An intramolecular disulphide bond in human 4E-T affects its binding to eIF4E1a protein.

The cap at the 5'terminus of mRNA is a key determinant of gene expression in eukaryotic cells, which among others is required for cap dependent translation and protects mRNA from degradation. These properties of cap are mediated by several proteins. One of them is 4E-Transporter (4E-T), which plays an important role in translational repression, mRNA decay and P-bodies formation. 4E-T is also one of several proteins that interact with eukaryotic initiation factor 4E (eIF4E), a cap binding protein which is a key component of the translation initiation machinery. The molecular mechanisms underlying the interactions of these two proteins are crucial for mRNA processing. Studying the interactions between human eIF4E1a and the N-terminal fragment of 4E-T that possesses unstructured 4E-binding motifs under non-reducing conditions, we observed that 4E-T preferentially forms an intramolecular disulphide bond. This "disulphide loop" reduces affinity of 4E-T for eIF4E1a by about 300-fold. Considering that only human 4E-T possesses two cysteines located between the 4E binding motifs, we proposed that the disulphide bond may act as a switch to regulate interactions between the two proteins.

[Expression and Clinical Significance of Eukaryotic Initiation Factor 4E in Newly Diagnosed Patients with Multiple Myeloma].

OBJECTIVE: To explore the expression and clinical significance of eukaryotic translation initiation factor 4E(eIF4E) in bone marrow samples of newly diagnosed multiple myeloma (NDMM) patients. METHODS: Immunohistochemical staining was used to analyze the expression of eIF4E protein in bone marrow biopsy samples from 75 NDMM patients and 25 patients with benign bone marrow disease. Clinical data were collected to analyze the correlation between eIF4E protein expression and clinical features and its prognostic significance in bone marrow samples of NDMM patients. RESULTS: The positive rate of eIF4E protein expression in bone marrow samples of NDMM patients was higher than that in patients with benign bone marrow disease (P<0.001). Positive expression of eIF4E protein was correlated with elevated serum lactate dehydrogenase in MM patients. Univariate and multivariate analysis showed that positive expression of eIF4E protein was significantly associated with shortened overall survival (OS) in NDMM patients, and was an independent risk factor affecting OS in NDMM patients. CONCLUSION: Positive expression of eIF4E protein is an independent poor prognostic factor for OS in NDMM patients.

RIP3/MLKL regulates necroptosis via activating 4EBP1-eIF4E pathway. / RIP3/MLKL通过激活4EBP1-eIF4E通路诱导程序性坏死.

OBJECTIVES: Necroptosis is a cell death type mediated by receptor interacting protein 3 (RIP3)/mixed lineage kinase domain-like protein (MLKL). It has been reported that mammalian target of rapamycin plays a regulatory role in necroptosis. Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)-eukaryotic initiation factor 4E (eIF4E) pathway is a key down streamer of mammalian target of rapamycin. However, whether 4EBP1-eIF4E pathway is involved in necroptosis is still unknown. This study aims to investigate the changes of 4EBP1-eIF4E pathway in necroptosis. METHODS: TNF-α/SM-164/Z-VAD-FMK (TSZ), a necroptosis inducer, was used to induce necroptosis in murine fibroblastoid cell line L929. Cell necrosis was observed under an optical microscope. Then, TSZ was added to L929 cells with RIP3 and MLKL gene knockout. Propidium iodide (PI) staining was used to observe cell necrosis. Real-time fluorescence quantitative PCR and Western blotting were used to determine the mRNA and protein expression of 4EBP1 and eIF4E, respectively. RESULTS: After treating L929 cells with TSZ, the number of necrotic cells was increased, the mRNA and protein expression levels of 4EBP1 were significantly downregulated, and the ratio of phosphorylated 4EBP1 (p-4EBP1) to 4EBP1 was increased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly upregulated, and the ratio of phosphorylated eIF4E (p-eIF4E) to eIF4E was increased (both P<0.01). After knocking out RIP3 and MLKL in L929 cells, PI positive necrotic cells were significantly reduced, the mRNA and protein expression levels of 4EBP1 were significantly upregulated, and the ratio of p-4EBP1 to 4EBP1 was decreased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly downregulated, and the ratio of p-eIF4E to eIF4E was decreased (both P<0.01). CONCLUSIONS: 4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis.

