Protein S6 Kinase Research Articles

Protective effects of glucagon-like peptide-1 on cardiac remodeling by inhibiting oxidative stress through mammalian target of rapamycin complex1/p70 ribosomal proteinS6 kinase pathway in diabetes mellitus.

Aims/IntroductionAlthough increased reactive oxygen species (ROS) generation is a major mechanism leading to cardiac remodeling in diabetes mellitus, research into the effects of anti‐oxidation on diabetic cardiac remodeling remains scarce and controversial. Glucagon‐like peptide‐1 (GLP‐1) shows potential anti‐oxidative effects besides lowering blood glucose. The objective of this research was to investigate the effects of GLP‐1 on cardiac remodeling and the molecular mechanism involved in diabetes mellitus.Materials and MethodsStreptozotocin‐induced diabetic rats received exenatide treatment for 3 months. Cardiac function, cardiac weight index and myocardial interstitial fibrosis were measured. Cardiomyocytes were cultured in high‐glucose medium with GLP‐1 treatment. The ROS production, apoptosis and the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase protein expression in cardiomyocytes were analyzed.ResultsExperimental diabetes mellitus showed impaired cardiac diastolic function, increased brain natriuretic peptide expression and increased interstitial collagen deposition in the myocardium, which were ameliorated by exenatide treatment. Exenatide reduced myocardial ROS production and apoptosis in diabetes mellitus. Also, high glucose‐induced ROS generation and apoptosis in cardiomyocytes were inhibited by GLP‐1, as well as the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase phosphorylation. Furthermore, GLP‐1 treatment upregulated adenosine monophosphate‐activated protein kinase activity in high‐glucose‐induced cardiomyocyte.ConclusionsGlucagon‐like peptide‐1 protects the cardiomyocytes from oxidative stress and apoptosis in diabetes mellitus, which might contribute to the improvement of cardiac remodeling. The cardiac protection of GLP‐1 might be dependent on inhibition of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase, through an adenosine monophosphate‐activated protein kinase‐mediated pathway.

Simultaneous activation of impaired autophagy and the mammalian target of rapamycin pathway in oral squamous cell carcinoma.

The aim of this study was to conduct a comparative evaluation of the expression levels of autophagy markers and proteins of the mammalian target of rapamycin (mTOR) pathway in normal, margin and tumour tissues of patients with oral squamous cell carcinoma (OSCC). Three regional specimens, including normal, margin and tumour tissues, were collected from 26 patients with OSCC. Western blotting, immunohistochemistry and immunofluorescence staining were performed to detect mTOR, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p70 ribosomal S6 protein kinase (p70S6K) and the corresponding phosphorylated proteins, as well as the light chain 3 (LC3) and sequestosome-1 (SQSTM1, also known as p62) autophagy indicators. LC3-II, p62, mTOR, phospho-mTOR, 4E-BP1 and phospho-4E-BP1 were highly expressed in the margin and tumour groups. There were positive correlations between mTOR/phospho-mTOR, mTOR/4E-BP1, mTOR/phospho-4E-BP1, mTOR/p70S6K, LC3-II/p62, LC3-II/p70S6K, p62/4E-BP1 and p62/phospho-4E-BP1 in normal group, while LC3-II/p62, LC3-II/mTOR, LC3-II/4E-BP1, LC3-II/phospho-4E-BP1, phospho-4E-BP1/mTOR, phospho-4E-BP1/4E-BP1 and p62/4E-BP1 showed positive relationships in margin group; however, in tumour group, only mTOR/phospho-mTOR, 4E-BP1/phospho-4E-BP1 and phospho-mTOR/p70S6K showed positive correlations. The study suggests that autophagy is impaired in patients with OSCC and impaired autophagy and the mTOR pathway are simultaneously activated in OSCC cells.

Responses of bladder smooth muscle to the stretch go through extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor-κB (NF-κB) Pathway.

The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch. Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real-time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO. The bioinformatics analysis showed that chronic obstruction activated mitogen-activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor-κB (NF-κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF-κB in HBSMCs in a time-dependent manner. The mRNA expressions of α-smooth muscle actin and SM22 also increased and filamentous actin (F-actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF-κB pathway reversed mechanical stretch-induced changes of contractile phenotypic expression and F-action polymerization. Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F-actin. This process partially goes through ERK/p90RSK/NF-κB pathway.

