Eltrombopag with a platelet count ≥ 50,000/μL, and 6 of the 8 responders at least doubled their counts during the 2 weeks of treatment. All 3 patients treated with Romiplostim responded with platelet count ≥ 50,000/μL. In both treatment groups there was a significant increase in median platelet count (p<0.001), median large platelet count (p<0.01), and median absolute IPF (A-IPF, p<0.01), while there was no significant change in median % IPF. The dose of ABT-737 required to kill half of the platelets in the sample (IC50) in the Eltrombopag group was lower in patients at day 1 than in non-ITP controls, and there was an increase in resistance to apoptosis between days 1 and 8, but these changes did not reach statistical significance. Between days 8 and 15 the IC50 declined to pre-treatment levels. In the Romiplostim group there was no significant difference in IC50 between the control and the patients over the 2 weeks of study. There was no significant correlation between the platelet counts and the IC50 values. FACS analysis of members of the AKT signal transduction pathway revealed increased activation of each of the markers between days 1 and 8, followed by a decrease between days 8 and 15. The levels of Bcl-xL and phosphor-AKT(308) decreased from day 1 to day 15. The other lab tests are pending. DISCUSSION:Because the A-IPF increased by less than the platelet increase and because the lifespan of the A-IPF is not known, it is unclear if the platelet count increase is solely a result of increased platelet production. Platelet lifespan may be enhanced by Eltrombopag treatment as there was a parallel albeit transient increase in AKT activation markers and platelet apoptosis resistance in the Eltrombopag group. Treatment with Romiplostim did not appear to affect apoptosis resistance although it did result in transient AKT activation. Our data suggest that platelets are more resistant to apoptosis when the levels of anti-apoptotic factors (eg. PTEN, Phospho-GSK3β) involved in the AKT/Bcl-xL pathway are greatest despite a concomitant increase in pro-apoptotic factors (eg. Bak, Bax). Since both the increased AKT activation and apoptotic resistance returned to baseline at day 15, megakaryocytes and platelets already present at the start of treatment may respond differently than those generated de novo in the presence of TPO mimetics. [Display omitted] Disclosures:Bussel:Portola: Consultancy; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cangene: Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding.

E3 ubiquitin ligase Cbl-b potentiates the apoptotic action of arsenic trioxide by inhibiting the PI3K/Akt pathway

Arsenic trioxide (ATO) is a strong inducer of apoptosis in malignant hematological cells. Inducible phosphatidyl inositol 3 kinase (PI3K)-Akt activation promotes resistance to ATO. In the present study, we evaluated whether E3 ubiquitin ligase Cbl-b, a negative regulator of PI3K activation, is involved in the action of ATO. The effect of ATO on cell viability was measured by the Trypan blue exclusion assay or by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry and protein expression was assayed by Western blotting. ATO decreased the viability of HL60 cells and induced cellular apoptosis, which was accompanied by transient activation of Akt. The PI3K/Akt inhibitor, LY294002, significantly increased ATO-induced apoptosis (P < 0.05). In addition, ATO up-regulated the expression of Cbl-b proteins. Furthermore, ATO inhibited cell viability with an IC50 of 18.54 μM at 24 h in rat basophilic leukemia-2H3 cells. ATO induced cellular apoptosis with transient activation of Akt and Cbl-b was also up-regulated. Rat basophilic leukemia-2H3 cells transfected with a dominant negative (DN) Cbl-b mutation showed overexpression of Cbl-b (DN) and enhanced Akt activation. Compared with cells transfected with vector, ATO-induced apoptosis was decreased and G2/M phase cells were increased at the same concentration (P < 0.05). The PI3K/Akt inhibitor, LY294002, re-sensitized Cbl-b (DN) overexpressing cells to ATO and reversed G2/M arrest (P < 0.05). Taken together, these results suggest that Cbl-b potentiates the apoptotic action of ATO by inhibition of the PI3K/Akt pathway.

Suppression of human prostate cancer PC-3 cell growth by N-acetylcysteine involves over-expression of Cyr61

N-acetylcysteine (NAC), sulfidryl-containing thiol antioxidant, has been heralded as chemopreventive agent, generally because of its ability to scavenge free radicals. It also suppresses the proliferation of many cancer cells; however, the antiproliferative mechanism(s) remain to be fully elucidated. In this study, we investigated a growth-suppressive mechanism of NAC action in androgen-independent prostate carcinoma PC-3 cells. NAC (⩾1 mM) inhibited the proliferation of PC-3 cells in a dose- and time-dependent manner. Moreover, NAC treatment suppressed the activation of NF-κB induced by IKK-β as detected by the NF-κB reporter gene assay. NAC exerted a biphasic effect on the intracellular ROS levels depending on incubation time; the antioxidant effect was seen within 2 h after NAC treatment, however, a pro-oxidant effect was evident after 48 h treatment. In addition to these effects, NAC treatment elicited a dose- and time-dependent increase in the Cyr61 expression that was accompanied by an increase in its mRNA and blocked by cycloheximide pretreatment. Importantly, NAC treatment caused an early but transient activation of Akt and Erk1/2. The NAC-induced increase in Cyr61 protein levels was suppressed by the PI3K inhibitor (Ly294002) and, to a lesser extent, MEK/Erk1/2 inhibitor (PD98059). Taken together, our data suggest that the antiproliferative effect of NAC is partially mediated by intracellular ROS production, the inhibition of NF-κB activity, and the activation of PI3K- and/or MEK/Erk-related intracellular signaling pathways, which lead to up-regulation of Cyr61 expression.

372 Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting the dependence receptor TrkC-induced apoptosis

Neurotrophin-induced neuroprotection against apoptosis was investigated using immature cultured cerebellar granule cells (CGC) from newborn rat pups. Apoptotic cell death induced by treatment with cytosine arabinoside (AraC) was confirmed by DNA fragmentation and quantified by cell survival assays. AraC was most effective in inducing apoptosis when added to CGC on the day of culture preparation, while less or no effect was observed when added at 24 or 48 h after plating, respectively. Pretreatment of CGC cultures for 24 h with brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3), robustly protected against AraC neurotoxicity. K252a, an inhibitor of the tropomyosin-related kinase (Trk) tyrosine kinase receptor family which showed no toxicity by itself, blocked BDNF protection of AraC-induced apoptosis in a concentration-dependent manner. Neither protein kinase C activation nor inhibition mimicked or affected BDNF protection against AraC neurotoxicity. BDNF, but not NT-3, treatment of immature CGC caused a marked, but transient activation of Akt through phosphatidylinositol (PI) 3-kinase. The neuroprotective effects of BDNF were suppressed by pretreatment with LY 294002 (a PI 3-kinase inhibitor). BDNF neuroprotection was also preceded by activation of mitogen activated protein kinase (MAPK) and suppressed by two MAPK/ERK (MEK)-selective inhibitors, PD 98059 and U-0126. Moreover, inhibitors of PI 3-kinase and MEK potentiated AraC-induced neurotoxicity. These results show that neurotrophins protect against AraC-induced apoptosis, at least in part, through TrkB-mediated activation of the PI 3-kinase/Akt and MEK signaling pathways.