Phosphorylation of T897 in the dimerization domain of Gemin5 modulates protein interactions and translation regulation.

Gemin5 is a multifunctional RNA binding protein (RBP) organized in domains with a distinctive structural organization. The protein is a hub for several protein networks performing diverse RNA-dependent functions including regulation of translation, and recognition of small nuclear RNAs (snRNAs). Here we sought to identify the presence of phosphoresidues on the C-terminal half of Gemin5, a region of the protein that harbors a tetratricopeptide repeat (TPR)-like dimerization domain and a non-canonical RNA binding site (RBS1). We identified two phosphoresidues in the purified protein: P-T897 in the dimerization domain and P-T1355 in RBS1. Replacing T897 and T1355 with alanine led to decreased translation, and mass spectrometry analysis revealed that mutation T897A strongly abrogates the association with cellular proteins related to the regulation of translation. In contrast, the phosphomimetic substitutions to glutamate partially rescued the translation regulatory activity. The structural analysis of the TPR dimerization domain indicates that local rearrangements caused by phosphorylation of T897 affect the conformation of the flexible loop 2-3, and propagate across the dimerization interface, impacting the position of the C-terminal helices and the loop 12-13 shown to be mutated in patients with neurological disorders. Computational analysis of the potential relationship between post-translation modifications and currently known pathogenic variants indicates a lack of overlapping of the affected residues within the functional domains of the protein and provides molecular insights for the implication of the phosphorylated residues in translation regulation.

Suppression of the doxorubicin response by hypoxia-inducible factor-1α is strictly dependent on oxygen concentrations under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and p53 are involved in anticancer drug resistance under hypoxic conditions. Here, we found that the cytotoxicity of anticancer drugs (doxorubicin, gemcitabine, and cisplatin) was lower at 1% O2 than at 5% O2. We examined the effects of these drugs on HIF-1α and p53 expression under different hypoxic oxygen concentrations. At 5% O2, the drugs decreased HIF-1α expression and increased p53 levels. At 1% O2, the drugs increased HIF-1α expression but did not alter p53 levels. When the HIF-1α protein was stabilized by DMOG under normoxic conditions, doxorubicin did not increase the level of p53 expression. These results show that the maintenance of HIF-1α expression blocked doxorubicin-dependent increases in p53 expression. We hypothesized the mechanism of HIF-1α protein translation might be different between at 5% and at 1% O2, because many reports indicate that the same mechanism of HIF-1α protein stabilization occurs under hypoxic conditions, such as 5% and 1% O2. The level of phosphorylated-4E-BP1, which causes translation of HIF-1α, was higher at 1% O2 than at 5% O2. Our results suggest that the sensitivity of tumor cells to anticancer drugs is dependent oxygen concentrations under hypoxic conditions, and involves 4E-BP1-dependent stabilization of the HIF-1α protein.

Computational study on the allosteric mechanism of Leishmania major IF4E-1 by 4E-interacting protein-1: Unravelling the determinants of m7GTP cap recognition.

