Reduction Of Akt Research Articles

Efficacy of silver-doped Carbon dots in Chemical Castration: a rat model study

This study evaluates silver-doped carbon dots (AgCDs) as a novel agent for chemical castration using a rat model. Six groups of rats (five males and ten females each, except for the surgical group which had only males) were utilized to compare the effects of different concentrations of AgCDs. The groups included control, sham, and three experimental groups injected with 1.25, 50, and 200 µg/mL AgCDs, respectively, along with a surgical castration group. Testosterone levels, sperm parameters, fertility index, oxidative damage, histopathological parameters, and gene expression of P53, Bax, Bcl-2, caspase-3, AKT, and PI3K were analyzed. Results demonstrated that the high-dose AgCDs group significantly reduced testosterone levels, sperm concentration, and motility, resulting in a decreased fertility index. MDA and NO significantly increased, while CAT, SOD, GPx, and TAC significantly reduced in the chemically castrated groups. Histological and genes expression analysis also revealed apoptosis and testicular damage in the AgCDs groups, indicated by significant increases in P53, Bax, and Caspase-3 levels, and significant reductions in AKT, PI3K, and Bcl-2. Based on these findings, AgCDs could be considered a potent and efficient agent for chemical castration, offering a less invasive, cost-effective solution with potential applications for population control.

Duhuo Jisheng Decoction regulates intracellular zinc homeostasis by enhancing autophagy via PTEN/Akt/mTOR pathway to improve knee cartilage degeneration.

Articular cartilage and cartilage matrix degradation are key pathological changes occurring in the early stage of knee osteoarthritis (KOA). However, currently, there are limited strategies for early prevention and treatment of KOA. Duhuo Jisheng Decoction (DHJSD) is a formula quoted in Bei Ji Qian jin Yao Fang, which was compiled by Sun Simiao in the Tang Dynasty of China. As a complementary therapy, it is widely used to treat early-stage KOA in China; however, its mechanism has not been completely elucidated. This study investigated the potential role of DHJSD in preventing cartilage degradation and the underlying mechanism. A rat model of KOA model was established via the Hulth method. Subsequently, 25 rats were randomized into sham (saline), model control (saline), high-DHJSD (1.9g/mL of DHJSD), medium-DHJSD (1.2g/mL of DHJSD), and low-DHJSD groups (0.6g/mL of DHJSD). After 4 weeks of treatment, all rats were sacrificed and the severity of the cartilage degeneration was evaluated by a series of histological methods. The autophagosome was observed using transmission electron microscopy, and the related functional proteins were detected by the western blotting and real-time polymerase chain reaction. Next, the mechanism by which DHJSD improves knee cartilage degeneration was further clarified the in vitro by gene silencing technology combined with a series of functional experiments. The proteins levels of PTEN, Akt, p-Akt, mTOR, and p-mTOR, as well as the marker proteins of autophagy and apoptosis were determined. Zinc levels in chondrocytes were determined using inductively coupled plasma mass spectrometry. Histopathological staining revealed that DHJSD had a protective effect on the cartilage. DHJSD increased autophagosome synthesis and the expression of autophagy proteins LC3 and Beclin-1 in chondrocytes. Moreover, it reduced the phosphorylation levels of Akt and mTOR and the levels of zinc, MMP-13, Bax, and Bcl-2. Following PTEN silencing, this DHJSD-mediated reduction in Akt and mTOR phosphorylation and Bax, Bcl-2, and zinc levels were further decreased; in addition, DHJSD-mediated increase in LC3 and Beclin-1 levels was decreased. DHJSD inhibits the Akt/mTOR signaling pathway by targeting PTEN to promote autophagy in chondrocytes, which may help reduce MMP-13 production by regulating zinc levels in chondrocytes.

Naringenin alleviate reproductive toxicity evoked by lead acetate via attenuation of sperm profile and biochemical alterations in male Wistar rat: Involvement of TGFβ/AKT/mTOR pathway.

Exposure to Lead -causes testicular dysfunction through oxidative stress, inflammation, and apoptosis; however, naringenin (NGN) therapeutic impact against lead-evoked testicular dysfunction remains elusive. Herein, the point of the study was to examine the defensive impact of NGN on testicular dysfunction initiated by lead. Seventy-Two male Wistar rats were allotted into nine groups; control group, drug control groups, lead acetate group, as well as NGN treated groups (10, 25, and 50 mg/kg) respectively, given 5 days before lead acetate treatment. The result showed clearly the impact of lead on reduced sperm count, sperm motility as well as serum testosterone and LH levels. Additionally, it caused a significant rise in testicular inflammatory markers TNF-α, IL-1β, and TGFβ, effects that were accompanied by a reduction of AKT and mTOR levels. Lead acetate also caused degenerative changes in the testis, atrophy, and loss of spermatogenic series. Our findings revealed that NGN in a dose-dependent manner improved spermiotoxicity induced by lead acetate via restoration of the testicular function, preservation of spermatogenesis, halting inflammatory cytokines along with the enhancement of germ cell survival using upregulation of AKT/mTOR expressions. The present study discloses that NGN suppresses lead acetate toxicity that is involved in the antioxidant effect in a dose-dependent manner,besides its anti-inflammatory property.

Alpha-Synuclein Clearance through Inhibiting Akt/mTOR Pathway by Microfluidic Encapsulated Induced Conjunctival MSCs in a Parkinsonian Model.

Background & Objective:Parkinson's disease (PD) is a progressive neurodegenerative disorder in which the cause is attributed to the alpha-synuclein (α-Syn) accumulation due to the decreased rate of autophagy. Due to the many advantages, mesenchymal stem cells (MSCs), such as the secretion of neurotrophic factors, have been proposed for PD cell therapy. The present study, in continuation of the previous study, aimed to investigate the therapeutic effect of human-derived Conjunctival MSCs (CJ-MSCs) on the clearance of α-Syn by the microRNA-149(miR-149)/Akt/mTOR/ pathway. Methods:Stereotaxic 6-hydroxy dopamine (6-OHDA) was injected directly into the medial forebrain bundle (MFB) to induce Parkinson's disease. An apomorphine-induced rotation test was used to confirm the model establishment. CJ-MSCs were encapsulated in alginate microgel using a microfluidic system. The green fluorescent protein (GFP) labeled CJ-MSCs were encapsulated, and free cells were transplanted into the rats' right striatum. Behavioral and molecular analyses evaluated the potency of CJ-MSCs (encapsulated and free cells) in PD rats. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was performed to investigate the expression of the miR-149-5p, Akt, mTOR, and α-Syn.Results:Our obtained results indicated that transplantation of CJ-MSCs leads to a decrease in the number of rotations while raising the balance and motor abilities. The gene expression evaluation showed a significant reduction in Akt, mTOR, and α-Syn mRNA levels and a significant increase in the level of miR-149-5p compared to the control group. Conclusion:It seems that CJ-MSCs can promote the degradation of intracellular α-Syn by miR-149-5p/Akt/mTOR pathway and improve rats' motor functions.

Endothelial thioredoxin interacting protein (TXNIP) modulates endothelium-dependent vasorelaxation in hyperglycemia

