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The development and maintenance of skeletal muscle is crucial for the support of daily function. Recent evidence suggests that genes coded for proteins associated with the human muscle growth program (myogenic and proteolytic genes) are sensitive to local heat application. Therefore, the purpose of this investigation was to determine the effect of 4 h of local heat application to the vastus lateralis at rest on acute phosphorylation (mTOR Ser2448 , p70-S6K1 Thr389 , and 4E-BP1 Thr47/36 ) and gene expression changes for proteins associated with the muscle growth program. Intramuscular temperature of the HOT limb was 1.2 ± 0.2 °C higher than CON limb after 4 h of local heating. However, this local heat stimulus did not influence transcription of genes associated with myogenesis (MSTN, p = 0.321; MYF5, p = 0.445; MYF6, p = 0.895; MEF2a, p = 0.809; MYO-G, p = 0.766; MYO-D1, p = 0.118; RPS3, p = 0.321; and RPL-3L, p = 0.577), proteolysis (Atrogin-1, p = 0.573; FOXO3a, p = 0.452; MURF-1, p = 0.284), nor protein phosphorylation (mTOR Ser2448 , p = 0.981; P70-S6K1 Thr389 , p = 0.583; 4E-BP1 Thr37/46 , p = 0.238) associated with the muscle growth program. These findings suggest little to no association between the local application of heat, at rest, and the activation of the observed muscle growth program-related markers., Competing Interests: Declaration of competing interest The authors declare no conflicts of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph + ) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph + ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells. Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph + ALL which will assist in developing novel targeted therapy for Ph + ALL patients with BCR-ABL1 independent nonmutational resistance., Competing Interests: Competing interests The authors declare no competing financial interests., (Copyright © 2021. Published by Elsevier Inc.)

Caffeine is a popular psychostimulant, which is frequently consumed with ethanol. However, the effects of caffeine on neuronal cells constantly exposed to ethanol have not been investigated. Apoptosis and oxidative stress occurring in ethanol-induced neurotoxicity were previously associated with decreased phosphorylation of the mTOR/p70S6K/4E-BP1 signaling proteins. Evidence also suggested that caffeine inhibits the mTOR pathway. In this study, human SH-SY5Y neuroblastoma cells were exposed to caffeine after pretreatment for 24 hours with ethanol. Results indicated that both ethanol and caffeine caused neuronal cell death in a dose- and time-dependent manner. Exposure to 20-mM caffeine for 24 hours magnified reduced cell viability and enhanced apoptotic cell death induced by 200 mM of ethanol pretreatment. The phosphorylation of mTOR, p70S6K, and 4E-BP1 markedly decreased in cells exposed to caffeine after ethanol pretreatment, associated with a decrease of the mitochondrial membrane potential (ΔΨm). These findings suggested that caffeine treatment after neuronal cells were exposed to ethanol resulted in marked cell damages, mediated through enhanced inhibition of mTOR/p70S6K/4E-BP1 signaling leading to impaired ΔΨm and, eventually, apoptotic cell death.

Acute lower extremity ischemia (ALEXI) is known worldwide as an urgent condition, occurring when there is an abrupt interruption in blood flow into an extremity. This study aims to investigate whether microRNA-224 (miR-224) affects the ALEXI mice and the underlying mechanism. The miR-224 expression and C/EBP homologous protein (CHOP), mammalian target of rapamycin (mTOR), translation initiation factor 4E-binding protein 1 (4E-BP1), and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) messenger RNA (mRNA), as well as protein expressions, were determined. The target gene of miR-224 was also verified by using a luciferase reporter gene assay. The vascular endothelial cells from the ALEXI mice were transfected with miR-224 mimics, miR-224 inhibitors, or small-interfering RNA against CHOP. Cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle distribution along with the cell apoptosis were both evaluated by using a flow cytometry. The muscle fibers of the lower extremities found in the ALEXI mice were evidently swollen and rounded, presenting with a remarkably narrowed gap. The positive CHOP expression increased in ALEXI mice than normal mice, while the miR-224 expression and mTOR, 4E-BP1, and p70S6K mRNA, as well as the protein expression, decreased. Luciferase reporter gene assay validated that the miR-224 gene directly targeted CHOP. MiR-224 facilitated cell proliferation but inhibited cell apoptosis; by contrast, CHOP increased cell apoptosis. Moreover, the cells transfected along with miR-224 mimic exhibited a lower CHOP expression as well as increased mTOR, 4E-BP1, and p70S6K expression. Our study provided evidence that miR-224 could alleviate the occurrence and development of ALEXI in mice through activation of the mTOR signaling pathway by downregulating CHOP., (© 2018 Wiley Periodicals, Inc.)

