| Section 1 BBD9 induced cell apoptosis and cycle arrest which inhibited cell proliferation in AML Objective:To explore the effects and mechanisms of of BBD9 on acute myeloid leukemia(AML)cell growth.Methods:(1)The proliferation was measured by MTT assay in primary AML cells and AML cell lines KG-1α,Kasumi-1 and THP-1 cells.(2)The apoptosis and cell cycle were detected by flow cytometry in primary AML cells and AML cell lines KG-1α,Kasumi-1 and THP-1 cells(3)The Western blot was used to detect the expression of caspase family proteins caspase-3,caspase-9 and PARP,apoptosis regulatory protein and cell cycle related proteins in AML cells.results:(1)The proliferation of AML cells were inhibited by BBD9:At the concentration of 0～3μg/ml,BBD9 can significantly inhibit the proliferation of three AML cell lines in a dose-and time-dependent manner.The 24 h IC50 of BBD9 on KG-1α、Kasumi-1and THP-1 cells were 1.75μg/ml and 2.57μg/ml and the 48 h IC50 are 1.40μg/ml 、1.34μg/ml and 1.777μg/ml.Primary cells from 7 patients with AML were treated with BBD9 for 24 h,and showed growth inhibition in a dose dependent manner.The average IC50 for 24 hours was 1.794 μg/ml.(2)AML cells were induceed apoptosis by BBD9:After 0μg/ml,1μg/ml,1.5μg/ml,2μg/ml BBD9 treatment for 24 h,the early cell apoptosis rate of AML cell line Kasumi-1 cells was 1.88±0.29%,3.19,respectively ±0.40%,7.68±1.32% and26.06±5.23%.After 0μg/ml,1μg/ml,1.5μg/ml,2μg/ml BBD9 treatment for 24 h,the early apoptosis rate of patient 1 was 3.98%,13.13%,20.85% and 28.99%;The early apoptosis rates of Patient 2 were 7.07%,9.66%,18.69% and 22.14%;The early apoptosis rates of Patient 3 were 4.02% 、 6.10% 、 8.23% and 11.45%.Western blot results showed that BBD9 increased the expression of p-Bad and Bim,and decrease the expression of Mcl-1 in Kasumi-1 cells.There was no significant change in the expression of Bad,Bax and Bcl-xl.Caspase-3,caspase-9 and PARP were spliced and activated,with the increase of BBD9 concentration.BBD9 induces the expression of caspase-3,caspase-9 and PARP cleavage activation,and down-regulates the expression of anti-apoptotic protein Mcl-1 in primary AML cells.(3)BBD9 induced cell cycle arrested in G1 phase : After treatment with 0μg/ml and 2μg/ml BBD9 for 24 hours,the proportion of Kasumi-1 cells in G1 phase was52.53±0.69% and 63.74±2.34%.Compared with the control group,the difference was statistically significant(p<0.05).Western blot shown that after the treatment of BBD9 for 24 hours in Kasumi-1 cells,the expression of cyclin D1,CDK4,CDK6,E2F1 and p21 proteins were down-regulated,while p27 protein was up-regulated.Conclusions:(1)BBD9 inhibits the proliferation of AML cell lines and primary AML cells.(2).Pro-apoptotic protein,Bax,p-Bad and Bim were up-regulated and anti-apoptotic protein Mcl-1 was down-regulated and caspase-3,caspase-9 and PARP cleavage activation induce degradation of by BBD9 in AML cell lines and primary AML cells.(3)BBD9 inhibits AML cell lines in G1 phase by down-regulating cyclin D1,CDK4,CDK6 and E2F1 protein and up-regulating p27 protein.Section 2 BBD9 Effects on AML IGF-1R/PI3K/AKT signaling pathways Objective:Study BBD9 Effects on IGF-1R/PI3K110/AKT Signal Pathway in AML Cells.Method:The Western blot was used to detect the expression of IGF-1R/PI3K/AKT signaling pathway protein in AML cells Kasumi-1 cell and primary AML cell.results:(1)With 0μg/ml、1μg/ml、1.5μg/ml and 2μg/ml BBD9 treatment in AML cell lines for 24 hours,Western blot assay showed that the expression of IGF-1R and p-AKT proteins were downr-egulated.There was no significant change in AKT protein expression.(2)With 0μg/ml、1μg/ml、1.5μg/ml and 2μg/ml BBD9 treatment in primary AML cells for 24 hours,Western blot assay showed that expresstion of IGF-1R、PI3K110and p-AKT proteins were downregulated.Conclusion:BBD9 inhibits IGF-1R/PI3K/AKT signaling pathways in AML cell lines and primary AML cells from patients with newly diagnosed AML.Section 3 The effect of BBD9 on autophagy of AML cells Objective:To explore the effect of BBD9 on autophagy of AML cells.Methods:(1)Observe the morphological changes of autophagy after BBD9 acts on AML cell line Kasumi-1 with the electron microscope.(2)The Western blot was used to detect the expression of the influence of m TOR protein in primary AML cells.(3)The Western blot was used to detect the expression of the changes of autophagy protein in the AML cell line Kasumi-1 and the initial primary AML cells.results:(1)After 0μg/ml and 2μg/ml BBD9 treatment in Kasumi-1 cells for 24 hours.Compared with the 0μg/ml control group,Kasumi-1 cells showed obvious autophagic vesicles 24 hours after BBD9 treatment.(2)0μg/ml,1μg/ml,1.5μg/ml and 2μg/ml BBD9 acted on the primary AML cells for 24 hours,Western blot detection showed that the expression of m TOR and p-m TOR protein increased with BBD9 The increase of the concentration gradually decreased,and the decrease of p-m TOR protein was more prominent.(3)0μg/ml,1μg/ml,1.5μg/ml and 2μg/ml BBD9 treated Kasumi-1 cells for 24 hours,Western blot tests showed that autophagy protein Beclin-1,ATG5 expression and the proportion of LC3-II/LC3-I gradually increased.(4)With 0μg/ml,1μg/ml,1.5μg/ml and 2μg/ml BBD9 treatment in primary AML cells for 24 hours,Western blot tests showed that autophagy protein Beclin-1 and the proportion of LC3-II/LC3-I is gradually increasing.Conclusions:BBD9 inhibits IGF-1R/PI3K/AKT/m TOR signaling pathway and induces autophagy in AML cells. |
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