Role of the mTOR Signalling Pathway in Human Sepsis-Induced Myocardial Dysfunction

BackgroundSepsis-induced myocardial dysfunction (SIMD) is a life-threatening complication of sepsis. Mammalian target of rapamycin (mTOR) signalling pathway is significantly associated with SIMD in an animal model; however, there have been no clinical studies of the association in humans. MethodsWe enrolled 88 patients with sepsis who were admitted to the intensive care unit (ICU) between April 2017, and April 2018. Biochemical indexes, hemodynamic parameters, and bedside echocardiographic parameters were recorded. Serum levels of mTOR, phosphorylated ribosome S6 protein kinase (PS6K), microtubule-associated protein light chain 3 type II (LC3B), Bcl-2-interacting mediator of cell death (BIM), interleukin 6, interleukin 10, and interferon-γ were examined. ResultsCompared with non-SIMD patients, patients with SIMD had higher ICU and 28-day mortality, PS6K and BIM levels, but lower LC3B levels. Serum PS6K levels in patients with SIMD were significantly negatively and positively correlated with LC3B and BIM, respectively. Multivariate regression analysis revealed that PS6K concentration at admission was an independent predictor of 28-day mortality. Receiver operating characteristic curve analysis indicated that a PS6K concentration cutoff of 42.43 pg/mL at ICU admission could predict the incidence of SIMD with a sensitivity and specificity of 91.7% and 96.2%, whereas a cutoff concentration of 41.17 pg/mL PS6K could predict 28-day mortality with a sensitivity and specificity of 83.3% and 54.3%, respectively. ConclusionsPatients with sepsis and SIMD had higher ICU and 28-day mortality. Higher serum PS6K concentrations were significantly associated with SIMD incidence and 28-day mortality, suggesting that activation of the mTOR pathway may play a major role in SIMD.

The effect of downregulation of MCT1 on the proliferation of glioma cells

Objective: To investigate the molecular mechanism of down-regulation of monocarboxylic acid transporter 1 (MCT1) on the proliferation inhibition of glioma cell. Methods: siMCT1, siMCT4 and negative control siRNA were transfected into glioma cell lines including U-251 and U-87. The proliferation activities of U-251 and U-87 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and clonogenic assay. Glucose consumption and lactic acid efflux of U-251 and U-87 cells were determined by spectrophotometry.Western blot was used to detect the expressions of MCT1, MCT4, human glucose transporter 1 (GLUT1), GLUT4, tuberous sclerosis associated protein (TSC2), p-TSC2, 4E binding protein 1 (4EBP1), p-4EBP1, ribosomal S6 protein kinase (S6) and p-S6 protein in U-251 and U-87 cells. Results: Compared with negative control group, siMCT1 and siMCT4 significantly inhibited the expressions of MCT1 and MCT4 protein in U-251 and U-87 cells (both P<0.05). However, only knockdown of MCT1, the proliferation activities of U-251 and U-87 cells significantly decreased (P<0.05). The clone formation rates of U-251 and U-87 cells decreased to (55.20±3.27)% and (68.33±4.58) %, respectively (P<0.05). The glucose consumption of U-251 and U-87 cells in the negative control group at 72 hours were (82.65±6.66) pmol/L and (63.33±5.27) pmol/L, respectively, significantly higher than (31.70±3.17) pmol/L and (26.41±3.19) pmol/L of the siMCT1 transfected group (P<0.05). The extracellular lactate flow of U-251 and U-87 cells in negative control group at 72 h were (155.49±8.15) mmol/L and (135.37±8.21) mmol/L, respectively, significantly higher than (42.69±4.66) mmol/L and (38.91±4.83) mmol/L of the siMCT1 transfected group (P<0.05). Western blot analysis showed that knockdown of MCT1 significantly decreased the protein levels of GLUT1 p-TSC2, p-4EBP1 and p-S6 in U-251 and U-87 cells. Conclusions: Downregulation of MCT1 expression can inhibit the proliferation of glioma cells. Deletion of MCT1 inhibits the glycolysis and metabolism of glioma cells through regulating the mTOR signaling pathway.

