The Study Of Anti-leukemia Mechanism Of Polyphyllin Ⅰ

Backgroudleukemia is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance toapoptosis. Although its prognosis has been taken anew look along with the development of combination chemotherapy , targeted therapy and hematopoietic stem cell transplantation, there are still many limitations. Plant of Paris polyphyllin , a famous folk medicinal herb in south of China , has been used not only as an anti-cancer, anti-biotic, and anti-inflammatory drug but also to treat snake bite , chronic bronchitis and bone fracture , as well as to stop bleeding. It was found that treatment of K562 cells with polyphyllin I (extracted from Paris polyphyllin)resulted in the inhibition of viability and induction of apoptosis in a dose-dependent manner. While it didn't affect the proliferation of normal bone marrow cells and normal peripheral blood cells. We investigated the mechanisms by which polyphyllin I elicits theseeffects by means of various techniques, including cell culture system, flow cytometry and semi-quantity RT-PCR.MethodsHuman leukemic cells K562-G01,K562,THP1,U937,Raji,H9 and KG-1 were cultured in RPMI 1640 or IMDM and treated with polyphyllin I as indicated time and dose. Control and treated cells were harvested at various times and analyzed for changes in: (1) cell proliferation: MTT were used to gain the values of IC50 of polyphyllin I when it was treated on different cell lines. (2) cell cycle phase: Flow cytometry (FCM) and Annexin V-FLUOS/PI staining kit were used to evaluate apoptosis of leukemic cells. (3) induction of apoptosis : FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure leukemic cells with activated Caspase-3. (4)gene expression: The mRNA levels of P53, GADD45-a in leukemic cells K562-G01 were determined by semi-quantity RT-PCR.Results(1) After treatment with polyphyllin I at different concentrations for 48 h, the values of IC₅₀ of K562-G01,K562, THP1,U937, Raji,H9,KG-1 were 1.86μM,1.34μM,1.18μM, 1.72μM,1.88μM, 0.89μM and 1.38μM , respectively.(2) After treatment with polyphyllin I at 5μM for 24 h, the percentages of leukemic cells K562-G01 expressing activated caspase-3 and apoptotic cells were 89.1% and 84.32%, respectively.(3) The semi-quantity RT-PCR results showed that the mRNA levels of P53, GADD45-a were obviously up-regulated after K562-G01 were treated with polyphyllin I .Conclution(1) polyphyllin I could inhibit the proliferation of several kinds of human leukemic cells through inducing apoptosis.(2) polyphyllin I could induce caspase-3-mediated apoptosis of K562-G01 cells , suggesting that polyphyllin I may be a novel antileukemia, and further study is required.