Upregulation of MTOR, RPS6KB1, and EIF4EBP1 in the whole blood samples of Iranian patients with multiple sclerosis compared to healthy controls.

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.

Silent Information Regulator 1 Negatively Regulates Atherosclerotic Angiogenesis via Mammalian Target of Rapamycin Complex 1 Signaling Pathway.

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 (CoCl2). 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 ± 0.02mm2 versus 5.44 ± 0.50mm2, P < 0.05). This effect was attenuated by nicotinamide (0.76 ± 0.15mm2 versus 0.05 ± 0.02mm2, 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.

Suppression of ribosomal protein RPS6KB1 by Nexrutine increases sensitivity of prostate tumors to radiation.

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.

Expression of miRNAs Targeting mTOR and S6K1 Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer.

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.

Effect of endurance training and branched-chain amino acids on the signaling for muscle protein synthesis in CKD model rats fed a low-protein diet.

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.

Expression of mTOR/70S6K signaling pathway in pathological scar fibroblasts and the effects of resveratrol intervention.

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 dose­dependent 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.

Metformin inhibited esophageal squamous cell carcinoma proliferation in vitro and in vivo and enhanced the anti-cancer effect of cisplatin.

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.

Physical and Flavor Characteristics, Fatty Acid Profile, Antioxidant Status and Nrf2-Dependent Antioxidant Enzyme Gene Expression Changes in Young Grass Carp (Ctenopharyngodon idella) Fillets Fed Dietary Valine.

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.

Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells.

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.

Correlation between Ribosome Biogenesis and the Magnitude of Hypertrophy in Overloaded Skeletal Muscle.

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.

Reciprocal expression of p-AMPKa and p-S6 is strongly associated with the prognosis of gastric cancer.

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.

MARCKSL1 exhibits anti-angiogenic effects through suppression of VEGFR-2-dependent Akt/PDK-1/mTOR phosphorylation.

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 VEGF­induced 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 3ß (GSK-3ß) 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.

Neuroprotective Effect of Simvastatin via Inducing the Autophagy on Spinal Cord Injury in the Rat Model.

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.

Extensive analysis of signaling pathway molecules in breast cancer: association with clinicopathological characteristics.

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.

Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

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.

MEK1/2 overactivation can promote growth arrest by mediating ERK1/2-dependent phosphorylation of p70S6K.

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.

Metformin inhibits growth and sensitizes osteosarcoma cell lines to cisplatin through cell cycle modulation.

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.

Expression of molecular markers associated with the mammalian target of rapamycin pathway in nonmetastatic renal cell carcinoma: Effect on prognostic outcomes following radical nephrectomy.

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.

[MicroRNA-223-3p inhibits the angiogenesis of ischemic myocardial microvascular endothelial cells via modulating Rps6kb1/HIF-1α signal pathway].

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.

MiR-145 is downregulated in human ovarian cancer and modulates cell growth and invasion by targeting p70S6K1 and MUC1.

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.