mTOR Protein Expression Research Articles

MiR-410 exerts neuroprotective effects in a cellular model of Parkinson's disease induced by 6-hydroxydopamine via inhibiting the PTEN/AKT/mTOR signaling pathway

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by loss of dopaminergic neurons in the substantia nigra. Recently, microRNAs (miRNAs) were emerging as important mediators in dopaminergic neuron biology. This study determined miR-410 expression in the 6-hydroxydopamine (6-OHDA)-induced in vitro cellular model of PD and explored the mechanistic role of miR-410 in the modulation of neuronal cell viability and apoptosis. Our data showed that 6-OHDA concentration-dependently suppressed neuronal cell viability and miR-410 expression in SH-SY5Y and PC12 cells. Overexpression of miR-410 partially restored the effects of 6-OHDA on neuronal cell viability, apoptosis, capsase-3 activity as well as reactive oxygen species (ROS) production. On the other hand, inhibition of miR-410 decreased neuronal cell viability and increased apoptotic rates, capase-3 activity as well as ROS production. Furthermore, the potential targets of miR-410 were predicted by TargetScan tool, and we verified that phosphatase and tensin homolog (PTEN) was a target of miR-410 as confirmed by the dual-luciferase reporter assay. MiR-410 overexpression attenuated PTEN expression and mediated the effects in the 6-OHDA-treated cells via targeting PTEN in SH-SY5Y and PC12 cells. Furthermore, 6-OHDA treatment suppressed the protein expression of phosphorylated AKT and phosphorylated mTOR, which was partially attenuated by miR-410 overexpression in SH-SY5Y and PC12 cells. MiR-410 overexpression increased phosphorylated AKT and phosphorylated mTOR protein expression, and this effect was attenuated by PTEN overexpression in both SH-SY5Y and PC12 cells. Collectively, this is the first study to demonstrate the neuroprotective effects of miR-410 in a 6-OHDA-induced cellular model of PD, and our data implied that miR-410 exerted its neuroprotective effects via regulating PTEN/AKT/mTOR signaling axis. The present study may suggest new paradigm to study the pathology of PD.

Activation of mammalian target of rapamycin signaling pathway in placental tissues of gravidas with gestational diabetes mellitus

Objective To investigate the activation of mammalian target of rapamycin (mTOR) signaling pathway in placental tissues of gestational diabetes mellitus (GDM) women and in JEG3 cells after high glucose treatment. Methods Placental tissues were collected from 60 singleton pregnant women at term, who underwent elective cesarean section in Peking University First Hospital from December 2016 to June 2017, including 30 GDM women (GDM group) and 30 normal glucose tolerance (NGT) women (NGT group). Expressions of mTOR, Rictor, Raptor, S6 kinase (S6K) and phosphorylated-S6K (p-S6K), which were mTOR signaling pathway-related molecules in those placental tissues were detected by real-time fluorescence quantitative polymerase chain reaction, Western blotting and immunohistochemistry. At the same time, JEG3 cells were treated with glucose of different concentrations (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) for 24 h, and the expressions of mTOR, S6K and p-S6K were detected by Western blotting. Two independent samples t-test, one-way analysis of variance test and Spearman correlation analysis were used for statistical analysis. Results Compared with the NGT group, the pre-pregnancy body mass index(BMI), neonatal birth weight, fasting blood glucose(FBG), and 1 h and 2 h post-load blood glucose in oral glucose tolerance test (OGTT) of the GDM group were significantly increased [pre-pregnant BMI: (21.6±2.7) vs (23.4±3.5) kg/m2, t=-2.192; neonatal birth weight: (3 337.5±347.7) vs (3 618.3±580.9) g, t=-2.727; FBG: (4.6±0.4) vs (5.2±0.8) mmol/L, t=-3.947; 1 h glucose level: (7.4±1.2) vs (9.6±1.9) mmol/L, t=-5.332; 2 h glucose level: (6.3±1.0) vs (8.1±1.5) mmol/L, t=-5.314; all P<0.05]. The mRNA expressions of mTOR, Rictor and Raptor were significantly higher in the GDM group than in the NGT group (0.051±0.015 vs 0.031±0.011, t=-5.635; 0.038±0.017 vs 0.026±0.010, t=-3.485; 0.036±0.012 vs 0.025±0.011, t=-3.312; all P<0.05). The expression of mTOR, Rictor, Raptor and p-S6K protein in the GDM group were enhanced as compared with those in the NGT group (0.834±0.432 vs 0.386±0.361, t=-2.249; 0.589±0.236 vs 0.262±0.075, t=-3.726; 0.767±0.345 vs 0.323±0.109, t=-3.472; 1.847±1.025 vs 0.834±0.432, t=-2.575; all P<0.05). The results of immunohistochemistry also showed that the p-S6K in the placental tissues of the GDM group was higher than that of the NGT group (0.29±0.09 vs 0.18±0.08, t=-3.122, P=0.004). The expressions of mTOR protein (0.190±0.085, 0.301±0.089, 0.419±0.065, 0.562±0.108, 0.412±0.058, F=18.351, P<0.05) and p-S6K protein (0.753±0.150, 1.146±0.289, 2.148±0.188, 2.248±0.115, 2.134±0.214, F=66.242, P<0.05) in JEG3 cells treated with different concentrations of glucose (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) were significantly different and increased with increasing concentrations of glucose (r=0.314, P=0.035; r=0.457, P=0.002). Conclusions The up-regulated expressions of mTOR signaling pathway-related molecules in GDM placenta and high glucose-treated trophoblast cells (JEG3 cells) indicate a possible correlation between mTOR signaling pathway and GDM. However, the specific mechanisms need further study. Key words: Diabetes, gestational; Placenta; Mammalian target of rapamycin, serine/threonine kinases; Signal transduction

