| Objective: To observe the killing effect of As2S2 on leukemic cells, explore the mechanism of the effect, and probe the therapy benefits from treatment combining As2S2 with a PI3K inhibitor, PI-103. Methods: After leukemia cells were treated with different concentrations of As2S2 for different times, MTT assay was used to test the effect of As2S2 on proliferation inhibition of treated cells; flow cytometry and MGG staining were used to examine the differentiation and apoptosis of leukemia cells, cord blood mononuclear mononuclear cells and bone marrow mononuclear cells of leukemia patients after being treated with As2S2, PI-103 or both. Colony-forming assay was used to asses the effect of cloning scavenging of As2S2 on leukemic cells. Cell immunofluorescence, western blot, real-time PCR and real-time PCR array were used to determine the regulation of PI3K signal pathway by As2S2 at the levels of transcription and protein. Results: As2S2 inhibited the proliferation and induced apoptosis of leukemia cells in a time- and dose-dependent manner, and supressed clone formation of leukemia cells significantly. After treatment with As2S2, PML protein expression was reduced significantly in leukemia cells, however no significant changes were seen in the level of transcription. The phosphorylation levels of key proteins in PI3K signaling pathway were activated in a short period of time, but they were downregulated in general with the extension of treatment time. As2S2 and PI-103 showed a synergistic enhanced effect on inducing apoptosis of leukemia cells, while showed no apparent toxicity on normal mononuclear cells. Conclusions: As2S2 shows a significant dose-dependent killing effect on leukemic cells through depletion of PML protein and downregulation of PI3K signaling pathway, but no obvious toxity on ormal mononuclear cells were seen, PI-103 can be coordinated to enhance the above effect. Therefore, As2S2 should hve a high therapeutic index. Objectives TO determine the effect of small molecule compounds As2S2 alone or in combination with PI-103 on the leukemia stem cells and conduct. Methods Leukemia stem cells were sorted by flow cytometry, and treated with As2S2 or / and PI-103. Then the apoptosis and differentiation of treated cells was detected by flow cytometry. The effect of As2S2 or / and PI-103 on the colony-forming ability of leukemia stem cells and normal hematopoietic stem cell cells was assessed by colony forming assay. The dynamic changes of p-AKT473 expression in leukemia stem cells treated with As2S2 was determined by multi-color flow cytometry. Results CD34 + CD38-cells sorted from the bone marrow cells of patients accounted for 75.4 percent of the total number, and sorting purity was 96.4%. Anti-ki67-Hoechst staining analysis showed that the cells in GO phase accounted for 83.9%, that in G1 phase accounted for 7.93%., respectively, the cell apoptosis-inducing effect of 1μmol / L of As2S2 or PI-103 was not apparent, but when used together, the apoptosis rate of leukemia stem cells jumped to 31.02% from 17.26%. As2S2 or PI-103 had a mild effect on inhibiting colony formation of leukemia cell when used alone, but the effect was significantly enhanced when used together. They had no significant effect on the colony-forming capacity of normal hematopoietic stem cells when used at concentrations above. The number of cells expressing CD11b was significantly increased in As2S2 treatment group, but the number of cells expressing CD11b in PI-103 treatment group was not changed significantly. The number of cells expressing CD11b was increased more significantly in group treated with As2S2 and PI-103 than with As2S2 alone. The expression of p-AKT473 in CD34+CD38-Lin- cells from specimens of six leukemia patients was increased after being treated with As2S2 for 1 h, but after 24 h, it was downregulated. Conclusion As2S2 could effectively induce apoptosis of leukemia stem cells, promote differentiation, inhibit their ability of self-renewal and proliferation, PI-103 can be coordinated to enhance the above effect.
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