Growth Suppression Of Human Burkitt Lymphoma Cells By Rapamycin

Objective:In this project, we investigate the effects of mTOR inhibitor-rapamycin on human Burkitt lymphoma cell line Raji cells growth and study the molecular mechanism.Methods : Raji cell lines were incubated in culture medium in vitro. Using MTT assay to detect the growth rate among different rapamycin concentration groups (0,1,5,10,20,40,50,100nmol/ml) and different time groups (24h,48h,72h), in order to choose proper drug concentration and action time. According to the result of MTT, establishing control and experimental groups: different rapamycin concentration groups (5,10,20,40,50,100nmol/ml) and different time groups (12h,24h,48h). Apoptosis and cell cycle were analyzed via flow cytometry using a Coulter Epics XL cytofluorometer. The morphological alterations were confirmed by the optical microscope. Extracting total cell protein of each experimental group, protein quantity was measured by Bradford, expressed in mg/ml.β-actin was regarded as internal reference.The expression of cyclinD1,cyclinE,cyclinA,p27,survivin protein was examined by western blot technique in the rapamycin-treated and untreated Raji cell.Results: 1 MTT assay results: rapamycin was found to be able to inhibit the proliferation and survival of Raji cells at concentration greater than 5nmol/L. After rapamycin-treated for 24,48,72 hours, compared with control group, the OD value of rapamycin -treated groups decreased, and there was statistically significant difference between control group and every experimental groups (p>0.05). Furthermore, with the increasing concentration of rapamycin and prolonging of treatment time, the OD value decreased gradually, that was, the effect rapamycin inhibited the proliferation of Raji cell was dose and time dependent.2 When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the result were: with the increasing concentration of rapamycin and prolonging of treatment time, the number of cells in S phase and G2/M phase decreased gradually, but, increased in G0/G1 phase. That was, rapamycin could induce G0/G1 arrest in dose- and time- dependent manners. However, it could not cause apoptosis in Raji cells evidently.3 Results of using western blot technique shown the expression of cyclinD1,p27,cyclinE had no obvious change and there were not expressions of cyclinA and survivin protein in Raji cells after treated with rapamycin at the concentrate of 50nmol/L.Conclusions:1 Rapamycin inhibits Raji cells proliferation by cell cycle arrest in G0/G1, which down regulate cyclinA and survivin. 2 Rapamcin may be useful in treatment of Burkitt lymphoma.