Synergistic Killing Effects of Arsenic Trioxide Combined with Itraconazole on KG1a Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of arsenic trioxide combined with itraconazole on proliferation and apoptosis of KG1a cells and its potential mechanism.</p><p><b>METHODS</b>The cell morphology was observed with Wrighe-Giemsa staining; cell survival rate was examined by CCK-8; and colony formation capacity was measured by methylcellulose colony formation test; the flow cytometry was used to analyse the cell apoptosis rate and cell cycle; the protein expressions of BCL-2,caspase-3,BAX,SMO,Gli1 and Gli2 were detected by Western-blot.</p><p><b>RESULTS</b>The arsenic trioxide and itraconazole alone both could inhibit the KG1a cell proliferation in dose-and time-dependent manner. In comparison between single and combined drug-treatment group, both the cell survival rate and the colony number of the single drug-treatment group were significantly lower(P<0.05), and the apoptosis rate was higher in the combined drug-treatment group. In the combined-treatment group, the protein expression of Caspase-3 and BAX was upregulated, while the protein expression of BCL-2,SMO,Gli1 and Gli2 was downregulated.</p><p><b>CONCLUSION</b>Arsenic trioxide combined with itraconazole can inhibit the KG1a cell proliferation and induce apoptosis, which may be related with the inhibition of Hh signaling pathway and upregulation of both Caspase-3 and BAX protein expression, and provided experimental data of arsenic trioxide combined with itraconazole for the treatment of refractory AML.</p>

Establishment of a Xenografted Acute Myeloid Leukemia Model by using Zebrafish / 中国实验血液学杂志

<p><b></b>Objectve: To investigate the feasibility of establishing xenografted leukemia model by zebrafish, so as to provide the more direct model in vitro and experimental evidence for study of acute myeloid leukemia and screening of the drugs for targeting therapy.</p><p><b>METHODS</b>Acute myeloid leukemia cell line KG-1a was labeled with red fluorescent dye-MitoRed, then the labeled cells were injected into the yolk sac of zebrafish embryos. Morphological observation, cell count and histopathological detection were used to analyse the infiltration and metastasis of KG-1a cells in zebrafish.</p><p><b>RESULTS</b>KG1a cells could proliferate and gradually spread to the entire abdominal cavity of the zebrafish after KG-1a cells were injected into the yolk sac during 1-7, the results of cell counting in vitro also proved a significant proliferation of KG-1a cells in zebrafish, suggesting that the implanted leukemia stem cells could survive, proliferate and spread in zebrafish. Further study showed that the implanted cells could be transfered to the liver of zebrafish, these cells displayed the signature of KG-1a cells by hematoxylin-eosin(HE) staining.</p><p><b>CONCLUSIONS</b>Human acute myeloid leukemia cells KG1a can survive, proliferate and migrate in zebrafish, suggesting xenografted leukemia model of zebrafish has been successfully established. This model may be benefitcial for the study of acute myeloid leukemia and the screening of the drugs for targeting therapy of acute myeloid leukemia.</p>

Honokiol combined with Gemcitabine synergistically inhibits the proliferation of human Burkitt lymphoma cells and induces their apoptosis / 中国实验血液学杂志

This study was aimed to investigate the effect of Honokiol (HNK) combined with Gemcitabine (GEM) on the proliferation and apoptosis of human Burkitt lymphoma Raji cells. Cell proliferation was detected by CCK-8 method to study the role of Honokiol and Gemcitabine in Raji cells. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The level of apoptosis-related protein BCL-2 was measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (P < 0.01) and the inhibition rate was related to the concentration and action time of HNK; and apoptosis rate markedly increased (P < 0.01), while most Raji cells were arrested at G0/G1 phase and decreased in S phase after treatment with combination of two drugs; the expression of BCL-2 protein decreased (P < 0.01). It is concluded that Honokiol combined Gemcitabine can synergistically inhibit the proliferation, induce cell apoptosis, and down-regulate the expression of BCL-2 in Raji cells. The possible mechanism of synergistic effect may be related with arrest of cell cycle at G0/G1 phase and downregulation of the expression of BCL-2.

Expression and antibody preparation of FKBP38 and its application / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) analyses.</p><p><b>METHODS</b>The N-terminal (1-207 aa) and C-terminal (209-387 aa) cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C. After sequencing, the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG. The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE. Rabbits were immunized with the purified recombinant protein to prepare the antiserum, which were analyzed by WB, IHC and IF.</p><p><b>RESULTS</b>The recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed. After IPTG induction, the E.coli transformed with these plasmids expressed GST-tagged protein, which was successfully purified. Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines. Immunofluorescence assay showed that FKBP38 was located mainly on the mitochondria. Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells.</p><p><b>CONCLUSION</b>We successfully expressed and purified N- and C-terminal of FKBP38, and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB, IF and IHC assays, which facilitates further functional investigation of FKBP38.</p>

Gamma secretase inhibitor-I exerted cytotoxic effects on malignant glionm cell lines by inducing cell cycle arrest and apoptosis / 中华神经医学杂志

Objective To investigate the role of gamma secretase inhibitor-I (GSI-I) in cell proliferation and apoptosis of human glioma cell lines U87 and U251.Methods RT-PCR and fluorescent quantitative RT-PCR (qRT-PCR) were employed to evaluate the expressions of Notch receptors and their target gene Hes-I in both U87 and U251 cells treated by GSI-I,respectively.Then,MTT assay was used to examine the effects of GSI-I on cell proliferation of the 2 glioma cells.Meanwhile,flow cytometry technique was also employed to detect the cell cycle changes and apoptosis induced by GSI-I treatment.Results The activity of Notch pathway was inhibited by GSI-I treatment through down-regulating the expression of Notch receptors target gene Hes-I in both U87 and U251 cells.Treatment with 2.5μmol/L GSI-I or above concentrations could significantly induce the cell cycle arrest of U87 and U251 cells and these effects were positively concentration-dependent.Flow cytometry technique showed that GSI-I inhibited the cell proliferation by inducing the cell cycle arrest of U87 cells at GI phase and inducing the apoptosis of U251 cells.Conclusion GSI-I can dramatically inhibit the cell proliferation and induce the apoptosis of U87 and U251 cells,providing a reliable evidence for clinical glioma treatment.