During their life cycle, Leishmania parasites display a fine-tuned regulation of the mRNA translation through the differential expression of isoforms of eukaryotic translation initiation factor 4E (LeishIF4Es). The interaction between allosteric modulators such as 4E-interacting proteins (4E-IPs) and LeishIF4E affects the affinity of this initiation factor for the mRNA cap. Here, several computational approaches were employed to elucidate the molecular bases of the previously-reported allosteric modulation in L. major exerted by 4E-IP1 (Lm4E-IP1) on eukaryotic translation initiation factor 4E 1 (LmIF4E-1). Molecular dynamics (MD) simulations and accurate binding free energy calculations (ΔGbind ) were combined with network-based modeling of residue-residue correlations. We also describe the differences in internal motions of LmIF4E-1 apo form, cap-bound, and Lm4E-IP1-bound systems. Through community network calculations, the differences in the allosteric pathways of allosterically-inhibited and active forms of LmIF4E-1 were revealed. The ΔGbind values show significant differences between the active and inhibited systems, which are in agreement with the available experimental data. Our study thoroughly describes the dynamical perturbations of LmIF4E-1 cap-binding site triggered by Lm4E-IP1. These findings are not only essential for the understanding of a critical process of trypanosomatids' gene expression but also for gaining insight into the allostery of eukaryotic IF4Es, which could be useful for structure-based design of drugs against this protein family.

Acetate regulates milk fat synthesis through the mammalian target of rapamycin/eukaryotic initiation factor 4E signaling pathway in bovine mammary epithelial cells.

Acetate is a short-chain fatty acid (SFA) that is the major substrate for de novo fatty acid synthesis. The mammalian target of rapamycin/eukaryotic initiation factor 4E (mTOR/eIF4E) signaling pathway is involved in fat synthesis. However, the effect and mechanism of acetate on fatty acid synthesis by the mTOR/eIF4E signaling pathway is unclear in bovine mammary epithelial cells (BMECs). The objectives of this study were to investigate the effect of acetate on cell viability, triacylglycerol (TG), and mRNA expression of the genes related to lipid synthesis. The mechanism of acetate regulation milk fat synthesis through the mTOR/eIF4E signaling pathway was assessed by blocking the mTOR signaling pathway and silencing eIF4E in BMECs. Third-passage BMECs were allocated to 6 treatments including 0, 4, 6, 8, 10, and 12 mM acetate to evaluate the effect of acetate on lipid synthesis; the optimum concentration in the first study was selected for the subsequent study. Subsequently, cells were randomly allocated to 4 treatments, 1 control group and 3 treated groups, consisting of acetate (6 mM), rapamycin (100 nM), and acetate + rapamycin to test the role of mTOR signaling pathway response to acetate in milk lipid synthesis. Finally, eIF4E was silenced by small interfering RNA (siRNA) to detect the role of eIF4E in milk lipid synthesis. Treatments included control, eIF4E siRNA, acetate (6 mM), and acetate+ eIF4E siRNA. Results showed that acetate increased TG accumulation and the relative expression of fatty acid synthase (FASN), acetyl-coenzyme A carboxylase α (ACACA), fatty acid-binding protein 3 (FABP3), sterol regulatory element binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma (PPARG), mTOR, eIF4E, P70 ribosomal protein S6 kinase-1 (S6K1), and 4E-binding protein-1 (4EBP1) in a dose-dependent manner. Rapamycin effectively inhibited the positive effect of acetate on the relative expression of mTOR, eIF4E, S6K1, 4EBP1, FASN, ACACA, FABP3, stearoyl-CoA desaturase (SCD1), SREBP1, and PPARG. The upregulation of acetate on the relative expressions of FASN, ACACA, SCD1, and SREBP1 was suppressed when eIF4E was knocked down. It suggested that acetate regulated milk fat synthesis through mTOR/eIF4E signaling pathway in BMECs.

Insights into the Functions of eIF4E-Biding Motif of VPg in Potato Virus A Infection.

The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXLΦ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVAVPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3'RLUC) from the 3' end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3'RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXLΦ motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.

O-GlcNAc transferase activates stem-like cell potential in hepatocarcinoma through O-GlcNAcylation of eukaryotic initiation factor 4E.