Theobromine is a caffeine derivative and the primary methylxanthine in Theobroma cacao. We have shown previously that theobromine inhibits the Akt-mammalian target of rapamycin (mTOR) signal in vitro. In this study, we investigated whether orally administered theobromine could inhibit mTOR activity in rats. mTOR is phosphorylated by Akt. Thus, the level of phosphorylated mTOR was used as an index of mTOR activity. Male Wistar rats were divided into two groups. The control group (CN) was fed a normal diet, while the theobromine group (TB) was fed a diet supplemented with 0.05% theobromine for 40 days. We measured body-weights and tissue weights, food and water intake, blood count, concentrations of theobromine in the plasma, liver and brain, and the levels of phosphorylated mTOR in the liver and brain. Orally administered theobromine did not affect the body-weights and tissue weights, food and water intake, and blood count as determined by comparison with levels in rats that were fed standard chow. Theobromine was detected in the plasma, liver and brain obtained from TB rats, but was not detected in tissues obtained from CN rats. The phosphorylated mTOR levels in the liver and brain were significantly lower in TB rats than in CN rats. The results suggest that oral theobromine inhibits mTOR signalling in vivo., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Objective: To study the expression of mTOR and 4E-BP1 in extranodal Nasal-type NK/T-cell Lymphoma(ENKTCL) and the correlation with the clinicopathological factors and prognosis of ENKTCL. Method: Immunohistochemistry was used to detect the expresstion of mTOR and 4E-BP1 in the tissues of ENKTCL and nasal pharyngeal lymphoid hyperplasia. The relationship between the expression of mTOR and 4E-BP1 and clinicopathological features was analyzed using the Chi-square test, which including sorting clinical stage, general condition score and international prognostic index(IPI). Result: The expression rates of mTOR and 4E-BP1 in ENKTCL were 63.9% and 58.3% respectively, and significantly higher than those（15.0% and 10.0%）in patients with nasal pharyngeal lymphoid hyperplasia(χ ²=12.355, P =0.0001; χ ²=12.410, P =0.0001). The positive expression rate of mTOR was linked to clinical staging, and the positive protein expressions in Stage Ⅲ and Ⅳwere higher than those in stageⅠand Ⅱ(χ ²=17.902, P =0.0001). mTOR expression was related to 4E-BP1( r =0.655, P =0.001). The positive expression rates of 4E-BP1were linked to IPI of lymphoma(χ ²=4.051, P =0.044). Conclusion: High-expressions of mTOR and 4E-BP1 in patients with ENKTCL are related to the biological progression and recurrence of ENKTCL and are malignant indicators to identify ENKTCL from nasal pharyngeal lymphoid hyperplasia. mTOR and 4E-BP1 may play important roles to assess ENKTCL prognosis.., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Fully grown mammalian oocytes utilize transcripts synthetized and stored during earlier development. RNA localization followed by a local translation is a mechanism responsible for the regulation of spatial and temporal gene expression. Here we show that the mouse oocyte contains 3 forms of cap-dependent translational repressor expressed on the mRNA level: 4E-BP1, 4E-BP2 and 4E-BP3. However, only 4E-BP1 is present as a protein in oocytes, it becomes inactivated by phosphorylation after nuclear envelope breakdown and as such it promotes cap-dependent translation after NEBD. Phosphorylation of 4E-BP1 can be seen in the oocytes after resumption of meiosis but it is not detected in the surrounding cumulus cells, indicating that 4E-BP1 promotes translation at a specific cell cycle stage. Our immunofluorescence analyses of 4E-BP1 in oocytes during meiosis I showed an even localization of global 4E-BP1, as well as of its 4E-BP1 (Thr37/46) phosphorylated form. On the other hand, 4E-BP1 phosphorylated on Ser65 is localized at the spindle poles, and 4E-BP1 phosphorylated on Thr70 localizes on the spindle. We further show that the main positive regulators of 4E-BP1 phosphorylation after NEBD are mTOR and CDK1 kinases, but not PLK1 kinase. CDK1 exerts its activity toward 4E-BP1 phosphorylation via phosphorylation and activation of mTOR. Moreover, both CDK1 and phosphorylated mTOR co-localize with 4E-BP1 phosphorylated on Thr70 on the spindle at the onset of meiotic resumption. Expression of the dominant negative 4E-BP1 mutant adversely affects translation and results in spindle abnormality. Taken together, our results show that the phosphorylation of 4E-BP1 promotes translation at the onset of meiosis to support the spindle assembly and suggest an important role of CDK1 and mTOR kinases in this process. We also show that the mTOR regulatory pathway is present in human oocytes and is likely to function in a similar way as in mouse oocytes.

