P70 Ribosomal Protein S6 Kinase Research Articles

L-Leucine stimulates proliferation and casein secretion in porcine mammary epithelial cells through mTOR-p70S6K-4EBP1 signalling pathway

Milk production determines health status and optimal growth of suckling neonates. Recent studies revealed that the secretion of leucine into sow milk from the mammary gland is significantly lower than the amount taken up by the mammary gland from the blood, suggesting the potential and fundamental role in udder metabolism. However, little is known about the underlying mechanism. In this study, porcine mammary epithelial cells (PMECs) were employed to investigate the effects of leucine on protein synthesis and mammalian target of rapamycin (mTOR) signalling. Results showed that leucine supplementation significantly increased the proliferation of PMECs in a dose-dependent manner from 0 to 1 mmol/l. Secretions of four casein subunits were significantly increased by addition of leucine. The phosphorylation of proteins related to mTOR signalling, including mTOR, eukaryotic translation initiation factor 4E-binding protein, and p70 ribosomal protein S6 kinase, were accordingly upregulated by leucine supplementation. Casein secretion was not affected by leucine treatment when mTOR signalling was inhibited by rapamycin. This suggests that leucine stimulates protein synthesis through the mTOR signalling pathway. These findings facilitate the utilization of dietary leucine supplementation to improve milk production in lactating sows.

Novel Repositioning Therapy for Drug-Resistant Glioblastoma: In Vivo Validation Study of Clindamycin Treatment Targeting the mTOR Pathway and Combination Therapy with Temozolomide.

Simple SummaryGiven the significant costs and lengthy timelines of drug development and clinical trials, drug repositioning is a promising alternative to find effective treatments for brain tumors quickly and inexpensively. In the present study, using a simple drug screen of macrolides, we found that clindamycin (CLD) had cytotoxic effects on glioblastoma (GBM) cells. Further studies showed the inhibition of the mammalian target of rapamycin (mTOR) pathway as the key mechanism of action. Interestingly, we found that co-treatment with temozolomide (TMZ), the alkylating agent considered as standard therapy in GBM, enhanced these effects and proposed the inhibition of O6-methylguanine-DNA methyltransferase (MGMT) protein by CLD as a potential mechanism for this combination effect.Multimodal therapy including surgery, radiation treatment, and temozolomide (TMZ) is performed on glioblastoma (GBM). However, the prognosis is still poor and there is an urgent need to develop effective treatments to improve survival. Molecular biological analysis was conducted to examine the signal activation patterns in GBM specimens and remains an open problem. Advanced macrolides, such as azithromycin, reduce the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K), a downstream mammalian target of rapamycin (mTOR) effector, and suppress the proliferation of T-cells. We focused on its unique profile and screened for the antitumor activity of approved macrolide antibiotics. Clindamycin (CLD) reduced the viability of GBM cells in vitro. We assessed the effects of the candidate macrolide on the mTOR pathway through Western blotting. CLD attenuated p70S6K phosphorylation in a dose-dependent manner. These effects on GBM cells were enhanced by co-treatment with TMZ. Furthermore, CLD inhibited the expression of the O6-methylguanine-DNA methyltransferase (MGMT) protein in cultured cells. In the mouse xenograft model, CLD and TMZ co-administration significantly suppressed the tumor growth and markedly decreased the number of Ki-67 (clone MIB-1)-positive cells within the tumor. These results suggest that CLD suppressed GBM cell growth by inhibiting mTOR signaling. Moreover, CLD and TMZ showed promising synergistic antitumor activity.

Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat

Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat

TAKTIC: A prospective, multicentre, uncontrolled, phase IB/II study of LY2780301, a p70S6K/AKT inhibitor, in combination with weekly paclitaxel in HER2-negative advanced breast cancer patients

BackgroundHormone-resistant HER2-negative or triple-negative advanced breast cancers (ABC) are routinely treated with paclitaxel chemotherapy. LY2780301 is a dual inhibitor of p70 ribosomal protein S6 kinase and AKT. The TAKTIC study aimed at exploring the combination of paclitaxel and LY2780301 in this population. MethodsIn this multicentric phase Ib/II trial, we enrolled patients with HER2-negative ABC, with (phase IB) or without (phase II) prior to cytotoxic treatment for advanced disease. Oral LY2780301 was administered once daily in combination with intravenous weekly paclitaxel. Primary endpoints were to determine the recommended phase II dose (RP2D) of the combination of LY2780301 with weekly paclitaxel (phase Ib), and to estimate a 6 months objective response rate (ORR) (phase II) in patients with HER2-negative ABC, both in the overall patient population and in cases with activation of the PI3K/AKT pathway (PI3KAKT+). ResultsA total of 51 patients were enrolled; RP2D was LY2780301 500 mg QD+ paclitaxel 80 mg/m2. Main drug-related adverse events noted in phase Ib included neuropathy (75% of patients, grade 3–4 in 8%), asthenia (58% of patients, no grade 3–4), and ungual toxicity (50% of patients, grade 3–4 in 25%). They were similar in the phase II part, except that 14% of patients experienced pneumonia (grade 3–4 in 6%). In the phase II part, 6-month ORR in the overall population and in PI3KAKT+ subgroup were, respectively, 63.9% [48.8–76.8] and 55% [35–73.7]. ConclusionCombining LY2780301 and weekly paclitaxel in patients with HER2-negative ABC was feasible with preliminary evidence of efficacy in both the overall population and the PI3KAKT+ subgroup. Trial registration idNCT01980277.

P70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

Background & AimsProgression of chronic liver disease (CLD) to liver cirrhosis and liver cancer is a major global cause of morbidity and mortality. Treatment options capable of inhibiting progression of liver fibrosis when etiological treatment of CLD is not available or fails have yet to be established. We investigated the role of serine/threonine kinase p70 ribosomal protein S6 kinase (p70S6K) as checkpoint of fibrogenesis in hepatic stellate cells (HSCs) and as target for the treatment of liver fibrosis.Approach & ResultsImmunohistochemistry was used to assess p70S6K expression in liver resection specimen. Primary human or murine HSCs from wild-type or p70S6K–/– mice as well as LX-2 cells were used for in vitro experiments. Specific small interfering RNA or CEP-1347 were used to silence or inhibit p70S6K and assess its functional relevance in viability, contraction and migration assays, fluorescence-activated cell sorting, and Western blot. These results were validated in vivo by a chemical model of fibrogenesis using wild-type and p70S6K–/– mice. Expression of p70S6K was significantly increased in human cirrhotic vs noncirrhotic liver-tissue and progressively increased in vitro through activation of primary human HSCs. Conversely, p70S6K induced fibrogenic activation of HSCs in different models, including the small interfering RNA–based silencing of p70S6K in HSC lines, experiments with p70S6K–/– cells, and the pharmacological inhibition of p70S6K by CEP-1347. These findings were validated in vivo as p70S6K–/– mice developed significantly less fibrosis upon exposure to CCl4.ConclusionsWe establish p70S6K as a checkpoint of fibrogenesis in vitro and in vivo and CEP-1347 as potential treatment option that can safely be used for long-term treatment.

Transcranial Laser Photobiomodulation Improves Intracellular Signaling Linked to Cell Survival, Memory and Glucose Metabolism in the Aged Brain: A Preliminary Study.

Aging is often accompanied by exacerbated activation of cell death-related signaling pathways and decreased energy metabolism. We hypothesized that transcranial near-infrared laser may increase intracellular signaling pathways beneficial to aging brains, such as those that regulate brain cell proliferation, apoptosis, and energy metabolism. To test this hypothesis, we investigated the expression and activation of intracellular signaling proteins in the cerebral cortex and hippocampus of aged rats (20 months old) treated with the transcranial near-infrared laser for 58 consecutive days. As compared to sham controls, transcranial laser treatment increased intracellular signaling proteins related to cell proliferation and cell survival, such as signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB), also known as Akt that is linked to glucose metabolism. In addition, ERK is linked to memory, while ERK and JNK signaling pathways regulate glucose metabolism. Specifically, the laser treatment caused the activation of STAT3, ERK, and JNK signaling proteins in the cerebral cortex. In the hippocampus, the laser treatment increased the expression of p70S6K and STAT3 and the activation of Akt. Taken together, the data support the hypothesis that transcranial laser photobiomodulation improves intracellular signaling pathways linked to cell survival, memory, and glucose metabolism in the brain of aged rats.