O-GlcNAcylation catalysed by O-GlcNAc transferase (OGT) is a reversible post-translational modification. O-GlcNAcylation participates in transcription, epigenetic regulation, and intracellular signalling. Dysregulation of O-GlcNAcylation in response to high glucose or OGT expression has been implicated in metabolic diseases and cancer. However, the underlying mechanisms by which OGT regulates hepatoma development remain largely unknown. Here, we employed the lentiviral shRNA-based system to knockdown OGT to analyse the contribution of OGT in hepatoma cell proliferation and stem-like cell potential. The sphere-forming assay and western blot analysis of stem-related gene expression were used to evaluate stem-like cell potential of hepatoma cell. We found that the level of total O-GlcNAcylation or OGT protein was increased in hepatocellular carcinoma. OGT activated stem-like cell potential in hepatoma through eukaryotic initiation factor 4E (eIF4E) which bound to stem-related gene Sox2 5'-untranslated region. O-GlcNAcylation of eIF4E at threonine 168 and threonine 177 protected it from degradation through proteasome pathway. Expression of eIF4E in hepatoma was determined by immunostaining in 232 HCC patients, and Kaplan-Meier survival analysis was used to determine the correlation of eIF4E expression with prognosis. High glucose promoted stem-like cell potential of hepatoma cell through OGT-eIF4E axis. Collectively, our findings indicate that OGT promotes the stem-like cell potential of hepatoma cell through O-GlcNAcylation of eIF4E. These results provide a mechanism of HCC development and a cue between the pathogenesis of HCC and high glucose condition.

Ferulic acid ameliorates cerebral infarction by activating Akt/mTOR/4E­BP1/Bcl­2 anti­apoptotic signaling in the penumbral cortex following permanent cerebral ischemia in rats.

The aim of the present study was to determine the effects of ferulic acid (FerA) administered immediately following the onset of permanent middle cerebral artery occlusion (MCAo) and then 7 days of ischemia, and also to explore the involvement of protein kinase B (Akt)­induced signaling in the penumbral cortex. Immediately following the onset of MCAo, FerA was intravenously administered to rats at a dose of 60 mg/kg (FerA­60 mg), 80 mg/kg (FerA­80 mg), or 100 mg/kg (FerA­100 mg). FerA­80 mg and FerA­100 mg effectively ameliorated cerebral infarction and neurological deficits 7 days following permanent cerebral ischemia. FerA­80 mg and FerA­100 mg significantly upregulated the expression of phospho­Akt (p­Akt), phospho­mammalian target of rapamycin (p­mTOR), and eukaryotic initiation factor 4E (eIF4E)­binding protein 1 (4E­BP1), and the phospho­4E­BP1 (p­4E­BP1)/4E­BP1 and mitochondrial Bcl­2/Bax ratios, and markedly downregulated the levels of cytochrome c­, cleaved caspase­3­, and terminal deoxynucleotidyl transferase­mediated dUTP­biotin nick­end labeling­immunoreactive cells in the penumbral cortex at 7 days post­ischemia. LY294002, a selective inhibitor of phosphoinositide 3­kinase/Akt signaling, was administered 30 min prior to ischemia, which abrogated the upregulating effects of FerA­100 mg on the expression of p­Akt, p­mTOR, 4E­BP1, p­4E­BP1 and eIF4E, the mitochondrial Bcl­2/Bax ratio and the ameliorating effect of FerA­100 mg on cerebral infarction. FerA administered at doses of 80 and 100 mg/kg exerted beneficial effects against cerebral ischemia by activating Akt­induced signaling. The effects of FerA at doses of 80 and 100 mg/kg on mitochondrial B­cell lymphoma-2 (Bcl­2)­associated X protein­related apoptosis were attributed to the activation of Akt/mTOR/4E­BP1/Bcl­2 anti­apoptotic signaling, and eventually contributed to suppression of the cytochrome c/caspase­3 activation pathway in the penumbral cortex 7 days following permanent cerebral ischemia.

Maintenance of Proteostasis by P Body-Mediated Regulation of eIF4E Availability during Aging in Caenorhabditis elegans.

