The Effects Of As₂O₃ On Cell Proliferation、apotosis And Autophagy In L1210 Cells

Leukemia, also known as“blood cancer”,is a kind of malignant disease of hematological system caused by abnormal hemopoietic stem cells.The number of leukemia patients ranked eighth among malignant tumors in our country,its total incidence rate accounted for about 5 percent of malignant tumors,the death rate of leukemia ranked sixth among malignant tumors,and showed a rising trend.In the 1970 s,experts of affiliated hospital of of Harbin Medical University creatievely applied As2O3 on treatment of acute promyelocytic leukemia(APL)patients with M3 type,then expanded to the treatment of recurrent cases of M3 type with retinoic acid, the complete remission rate could reach more than 93 percent, it has been considered as the fisrt choice of application on M3 type of leukemia. It was generally believed that the main mechanism of As2O3 in the treatment of leukemia was its induction of apoptosis in leukemia cells, its effect on autophagy was seldom considered. This study explored the more likely to be hidden mechanism of As2O3’s anti-leukemia effect from its effects on proliferation、apoptosis、and autophagy in L1210 cells.In this study, MTT and Brdu-ELISA assay were used to detect the influence of different concentration of As2O3 on L1210 cell proliferation. The situation of apoptosis were detected by flow cytometry and AO/EB double staining,AO staining and MDC staining were used to observe the autophagy situation.Proliferation related protein PCNA、apoptosis related proteins BAX and Bcl-2、autophagy related proteins LC3 B were detecte by immunoblotting.In addition, aplastic anemia model mice were induced by benzene to investigate if As2O3 could stimulate the hemopoiesis function of marrow cells.The results of cell experiments showed that As2O3 suppressed L1210 cells in a dose and tine dependent matter, after treatment of 24h、48h and 72 h with As2O3,the half inhibitory concentration(IC50) were 11.61μmol/L±0.05、3.34μmol/L±0.03、0.13μmol/L±0.08. Apoptois rate increased with the increased concentration of As2O3 after treatment of 24 h, apoptosis rate were 26.52% ±0.02、30.99% ±0.06、74.48% ±0.05 when the concentration of As2O3 were 5、10、20μmol/L.AO/EB staining showed obvious change of cell morpho logy,such as nuclear condensation、cell surface memberane blebbling、smaller size、cell rupture and the emergence of apoptotic bodies in a dose dependent matter.AO staining and MDC staining showed autophagic positive color,when the concentration of As2O3 was higher,the positive rate was higher. Western-Blot showed the expression of autophagy related proteins LC3 B 、pro-apoptosis protein BAX were increased in a a dose dependent matter, the level of proliferation related protein PCNA、apoptosis-suppressed protein Bcl-2 were decreased in a dose dependent matter.In vivo tests showed that As2O3 could increase the number of peripheral blood cells、marrow cells、The absolute value of reticulocyte and hemoglobin content,improve the damage of liver and kidney tissue.In summary, As2O3 could cure leukemia through inducing cells apoptosis and autophagy,it also could stimulate the hematopoietic function of aplastic anemia modle mice.