Novel Anti-Angiogenic and Anti-Tumour Activities of the N-Terminal Domain of NOEY2 via Binding to VEGFR-2 in Ovarian Cancer.

The imprinted tumour suppressor NOEY2 is downregulated in various cancer types, including ovarian cancers. Recent data suggest that NOEY2 plays an essential role in regulating the cell cycle, angiogenesis and autophagy in tumorigenesis. However, its detailed molecular function and mechanisms in ovarian tumours remain unclear. In this report, we initially demonstrated the inhibitory effect of NOEY2 on tumour growth by utilising a xenograft tumour model. NOEY2 attenuated the cell growth approximately fourfold and significantly reduced tumour vascularity. NOEY2 inhibited the phosphorylation of the signalling components downstream of phosphatidylinositol-3’-kinase (PI3K), including phosphoinositide-dependent protein kinase 1 (PDK-1), tuberous sclerosis complex 2 (TSC-2) and p70 ribosomal protein S6 kinase (p70S6K), during ovarian tumour progression via direct binding to vascular endothelial growth factor receptor-2 (VEGFR-2). Particularly, the N-terminal domain of NOEY2 (NOEY2-N) had a potent anti-angiogenic activity and dramatically downregulated VEGF and hypoxia-inducible factor-1α (HIF-1α), key regulators of angiogenesis. Since no X-ray or nuclear magnetic resonance structures is available for NOEY2, we constructed the three-dimensional structure of this protein via molecular modelling methods, such as homology modelling and molecular dynamic simulations. Thereby, Lys15 and Arg16 appeared as key residues in the N-terminal domain. We also found that NOEY2-N acts as a potent inhibitor of tumorigenesis and angiogenesis. These findings provide convincing evidence that NOEY2-N regulates endothelial cell function and angiogenesis by interrupting the VEGFR-2/PDK-1/GSK-3β signal transduction and thus strongly suggest that NOEY2-N might serve as a novel anti-tumour and anti-angiogenic agent against many diseases, including ovarian cancer.

One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency.

BackgroundInborn errors of immunity (IEI) present with a large phenotypic spectrum of disease, which can pose diagnostic and therapeutic challenges. Suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator of cytokine signaling, and has recently been associated with a novel IEI. Of patients described to date, it is apparent that SOCS1 haploinsufficiency has a pleiotropic effect in humans.ObjectiveWe sought to investigate whether dysregulation of immune pathways, in addition to STAT1, play a role in the broad clinical manifestations of SOCS1 haploinsufficiency.MethodsWe assessed impacts of reduced SOCS1 expression across multiple immune cell pathways utilizing patient cells and CRISPR/Cas9 edited primary human T cells.ResultsSOCS1 haploinsufficiency phenotypes straddled across the International Union of Immunological Societies classifications of IEI. We found that reduced SOCS1 expression led to dysregulation of multiple intracellular pathways in immune cells. STAT1 phosphorylation is enhanced, comparably with STAT1 gain-of-function mutations, and STAT3 phosphorylation is similarly reduced with concurrent reduction of Th17 cells. Furthermore, reduced SOCS1 E3 ligase function was associated with increased FAK1 in immune cells, and increased AKT and p70 ribosomal protein S6 kinase phosphorylation. We also found Toll-like receptor responses are increased in SOCS1 haploinsufficiency patients.ConclusionsSOCS1 haploinsufficiency is a pleiotropic monogenic IEI. Dysregulation of multiple immune cell pathways may explain the variable clinical phenotype associated with this new condition. Knowledge of these additional dysregulated immune pathways is important when considering the optimum management for SOCS1 haploinsufficient patients.

Glucagon-like peptide-1 attenuates cardiac hypertrophy via the AngII/AT1R/ACE2 and AMPK/mTOR/p70S6K pathways.

Glucagon-like peptide-1 (GLP-1), a novel type of glucose-lowering agent, has been reported to exert cardioprotective effects. However, the cardioprotective mechanism of GLP-1 on spontaneous hypertension-induced cardiac hypertrophy has not been fully elucidated. In this study, we revealed that liraglutide or alogliptin treatment ameliorated spontaneous hypertension-induced cardiac hypertrophy, as evidenced by decreased levels of cardiac hypertrophic markers (atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain), as well as systolic blood pressure, diastolic blood pressure, mean arterial pressure, and histological changes. Both drugs significantly reduced the levels of angiotensin II (AngII) and AngII type 1 receptor (AT1R) and upregulated the levels of AngII type 2 receptor (AT2R) and angiotensin-converting enzyme 2 (ACE2), as indicated by a reduced AT1R/AT2R ratio. Simultaneously, treatment with liraglutide or alogliptin significantly increased GLP-1 receptor expression and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and downregulated the phosphorylation of mammalian target of rapamycin (mTOR), p70 ribosomal S6 protein kinase, and eukaryotic translation initiation factor 4E binding protein 1 in spontaneous hypertension rats. Furthermore, our data demonstrated that the AMPK inhibitor compound C or mTOR activator MHY1485 inhibited the anti-hypertrophic effect of GLP-1. In summary, our study suggests that liraglutide or alogliptin protects the heart against cardiac hypertrophy by regulating the expression of AngII/AT1R/ACE2 and activating the AMPK/mTOR pathway, and GLP-1 agonist can be used in the treatment of patients with cardiac hypertrophy.

DIF-1 inhibits growth and metastasis of triple-negative breast cancer through AMPK-mediated inhibition of the mTORC1-S6K signaling pathway.

We have previously reported that the differentiation-inducing factor-1 (DIF-1), a compound identified in Dictyostelium discoideum, suppresses the growth of MCF-7 breast cancer cells by inactivating p70 ribosomal protein S6 kinase (p70S6K). Therefore, we first examined whether the same mechanism operates in other breast cancer cells, especially triple-negative breast cancer (TNBC), the most aggressive and refractory phenotype of breast cancer. We also investigated the mechanism by which DIF-1 suppresses p70S6K by focusing on the AMPK-mTORC1 system. We found that DIF-1 induces phosphorylation of AMPK and Raptor and dephosphorylation of p70S6K in multiple TNBC cell lines. Next, we examined whether AMPK-mediated inhibition of p70S6K leads to the suppression of proliferation and migration/infiltration of TNBC cells. DIF-1 significantly reduced the expression levels of cyclin D1 by suppressing the translation of STAT3 and strongly suppressed the expression levels of Snail, which led to the suppression of growth and motility, respectively. Finally, we investigated whether DIF-1 exerts anticancer effects on TNBC in vivo. Intragastric administration of DIF-1 suppressed tumor growth and spontaneous lung metastasis of 4T1-Luc cells injected into the mammary fat pad of BALB/c mice. DIF-1 is expected to lead to the development of anticancer drugs, including anti-TNBC, by a novel mechanism.

2,6-Dimethoxy-1,4-benzoquinone increases skeletal muscle mass and performance by regulating AKT/mTOR signaling and mitochondrial function