Aging is accompanied by a pervasive collapse of proteostasis, while reducing general protein synthesis promotes longevity across taxa. Here, we show that the eIF4E isoform IFE-2 is increasingly sequestered in mRNA processing (P) bodies during aging and upon stress in Caenorhabditis elegans. Loss of the enhancer of mRNA decapping EDC-3 causes further entrapment of IFE-2 in P bodies and lowers protein synthesis rates in somatic tissues. Animals lacking EDC-3 are long lived and stress resistant, congruent with IFE-2-deficient mutants. Notably, neuron-specific expression of EDC-3 is sufficient to reverse lifespan extension, while sequestration of IFE-2 in neuronal P bodies counteracts age-related neuronal decline. The effects of mRNA decapping deficiency on stress resistance and longevity are orchestrated by a multimodal stress response involving the transcription factor SKN-1, which mediates lifespan extension upon reduced protein synthesis. Our findings elucidate a mechanism of proteostasis control during aging through P body-mediated regulation of protein synthesis in the soma.

Levels of 4EBP1/eIF4E Activation in Renal Cell Carcinoma Could Differentially Predict Its Early and Late Recurrence.

BACKGROUND: The objective was to explore the predictive markers of late recurrence (LR) > 5 years after curative nephrectomy for renal cell carcinoma (RCC). PATIENTS AND METHODS: We retrospectively examined the data from 303 patients with localized clear cell RCC treated surgically at our institution from 1993 to 2011. Activation of the eukaryotic initiation factor (eIF)4E-binding protein 1 (4EBP1)/eIF4E axis at the mammalian target of rapamycin complex 1 (mTORC1) was evaluated in the tumor specimens. Weak, intermediate, and strong immunohistochemistry staining grades were defined for 4EBP1, phosphorylated 4EBP1, and eIF4E. The effects of clinicopathologic factors and activation level grades on tumor recurrence were analyzed using multivariate Cox regression models. To validate the present findings, we investigated clinical data from The Cancer Genome Atlas and protein/phosphoprotein data from corresponding patients from The Cancer Proteome Atlas. RESULTS: Of the 303 patients, 31 and 16 patients developed early recurrence (ER, ≤ 5 years) and LR, respectively. The activation levels were comparable among the subcategories of pathologic TN stage, Fuhrman grade, and microvascular and capsular invasion. Pathologic stage ≥ T1b, Fuhrman grade 3/4, and an intermediate or strong activation level correlated significantly with overall recurrence and ER. Strong activation of the axis and pathologic stage ≥ T1b were identified as independent predictors of LR. Only 2 patients with weak activation experienced recurrence (1 each with ER and LR). Similar results were confirmed by the analyses of The Cancer Genome Atlas and The Cancer Proteome Atlas data. CONCLUSION: The activation level of the axis in RCC tissues could independently predict for recurrence and differentially affect the timing of recurrence.

Dietary cystine is important to maintain plasma mercaptalbumin levels in rats fed low-protein diets.

The oxidized/reduced state of plasma albumin in rats is influenced by the quantity of dietary protein. However, the effects of the protein quality on the oxidized/reduced state of plasma albumin are not clear. We hypothesized that the quality of dietary protein might modulate the oxidized/reduced state of plasma albumin. The aim of the present study was to examine whether the amino acid composition of dietary protein modulates the oxidized/reduced state of plasma albumin in rats. Male Sprague-Dawley rats were fed low-protein diets containing 5% casein (CA), 5% egg white (EW), or 6% wheat gluten (WG) for 2 weeks. The plasma albumin concentration gradually decreased in rats fed each diet; however, there was no significant difference among the groups. In rats fed the 5% CA diet, the percentage of mercaptalbumin within the total plasma albumin was significantly lower than in those fed the EW or WG diet. Compared with EW or WG, CA contains lower amounts of glycine and cystine. In rats fed a 5% CA diet supplemented with cystine, the percentage of mercaptalbumin was significantly higher than that in rats fed a 5% CA diet supplemented with glycine. The expression of hepatic eukaryotic initiation factor 4E-binding protein 1 was significantly lower in rats fed the cystine-supplemented diet than in those fed the glycine-supplemented diet. These results suggest that dietary protein with a high cystine content maintains plasma mercaptalbumin levels in rats fed low-protein diets.