Endothelial dysfunction, hallmarked by an imbalance between vasoconstriction and vasorelaxation, is associated with diabetes. Thioredoxin Interacting protein (TXNIP), controlled by an exquisitely glucose sensitive gene, is increasingly recognized for its role in diabetes. However, the role of TXNIP in modulating diabetes-related endothelial dysfunction remains unclear. To elucidate the role of TXNIP, we generated two novel mouse strains; endothelial-specific TXNIP knockout (EKO) and a Tet-O inducible, endothelial-specific TXNIP overexpression (EKI). Hyperglycemia was induced by streptozotocin (STZ) treatment in floxed control (fl/fl) and EKO mice. Doxycycline (DOX) was given to EKI mice to induce endothelial TXNIP overexpression. The ablation of endothelial TXNIP improved glucose tolerance in EKO mice. Acetylcholine-induced, endothelium-dependent vasorelaxation was impaired in STZ-treated fl/fl mice while this STZ impaired vasorelaxation was attenuated in EKO mice. Hyperglycemia induction of NLRP3 and reductions in Akt and eNOS phosphorylation were also mitigated in EKO mice. Overexpression of endothelial TXNIP did not impair glucose tolerance in DOX-treated EKI mice, however induction of endothelial TXNIP led to impaired vasorelaxation in EKI mice. This was associated with increased NLRP3 and reduced Akt and eNOS activation. In conclusion, deletion of endothelial TXNIP is protective against and overexpression of endothelial TXNIP induces endothelial dysfunction; thus, endothelial TXNIP plays a critical role in modulating endothelial dysfunction.

Interferon Regulatory Factor 4 Orchestrates the Response to B-Cell Receptor in Chronic Lymphocytic Leukemia Cells By Regulating Ikaros

Chronic lymphocytic leukemia (CLL) accounts for about 30% of all adult leukemias and is the most common hematologic malignancy in the Western countries. Interferon regulatory factor 4 (IRF4, also known as MUM1) is a transcriptional regulator of immune system development and function. Different IRF4 concentrations underlie the generation of alternative cell fate in mature B cells localized in secondary lymphoid organs, i.e. IRF4 “kinetic control” model. IRF4 at lower levels promotes germinal center formation and class switch recombination, whereas at higher levels IRF4 inhibits Bcl-6 and induces Blimp-1 to facilitate plasma cell development. Recent studies suggest a possible role of IRF4 in CLL pathogenesis. In New Zealand Black (NZB) IRF4+/- mouse model, CLL development is dramatically accelerated and IRF4+/- CLL cells showed hyper-responsiveness to B-cell receptor (BCR) activation.In the present study, we investigated the role of IRF4 in controlling CLL responsiveness to BCR stimulation.We first inspected IRF4 expression in a cohort of 225 CLL, finding reduced levels of expression in almost all cases. Then, we modulated IRF4 levels in vitro in leukemic cells collected from CLL patients by transfecting cells using Human IRF4 pCMV6-Entry vector or by silencing with siRNA strategy. CLL cells were then stimulated with F(AB')2 fragment to human IgM (5FCµ). Higher levels of IRF4 attenuated BCR signaling as assessed by a reduction in AKT and ERK phosphorylation and calcium release. Conversely, IRF4 silencing improved CLL responsiveness to BCR stimulation. The expression levels of IgM, CD79A and CD79B were not affected by IRF4 modulation. Ikaros (IKZF1), a zinc finger, DNA-binding protein that regulates gene transcription and chromatin remodeling, is a master regulator of lymphocyte development. Our results indicate that IRF4 induction in CLL cells down-regulates IKZF1 levels throughout post-transcriptional mechanisms. We wondered if IKZF1 may mediate BCR activation in CLL cells. To demonstrate this hypothesis, we interfered with IKZF1 by silencing strategies and pharmacologically by treating CLL cells with IMIDs, lenalidomide and avadomide (CC-122). We found that IKZF1 down-regulation attenuates BCR signaling by inducing the expression of the inositol 5-phosphatase SHIP, which dephosphorylates PIP3 blocking AKT activation. Concomitantly, IRF4 up-regulation promotes ZAP70 degradation. These effects were also obtained by treating CLL cells with both lenalidomide and avadomide, which up-regulated IRF4 expression while reducing IKZF1 levels. Both IMIDs interfered with BCR response and mediated apoptosis in CLL cells in combination with ibrutinib.Overall, the results elucidate the mechanism by which IRF4 regulates BCR responsiveness in CLL cells. Low IRF4 levels maintains a permissive state in CLL cells, allowing an effective transmission of BCR signals throughout the accumulation of IKZF1. DisclosuresNo relevant conflicts of interest to declare.

