Arsenious Acid, Gleevec Lymphoma Cell Mechanism

Objective To investigate the effect of arsenic trioxide and gleevec or in combination with gleevec with different concentration on Raji cells and the mechanisms for applying with a experience for lymphoma in clinical as a new method.Methods1. MTT assay :The inhibition of cell proliferation was measured by MTT assay to assess the cells survival after As₂O₃ and STI571 or in combination with STI571 treatment with different concentration at indicated time. Raji cells (1×106/ml) were transferred to 96-well microtiter plates in 200μl culture medium after 24h, 48h and 72h incubation. Absorbance value was measured with a microliter plate reader at a wavelength of 490nm.2. Morphological observation:Raji cells morphologic characteristics of apoptosis were observed by Wright staining with light microscope after treatment with As₂O₃ and STI571 or in combination with STI571 in different concentration at indicated time(24h,48h,72h).3. Flow cytometry analysis:Caspase-3 activeness changes were determined by flow cytometry (FCM) after treatment with As₂O₃ and STI571 or in combination with STI571 in different concentration at indicated time(24h,48h,72h).4. SABC immunohistochemistry method:The expression of p16 protein was analysed by SABC immunohistochemical method after treatment with As₂O₃ and STI571 or in combination with STI571 in different concentration at indicated time(24h,48h,72h).5. Flow cytometry analysis:The relative number of cells in different phases and the percentages of cells calculated in G1 and S phase of the cell cycle were assessed by FCM after treatment with As₂O₃ and STI571 or in combination with STI571 in different concentration at indicated time(24h,48h,72h).Results1. The results of MTT assay showed that As₂O₃ and STI571 or in combination with STI571 could inhibit Raji cells growth. There was clear statistical significance between the union groups and the single-drug group(P<0.05).2. Morphological observation showed characteristic apoptosis changes including cell shrinkage,chromatin condensation,DNA fragmentation,membrane bleb and formation of apoptotic bodies after treatment with As₂O₃.