Total Saponins of Rubus Parvifolius L. Exhibited Anti-Leukemia Effect in vivo through STAT3 and eIF4E Signaling Pathways.

OBJECTIVE: To investigate the anti-leukemia effect of total saponins of Rubus parvifolius L. (TSRP) on K562 cell xenografts in nude mice and the mechanisms of action. METHODS: The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group(Ara-c) and 3 TSRP groups (20, 40 and 100 mg/kg). The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 (STAT3), eukaryotic initiation factor 4E (eIF4E) and B-cell lymphoma-2 (bcl-2) were determined with immunohistochemistry tests. RESULTS: Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a signifificant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a signifificant decrease in tumor growth rate and tumor weight in comparison to the control group (all P<0.05). The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of eIF4E and STAT3 were decreased obviously after the treatment of TSRP. CONCLUSION: TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.

Integrin ß1 mediates 5-fluorouracil chemoresistance under translational control of eIF4E in colorectal cancer.

PURPOSE: In recent years, aberrant mRNA translational control has gained much attention as a critical player in the malignant process of tumors. Eukaryotic initiation factor 4E (eIF4E), by binding to the mRNA cap, can regulate specific protein synthesis, contributing to malignancy in human tumors. However, integrin ß1 mediated chemoresistance under translational control remains unknown in colorectal cancer. PATIENTS AND METHODS: The expression relationship between eIF4E and Integrin ß1, along with their clinical significance was investigated in colorectal cancerous tissues of 118 cases using immunohistochemistry. Cell transfection techniques of small interfering RNA (siRNA) and cDNA expression plasmid were applied to investigate the molecular relationship of integrin ß1 and eIF4E and their biological effects on 5FU resistance in SW480 and LoVo cell lines. RESULTS: The expression of eIF4E and integrin ß1 was positively correlated in colorectal cancer, and patients with high expressions of both markers tended to have a worse prognosis according to a Kaplan-Meier survival analysis. Integrin ß1 could contribute to 5-fluorouracil (5FU) resistance in colorectal cancer cell lines. Moreover, the protein expression of ß1 could be regulated by eIF4E, interestingly, without any change of mRNA expression level. Significantly, Hoechst/PI double staining and an MTT assay proved integrin ß1 could contribute to cellular survival and 5FU resistance under translational control of eIF4E in these cells. CONCLUSION: We conclude that integrin ß1 mediated 5FU chemo resistance in colorectal cancer could be translationally regulated by eIF4E. Promisingly, targeting key molecules of this translational apparatus may provide an innovative therapeutic strategy for colorectal cancer.

Total Saponins of Rubus Parvifolius L. Exhibited Anti-Leukemia Effect in vivo through STAT3 and eIF4E Signaling Pathways / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-leukemia effect of total saponins of Rubus parvifolius L. (TSRP) on K562 cell xenografts in nude mice and the mechanisms of action.@*METHODS@#The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group(Ara-c) and 3 TSRP groups (20, 40 and 100 mg/kg). The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 (STAT3), eukaryotic initiation factor 4E (eIF4E) and B-cell lymphoma-2 (bcl-2) were determined with immunohistochemistry tests.@*RESULTS@#Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a signifificant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a signifificant decrease in tumor growth rate and tumor weight in comparison to the control group (all P<0.05). The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of eIF4E and STAT3 were decreased obviously after the treatment of TSRP.@*CONCLUSION@#TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.

The activation of autophagy protects neurons and astrocytes against bilirubin-induced cytotoxicity.

