mRNA Expression Of mTOR Research Articles

Effect of HeLa cells infected with Coxsackie virus B3 on mTOR and p70S6K mRNA expression under different nutritional conditions

Objective To explore the effect of HeLa cells infected with Coxsackie virus B3 (CVB3) on the changes of mTOR signal pathway under different nutritional conditions. Methods The HeLa cells were cultured under conventional culture and serum starvation culture.(1) For the conventional method, the medium with 10 g/L fetal bovine serum was added for 24 h after the Hela cells were fused into 40% to 50%, and the medium was changed on the next day, then the virus group was infected with CVB3 of 50% tissue culture infective dose (TCID50). However, the control group was cultured by 2 g/L fetal bovine serum.(2) For the serum starvation method, HeLa cells were cultured with the medium without fetal bovine serum for 24 h. Then the virus group was infected with CVB3 of TCID50.The cells in control group were cultured by 2 g/L fetal bovine serum.Cell morphology changes were observed by inverted microscope, and the expressions of the mTOR, p70S6K mRNA were detected with Real-time PCR at 3 h, 6 h, 9 h, 12 h, 24 h respectively in both conventional culture and serum starvation groups. Results The expressions of mTOR and p70S6K mRNA were lower in the virus group than those in control group at 12 h and the 24 h (all P 0.05). Conclusion CVB3 may be able to down-regulate the expressions of mTOR and p70S6K mRNA. Key words: Coxsackie virus B3; Hela cell; Serum deprivation; mTOR

Proliferation and apoptosis of choriocarcinoma cell JEG-3 induced by VB2 and its in vitro mechanism

To investigate the effect of purified vitexin compound 2 (VB2), a noval lignanoid from the acetoacetate extract of Vitex negundo seed on the proliferation and apoptosis as well as the expression of mTOR and 4E-BP1 mRNA signal pathway in human choriocarcinoma JEG-3 cell lines in vitro. The inhibitory effect of different concentrations of VB2 on JEG-3 cells was examined by methyl thiazolyl tetrazolium (MTT) assay. Flow cytormetry was used to analyze the apoptosis after using different concentrations of VB2, and the expression of mTOR and 4E-BP1 mRNA was determined by RT-PCR. The inhibitory rate of JEG-3 cell growth which was cultured with different concentrations of VB2 (2.5, 5.0, 10.0, 20.0, 40.0, 80.0, and 160.0 μmol/L) for 24, 48, or 72 hours increased from (6.34±0.41)% to (85.89±0.81)%, and it was positively correlated with the dose and time of culture (P<0.05). VB2 at 5.0, 10.0, or 20.0 μmol/L increased the rate of JEG-3 cell apoptosis in vitro from (9.26±1.02)% to (35.55±1.24)% after 48 hour culture, which was in a dose dependent manner (P<0.05), while 5.0, 10.0, or 20.0 μmol/L of VB2 down-regulated the mRNA levels of mTOR and 4E-BP1 after 48 hour culture, which presented a significant negative correlation between VB2 and the mRNA levels of mTOR and 4E-BP1(P<0.05). VB2 can restrain the proliferation of choriocarcinoma cell JEG-3 and induce its apoptosis. This effect may be related to the inhibition of VB2 on the mRNA expression of JEG-3 cell mTOR and 4E-BP1.

Abstract 1800: Expression analysis of PI3K pathway genes in head and neck cancer.

Abstract Head and neck cancer (HNC) is a heterogenous group of malignant tumors which includes neoplasias of the oral cavity, the pharynx, the larynx and paranasal sinuses. Squamous cell carcinomas comprise more than 95% of all head and neck cancers. The vast majority of HNC is related to exposure to carcinogens, including tobacco, alcohol, betel nut, and sexually transmitted viral pathogens such as human papilloma viruses (HPV). However, only a small fraction of tobacco and alcohol users develop the disease suggesting that genetic factors may play a key role in HNC. Increasing evidence indicate that mTOR is a central regulator of cell growth, proliferation, survival and dysregulation of the PI3K/Akt/mTOR signaling pathway that occurs in many tumors including head and neck cancer. The TSC complex (TSC1/TSC2) negatively regulates mTOR by inhibiting a small GTP binding protein, Rheb, considered to be the direct activator of mTOR. On the other hand, the Ras protein upregulates PI3K signaling which is an upstream effector of mTOR. When PI3K activates Akt, the dual function phosphatase PTEN negatively regulates it. Human cancers frequently harbor activating mutations in the p110 catalytic subunit of PI3K or genomic amplification of Akt. Activated Akt also promotes survival under conditions of cellular stress. In this study we investigated the expression of the critical genes (mTOR, K-ras, PI3KCA, TSC1, RHEB, PTEN and AKT1) acting in this pathway. For this purpose, we analyzed expression levels of these genes in 49 matched HNC tumor and non-cancerous tissue samples by quantitative RT-PCR. We observed a significant downregulation of the mTOR (p=0.005), TSC1 (p=0.001), and PTEN (p=0.000) mRNA expressions. On the other hand, a slight decrease was also observed in K-ras, Akt1 and PI3KCA mRNA levels in tumor tissues compared to the non-cancerous ones. However, the difference in the expression levels of these genes between the two groups were not statistically significant. Whereas we didn't observe any difference in Rheb mRNA expression level in tumor tissue when compared to the non-cancerous tissue samples. mTOR, TSC1, and PTEN mRNA expression levels were decreased 0.48, 0.44 and 0.32 % in HNC tumor tissue samples compared to non-cancerous tissue samples, respectively . Our result indicate that, the PI3K pathway has an important function in HNC progression with the contribution of more than one gene of this pathway. Citation Format: Nur Buyru, Seda Ekizoglu, Emin Karaman, Ozgun Enver. Expression analysis of PI3K pathway genes in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1800. doi:10.1158/1538-7445.AM2013-1800 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Inhibitory effect and significance of rapamycin on the mammalian target of rapamycin signaling pathway in osteosarcoma stem cells and osteosarcoma cells

To observe the effect of rapamycin on the MG-63 osteosarcoma cells (OC), osteosarcoma stem cells (OSC) and on mTOR signaling pathway, and explore the feasibility of rapamycin as a novel therapeutic measure in osteosarcoma chemotherapy regimens. OC and OSC were cultured in vitro. Immunofluorescence assay was used to detect the expression of Nanog and Oct4 in OC and OSC. OC and OSC were treated with rapamycin in concentrations of 0, 20, 50 and 100 nmol/L. Semi-quantitative PCR and RT-PCR were used to detect the mTOR mRNA and CCK-8 assay was used to detect cell proliferation, and the cell morphology was observed under an inverted microscope. The cores of MG-63 cellular spheres exhibited embryonic stem cell characteristics such as Nanog and Oct4 expession. The mTOR pathway was activated in the OSC and the expression of mTOR mRNA was higher in OSC (0.761 ± 0.080) than that in OS (0.406 ± 0.090, P < 0.05) by semi-quantitative PCR. RT-PCR showed that the expression of mTOR mRNA was lower in OSCs treated with 100 nmol/L rapamycin (0.961 ± 0.060) than that with 0 nmol/L rapamycin (1.654 ± 0.246, P < 0.05). Cell counting kit-8 (CCK-8) assay showed that the proliferation of OC treated with 20, 50 and 100 nmol/L rapamycin was significantly inhibited, compared with that with 0 nmol/L rapamycin (P < 0.05). Compared with 0 nmol/L rapamycin, the proliferation of OSC treated with 20 and 50 nmol/L rapamycin was not significantly inhibited (P > 0.05), but that with 100 nmol/L rapamycin was significantly inhibited (P < 0.05). The invert microscopic observation revealed that rapamycin inhibited the formation of OSC spheres. Rapamycin can effectively inhibit cell proliferation and the ability of sphere formation of OSCs. It will provide a basis for a novel therapeutic approach in osteosarcoma chemotherapy regimens.

Cyclin D1, EGFR, and Akt/mTOR pathway

EGFR (epidermal growth factor receptor), cyclin D1 and Akt/mTOR pathways are active in head and neck cancer. The aim of this study was to explore biomarker expression, their correlations with clinicopathological parameters and their prognostic utility in a cohort of patients with localized squamous laryngeal carcinoma. We assessed relative messenger RNA expression of EGFR, Akt1, 2, and 3, mTOR and CCND1, copy number variants of the EGFR and CCND1 genes and immunohistochemical protein expression of EGFR, p-Akt308, p-Akt473, pmTOR, PTEN, p53 and cyclin D1 in paraffin-embedded tissue samples of localized laryngeal carcinomas. In 289patients with T3-4 (77.8%), node-negative (84.1%) tumors of the larynx, high EGFR and CCND1 mRNA correlated with no or ex-smoking, (p = 0.003 and p = 0.029, respectively), while low Akt3 mRNA correlated with alcohol abuse, N0 stage, total laryngectomy, and absence of neck dissection. At a median follow-up of 74.5months, high mTOR mRNA expression was marginally associated with shorter disease-free survival (hazard ratio [HR] = 1.54; p = 0.093) and high Akt3 mRNA with shorter overall survival (HR = 1.49; p = 0.0786), in univariate analysis. In multivariate analysis, node-positive status, subglottic-transglottic location, surgery other than total laryngectomy and mTOR/CCND1 mRNA interaction with a hazard ratio of 2.16 (p value for interaction: 0.0010) were independent predictors of relapse, while node-positive status and subglottic-transglottic location were associated with higher risk for death. In localized laryngeal cancer, clinicopathological parameters and an interaction of high mTOR and CCND1 mRNA expression were found to be associated with poor patient outcome.

Regulation of mTOR signal pathway in HeLa cells under different nutritional conditions by Coxsackie virus B3

To explore the changes of mTOR signal pathway in HeLa cells under different nutritional conditions infected with Coxsackie virus B3 (CVB3). The HeLa cells were cultured with two methods: the conventional culture method cultured HeLa cells with medium with 10% fetal bovine serum for 24 h and changed the medium next day, and then infected with CVB3; the serum starvation method cultured HeLa cells with medium without fetal bovine serum for 24 h, and then infected with CVB3. The expression of the coat protein of CVB3, mTOR, p70S6K mRNA was detected with RT-PCR at different time points. The virus group showed the expressions of mTOR and p70S6K mRNA were significantly higher than those in the control group at 12 h and 24 h (P<0.05) in the conventional culture. The virus group showed the expressions of mTOR and p70S6K mRNA were lower than those in the control group (all P<0.05) in the starvation serum. The expression of mTOR mRNA in the starvation serum virus group was higher than that in the conventional culture virus group (all P<0.05) and the control group. The expression of p70S6K mRNA was not significantly different in the two groups (P>0.05). CVB3 can down-regulate the expressions of mTOR and p70S6K mRNA. The mTOR expression in the starvation serum is higher than that in the conventional culture.

Abstract P1-04-09: mTORC1 and Rictor expression in human breast cancer: correlations with clinicopathological parameters and disease outcome

Abstract BACKGROUND: mTOR (the mammalian target of rapamycin) plays a key role in regulation of cellular metabolism, growth, and proliferation. It forms two multi-protein complexes known as complex 1 (mTORC1) and 2 (mTORC2). Raptor and Rictor are the core proteins for mTORC1 and mTORC2 respectively. There is a growing body of evidence that mTORC1 is up-regulated in many cancers and plays a role in carcinogenesis. The aim of the study was to investigate the mRNA expression of mTOR and Rictor in human breast cancer and examine the relationship between their expression and clinicopathological parameters. METHODS: Specimens of breast cancer (BC) tissues (N = 150) and normal tissues (N = 31) underwent RNA extraction and reverse transcription. mTOR and Rictor transcript levels were determined using real-time quantitative PCR. Expression levels were analyzed against tumor size, grade, nodal involvement, TNM stage, and clinical outcome over a 10 year follow-up period. RESULTS: Significantly higher mRNA transcript levels of mTOR were found in the breast cancer specimens compared to normal glandular tissue (p = 0.0018). The expression of mTOR mRNA was demonstrated to increase with increasing NPI (53 for NPI1 to 219 for NPI3) and tumor grade (37 for grade 2 vs. 159 for grade 3, p = 0.047). mTOR expression was found to be higher in ductal tumors compared with non-ductal tumors (p = 0.0014). The patients who developed recurrent disease or died from breast cancer had higher expression levels than those who had been disease-free after a median follow-up period of 10 years (p = 0.17). Higher expression levels were significantly associated with worse overall survival (p = 0.01). In contrast, higher levels of Rictor mRNA expression were found in normal breast tissue, lower NPI stage (NPI1 vs. 2: p = 0.03) and lower tumor grade (grade 1 vs. grade 3: p = 0.01). Patients with higher Rictor expression had a significantly better overall (p = 0.037) and disease-free (p = 0.048) survival. CONCLUSIONS: mTOR expression was found to be significantly higher in BC specimens compared to normal breast tissue. Higher transcript levels were significantly associated with unfavorable pathological parameters and adverse clinical outcomes. The core protein of the less predominant mTORC2 was associated with favorable pathological parameters and clinical outcome. Taken together these observations are consistent with mTORC1 role in the anti-apoptosis pathway and suggest that selective inhibitors of mTORC1 are likely to be a more effective therapeutic strategy than dual inhibitors in human breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-09.

1031P - Cyclin D1, EGFR and AKT/MTOR Signalling in Localized Laryngeal Squamous Cell Carcinoma

ABSTRACT Introduction EGFR pathway, Akt/mTOR and Cyclin D1 are activated and interact in squamous cell head and neck cancer. Patients and methods We assessed relative mRNA expression of EGFR, Akt1, 2, 3, mTOR and cyclin D1, copy number variants of EGFR and CCND1 genes and immunohistochemical (IHC) expression of EGFR, pAKT308, pAKT473, pmTOR, PTEN, p53 and cyclin D1 in paraffin-embedded tissue of localized laryngeal carcinomas. Results In 289 patients with T3-4 (77.8%), node-negative (84.1%) tumours of the larynx, high EGFR and CCND1 mRNA correlated with never or ex smoking, (p = 0.003) and (p = 0.029) respectively, while low AKT3 correlated with alcohol abuse. At a median follow-up of 74.5 months, high mTOR RNA was marginally associated with shorter DFS, HR = 1.54, p = 0.093 and high Akt3 mRNA with shorter OS, HR = 1.49, p = 0.0786 whereas high EGFR IHC expression was associated with shorter DFS HR = 1.60, p = 0.0311 and OS HR = 1.57, p = 0.056, pAKT308 with shorter OS, HR = 1.003, p = 0.0586 and pAKT473 with shorter DFS HR = 1.004, p = 0.021 and OS HR = 1.004, p = 0.033, all in univariate analysis. An interaction between mRNA levels of mTOR and cyclin D1 was found to be associated with shorter DFS, namely at high mTOR mRNA expression, one unit increase of CCND1 mRNA expression had aHR = 2.16 and this remained statistically significant in multivariate analysis (p-value for interaction = 0.0010). In multivariate analysis, node-positive status, subglottic-transglottic localization, surgery other than total laryngectomy and mTOR/cyclin D1 mRNA interaction, were independent predictors of relapse, while node-positive status, and subglottic-transglottic localization were associated with higher risk of death. Conclusions In laryngeal cancer, an interaction of high mTOR and cyclin D1 mRNA expression is associated with poor patient outcome. Disclosure All authors have declared no conflicts of interest.

Impact of glucagon‐like peptide‐1 (7‐36) amide, isosteviol and 5‐aminoimidazole‐4‐carboxamide 1‐β‐d‐ribofuranoside on leucine‐mediated α‐cell dysfunction

To investigate the acute and chronic effects of l-leucine on pancreatic α-cell function in vitro. Furthermore, we wanted to explore if glucagon-like peptide-1 (GLP-1), isosteviol (ISV) and 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) counteract changes in α-cell function induced by chronic exposure to leucine. Isolated mice islets were incubated with 10 mM leucine for 2 or 72 h. We investigated glucagon and insulin secretion at 2 mM and 16.7 mM glucose. In addition, we cultured clonal α-TC1-6 cells with 5 mM leucine, 5 mM leucine plus GLP-1 (10(-6) M), or ISV (10(-6) M) or AICAR (10(-5) M) at high glucose for 72 h. We measured the glucagon secretion, cholesterol (CHO) and triglyceride (TG) content, cell proliferation as well as gene expression. Ten millimolar of leucine for 2 h significantly stimulated glucagon and insulin secretion both at 2 and 16.7 mM glucose in mice islets. After 72 h incubation with 10 mM leucine the glucagon secretion was enhanced at both 2 and 16.7 mM glucose, whereas the glucose-stimulated insulin secretion (16.7 mM glucose) was inhibited. Chronic exposure to 5 mM leucine increased glucagon secretion, CHO and TG content, cell proliferation and Pcsk2 (p < 0.001), MafB (p < 0.05), Gcg (p < 0.001), Prkaa1 (p < 0.01), Hmgcr (p < 0.001), Srebf2 (p < 0.001), Acaca (p < 0.001), Mtor (p < 0.05) mRNA expression in clonal α-TC1-6 cells. While GLP-1 was cable of reducing glucagon hypersecretion and Pcsk2 (p < 0.05) mRNA expression. ISV and AICAR had no effect on leucine-induced glucagon hypersecretion. Long-term exposure to leucine induces hypersecretion of glucagon secretion, that is, aminoacidotoxicity and influences some key genes of pancreatic α-cells. Interestingly, GLP-1 counteracts the leucine-induced α-cell dysfunction.

Rapamycin Inhibits the mTOR/p70S6K Pathway and Attenuates Cardiac Fibrosis in Adriamycin-induced Dilated Cardiomyopathy

The objective of the present study was to investigate whether mTOR is involved in cardiac fibrosis evident in dilated cardiomyopathy, and whether rapamycin provides therapeutic potential for cardiac fibrosis. Forty-five rats were divided into three groups. Fifteen rats in the Adriamycin group underwent 8 weeks of Adriamycin treatment (2.5 mg/kg, twice per week; i.v.) to induce cardiac fibrosis and dilated cardiomyopathy. Fifteen rats in the rapamycin group received rapamycin (2 mg/kg, per day, orally) and i.v. Adriamycin simultaneously for 8 weeks. Fifteen untreated rats served as controls. Cardiac morphology and function were quantified using echocardiography. mTOR and p70S6K1 mRNA expression were assessed using reverse transcription-PCR. Collagen volume fraction (CVF) was significantly elevated in the adriamycin group (3.36 ± 0.75) compared with controls (1.51 ± 0.31), whereas mTOR and p70S6K mRNA expression were significantly increased in the adriamycin group (0.68 ± 0.03 and 0.69 ± 0.03) compared with controls (0.38 ± 0.03 and 0.34 ± 0.02). The Adriamycin group was associated with cardiac dilation and decreased contractile function. The rapamycin group showed significantly decreased CVF (1.87 ± 0.45), accompanied with a significant decrease in mTOR and p70S6K mRNA expression (0.42 ± 0.05 and 0.45 ± 0.04) relative to the Adriamycin group. In addition, treatment with rapamycin recovered impairments in cardiac morphology and function. The mTOR/p70S6K pathway plays an important role in adriamycin-induced cardiac fibrosis resulting from dilated cardiomyopathy. Rapamycin is a potential therapeutic treatment that can be used to attenuate cardiac fibrosis and improve cardiac function.

Effects of 2 weeks lower limb immobilization and two separate rehabilitation regimens on gastrocnemius muscle protein turnover signaling and normalization genes

BackgroundLimb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Whereas the knowledge of muscle mass signaling with immobilization has increased in recent years, the molecular regulation in the rehabilitation of immobilization-induced muscle atrophy is only sparsely studied. To investigate the phosphorylation and expression of candidate key molecular muscle mass regulators after immobilization and subsequent rehabilitation we performed two separate studies.MethodsWe immobilized the lower limb for 2 weeks followed by the in-house hospital standard physiotherapy rehabilitation (Study 1). Secondly, we conducted an intervention study using the same 2 weeks immobilization protocol during which protein/carbohydrate supplementation was given. This was followed by 6 weeks of rehabilitation in the form of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1, GSK3β, ubiquitin and MURF1 and mRNA expression of Atrogin-1, MURF1, FOXO1, 3 and 4 as well as appropriate housekeeping genes.ResultsIn both studies, no changes in protein expression or phosphorylation for any measured protein were observed. In Study 1, FOXO3 and FOXO4 mRNA expression decreased after IMMO and REHAB compared to PRE, whereas other mRNAs remained unchanged. Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies.ConclusionsIn neither study, the changes in muscle mass associated with immobilization and rehabilitation were accompanied by expected changes in expression of atrophy-related genes or phosphorylation along the Akt axis. Unexpectedly, we observed significant changes in several of the so-called housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies, thereby questioning the usefulness of these genes for normalization of RNA data purposes in muscle immobilization studies.

Targeted Inhibition of Mammalian Target of Rapamycin (mTOR) Signaling Pathway Inhibits Proliferation and Induces Apoptosis of Laryngeal Carcinoma Cells in vitro

Laryngeal carcinoma is one of the most aggressive cancers of the head and neck region. The survival rate of patients with laryngeal carcinoma is low due to its late metastases and resistance to chemotherapy and radiotherapy. It was reported that mTOR was involved in the growth and apoptosis of various cancer cells. The aim of this study was to detect the effects of mTOR inhibition by mTOR shRNA on the proliferation, apoptosis and invasive ability of Hep-2 human laryngeal carcinoma cells in vitro. mTOR shRNA was designed and transfected into Hep-2 human laryngeal carcinoma cells. Untreated cells and cells treated with control vector (non-targeted shRNA) were used as control. The proliferation and apoptosis of Hep-2 cells were detected by MTT and flow cytometry. A transwell assay was used to measure the invasive ability of Hep-2. The inhibition effects on the mTOR signaling pathway by mTOR shRNA were studied using RT-PCR and Western blot. Our results showed that the mRNA and protein expression of mTOR and Akt were high in laryngeal carcinoma cells and could be inhibited by mTOR shRNA. At the same time, low expression of PTEN mRNA and protein was observed in Hep-2 cells. The expression increased when the cells were transfected with mTOR shRNA. This showed that mTOR shRNA could inhibit the proliferation and invasive ability of Hep-2 cells. It also could induce the apoptosis of Hep-2 cells in vitro. The mTOR signaling pathway plays an important role in the development of laryngeal carcinoma. The mTOR shRNA we designed in this experiment effectively inhibited the mTOR signaling pathway. It inhibited the proliferation and invasive ability of the studied laryngeal carcinoma cells and induced their apoptosis in vitro. mTOR might therefore be a useful target in the therapy of laryngeal carcinoma.

Supplementation with BCAA Reduces MAFbx Expression and Phenylalanine Concentration in Rested and Exercised Human Muscle

Resistance exercise and amino acids are each capable of stimulating skeletal muscle protein synthesis through the mammalian target of rapamycin (mTOR) pathway. When these two are combined the effect is additive and a positive net muscle protein balance can be achieved. Resistance exercise also elevates muscle protein breakdown, suggested to revolve around the genes MuRF-1 and MAFbx, which are molecularly linked to the mTOR pathway. PURPOSE: To determine the individual and combined effect of resistance exercise and branched-chain amino acids on the expression of anabolic and catabolic genes in human skeletal muscle. METHODS: Seven healthy male subjects went through two sessions of unilateral leg exercise and were randomly supplemented with BCAA or flavored water. Muscle biopsies were collected before and 3h after exercise in both resting (R) and exercising (E) muscle for determination of mRNA expression of proteolytic and mTOR associated genes. Fold changes in mRNA expression at 3h compared to pre-exercise were compared with a two-way ANOVA. Blood and muscle samples were taken for analysis of amino acid concentration. RESULTS: Supplementation generated a decreased (P < 0.05) MAFbx expression. Fold changes from pre-exercise were 0.5 (E) and 0.7 (R) in the BCAA session compared to 1.4 (E) and 1.5-fold (R) in the placebo session. MuRF-1 expression increased (P < 0.05) 2 and 3-fold in (E) in the placebo and BCAA session respectively, in relation to the unaltered expression in (R). Plasma and muscle BCAA concentration were elevated (P < 0.05) during the recovery period. Muscle phenylalanine concentrations decreased 19% (E) and 14% (R) (P < 0.05) 3h following exercise in the placebo session, with greater reductions (P < 0.05) 44% (E) and 37% (R) after BCAA supplementation. CONCLUSIONS: There is a divergent response to BCAA supplementation and exercise on the expression of the proteolytic genes MAFbx and MuRF-1. This may be due to their diverse cellular function and suggests a somewhat dissimilar control mechanism. The decrease in MAFbx expression together with a more pronounced reduction in muscle phenylalanine concentration during recovery with BCAA supplementation strongly suggests an increased net muscle protein balance. Supported by Swedish National Center for Research in Sports

MTORC1 and mTORC2 Regulate EMT, Motility, and Metastasis of Colorectal Cancer via RhoA and Rac1 Signaling Pathways

Activation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mTOR kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners toward regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor, and Rictor mRNA was noted with advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor, and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastases compared with normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition (MET) and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact and decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating epithelial-mesenchymal transition (EMT), motility, and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.

Antiproliferation of cardamonin associated with mRNA expression of mTOR, Raptor and Rictor

To investigate the antiproliferation effect of cardamonin (CAR) and its possible mechanisms on human umbilical artery smooth muscle cells (HUASMCs) cultured in the mimicking insulin resistance (IR) medium. Proliferation of HUASMCs was assayed by MTT method. The mRNA expression of mTOR, Raptor and Rictor was detected by a real-time PCR. The expression content was calculated by Livak method using internal control of beta-actin. The proliferation of HUASMCs cultured in the mimicking IR medium was significantly increased. Both in normal and mimic IR culture medium, cells proliferation was inhibited by CAR (1 x 10(-5), 1 x 10(-4) mol x L(-1)). Pretreated with PD98059 and LY294002, cell proliferation induced by phosphatidic acid (PA) was inhibited, and the mRNA expression of mTOR, Raptor and Rictor was significantly decreased by CAR in the mimic IR medium. It is implicated that antiproliferation of CAR is involved in mRNA expression decrease of mTOR and its relative protein Raptor and Rictor.

Antiproliferation of cardamonin is involved in mTOR on aortic smooth muscle cells in high fructose-induced insulin resistance rats

The objective of this study was to determine possible effects and potential mechanisms of cardamonin on improving insulin resistance and vascular proliferative lesions in the rat's model system. Fed with 60% fructose-enriched diet for 12 weeks, male Sprague–Dawley (SD) rats developed insulin resistance and hyperinsulinemia. They also showed excessive proliferation of the vascular smooth muscle cells (VSMCs) and activation of the mammalian target of rapamycin (mTOR)/translation control proteins p70 ribosomal S6 kinase (P70S6K1)/eukaryotic initiation factor 4E binding protein 1 (4E-BP1) signaling in the rat thoracic aorta. From weeks 9–12, cardamonin was injected into the peritoneal cavity once daily. Under the detection of microscopy and electron microscopy, cardamonin improved hyperinsulinemia and inhibited proliferation of VSMCs in the thoracic aorta of rats in a dose-dependent manner. By the Real-Time RT-PCR, mRNA expression of mTOR, P70S6K1 and 4E-BP1 was significantly reduced in cardamonin treated rats. Similarly, protein over-expression of mTOR and p-P70S6K1 was obviously inhibited by immunohistochemical analyses. These findings suggest that cardamonin may play a role in ameliorating insulin resistance and smooth muscle hyperplasia of major vessels in fructose-induced rats, possibly via a mechanism that involves the modulation of insulin/mTOR signaling.

Prognostic significance of PI3K mRNA expression in patients with operable high-risk breast cancer

577 Background: The aim of the study was to examine the prognostic and predictive significance of PI3K, AKT-1, mTOR, and PTEN mRNA expression and PIK3CA mutations in patients with operable high-risk breast cancer. Methods: Formalin-fixed paraffin-embedded tumor tissue blocks were available from 279 out of 595 operable high-risk breast cancer patients enrolled in a phase III clinical trial of E-T-CMF vs E-CMF. Updated clinical data were retrospectively used in the present translational research study. RNA was isolated followed by kinetic one-step quantitative RT-PCR for assessment of the expression of PI3K, AKT-1, mTOR, and PTEN. Mutations (E542K, E545K and H1047R) in the helical (exon 9) and the kinase (exon 20) domains of the PIK3CA gene encoding the catalytic p110 subunit of PI3K were investigated with custom Taqman SNP genotyping assays in 223 tumor DNA samples from the patient population enrolled in this study. Results: The two groups were well balanced concerning their basic characteristics except for grade (p = 0.015). The vast majority of hormone receptor positive patients (92%) received tamoxifen for 5 years, with LHRH analogs for premenopausal patients. The distribution of AKT-1 values was bimodal and therefore the 25th percentile was a natural cut-off. The median was used as a cut-off for all other genes. Mutations of the PIK3CA gene were detected in 51 patients (23%) (E542K in 8, E545K in 17 and H1047R in 26 patients). After a median follow-up time of 97 months, 93 patients (33%) had relapsed and 65 (23%) had died. High PI3K mRNA expression was associated with significantly shorter DFS (p = 0.044), irrespectively of the treatment arm. Multivariate analysis showed that high PI3K mRNA expression was an adverse prognostic factor for OS (HR = 2.14, 95% CI = 1.16–3.94, p = 0.015) and DFS (HR = 1.71, 95% CI = 1.09–2.68, p = 0.02), while PIK3CA mutations negatively affected OS (HR = 2.05, 95% CI = 1.07–3.95, p = 0.031). None of the other genes assessed was found to be of prognostic significance. Conclusions: High PI3K mRNA expression and PIK3CA mutations appear to be of adverse prognostic significance in patients with high-risk operable breast cancer. No significant financial relationships to disclose.

Expression of genes related to muscle plasticity after strength and power training regimens

The purpose of our study was to compare the effects of 8-week progressive strength and power training regimens on strength gains and muscle plasticity [muscle fiber hypertrophy and phenotype shift, mammalian target of rapamycin (mTOR), regulatory-associated protein of mTOR (RAPTOR), rapamycin-insensitive companion of m-TOR (RICTOR), calcineurin and calcipressin gene expression]. Twenty-nine physically active subjects were divided into three groups: strength training (ST), power training (PT) and control (C). Squat 1 RM and muscle biopsies were obtained before and after the training period. Strength increased similarly for both ST and PT groups (P<0.001). Fiber types I, IIa and IIb presented hypertrophy main time effect (P<0.05). Only type IIb percentage decreased from pre- to post-test (main time effect, P<0.05). mTOR and RICTOR mRNA expression increased similarly from pre- to post-test (P<0.01). RAPTOR increased after training for both groups (P<0.0001), but to a greater extent in the ST (P<0.001) than in the PT group. 4EBP-1 decreased after training when the ST and PT groups were pooled (P<0.05). Calcineurin levels did not change after training, while calcipressin increased similarly from pre- to post-test (P<0.01). In conclusion, our data indicate that these training regimens produce similar performance improvements; however, there was a trend toward greater hypertrophy-related gene expression and muscle fiber hypertrophy in the ST group.

The expression of mammalian target of rapamycin and its substrates in autogenous vein graft in rats

To investigate the expression of mammalian target of rapamycin (mTOR) and its substrates including p70s6k and 4E-BP1 in autogenous vein graft. Autogenous vein graft model was established in 64 Wistar rats by transplanting the right common jugular vein to infrarenal abdominal aorta. Vein graft samples were harvested 6 hours, 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks after surgery. The mRNA expression of mTOR, p70s6k and 4E-BP1 were measured by RT-PCR and in situ hybridization. Western blot and immunohistochemistry methods also were used to detect the protein expression of mTOR, p70s6k and 4E-BP1. Proliferating cell nuclear antigen (PCNA) was also detected at the same time. The mRNA expression of mTOR and p70s6k increased soon after vein graft transplanting, rose quickly and reached the peak 3 days to 2 weeks after surgery, which recovered 6 to 8 weeks after surgery. The expression of 4E-BP1 mRNA decreased soon after surgery and reached the lowest at 1 week, then rose to the peak 4 to 6 weeks after transplantation. Protein expression of mTOR and p70s6k reached the peak 2 to 4 weeks and recovered to normal level 8 weeks after surgery, but the expression of 4E-BP1 decreased to the lowest during 1 to 2 weeks and reached the peak 4 to 6 weeks after transplanting. The positive cells mostly located in vascular smooth muscle cell (VSMC) just like PCNA. The expression of mTOR and its substrates were activated in vein graft soon after transplantation, which means that mTOR and its substrates might become new targets for the prevention and therapy of stenosis or obliteration after vein graft transplanting.