Differentiation-Inducing Effects And Molecular Mechanism Of Pulsatilla Saponin A On Acute Myeloid Leukemia Cells

【Objectives】1. To investigate the differentiation-inducing protentiality of Pulsatilla saponin A on K562 cells.2. To investigate the differentiation-inducing protentiality of Pulsatilla saponin A on acute myeloid leukemia(AML) cell lines.3. To explore the molecular mechanism of differentiation-inducing effects of Pulsatilla saponin A on K562 cells.4. To investigate the effects of Pulsatilla saponin A on AML cells.【Methods】1. Differentiation induction experiments were performed by culturing K562 cells with the treatment of Pulsatilla saponin A. For observation of cytochemistry and cytophyletic characteristic makers(CD71 and GPA) changes, K562 cells were treated with Pulsatilla saponin A and then stained with benzidine stain and analyzed by flow cytometry.2. Differentiation induction experiments were performed by culturing AML cell lines(K562 cells, U937 cells and HL-60 cells) with the presence of Pulsatilla saponin A. For observation of morphology and cytophyletic characteristic maker(CD15) changes, AML cell lines were treated with Pulsatilla saponin A and then stained with Liu’s stain, observed microscopically, and analyzed by flow cytometry.3. After treatment of K562 cells with Pulsatilla saponin A and U0126 for different time intervals, the changes of protein expression, including ERK and p-ERK, were detected by Western blot, and the changes of cytophyletic characteristic maker(CD15) was analyzed by flow cytometry.4. The effects of Pulsatilla saponin A on AML cells were analyzed by flow cytometry.【Results】1. Pulsatilla saponin A induces erythroid lineage differentiation on K562 cellsK562 cells treated with 4μg/ml Pulsatilla saponin A were found differentiated. With the treatment of Pulsatilla saponin A, hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the surface of K562 cells up-regulated.2. Pulsatilla saponin A induces granulocyte lineage differentiation on AML cell lines(K562 cells, U937 cells and HL-60 cells)K562 cells, U937 cells and HL-60 cells treated with 5μg/ml, 4μg/ml and 3μg/ml Pulsatilla saponin A were found differentiated and morphologically changed to mature granulocytes. After treatment with Pulsatilla saponin A, CD15 expression on the surface of K562 cells, U937 cells and HL-60 cells all up-regulated in a dose-dependent manner.3. Molecular mechanism of the differentiation-inducing effects of Pulsatilla saponin A on K562 cellsWestern blot results showed the phosphorylation of ERK was up-regulated on K562 cells after administration of Pulsatilla saponin A; U0126, a specific MEK/ERK activation inhibitor, abrogated Pulsatilla saponin A–induced granulocyte lineage differentiation.4. The effects of Pulsatilla saponin A on AML cells.Treatment of AML cells with Pulsatilla saponin A up-regulated expression of CD15, and partly decreased the CD34+ AML cells.【Conclusions】1. Pulsatilla saponin A can induce erythroid lineage differentiation of K562 cells.2. Pulsatilla saponin A can induce granulocyte lineage differentiation of AML cell lines(K562 cells, U937 cells and HL-60 cells).3. The MEK/ERK signaling pathway is essential for the Pulsatilla saponin A-induced differentiation of K562 cells.4. Pulsatilla saponin A can induce granulocyte lineage differentiation of AML cells.