Prevention of kidney cell damage in hyperglycaemia condition by adiponectin.

Adiponectin (APN) is an adipocytokine, secreted from adipose tissue and has anti-inflammatory, anti-ageing, and antidiabetic properties. Hyperglycaemia can damage the renal cells, and mammalian target of rapamycin (mTOR), along with Sirtuin 1 (SIRT1), have an important role in kidney cell response to hyperglycaemia. Therefore, understanding the relationship between adiponectin, mTOR, and SIRT1 proteins is beneficial for deciphering the mechanism of adiponectin function. In this study, Human Embryonic Kidney-293 (HEK-293) cells were cultured under normal and high-glucose condition, with and without APN (1, 10, and 100ng/mL) for 48hours. mTOR protein expression was evaluated by western blot analysis, and SIRT1 protein was assessed using ELISA method. To evaluate hyperglycaemia-mediated cytotoxicity, cell viability was determined using MTT assay. Data showed that APN in high dose (100ng/mL) significantly reduced the expression of mTOR and p-mTOR, increased SIRT1 protein, and also improved cell viability compared with the control high glucose (p≤0.05). According to this results, APN can be useful in preventing renal cell damage, by affecting on the expression of mTOR and SIRT1 proteins, as well as increasing the survival of kidney cells in hyperglycaemia conditions. SIGNIFICANCE OF THE STUDY: Adiponectin triggered mTOR/p-mTOR/SIRT1 pathway and decreased cell death in human kidney cells. Our findings provide preliminary experimental data that support further studies on the potential therapeutic role of adiponectin in diabetes and diabetic-induced metabolic complications.

MiRNA-214 suppresses oxidative stress in diabetic nephropathy via the ROS/Akt/mTOR signaling pathway and uncoupling protein 2.

In the present study, the function of microRNA (miR)-214 on diabetic nephropathy (DN) and diabetes of proximal tubular cells was investigated. Reverse transcription-quantitative polymerase chain reaction was used measure the expression of miR-214 in rats with DN and ELISA was performed to measure oxidative stress and ROS levels. Results indicated that miR-214 expression in the peripheral blood was significantly decreased in rats with DN. The in vitro model of DN indicated that miR-214 upregulation significantly decreased oxidative stress and reactive oxygen species (ROS) levels, but significantly increased uncoupling protein 2 (UCP2), phosphorylated (p)-Akt and p-mammalian target of rapamycin (mTOR) protein expression levels. The administration of genipin, a UCP2 inhibitor, significantly attenuated the effects of miR-214 upregulation on oxidative stress in the in vitro DN model by regulating ROS, Akt and mTOR protein expression levels. Notably, Akt inhibitor suppressed p-Akt protein expression and attenuated the effects of miR-214 upregulation on oxidative stress in the in vitro DN model. Collectively, these data suggest that miR-214 regulates diabetes through a ROS/Akt/mTOR signaling pathway by UCP2 in proximal tubular cells.

Exercise initiated during pregnancy in rats born growth restricted alters placental mTOR and nutrient transporter expression.