Different glycoforms of alpha-1-acid glycoprotein contribute to its functional alterations in platelets and neutrophils.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12-20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.

FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors

BackgroundBreast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer.MethodsTo understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested.ResultsWe found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival.ConclusionsIn summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.

Downregulated AKT-mTOR signaling pathway proteins in dorsolateral prefrontal cortex in Schizophrenia.

Abnormal neurotransmission is central to schizophrenia (SZ). Alterations across multiple neurotransmitter systems in SZ suggest that this illness may be associated with dysregulation of core intracellular processes such as signaling pathways that underlie the regulation and integration of these systems. The AKT-mTOR signaling cascade has been implicated in SZ by gene association, postmortem brain and animal studies. AKT and mTOR are serine/threonine kinases which play important roles in cell growth, proliferation, survival, and differentiation. Both AKT and mTOR require phosphorylation at specific sites for their complete activation. mTOR forms two functionally distinct multiprotein complexes, mTOR Complex 1 (mTORC1) and Complex 2 (mTORC2). mTORC1 mediates ribosome biogenesis, protein translation, and autophagy, whereas mTORC2 contributes to actin dynamics. Altered protein synthesis and actin dynamics can lead to an abnormal neuronal morphology resulting in deficits in learning and memory. Currently, there is a lack of direct evidence to support the hypothesis of disrupted mTOR signaling in SZ, and we have addressed this by characterizing this signaling pathway in SZ brain. We found a reduction in AKT and mTOR protein expression and/or phosphorylation state in dorsolateral prefrontal cortex (DLPFC) from 22 pairs of SZ and matched comparison subjects. We also found reduced protein expression of GβL, a subunit protein common to both mTOR complexes. We further investigated mTOR complex-specific subunit composition and phosphorylation state, and found abnormal mTOR expression in both complexes in SZ DLPFC. These findings provide evidence that proteins associated with the AKT-mTOR signaling cascade are downregulated in SZ DLPFC.

The role of skeletal muscle Akt in the regulation of muscle mass and glucose homeostasis

ObjectiveSkeletal muscle insulin signaling is a major determinant of muscle growth and glucose homeostasis. Protein kinase B/Akt plays a prominent role in mediating many of the metabolic effects of insulin. Mice and humans harboring systemic loss-of-function mutations in Akt2, the most abundant Akt isoform in metabolic tissues, are glucose intolerant and insulin resistant. Since the skeletal muscle accounts for a significant amount of postprandial glucose disposal, a popular hypothesis in the diabetes field suggests that a reduction in Akt, specifically in skeletal muscle, leads to systemic glucose intolerance and insulin resistance. Despite this common belief, the specific role of skeletal muscle Akt in muscle growth and insulin sensitivity remains undefined. MethodsWe generated multiple mouse models of skeletal muscle Akt deficiency to evaluate the role of muscle Akt signaling in vivo. The effects of these genetic perturbations on muscle mass, glucose homeostasis and insulin sensitivity were assessed using both in vivo and ex vivo assays. ResultsSurprisingly, mice lacking Akt2 alone in skeletal muscle displayed normal skeletal muscle insulin signaling, glucose tolerance, and insulin sensitivity despite a dramatic reduction in phosphorylated Akt. In contrast, deletion of both Akt isoforms (M-AktDKO) prevented downstream signaling and resulted in muscle atrophy. Despite the absence of Akt signaling, in vivo and ex vivo insulin-stimulated glucose uptake were normal in M-AktDKO mice. Similar effects on insulin sensitivity were observed in mice with prolonged deletion (4 weeks) of both skeletal muscle Akt isoforms selectively in adulthood. Conversely, short term deletion (2 weeks) of skeletal muscle specific Akt in adult muscles impaired insulin tolerance paralleling the effect observed by acute pharmacological inhibition of Akt in vitro. Mechanistically, chronic ablation of Akt induced mitochondrial dysfunction and activation of AMPK, which was required for insulin-stimulated glucose uptake in the absence of Akt. ConclusionsTogether, these data indicate that chronic reduction in Akt activity alone in skeletal muscle is not sufficient to induce insulin resistance or prevent glucose uptake in all conditions. Therefore, since insulin-stimulated glucose disposal in skeletal muscle is markedly impaired in insulin-resistant states, we hypothesize that alterations in signaling molecules in addition to skeletal muscle Akt are necessary to perturb glucose tolerance and insulin sensitivity in vivo.