Mesenchymal stem cell therapy inhibited inflammatory and profibrotic pathways induced by partial bladder outlet obstruction and prevented high-pressure urine storage

Partial bladder outlet obstruction (pBOO) is characterized by an initial inflammatory response that progresses to smooth muscle hypertrophyand fibrosis. Current treatment modalities carry high risk of morbidity. Mesenchymal stem cells (MSCs) are undifferentiated adult cells with reparative, immunomodulatory, and anti-inflammatory capacities. The ability of MSCs to inhibit inflammatory and profibrotic pathways in bladder cells has been recently reported. This study aimed to investigate the therapeutic effects of MSCs on pBOO-induced inflammatory, profibrotic signaling pathways and end-organ physiology. Twenty Sprague Dawley rats were randomly assigned to 5 groups: unobstructed controls, pBOO for 2 and 4 weeks, pBOO+MSCs for 2 and 4 weeks. Partial bladder outlet obstruction was surgically induced followed by intravenous injection of MSCs. Endpoint urodynamics was performed, and bladder tissueharvested for analysis. Reverse transcription real time polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to study gene and protein expression of major inflammatory and profibrotic markers. Partial bladder outlet obstruction resulted in an upregulation of transforming growth factor beta (TGFβ1), mothers against decapentaplegic homolog 2/3 (SMAD2/3), hypoxia inducible factor 1 alpha (HIF1α), hypoxia inducible factor 3 alpha (HIF3α), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNFα), mechanistic target of rapamycin (mTOR), p70 ribosomal S6 protein kinase (p70 S6K), collagen 1 (COL1), and collagen 3 (COL3) expression in a time-dependent manner. This was coupled with a downregulation of interleukin (IL)-10 expression. Increase of bladder fibrosis was directly related to the duration of pBOO and associated with high urine storage pressure. Injected MSCs were identified in the bladder 4 weeks after therapy. The immunomodulatory effect of MSCs(defined by reduced TNFα and increased IL-10 and VEGF) was most predominant 2 weeks after therapy. Significant downregulation of profibrotic genes occurred 4 weeks after therapy. End filling pressure, hypertrophy, and fibrosis were significantly reduced after MSCtherapy (P<0.05). Mesenchymal stem cell therapy led to a profound systematic improvement of the obstructed bladder. This included an initial anti-inflammatory response and a subsequent antifibrotic reaction. Essentially, both phases were associated with a reduction of urine storage pressure. The intravenously injected MSCs were tracked in the bladder. However, their presence in non-target organs such as the lungs, spleen, and liver was not tracked. Partial bladder outlet obstruction induced significant upregulation of hypoxic, inflammatory, and profibrotic markers. Mesenchymal stem cell therapy potently inhibited these pathways and improved bladder function.

Arabidopsis KIN gamma subunit 1 has a potential to regulate activity of sucrose nonfermenting 1-related protein kinase 2s (SnRK2s) in vitro

Plants must precisely regulate their signalling pathways to respond to environmental changes promptly. Sucrose non fermenting1 (SNF1)-related protein kinases (SnRK) 2 are essential kinases in abiotic stress responses, including responses to abscisic acid. Although homologs of SnRKs in yeast require a γ-subunit for full activation, it has been unclear whether SnRK2s in higher plants are affected by γ-subunits. In this report, we aimed to show the effect of Arabidopsis KIN γ-subunit 1 (KING1), which is a potential γ-subunit, on the activity of SnRK2. A recombinant KING1 bound to SnRK2.6 and functionally inhibited its activity in vitro. On the other hand, KING1 facilitated the activity of SnRK2.2. Structural models suggest that significant structural changes occurred as a result of KING1 binding to the C-terminal tail of SnRK2s. Since KING1 inhibited the kinase activity of a chimeric protein consisting of the N-terminal domain of SnRK2.6 and the C-terminal domain of SnRK2.2, regulation by KING1 was determined by the N-terminal domain of SnRK2s. Together, these results show that KING1 can mediate activity of SnRK2s in vitro.

The Role of Abscisic Acid Signaling in Maintaining the Metabolic Balance Required for Arabidopsis Growth under Nonstress Conditions.

Abscisic acid (ABA) is a plant hormone that regulates a diverse range of cellular and molecular processes during development and in response to osmotic stress. In Arabidopsis (Arabidopsis thaliana), three Suc nonfermenting-1-related protein kinase2s (SnRK2s), SRK2D, SRK2E, and SRK2I, are key positive regulators involved in ABA signaling whose substrates have been well studied. Besides reduced drought-stress tolerance, the srk2d srk2e srk2i mutant shows abnormal growth phenotypes, such as an increased number of leaves, under nonstress conditions. However, it remains unclear whether, and if so how, SnRK2-mediated ABA signaling regulates growth and development. Here, we show that the primary metabolite profile of srk2d srk2e srk2i grown under nonstress conditions was considerably different from that of wild-type plants. The metabolic changes observed in the srk2d srk2e srk2i were similar to those in an ABA-biosynthesis mutant, aba2-1, and both mutants showed a higher leaf emergence rate than wild type. Consistent with the increased amounts of citrate, isotope-labeling experiments revealed that respiration through the tricarboxylic acid cycle was enhanced in srk2d srk2e srk2i These results, together with transcriptome data, indicate that the SnRK2s involved in ABA signaling modulate metabolism and leaf growth under nonstress conditions by fine-tuning flux through the tricarboxylic acid cycle.

