Effect of Silencing Eps8 Gene Expression on the Biology Activity of Human Leukemia K562 Cells and Its Molecular Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>The study was to investigate the effect of silencing Eps8 gene expression on proliferation and apoptosis of human leukemia K562 cells and its molecular mechanism.</p><p><b>METHODS</b>The expetriments were divided into 3 groups, including blank control group(K562 cells without treatment), K562-shRNA group(K562 cells transfected by specific Eps8-shRNA lenticiral vector) and K562-NC group(K562 cells transfected by negtive control lenticiral vector). K562 cells with stably-silenced Eps8 gene were constucted by lentibirus-mediated RNA technology. The efficacy of transfection was observed by fluorescence microscopy and the changes of Eps8 mRNA and protein level were detected by RT-PCR and Western blot respectively. Cell proliferation was confirmed by typan blue exclusion and MTT method. The apoptosis rate of cells was analysed by the flow cytometry, and colony forming was detected by methylcellulose colony forming assay. The protein level change of phosphrylated-AKT were detected by Western blot.</p><p><b>RESULTS</b>Stably-silenced Eps8 gene K562 cells and the negative control cells were successfully constructed. Compared with the blank control group and the K562-NC group, the proliferation of K562-shRNA cells were siginificantly inhibited(P<0.05); the apoptosis of K562-shRNA cells increased(P<0.05). In addition, the methylcellulose colony forming assay showed that the colony forming was dramatically suppressed in K562-shRNA cells (P<0.05). Furthermore, knocking down Eps8 gene reduced the protein level of AKT phosphrylation at both residue Ser437 and Thr308(P<0.05), while there was no obvious change in the level of total-AKT(P>0.05). Knocking down Eps8 gene reduced the protein level of m-TOR phosphrylation and PRAS40 phosphrylation (P<0.05), while there was no obvious change in the level of total-mTOR and PRAS40 (P>0.05).</p><p><b>CONCLUSION</b>Silencing Eps8 gene through lentvirus can inhibit the cell proliferation and promote the apoptosis of human leukemia K562 cells, which possibly relates with the inhibition of AKT/mTOR activation.</p>

ApoE4 increases glycogen synthase kinase 3β expression and Tau phosphorylation in U87 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the relations among apolipoprotein E4, Tau protein and glycogen synthase kinase 3β (GSK-3β).</p><p><b>METHODS</b>U87 cells were transfected with pIRES-EGFP (control) or the recombinant plasmids ApoE4/pIRES-EGFP or ApoE3/pIRES-EGFP, and the expression levels of p-Tau/Tau and GSK-3β in the cells were examined with Western blotting. To further confirm the effect of ApoE on GSK-3β and p-Tau expressions, a short interfering RNA (siRNA) targeting ApoE (ApoE-siRNA) was transfected into U87 cells via Lipofectamine 2000 and the protein expressions were examined 24 h later.</p><p><b>RESULTS</b>Compared with those in the control group, the expressions levels of both GSK-3β and p-Tau/Tau increased significantly in the cells transfected with ApoE4 and ApoE3 plasmids (P<0.01), and the ApoE4 plasmid produced a more potent effect than the ApoE3 plasmid on the protein expressions (P<0.01). ApoE knockdown resulted in significantly reduced expressions of GSK-3β (P<0.001) and p-Tau (P<0.01) in the cells.</p><p><b>CONCLUSION</b>ApoE4 can enhance Tau phosphorylation though upregulating GSK-3β, which sheds light on a new role of ApoE4 in Alzheimer's disease.</p>

Prediction and Identification of HLA-A*0201 Restricted CTL Epitopes from Eps8 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To find and identify HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes from epidermal growth factor pathway substrate number 8 (Eps8) for specific immunotherapy based on Eps8-derived epitopes in clinic.</p><p><b>METHODS</b>Online biological softwares involved C-proteasomal cleavage, MHC class I binding affinity and TAP transport efficiency were used for prediction of HLA-A*0201 restricted epitopes from Eps8. Then, T2-binding assays and peptide/MHC complex stability tests were used to further verify the predicted epitopes. Specific secretion of IFN-γ from human CTL was assayed using the IFN-γ ELISPOT kit, and cytolytic activity was measured by a 4-h lactate dehydrogenase (LDH) release assay. Finally, the functional effects in vivo were measured in HLA-A*0201/Kb transgenic (Tg) mice.</p><p><b>RESULTS</b>Four natural epitopes were designed through online biological softwares. Of the four epitopes selected, p360-368 was found to have the high binding affinity to HLA-A*0201, while p101-109 and p276-284 showed moderate affinities. DC50 of peptide/MHC complexes of the natural epitopes mentioned were all longer than 8 h. In functional assays with human PBMNC in vitro and in HLA-A*0201/Kb transgenic mice in vivo, CTLs primed by each epitope (p101-109, p276-284 and p360-368) secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A*0201-restricted and Eps8-specific manner.</p><p><b>CONCLUSION</b>Natural epitopes (p101-109, p276-284 and p360-368) may be the HLA-A*0201 restricted epitope derived from Eps8.</p>

Cytotoxicity of Naja Naja Actra Venom Component combined with activated immune cells on leukemia cell line KG1a / 中国实验血液学杂志

This study was aimed to investigate the cytotoxic effect of the Naja Naja Actra Venom Component (NNAVC) combined with activated immune cells on human acute myeloblastic leukemia line KG1a cells. The cytotoxic effects of NNAVC at different concentrations on KG1a cells were measured by CCK-8 method. LDH releasing assay was used to detect the cytotoxic effects of activated immune cells, NNAVC combined with activated immune cells on KG1a cells and the sensitivity of KG1a treated with NNAVC to activated immune cells. The results showed that the inhibitory rate of NNAVC on KG1a cells increased with the concentration enhancement, the cytotoxicity of activated immune cells at the different effector to target (E:T) ratios(6.25:1, 12.5:1, 25:1) on KG1a cells were 12.30%, 24.85% and 52.26%. The cytotoxicity of NNAVC combined with activated immune cells at the different E:T cell ratios (10:1, 20: 1) on KG1a cells were 56.21% and 85.59%, which were higher than that of NNAVC or activated immune cells alone. The cytotoxicity of activated immune cells at the E: T cell ratio of 10:1 on KG1a cells treated with NNAVC at different concentrations were 25.65%, 31.33%, 28.63% and 16.78%, respectively, and that at the E:T cell ratio of 20: 1 were 40.62%, 44.70%, 44.62% and 40.72%. It is concluded that:both of NNAVC and activated immune cells have lethal effect on KG1a cells, and the combination of NNAVC and activated immune cells can strengthen their effect on KG1a.

Advancement of peptide vaccines for hematologic malignancies / 中国实验血液学杂志

Despite recent significant advances in the treatment of hematological malignancies, relapse of this disease is of great note with the existence of the minimal residual disease (MRD). Tumour peptide vaccine seems to be one of the effective immunotherapies for eliminating tumor cells of MRD. This review focuses on the late results of clinical trails of peptide vaccination protocols targeting WT1, RHAMM, BCR-ABL, PR1 in hematological malignancies and the development of specific immune responses to PRAME and Survivin peptides. An outlook to heteroclitic peptides, new adjuvants, combined peptide vaccines and Ad-tWT1 vaccine is also given to further explore the possibility to enhance the efficacy of the peptide vaccine.