Unconjugated bilirubin (UCB) neurotoxicity involves oxidative stress, calcium signaling and ER-stress. The same insults can also induce autophagy, a process of "self-eating", with both a pro-survival or a pro-apoptotic role. Our aim was to study the outcome of autophagy activation by UCB in the highly sensitive neuronal SH-SY5Y cells and in the resistant astrocytoma U87 cells. Upon treatment with a toxic dose of UCB, the conversion of LC3-I to LC3-II was detected in both cell lines. Inhibition of autophagy by E64d before UCB treatment increased SH-SY5Y cell mortality and made U87 cells sensitive to UCB. In SH-SY5Y autophagy related genes ATG8 (5 folds), ATG18 (5 folds), p62 (3 folds) and FAM 129A (4.5 folds) were induced 8h after UCB treatment while DDIT4 upregulation (13 folds) started at 4h. mTORC1 inactivation by UCB was confirmed by phosphorylation of 4EBP1. UCB induced LC3-II conversion was completely prevented by pretreating cells with the calcium chelator BAPTA and reduced by 65% using the ER-stress inhibitor 4-PBA. Pretreatment with the PKC inhibitor reduced LC3 mRNA by 70% as compared to cells exposed to UCB alone. Finally, autophagy induction by Trifluoroperazine (TFP) increased the cell viability of rat hippocampal primary neurons upon UCB treatment from 60% to 80%. In SH-SY5Y cells, TFP pretreatment blocked the UCB-induced cleaved caspase-3 protein expression, decreased LDH release from 50% to 23%, reduced the UCB-induction of HO1, CHOP and IL-8 mRNAs by 85%, 70% and 97%. Collectively these data indicate that the activation of autophagy protects neuronal cells from UCB cytotoxicity. The mechanisms of autophagy activation by UCB involves mTOR/ER-stress/PKC/calcium signaling.

PLK1 regulates spindle association of phosphorylated eukaryotic translation initiation factor 4E-binding protein and spindle function in mouse oocytes.

Oocyte meiotic spindles are associated with spindle-enriched mRNAs, phosphorylated ribosome protein S6, and phosphorylated variants of the key translational regulator, eukaryotic translation initiation factor 4E-binding protein 1 (eIF4E-BP1), consistent with translational control of localized mRNAs by eIF4E-BP1 in facilitating spindle formation and stability. Using specific kinase inhibitors, we determined which kinases regulate phosphorylation status of eIF4E-BP1 associated with meiotic spindles in mouse oocytes and effects of kinase inhibition on chromosome congression and spindle formation. Neither ataxia telangiectasia-mutated kinase nor mechanistic target of rapamycin inhibition significantly affected phosphorylation status of spindle-associated eIF4E-BP1 at the phosphorylation sites examined. Spindle-associated phospho-eIF4E-BP1, spindle formation, and chromosome congression were strongly disrupted by polo-like kinase I (PLK1) inhibition at both metaphase I (MI) and MII. In addition, direct inhibition of eIF4E-BP1 via 4EGI led to spindle defects at MI, indicating a direct role for eIF4E-BP1 phosphorylation in meiotic spindle formation. PLK1 also regulated microtubule dynamics throughout the ooplasm, indicating likely coordination between spindle dynamics and broader ooplasm cytoskeletal dynamics. Because diverse upstream signaling pathways converge on PLK1, these results implicate PLK1 as a major regulatory nexus coupling endogenous and exogenous signals via eIF4E-BP1 to the regulation of spindle formation and stability.

Targeting eukaryotic protein translation in mesothelioma.

The default mechanism for protein translation in eukaryotes involves activation of the eIF4 complex at the 5' end of mRNA. This activity is upregulated in cancers, resulting in the expression of a variety of proteins necessary for the development and maintenance of the neoplastic state. Not surprisingly, mesothelioma demonstrates this same reliance on activation of 5' cap mediated translation. Efforts are ongoing to target and exploit our knowledge of this key molecular switch for cancer therapy. Agents targeting the critical eIF4E cap binding protein, disruption of the eIF4 complex, and exploitation for oncolytic virotherapy are some of the important areas of current research in mesothelioma protein translational research.

Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies.

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.