Fetal growth is dependent on effective placental nutrient transportation, which is regulated by mammalian target of rapamycin (mTOR) complex 1 modulation of nutrient transporter expression. These transporters are dysregulated in pregnancies affected by uteroplacental insufficiency and maternal obesity. Nutrient transporters and mTOR were altered in placentae of mothers born growth restricted compared to normal birth weight dams, with maternal diet- and fetal sex-specific responses. Exercise initiated during pregnancy downregulated mTOR protein expression, despite an increase in mTOR activation in male associated placentae, and reduced nutrient transporter gene abundance, which was also dependent on maternal diet and fetal sex. Limited changes were characterized with exercise initiated before and continued throughout pregnancy in nutrient transporter and mTOR expression. Maternal exercise during pregnancy differentially regulated mTOR and nutrient transporters in a diet- and sex-specific manner, which likely aimed to improve late gestational placental growth and neonatal survival. Adequate transplacental nutrient delivery is essential for fetoplacental development. Intrauterine growth restriction and maternal obesity independently alter placental nutrient transporter expression. Although exercise is beneficial for maternal health, limited studies have characterized how the timing of exercise initiation influences placental nutrient transport. Therefore, this study investigated the impact of maternal exercise on placental mechanistic target of rapamycin (mTOR) and nutrient transporter expression in growth restricted mothers and whether these outcomes were dependent on maternal diet or fetal sex. Uteroplacental insufficiency or sham surgery was induced on embryonic day (E) 18 in Wistar-Kyoto rats. F1 offspring were fed a chow or high-fat diet from weaning and at 16 weeks were randomly allocated to an exercise protocol: sedentary, exercised prior to and during pregnancy, or exercised during pregnancy only. Females were mated with normal males (20 weeks) and F2 placentae collected at E20. Exercise during pregnancy only, reduced mTOR protein expression in all groups and increased mTOR activation in male associated placentae. Exercise during pregnancy only, decreased the expression of amino acid transporters in a diet- and sex-specific manner. Maternal growth restriction altered mTOR and system A amino acid transporter expression in a sex- and diet-specific manner. These data highlight that maternal exercise initiated during pregnancy alters placental mTOR expression, which may directly regulate amino acid transporter expression, to a greater extent than exercise initiated prior to and continued during pregnancy, in a diet- and fetal sex-dependent manner. These findings highlight that the timing of exercise initiation is important for optimal placental function.

MTORC1 inhibitor RAD001 (everolimus) enhances non-small cell lung cancer cell radiosensitivity in vitro via suppressing epithelial-mesenchymal transition.

Resistance to radiotherapy causes non-small cell lung cancer (NSCLC) treatment failure associated with local recurrence and metastasis. Thus, understanding the radiosensitization of NSCLC cells is crucial for developing new treatments and improving prognostics. mTORC1 has been shown to regulate tumor cell radiosensitivity, but the underlying mechanisms are unclear. Moreover, mTORC1 also regulates epithelial-mesenchymal transition (EMT) that is important to metastasis and recurrence. In this study we explored whether mTORC1 regulated NSCLC cell radiosensitivity by altering EMT. We performed immunohistichemical analysis using tumor, adjacent and normal tissues from 50 NSCLC patients, which confirmed significantly elevated mTOR protein expression in NSCLC tissue. Then we used NCI-H460 and NCI-H661 cell lines to examine the effects of the mTORC1 inhibitor RAD001 (everolimus) on in vitro radiosensitivity, protein expression and dose-survival curves. RAD001 (10 nmol/L) significantly inhibited the mTORC1 pathway in both the cell lines. Pretreatment with RAD001 (0.1 nmol/L) enhanced the radiosensitivity in NCI-H661 cells with wild-type PIK3CA and KRAS but not in NCI-H460 cells with mutant PIK3CA and KRAS; the sensitivity enhancement ratios in the two NSCLC cell lines were 1.40 and 1.03, respectively. Furthermore, pretreatment with RAD001 (0.1 nmol/L) significantly decreased the migration and invasion with altered expression of several EMT-associated proteins (significantly increased E-cadherin and decreased vimentin expression) in irradiated NCI-H661 cells. Publicly available expression data confirmed that irradiation affected mTOR and EMT-associated genes at the transcript level in NSCLC cells. These results suggest that mTORC1 inhibition enhances the in vitro radiosensitivity of NSCLC cells with wild-type PIK3CA and KRAS by affecting EMT. Our preclinical data may provide a potential new strategy for NSCLC treatment.

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

Hbs1 has been established as a central component of the cell’s translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

MTOR-S6K1 pathway mediates cytoophidium assembly

CTP synthase (CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin (mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1 (mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing mTOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells. Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.

Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-\u03b1 mediated NF-\u03baB and HDAC-3 nuclear translocation

Acute respiratory distress syndrome (ARDS) is characterized by an excessive acute inflammatory response in lung parenchyma, which ultimately leads to refractory hypoxemia. One of the earliest abnormalities seen in lung injury is the elevated levels of inflammatory cytokines, among them, the soluble tumor necrosis factor (TNF-α) has a key role, which exerts cytotoxicity in epithelial and endothelial cells thus exacerbates edema. The bacterial lipopolysaccharide (LPS) was used both in vitro (RAW 264.7, THP-1, MLE-12, A549, and BEAS-2B) and in vivo (C57BL/6 mice), as it activates a plethora of overlapping inflammatory signaling pathways involved in ARDS. Nimbolide is a chemical constituent of Azadirachta indica, which contains multiple biological properties, while its role in ARDS is elusive. Herein, we have investigated the protective effects of nimbolide in abrogating the complications associated with ARDS. We showed that nimbolide markedly suppressed the nitrosative-oxidative stress, inflammatory cytokines, and chemokines expression by suppressing iNOS, myeloperoxidase, and nitrotyrosine expression. Moreover, nimbolide mitigated the migration of neutrophils and mast cells whilst normalizing the LPS-induced hypothermia. Also, nimbolide modulated the expression of epigenetic regulators with multiple HDAC inhibitory activity by suppressing the nuclear translocation of NF-κB and HDAC-3. We extended our studies using molecular docking studies, which demonstrated a strong interaction between nimbolide and TNF-α. Additionally, we showed that treatment with nimbolide increased GSH, Nrf-2, SOD-1, and HO-1 protein expression; concomitantly abrogated the LPS-triggered TNF-α, p38 MAPK, mTOR, and GSK-3β protein expression. Collectively, these results indicate that TNF-α-regulated NF-κB and HDAC-3 crosstalk was ameliorated by nimbolide with promising anti-nitrosative, antioxidant, and anti-inflammatory properties in LPS-induced ARDS.

Effects and mechanism of epigallocatechin-3-gallate on apoptosis and mTOR/AKT/GSK-3β pathway in substantia nigra neurons in Parkinson rats.

The aim of this study is to investigate the protective effect of epigallocatechin-3-gallate (EGCG) on apoptosis and mTOR/AKT/GSK-3β pathway in substantia nigra neurons in 6-dopamine-induced Parkinson rats. A total of 30 healthy male SD rats were randomly divided into control group, the Parkinson model group, and Parkinson model+EGCG treatment group. The model and EGCG groups were injected into the right striatum with 6-OHDA to establish the Parkinson model, and the control group was injected with saline only. The EGCG group was intragastrically administered with EGCG 50 mg/kg daily for 4 weeks. The rats' turns, speed, and left forelimb usage; neuron apoptosis by TUNEL; and the α-synuclein protein expression in substantia nigra by immunohistochemical staining were studied. Western blotting was used to detect the relative protein (mTOR, AKT and GSK-3β) expressions. Compared with the model group, the EGCG group significantly reduced the rotation speed; increased the left forelimb usage (P<0.01); reduced the neuron apoptosis (P<0.01); decreased α-synuclein expression (P<0.01); and decreased the mTOR, AKT, and GSK-3β protein expressions (P<0.01). EGCG can reduce neuron cell apoptosis in substantia nigra neurons in 6-OHDA-induced Parkinson rats. The mechanism might be related to mTOR/AKT/GSK-3β activation.

