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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.
Subject(s)
Animals , Humans , Male , Mice , Apoptosis , Eukaryotic Initiation Factor-4E , Physiology , K562 Cells , Leukemia , Drug Therapy , Pathology , Rubus , Chemistry , STAT3 Transcription Factor , Physiology , Saponins , Pharmacology , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
Objective To investigate the function of tripartite motif protein 22 (TRIM22) and the interaction with eukaryotic translation initiation factor-4E (eIF4E) in the differentiation of NB4 cells, one kind of acute promyelocytic leukemia cells, which elucidates the mechanism of TRIM22 targeting to regulate eIF4E.Methods The model of NB4 cells inducing differentiation was established in vitro.The expression changes of gene and protein of TRIM22 and eIF4E were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.In addition, the effect on cell function and protein expression level of eIF4E after adopting electroporation technology to depress or over-express TRIM22 was detected by CCK-8 and flow cytometry.Finally, the interaction of TRIM22 and eIF4E was verified by using co-immunoprecipitation (CO-IP).Results The mRNA relative expression level of TRIM22 was gradually increasing from 1.01±0.15 to 30.98±2.79 (F=280.700, P=0.000), and the protein relative expression level was gradually increasing from 0.22±0.03 to 0.51±0.05 (F=51.430, P=0.000) after the all-trans-retinoic acid (ATRA) induction for NB4 cell.However, the mRNA relative expression level of eIF4E was gradually decreasing from 1.01±0.09 to 0.47±0.06 (F=20.520, P=0.000), with the same trend, the protein relative expression level was gradually decreasing from 0.97±0.02 to 0.64±0.09 (F=14.700, P=0.001).The expression level of PE-CD11b in the TRIM22 over-expression group with ATRA detected by flow cytometry [(78.80±2.00)%] was higher than that in the transfection group of empty vetor with ATRA [(58.70±2.70)%] (t=9.535, P=0.000) and the cotransfection group with ATRA [(61.60±3.80)%] (t=8.187, P=0.000).Meanwhile, the protein level of eIF4E changed reversely after over-expressing the gene level of TRIM22 (t=4.985, P=0.007).The CO-IP experiment was used to verify the interaction of TRIM22 and eIF4E.ConclusionTRIM22 is able to promote the cell differentiation during the process of NB4 cells differentiation.Furthermore, eIF4E is a target of TRIM22 for binding with, which plays an important role in depressing the expression of eIF4E.
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AIM: To investigate the expression and significance of MAPK-interacting kinase-2 ( Mnk2 ) and eukaryotic initiation factor 4E ( eIF4E) in the patients with resected esophageal squamous cell carcinoma ( ESCC ). METHODS:The protein expression of Mnk2 and eIF4E in ESCC tissues (98 cases) and normal esophageal tissues (20 cases) were assessed by immunohistochemistry (IHC), and their correlations with clinicopathological features were statisti-cally analyzed.RESULTS:The over-expression rate of Mnk2 and eIF4E was 68.4%(67/98) and 61.2%(60/98), re-spectively.The expression of Mnk2 had a positive correlation with eIF4E (P<0.05).Clinicopathologic analysis showed that Mnk2 expression was significantly correlated with T classification ( P<0.05 ) and clinical stage ( P<0.05 ) .CON-CLUSION:The over-expression of Mnk2 was significantly related to the tumor invasive depth , TNM stages and expression of eIF4E in ESCC.Expression of Mnk2 and eIF4E may have a cooperative formation mechanism in the development of ESCC.
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The phosphatidylinositide-3-kinase(PI3K)/ protein kinase B(Akt)/ mammalian target of rapamycin(mTOR)pathway is recognized to have an important role in the development and progression of colo-rectal cancer(CRC). The most extensively characterized downstream targets of mTORC1 are ribosomal protein S6 kinase 1(p70S6K1)and eukaryotic translation initiation factor(eIF)4E-binding protein 1(4EBP1),both of which are crucial to the regulation of protein synthesis. The abnormal expression of p70S6K1 and 4EBP1 in CRC has become the focus of attention.
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Objective To investigate the activation and clinical significance of the mammalian target of rapamycin (mTOR) pathway related protein and eukaryotic translation factor 4E-binding protein 1 (4E-BP1) in gastric cancer.Methods The activation of mTOR and 4E-BP1 in gastric cancer tissues of 38 surgical patients were detected by immunohistochemical method.The differences of phosphorylated mTOR and 4E-BP1 expression among cancerous tissues,para-cancerous tissues and normal gastric mucosa tissues and dinicopathological variables were analyzed by Chi square test and Kruskal-Wallis test.Results The positive expression rate of phosphorylated mTOR in the gastric cancerous tissues was significantly higher than that of para-cancerous tissues and normal tissues [71.1％ (27/38),50.0 ％ (19/38) and 44.7 ％ (17/38),x2 =11.031,P =0.026].The positive expression rate of downstream protein 4E-BP1 in the gastric cancer tissues was also significantly higher than that of paracancerous tissues and normal tissues [68.4％(26/38),57.9％(22/38) and 28.9％ (11/38),x2 =13.943,P=0.007].There was no correlation between phosphorylated mTOR and 4E-BP1 expression and tumor Lauren's sub-type,infiltration,differentiation degree,lymph node metastasis and patient's age.There was statistical significant difference between activated 4E-BP1 expression and tumor size in gastric cancer (H=3.86,P＜0.05).Conclsions mTOR pathway was over activated in gastric cancer.There was difference between phosphorylation degree of its downstream protein 4E-BP1 and the tumor size.
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Objective: To observe the effects of low concentration of CGP57380, an inhibitor of mitogen-activated protein kinase-interacting kinase 1 (MNK1) on the proliferation and apoptosis of human lung adenocarcinoma A549 cells. Methods: A549 cells were treated with low concentrations (1-4 μmol/L) CGP57380 for 24-72 hours, then the cell proliferation was assessed by MTT assay, and the apoptosis and cell cycle distribution were detected by flow cytometry. The expression levels of phosphorylated MNK1 (p-MNK1) and phosphorylated human eukaryotic translation initiation factor 4E (p-eIF4E) proteins in A549 cells treated with 1 μmol/L CGP57380 for 48 hours were examined by Western blotting. Results: Comparing to the control, the proliferation of A549 cells was inhibited after treatment with different concentrations of CGP57380 for 48 hours (all P < 0.05). A549 cells treated with 2-4 μmol/L CGP57380 for 72 hours were induced G2/M cell cycle arrest (both P < 0.05). CGP57380 at different concentrations could induce dose-dependent apoptosis in A549 cells. The expression levels of p-MNK1 and p-eIF4E were significantly lower in A549 cells treated with 1 μmol/L CGP57380 than those in the control (P < 0.05). Conclusion: Low concentration of CGP57380 can inhibit the proliferation of human lung adenocarcinoma A549 cells and induce their apoptosis, implying that MNK1 gene may be a candidate target for the treatment of lung cancer. Copyright © 2013 by TUMOR.
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Objective To explore the influence on the expression of elF4E and heparanase by antisense oligodeoxyribonucleotides (ASODN) transfection in human bladder carcinoma BIU-87 cells.Methods After transfecting the 2.5,5.O,7.5 μg/ml eIF4E ASODN into BIU-87 cells,at 24 h,48 h and 72 h,a cell control group (no transfection),a blank control group (empty liposomes) and a non-sense control group (transfected with non-sense ASODN) were established.The expression of eIF4E,HPA and mRNA were detected by in situ hybridration.The expression of eIF4 and HPA protein were detected by immunocytochemistry.SPSS 13.0 statistical software was used for statistical analysis.Results The expression quantity of eIF4E protein and mRNA were lower in the experimental groups ( protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:3.55 ±0.52,3.52 ±0.51,3.22.±0.44 respectively; 5.0 μg/ml group:3.43 ±0.47,3.41 ± 0.46,3.19 ± 0.41 respectively ; 7.5 μg/ml group:2.36 ± 039,2.33 ± 0.37,2.05 ± 0.30 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:3.19 ±0.41,3.13 ±0.39,2.90 ±0.38 respectively ; 5.0 μg/ml group:3.07 ± 0.39,2.94 ± 038,2.27 ± 0.37 respectively ; 7.5 μg/ml group:2.22 ± 037,2.06 ± 0.30,1.95 ± 0.29 respectively.All data were less than those in the control groups (P ＜0.05).The expression quantity of HPA protein and mRNA were lower in experimental groups (protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:4.44 ±0.55,4.40 ±0.54,3.99 ±0.52 respectively; 5.0 μg/ml group:4.41 ±0.55,4.21 ±0.53,3.77 ±0.50 respectively; 7.5 μg/ml group:4.02 ±0.52,3.98 ±0.52,2.32 ±0.37 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:4.12 ±0.51,3.75 ± 0.50,3.63 ± 0.45 respectively ; 5.0 μg/ml group:4.00 ± 0.51,3.71 ± 0.50,3.54 ± 0.44respectively ; 7.5 μg/ml group:3.87 ± 0.52,3.61 ± 0.49,2.08 ± 0.30 respectively).All data were less than in control groups ( P ＜ 0.05 ).There was a positive correlation on expression of HPA protein and eIF4E protein by inhibitory effect of eIF-4E ASODN (protein r=9.23,mRNA r=9.59,P ＜0.01).Conclusion eIF-4E ASODN might be used to inhibit the expression of eIF-4E gene and HPA gene in bladder cancer BIU-87 cells.
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Activity-dependent dendritic translation in CNS neurons is important for the synapse-specific provision of proteins that may be necessary for strengthening of synaptic connections. A major rate-limiting factor during protein synthesis is the availability of eukaryotic translation initiation factor 4E (eIF4E), an mRNA 5'-cap-binding protein. In this study we show by fluorescence in situ hybridization (FISH) that the mRNA for eIF4E is present in the dendrites of cultured rat hippocampal neurons. Under basal culture conditions, 58.7 +/- 11.6% of the eIF4E mRNA clusters localize with or immediately adjacent to PSD-95 clusters. Neuronal activation with KCl (60 mM, 10 min) very significantly increases the number of eIF4E mRNA clusters in dendrites by 50.1 and 74.5% at 2 and 6 h after treatment, respectively. In addition, the proportion of eIF4E mRNA clusters that localize with PSD-95 increases to 74.4 +/- 7.7% and 77.8 +/- 7.6% of the eIF4E clusters at 2 and 6 h after KCl treatment, respectively. Our results demonstrate the presence of eIF4E mRNA in dendrites and an activity-dependent increase of these clusters at synaptic sites. This provides a potential mechanism by which protein translation at synapses may be enhanced in response to synaptic stimulation.