Enhancing the anti‐tumor efficacy of chimeric antigen receptor‐expressing T cells with naturally occurring plant stilbenes

Despite advances in treatment options, cancer remains the second leading cause of death in the United States. Many therapies use immune cells, specifically T cells, for cancer therapy. To enhance tumor recognition by T cells, chimeric antigen receptors consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Our lab has genetically engineered a chimeric PD1 (chPD1) receptor and has shown that chPD1‐expressing T cells kill tumor cells, secrete inflammatory cytokines, reduce tumor burden, and increase survival in multiple tumor types. A current focus of cancer research is the development of combination therapies that enhance anti‐tumor efficacy without increasing harmful side effects. Recently, a family of naturally occurring plant compounds called stilbenes have been shown to enhance T cell function. Therefore, we tested if stilbenes enhanced chPD1 T cell function in breast cancer. Addition of resveratrol, a stilbene found in berries, grapes, peanuts, and other plants, decreased proliferation, enhanced tumor cell killing, and increased secretion of proinflammatory cytokines (IFNγ, GM‐CSF, TNFα, IL‐17, IL‐2, and IL‐21) from chPD1 T cells. Presence of resveratrol also increased skewing of T cell differentiation to a central memory phenotype through reduction of AKT and mTORc1 signaling, reduced expression of T‐bet and BLIMP‐1 transcription factors, and increased expression of Bcl‐6 and Eomes transcription factors. Hence, combination of resveratrol and chimeric antigen receptor‐expressing T cells may enhance anti‐tumor immune responses through inhibition of the AKT and mTORc1 pathways and skewing to a long‐lived central memory phenotype.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract LB-014: Pirfenidone as a new potential therapy for malignant mesothelioma

Abstract The purpose of this study was to examine pirfenidone (PFD) as a potential new therapy for malignant pleural mesothelioma. PFD is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis (IPF). PFD reduces fibrous tissue formation e.g. by downregulating the production of fibroblasts and procollagens and inhibiting TGFß, TNFα and PDGF pathways. Since mesothelioma shares characteristics with IPF alongside with the activation of PI3K/Akt/mTOR, EGF, PDGF, HGF, VEGF, FGF and TGF-β pathways, we assessed the effect of PFD on mesothelioma cells in vitro and in vivo. In vitro studies were carried out using mesothelioma cell lines (JL-1, H2052, AB12) and primary mesothelioma cells. Cells treated with PFD and/or other agents (cisplatin, pemetrexed, MK-2206, PD98059, MG132) were subjected to cell viability assays, Transwell migration assays and 3D collagen or Matrigel growth and invasion assays. TGFβ/BMP, MAPK/Erk and Akt pathway activities were studied using luciferase reporter assays and human phospho-kinase array and validated with ELISA, RT-qPCR and Western blotting. For in vivo xenograft studies mice were injected subcutaneously with primary human mesothelioma cells expressing luciferase marker. Mice were treated with pirfenidone daily for 60 days and tumor growth was followed by bioluminescent imaging. Proliferation was studied by IHC. Differential gene expression in treated vs. untreated tumors was analyzed using RNA sequencing. We demonstrated that PFD treatment reduced significantly mesothelioma cell proliferation, migration and invasive growth in 3D in a concentration-dependent manner. The inhibitory effect of PFD increased when combined with cisplatin treatment in vitro. We also observed reduction of Akt and MAPK/Erk pathway activities and increase in BMP signaling in PFD treated tumor cells. Preliminary analyses of in vivo experiment revealed differential expression of multiple genes in pirfenidone treated vs. untreated tumors associated with a shift to more anti-fibrotic tumor microenvironment in PFD treated tumors. Expression profiles are currently under closer investigation. We identified pirfenidone as an inhibitor of mesothelioma invasive growth and showed that it has anti-fibrotic effects to tumor microenvironment. Since mesotheliomas are often inherently chemoresistant in fibrotic microenvironment, the use of pirfenidone could aid in overcoming chemoresistance by loosening ECM barrier for better drug penetration into the tumor tissue. Based on the current research we suggest that pirfenidone should be subjected to more intensive study as potential adjuvant-therapy for malignant mesotheliomas. Citation Format: Chang Li, Veronika Rezov, Emmi I. Joensuu, Ville Vartiainen, Mikko Rönty, Miao Yin, Marjukka Myllärniemi, Katri Koli. Pirfenidone as a new potential therapy for malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-014.