The p21-activated kinases (Paks) interact with Rac/Cdc42 GTPases to regulate the actin cytoskeleton as well as various signaling pathways. Although activation of Paks in many human cancers is known to mediate cancer progression, the role of Pak proteins in hypoxia is poorly understood. In this study, we found that both Pak1 and Pak4 are highly expressed in HeLa cervical cancer cells, but only Pak4 knockdown attenuates expression of hypoxia-inducible factor-1α (HIF-1α) in hypoxia. We further discovered that Pak4 regulates HIF-1α translation via the Akt-mTOR-4E-BP1 pathway under hypoxic conditions. These results support a novel connection between HIF-1α and Pak4 in hypoxic cancer cells, and provide insights into mechanisms whereby tumors respond to and thrive under oxygen-deficient conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The characteristic clinical heterogeneity and mostly slow-growing behavior of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) cause problems in finding appropriate treatments. Thus, the current therapy options are not satisfactory. PKI-587 is a highly potent, novel dual inhibitor of PI3K and mTORC1/C2., Aim: We assessed the effects of PKI-587 in different GEP-NEN tumor models, including the poorly differentiated cell line LCC-18, and compared them with those of the established mTORC1 inhibitor everolimus., Methods: We treated BON, QGP-1, KRJ-I, and LCC-18 cell lines with increasing concentrations of the inhibitor PKI-587, and compared the results with those of everolimus and DMSO. We assessed the impact of the treatments on viability (WST-1 assay), on apoptotic processes (caspase 3/7 assay, JC-1), and on cell cycle regulation (flow cytometry). We determined alterations in signaling mediators by phosphor-specific Western blot analysis and conducted multiplexed gene expression analysis (nCounter® technology)., Results: In all cell lines, PKI-587 dose-dependently inhibited proliferation, whereas everolimus was less effective. Treatment with PKI-587 led to cell cycle arrest and induction of apoptosis and successfully suppressed activity of the direct mTORC1 target 4E-BP1, a crucial factor for tumor genesis only partially inhibited by everolimus. Gene expression analyses revealed relevant changes of RAS, MAPK, STAT, and PI3K pathway genes after treatment. Treatment-dependent and cell line-characteristic effects on AKT/Rb/E2F signaling regarding cell cycle control and apoptosis are extensively discussed in this paper., Conclusion: PI3K/mTOR dual targeting is a promising new therapeutic approach in neuroendocrine tumor disease that should be evaluated in further clinical trials., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Generation of new HIV-1 virions requires the constant supply of proteins, nucleotides, and energy; however, it is not known which cellular pathways are perturbed and what molecular mechanisms are employed. We hypothesized that HIV-1 may regulate pathways that control synthesis of biomolecules in the cell. In this study, we provide evidence that HIV-1 hyperactivates mammalian target of rapamycin complex 1 (mTORC1), the central regulator of biosynthesis. Mechanistically, we identify the viral regulatory gene tat (transactivator) as being responsible for increasing mTORC1 activity in a PI3K-dependent manner. Furthermore, we show that hyperactivation of mTORC1 leads to activation of the enzyme, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase, and repression of initiation factor 4E-binding protein 1 activity. These are regulators of nucleotide biogenesis and protein translation, respectively. Moreover, we are able to replicate these results in HIV-1 latent cell line models. Finally, we show that inhibition of mTORC1 or PI3K inhibits viral replication and viral reactivation as a result of a decrease in biosynthesis. Overall, our study identifies a new avenue in HIV-1 biology that can lead to development of novel therapeutic targets.-Kumar, B., Arora, S., Ahmed, S., Banerjea, A. C. Hyperactivation of mammalian target of rapamycin complex 1 by HIV-1 is necessary for virion production and latent viral reactivation., (© FASEB.)

Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA--strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The regulator of G protein signaling 10 (RGS10) protein is a GTPase activating protein that accelerates the hydrolysis of GTP and therefore canonically inactivates G proteins, ultimately terminating signaling. Rheb is a small GTPase protein that shuttles between its GDP- and GTP-bound forms to activate mTOR. Since RGS10 suppression augments ovarian cancer cell viability, we sought to elucidate the molecular mechanism. Following RGS10 suppression in serum-free conditions, phosphorylation of mTOR, the eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), p70S6K and S6 Ribosomal Protein appear. Furthermore, suppressing RGS10 increases activated Rheb, suggesting RGS10 antagonizes mTOR signaling via the small G-protein. The effects of RGS10 suppression are enhanced after stimulating cells with the growth factor, lysophosphatidic acid, and reduced with mTOR inhibitors, temsirolimus and INK-128. Suppression of RGS10 leads to an increase in cell proliferation, even in the presence of etoposide. In summary, the RGS10 suppression increases Rheb-GTP and mTOR signaling in ovarian cancer cells. Our results suggest that RGS10 could serve in a novel, and previously unknown, role by accelerating the hydrolysis of GTP from Rheb in ovarian cancer cells., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Phytoecdysteroids such as 20-hydroxyecdysone (20HE) are nutritional supplements marketed as enhancers of lean body mass. In this study the impact of 20HE ingestion on protein kinase B/Akt-mechanistic target of rapamycin complex 1 signaling in the skeletal muscle and liver of male rats was found to be limited. Bioavailability of 20HE, whether consumed alone or with leucine, also remained low at all doses ingested. Additional work is necessary to clarify 20HE mechanism of action in vivo.

