Effectiveness of low dose of rapamycin in preventing seizure-induced anxiety-like behaviour, cognitive impairment, and defects in neurogenesis in developing rats.

Aims: Previous studies have demonstrated that rapamycin prevents seizure-induced anxiety-like behaviors. However, rapamycin had been used at a higher dose of 3 mg/kg and resulted in side effects in immature animals. This work was designed to explore whether a lower dose of rapamycin has similar efficacy but has milder side effects.Methods: Acute seizures were induced by injection of pilocarpine at postnatal 10-day Sprague-Dawley rats. Western blot analysis was used to detect changes in mammalian target of rapamycin (mTOR) pathway after seizure. Immunofluorescent intensity of doublecortin (DCX) was conducted to evaluate the development of neurons in hippocampus. Morris water maze and Y-maze test were used to assess cognitive functions and open-field test and elevated plus maze were used to detect anxiety-like behaviors 4 weeks after seizure onset.Results: mTOR pathway was abnormally activated with two peaks after pilocarpine-induced seizures, and no difference of DCX-positive cells and body weight were noticed between control and pilocarpine-induced seizure rats. Pilocarpine-induced seizure in postnatal 10 days rats did not exert impairment on cognitive functions, but resulted in obvious anxiety-like behaviors. Low dose of rapamycin at 0.3 mg/kg significantly reversed seizure-induced increase of p-S6 levels as well as abnormal anxiety-like behaviors. In addition, rapamycin at the dose of 0.3mg/kg did not affect normal development and cognitive functions.Conclusion: lower doses of rapamycin should be used in infants compared with older children or adults.

Evaluation of the Effect of Everolimus on Retinal Pigment Epithelial Cells and Experimental Proliferative Vitreoretinopathy.

PURPOSE: Failure of retinal detachment surgery is most commonly due to the development of proliferative vitreoretinopathy (PVR). Everolimus is an inhibitor of mammalian target of rapamycin (mTOR), and is available as oral tablets. In this study, we investigated the effect of everolimus on retinal pigment epithelial cells and modification of the severity of experimental PVR. METHODS: In our in vitro studies, primary culture of retinal pigment epithelium (RPE) cells was obtained from pigmented Rex rabbits. Cell proliferation was assayed with the tetrazolium dye cytotoxicity test, and cell migration assay was performed in 24-well transwell units with 8-µm filters. In the in vivo study, pigmented Rex rabbits weighing between 2 and 2.5 kg were used. Each rabbit eye underwent gas compression; one week later, 5 × 104 RPE cells were injected into the vitreous cavity to induce PVR, and each eye was graded with indirect ophthalmoscopy on days 1, 3, 7, 14, 21, and 28. The rabbits were administered everolimus (0.5 mg/day orally) from the day of PVR induction. Total proteins extracted from RPE cells and dissected retinal samples were processed for Western blotting analysis of mTOR and ribosomal protein S6 (RPS6). RESULTS: The in vitro studies showed that everolimus significantly inhibited the proliferation of RPE cells at 0.1 µg/ml; additionally, at 10 µg/ml, it suppressed the migration of RPE cells and significantly suppressed the expression of mTOR and RPS6 in RPE cells. The in vivo study did not show any benefit of oral everolimus (0.5 mg/day) in suppressing experimental PVR. Thus, everolimus significantly suppressed the expression of mTOR and RPS6 in PVR. CONCLUSIONS: Everolimus suppressed the proliferation and migration of RPE cells in vitro. Oral everolimus (0.5 mg/day) suppressed the expression of mTOR and RPS6 in the retina, but showed no effect in suppressing experimental PVR.

Extinction, applied after retrieval of auditory fear memory, selectively increases zinc-finger protein 268 and phosphorylated ribosomal protein S6 expression in prefrontal cortex and lateral amygdala.

Retrieval of consolidated memories induces a labile phase during which memory can be disrupted or updated through a reconsolidation process. A central component of behavioral updating during reconsolidation using a retrieval-extinction manipulation (Ret+Ext) is the synaptic removal of a calcium-permeable-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (CP-AMPARs) in the lateral amygdala-a metabotropic GluR1 receptor (mGluR1) dependent mechanism. In the present study, we investigate the effect of Ret+Ext on the expression of molecular markers that could play a role in the reconsolidation process. Specifically, we tested the effects of Ret+Ext on the global expression of zinc-finger 268 protein (Zif268), a marker previously found to be implicated in memory reconsolidation, to confirm its occurrence after retrieval (Ret) and Ret+Ext. We also evaluated the global expression of phosphorylated ribosomal protein S6 (rpS6P), here proposed as a marker of the mGluR1-mediated memory process induced by Ret+Ext. The expression of both markers (zif268, rpS6P) was assessed by immunolocalization in prelimbic cortex (PRL), infralimbic cortex (IL), ventral subdivision of the lateral amygdala (LA) and hippocampus CA1 (CA1) in fear-conditioned rats. Our results showed that retrieval and Ret+Ext, but not extinction alone, increased Zif268 expression in prefrontal cortex and lateral amygdala. Ret+Ext, but not retrieval, retrieval followed by context exposure or extinction alone, increased the expression of rpS6P in prefrontal cortex and LA. In summary, (i) Zif268 increased after retrieval confirming that reconsolidation is engaged in our conditions, (ii) Zif268 increased after Ret+Ext confirming that it does not simply reflect an extinction or reconsolidation disruption (Zif268 level of expression should be lower in both cases) and (iii) rpS6P increased after Ret+Ext, but not after extinction, suggesting, as expected, a potential mGluR1 mediated molecular mechanism specific for Ret+Ext. Together with the Zif268 increase, our results suggest that the Ret+Ext induced memory process is more similar to reconsolidation updating than extinction facilitation.

Downregulation of ribosomal protein S6 inhibits the growth of non-small cell lung cancer by inducing cell cycle arrest, rather than apoptosis.

Ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit, has been found to be associated with multiple physiological and pathophysiological functions. However, its effects and mechanisms in non-small cell lung cancer (NSCLC) still remain unknown. Here, we showed that expressions of total rpS6 and phosphorylation rpS6 (p-rpS6) were both significantly overexpressed in NSCLC. Further survival analysis revealed the shortened overall survival (OS) and relapse-free survival (RFS) in p-rpS6 overexpressed patients and confirmed it as an independent adverse predictor. Stable downregulation of rpS6 in lung adenocarcinoma A549 and squamous cell carcinoma H520 cell lines was then achieved by two specific small hairpin RNA (shRNA) lentiviruses separately. Subsequent experiments showed that downregulation of rpS6 dramatically inhibited cell proliferation in vitro and tumorigenicity in vivo. Moreover, loss of rpS6 promoted cells arrested in G0-G1 phase and reduced in G2-M phase, along with the expression alterations of relative proteins. However, no notable change in apoptosis was observed. Collectively, these results suggested that rpS6 is overactivated in NSCLC and its downregulation suppresses the growth of NSCLC mainly by inducing G0-G1 cell cycle arrest rather than apoptosis.

PI3K/AKT pathway activation in bladder carcinogenesis.

The PI3K/AKT pathway is considered to play a major role in bladder carcinogenesis, but its relationships with other molecular alterations observed in bladder cancer remain unknown. We investigated PI3K/AKT pathway activation in a series of human bladder urothelial carcinomas (UC) according to PTEN expression, PTEN deletions and FGFR3, PIK3CA, KRAS, HRAS, NRAS and TP53 gene mutations. The series included 6 normal bladder urothelial samples and 129 UC (Ta n = 25, T1 n = 34, T2-T3-T4 n = 70). Expression of phospho-AKT (pAKT), phospho-S6-Ribosomal Protein (pS6) (one downstream effector of PI3K/AKT pathway) and PTEN was evaluated by reverse phase protein Array. Expression of miR-21, miR-19a and miR-222, known to regulate PTEN expression, was also evaluated. pAKT expression levels were higher in tumors than in normal urothelium (p < 0.01), regardless of stage and showed a weak and positive correlation with pS6 (Spearman coefficient RS = 0.26; p = 0.002). No association was observed between pAKT or pS6 expression and the gene mutations studied. PTEN expression was decreased in PTEN-deleted tumors, and in T1 (p = 0.0089) and T2-T3-T4 (p < 0.001) tumors compared to Ta tumors; it was also negatively correlated with miR-19a (RS = -0.50; p = 0.0088) and miR-222 (RS = -0.48; p = 0.0132), but not miR-21 (RS = -0.27; p = 0.18) expression. pAKT and PTEN expressions were not negatively correlated, and, on the opposite, a positive and moderate correlation was observed in Ta (RS = 0.54; p = 0.0056) and T1 (RS = 0.56; p = 0.0006) tumors. Our study suggests that PI3K/AKT pathway activation occurs in the entire spectrum of bladder UC regardless of stage or known most frequent molecular alterations, and independently of low PTEN expression.

Differential expression of HIF-1 in glioblastoma multiforme and anaplastic astrocytoma.

Hypoxia is an important factor mediating tumor progression and therapeutic resistance, in part through proteome changes mediated by the transcription factor hypoxia-inducible factor (HIF)-1. Since glioblastoma multiforme is the epitome of a highly aggressive tumor entity, while lower-grade astrocytomas often show a prolonged clinical course, a profound difference in the extent of hypoxic tissue areas and corresponding magnitude of HIF-1 activity may exist between these entities. In this study, to address this question, serial sections of 11 glioblastomas and 10 anaplastic astrocytomas were immunostained for HIF-1α, glucose transporter (GLUT)-1, carbonic anhydrase (CA) IX (i.e., hypoxia-related markers), Ki67 (proliferation), phosphorylated ribosomal protein S6 [p-rpS6; mammalian target of rapamycin (mTOR) activity] and CD34 (microvascular endothelium). Digital scans of whole tumor sections were registered to achieve geometric correspondence for subsequent morphometric operations. HIF-1α-, GLUT-1- and CA IX-positive staining was found in all 11 glioblastomas, showing a preferential expression in tissue areas adjacent to necroses. A considerable spatial overlap between GLUT-1 and CA IX, and a colocalization of these proteins with areas of enlarged mean diffusion distances were observed. Conversely, 8 of the 10 anaplastic astrocytomas were completely negative for hypoxia-related markers. The glioblastomas also showed significantly greater heterogeneity of intercapillary distances, larger diffusion-limited tissue fractions, significantly higher mTOR activity and a trend for higher proliferation rates. Microregionally, mTOR and proliferation showed a significant spatial overlap with areas of shorter mean diffusion distances. In conclusion, diffusion-limited hypoxia, leading to the expression of hypoxia-related markers is a pivotal element of the glioblastoma phenotype and may be driven by dysregulated growth and proliferation in normoxic subregions.

The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels.

Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein.

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.

Temsirolimus in advanced leiomyosarcomas: patterns of response and correlation with the activation of the mammalian target of rapamycin pathway.

Preclinical data have indicated that alteration of PTEN and activation of the mammalian target of rapamycin (mTOR) pathway play a crucial role in the oncogenesis of leiomyosarcoma. The objective of this exploratory study was to assess the clinical role of mTOR inhibition in patients with advanced leiomyosarcoma refractory to standard chemotherapy. Patients with advanced leiomyosarcoma were treated with temsirolimus and consented to retrospective collection of data from their medical records and analysis of archival tumor specimens. Tumor response was determined according to the response evaluation criteria in solid tumor (RECIST) and Choi criteria. Tumors were assessed for immunohistochemical evidence of PTEN loss of expression and mTOR activation. Six patients participated in the study. According to the RECIST, three patients had stable disease and three patients had progressive disease. The three patients with RECIST stable disease had partial response according to the Choi criteria. Partial response according to the Choi criteria was associated with clinical improvement and biological signs of temsirolimus antitumor activity. The immunohistochemical status of PTEN and phosphorylated S6 ribosomal protein was not predictive of the outcome. This exploratory study indicates antitumor activity of temsirolimus in leiomyosarcoma, possibly through a mechanism involving aberration of the PTEN gene. Further investigations of the phosphoinositide 3-kinases/PTEN/Akt/mTOR pathway are needed to explore the role of mTOR inhibitors, either alone or in combination, in patients with advanced sarcoma.

Differential requirement of ribosomal protein S6 by plant RNA viruses with different translation initiation strategies.

Potyvirus infection has been reported to cause an increase in the mRNA transcripts of many plant ribosomal proteins (r-proteins). In this study, increased expression of r-protein mRNA transcripts was determined to occur in Nicotiana benthamiana during infection by potyviruses as well as a tobamovirus demonstrating that this response is not unique to potyviruses. Five r-protein genes, RPS6, RPL19, RPL13, RPL7, and RPS2, were silenced in N. benthamiana to test their roles in viral infection. The accumulation of both Turnip mosaic virus (TuMV), a potyvirus, and Tobacco mosaic virus (TMV), a tobamovirus, was dependent on RPL19, RPL13, RPL7, and RPS2. However, TMV was able to accumulate in RPS6-silenced plants while accumulation of TuMV and Tomato bushy stunt virus (TBSV) was abolished. These results demonstrate that cap-independent TuMV and TBSV require RPS6 for their accumulation, whereas accumulation of TMV is independent of RPS6.

AMPK represses TOP mRNA translation but not global protein synthesis in liver.

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5'-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1alpha. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or beta-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.