The Effect And Mechanism Of Bufotalin On Malignant Glioma

Part one:The effect of bufotalin onhuman glioma cell lines in vitroObjective:To investigate the proliferation inhibitory effect of bufotalin on different glioma cell lines in vitro and detect the50%inhibitoryconcentrationof these cell lines.To preliminarily study the effect of bufotalin on the invasiveness and the cell cycle of U87cell line in vitro.Methods:The dose-effect and time-effect relationship of bufotalin were evaluated in U87and U251cell lines with XTT assay.4different glioma cells (the primarily cultured human glioma cell JZ001and JZ002, the human glioma cell lines U87and U251) were treated with different concentration of bufotalin for72hours, the growth inhibition rate was measured with XTT assay and50%inhibitoryconcentration of each cell linewas calculated with Bliss’ method. The invasiveness of U87cell line was measured with TRANSWELL assay in vitro. The effect of bufotalin on the cell cycle of U87cell line was determined with flow cytometry analysis.Results:The similar trend of growth inhibition on glioma cell lines was found in different concentration of bufotalin after24,48and72hours treatment. The72-hour growth inhibitory activity to human glioma cells varied withdifferentconcentration of bufotalin. The IC50of bufotalin for U87cell line, U251cell line and primarily cultured human glioma cell JZ001and JZ002was0.071ug/ml,0.146ug/ml,0.063ug/ml, and0.084ug/ml respectively. The invasiveness of U87cell line in vitro could be suppressed by bufotalin at the concentration of2-5ug/ml. Proportion of the cells in G2phase and S phaseof U87cells elevated remarkably after24hour treatment of2-2ug/ml bufotalin.Conclusions:A strong proliferation inhibitory activityof bufotalin was observed to different human glioma cells. The growth inhibitory activity of bufotalin is dose-dependent but not time-dependent. There wasinverse correlation between the invasiveness of U87cell line and the concentration of Bufotalin. Bufotalin could induce cell cycle arrest of U87cells in G2/M and S phase.Part two:Evaluation of the antitumor effect of bufotalin in a xenograft animal model of human glioma in vivoObjective:To establish an orthotopic xenograft animal model of human glioma. To detect the distribution of bufotalin in the brain of mice after intravenous administration at different time points(15min,30min,1h,2h,4h,8h,16h,24h) and compare the curative effect of bufotalin and positive control temozolomide on U87xenograft model mice.Methods:To detect the distribution of bufotalin in the brain of mice after a single bolus intravenous injection through high performance liquid chromatography determination at different time points. An orthotopic U87xenograft model was established with stereotaxic injection technology. The model mice were divided into3groups, the control group (n=15) received intravenous injection of drug solvent biw, the experimental group (n=14) receivedintravenous injection of6mg/kg bufotalin biw and the positive control group (n=18) received intraperitoneal injection of82.5mg/kg temozolomide (TMZ) qd in5consecutive days. Through series enhanced MR, the difference in growth of different intervention was monitored; the weight, biological behavior and survival of mice were observed and recorded. To compare the influence of different intervention on survival period of model animals through Kaplan-Meier survival curve according to the recorded results. To validate the effect of bufotalin on the implanted tumor with histological studyand immunohistochemical staining.Results:The establishment of stable and reproducible orthotopic U87xenograft model was achieved. Bufotalin could enter brain tissue through the blood brain barrier with a steady concentration over0. lug/g for more than16hours. Progressive weight loss, which could be regained after the course of TMZ treatment, occurred in the most mice of positive control group in the first post-administration week. Since the second week after the inoculation procedure, progressive weight loss could be observed in the control group and the experimental group with the manifestation of hypopraxia, hemiplegia and eventually dyscrasia. Tumor volume in blank control group and bufotalin group was found to be in progressive increase through series enhanced MRI scan, while no significant enhancement could be found in temozolomide group after a4-week period. The median overall survival of blank control group and experimental group was21days and25days respectively. From the cumulative survival curve according to the observation record, we could found a significant difference between the blank control group and experimental group (p=0.038). There was no death within temozolomide group in4weeks.Conclusions:Stable and reproducible orthotopic U87xenograft model, which was convenient to manipulate with a constant tumor formation rate for the in vivo evaluation of chemotherapy agents, was successfully established. High distribution of bufotalin after single bolus intravenous and intraperitoneal administration was observed, a relative stable concentration above IC50for most glioma cell could be maintained for more than16hours. Enhanced magnetic resonance imaging scan was safe and intuitionistic in the observation of tumor-bearing mice. The monotherapy of bufotalin could significantly improve the overall survival of U87bearing mice Part three:Explorationof the inhibitive mechanism of bufotalin on human glioma cellsObjective:To preliminarily study the mechanism of the growth inhibition effect of Bufotalin to glioma cell.Methods:To detect the DNA fragmentation caused by bufotalin with TUNEL/PI double stainingapoptosis assay. The effect of bufotalin on the mitochondrial membrane potential was measured with JC-1staining through flow cytometry. The influence of bufotalin on cell apoptosis related proteins caspase-3(pro-form), caspase-8, caspase-9, p53, and survivin levels were studied with Western-Blot analysis. The difference of expression of P-Glycoprotein (P-Gp) which was encoded by MDR1between control and experiment group in section Ⅱ was observed with immunohistochemical staining. Results:Mitochondrial membrane potential decreaseand DNA fragmentation could be inducedin U87cell line after24-hour treatment of bufotalin.Most of the U87cells were induced into an irreversible apoptosis process after36-hour treatment of bufotalin. With the elevation of concentration of bufotalin treated, content of Caspase-3pro-form decreased and content of Caspase-8or Caspase-9increased. The expression of p53and survivin in U87cells, which was in a relatively low level, was not influenced by bufotalin. The expression of P-Gp of bufotalin treated tumor tissue was higher than that of control group.Conclusions:The short period treatment of bufotalin can induce glioma cells into apoptosis process in which the death receptor pathway and mitochondrial pathway were both involved, while p53and survivin might not participate in this apoptosis induction process. U87cells might resist bufotalin by increasing drug efflux.