mTOR (mechanistic target of rapamycin) functions as the central regulator for cell proliferation, growth and survival. Up-regulation of proteins regulating mTOR, as well as its downstream targets, has been reported in various cancers. This has promoted the development of anti-cancer therapies targeting mTOR, namely fungal macrolide rapamycin, a naturally occurring mTOR inhibitor, and its analogues (rapalogues). One such rapalogue, everolimus, has been approved in the clinical treatment of renal and breast cancers. Although results have demonstrated that these mTOR inhibitors are effective in attenuating cell growth of cancer cells under in vitro and in vivo conditions, subsequent sporadic response to rapalogues therapy in clinical trials has promoted researchers to look further into the complex understanding of the dynamics of mTOR regulation in the tumour environment. Limitations of these rapalogues include the sensitivity of tumour subsets to mTOR inhibition. Additionally, it is well known that rapamycin and its rapalogues mediate their effects by inhibiting mTORC (mTOR complex) 1, with limited or no effect on mTORC2 activity. The present review summarizes the pre-clinical, clinical and recent discoveries, with emphasis on the cellular and molecular effects of everolimus in cancer therapy., (© 2015 Authors; published by Portland Press Limited.)

Among the numerous signaling pathways involved in tumorigenesis, PI3K-AKT-mTOR is a key one that regulates diverse cellular functions. However, its prognostic value in esophageal carcinoma remains unclear. In our study, we examined the immunohistochemical expression of phosphorylated (p-) AKT, mTOR, p70S6K and 4E-BP1 along with the mutational status of PIK3CA and AKT1 genes by High Resolution Melting Analysis and Pyrosequencing in 44 esophageal carcinomas. The results were correlated with the clinicopathological characteristics of the patients in an effort to define their possible prognostic significance. Total p-mTOR cytoplasmic expression, assessed in 10 random areas, was positively correlated with tumor stage (Kruskal-Wallis ANOVA, I/II vs III/IV, p = 0.0500). Μoreover, maximum p-mTOR cytoplasmic immunoexpression, estimated in hot spot areas, was positively associated with tumor grade (Mann-Whitney U test, I/II vs III, p = 0.0565). Interestingly, p-4E-BP1 immunoreactivity was negatively correlated with tumor histological grade (Mann-Whitney U test, I/II vs III, p = 0.0427). No mutation was observed in exons 9 and 20 of PIK3CA gene and in exon 4 of AKT1 gene. In conclusion, our findings depict the presence of activated PI3K/AKT/mTOR pathway in esophageal cancer bringing forward p-mTOR and p-4E-BP1 for their potential role in esophageal carcinogenesis. Additional studies are warranted to validate our findings., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.

Rapamycin, a highly specific inhibitor of mammalian target of rapamycin (mTOR), exhibits significant antitumor/antiangiogenic activity in human cancer cells. Its effect on the retinal pigment epithelial (RPE) cells was rarely investigated. This study assessed the proliferation of hypoxia-induced RPE and the inhibitory effects of rapamycin using 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and examined the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells with or without rapamycin under normoxic and hypoxic conditions using real-time PCR and Western blot. We found that hypoxia increased the levels of mTOR, HIF-1α, and VEGF. The suppression of HIF-1α and VEGF by rapamycin was associated with dephosphorylation of mTOR and the downstream effector ribosomal protein S6 kinase (P70S6K) and 4E-binding protein-1 (4E-BP1) of mTORC1. Rapamycin only inhibited the protein levels and did not change the mRNA expression of HIF-1α. No cytotoxicity to the RPE cells by rapamycin was caused under either normoxia or hypoxia. Our data suggest that rapamycin suppresses hypoxia-induced RPE cell proliferation through a mechanism related to the targeting of mTOR/HIF-1α/VEGF signaling. Rapamycin may potentially provide a safe and effective novel treatment for choroidal vascular disease., (© 2015 International Union of Biochemistry and Molecular Biology.)

Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells., (Copyright © 2014. Published by Elsevier Ltd.)

Protein synthesis is one of the most energy consuming processes in the cell. The mammalian/mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates a multitude of extracellular signals and intracellular cues to drive growth and proliferation. mTOR activity is altered in numerous pathological conditions, including metabolic syndrome and cancer. In addition to its well-established role in regulating mRNA translation, emerging studies indicate that mTOR modulates mitochondrial functions. In mammals, mTOR coordinates energy consumption by the mRNA translation machinery and mitochondrial energy production by stimulating synthesis of nucleus-encoded mitochondria-related proteins including TFAM, mitochondrial ribosomal proteins and components of complexes I and V. In this review, we highlight findings that link mTOR, mRNA translation and mitochondrial functions.

We treated the thymoma cell line (EL4) with two model immunosuppressants, rapamycin and tributyltin oxide (TBTO), and compared their effects on the expression levels of proteins that are downstream targets of mTOR kinase 1 (mammalian target of rapamycin, known also as mechanistic target of rapamycin): p70 ribosomal S6 kinase1 and 4E-binding protein 1, a repressor of the cap-binding protein eIF4E. In addition, we evaluated the levels of ribosomal protein S6, p-eIF4B, substrates of p70S6 kinase1, matrin 3 and ribonucleotide reductase, subunit RRM2. The levels of these proteins were evaluated in cell lysates by immunoblot. We found that both compounds inhibited the phosphorylation state of p70S6 kinase 1 and its substrates; however, TBTO, in contrast to rapamycin, reduced the level of the total p70S6k1. Besides, we detected a band with a molecular weight of c. 32 kDa only in the TBTO-treated lysates. This band was detected with a monoclonal antibody specific for S6k1, suggesting that this band might be a degradation product of the kinase. Further, TBTO and rapamycin differentially affected 4E-binding protein 1; the former compound stimulated its phosphorylation state whereas the latter inhibited it. The two immunosuppressants did not affect the level of ribonucleotide reductase, but TBTO downregulated matrin3, in agreement with a previous report, whereas rapamycin had no effect on the expression level of this latter protein. We conclude that TBTO inhibits, like rapamycin, the p70 S6 kinase 1 pathway, but with a different mechanism. However, in contrast to rapamycin, which inhibits the cap-dependent translation, TBTO increases the phosphorylation of 4E-binding protein1., (Copyright © 2013 John Wiley & Sons, Ltd.)

The phosphoinositide 3-kinase (PI3K) related protein kinases (PIKKs) are a family of protein kinases with a diverse range of vital cellular functions. Recent high-resolution crystal structures of the protein kinase mTOR suggest general architectural principles that are likely to be common to all of the PIKKs. Furthermore, the structures make clear the close relationship of the PIKKs to the PI3Ks. However, the structures also make clear the unique features of mTOR that enable its substrate specificity. The active site is deeply recessed and flanked by structural elements unique to the PIKKs, namely, the FRB domain, the LST8 binding element, and a C-terminal stretch of helices known as the FATC domain. The FRB has a conserved element in it that is part of a bipartite substrate recognition mechanism that is probably characteristic of all of the PIKKs. The FRB also binds the mTOR inhibitor rapamycin that has been referred to as an allosteric inhibitor, implying that this inhibitor is actually a competitive inhibitor of the protein substrate. This bipartite substrate-binding site also helps clarify how rapamycin can result in substrate-specific inhibition., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Drug resistance is one of the most formidable obstacles for treatment of glioma. Eukaryotic initiation factor 4E-binding protein (4E-BP1), a key component in the rate-limiting step of protein translation initiation, is closely associated with poor prognosis in multiple tumor types. However, it is unclear whether 4E-BP1 is involved in the drug resistance of human glioma. Herein we show that the expression of 4E-BP1 in human SWOZ2-BCNU drug-resistant glioma cells is significantly lower than that of the parent SWOZ2 cell line. Moreover, down-regulation of 4E-BP1 by short interfering RNA significantly impaired the sensitivity of SWOZ2 and U251 cells to carmustine (BCNU). Furthermore, overexpression of 4E-BP1 with plasmid transfection regained this sensitivity. Clinical studies showed that the expression levels of 4E-BP1 in primary glioma tissues were markedly higher than those of recrudescent glioma tissues. Taken together, our results suggest that 4E-BP1 is a novel protein that contributes to acquired drug resistance and it may be a potential target for reversing drug resistance in human glioma., (© 2013 Japanese Society of Neuropathology.)

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Studies have shown that excess body fat negatively affects reproductive functions in females. However, whether obesity affects the ovarian follicle development and ovarian lifespan and the underlying mechanism has not been well elucidated. The aim of the present study was to investigate the association between obesity and ovarian follicle development., Methods: Adult female Sprague-Dawley rats (n = 36) were randomly divided into three groups: the normal control (NC) group, the caloric restriction (CR) group (fed 70% food of the NC group) and the high-fat diet (HF) group. They were maintained on these regimens for 18 weeks., Results: The body weight, ovary weight and visceral fat in the HF group were significantly higher than those in the NC group and the CR group at the end of treatment. Histological analysis showed that the HF rats had significantly less number and percentage of primordial follicles, but greater number and percentage of developing and atretic follicles than the NC rats and CR rats. Western blot analysis demonstrated that the level of mTORC1 and p-S6K1 proteins significantly increased in the ovaries of HF rats, whereas that of SIRT1, SIRT6, FOXO3a and NRF-1 decreased compared to the NC rats. In contrast, the expression of mTORC1 and p-S6K1 dramatically declined, while that of SIRT1, SIRT6, FOXO3a and NRF1 increased in the ovaries of CR rats., Conclusions: Our study suggests that the HF diet induced obesity may accelerate the ovarian follicle development and rate of follicle loss through activating mTOR and suppressing SIRT1 signaling, thus leading to POF, and that CR may inhibit the activation of primordial follicles, follicular development and loss, thus extending the ovarian lifespan through suppressing mTOR and activating SIRT1 signaling., (© 2013.)

Objectives: To evaluate the expression of multiple molecular markers involved in mammalian target of rapamycin (mTOR) signaling pathway in renal cell carcinoma (RCC) to determine the prognostic significance of these markers following radical nephrectomy., Material and Methods: The expression levels of 5 markers, including PTEN, phosphorylated (p)-Akt, p-mTOR, p-p70 ribosomal S6 kinase, and p-4E-binding protein 1 (4E-BP1), were measured in radical nephrectomy specimens from 137 patients with nonmetastatic RCC by immunohistochemical staining., Results: During the follow-up period of this series (median, 63.5 mo), disease recurrence occurred in 59 of the 137 patients (43.0%), with a 5-year recurrence-free survival rate of 58.3%. On Univariate analysis, expression levels of p-mTOR and p-4E-BP1, in addition to the C-reactive protein level, pathological stage, and microvascular invasion, were identified as significant predictors for disease recurrence. Of these factors, the expression of p-4E-BP1, C-reactive protein level, and pathological T stage appeared to be independently related to recurrence-free survival on multivariate analysis. Moreover, significant differences were observed in recurrence-free survival according to the positive numbers of these 3 independent factors; that is, disease recurrence developed in 5 of 42 patients with negative results for any risk factor (11.9%), 23 of 50 patients with positive results for a single risk factor (46.0%), and 31 of 45 patients with positive results for 2 or 3 risk factors (68.8%)., Conclusions: The combined evaluation of the expression levels of potential markers in the mTOR signaling pathway, particularly p-4E-BP1, in RCC specimens with conventional prognostic parameters would contribute to the accurate prediction of disease recurrence following radical nephrectomy for nonmetastatic RCC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

Regulated in DNA damage and development 1 (REDD1) functions to repress signaling through the mechanistic target of rapamycin (mTOR) protein kinase in complex 1 (mTORC1) in response to diverse stress conditions. In the present study, we investigated the role of REDD1 in the response of cells to growth cessation induced by serum deprivation. REDD1 expression was induced within 2h of depriving cells of serum, with the induction being mediated through ER stress, as evidenced by activation of PERK, enhanced eIF2α phosphorylation, and ATF4 facilitated transcription of the REDD1 gene. In wild-type cells, signaling through mTORC1 was rapidly (within 30min) repressed in response to serum deprivation and the repression was sustained for at least 10h. In contrast, in REDD1 knockout cells mTORC1 signaling recovered toward the end of the 10h-deprivation period. Interestingly, Akt phosphorylation initially declined in response to serum deprivation and then recovered between 2 and 4h in wild-type but not REDD1 knockout cells. The recovery of mTORC1 signaling and the failure of Akt phosphorylation to do so in the REDD1 knockout cells were accompanied by a dramatic increase in caspase-3 cleavage and cell death, both of which were blocked by rapamycin. Furthermore, overexpression of constitutively active Akt rescued REDD1 knockout cells from serum deprivation induced cell death. Overall, the results implicate REDD1 as a key regulatory checkpoint that coordinates growth signaling inputs to activate pro-survival mechanisms and reduce susceptibility to cell death., (© 2013.)