Rapamycin alleviates proinflammatory cytokines and nociceptive behavior induced by chemotherapeutic paclitaxel

ABSTRACTBackground/Aims: Paclitaxel is largely used as a chemotherapeutic agent for the treatment of several types of cancers. However, one of the significant limiting complications of paclitaxel is painful peripheral neuropathy during its therapy. The purposes of this study were to examine (1) the effects of blocking mammalian target of rapamycin (mTOR) on mechanical and thermal hypersensitivity evoked by paclitaxel; and (2) the underlying mechanisms responsible for the role of mTOR in regulating paclitaxel-induced neuropathic pain.Methods: Behavioral test was performed to determine mechanical and thermal sensitivity in rats. ELISA was used to examine the levels of proinflammatory cytokines (PICs including IL-1β, IL-6, and TNF-α) and substance P and calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DGR); and Western blot analysis was used to examine expression of mTOR signal pathway.Results: Paclitaxel increased mechanical and thermal sensitivity as compared with vehicle control animals (P < 0.05 vs. controls). Paclitaxel also amplified the expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4E-binding protein 1 (p-4E-BP1) in the DRG. Blocking mTOR using rapamycin attenuated peripheral painful neuropathy observed in paclitaxel rats (P < 0.05 vs. without rapamycin). This inhibitory effect was accompanied with decreases of IL-1β, IL-6, and TNF-α as well as substance P and CGRP. In addition, inhibition of phosphatidylinositide 3-kinase (p-PI3K) attenuated expression of p-mTOR and PICs/substance P/CGRP in paclitaxel rats and this further attenuated mechanical and thermal hypersensitivity.Conclusions: The data revealed specific signaling pathways leading to paclitaxel-induced peripheral neuropathic pain, including the activation of PI3K-mTOR, PIC signal, and substance P and CGRP. Inhibition of these pathways alleviates neuropathic pain. Targeting one or more of these molecular mediators may present new opportunities for treatment and management of neuropathic pain observed during chemotherapeutic application of paclitaxel.

AMPK/TSC2/mTOR pathway regulates replicative senescence of human vascular smooth muscle cells.

Age-associated diseases, including vascular diseases, are on the rise with the increase in the aging population. However, the mechanisms of aging and age-associated vascular dysfunction remain to be fully elucidated. Replicative senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-associated vascular diseases. Rapamycin may delay aging-associated diseases via inhibition of the mammalian target of rapamycin (mTOR), but its role in VSMC aging has remained elusive. The present study investigated the involvement of mTOR signaling in replicative senescence of VSMCs. Replicative senescence was induced in human VSMCs by extended passages and identified by assessing the cell morphology, senescence-associated β-galactosidase activity, and p53 and p21 protein expression. Protein expression and phosphorylation were determined by western blot analysis. Significant senescence of VSMCs was observed in cells subjected to extended passaging (until passage 15). Significant decreases in adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2) phosphorylation, but significant increases in mTOR/ribosomal protein S6 kinase 1 (S6K1) phosphorylation, were observed in cells with replicative senescence compared with those in young cells. Pre-treatment of VSMCs with AMPK activator and mTOR inhibitor delayed replicative senescence and reversed changes in AMPKα, TSC2, mTOR and S6K1 phosphorylation in senescent VSMCs. The AMPK/TSC2/mTOR/S6K1 signaling axis was found to have an important role in regulating replicative senescence of human VSMCs.

Downregulation of long noncoding RNA Sox2ot protects PC-12 cells from hydrogen peroxide-induced injury in spinal cord injury via regulating the miR-211-myeloid cell leukemia-1 isoform2 axis.

