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Research on Oridonin Induce Apoptosis and Differentiation in Actute Promyelocytic Leukemia Cells

Author: TuXueLiang
Tutor: DuYing；QinDongChun
School: Zhengzhou University
Course: Immunology
Keywords: APL leukemia oridonin apoptosis ROS PML-RARα
CLC: R285
Type: Master's thesis
Year: 2008
Downloads: 23
Quote: 0
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Abstract


Objective: The malignant disease leukemia badly threaten the health of human beings . It is the high point for the research to find the agents of induction involving apoptosis and differentiation in leukemia cells. The disease of APL occupies 5%-8%in actute myeloid leukemia (AML). 95% sufferers of the disease characterized by a translocation between the promyelocytic leukemia gene (PML) on chromosome 15 and the retinoic acid receptor-alpha (RAR alpha ) gene on chromosome 17,leading to the expression of a leukemia-generating fusion protein , PML-RAR alpha . The thesis is developed preliminary research based on the two aspects , that is , how the oridonin at high dose trigger apoptosis in APL cells , and how the oridonin synergize ATRA trigger the differentiation in APL cells .Methods: Routine culture actue mono-nucleus leukemia cell system NB4and NB4-R1, using oridonin from different concentration(1-6ug) treat NB4 and NB4-R1 cell 3d . Respectively using trypan blue staining method tests living rate of the cells ;Using AnnetinV-FITC and PI double staining FCM method tests the apoptosis and period of the cells ; Using DCFH-DA tests reactive oxygen species(ROS) in cells .According to the detection of morphological observation , the narration of FCM tests CD11b , NBT reduction test etc . Showed that oridonin at low dose cooperating with ATRA trigger the diferentiation in leukemia cells . Meanwhile ,adopting some techenicals such as SDS-PAGE and Western Blot observe poly ADP-ribose polymerase(PARP) in cells . With the help of the above methods the study Oridonin trigger apopsis at higher dose and sunergize ATRA induce differentiation at low dose in promyelocytic leukemia cells was developed.Results: It was found that ROS have been implicated in the induction or enhancement of apoptosis , while the ROS scavenger reagent NAC could completely protect NB4 from Ori induced apoptosis . Neither Ori at low dose nor ATRA at low dose can trigger the cell differentiation in NB4 and NB4-R1 . Interesting , Ori alone could increase the amount of RAR protein and inhibits ATRA induced degradation of RAR not only in NB4 cells but also in NB-R1 cells .For the further observation ,Ori can increase RARa and RARa target gene.Conclusions: It suggest that ROS signaling is involved in the induction of apoptosis by oridonin. Research suggested that suboptimal concentration of Ori could enhance ATRA induced apoptosis. To our knowledge, this is the first report to show RARαcould be accumulated by a small compound. Based on the important role of RARαin the physiology and patho, future investigation on the mechanism of RARαaccumulation is valuable. These results suggested that oridonin is a potential candidate for APL cancer therapy.

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