Background2,6-Dimethoxy-1,4-benzoquinone (DMBQ), a natural phytochemical present in fermented wheat germ, has been reported to exert anti-cancer, anti-inflammatory, and anti-adipogenic effects. However, the effect of DMBQ on muscle hypertrophy and myoblast differentiation has not been elucidated. PurposeWe investigated the effect of DMBQ on skeletal muscle mass and muscle function and then determined the possible mechanism of DMBQ. MethodsTo examine myogenic differentiation and hypertrophy, confluent C2C12 cells were incubated in differentiation medium with or without various concentrations of DMBQ for 4 days. In animal experiments, C57BL/6 mice were fed DMBQ-containing AIN-93 diet for 7 weeks. Grip strength, treadmill, microscopic evaluation of muscle tissue, western blotting, and quantitative real-time PCR were performed. ResultsDMBQ significantly increased fusion index, myotube size, and the protein expression of myosin heavy chain (MHC). DMBQ increased the phosphorylation of protein kinase B (AKT) and p70 ribosomal protein S6 kinase (S6K), whereas the phosphorylation of these proteins was abolished by the phosphoinositide 3-kinase inhibitor LY294002 in C2C12 cells. In addition, DMBQ treatment increased peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), which programs mitochondrial biogenesis, protein levels compared with control C2C12 cells. DMBQ significantly increased maximal respiration and spare respiratory capacity in C2C12 cells. In animal experiments, DMBQ increased skeletal muscle weights and skeletal muscle fiber size compared with the control group values. In addition, the DMBQ group showed increased grip strength and running distance on an accelerating treadmill. The protein expression of total MHC, MHC1, MHC2A, and MHC2B in skeletal muscle was upregulated by DMBQ supplementation. We found that DMBQ increased the phosphorylation of AKT and mammalian target of rapamycin (mTOR), as well as downstream S6K and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in skeletal muscle. DMBQ also stimulated mRNA expression of PGC1α, accompanied by an increase in mitochondrial DNA content, oxidative phosphorylation (OXPHOS) proteins, and oxidative enzyme activity. ConclusionCollectively, DMBQ was shown to increase skeletal muscle mass and performance by regulating the AKT/mTOR signaling pathway and enhancing mitochondrial function, which might be useful for the treatment and prevention of skeletal muscle atrophy.

Adhesion to E-selectin primes macrophages for activation through AKT and mTOR.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

Effects of electroacupuncture on proangiogenesis process and protein turnover in a mouse model of sarcopenia

To observe the effect of electroacupuncture(EA) on proangiogenesis process and protein turn-over in a mouse model of sarcopenia, so as to explore its potential molecular mechanism anti-aging. Fourteen 30-week-old male SAMP8 mice were randomly divided into a model group (n=7) and an EA group (n=7). Seven anti-rapidly aging SAMR1 mice of the same age were used as the control group (n=7). EA (1 mA, 4 Hz) was applied to bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 20 minutes each time once a day, 6 times a week for 4 weeks. The exhausted running platform was used to test the sports function. Gastrocnemius muscle mass and relative ratio of gastrocnemius muscle mass to body mass were measured. HE staining and transmission electron microscope were used to observe the morphology, and the cross-sectional area of gastrocnemius muscle was calculated. Relative protein expressions of protein kinase B (AKT) , phosphorylated (p) -AKT, mammalian target of rapamycin (mTOR) , p-mTOR, p70 ribosomal protein S6 kinase (p70S6K) , p-p70S6K,hypoxia inducible factor-1α (HIF-1α) and relative mRNA expressions of HIF-1α, vascular endothelial growth factor A (VEGF-A) , muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx) were detected by Western blot and real-time fluorescence quantitative PCR, seperatively. Compared with the control group, the running time and distance, body mass and gastrocnemius mass, and the ratio of gastrocnemius mass to body mass decreased(P<0.01, P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A decreased (P<0.01), while mRNA expression of MuRF1 and MAFbx increased (P<0.01) in the model group. Following EA intervention, the running time and distance, body mass and gastrocnemius mass and the ratio of gastrocnemius mass to body mass increased (P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A were significantly up-regulated (P<0.01), mRNA expression of MuRF1 and MAFbx down-regulated (P<0.01, P<0.05) in the EA group compared with the model group. EA may delay the aging muscle atrophy in mice by regulating the gastrocnemius muscle's proangiogenesis process and protein turnover.

Inhibition of p70 isoforms of S6K1 induces anoikis to prevent transformed human hepatocyte growth

AimsThe mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation. Main methodsTHH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA. Key findingsp70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-β1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth. SignificanceOur results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.

