Resveratrol Induces Differentiation And Apoptosis Of Medulloblastoma Cells And Their Corresponding Gene Analysis

Objective 1) To observe the changes of cell growth, differentiation, survival and morphological character of four human medulloblastoma cell lines(UW228-1,-2,-3 and Med-3) treated by different concentrations of resverartrol. 2) To elucidate the expression patterns of CYP1A1, CYP1B1 in four different medulloblastoma cell lines after the treatment of resveratrol and to explore the cell cycle distribution changes in UW228-3 cell line. 3) To determine the potential genetic mechanism of resveratrol on medulloblastoma cell lines. 4) To provide an adjuvant therapeutic clue for better treatment of human medulloblastomas. Methods Med-3 and UW228-1, 2, 3 cells were cultured separatedly in EMEM(Eagle's Minimum Essential Medium) and DMEM(Ducbecoo's Modified Eagle's Medium) culture medium containing 10% Fetal Bovine Serum. Treat the four cell lines with 0,50,75,100,150μM concentrations of resveratrol respectively when cells were in good stage and observe the character at different time intervals 0hrs, 24hrs, 36hrs, 48hrs, 60hrs and 72hrs. Put sterilized cover slips in the culture dish before cell inoculated whenever cell shape observation or cytochemical staining analyse were needed. After treatment, cover slips were collceted and fixed properly according to the experiment at purposes. Effects of resveratrol on the cell proliferation and survival were examined through MTT assay and Typran Blue accounting methods. Immunocytochemical staining, TdT-mediated dUTP-biotin nick end-labeling test (TUNEL) and Flow cytometry (FCM) were used to determine the cell death pattern. Immunocytochemistry (ICC) and RT-PCR, Western-blot hybridization were used to analyze the statuses of apoptosis related gene Fas, FasL and Caspase-3. The expression level of metabolitic enzyme (CYP1A1, CYP1B1) and its correlation with cell differentiation /apoptosis were studied as well. The influence of resveratrol in cell cycle was determined with FCM. Results 1) Resveratrol not only suppresses the growth of medulloblastoma cells in a dose and time related fashion but also induces cell differentiation and apoptosis. Resveratrol promotes differention of UW228-1, -2 cells to forward glia, UW228-3 and Med-3 cells to neuron. A treatment of resveratrol in the concentration of 100μM for 48hrs comit most of cells die of apoptosis. 2) Among the four cell lines Fas and Caspase-3 were constantly expessed, whereas FasL was absent irrespective to the drug treatment. 3) ICC, RT-PCR, Western-blot hybridization and EROD enzymatic activity assay demonstrated that expression of CYP1A1 is different after treatment with resveratrol in four cell lines, up-regulated in three UW228 cell lines in a dose dependent manner but down-regulated in Med-3 cells. Suppressive effect of resveratrol on CYP1B1 expression is the same among four medulloblatoma cell lines. 4) Cell cycle distribution of UW228-3 was greatly changed after treatment. That is proportion of G0/G1 phase in the whole cell cycle is increased, the other phase are decreased accordingly. Conclusion 1) These findings suggest for the first time that nontoxic compound resveratrol is an effective anti-medulloblastoma agent that kills medulloblastoma cells through a Fas-independent apoptosis pathway. It significantly inhibits the growth of four medulloblastoma cell lines in a dose and time dependent manner. And this effect may be relevant to the cell cycle arrest at G0/G1. 2) Although the morphological phenotypes are different among the four cell lines, resveratrol promotes all of them to forward differentiation and, finaly, apoptosis. These data suggest that resveratrol may be an ideal anti-medulloblastoma drugs for clinical purposes. 3) CYP1A1, rather than CYP1B1 probably involve in the activation of resveratrol in medulloblatoma cells. Therefore CYP1A1 can be of the determiner of chemotherapy and the essential biomarker of patients' prognosis.