Consecutive-Day Intake of Whey Protein Upregulates mTOR mRNA and Protein Expression in Resting Skeletal Muscle of Mice

Background: Although the effect of whey protein intake on protein metabolism in exercise-loaded skeletal muscle has been well documented, little has been reported on its effect on resting muscle. The effects of whey protein intake on protein metabolism in resting mouse skeletal muscle were investigated. Methods: Mice were fed AIN-93G composed of either casein or whey protein as the protein source for 3 or 7 consecutive days. The gastrocnemius muscle was excised, and the expression levels of the regulatory factor, mTOR, and its subunits, Raptor and Rictor, were measured by real-time PCR. The protein expression levels of mTOR and its phosphorylated form were measured by immunofluorescent western blotting. The effects of whey protein were compared to those of the case in control. Results: mTOR expression increased in the gastrocnemius muscle of mice fed whey protein for 7 consecutive days. The expression of Raptor significantly increased, whereas that of Rictor did not change, suggesting a dominant formation of mTORC1 relating to the upregulation of protein synthesis. The protein levels of mTOR and its phosphorylated form significantly increased in mice fed whey protein, indicating enhanced protein synthesis. Increased mTOR expression was not seen in the gastrocnemius muscle of mice fed whey protein for 3 consecutive days. Conclusions: These results indicate that the intake of whey protein for 7 consecutive days, but not 3 days, upregulates the mRNA and protein expression of mTOR in the resting gastrocnemius muscle of mice, suggesting its ability to enhance protein synthesis. Consecutive-day intake of whey protein may induce constitutive alteration of the skeletal muscle, including continuous upregulation of muscle protein synthesis.

Identifying Biomarkers of Autophagy and Apoptosis in Transfected Nuclear Donor Cells and Transgenic Cloned Pig Embryos

Abstract In this study, we first investigated the effects of 3-methyladenine (3-MA), an autophagy inhibitor, and the inducer – rapamycin (RAPA) on the incidence of programmed cell death (PCD) symptoms during in vitro development of porcine somatic cell nuclear transfer (SCNT)-derived embryos. The expression of autophagy inhibitor mTOR protein was decreased in porcine SCNT blastocysts treated with 3MA. The abundance of the autophagy marker LC3 increased in blastocysts following RAPA treatment. Exposure of porcine SCNT-derived embryos to 3-MA suppressed their developmental abilities to reach the blastocyst stage. No significant difference in the expression pattern of PCD-related proteins was found between non-transfected dermal cell and transfected dermal cell groups. Additionally, the pattern of PCD in SCNT-derived blastocysts generated using SC and TSC was not significantly different, and in terms of porcine SCNT-derived embryo development rates and total blastocyst cell numbers, there was no significant difference between non-transfected cells and transfected cells. In conclusion, regulation of autophagy affected the development of porcine SCNT embryos. Regardless of the type of nuclear donor cells (transfected or non-transfected dermal cells) used for SCNT, there was no difference in the developmental potential and quantitative profiles of autophagy/apoptosis biomarkers between porcine transgenic and non-transgenic cloned embryos. These results led us to conclude that PCD is important for controlling porcine SCNT-derived embryo development, and that transfected dermal cells can be utilized as a source of nuclear donors for the production of transgenic cloned progeny in pigs.

Hyperbaric oxygen relieves neuropathic pain through AKT/TSC2/mTOR pathway activity to induce autophagy.

BackgroundOur previous study suggested that HBO treatment attenuated neuropathic pain by inhibiting mTOR to induce autophagy in SNL neuropathic pain model. The aim of this study was to evaluate the role of AKT/TSC2/mTOR pathway in SNL and autophagy and determine whether HBO treatment could relieve neuropathic pain via modulating AKT/TSC2/mTOR pathway.Materials and methodsRats were randomly divided into sham, SNL, SNL + HBO treatment, SNL + vehicle, and SNL + AKT inhibitor groups. Neuropathic pain was induced following SNL procedure. Rats in the SNL + HBO group received HBO treatment for 7 consecutive days beginning on postoperative day 1. The SNL + vehicle group received 10 µL of 3% dimethyl sulfoxide in saline. SNL + AKT inhibitor group received 10 µL AKT inhibitor IV intrathecally. Mechanical withdrawal threshold tests were performed to evaluate mechanical hypersensitivity. AKT, p-AKT, TSC2, mTOR, p-mTOR, and LC3-II protein expressions were examined by Western blot analysis.ResultsHBO reversed AKT/TSC2/mTOR upregulation induced by SNL and attenuated neuropathic pain. Intrathecal injection of AKT inhibitor IV decreased the activity of AKT/TSC2/mTOR pathway and increased LC3-II expression accompanied by analgesic effect in SNL rats.ConclusionTaken together, our findings demonstrated AKT/TSC2/mTOR pathway was activated in SNL-induced neuropathic pain, and HBO treatment attenuated neuropathic pain via neutralizing AKT/TSC2/mTOR pathway activation.