Insulin potentiates essential amino acids effects on mechanistic target of rapamycin complex 1 signaling in MAC-T cells

Different models of lactation offer conflicting evidence as to whether insulin signaling is required for AA to stimulate mechanistic target of rapamycin complex 1 (mTORC1) activity. We hypothesized that insulin potentiates essential AA stimulation of mTORC1 activity in the MAC-T mammary epithelial cell line. Here, our objective was to assess mTORC1 signaling activity in response to insulin and individual or grouped essential AA. Insulin and essential AA concentrations in the treatment medium ranged from normo- to supraphysiological, with insulin at 0, 1, 10, or 100 nmol/L and essential AA at approximately 0, 0.01, 0.05, 0.1, 1, or 3× reference plasma levels. Effects and interaction of insulin and total essential AA were tested in a 3 × 5 factorial design (n = 3 replicates/treatment); insulin and the individual AA Leu, Met, Ile, and Arg were likewise tested in 3 × 4 factorials (n = 4). As the remaining individual AA His, Lys, Phe, Thr, Trp, and Val were expected to not affect mTORC1, these were tested only at the highest insulin level, 100 nmol/L (n = 4). For all of these, linear and quadratic effects of total and individual AA were evaluated. Essential AA were subsequently grouped by their positive (Leu, Met, Ile, Arg, and Thr; TOR-AA) or absent-to-negative effects (His, Lys, Phe, Trp, and Val; NTOR-AA), and tested for interaction in a 2 × 2 factorial design (n = 4), with each AA at its respective 1× plasma level, and insulin held at 100 nmol/L. All experiments consisted of 1 h treatment incubation, followed by Western blotting of cell lysates to measure phosphorylation and abundance of the mTORC1 pathway proteins Akt (Ser473); ribosomal protein S6 kinase p70 (S6K1, Thr389); eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Ser65); and ribosomal protein S6 (S6, Ser240/244). The Akt phosphorylation was overall increased by insulin, with a possible negative interaction with both total essential AA and the individual AA Leu. Total essential AA also increased S6K1 and 4E-BP1 phosphorylation in an insulin-dependent manner. The individual AA Leu, Met, Ile, and Arg increased S6K1 phosphorylation in an insulin-dependent manner. Similarly, Met and Arg increased 4E-BP1 phosphorylation in an insulin-dependent manner. Histidine, Lys, Trp, and Val did not affect S6K1 phosphorylation. However, S6K1 phosphorylation was linearly increased by Thr and quadratically decreased by Phe. Relative to the phosphorylation of S6K1 when cells were incubated with no essential AA, the NTOR-AA group had no effect, whereas the TOR-AA increased phosphorylation to the same degree observed with all 10 essential AA. Overall, we have found that insulin is required for essential AA to stimulate mTORC1 activity in MAC-T cells. In addition, the AA responsible for the bulk of mTORC1 activation in MAC-T are limited to Leu, Met, Ile, Arg, and Thr.

MTORC1/2 signaling is downregulated by amino acid-free culture of mouse preimplantation embryos and is only partially restored by amino acid readdition.

Development of the mammalian preimplantation embryo is influenced by autocrine/paracrine factors and the availability of nutrients. Deficiencies of these during in vitro culture reduce the success of assisted reproductive technologies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway integrates external and internal signals, including those by amino acids (AAs), to promote normal preimplantation development. For this reason, AAs are often included in embryo culture media. In this study, we examined how withdrawal and addition of AAs to culture media modulate mTORC1 pathway activity compared with its activity in mouse embryos developed in vivo. Phosphorylation of signaling components downstream of mTORC1, namely, p70 ribosomal protein S6 kinase (p70S6K), ribosomal protein S6, and 4E binding protein 1 (4E-BP1), and that of protein kinase B (Akt), which lies upstream of mTORC1, changed significantly across stages of embryos developed in vivo. For freshly isolated blastocysts placed in vitro, the absence of AAs in the culture medium, even for a few hours, decreased mTORC1 signaling, which could only be partially restored by their addition. Long-term culture of early embryos to blastocysts in the absence of AAs decreased mTORC1 signaling to a greater extent and again this could only be partially restored by their inclusion. This failure to fully restore is probably due to decreased phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC2 signaling in culture, as indicated by decreased P-AktS473. mTORC2 lies upstream of mTORC1 and is insensitive to AAs, and its reduced activity probably results from loss of maternal/autocrine factors. These data highlight reduced mTORC1/2 signaling activity correlating with compromised development in vitro and show that the addition of AAs can only partially offset these effects.