Puquitinib, a novel orally available PI3Kδ inhibitor, exhibits potent antitumor efficacy against acute myeloid leukemia.

The PI3Kδ isoform (PIK3CD), also known as P110δ, is predominately expressed in leukocytes and has been implicated as a potential target in the treatment of hematological malignancies. In this report, we detailed the pharmacologic properties of puquitinib, a novel, orally available PI3Kδ inhibitor. Puquitinib, which binds to the ATP‐binding pocket of PI3Kδ, was highly selective and potent for PI3Kδ relative to other PI3K isoforms and a panel of protein kinases, exhibiting low‐nanomolar biochemical and cellular inhibitory potencies. Additional cellular profiling demonstrated that puquitinib inhibited proliferation, induced G1‐phase cell‐cycle arrest and apoptosis in acute myeloid leukemia (AML) cell lines, through downregulation of PI3K signaling. In in vivo AML xenografts, puquitinib alone showed stronger efficacy than the well‐known p110δ inhibitor, CAL‐101, in association with a reduction in AKT and ERK phosphorylation in tumor tissues, without causing noticeable toxicity. Furthermore, the combination of puquitinib with cytotoxic drugs, especially daunorubicin, yielded significantly stronger antitumor efficacy compared with each agent alone. Thus, puquitinib is a promising agent with pharmacologic properties that are favorable for the treatment of AML.

Targeting group I p21-activated kinases to control malignant peripheral nerve sheath tumor growth and metastasis.

Malignant peripheral nerve sheath tumors (MPNSTs) are devastating sarcomas for which no effective medical therapies are available. Over 50% of MPSNTs are associated with mutations in NF1 tumor suppressor gene, resulting in activation of Ras and its effectors, including the Raf/Mek/Erk and PI3K/Akt/mTORC1 signaling cascades, and also the WNT/β-catenin pathway. As Group I p21-activated kinases (Group I Paks, PAK1/2/3) have been shown to modulate Ras-driven oncogenesis, we asked if these enzymes might regulate signaling in MPNSTs. In this study we found a strong positive correlation between the activity of PAK1/2/3 and the stage of human MPNSTs. We determined that reducing Group I Pak activity diminished MPNST cell proliferation and motility, and that these effects were not accompanied by significant blockade of the Raf/Mek/Erk pathway, but rather by reductions in Akt and β-catenin activity. Using the small molecule PAK1/2/3 inhibitor Frax1036 and the MEK1/2 inhibitor PD0325901, we showed that the combination of these two agents synergistically inhibited MPNST cell growth in vitro and dramatically decreased local and metastatic MPNST growth in animal models. Taken together, these data provide new insights into MPNST signaling deregulation and suggest that co-targeting of PAK1/2/3 and MEK1/2 may be effective in the treatment of patients with MPNSTs.

Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

Idelalisib Impacts Cell Growth through Inhibiting Translation-Regulatory Mechanisms in Mantle Cell Lymphoma.

PI3K is a critical node in the B-cell receptor pathway, which is responsible for survival and proliferation of B-cell malignancies. Idelalisib, a PI3Kδ-isoform-specific inhibitor, has been approved to treat B-cell malignancies. Although biological activity of the drug has been evaluated, molecular mechanisms and signaling pathway disruption leading to the biological effects of idelalisib are not yet well defined. Prior laboratory reports have identified transcription and translation as the primary events for attenuation of PI3Kα isoform. We hypothesized that PI3Kδ-isoform inhibition by idelalisib should also affect gene transcription and protein translation. Using three mantle cell lymphoma cell lines and primary cells from patients, biological consequences such as apoptosis/cell-cycle analysis, as well as RNA/protein synthesis were evaluated. Proteomics analyses (RPPA and immunoblot assays) defined molecular events downstream of PI3K/AKT cassette. Idelalisib treatment resulted in inhibition of protein synthesis, which correlated with reduction in cell size and cell growth. A moderate loss of viability without any change in cell-cycle profile was observed. Idelalisib treatment inhibited AKT activation, an immediate downstream PI3K effector, and also reduced phosphorylation levels of downstream AKT/mTOR pathway proteins such as PRAS40. In addition, idelalisib treatment impeded activation of the MAPK pathway, and MEK, ERK and p90RSK phosphorylation levels were reduced. Reduction in AKT, PDK1, and MEK phosphorylation correlated with protein synthesis inhibition. Collectively, these results clarify the molecular mechanisms of actions and may provide biomarkers and targets for combination with idelalisib in B-cell malignancies. Clin Cancer Res; 23(1); 181-92. ©2016 AACR.

Long-term Leucine Supplementation Improves Metabolic But Not Molecular Responses in the Skeletal Muscle of Trained Rats Submitted to Exhaustive Exercise

Aim: Although there is some evidence of an ergogenic effect of leucine supplementation on acute response to exercise, there is a paucity of information on whether long-term leucine supplementation influences the adaptive response to chronic endurance training and performance. The main aim of our study was to assess the role of long-term leucine supplementation on molecular and metabolic response in skeletal muscle of trained rats after an exhaustion test.Methods: Twenty-four male Wistar rats were randomly allocated into 4 groups. Two of them (control and trained groups) received a balanced control diet (18% protein) and the other 2 (control leucine and trained leucine groups) received a leucine-rich diet (15% protein with 3% leucine) for 6 weeks. The trained groups were submitted to 1 hour of swimming exercise, 5 d/wk for 6 weeks. Three days after the exercise training period, trained groups were submitted to swimming exercise until exhaustion and muscle metabolic and molecular parameters were assessed.Results: Endurance training increased citrate synthase activity significantly, whereas exercise until exhaustion increased cytokine levels and led to a lack of activation of phosphorylation of the signaling intermediates assessed. Long-term leucine supplementation enhanced muscle glycogen level in trained rats and citrate synthase activity in sedentary ones. However, it failed to enhance endurance performance of trained rats submitted to an exhaustion test and did not prevent exercise-induced reduction in Akt and mTOR activation.Conclusion: Long-term leucine supplementation can enhance citrate synthase activity by itself in sedentary individuals and glycogen content when combined with exercise; however, it does not improve endurance performance or prevent Akt and mTOR exercise-induced inhibition.

Interference with Akt signaling pathway contributes curcumin-induced adipocyte insulin resistance

Previous study has shown that curcumin directly or indirectly suppresses insulin signaling in 3T3-L1 adipocytes. However, the underlying mechanism remains unclear. Here we experimentally demonstrate that curcumin inhibited the ubiquitin-proteasome system (UPS) function, activated autophagy, and reduced protein levels of protein kinase B (Akt) in a dose- and time-dependent manner in 3T3-L1 adipocytes, accompanied with attenuation of insulin-stimulated Akt phosphorylation, plasma membrane translocation of glucose transporter type 4 (GLUT4), and glucose uptake. These in vitro inhibitory effects of curcumin on Akt protein expression and insulin action were reversed by pharmacological and genetic inhibition of autophagy but not by inhibition of the UPS and caspases. In addition, Akt reduction in adipose tissues of mice treated with curcumin could be recovered by administration of autophagy inhibitor bafilomycin A1 (BFA). This new finding provides a novel mechanism by which curcumin induces insulin resistance in adipocytes.

