Pulsatilla Saponin A Induces Apoptosis And Differentiation Of Multiple Myeloma Cells In Vitro

【Objective】1.To test the anticancer activity of Pulsatilla saponin A on U266 cells2.To explore the molecular mechanism of apoptosis effects of Pulsatilla saponin A on U266 cells.3.To investigate the differentiation-inducing protentiality of Pulsatilla saponin A on U266 cells.4.To investigate the effects of Pulsatilla saponin A on MM cells.【Methods】1.Proliferation experiments were performed by culturing U266 cells after Pulsatilla saponin A treating.The CCK-8 test was used to determine the inhibition of PsA on the proliferation of U266 cells.The cycle distribution and apoptosis rates of U266 cells were examined by flow cytometry.2.The protein levels of Bcl2,cyclin-B1 were detected by Western blot,after different concentration Pulsatilla saponin A treated in U266 cells.3.Differentiation induction experiments were performed by culturing MM cell line U266 cells with the presence of Pulsatilla saponin A.For observation of morphology and cytophyletic characteristic maker(CD49e)changes,U266 cells were treated with Pulsatilla saponin A and then stained with Liu’s stain,observed microscopically,and analyzed by flow cytometry.4.The effects of Pulsatilla saponin A on primary MM cells were analyzed by flow cytometry.【Results】1.The anticancer activity of Pulsatilla saponin A on U266 cellsPulsatilla saponin A obviously inhibited the growth of U266 cell in dose and time dependent manners.By flow cytometric analysis of cell cycle distribution and annexin V expression,G2 arrest and apoptosis were identified in Pulsatilla saponin A-treated U266 cells for 24 hours.2.Molecular mechanism of the apoptosis-inducing effects of Pulsatilla saponin A on U266 cellsWestern blot results showed Bcl-2 protein expression was significantly reduced in the Pulsatilla saponin A-treated cells in a dose-dependent manner.By contrast,cyclin B protein expression was increased in these cells.3.Pulsatilla saponin A induces differentiation on U266 cellsThe CD49 e positive rate of U266 cells,after PsA treatment 96 h CD49e positive rate was significantly increased compared with the control group.Meanwhile,concentration of light chain protein of the cell culture supernatant in the Pulsatilla saponin A-treated U266 cells increased as well.4.The effects of Pulsatilla saponin A on primary MM cells.Treatment of primary MM cells with Pulsatilla saponin A up-regulated expression of CD49 e.【Conclusions】1.Pulsatilla saponin A may exert its anti-tumor effect by inhibiting proliferation and inducing apoptosis of U266 cells in dose dependent and time dependent manners2.The Bcl-2,cyclin-B1 protein is essential for the Pulsatilla saponin A-induced differentiation of U266 cells.Pulsatilla saponin A can induced functional maturation of U266 cells.3.Pulsatilla saponin A can induce differentiation of primary MM cells.