This study aimed to investigate the effects and possible mechanisms of long noncoding RNA (lncRNA) Sox2 overlapping transcript (Sox2ot) on hydrogen peroxide (H2 O2 )-induced injury in pheochromocytoma (PC-12) cells. PC-12 cells were treated with H2 O2 to cell injury. The cells were transfected with short-hairpin RNA directed against Sox2ot (sh-Sox2ot), small interfering RNA directed against myeloid cell leukemia-1 (MCL-1) isoform2 (si-MCL-1), a miR-211 mimic, a miR-211 inhibitor, and their negative controls. Under different transfected treatments, cell viability, migration, invasion, and apoptosis as well as the expressions of apoptosis- and autophagy-related proteins were investigated. Besides, the regulatory relationships between Sox2ot and miR-211, miR-211 and MCL-1, as well as between MCL-1 and the protein kinase B (Akt)/mammalian target of the rapamycin (mTOR)/p70 ribosomal S6 protein kinase (p70S6K) signaling pathway were explored. Suppression of Sox2ot inhibited H2 O2 -induced PC-12 cell injury by increasing cell viability, migration, invasion, and decreasing apoptosis and autophagy. Moreover, suppression of Sox2ot increased miR-211 expression and alleviated H2 O2 -induced injury in PC-12 cells possibly via upregulation of miR-211. Furthermore, MCL-1 isoform2 was identified as a direct target of miR-211 and could be negatively regulated by miR-211. Suppression of miR-211 aggravated H2 O2 -induced cell injury by regulation of MCL-1 isoform2. Besides, inhibition of miR-211 suppressed the activation of the Akt/mTOR/p70S6K signaling pathway in H2 O2 -treated PC-12 cells, which was reversed after knockdown of MCL-1 isoform2 at the same time. Our findings indicate that downregulation of Sox2ot may protect PC-12 cells from H2 O2 -induced injury in SCI via targeting the miR-211/MCL-1 isoform2 axis. MCL-1 isoform2 may further regulate the activation of the Akt/mTOR/p70S6K pathway to mediate H2 O2 -induced injury. The Sox2ot-miR-211-MCL-1 isoform2 axis may be a promising therapeutic strategy for SCI.

Abstract 3984: Combination S6K and TAM tyrosine kinase targeting in PTEN-deficient glioma

Abstract Loss of PTEN is one of the most frequent driver events in primary glioblastoma, conferring the ability to reconfigure metabolism and enhance apoptosis resistance to transformed glial cells. The ribosomal S6 protein kinases (S6Ks) are hyperactivated in PTEN-deficient settings, and genetic inactivation studies have shown that S6K1 deficiency can reduce the oncogenic growth of PTEN-deficient neoplasia. Investigating pharmacologic agents, we found that the S6K inhibitor LY-2584702 selectively elicits cytotoxic responses in PTEN-deficient cells when combined with the TAM kinase inactivating agent BMS-777607. Combined knockdown of TAM kinases sensitized cells to single agent treatment with LY-2584702. Conversely, expression of activated S6K1 or elevated AXL protected from combination treatment. These results indicate that both the TAMS and S6K1 are key regulators of survival in PTEN deficient cells. Citation Format: Kelli N. Ennis, Pranjal Sarma, Catherine A. Behrmann, Jie Wang, David R. Plas. Combination S6K and TAM tyrosine kinase targeting in PTEN-deficient glioma [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 3984.

Muscle Protein Anabolic Resistance to Essential Amino Acids Does Not Occur in Healthy Older Adults Before or After Resistance Exercise Training

The muscle protein anabolic response to contraction and feeding may be blunted in older adults. Acute bouts of exercise can improve the ability of amino acids to stimulate muscle protein synthesis (MPS) by activating mechanistic target of rapamycin complex 1 (mTORC1) signaling, but it is not known whether exercise training may improve muscle sensitivity to amino acid availability. The aim of this study was to determine if muscle protein anabolism is resistant to essential amino acids (EAAs) and whether resistance exercise training (RET) improves muscle sensitivity to EAA in healthy older adults. In a longitudinal study, 19 healthy older adults [mean ± SD age: 71±4 y body mass index (kg/m2): 28±3] were trained for 12 wk with a whole-body program of progressive RET (60-75% 1-repetition maximum). Body composition, strength, and metabolic health were measured pre- and posttraining. We also performed stable isotope infusion experiments with muscle biopsies pre- and posttraining to measure MPS and markers of amino acid sensing in the basal state and in response to 6.8 g of EAA ingestion. RET increased muscle strength by 16%, lean mass by 2%, and muscle cross-sectional area by 27% in healthy older adults (P<0.05). MPS and mTORC1 signaling (i.e., phosphorylation status of protein kinase B, 4E binding protein 1, 70-kDa S6 protein kinase, and ribosomal protein S6) increased after EAA ingestion (P<0.05) pre- and posttraining. RET increased basal MPS by 36% (P<0.05); however, RET did not affect the response of MPS and mTORC1 signaling to EAA ingestion. RET increases strength and basal MPS, promoting hypertrophy in healthy older adults. In these subjects, a small dose of EAAs stimulates muscle mTORC1 signaling and MPS, and this response to EAAs does not improve after RET. Our data indicate that anabolic resistance to amino acids may not be a problem in healthy older adults. This trial was registered at www.clinicaltrials.gov as NCT02999802.