Abstract 12702: A Novel Human S10F-Hsp20 Mutation Induces Lethal Peripartum Cardiomyopathy (Best of Basic Science Abstract)

Background: Heat shock protein 20 (Hsp20) has emerged as a novel cardioprotector against stress-induced injury. In this study, we investigated the functional significance of a novel human Hsp20 mutation (S10F) in peri-partum cardiomyopathy. Methods and Results: We identified a human S10F-Hsp20 mutant with a frequency of 2.8% in dilated cardiomyopathy patients (470 screened), while we did not find any normal subjects carrying this mutation (282 screened). To determine the functional significance of S10F-Hsp20, transgenic mice with cardiac-specific overexpression of this mutant were generated. We observed that female TGs were fertile, and delivered normal litter sizes, but they invariably died after 2, 3 or 4 pregnancies (Fig. 1a). However, overexpression of WT-Hsp20 to similar levels as S10F was not associated with any deaths following multiple pregnancies. Further histological examination of S10F females revealed significantly dilated hearts and increased heart weight/tibia length after the third delivery, compared to WT (non transgenic) mice. (Fig. 1b,c). Echocardiography assessment showed that this dilation (Fig. 1d) is associated with increased left ventricular end-systolic volume (Fig. 1e), increased left ventricular end-diastolic volume, depressed ejection fraction (Fig. 1f) and decreased fractional shortening. Further studies revealed that cardiomyocyte apoptosis was increased by 4 fold in S10F hearts after the third delivery(Fig. 1g,h). The mechanisms associated with the detrimental remodelling in S10F females included decreased Akt (Fig. 1i) and ERK phosphorylation under both baseline and pregnancy conditions. As a result, the activities of Akt and ERK were reduced, contributing to increased cell death in the S10F hearts of pregnant females. Conclusion: The human S10F mutation may compromise the heart’s coping with pregnancy induced stress conditions mainly through increased apoptosis by reduction of Akt and ERK activities.

Reduced cortical BACE1 content with one bout of exercise is accompanied by declines in AMPK, Akt, and MAPK signaling in obese, glucose-intolerant mice.

Obesity and type 2 diabetes are significant risk factors in the development of neurodegenerative diseases, such as Alzheimer's disease. A variety of cellular mechanisms, such as altered Akt and AMPK and increased inflammatory signaling, contribute to neurodegeneration. Exercise training can improve markers of neurodegeneration, but the underlying mechanisms remain unknown. The purpose of this study was to determine the effects of a single bout of exercise on markers of neurodegeneration and inflammation in brains from mice fed a high-fat diet. Male C57BL/6 mice were fed a low (LFD; 10% kcal from lard)- or a high-fat diet (HFD; 60% kcal from lard) for 7 wk. HFD mice underwent an acute bout of exercise (treadmill running: 15 m/min, 5% incline, 120 min) followed by a recovery period of 2 h. The HFD increased body mass and glucose intolerance (both P < 0.05). This was accompanied by an approximately twofold increase in the phosphorylation of Akt, ERK, and GSK in the cortex (P < 0.05). Following exercise, there was a decrease in beta-site amyloid precursor protein cleaving enzyme 1 (BACE1; P < 0.05) and activity (P < 0.001). This was accompanied by a reduction in AMPK phosphorylation, indicative of a decline in cellular stress (P < 0.05). Akt and ERK phosphorylation were decreased following exercise in HFD mice to a level similar to that of the LFD mice (P < 0.05). This study demonstrates that a single bout of exercise can reduce BACE1 content and activity independent of changes in adiposity. This effect is associated with reductions in Akt, ERK, and AMPK signaling in the cortex.