Effects Of BCNU On Cell Proliferation And Cell Cycle In Human Brain Tumor Stem Cells

ObjectiveTo investigate the effects of BCNU on the proliferation and cell cycle of tumor stem cells derived from human brain.And to study the primary mechanism.It will provide the experiment rational for chemotherapy of BCNU on brain tumor.MethodsFresh surgical specimen was obtained from one case with ependymocytoma,and was prepared in single cell suspensions firstly,then cultured in serum-free medium and in serum supplemented medium in order to acquire BTSCs and BTCs respectively.7d, cell will be identifid by immunocytochemistry method(CD133 and nestin),Cells were be treated with different concentrations of BCNU(1ug/ml,0.1ug/ml,0.01ug/ml,0.001ug/ml,0.001 ug/ml)for 72h,then the viability of each group was measured by MTT method.Cells were also treated with 0.01ug/mL BCNU for a variety of time spans,then cell cycle was examined by PI fluorescence flow cytometry.Results1.in the serum-free medium,a minority of ependymocytoma cells showed the capacity of self-renew and proliferation,these BTSCs generated free-floating neurosphere-like BTSs,the espression of CD133 and nestin was detected in BTSCs. In the serum-supplement medium,all of ependymocytoma cells was adherent,and don't detect the expression of CD133 and nestin by immunocytochemistry stain.2.BCNU could inhibit both the growth of BTSCs and BTCs,while BTCs (IC50=0.157μg/ml)was more sensitive than BTSCs(IC50=0.072μg/ml)(P＜0.05)3.With the prolonged treatment of BCNU,flow cytometry analysis showed that not only cells at S phase but also apoptosis cells increased gradually,but these changes of BTSCs occurred(24h)later than that of BTCs(6h).Conclusions1.The result of experiment indicate that BTSCs exist in ependymocytoma,which is correspond with the theory of brain tumor stem cell.2.we prove that CD133 and nestin,which is specificness marker of nerve stem cell, also can mark brain tumor stem cell.3.BCNU could inhibit the growth of BTSC in vitro,The mechanism was probably through blocking the cell cycle on S phase to induce BTSC apoptosis4.Compared with BTC,BTSC was resistant to BCNU by MTT method and flow cytometry.