Nervilifordin F alleviates intestinal ischemia/reperfusion-induced acute lung injury via inhibiting inflammasome and mTOR pathway

Acute lung injury (ALI) is a life-threatening disorder with high rates of morbidity and mortality. Up to now, there are still no effective drugs for its therapies due to the complexity of its etiology and pathogenesis. In this present study, we investigated the protective effect of Nervilifordin F (NF) on ALI induced by intestinal ischemia/reperfusion (II/R) and its related mechanism. Firstly, the ALI model rats were induced through II/R, and treated with NF. Then, the pathological and cytokine level changes in the lung tissue of ALI rats were evaluated by hematoxylin and eosin and enzyme-linked immunosorbent assay (ELISA). The related genes expression level of mammalian target of rapamycin (mTOR) pathway and inflammasome were measured by real-time quantitative polymerase chain reaction (RT-qPCR), western blot and immunohistochemistry. Finally, the NF-protein complexes were predicted by SYBYL-X 2.0. The results indicated that NF can significant reduces the levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1β, and inhibits the expression of inflammasome related genes (such as toll-like receptor 4 (TLR4), p65, NOD-like receptor protein 3 (NLRP3) and Caspase 1), thereby reduce inflammation in II/R-induced ALI rats. Moreover, NF can activate the expression of FK506 binding protein 25 (FKBP25) and down-regulate the expression of mTOR and p70 ribosomal protein S6 kinase 1 (p70S6K). In addition, molecular docking results showed that NF can be combined well with p70S6K, TLR4, mTOR and NLRP3, which further verified the inhibitory effect of NF on ALI inflammation. Therefore, the findings indicate that NF can alleviates II/R-induced inflammation of ALI rats by inhibiting inflammasome related genes and mTOR pathway, which expected to use as a potential drug for the treatment of ALI.

Abstract B19: Susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition via PDK1

Abstract Genomic alterations in the PI3K/mTOR pathway occur in 54% of head and neck squamous cell carcinoma (HNSCC) patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors in HNSCC, we tested the efficacy of seven PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and genomic alterations. We identified that NOTCH1MUT lines were significantly sensitive to the following PI3K/mTOR pathway inhibitors: GSK2126458 (12/14 lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14), and GDC0980 (5/14). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis in addition to G1/S cell cycle arrest after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth in vitro and significant tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After GSK2126458 treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. Both NOTCH1 KO lines also showed increased cleaved PARP and cleaved caspase 3 by Western blot analysis after PI3K/mTOR inhibition. With GSK2126458 and BEZ235 treatment, both PJ34 and UMSCC49 NOTCH1 knock out lines showed significantly decreased number of colonies compared to the parental lines. As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in three NOTCH1MUT and three NOTCH1WT lines after GSK2126458 treatment. Several proteins were differentially regulated in NOTCH1MUT cells compared with wild-type lines including PDK1, FoxM1 and phospho-ERK. We then investigated PDK1 because it is activated by PI3K and can regulate FOXM1, ERK, and p70 ribosomal protein S6 kinase (p70S6K), which were also inhibited more robustly in NOTCH1MUT HNSCC. To test the role of PDK1 in PI3K/mTOR inhibition, we overexpressed PDK1 in NOTCH1MUT cells and the presence of PDK1-GFP was confirmed by Western blot analysis. PDK1 overexpression in NOTCH1MUT cells successfully led to decreased apoptosis after GSK2126458 treatment as compared to the parental lines. The combination of AKT and PDK1 inhibition led to decreased cell viability in all four NOTCH1WT lines tested as compared to single agents. The combination also led to significantly increased apoptosis in all NOTCH1WT cell lines tested as demonstrated by cleaved PARP and cleaved Caspase 3 levels by Western blot analysis. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and in squamous cell carcinomas of the lung, esophagus, and other sites, may serve as a biomarker for response. Our present work may uncover important combination therapies for HNSCC. Citation Format: Vaishnavi Sambandam, Mitchell J. Frederick, Li Shen, Pan Tong, Xiayu Rao, Shaohua Peng, Tuhina Mazumdar, Quili Li, Curtis R. Pickering, Jeffery N. Myers, Jing Wang, Faye M. Johnson. Susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition via PDK1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B19.