PI3K-PKB-mTOR hyperactivation in relation to nasopharyngeal carcinoma progression and prognosis.

Nasopharyngeal carcinoma (NPC) has a unique and complex etiology, which is not completely understood. The aim of this study is to investigate the expression patterns of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and mammalian target of rapamycin (mTOR) proteins in patients with NPC and their relationship with NPC progression and prognosis. Between January 2008 and March 2010, PI3K, PKB, and mTOR protein expressions were detected using immunohistochemistry among 119 patients with NPC and 30 healthy people. A 5-year follow-up was conducted for all patients. Correlations of PI3K, PKB, and mTOR proteins with the clinicopathological features and prognosis of NPC were evaluated using Spearman's rank correlation coefficient and Kaplan-Meier curve. Cox's regression analysis was performed to analyze the risk factors for the prognosis of NPC. First, PI3K, PKB, and mTOR were highly expressed in patients with NPC. The expressions of PI3K, PKB, and mTOR proteins were associated with T stage, N stage, clinical stage, relapse, and distant metastasis. Meanwhile, PI3K is positively correlated with PKB and PKB is positively correlated with mTOR in NPC. Higher PI3K, PKB, and mTOR protein expressions were related to a shorter survival time and a lower survival rate in NPC. Cox regression analysis revealed that age, T stage, N stage, PI3K, PKB, and mTOR were independent risk factors for NPC patient survival. Altogether, our data suggest that overexpression of PI3K, PKB, and mTOR proteins is an important indicator of poor survival in NPC. In addition, inhibition of PI3K-PKB-mTOR signaling may also contribute to the development of new therapeutic strategies for NPC.

Effect of cuecumin on mammalian target of rapamyein signaling pathway during ischemia-reperfusion injury in isolated rat lungs

Objective To evaluate the effect of curcumin on the mammalian target of rapamyein(mTOR)signaling pathway during ischemia-reperfusion(I/R)injury in isolated rat lungs. Methods Sixty-four clean-grade healthy male Sprague-Dawley rats, aged 3-4 months, weighing 250-320 g, were divided into 4 groups(n=16 each)using a random number table method: sham operation group(S group), I/R group, low-dose curcumin group(LC group)and high-dose curcumin group(HC group). The rats only received in vitro perfusion without ischemia in S group.Isolated rat lungs were subjected to 60 min of ischemia followed by 75-min reperfusion to establish the lung I/R injury model in I/R group.Curcumin 5 and 10 μmol/L were added to perfusion fluid from the beginning of reperfusion in LC and HC groups, respectively.Airway resistance(Res), lung compliance, perfusion flow(Flow)and pulmonary venous partial pressure of oxygen(PaO2)were recorded at 10 min of first perfusion(T0)and 15, 45 and 75 min of reperfusion(T1-3). Wet/dry lung weight ratio(W/D ratio)was measured at the end of reperfusion.The morphological structure and ultrastructure of lung tissues were observed by using a light microscope and a transmission electron microscope, respectively.The expression of mTOR, Tau protein, nuclear factor kappa B(NF-κB)and tumor necrosis factor-alpha(TNF-α)mRNA in lung tissues was detected by real-time polymerase chain reaction.The expression of mTOR, phosphorylated Tau protein(pS396 Tau protein), NF-κB and TNF-α protein in lung tissues was determined by Western blot. Results Compared with S group, Res at T1-3 and W/D ratio at T3 were significantly increased, lung compliance, Flow and PaO2 were decreased at T1-3, and the expression of mTOR, NF-κB and TNF-α protein and mRNA, Tau protein mRNA and pS396 Tau protein was up-regulated at T3 in I/R, LC and HC groups(P<0.05). Compared with I/R group, Res at T1-3 and W/D ratio at T3 were significantly decreased, lung compliance, Flow and PaO2 were increased at T1-3, and the expression of mTOR, NF-κB and TNF-α protein and mRNA, Tau protein mRNA and pS396 Tau protein was down-regulated at T3 in LC and HC groups(P<0.05). Compared with LC group, Res at T1-3 and W/D ratio at T3 were significantly decreased, lung compliance, Flow and PaO2 were increased at T1-3, and the expression of mTOR, NF-κB and TNF-α protein and mRNA, Tau protein mRNA and pS396 Tau protein was down-regulated at T3 in HC group(P<0.05). The microscopic examination showed that the injury to lung tissues was significantly attenuated in LC and HC groups as compared with I/R group. Conclusion The mechanism by which curcumin reduces I/R injury in isolated rat lungs is related to inhibiting mTOR signaling pathway. Key words: Curcumin; Lung; Reperfusion injury; Protein kinase C

An Integrated Score and Nomogram Combining Clinical and Immunohistochemistry Factors to Predict High ISUP Grade Clear Cell Renal Cell Carcinoma.