Pulsed electromagnetic fields inhibit osteoclast differentiation in RAW264.7 macrophages via suppression of the protein kinase B/mammalian target of rapamycin signaling pathway.

When bone resorption, aided by the activity of osteoclasts, exceeds bone formation induced by osteoblasts, bone metabolism loses equilibration, which results in the development of bone diseases, including osteoporosis. Pulsed electromagnetic fields (PEMFs) are known to be involved in various biological processes, including cell proliferation, differentiation and apoptosis. However, the exact mechanism of action of osteoclasts remains poorly understood. In the present study, the effects of PEMFs on osteoclast differentiation and associated signaling pathways were systematically investigated in RAW264.7 macrophages. RAW264.7 cells were induced by receptor activator of nuclear factor‑κB ligand (RANKL) to obtain osteoclasts invitro. The results of the present study demonstrated that PEMF exposure decreased osteoclast formation, limited tartrate‑resistant acid phosphatase activity, contracted bone resorption area and inhibited osteoclastic specific gene and protein expression. Furthermore, western blot analysis indicated that PEMFs distinctly abolished the upregulation of phosphorylated‑protein kinase B (Akt), ‑mammalian target of rapamycin (mTOR) and ‑ribosome S6 protein kinase (p70S6K) induced by RANKL, which was consistent with the effects of pharmacological inhibitor perifosine and rapamycin. Therefore, the present study suggested that PEMFs reduced osteoclast formation from RAW264.7 macrophages via inhibition of the Akt/mTOR signaling pathway. These findings provided novel insight into the mechanisms through which PEMFs suppress osteoclast differentiation.

Multiscale systems pharmacological analysis of everolimus action in hepatocellular carcinoma.

Dysregulation of mTOR pathway is common in hepatocellular carcinoma (HCC). A translational quantitative systems pharmacology (QSP), pharmacokinetic (PK), and pharmacodynamic (PD) model dissecting the circuitry of this pathway was developed to predict HCC patients' response to everolimus, an mTOR inhibitor. The time course of key signaling proteins in the mTOR pathway, HCC cells viability, tumor volume (TV) and everolimus plasma and tumor concentrations in xenograft mice, clinical PK of everolimus and progression free survival (PFS) in placebo and everolimus-treated patients were extracted from literature. A comprehensive and multiscale QSP/PK/PD model was developed, qualified, and translated to clinical settings. Model fittings and simulations were performed using Monolix software. The S6-kinase protein was identified as critical in the mTOR signaling pathway for describing everolimus lack of efficacy in HCC patients. The net growth rate constant (kg) of HCC cells was estimated at 0.02h-1 (2.88%RSE). The partition coefficient of everolimus into the tumor (kp) was determined at 0.06 (12.98%RSE). The kg in patients was calculated from the doubling time of TV in naturally progressing HCC patients, and was determined at 0.004day-1. Model-predicted and observed PFS were in good agreement for placebo and everolimus-treated patients. In conclusion, a multiscale QSP/PK/PD model elucidating everolimus lack of efficacy in HCC patients was successfully developed and predicted PFS reasonably well compared to observed clinical findings. This model may provide insights into clinical response to everolimus-based therapy and serve as a valuable tool for the clinical translation of efficacy for novel mTOR inhibitors.

Down-regulation of TRAF4 targeting RSK4 inhibits proliferation, invasion and metastasis in breast cancer xenografts

Ribosomal S6 protein kinase 4 (RSK4) was known as a novel tumor suppressor gene, and the tumor necrosis factor receptor-associated factor 4 (TRAF4) was linked to carcinogenesis. The purpose of this study is to further investigate the effect of the TRAF4 gene on cell proliferation, invasion and metastasis in vivo and explore whether there is an interaction between TRAF4 and RSK4 in breast cancer. MDA-MB-231 cells were transfected with lentivirus TRAF4-shRNA to specifically block the expression of TRAF4, or transfected with lentivirus negative-shRNA as a negative control. Four-six weeks female BALB/c nude mice were randomly assigned to three groups (n = 14): TRAF4-shRNA, negative and control, and then inoculated subcutaneously with the corresponding cells. In-vivo metastasis model was constructed by injecting above cells into tail vein. Tumor proliferation was assessed in terms of the tumor growth curve, tumor size and weight. Invasion and metastasis were evaluated by the histopathologic examination in lung or/and liver tissues. Measurement of TRAF4 and RSK4 expression and their correlation factors (AKT, NF-κB, TGF-β1, TNF-α, MMP2 and MMP9) were performed by immunohistochemistry, western blot or fluorescence quantitative RT-PCR. We found that the size and weight of tumors in TRAF4-shRNA group was significantly smaller than the negative and blank group, and the number of the lung and liver metastases lesions was also fewer (P < 0.05). And TRAF4 and its correlation factors (P-AKT, P-NF-κB, TGF-β1, TNF-α, MMP2 and MMP9) in the TRAF4-shRNA group were significantly decreased compared with the negative and blank group. However, the expression of RSK4 mRNA and protein in TRAF4-shRNA group were significantly increased. Collectively, TRAF4 knockdown significantly inhibited proliferation, invasion and metastasis in the xenograft nude mouse model, possibly involving in the interaction with RSK4 through down-regulation of AKT signaling pathway and then inactivating NF-κB pathway.

Autophagy induced by cardamonin is associated with mTORC1 inhibition in SKOV3 cells.

The mammalian target of rapamycin (mTOR) integrates energy level to modulate cell proliferation and autophagy. Cardamonin exhibits anti-proliferative activity through inhibiting mTOR. In this study, the effect of cardamonin on autophagy and its mechanism on mTOR inhibition were investigated. Cell viability and proliferation were measured by MTT assay and BrdU incorporation, respectively. Cell apoptosis was assayed by flow cytometry and cell autophagy was detected by electron microscopy and GFP-LC3 fluorescence. The mechanism of cardamonin on mTORC1 inhibition was investigated by Raptor siRNA and Raptor over-expression. The cell viability and proliferation were inhibited by cardamonin. The autophagosomes and the protein level of LC3-II were increased by cardamonin. Cell apoptosis and the levels of cleaved PARP and Caspase-3 were increased by cardamonin. Cardamonin inhibited the phosphorylation of mTOR and ribosome S6 protein kinase 1 (S6K1) as well as the protein level of regulatory associated protein of mTOR (Raptor). However, cardamonin had no effect on the component of mTORC2 and its downstream substrate Akt. The inhibitory effect of cardamonin on the phosphorylation of mTOR and S6K1 was eliminated by Raptor knockdown with siRNA, whereas this effect of cardamonin was stronger than that of rapamycin and AZD8055 in Raptor over-expression cells. Cell viability was inhibited by cardamonin in both Raptor knockdown and Raptor over-expression cells, which was consistent with the inhibitory effect of cardamonin on mTOR. These findings demonstrated that the autophagy induced by cardamonin was associated with mTORC1 inhibition through decreasing the protein level of Raptor in SKOV3 cells.

Paclitaxel suppresses proliferation and induces apoptosis through regulation of ROS and the AKT/MAPK signaling pathway in canine mammary gland tumor cells.

Paclitaxel is a diterpenoid compound, derived from the pacific yew (Taxus brevifolia) berry, which exhibits antineoplastic effects against various types of cancer. However, the antitumor effects and the molecular mechanisms of paclitaxel on canine CHMm cells remain to be elucidated. The aim of the present study was to investigate the antitumor effects of paclitaxel on CHMm cells and identify relevant signal transduction pathways modulated by paclitaxel using multiple methods including MTT assay, flow cytometry, acridine orange/ethidium bromide staining, transmission electron microscopy, determination of cellular reactive oxygen species (ROS), superoxide dismutase (SOD) and malondiadehyde (MDA) and western blotting, the data indicated that paclitaxel decreased cell viability, induced G2/M-phase cell cycle arrest, suppressed the expression of cyclin B1 and induced apoptosis in a dose-dependent manner. In addition, paclitaxel upregulated the expression of Bax and cytochrome c, but reduced expression of apoptosis regulator Bcl-2, resulting in activation of caspase-3, chromatin condensation, karyopyknosis, intracellular vacuolization, increased production of ROS and MDA, and decreased activity of SOD. However, these effects were inhibited when CHMm cells were treated with N-acetyl-L-cysteine. Furthermore, treatment with paclitaxel inhibited the level of of phospho (p)-RAC-α serine/threonine-protein kinase (AKT) and p-ribosomal protein S6 kinase proteins, and promoted phosphorylation of P38 mitogen-activated protein kinase (MAPK) and p-90 kDa ribosomal protein S6 kinase 1 proteins in CHMm cells. It was observed that paclitaxel in combination with pharmacological inhibitors of the P38 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathways (SB203580 and LY294002, respectively) exerted synergistic inhibitory effects on the proliferation of the CHMm cells. The results of the present study demonstrated that paclitaxel inhibited tumor cell proliferation by increasing intrinsic apoptosis through inhibition of the PI3K/AKT signaling pathway and activation of MAPK signaling pathway in CHMm cells.

Early dietary restriction in rats alters skeletal muscle tuberous sclerosis complex, ribosomal s6 and mitogen-activated protein kinase

Intrauterine growth restriction is linked to decreased lean body mass and insulin resistance. The mammalian target of rapamycin (mTOR) regulates muscle mass and glucose metabolism; however, little is known about maternal dietary restriction and skeletal muscle mTOR in offspring. We hypothesized that early dietary restriction would decrease skeletal muscle mass and mTOR in the suckling rat. To test this hypothesis, ab libitum access to food or dietary restriction during gestation followed by postnatal cross-fostering to a dietary-restricted or ad libitum–fed rat dam during lactation generated 4 groups: control (CON), intrauterine dietary restricted (IUDR), postnatal dietary restricted (PNDR), and IUDR+PNDR (IPDR). At day 21, when compared to CON, the IUDR group demonstrated “catchup” growth, but no changes were observed in the mTOR pathway. Despite having less muscle mass than CON and IUDR (P < .001), in IPDR and PNDR rats mTOR remained unchanged. IPDR and PNDR (p)-tuberous sclerosis complex 2 was less than the IUDR group (P < .05). Downstream, IPDR's and PNDR's phosphorylated (p)-ribosomal s6 (rs6)/rs6 was less than that of CON (P < .05). However, male IPDR's and PNDR's p-mitogen activated protein kinase MAPK/MAPK was greater than CON (P < .05) without a change in p90 ribosomal s6 kinase (p90RSK). In contrast, in females, MAPK was unchanged, but IPDR p-p90RSK/p90RSK was less than CON (P = .01). In conclusion, IPDR and PNDR reduced skeletal muscle mass but did not decrease mTOR. In IPDR and PNDR, a reduction in tuberous sclerosis complex 2 may explain why mTOR was unchanged, whereas, in males, an increase in MAPK with a decrease in rs6 may suggest a block in MAPK signaling.

Transcriptome and physiological analyses reveal that AM1 as an ABA-mimicking ligand improves drought resistance in Brassica napus

Abscisic acid (ABA) is the most important stress hormone in the regulation of plant adaptation to drought. Owing to the chemical instability and rapid catabolism of ABA, ABA mimic 1 (AM1) is frequently applied to enhance drought resistance in plants, but the molecular mechanisms governed by AM1 on improving drought resistance in Brassica napus are not entirely understood. To investigate the effect of AM1 on drought resistance at the physiological and molecular levels, exogenous ABA and AM1 were applied to the leaves of two B. napus genotypes (Q2 and Qinyou 8) given progressive drought stress. The results showed that the leaves of 50 µM ABA- and AM1-treated plants shared over 60% differential expressed genes and 90% of the enriched functional pathways in Qinyou 8 under drought. AM1 affected the expression of the genes involved in ABA signaling; they down-regulated pyrabactin resistance/PYR1-like (PYR/PYLs), up-regulated type 2C protein phosphatases (PP2Cs), partially up-regulated sucrose non-fermenting 1-related protein kinase 2s (SnRK2s), and down-regulated ABA-responsive element (ABRE)-binding protein/ABRE-binding factors (AREB/ABFs). Additionally, AM1 treatment repressed the expression of photosynthesis-related genes, those mainly associated with the light reaction process. Moreover, AM1 decreased the stomatal conductance, the net photosynthetic rate, and the transpiration rate, but increased the relative water content in leaves and increased survival rates of two genotypes under drought stress. Our findings suggest that AM1 has a potential to improve drought resistance in B. napus by triggering molecular and physiological responses to reduce water loss and impair growth, leading to increased survival rates.