Metformin: A Prospective Alternative for the Treatment of Chronic Pain.

Metformin (biguanide) is a drug widely used for the treatment of type 2 diabetes. This drug has been used for 60 years as a highly effective antihyperglycemic agent. The search for the mechanism of action of metformin has produced an enormous amount of research to explain its effects on gluconeogenesis, protein metabolism, fatty acid oxidation, oxidative stress, glucose uptake, autophagy and pain, among others. It was only up the end of the 1990s and beginning of this century that some of its mechanisms were revealed. Metformin induces its beneficial effects in diabetes through the activation of a master switch kinase named AMP-activated protein kinase (AMPK). Two upstream kinases account for the physiological activation of AMPK: liver kinase B1 and calcium/calmodulin-dependent protein kinase kinase 2. Once activated, AMPK inhibits the mechanistic target of rapamycin complex 1 (mTORC1), which in turn avoids the phosphorylation of p70 ribosomal protein S6 kinase 1 and phosphatidylinositol 3-kinase/protein kinase B signaling pathways and reduces cap-dependent translation initiation. Since metformin is a disease-modifying drug in type 2 diabetes, which reduces the mTORC1 signaling to induce its effects on neuronal plasticity, it was proposed that these mechanisms could also explain the antinociceptive effect of this drug in several models of chronic pain. These studies have highlighted the efficacy of this drug in chronic pain, such as that from neuropathy, insulin resistance, diabetic neuropathy, and fibromyalgia-type pain. Mounting evidence indicates that chronic pain may induce anxiety, depression and cognitive impairment in rodents and humans. Interestingly, metformin is able to reverse some of these consequences of pathological pain in rodents. The purpose of this review was to analyze the current evidence about the effects of metformin in chronic pain and three of its comorbidities (anxiety, depression and cognitive impairment).

Effect of Modified Constraint-induced Movement Therapy on Neurotransmitter Levels of Motor Cortex in Cerebral Ischemia-Reperfusion Injured Rats

To study the effect and mechanism of modified constraint-induced movement therapy (mCIMT) on motor function recovery in cerebral ischemia-reperfusion rats. The rats were randomly divided into the control group and the mCIMT group, with 12 rats in each group. The left middle cerebral artery occlusion (MCAO) model was established by the Longa suture method. In the mCIMT group, the rats started continuous training for 14 d on the 7 th day after modeling. The unaffected limb was tied to the chest with elastic bandages, and the affected limb was trained in the compulsory runner equipment. In the control group, rats moved freely in the cage. The body mass of rats was recorded within 20 d after modeling, and behavior was assessed by the foot-fault test. Some of the rats were euthanized 18 d after modeling, and high performance liquid chromatography (HPLC) was used to detect monoamine neurotransmitters (5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIVV), homovanillic acid (HVA) ), and amino acid neurotransmitters (glutamic acid (Glu), asparaginic acid (ASP), glutamine (Gln), glycine (Gly), taurine (Tau), gamma aminobutyric acid (GABA) ) in the motor cortex and striatum, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of total P70 ribosomal protein S6 kinase (p70s6k) and p70s6k phosphorylated protein (p-p70s6k) in motor cortex and striatum, respectively. Compared with the control group, the body mass of rats in the mCIMT group was comparable (P >0.05) within 21 d after modeling, foot-fault rate of the mCIMT group was significantly lower at 17 d after modeling (P<0.05). At 18 d after modeling, compared with the control group, the level of 5-HIVV in the motor cortex increased significantly (P<0.05), and the relative content of amino acid neurotransmitters (the ratio of Glu) in the motor cortex including Gln, Gly, Tau and GABA to Glu increased significantly (P<0.05 or P<0.01) except for decreased ASP/Glu (P<0.05). Moreover, compared with the control group, the expression of p-p70s6k in the motor cortex of the mCIMT was significantly decreased (P<0.05). There were no significant differences in monoamine neurotransmitters and amino acid neurotransmitters in the striatum between two groups (P>0.05). mCIMT improved the motor function of MCAO rats, and the mechanism might be related to the increase of amino acid neurotransmitters and 5-HIVV and decrease of p-p70s6k expression in the motor cortex.