Objective: The International Society of Urological Pathology (ISUP) has proposed a grading system to classify renal cell carcinoma (RCC). However, classification using biopsy specimens remains problematic and, consequently, the accuracy of a biopsy-based diagnosis is relatively poor. This study aims to combine clinical and immunohistochemical (IHC) factors for the prediction of high ISUP grade clear cell RCC (ccRCC) in an attempt to complement and improve the accuracy of a biopsy-based diagnosis.Methods: A total of 362 ccRCC patients were enrolled in this study and used for the training set. We performed IHC analysis of 18 protein markers on standard tissue sections using an automated stainer. Multivariate logistic regression models were developed to evaluate independent predictors for high ISUP grade. We evaluated different prediction models using receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) analysis. A nomogram for the derivation of an integrated score for predicting high ISUP grade ccRCC and a calibration curve were also plotted. Finally, an internal validation cohort was examined to evaluate the performance of our integrated scoring system and nomogram.Results: Multivariate logistic analyses revealed seven credible candidates for predicting high grade ISUP. These were age, tumor diameter, surgery, and CK7, Ki-67, PTEN, and MTOR protein expression. The ROC curves for the clinical, IHC and integrated models were compared in the training set, and the AUC for each was 0.731, 0.744, and 0.801, respectively. DeLong's test showed that the integrated model was significantly better at predicting high ISUP grade, when compared with the other models. Internal validation confirmed the good performance of the integrated score in predicting ISUP grade.Conclusion: We have developed a nomogram integrating clinical and immunohistochemical parameters to predict high ISUP grade for M0 ccRCC patients. This nomogram may offer potentially useful information during preoperative individualized patient risk assessment, and consequently may help urologists when planning personalized management regimens.

Alteration in cardiac PI3K/Akt/mTOR and ERK signaling pathways with the use of growth hormone and swimming, and the roles of miR21 and miR133.

Athletes misuse recombinant human growth hormone (r-hGH) to enhance their performance. Although r-hGH is known to increase cardiac hypertrophy, the underlying molecular mechanism remains unclear. The aim of the present study was to investigate the role of r-hGH in cardiac intracellular signaling pathways and of miR-21 and miR-133 expression in rat hearts during exercise. A total of 36 adult male Sprague-Dawley rats were divided into sedentary control (SC, n=9), swimming exercise (SE, n=8), r-hGH (GH, n=10) and swimming exercise plus r-hGH (SE-GH, n=9) groups. The exercise groups completed a 1-h swimming exercise 5 times a week for 8 weeks. Subcutaneous r-hGH was administered as 0.3 mg/kg/day. Phosphoinositide-3-kinase (PI3K), serine/threonine protein kinase 1 (AKT1), extracellular signal-regulated kinase (ERK), microRNA (miR)-21 and miR-133 expression was evaluated in ventricular muscle by real-time quantitative polymerase chain reaction. Protein expression of PI3K, AKT1, ERK and mechanistic target of rapamycin (mTOR) was also assessed by immunohistochemistry. Statistical differences were analyzed by two-way ANOVA. PI3K and AKT1 expression and the gene and protein levels was notably increased in the SE-GH group compared with in SC ventricular tissues (P<0.05). mTOR protein expression was higher in the GH, SE and SE-GH groups compared with in the SC group (P<0.05, <0.05 and <0.001, respectively). ERK gene/protein expression was similar across all groups. miR-21 and miR-133 levels in ventricular muscle were higher in the SE and GH groups than those in the SC group. In summary, growth hormone application coupled with swimming exercise appeared to affect the PI3K/AKT/mTOR signaling pathway in the left ventricular tissue of rats; however, ERK signaling pathway appeared inactive in physiological left ventricular hypertrophy caused by swimming and GH administration over 8 weeks. Furthermore, GH treatment resulted in increased miR-21 and miR-133 expression. Future study by our group will aim to assess the effects of higher dose GH treatment.

Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

BackgroundGlioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients.MethodsIn vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro.ResultsOur results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients’ materials.ConclusionsBased on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.

Mtor-Independent Translation in Early Hematopoietic Development

Mechanisms of translational regulation are poorly understood in hematopoietic stem cells (HSCs) and committed progenitors (MP). In order to investigate the impact of translational regulation on early mouse hematopoietic development, we characterized the translatome using a combination of polysome profiling, RNA-sequencing (RNA-seq) of polysome-associated and total cellular mRNA, and whole proteome evaluation. Comparison of RNA-seq data from HSC-enriched LSK (Lin-Sca-1+c-Kit+) and MP (Lin-Sca-1-c-Kit+) cells showed more differentially expressed mRNAs in polysomal RNA than total RNA (412 vs 280 mRNAs, respectively) among ~15,000 mRNAs analyzed. In addition, polysomal mRNAs were enriched for a unique set of functional pathways (e.g. inflammatory response, apoptosis and p53) compared to total RNA in both LSK and MP cells. Interestingly, although LSK cells showed ~20% lower global translation than MPs as demonstrated by polysome profiling (Figure 1A), they exhibited significantly higher translational efficiency (TE = polysome RNA abundance/total RNA abundance) for mRNAs that support HSC maintenance (e.g. glycolysis, fatty acid metabolism, oxidative phosphorylation, mTOR signalling). Integration of proteomic and RNA-seq data demonstrated that 605 out of 784 (77.2%) differentially expressed genes (DEGs) between LSK and MP cells identified based on total RNA-seq data (Groups I & III; Fig 1B) also showed a corresponding change in protein expression, while remaining 179 DEGs (22.8%; Groups II & IV; Fig. 1B) showed an anti-correlation. Remarkably, in the latter group, expression of 129 proteins (72.1% of all differentially expressed proteins in LSKs vs MPs) correlated with their TEs (Figure 1C). While gene set enrichment analysis of published HSC regulators showed an enrichment in LSKs in total RNA-seq data, such an enrichment was not observed when evaluating mRNAs with differential TEs. However, mRNAs with high TE confirmed a surprising enrichment in mTOR-responsive genes independent of their total RNA expression in LSKs, but not in MP cells (Figure 1D). To investigate the biochemical basis of this observation, we performed western blot analysis of LSK and MP cells and observed decreased mTOR protein expression and signaling in purified MP cells, despite their higher global levels of translation. In addition, despite abundant expression of mTOR protein in LSK cells, 4E-BP1, a known target of mTOR, was only phosphorylated at the priming residues Thr-37/46 but not at the downstream Ser-65, a residue that initiates cap-dependent translation. In contrast, MP cells phosphorylated Ser-65, consistent with its increased translation despite the absence of mTOR signalling. mTOR inhibition with Torin-1 did not alter 4E-BP Ser-65 phosphorylation or translation in MPs ex vivo. The presence of mTOR-independent translation in MPs was corroborated by in vivo rapamycin treatment studies, which induced increased colony formation by LSKs, but not MPs. Decreased mTOR activity in MPs was due to degradation of mTOR protein mediated by the proteasome since mTOR protein expression was restored following treatment with the proteasome inhibitors bortezomib and MG132, as well as deletion of the E3 ubiquitin ligase, c-Cbl. Indeed, LSKs and MPs exhibit differential dependencies on mTOR signaling for translation, as mTOR protein is post-translationally downregulated in MPs by a previously undescribed mechanism for mTOR proteosomal degradation mediated by c-Cbl. These findings establish the presence of developmental stage-specific mechanisms of translational regulation in early hematopoiesis.Figure legend. (A) Representative polysome profiles from LSK (Lin-Sca-1+c-Kit+) and MP (Lin-Sca-1-c-Kit+) cells. Polysome/subpolysome ratios (poly/subpoly) were calculated by dividing total RNA abundance from polysomes (fractions 5-10) by subpolysomes (fractions 1-4) (n=3, p < 0.05). (B) Comparison of total RNA versus protein expression in LSK versus MP cells. Four groups of mRNAs were identified based on comparisons of their total mRNA and protein expression. Percentage of total analyzed mRNAs is indicated in each quadrant. (C) Comparison of TEs versus protein expression in LSK/MP cells (D) Enrichment plots comparing total RNA and TE in LSK and MP cells, using previously validated mTOR gene sets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P-mTOR, p-4EBP-1 and eIF4E expression in canine prostatic carcinoma

The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC.