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Various genetic and epigenetic mechanisms have been suggested to play roles as the underlying pathophysiology of Multiple Sclerosis (MS). Changes in different parts of the mTOR signaling pathway are among the potential suggested mechanisms based on the specific roles of this pathway in CNS. MTOR, RPS6KB1, and EIFEBP1 genes are among important genes in the mTOR pathway, responsible for the proper function of acting proteins in this signaling pathway. This study aimed to investigate the relative expression levels of these genes in the blood samples of relapsing-remitting MS (RRMS) patients compared to healthy controls. In this case-control study blood samples were collected from 30 newly diagnosed RRMS patients and 30 age and sex-matched healthy controls. mRNA level of MTOR, RPS6KB1, and EIFEBP1 genes were assessed using Real-Time PCR. The expression of MTOR, RPS6KB1, and EIF4EBP1 genes was up regulated in MS patients compared to healthy controls (p < 0.001 for all mentioned genes). Considering gender differences, expression of the mentioned genes was increased among female patients (all P < 0.001). However, no statistically significant changes were observed among male patients. Based on the receiver operating characteristic, MTOR gene had the highest diagnostic value followed by EIF4EBP1 and RPS6KB1 genes in differentiating RRMS patients from controls. In conclusion, we found the simultaneous upregulation of MTOR, RPS6KB1, and EIF4EBP1 genes among RRMS patients. MTOR showed to have the highest diagnostic value compared to other 2 genes in differentiating RRMS patients. Further studies evaluating the importance of these findings from pharmacological and prognostic perspectives are necessary.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3'-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.

Radiation therapy (XRT) is a standard treatment for prostate cancer (PCa). Although dose escalation increases local control, toxicity hampers further escalation. Broader improvement will be possible by the addition of adjuvant therapies, which can synergize with radiation and thus improve efficacy. We have identified a natural compound (Nexrutine, Nx) that inhibits the survival and growth of PCa cells in combination with radiation. Combination studies demonstrated strong interaction between Nx and radiation both in vitro in multiple PCa cell lines and in the Transgenic adenocarcinoma of mouse prostate (TRAMP) model. Nx potentiated growth inhibitory effects of IR by down regulating ribosomal protein S6K (RPS6KB1), CyclinD1, Chk1 and HIF-1 α and prolonging G2/M checkpoint block. RPS6KB1 is upregulated in prostate cancers and its expression is correlated with tumor grade. Knockdown of RPS6KB1 in PCa cells increased their sensitivity toward radiation-induced survival inhibition. Overall, we provide scientific evidence (i) in support of Nx as an adjuvant in PCa patients receiving XRT (ii) suggesting that RPS6KB1 is an important player in Nx-mediated combinatorial benefits and emphasizes that RPS6KB1 is a novel target for PCa treatment. These data underscore the need to test the agent in additional preclinical models to validate these observations., (Published by Elsevier B.V.)

Background: This study aimed to investigate the interactions between silent information regulator 1 (SIRT1) and mammalian target of rapamycin (mTOR) in intraplaque angiogenesis and their potential mechanisms through in vivo and in vitro studies., Methods: An atherosclerosis model was established in 12 rabbits on a high-cholesterol diet. The rabbits were equally divided into 3 groups: a control group (high-lipid diet), RAP group (high-lipid diet supplemented with rapamycin) and RAP + NAM group (high-lipid diet supplemented with rapamycin and nicotinamide). At the end of 4 weeks, the area of plaques in the aorta was determined and the protein expression of CD31 and vascular endothelial growth factor (VEGF) was detected through hematoxylin and eosin staining and immunohistochemical staining, respectively. For in vitro study, a hypoxia model was established in human umbilical vein endothelial cells (HUVECs) by using the chemical method (CoCl 2 ). The MTT assay, scratch assay and tube formation assay were performed to evaluate the proliferation and angiogenesis abilities of HUVECs. Reverse transcription polymerase chain reaction was used to examine the mRNA levels of SIRT1, hypoxia-inducible factor-1α (HIF-1α), mTOR and p70 ribosomal S6 kinase (p70S6K). Western blotting was used to examine the protein levels of SIRT1, HIF-1α, mTOR, p-mTOR, p-raptor and p-p70S6K., Results: The results of the in vivo study indicated a significant inhibitory effect of rapamycin on plaque size and intraplaque angiogenesis (0.05 ± 0.02mm 2 versus 5.44 ± 0.50mm 2 , P < 0.05). This effect was attenuated by nicotinamide (0.76 ± 0.15mm 2 versus 0.05 ± 0.02mm 2 , P < 0.05). Compared with the RAP group, CD31- and VEGF-positive vessels were abundant in the RAP + NAM group. The RAP group showed lower expression of p-mTOR, p-p70S6K and HIF-1α than did the control group (P < 0.05), whereas the RAP + NAM group showed slightly higher expression of these factors than did the RAP group (P < 0.05). Furthermore, in vitro studies revealed that the inhibitory effect of rapamycin on the angiogenic ability of HUVECs and its significant inhibitory effects on the protein level of HIF-1α and the phosphorylation of proteins involved in the mTORC1 pathway, including mTOR, raptor and p70S6K (P < 0.05), were enhanced by cotreatment with SRT1720 and rapamycin (P < 0.05). In contrast to mTOR and SIRT1, the mRNA levels of p70S6K and HIF-1α were reduced by rapamycin (P < 0.05) and further reduced by cotreatment with SRT1720 and rapamycin., Conclusions: The study results indicate that SIRT1 might negatively regulate atherosclerotic angiogenesis via mTORC1 and HIF-1α signaling pathway and cointervention of SIRT1 and mTOR may serve as a crucial therapeutic strategy in cardiovascular medicine., (Copyright © 2018. Published by Elsevier Inc.)

A low-protein diet (LPD) protects against the progression of renal injury in patients with chronic kidney disease (CKD). However, LPD may accelerate muscle wasting in these patients. Both exercise and branched-chain amino acids (BCAA) are known to increase muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) pathway. The aim of this study was to investigate whether endurance exercise and BCAA play a role for increasing muscle protein synthesis in LPD-fed CKD (5/6 nephrectomized) rats. Both CKD and sham rats were pair-fed on LPD or LPD fortified with a BCAA diet (BD), and approximately one-half of the animals in each group was subjected to treadmill exercise (15 m/min, 1 h/day, 5 days/wk). After 7 wk, renal function was measured, and soleus muscles were collected to evaluate muscle protein synthesis. Renal function did not differ between LPD- and BD-fed CKD rats, and the treadmill exercise did not accelerate renal damage in either group. The treadmill exercise slightly increased the phosphorylation of p70s6 kinase, a marker of mTOR activity, in the soleus muscle of LPD-fed CKD rats compared with the sham group. Furthermore, BCAA supplementation of the LPD-fed, exercise-trained CKD rats restored the phosphorylation of p70s6 kinase to the same level observed in the sham group; however, the corresponding induced increase in muscle protein synthesis and muscle mass was marginal. These results indicate that the combination of treadmill exercise and BCAA stimulates cell signaling to promote muscle protein synthesis; however, the implications of this effect for muscle growth remain to be clarified., (Copyright © 2017 the American Physiological Society.)

The aim of the study was to examine the expression of mammalian target of rapamycin (mTOR)/70S6K signaling pathway in pathological scar fibroblasts and the effects of resveratrol (Res) intervention. The mTOR and 70S6K in pathological scar and normal skin fibroblasts were detected by immunofluorescence following treatment with different concentrations of Res. RT-PCR and western blot analysis were used to detect the expression of mTOR and 70S6K mRNA and protein, respectively. Immunofluorescence showed that the expression of 70S6K and mTOR was significantly enhanced in pathological scar fibroblasts, and mainly expressed in the nucleus, but not in normal skin fibroblasts. RT-PCR and western blot analysis showed that after different concentrations of Res treatments, the mTOR and 70S6K mRNA and protein expression significantly (P<0.05) decreased in a dose‑dependent manner. In conclusion, the expression of mTOR/70S6K signaling pathway in pathological scar fibroblasts was significantly enhanced. Res can downregulate the expression of mTOR and 70S6K to achieve the inhibition of pathological scar fibroblast proliferation.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor prognosis in China. Chemotherapy now is one of the most frequently used treatments for patients with ESCC in middle or late stage, however the effects were often limited by increased chemoresistance or treatment toxicity. So it is urgent to find new drugs to treat ESCC patients. Metformin with low cost and toxicity has proved to have anti-cancer effects in a numerous cancers, while its role and mechanism in ESCC has seldom been studied. In the present study, we found that metformin exhibited not only an anti-proliferation ability in a dose and time dependent manner but also a proapoptosis effect in a dose dependent manner in ESCC cell line KYSE450. Our in vivo experiment also showed that metformin markedly inhibited KYSE450 xenograft tumors growth compared to those treated with normal saline. What's more, no obvious toxic reactions were observed. To further explore the underlying mechanism, we found that metformin treatment could significantly damp the expression of 4EBP1 and S6K1 in KYSE 450 cells in vitro and in vivo, furthermore, the p-4EBP1 and p-S6K1 expression in KYSE 450 cells were also inhibited greatly in vitro and in vivo. During the therapy of cancer, in order to overcome side effects, combination therapy was often used. In this paper, we demonstrated that metformin potentiated the effects of cisplatin via inhibiting cell proliferation and promoting cell apoptosis. Taken together, metformin owned the potential anti-cancer effect on ESCC in monotherapy or was combined with cisplatin and these results laid solid basis for the use of metformin in ESCC.

This study was conducted to examine the effects of dietary valine on the physical and flavor characteristics, fatty acid (FA) profile, antioxidant status and Nrf2-dependent antioxidant enzyme gene expression in the muscle of young grass carp (Ctenopharyngodon idella) fed increasing levels of valine (4.3, 8.0, 10.6, 13.1, 16.9 and 19.1 g/kg) for 8 weeks. Compared with the control group, the group fed valine showed improved physical characteristics of fish fillets (increased relative shear force, hydroxyproline, protein and lipid levels and decreased cathepsin B and L activities, as well as cooking loss, were observed). Moreover, valine improved the flavor of young grass carp fillets by increasing the amino acid (AA) concentration in fish muscle (increased aspartic acid, threonine, glutamine, cystine, methionine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and valine concentrations were observed). Additionally, optimal valine supplementation increased the potential health benefits to humans by decreasing the saturated FA (C15:0 and C16:0) concentration and increasing the unsaturated FA (monounsaturated FAs (MUFAs), such as C16:1, C18:1c+t and C20:1, and polyunsaturated FAs (PUFAs), such as C18:3n-3, C20:2 and C22:6) concentration. In addition, the reduced glutathione (GSH) content and the activities of Cu/Zn superoxide dismutase (SOD1), catalase (CAT) and Selenium-dependent glutathione peroxydase (Se-GPx) increased under valine supplementation (P < 0.05). Furthermore, the SOD1, CAT and Se-GPx mRNA levels increased with dietary valine levels, possibly due to the up-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the down-regulation of Kelch-like-ECH-associated protein 1 (Keap1) in muscle (P < 0.05). In conclusion, valine improved the physical and flavor characteristics, FA profile, and antioxidant status and regulated the expression of the antioxidant enzyme genes Nrf2, Keap1, TOR and S6K1 in fish fillets., Competing Interests: The authors have declared that no competing interests exist.

Activation of AMP-activated protein kinase (AMPK) suppressed mammalian target of rapamycin (mTOR) pathway, resulting in impaired cancer cell proliferation. Two cohorts (50 and 1072 cases) of patients with resected gastric adenocarcinoma were enrolled in the study. Immunohistochemical staining for p-AMPKa, p-ACC, p-mTOR, p-S6, and p-4EBP1 was performed on the 50-patient cohort. Tissue microarray blocks containing samples from 1072 patients of Chinese ethnicity were used for the immunohistochemical detection of p-AMPKa and p-S6 levels. p-AMPK and p-ACC were frequently inactivated in both cohorts of gastric cancer samples, while p-mTOR, p-S6, and p-4EBP1 were frequently activated in the small cohort of gastric cancer. However, only levels of p-AMPKa and p-S6 were associated with the overall survival of gastric cancer patients. In the larger 1072-patient cohort, downregulation of p-AMPKa and upregulation of p-S6 were associated with tumor progression and were independent predictors of survival after resection of primary gastric cancer. Therefore, reciprocal expression of p-AMPKa and p-S6 may be promising prognostic biomarkers in patients with gastric cancer.

Background: Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling., Materials and Methods: To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells., Results: AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation., Conclusion: The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1) plays a pivotal role in the regulation of apoptosis and has been shown to maintain antitumor and metastasis-suppressive properties. In the present study, we examined the effects of MARCKSL1 as a novel anti-angiogenic agent on the inhibition of angiogenesis-mediated cell migration. MARCKSL1 also reduced vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, as well as capillary-like tubular structure formation in vitro. MARCKSL1 disrupted phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) in ovarian tumorigenesis. In addition, MARCKSL1 showed potent anti-angiogenic activity and reduced the levels of VEGF and hypoxia-inducible factor 1α (HIF-1α) expression, an essential regulator of angiogenesis. Consistently, MARCKSL1 decreased VEGF‑induced phosphorylation of the PI3K/Akt signaling pathway components, including phosphoinositide-dependent protein kinase 1 (PDK-1), mammalian target of rapamycin (mTOR), tuberous sclerosis complex 2 (TSC-2), p70 ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase 3β (GSK-3β) protein. Collectively, our results provide evidence for the physiological/biological function of an endothelial cell system involved in angiogenesis through suppression of Akt/PDK-1/mTOR phosphorylation by interaction with VEGFR-2.

External loads applied to skeletal muscle cause increases in the protein translation rate, which leads to muscle hypertrophy. Although some studies have demonstrated that increases in the capacity and efficiency of translation are involved in this process, it remains unclear how these two factors are related to the magnitude of muscle hypertrophy. The present study aimed to clarify the roles played by the capacity and efficiency of translation in muscle hypertrophy. We used an improved synergist ablation in which the magnitude of compensatory hypertrophy could be controlled by partial removal of synergist muscles. Male rats were assigned to four groups in which the plantaris muscle was unilaterally subjected to weak (WK), moderate (MO), middle (MI), and strong (ST) overloading by four types of synergist ablation. Fourteen days after surgery, the weight of the plantaris muscle per body weight increased by 8%, 22%, 32% and 45%, in the WK, MO, MI and ST groups, respectively. Five days after surgery, 18+28S rRNA content (an indicator of translational capacity) increased with increasing overload, with increases of 1.8-fold (MO), 2.2-fold (MI), and 2.5-fold (ST), respectively, relative to non-overloaded muscle (NL) in the WK group. rRNA content showed a strong correlation with relative muscle weight measured 14 days after surgery (r = 0.98). The phosphorylated form of p70S6K (a positive regulator of translational efficiency) showed a marked increase in the MO group, but no further increase was observed with further increase in overload (increases of 22.6-fold (MO), 17.4-fold (MI), and 18.2-fold (ST), respectively, relative to NL in the WK group). These results indicate that increases in ribosome biogenesis at the early phase of overloading are strongly dependent on the amount of overloading, and may play an important role in increasing the translational capacity for further gain of muscular size.

Background: The aim of this study was to extensively analyze the signaling pathway molecules in breast cancer and to explore candidate biomarkers for clinicopathological relevance., Methods: We assessed the expression of key factors in cell signaling, namely p-AKT, cyclin D1, P27, p-p70S6 K, p-4EBP1, and p-MAPK/ERK, within 338 invasive breast cancer patients. These factors were immunohistochemically examined in tumor tissues and assessed by staining score. Staining scores were analyzed by a clustering method to devise a new classification based on pathway activity. We investigated the relationships among staining scores, the clustering classification, and patient characteristics., Results: The proportion of patients displaying high expression levels were as follows: p-AKT, 75%; cyclin D1, 12%; P27, 53%; p-p70S6 K, 37%; p-4EBP1, 19%; and p-MAPK/ERK, 3%. Patients were classified into two groups on the basis of staining scores. Group 1 (39%) included more positive cases for p-4EBP1, p-MAPK/ERK, and p-p70S6 K and fewer positive cases for P27 and cyclin D1 than Group 2 (61%). The clustering classification was significantly related to subgrouping by hormone receptor and HER2 (P < 0.001), nuclear grade (P < 0.001) and histological subtype (P = 0.034). A strong positive correlation was identified between p-AKT and P27, cyclin D1 and P27, p-p70S6 K and p-4EBP1, p-p70S6 K and p-MAPK/ERK, and between p-4EBP1 and p-MAPK/ERK. Levels of p-p70S6 K were significantly related to recurrence in both univariate (RR = 0.75, P < 0.001) and multivariate (RR = 0.71, P = 0.049) analyses., Conclusions: The present study helps us to understand the characteristics of signaling pathway status in breast cancers. Moreover, p-p70S6 K expression may be of use in predicting clinical outcome.

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is invariably used to treat cardiovascular diseases. Simvastatin has been recently demonstrated to have a neuroprotective effect in nervous system diseases. The present study aimed to further verify the neuroprotection and molecular mechanism of simvastatin on rats after spinal cord injury (SCI). The expression of Beclin-1 and LC3-B was evidently enhanced at postoperation days 3 and 5, respectively. However, the reduction of the mTOR protein and ribosomal protein S6 kinase p70 subtype (p70S6K) phosphorylation level occurred at the same time after SCI. Simvastatin significantly increased the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Meanwhile, immunofluorescence results indicated that the expression of chondroitin sulfate proteoglycan (CSPG) and caspase-3 protein was obviously reduced by simvastatin. Furthermore, Nissl staining and Basso, Beattie, and Bresnahan (BBB) scores showed that the quantity and function of motor neurons were visibly preserved by simvastatin after SCI. The findings of this study showed that simvastatin induced autophagy by inhibiting the mTOR signaling pathway and contributed to neuroprotection after SCI.

Objective: To explore the role of microRNA on the myocardial microvascular endothelial cells (CMECs) of ischemic heart rats in the process of angiogenesis and related regulation mechanism., Methods: Myocardial ischemic rats model was established by coronary ligation.Seven days after operation, the ischemic CMECs were cultured by the method of planting myocardium tissue and identified by immunocytochemistry to observe the biological characteristics of ischemic CMECs angiogenesis, to determine the window period of migration, proliferation, tube formation in the process of its angiogenesis. Dynamic expression changes of microRNA in the process of ischemic CMECs angiogenesis was detected using microRNA chip and further verified by real-time PCR, the core microRNA of the ischemic CMECs was defined and the predicted target genes of core microRNA were determined by bioinformatics methods and real-time PCR. At the same time, the protein expression of target gene and angiogenesis related genes of p38MAPK, PI3K,Akt,VEGF were measured by Western blot., Results: The CMECs of rats presented typical characteristics of microvascular endothelial cells, and factor VIII, CD31 related antigens were all positively stained by immunocytochemical analysis. The migration window period was on the first day, and the tube formation window period was on the second day of both control and ischemic groups, while the proliferation window period was on the third day for the normal group, and the sixth day for ischemic group. According to the expressional difference and their relationship with angiogenesis, miRNA-223-3p was ultimately determined as the core microRNA in the process of ischemic CMECs angiogenesis, real-time PCR verified this hypothesis. Bioinformatics methods predicted that Rps6kb1 is the target genes of miRNA-223-3p, the pathway analysis showed that Rps6kb1 could regulate angiogenesis via HIF-1α signal pathway. Moreover, the mRNA and protein expression of VEGF, p38MAPK, PI3K,Akt, which were the downstream molecules of Rps6kb1/HIF-1α signal pathway, were also significantly downregulated in ischemic CMECs from migration and proliferation stage., Conclusion: Our results show that the miRNA-223-3p is the core microRNA of ischemic CMECs angiogenesis. MiRNA-223-3p could regulate Rps6kb1/HIF-1α signal pathway, inhibit the process of migration and proliferation of ischemic CMECs angiogenesis. MiRNA-223-3p is thus likely to be a core target for enhancing angiogenesis of ischemic heart disease.

The extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway has been involved in the positive and negative regulation of cell proliferation. Upon mitogen stimulation, ERK1/ERK2 activation is necessary for G1- to S-phase progression whereas when hyperactived, this pathway could elicit cell cycle arrest. The mechanisms involved are not fully elucidated but a kinase-independent function of ERK1/2 has been evidenced in the MAPK-induced growth arrest. Here, we show that p70S6K, a central regulator of protein biosynthesis, is essential for the cell cycle arrest induced by overactivation of ERK1/2. Indeed, whereas MEK1 silencing inhibits cell cycle progression, we demonstrate that active mutant form of MEK1 or MEK2 triggers a G1 phase arrest by stimulating an activation of p70S6K by ERK1/2 kinases. Silencing of ERK1/2 activity by shRNA efficiently suppresses p70S6K phosphorylation on Thr421/Ser424 and S6 phosphorylation on Ser240/244 as well as p21 expression, but these effects can be partially reversed by the expression of kinase-dead mutant form of ERK1 or ERK2. In addition, we demonstrate that the kinase p70S6K modulates neither the p21 gene transcription nor the stability of the protein but enhances the translation of the p21 mRNA. In conclusion, our data emphasizes the importance of the translational regulation of p21 by the MEK1/2-ERK1/2-p70S6K pathway to negatively control the cell cycle progression., (© 2013 Wiley Periodicals, Inc.)

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

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.)

Osteosarcoma (OS) is the most common cancer that affects the bone and appears to be resistant to several chemotherapeutic drugs. The aim of the present study was to verify whether the combination of metformin and cisplatin has an effect on OS cell lines. OS cell lines U2OS, 143B and MG63 were treated with metformin, cisplatin or a combination of both drugs. Viability, apoptosis and cell cycle were evaluated to characterize the effects of the treatments. Western blot analyses were used to evaluate protein expression. All OS cell lines were found to be sensitive to metformin with different values of IC50, showing a slowdown of cell cycle associated or not with apoptosis. In particular, metformin was able to sensitize cells to cisplatin, to which all OS cell lines were resistant, demonstrating a synergistic effect in the combined treatment of the two drugs. The data obtained may have clinical relevance for novel therapeutic strategies for the treatment of OS; metformin inhibits tumor cell growth and amplifies the effect of cisplatin.

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules that regulate gene expression at post-transcriptional levels. Previous studies have shown that miR-145 is downregulated in human ovarian cancer; however, the roles of miR-145 in ovarian cancer growth and invasion have not been fully demonstrated. In the present study, Northern blot and qRT-PCR analysis indicate that miR-145 is downregulated in ovarian cancer tissues and cell lines, as well as in serum samples of ovarian cancer, compared to healthy ovarian tissues, cell lines and serum samples. Functional studies suggest that miR-145 overexpression leads to the inhibition of colony formation, cell proliferation, cell growth viability and invasion, and the induction of cell apoptosis. In accordance with the effect of miR-145 on cell growth, miR-145 suppresses tumor growth in vivo. MiR-145 is found to negatively regulate P70S6K1 and MUC1 protein levels by directly targeting their 3'UTRs. Importantly, the overexpression of p70S6K1 and MUC1 can restore the cell colony formation and invasion abilities that are reduced by miR-145, respectively. MiR-145 expression is increased after 5-aza-CdR treatment, and 5-aza-CdR treatment results in the same phenotype as the effect of miR-145 overexpression. Our study suggests that miR-145 modulates ovarian cancer growth and invasion by suppressing p70S6K1 and MUC1, functioning as a tumor suppressor. Moreover, our data imply that miR-145 has potential as a miRNA-based therapeutic target for ovarian cancer., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Siegesbeckia glabrescens (SG) Makino (Compositae) has been used as a traditional medicine for the treatment of a variety of diseases such as allergy, inflammation, acute hepatitis and hypertension. The primary aim of this study was to determine whether the ethanol extract of SG has antitumor activity against ovarian cancer and to identify molecular mechanisms and targets involved in the regulation of cell growth and progression. We demonstrate that SG treatment inhibits proliferation, adhesion, migration and invasion of SKOV-3 human ovarian cancer cells. The anti-proliferative effect of SG on SKOV-3 cells is accompanied by reduced expression of cyclin E and enhanced expression of the cyclin-dependent kinase inhibitor p27(Kip1), leading to inhibition of pRb phosphorylation. We also show that these antitumor activities are found to be mediated through suppression of FAK, ERK, Akt and p70(S6K)-dependent signaling pathways and downregulation of receptor tyrosine kinases such as EGFR, VEGFR-2 and FGFR-1 as well as the cell adhesion molecule N-cadherin. Taken together, our findings suggest further development and evaluation of SG for the treatment of ovarian cancer.

Background: 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., Methods: 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., Results: 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., Conclusion: 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., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: The gene amplification of ribosomal protein S6 kinase 1 and 2 (S6K1 and S6K2) and its clinical relevance in gastric cancer remain unclear., Materials and Methods: A comparative genomic hybridization analysis and DNA copy number assay were performed for nine cancer cell lines. The gene amplification of S6K1 and S6K2 were determined using a DNA copy number assay of 213 gastric cancer tissues., Results: S6K1 and S6K2 amplifications were observed in one and three cancer cell lines, respectively. No amplification of S6K1 was detected in the gastric cancer tissues, while S6K2 amplification was observed in 4.7% of the gastric carcinoma tissues. Patients with stage IV gastric cancer whose tumors exhibited amplification had a significantly shorter overall survival., Conclusion: S6K2 amplification was frequently observed in gastric cancer and was related to a poor prognosis. Our findings may provide novel insight into the dysregulation of mammalian target of rapamycin signaling by S6K2 amplification in gastric cancer.

RPS6KB1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) plays a key role in regulating protein translation. The role of RPS6KB1 in HCC (hepatocellular carcinoma) has not been fully investigated. This study was undertaken to determine the clinicopathological features and prognostic value of RPS6KB1 in HCC. We examined RPS6KB1 expression in 30 paired liver cancer tissues and adjacent noncancerous tissues by reverse transcription real-time PCR and Western blotting. In addition, we analyzed RPS6KB1 expression in 87 HCC samples by immunohistochemistry. The expression of RPS6KB1 was significantly increased in cancer tissues. Clinicopathological analysis showed that the expression of RPS6KB1 was significantly correlated with tumor size, histopathologic classifications, and serum alpha-fetoprotein (AFP). Kaplan-Meier survival curves revealed that increased expression of RPS6KB1 correlated with poor prognosis of HCC patients. RPS6KB1 expression was an independent prognostic marker for overall survival of HCC patients by multivariate analysis. Our data suggest that RPS6KB1 may play an important role in the progression of HCC and could serve as a potential molecular target for HCC therapy.

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The S6K1 and S6K2 kinases are considered important mTOR signaling effectors, yet their contribution to tumorigenesis remains unclear. Aberrant mTOR activation is a frequent event in cancer that commonly results from heterozygous loss of PTEN. Here, we show for the first time a differential protein expression between S6K1 and S6K2 in both mouse and human tissues. Additionally, the inactivation of S6k1 in the context of Pten heterozygosity (Pten(+/-)) suggests a differential requirement for this protein across multiple tissues. This tissue specificity appears to be governed by the relative protein expression of S6k2. Accordingly, we find that deletion of S6k1 markedly impairs Pten(+/-) mediated adrenal tumorigenesis, specifically due to low expression of S6k2. Concomitant observation of low S6K2 levels in the human adrenal gland supports the development of S6K1 inhibitors for treatment of PTEN loss-driven pheochromocytoma., (©2011 AACR)

Context: The rapidly increasing prevalence of gestational diabetes mellitus (GDM) globally places a growing population at risk for developing type 2 diabetes mellitus (T2DM), particularly those with persistent impaired glucose tolerance (IGT) postpartum., Objective: We sought to 1) identify dynamic insulin signaling abnormalities in vivo in a prospective, longitudinal study of GDM women compared to weight-matched pregnant controls both antepartum and postpartum; and 2) determine abnormalities that might distinguish GDM women who normalize their glucose tolerance postpartum from those with persistent IGT., Design: Skeletal muscle biopsies were obtained before and after a 75-g glucose load in nine overweight to obese GDM women and 10 weight-matched pregnant controls antepartum and postpartum. Postpartum biopsies were collected in five weight-matched GDM women with IGT (GDM/IGT)., Results: GDM women had decreased skeletal muscle insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine activation and reduced IRS1, concomitant with increased basal IRS1 serine phosphorylation and basal p70 S6-kinase (S6K1) activation, which resolved postpartum. However, GDM/IGT subjects had a persistent impairment in IRS1 activation and increased S6K1 phosphorylation compared to GDM subjects with normal glucose tolerance., Conclusions: This study reveals that women with GDM demonstrate impaired IRS1 signaling associated with increased S6K1 activation in skeletal muscle in vivo. This defect is maintained postpartum in GDM/IGT subjects, despite similar body weights and cytokine levels. Given that GDM women with persistent IGT are at a high risk of developing T2DM, understanding how the nutrient-sensitive mammalian target of rapamycin/S6K1 pathway is chronically activated in GDM may lead to important therapies that could prevent the progression to T2DM.

The protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 protein kinase (p70S6K) signaling pathway, as a central controller of cell growth, proliferation, survival, and differentiation in response to extracellular signals, growth factors, nutrient availability, energy status of the cell, and stress, has recently gained attention in neuroscience. The effects of this signaling pathway on repair of spinal cord injury (SCI), however, have not been well elucidated. ATP is increasingly recognized as an important regulator of signal transduction pathways, and plays important roles in functional recovery after nervous system injury. In the present study, we examined the ATP-induced changes of the Akt/mTOR/p70S6K signaling pathway in injured spinal cord of adult rats and potential therapeutic effects of this pathway on SCI-induced locomotor dysfunction. SCI was produced by extradural weight-drop using modified Allen's stall with damage energy of 50 g-cm force. The rats were divided into four groups: SCI plus ATP, SCI plus saline, SCI plus ATP and rapamycin, and sham-operated. Using immunostaining studies, Western blot analyses and real-time qualitative RT-PCR analyses, we demonstrated that the Akt/mTOR/p70S6K signaling pathway is present in the injured spinal cord and the expression of its components at the protein and mRNA levels is significantly elevated by exogenous administration of ATP following SCI. We observed the effectiveness of the activated Akt/mTOR/p70S6K signaling pathway in improving locomotor recovery, significantly increasing the expression of nestin, neuronal nuclei (NeuN), neuron specific enolase (NSE), and neurofilament 200 (NF200), and relatively inhibiting excessive reactive astrogliosis after SCI in a rapamycin-sensitive manner. We concluded that ATP injection produced a significant activation of the Akt/mTOR/p70S6K signaling pathway in the injured spinal cord and that enhancement of rapamycin-sensitive signaling produces beneficial effects on SCI-induced motor function defects and repair potential. We suggest that modulation of this protein kinase signaling pathway activity should be considered as a potential therapeutic strategy for SCI., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mammalian target of rapamycin (mTOR) controls protein translation and has an important role in the mechanism of pain hypersensitivity under persistent pain conditions. However, its expression and localization in pain-related regions of the nervous system is not completely understood. Here, we examined the expression and distribution of mTOR, eukaryotic initiation factor 4E-binding protein1/2 (4E-BP1/2), p70 ribosomal S6 protein kinase (p70S6K), and their phosphorylated (active) counterparts in two major pain-related regions, the dorsal root ganglion (DRG) and spinal cord dorsal horn. Reverse transcriptase-polymerase chain reaction showed that mTOR, 4E-BP1, and p70S6K mRNA are expressed in the DRG and dorsal horn. Western blot analysis further confirmed the existence of their protein products in these two regions, but expression of their phosphorylated counterparts was very low in dorsal horn and was not detected in the DRG. Immunohistochemistry revealed mTOR and p70S6K in the DRG neurons. Quantitative analysis showed that approximately 26.1% (+/- 3.2%) of DRG neurons were positive for mTOR and 19.1% (+/- 1.9%) were positive for p70S6K. Most of these neurons were small-less than 600 microm(2) in cross-sectional area-and some co-labeled with substance P or isolectin B4. Surprisingly, 4E-BP1 was observed only in the DRG satellite glial cells. In the dorsal horn, mTOR, p70S6K, and 4E-BP1 were detected in neurons, but not in astrocytes or microglia. They were distributed in the whole dorsal horn, especially in the superficial dorsal horn. Immunostaining for their phosphorylated counterparts was very low or undetectable in DRG and dorsal horn. Behavioral study showed that intrathecal mTOR inhibitor, rapamycin, did not affect acute nocicepetive transmission. The results indicate that although mTOR, p70S6K, and 4E-BP1 are highly expressed in the DRG and dorsal horn, their activate forms are very low in both regions under normal conditions. Our findings support the view that mTOR and its downstream effectors do not play a key role in acute pain., (Copyright 2010 Elsevier B.V. All rights reserved.)

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser(473) induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr(389) in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN.

The molecular mechanisms by which resistance exercise enlarges muscle mass, particularly the mass of fast-twitch type II fibers, are likely to involve enhanced phosphorylation/activation of key enzymes regulating protein synthesis. The hypothesis is that resistance exercise influences the phosphorylation of such key signaling proteins to a greater extent in type II than in type I fibers. Six recreationally active male subjects performed four sets of six maximal lengthening contractions with one leg. Muscle biopsies were taken from the vastus lateralis before and immediately after exercise and following 1 and 2 h of recovery. Samples were freeze-dried, and individual muscle fibers were dissected out and identified as type I or type II after staining for myosin ATPase. Phosphorylation of p70(S6k) on Thr(389) and S6 in type II fibers was increased three-to fourfold and six- to ninefold (P < 0.05), respectively, 1 and 2 h after exercise, whereas phosphorylation in type I fibers remained unchanged. Phosphorylation of Akt, mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) was unaltered in both fiber types, whereas that of eukaryotic elongation factor 2 (eEF2) was attenuated 20-45% (P < 0.05) in type II fibers during recovery. Phosphorylation of ERK1/2 was elevated six- to sevenfold (P < 0.05) immediately after exercise, and p38 MAPK phosphorylation was increased three- to fourfold (P < 0.05) for as long as 1 h after exercise in both types of fibers, although the level was markedly higher in type II fibers (P < 0.05). In conclusion, the elevation of p70(S6k) and the reduction of eEF2 phosphorylation in the type II fibers following resistance exercise suggest stimulation of protein synthesis, which may contribute to a more pronounced enlargement of these fibers. Our findings also suggest that p70(S6k) is activated, at least in part, via pathways not involving Akt-mTOR and MAPK.

Ischemic postconditioning (IPoC) reduces infarct size following ischemia/reperfusion. Whether or not phosphorylation of RISK (reperfusion injury salvage kinases) (AKT, ERK1/2, P70S6K, GSK3beta) is causal for protection by IPoC is controversial. We therefore studied the impact of RISK on IPoC in anesthetized pigs subjected to 90 minutes of left anterior descending coronary artery hypoperfusion and 120 minutes of reperfusion. In protocol 1, IPoC, by 6 cycles of 20/20 seconds of reperfusion/reocclusion (n=13), was compared with immediate full reperfusion (IFR) (n=15). In protocol 2, IPoC (n=4) or IFR (n=4) was performed with pharmacological RISK blockade by IC coinfusion of Wortmannin and U0126. Infarct size was determined by TTC staining, and the expression of phosphorylated RISK proteins by Western blot analysis in biopsies. In protocol 1, infarct size was 20+/-3% (percentage of area at risk; mean+/-SEM) with IPoC and 33+/-4% (P<0.05) with IFR. RISK phosphorylation increased with reperfusion but was not different between IPoC and IFR. In protocol 2, Wortmannin and U0126 blocked the increases in RISK phosphorylation during reperfusion, but infarct size was still smaller with IPoC (15+/-7%) than with IFR (35+/-6%; P<0.05).

Stimulation of cholinergic muscarinic receptors has been shown to provide substantial protection from DNA damage, oxidative stress and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. A study recently indicated that the activation of muscarinic receptors in astrocytoma cells modified the expression of the kinase p70S6K involved in the translational control. The translational control is in part regulated by a cascade of phosphorylation affecting proteins of the anti-apoptotic way controlled by mTOR (mammalian target of rapamycin) and the pro-apoptotic way controlled by PKR. The aim of our study was to investigate the effect of cholinergic muscarinic stimulation by an agonist oxotremorine on the anti-apoptotic way of translational control, in human neuroblastoma cells and in mice brain. Our results showed that muscarinic receptor activation significantly increased the expression of phosphorylated p70S6K, eIF4E and ERK without modification of mTOR activity in neuroblastoma cells and in cerebral cortex and hippocampus of mice, suggesting a stimulation of protein synthesis. Our findings support the notion that synaptic activity, through activation of neurotransmitter receptors, can provide substantial support of cellular survival mechanisms and suggest that loss of such synaptic input increases vulnerability to insult-induced programmed cell death.

In experimental animals, including rats, MK-801 produces characteristic behavioural changes that model schizophrenia. It has been hypothesized that these changes accompany long-term synaptic changes, which require protein neosynthesis. We observed the effect of MK-801 on the "mammalian target of rapamycin" (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway that regulates protein synthesis in the rat frontal cortex. A single injection of MK-801 (0.5, 1, or 2mg/kg) induced an acute increase in the phosphorylation of Akt (Ser-473) eIF4E-binding protein (4E-BP1) (Thr-37/46) and p70S6K (Thr-389). In contrast, after repeated treatment with MK-801 (1mg/kg for 5 or 10 days), the phosphorylation of Akt (Ser-473), mTOR (Ser-2481), 4E-BP1 (Thr-37/46), p70S6K (Thr-389), and S6 (Ser-240/244) increased. Thus, proteins in the mTOR/p70S6K pathway are modulated in chronic MK-801 animal models. These findings may suggest that repeated MK-801 treatment activates the signal transduction pathways involved in the initiation of protein synthesis in the rat frontal cortex.

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation., ((c) 2007 Wiley-Liss, Inc.)

Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the cap-binding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg-1, the worm homologue of eIF4G, which is a scaffold protein in the cap-binding complex; and rsks-1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg-1 or rsks-1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg-1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans.

The mammalian target of rapamycin kinase (mTOR) regulates protein synthesis in neurons at the translational level through phosphorylation of several intracellular targets. Recent work in invertebrates indicates that mTOR-dependent translational control may be critical for the induction and maintenance of activity-dependent synaptic plasticity underlying memory formation. Here, we report that training rats in a simple fear conditioning procedure evokes a time-dependent increase in the phosphorylation of p70s6 kinase, a major direct downstream target of mTOR. When the activation of mTOR was prevented by posttraining injection of rapamycin into the amygdala, formation of the memory and the increase in p70s6 kinase phosphorylation was attenuated. Furthermore, when rapamycin was applied to the amygdala after the recall of a previously stored fear memory, subsequent retention was disrupted, indicating that local translational control at active synapses is required for the stability as well as the formation of long-term memory in this system.

We studied antitumor effects of receptor tyrosine kinase inhibitor sunitinib (formerly SU11248) against a variety of hematologic malignancies including the following leukemias: eosinophilic (EOL-1), acute myeloid (THP-1, U937, Kasumi-1), biphenotypic (MV4-11), acute lymphoblastic (NALL-1, Jurkat, BALL-2, PALL-1, PALL-2), blast crisis of chronic myeloid (KU812, Kcl-22, K562), and adult T-cell (MT-1, MT-2, MT-4), as well as non-Hodgkin's lymphoma (KS-1, Dauji, Akata) and multiple myeloma (U266). Thymidine uptake studies showed that sunitinib was active against EOL-1, MV4-11, and Kasumi-1 cells, which possessed activating mutations of the PDGFRalpha, FLT-3, and c-KIT genes, respectively, with IC(50)s of <30 nmol/L. In addition, sunitinib inhibited the proliferation of freshly isolated leukemia cells from patients possessing mutations in FLT3 gene. Annexin V staining showed that sunitinib induced apoptosis of these cells. Sunitinib inhibited phosphorylation of FLT3 and PDGFRalpha in conjunction with blockade of mammalian target of rapamycin signaling in MV4-11 and EOL-1 cells, respectively. Interestingly, rapamycin analogue RAD001 enhanced the ability of sunitinib to inhibit the proliferation of leukemia cells and down-regulate levels of mammalian target of rapamycin effectors p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 in these cells. Taken together, sunitinib may be useful for treatment of individuals with leukemias possessing activation mutation of tyrosine kinase, and the combination of sunitinib and RAD001 represents a promising novel treatment strategy.

Purpose: Activation of mitogen-activated protein kinase (MAPK) and members of the Akt pathway have been shown to promote cell proliferation, survival, and resistance to radiation. This study was conducted to determine whether any of these markers are associated with survival time and response to radiation in glioblastoma., Experimental Design: The expression of phosphorylated (p-)Akt, mammalian target of rapamycin (p-mTOR), p-p70S6K, and p-MAPK were assessed by immunohistochemical staining in 268 cases of newly diagnosed glioblastoma. YKL-40, a prognostic marker previously examined in these tumors, was also included in the analysis. Expression data were tested for correlations with response to radiation therapy in 131 subtotally resected cases and overall survival (in all cases). Results were validated in an analysis of 60 patients enrolled in clinical trials at a second institution., Results: Elevated p-MAPK expression was most strongly associated with poor response to radiotherapy, a finding corroborated in the validation cohort. For survival, higher expressions of p-mTOR, p-p70S6K, and p-MAPK were associated with worse outcome (all P < 0.03). YKL-40 expression was associated with the expressions of p-MAPK, p-mTOR, and p-p70S6K (all P < 0.02), with a trend toward association with p-Akt expression (P = 0.095). When known clinical variables were added to a multivariate analysis, only age, Karnofsky performance score, and p-MAPK expression emerged as independent prognostic factors., Conclusions: p-MAPK and activated members of the Akt pathway are markers of outcome in glioblastoma. Elevated expression of p-MAPK is associated with increased radiation resistance and represents an independent prognostic factor in these tumors.

Proliferation of activated hepatic stellate cells (HSC) is an important event in the development of hepatic fibrosis. Insulin-like growth factor-1 (IGF-1) has been shown to be mitogenic for HSC, but the intracellular signaling pathways involved have not been fully characterized. Thus, the aims of the current study were to examine the roles of the extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3-K) and p70-S6 kinase (p70-S6-K) signaling pathways in IGF-1- and platelet-derived growth factor (PDGF)-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. Both IGF-1 and PDGF increased ERK, PI3-K and p70-S6-K activity. When evaluating potential crosstalk between these signaling pathways, we observed that PI3-K is required for p70-S6-K activation by IGF-1 and PDGF, and is partially responsible for PDGF-induced ERK activation. PDGF and IGF-1 also increased the levels of cyclin D1 and phospho-glycogen synthase kinase-3beta. Coordinate activation of ERK, PI3-K and p70-S6-K is important for perpetuating the activated state of HSC during fibrogenesis.

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires a cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6KDeltaCT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant (Km) values of S6K for ATP and the Biotin-S6 substrate peptide were determined to be 21.4+/-0.29 and 0.9+/-0.48 microM, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose-response and inhibition mechanism by these inhibitors were also studied. Our data provide a new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors.

In order to determine the function and possible relationship between Cdc2 and P(70)S6K, Western blot analysis and immunohistochemistry analysis were used to study the expression and kinase activity of Cdc2 and P(70)S6K in male mouse germ cells. With the maturation of germ cells in the testis, the expression of Cdc2 and P(70)S6K was relatively constant. However, the kinase activity of P(70)S6K was increased and the phosphorylation of Tyr15 residue of Cdc2 was enhanced, which suggests that the kinase activity of Cdc2 is decreasing. Immunohistochemistry analysis also showed that there was a P(70)S6K transfer from nucleus to cytoplasm during spermatogenesis. During spermatogenesis, cell division of the germ cell in male mouse is decelerated; nevertheless, cell growth is enhanced. Cdc2 and P(70)S6K are involved in these two processes. It could be an alternative mechanism to prepare for future fertilization that Cdc2 is able to maintain a subtle balance between the production and growth of male germ cells by regulating P(70)S6K., (Copyright 2005 John Wiley & Sons, Ltd.)

Previous studies have shown that freeze-dried black raspberry extract fractions inhibit benzo(a)pyrene [B(a)P]-induced transformation of Syrian hamster embryo cells and benzo(a)pyrene diol-epoxide [B(a)PDE]-induced activator protein-1 (AP-1) activity in mouse epidermal Cl 41 cells. The phosphotidylinositol 3-kinase (PI-3K)/Akt pathway is critical for B(a)PDE-induced AP-1 activation in mouse epidermal Cl 41 cells. In the present study, we determined the potential involvement of PI-3K and its downstream kinases on the inhibition of AP-1 activation by black raspberry fractions, RO-FOO3, RO-FOO4, RO-ME, and RO-DM. In addition, we investigated the effects of these fractions on the expression of the AP-1 target genes, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). Pretreatment of Cl 41 cells with fractions RO-F003 and RO-ME reduced activation of AP-1 and the expression of VEGF, but not iNOS. In contrast, fractions RO-F004 and RO-DM had no effect on AP-1 activation or the expression of either VEGF or iNOS. Consistent with inhibition of AP-1 activation, the RO-ME fraction markedly inhibited activation of PI-3K, Akt, and p70 S6 kinase (p70(S6k)). In addition, overexpression of the dominant negative PI-3K mutant delta p85 reduced the induction of VEGF by B(a)PDE. It is likely that the inhibitory effects of fractions RO-FOO3 and RO-ME on B(a)PDE-induced AP-1 activation and VEGF expression are mediated by inhibition of the PI-3K/Akt pathway. In view of the important roles of AP-1 and VEGF in tumor development, one mechanism for the chemopreventive activity of black raspberries may be inhibition of the PI-3K/Akt/AP-1/VEGF pathway.

The precise mechanisms by which imatinib mesylate (STI571) and interferon alpha (IFNalpha) exhibit antileukemic effects are not known. We examined the effects of IFNs or imatinib mesylate on signaling pathways regulating initiation of mRNA translation in BCR-ABL-expressing cells. Treatment of IFN-sensitive KT-1 cells with IFNalpha resulted in phosphorylation/activation of mammalian target of rapamycin (mTOR) and downstream activation of p70 S6 kinase. The IFN-activated p70 S6 kinase was found to regulate phosphorylation of S6 ribosomal protein, which regulates translation of mRNAs with oligopyrimidine tracts in the 5'-untranslated region. In addition, IFNalpha treatment resulted in an mTOR- and/or phosphatidyl-inositol 3'(PI 3') kinase-dependent phosphorylation of 4E-BP1 repressor of mRNA translation on sites that are required for its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. In contrast to the effects of IFNs, imatinib mesylate suppressed p70 S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. Moreover, the mTOR inhibitor rapamycin enhanced the suppressive effects of imatinib mesylate on primary leukemic granulocyte macrophage-colony-forming unit (CFU-GM) progenitors from patients with chronic myelogenous leukemia (CML). Taken altogether, our data demonstrate that IFNs and imatinib mesylate differentially regulate PI 3' kinase/mTOR-dependent signaling cascades in BCR-ABL-transformed cells, consistent with distinct effects of these agents on pathways regulating mRNA translation. They also support the concept that combined use of imatinib mesylate with mTOR inhibitors may be an appropriate future therapeutic strategy for the treatment of CML.

We have previously demonstrated that dietary protein induced pancreatic hypergrowth in pancreaticobiliary diverted (PBD) rats. Dietary protein and dietary amino acids stimulate protein synthesis by regulating translation initiation in the rat skeletal muscle and liver. The aim of the present study was to determine whether feeding a high-protein diet induces activation of translation initiation for protein synthesis in the rat pancreas. In PBD rats in which the bile-pancreatic juice was surgically diverted to the upper ileum for 11-13 days, pancreatic dry weight and protein content were doubled compared with those in sham rats and further increased with feeding of a high-protein diet (60% casein diet) for 2 days. These pancreatic growth parameters were maintained at high levels for the next 5 days and were much higher than those of sham rats fed a high-protein diet. In both sham and PBD rats, feeding of a high-protein diet for 2 days induced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 and 70-kDa ribosomal protein S6 kinase, indicating the activation of the initiation phase of translation for pancreatic protein synthesis. However, this increased phosphorylation returned to normal levels on Day 7 in PBD but not in sham rats. We concluded that feeding a high-protein diet induced pancreatic growth with increases in the translation initiation activities for pancreatic protein synthesis within 2 days and that prolonged feeding of a high-protein diet changed the initiation activities differently in sham and PBD rats.

In this study we compared the content and phosphorylation levels of several molecules believed to regulate muscle hypertrophy and fiber type changes in the extensor digitorum longus (EDL), soleus, diaphragm, and heart of adult (6 months), aged (30 months), and very aged (36 months) Fischer 344 x Brown Norway rats. With aging, the mass of the EDL and soleus decreased significantly (approximately 38% and approximately 36%, respectively), the diaphragm's mass remained unchanged while the mass of the heart increased (approximately 35%). Western blotting demonstrated that calcineurin (CnA), the 70-kDa ribosomal S6 kinase (p70(S6k)), glycogen synthase kinase-3beta (GSK-3beta), and the phosphorylated forms of GSK-3beta and p70(S6k) (p-GSK-3beta(Ser9) and p-p70(S6kThr389)) were regulated differently with aging and between muscle types. Total p70(S6k), GSK-3beta, and p-GSK-3beta(Ser9) decreased in the aged-atrophic EDL and soleus while p-p70(S6kThr389) increased. Although total p70(S6k) content diminished in the continuously active diaphragm, phosphorylation of p70(S6k )remained unchanged. Conversely, the expression of GSK-3beta and p-GSK-3beta(Ser9) increased in the diaphragm. With aging, the amount of p-p70(S6kThr389) decreased approximately 56% in the heart while p-GSK-3beta( Ser9) increased approximately 193%. Interestingly, CnA content remained unchanged in the diaphragm, increased approximately 204% in the EDL, and decreased approximately 30% and approximately 65% with aging in the soleus and heart, respectively. These results indicate remarkable differences in the regulation of molecules thought to govern protein synthesis and changes in contractile protein expression.

Aim: The aim of this study was to analyze the levels 70 kDa ribosomal protein S6 kinase 1 (S6K1) and 70 kDa ribosomal protein S6 kinase 2 (S6K2) expression and S6 ribosomal protein phosphorylation in endometrial adenocarcinomas., Methods: S6K1/2 expression and phosphorylated ribosomal S6 protein (phS6) content have been detected in formalin fixed, paraffin embedded sections of 50 human endometrial adenocarcinomas with different grade of differentiation and in 13 normal endometrial tissues using immunohistochemical approach with following semiquantitative analysis., Results: In normal endometrial epithelial cells both S6K1 and S6K2 were expressed on the low level. S6K1 and S6K2 has been detected predominantly in stromal elements. Increased phS6 level was found in superficial epithelial cells. In deeper parts of endometrial glands and vessels phS6 was discovered occasionally. In endometrial adenocarcinoma's tissues, overexpression of S6K1 was found in cytoplasm and nuclei in 8.0% of cases, overexpression of S6K2--in cytoplasm in 12.0% of cases and in nuclei in 18.0% of cases. Overexpression of S6K1 in endothelial cells of vessels was discovered in 58% of cases. Positive correlation has been determined between: 1) tumor stage and intensity of stromal staining for S6K1 (p = 0.027); 2) tumor differentiation grade and intensity of cytoplasm staining of cancer cells for S6K1 (p = 0.039); 3) intensity of stromal staining and vessel's staining for S6K1 (p = 0.019); 4) vessel's staining for S6K1 and staining for phS6 (p = 0.028)., Conclusion: Overexpression of S6K1 and S6K2 is a characteristic feature of parenchyma and vessels of endometrial adenocarcinomas. Phosphorylation of ribosomal S6 protein is not dependent from expression level of S6K1 and S6K2.

To determine the mechanism of meal-regulated synthesis of pancreatic digestive enzymes, we studied the effect of fasting and refeeding on pancreatic protein synthesis, relative mRNA levels of digestive enzymes, and activation of the translational machinery. With the use of the flooding dose technique with L-[3H]phenylalanine, morning protein synthesis in the pancreas of Institute for Cancer Research mice fed ad libitum was 7.9 +/- 0.3 nmol phenylalanine.10 min(-1).mg protein(-1). Prior fasting for 18 h reduced total protein synthesis to 70 +/- 1.4% of this value. Refeeding for 2 h, during which the mice consumed 29% of their daily food intake, increased protein synthesis to 117.3 +/- 4.9% of the control level. Pancreatic mRNA levels of amylase, lipases, trypsins, chymotrypsin, elastases, as well as those for several housekeeping genes tested were not significantly changed after refeeding compared with fasted mice. By contrast, the major translational control pathway involving Akt, mTOR, and S6K was strongly regulated by fasting and refeeding. Fasting for 18 h decreased phosphorylation of ribosomal protein S6 to almost undetectable levels, and refeeding highly increased it. The most highly phosphorylated form of the eIF4E binding protein (4E-BP1) made up the 14.6% of total 4E-BP1 in normally fed animals, was only 2.8% after fasting, and was increased to 21.4% after refeeding. This was correlated with an increase in the formation of the eIF4E-eIF4G complex after refeeding. By contrast, feeding did not affect eIF2B activity. Thus food intake stimulates pancreatic protein synthesis and translational effectors without increasing digestive enzyme mRNA levels.

We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration.