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Objective To investigate the effect of Angelica Sinensis polysaccharide (ASP) on proliferation, differentiation and transplantation of human leukemia stem cells (LSCs) . Methods 1. Effect of angelica sinensis polysaccharides on proliferation of CD34
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Objective:To investigate the expression of SHIP1 in the patients with acute myeloid leukemia and its effect on the apoptosis of human leukemia cells.Methods:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was detected by Westem blot.U937 cells was transfected with SHIP1 expression vector (pEGFP-SHIP1 group) and empty vector control (pEGFP group) respectively,U937 cells without transfection were used as the control group.Flow cytometry was used to detect the apoptosis of the cells,the expression of SHIP1,Bcl-2,Bax,Akt,p-Akt were detected by western blot.Results:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was significantly lower than that of the normal human bone marrow SHIP 1 (P＜0.01).The SHIP1 and Bax expressions as well as the apoptotic rate ofpEGFP-SHIP1 group were significantly higher than those of the control group(P＜0.01),while the Bcl-2 and p-Akt expressions were significantly lower than those in the control group(P＜0.01).Conclusions:SH-P1 expression was down regulated in the bone marrow of patients with acute myeloid leukemia.SHIP1 could promote the apoptosis of human leukemia cells via Akt signaling pathway.
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Aim To explore the proteomics mechanism of the differentiation induction effect of 4-amino-2-trif-luoromethyl-phenyl retinate(ATPR)on human leukemi-a K562 cells. Methods Human leukemia K562 cells were incubated with the same concentration (1 × 10 - 6 mol·L - 1 ) of ATPR or ATRA for 48 hours. The total cell proteins were collected, purified and digested by trypsin, solid phase extraction, and the peptides were detected by ESI-LC-MS / MS. The difference of the pro-tein expression between the cells treated with ATPR and ATRA was compared by using the Discoverer Pro-teome 1. 2 software, and the molecular function, the biological process and other information of those pro-teins were analyzed based on the DAVID, KEGG, STRING databases. Results 120 specific proteins were identified only in the ATPR group, 143 only in the ATRA group, and 422 other proteins in both groups. Results of DAVID analysis showed that ATPR-induced specific proteins were mainly involved in 39 biological processes of proteins and macromolecules metabolism, protein transport and localization and so on. Results of KEGG analysis revealed that ATPR-in-duced proteins participated in signal pathways, mainly metabolic pathways, PI3K-Akt signal pathway, TGF-beta signal pathway and other pathways in cancer. String protein interaction network analysis displayed that ATPR-induced proteins, like EIF3A, EIF6, RPL3, RPL8, RPL13, RPL7A, RPL21, RPS3, RPS14, NACA, BTF3, NHP2L1, PPP2CA proteins had direct interactions with more than or equal to 10 associated proteins. Conclusion The differentiation induction effect of ATPR on K562 cells might be as-cribed to the ATPR-induced proteins interaction net-work and the specific central proteins it induced, which are involved in the regulation of cell prolifera-tion, differentiation and apoptosis.
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Objective: To explore the effects and the possible mechanism of combined injection of Angelica sinensis polysaccharide (ASP) and cytarabine (Ara-C) on the bone marrow mononuclear cells (BMMCs) of the transplanted human leukemia mouse model. Methods: K562 cells (2×107)were transplanted into the tail vein of mice to establish the transplanted human leukemia NOD/SCID mouse model. Then the leukemia mice were randomly divided into model, ASP, Ara-C, and ASP + Ara-C groups. After the 30 d transplantation, the mice were ip injected with ASP (200 mg/kg/d), Ara-C (2.5 mg/kg/d), and ASP (200 mg/kg/d) + Ara-C (2.5 mg/kg/d), respectively for 14 d, and the mice in the model group were injected with saline (equal volume and time). The next day after the treatment, the eyeball blood was collected to detect the amount and classification of white blood cells (WBC). The femurs were taken to count BMMCs of each femur. The proliferation of BMMCs was detected by CCK-8; The distribution of cell cycles was analyzed by flow cytometry (FCM); The capability of colony forming was examined by CFU-Mix cultivation; The ratio of the SA-β-Gal staining positive BMMCs was counted; The aging related proteins of P16, Rb, CDK4, and CyclinD1 were detected by Western blotting. Results: Compared with the model group, ASP or Ara-C injected alone and their combined injection can obviously reduce the amount of the peripheral blood WBC, the percentage of neutrophiles, and the number of femur BMMCs; effectively inhibit the proliferation of BMMCs, CFU-Mix forming, and the ratio of S stages; markedly raise the percentage of lymphocytes, ratio of G1 stages, and the percentage of SA-β-Gal positive cells; down-regulate the expression of the aging related proteins of CDK4 and CyclinD1; and up-regulate the expression of P16 and Rb protein. The effects of ASP + Ara-C group were much better than those in the other groups. Conclusion: The aging mechanism of BMMCs for the transplanted human leukemia mice induced by ASP and Ara-C may be ascribed to the regulation of the expression of the aging related proteins of P16, Rb, CDK4, and CyclinD1. Our research provides a new idea to treat leukemia in clinic.
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Aim Toresearchthemolecularmecha-nisms of DADS-induced apoptosis in human leukemia K562cells.Methods Cellviabilitywasmeasuredby MTT. Levels of DADS-induced ROS were measured by 2ˊ, 7ˊ-dichlorofluorescein diacetate ( DCFH-DA) fluo-rescence. DADS-induced mRNA levels of components of the NADPH oxidase were detected by Real-time PCR. The combination of protein Rac2 and p67phox was measured by immunoprecipitation assays. Flow cy-tometry methods were used to determine the percentage of apoptosis cells. DADS-induced Rac2 levels were measuredbyWesternblot.Results TheDADS-trea-ted K562 cells showed a dose-and time-dependent de-crease in cell viability and proliferation. There was sig-nificant up-regulation of the mRNA level of components of the NADPH oxidase complex in K562 cells after treatment with 6 mg·L-1 DADS for 6 h. Western blot results revealed that, compared with the control group, there was a significant up-regulation of Rac2 protein in K562 cells treated with 5. 0 and 10. 0 mg·L-1 DADS for 24h. And Rac2 combined with p67phox in DADS-induced apoptosis in K562 cells. PMA markedly in-creased the percentage of apoptotic cells, and DPI re-duced the percentage of apoptotic cells in DADS-in-duced K562 cells. Levels of DADS-induced ROS, also showed enhancement when exposed in PMA, but there was no DADS-induced ROS production evident when exposed in DPI in DADS induced K562 cells. Conclu-sions TheseresultsindicatethatNADPHoxidaseis the main source of DADS-induced ROS production. Diallyl disulfide induces apoptosis in human leukemia K562 cells through activation of NADPH oxidase.
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Objective To observe the change of mitochondrion membrane potential(??m) in apoptosis induced by an iron chelator(deferoxamine,DFO),and to explore the mechanism of this apoptosis in human leukemia-60(HL-60) cells.Methods HL-60 cells were co-cultivated with various concentration of DFO and FeCl_3 for certain time,resultingin status of intracellular iron deprivation and rich iron.Cell vital force was determinded by the MTT method.The cells were examined by phase contrast microscopy,flow cytometry(FCM) for evidence of apoptosis,and also by FCM for ??m.The transcription of the apoptogene of bax was detected by hybridization in situ.Results The cell growth rate assumed descent tendency.DFO could induce the apoptosis and descent ??m of HL-60 cells(P
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Objective To explore the silence of B-cell specific Moloney murine leukaemia virus insertion site 1(Bmi-1)by RNA interference on the proliferation of human leukemia cell line K562 and its mechanisms.Methods Small interfering RNA(siRNA)targeting Bmi-1 gene was designed and double-stranded siRNA was chemically synthesized.After double-stranded siRNA was transfected into K562 cells with Lipofectamine 2000,the proliferation of K562 cells was detected by MTT colorimetry,cell cycle was determined by flow cytometry,and the expression of Bmi-1 and P16 were analyzed by Western blotting.Results The siRNA targeting human Bmi-1 gene effectively prolonged the double time of K562 cells,increased the percentage of cells at G1 phase,and the expression of Bmi-1 was significantly down-regulated but the expression of P16 was up-regulated.Conclusion The siRNA targeting human Bmi-1 gene inhibits the proliferation of K562 cells,and up-regulates the expression of P16 in the cells.
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Objective: To explore the effect of LBP X on apoptosis of human leukemia HL 60 cells. Methods: The inhibitory effect of LBP X on growth of HL 60 cells was assayed by MTT method. The membrance fludity was determined with DPH fluorigenic labeling technique. HL 60 cells were stained with Hoechst 33 258 for nuclear morphology analysis. Agarose gel electrophoresis and flow cytometry were used to analyze apoptosis qualitatively and quantitatively. Results: 20-1 000mg/L LBP X inhibited the growth of HL 60 cells in dose dependent manner and the membrance fludity decreased. The nuclei of HL 60 cells treated with LBP X for 48 h shrinked, condensed and cleaved. Agarose gel electrophoresis of DNA from cells treated with LBP X revealed "DNA ladder". HL 60 cells exposed to LBP X showed apoptotic peaks. Conclusion: LBP X can induce apoptosis in human leukemia HL 60 cells.
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The heterospecies hybrid cells(HL-N)from the fusion of human promyelocy-tic leukemia mutant cells(HL-60-AR)and mouse bone marrow nucleated red cellswere established in HAT selective medium.Malignant phenotype comparative analy-sis between parental tumor cells and hybrid cells showed that growth ability ofhybrid cells was decreased.The hybrid cells reduced their DNA synthesis rate andlost the ability of colony-forming in 0.3% soft agar medium.The cells lost tumor-producing ability when they were transplanted into nude mice also.Inhibition orreduction of c-myc oncogene expression was demonstrated by Northern molecularhybridization techniques.The ultrastructure of hybrid cells were also different fromtheir parental cells.These results mentioned above showed that the mouse bone mar-row nucleated red cells might provide some peculiar factors(both nuclear factorsand cytoplasmic factors)to inhibit the expression of HL-60-AR cell malignant phe-notypes.