Purpose: Penile squamous cell carcinoma is a rare neoplasm associated with a high risk of metastasis and morbidity. There are limited data on the role of the mTOR signaling pathway in penile squamous cell carcinoma carcinogenesis and tumor maintenance. We assessed a possible role for mTOR signaling pathway activation as a potential predictive biomarker of outcome and a therapeutic target for penile cancer., Material and Methods: A cohort of 67 patients diagnosed with invasive penile squamous cell carcinoma from 1987 to 2010 who had known HPV status were selected for study. Tissue microarrays were constructed with 67 primary penile squamous cell carcinomas, matched normal tissues and 8 lymph node metastases. Immunohistochemical staining was performed for p53, pmTOR, pERK, p4E-BP1, eIF4E and peIF4E. Expression was evaluated using a semiquantitative H-score on a scale of 0 to 300., Results: Expression of pmTOR, p4E-BP1, eIF4E and peIF4E was increased in penile tumors compared with matched adjacent normal tissues, indicating activation of the mTOR signaling pathway in penile tumorigenesis. Over expression of pmTOR, peIF4E and p53 was significantly associated with lymph node disease. peIF4E and p53 also correlated with a poor outcome, including recurrence, metastasis or disease specific death. In contrast, pERK and p4E-BP1 were associated with lower pT stages. pmTOR and intense p53 expression was associated with HPV negative tumors., Conclusions: Activation of mTOR signaling may contribute to penile squamous cell carcinoma progression and aggressive behavior. Targeting mTOR or its downstream signaling targets, such as peIF4E, may be a valid therapeutic strategy., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Administration of β2-agonists triggers skeletal muscle anabolism and hypertrophy. We investigated the time course of the molecular events responsible for rat skeletal muscle hypertrophy in response to 1, 3 and 10 days of formoterol administration (i.p. 2000μg/kg/day). A marked hypertrophy of rat tibialis anterior muscle culminated at day 10. Phosphorylation of Akt, ribosomal protein S6, 4E-BP1 and ERK1/2 was increased at day 3, but returned to control level at day 10. This could lead to a transient increase in protein translation and could explain previous studies that reported increase in protein synthesis following β2-agonist administration. Formoterol administration was also associated with a significant reduction in MAFbx/atrogin-1 mRNA level (day 3), suggesting that formoterol can also affect protein degradation of MAFbx/atrogin1 targeted substrates, including MyoD and eukaryotic initiation factor-3f (eIF3-f). Surprisingly, mRNA level of autophagy-related genes, light chain 3 beta (LC3b) and gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1), as well as lysosomal hydrolases, cathepsin B and cathepsin L, was significantly and transiently increased after 1 and/or 3 days, suggesting that autophagosome formation would be increased in response to formoterol administration. However, this has to be relativized since the mRNA level of Unc-51-like kinase1 (Ulk1), BCL2/adenovirus E1B interacting protein3 (Bnip3), and transcription factor EB (TFEB), as well as the protein content of Ulk1, Atg13, Atg5-Atg12 complex and p62/Sqstm1 remained unchanged or was even decreased in response to formoterol administration. These results demonstrate that the effects of formoterol are mediated, in part, through the activation of Akt-mTOR pathway and that other signaling pathways become more important in the regulation of skeletal muscle mass with chronic administration of β2-agonists., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Target of rapamycin complex 1 (TORC1) has a key role in cellular regulations in response to environmental conditions. In yeast, Tip41 downregulates TORC1 signaling via activation of PP2A phosphatase. We show here that overexpression of TIPRL, a mammalian Tip41, suppressed dephosphorylation of mechanistic TORC1 (mTORC1) substrates under amino acid withdrawal, and knockdown of TIPRL conversely attenuated phosphorylation of those substrates after amino acid refeeding. TIPRL associated with the catalytic subunit of PP2A (PP2Ac), which was required for the TIPRL action on mTORC1 signaling. Collectively, unlike yeast TIP41, TIPRL has a positive effect on mTORC1 signaling through the association with PP2Ac., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The unfolded protein response (UPR) is a largely cytoprotective signaling cascade that acts to re-establish homeostasis of the endoplasmic reticulum (ER) under conditions of stress by inducing an early and transient block in general protein synthesis and by increasing the folding and degradative capacity of the cell through an extensive transcriptional program. It is well established that the mechanism for the early translational attenuation during ER stress occurs through phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by activated PERK. Our data demonstrate that when eIF2α is dephosphorylated translation is not fully restored to pre-stressed levels. We found that this correlates with reduced mTOR activity and as a result decreases phosphorylation of 4E-BP1, which negatively regulates assembly of the eIF4F complex and cap-dependent translation. The decrease in mTOR activity and 4E-BP1 phosphorylation is associated with activation of AMP kinase, a negative regulator of mTOR, and in the case of some stress conditions, downregulation of signaling through key components of the PI3K pathway. Furthermore, we show that there is a subset of mRNAs that does not recover from UPR-induced translational repression, including those whose translation is particularly sensitive to loss of eIF4F, such as cyclin D1, Bcl-2 and MMP-9. Together these data implicate reduced mTOR activity and 4E-BP1 hypophosphorylation as a second, more restricted mechanism of translational control occurring somewhat later in the UPR.

Background: Although the expression pattern of phosphorylated (p)-mTOR pathway components has attracted scientific interest in several neoplasms, to our knowledge, there is no published information regarding its significance in mycosis fungoides (MF)., Objective: We sought to perform a comprehensive simultaneous assessment of key members of AKT/mTOR pathway along with p-extracellular signal-regulated kinase (ERK), NOTCH1, and p-STAT3 in patients with MF., Methods: In all, 54 skin biopsy specimens (21 tumors, 30 plaques, and 3 folliculotropic MF) from 50 patients with MF were analyzed immunohistochemically for p-mTOR, its upstream p-AKT, its downstream effectors p-p70S6K and p-4E-BP1, and for p-ERK1/2, NOTCH1, and p-STAT3., Results: p-mTOR was coexpressed with p-p70S6K in 67.3% of lesions, but coexpression with other molecules was less common. p-p70S6K and marginally NOTCH1 displayed higher H-scores in tumors than in plaques. Significant correlations were recorded between p-ERK and p-4E-BP1, as well as between NOTCH1 and p-p70S6K or p-4E-BP1. NOTCH1, p-4E-BP1, and p-p70S6K expression were associated with advanced stage. In survival analysis simultaneous overexpression of p-AKT and p-p70S6K, along with p-4E-BP1 positivity, adversely affected cancer-specific, disease-free, and progression-free survival in advanced-stage cases., Limitations: A limitation may be the small number of cases included in our investigation, precluding multivariate survival analysis., Conclusions: Activation of AKT/mTOR pathway in MF appears to be correlated with NOTCH1, p-ERK, and p-STAT3 and is implicated in the acquisition of a more aggressive phenotype. The combination of p-AKT, p-p70S6K, and p-4E-BP1 emerges as a significant potential prognostic marker in patients with advanced stage., (Copyright © 2013 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

7-Ketocholesterol (7-KC) is found at an elevated level in patients with cancer and chronic liver disease. The up-regulation of an efflux pump, P-glycoprotein (P-gp) leads to drug resistance. To elucidate the effect of 7-KC on P-gp, P-gp function and expression were investigated in hepatoma cell lines Huh-7 and HepG2 and in primary hepatocyte-derived HuS-E/2 cells. At a subtoxic concentration, 48-h exposure to 7-KC reduced the intracellular accumulation and cytotoxicity of P-gp substrate doxorubicin in hepatoma cells, but not in HuS-E/2 cells. In Huh-7 cells, 7-KC elevated efflux function through the activation of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. 7-KC activated the downstream protein synthesis initiation factor 4E-BP1 and induced P-gp expression post-transcriptionally. The stimulation of efflux was reversible and could not be prevented by N-acetyl cysteine. Total cellular ATP content remained the same, whereas the lactate production was increased and fluorescence lifetime of protein-bound NADH was shortened. These changes suggested a metabolic shift to glycolysis, but glycolytic inhibitors did not eliminate 7-KC-mediated P-gp induction. These results demonstrate that 7-KC induces P-gp through PI3K/mTOR signaling and decreased the cell-killing efficacy of doxorubicin in hepatoma cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The effect of resistance exercise with the ingestion of supplementary protein on the activation of the mTOR cascade, in human skeletal muscle has not been fully elucidated. In this study, the impact of a single bout of resistance exercise, immediately followed by a single dose of whey protein isolate (WPI) or placebo supplement, on the activation of mTOR signalling was analyzed. Young untrained men completed a maximal single-legged knee extension exercise bout and were randomized to ingest either WPI supplement (n = 7) or the placebo (n = 7). Muscle biopsies were taken from the vastus lateralis before, and 2, 4 and 24 h post-exercise. WPI or placebo ingestion consumed immediately post-exercise had no impact on the phosphorylation of Akt (Ser473). However, WPI significantly enhanced phosphorylation of mTOR (Ser2448), 4E-BP1 (Thr(37/46)) and p70(S6K) (Thr389) at 2 h post-exercise. This study demonstrates that a single dose of WPI, when consumed in modest quantities, taken immediately after resistance exercise elicits an acute and transient activation of translation initiation within the exercised skeletal muscle.