(Studies Of Purified Vitexin Compound1Suppressing Tumor Growth And Inducing Cell Apoptosis In Human Choriocarcioma)

Part Ⅰ The antitumor effect of purified vitexin compound1(VB1) on human choriocarcinoma cell JEG-3objective:In our previous study, we had isolated a series of lignan compounds, ermed vitexins, from the seed of Chinese herb Vitex Negundo and found Droad antitumor activities of these compounds in many cancer xenograft nodels and cell lines. This study aimed to determine the antitumor effect of purified vitexin compound1(VB1) alone and combinating with5-FU on human choriocarcinoma cell JEG-3.Methods:1. The choriocarcinoma JEG-3cell culture system was established.2. The proliferation in JEG-3cell line was evaluated by MTT assay.3. Colony formation assay was used to get the cloning formation (?)te.4. The apoptotic morphology was observed by Hoechst33258stain-ng and the apoptosis rate was detected by flow cytometry.Results:1. After the treatment of different concentrations of VB1(0.1,0.5,(?).0,5.0,10.0,20.0,50.0) μmol/L in JEG-3cells, the result shows that with the increase of the the VB1concentration and time, the cell viability decreased (P<0.05); IC50of24,48and72hours was13.537,5.170,1.926μmol/L respectively.2. The IC50(48h) of5-FU was25.257μg/ml;while combined with5μmol/L VB1the IC50decreased to4.078μg/ml. Guinness formula calculated the values of Q were greater than0.85, suggesting that the VB1combined with5-FU associated with a synergistic effect.3. The colony formation rate after treatment of different concentra-tions of was significantly lower than the control group(P<0.05).,and the higher the drug concentration increased, the lower the colony formation rate decreased. The colony formation rate of the combined group was lower than each of the two drugs treated alone (P<0.05).4. Hoehest33258staining showed the typical apoptotic changes of the cell nuclei. The flow cytometry results showed that apoptosis rate of each treated group was significantly higher than the control group (P <0.05). The apoptosis rate of each group treated by VB1alone positively correlated with the drug concentration (P<0.05), and of the combined group of was significantly higher than each of the two drugs treated alone (P<0.05).Conclusion:1. VB1in certain concentration range could significantly inhibit the proliferation of JEG-3choriocarcinoma cell,and the results showed a dose -time-dependent.2. VB1can promote apoptosis in JEG-3choriocarcinoma cell within a certain concentration range, with a dose-dependent.Part Ⅱ The possible mechanism of the VB1effect on inhibition of proliferation and promoting of apoptosis in choriocarcinoma cell lineObjective:To study the possible mechanism of the VB1effect on inhibition of proliferation and promoting of apoptosis in choriocarcinoma cell line JEG-3.Methods:1. The mRNA expression level of AKT, mTOR, P70S6K was detec-ted by RT-PCR.2. The protein expression level of AKT, mTOR, P70S6K and p-AKT, p-mTOR, p-P70S6K was detected by Western blot.3. Caspase-3, Bcl-2protein expression was detected by Western blot.Results:1. After treated with different concentrations of VB1in JEG-3cells, the relative mRNA expressions of AKT, mTOR and P70S6K were signi-ficantly lower than the control group; and to some extent, showed a dose-dependent manner (P<0.05).2. Western blot results showed that with the increasing of VB1concentration, the relative protein expressions of AKT and mTOR, P70S6K and their corresponding phosphorylated protein were lower than the control group (P<0.05).3. As concentration of VB1increased, the pro-caspase-3expression in JEG-3cells had no significant changes, but cleaved-caspase-3was cor-responded increasd; and Bcl-2expression was gradually decreased com-paring with the control group. The difference was significant (P<0.05).Conclusion:The inhibition effect of VB1to human choriocarcinoma JEG-3cell was refered to mTOR signaling pathways, and Caspase-3and Bcl-2family proteins were involved in the inducing apoptosis process.Part Ⅲ The effect of VB1treating the tumor-bearing miceObjective:To establish human choriocarcinoma JEG-3cell xenograft model in SCID mice and to study the effect of VB1the alone and combined with5-FU treating the tumor-bearing mice, and to detect preliminary observations of its toxicity. Methods:1. The human choriocarcinoma JEG-3cells xenograft model was tried to establish using SCID mice and the biological characteristics of the tumor model were observed.2. The tumor volume, weight of the xenograft was observed and calculated after the mice was treated with VB1alone or combined with5-FU. The serum (3-HCG level was examined by chemoluminescence. White blood cell were count in peripheral blood, bone marrow smears and liver enzyme were examined in order to understand its toxicity.Results:1. The human choriocarcinoma JEG-3cells xenograft model was successfully establishe using the SCID mice.2. VB1significantly inhibited the growth of choriocarcinoma in SCID mice both in volume and weight.3. The Volumes and weights of the xenograf tumors in SCID mice were obviously reduced by VB1and VB1combined with5-FU compa-ring with non-treated tumors. More reduction occurred in xenogmfted tumors in combined group (P<0.05).4. The serum (3-HCG level was reduced after the drug treatment.5. The tumor-bearing mice bone marrow suppression and liver damage was not obvious; high doses the VB1may cause tumor-bearing mice weight loss. Conclusion:1. VB1can suppress the growth of human choriocarcinoma and decreased the serum level of β-HCG in tumor-bearing mice.2. VB1combined with5-FU can enhance its suppression effect in mice model.3. VB1has no significant inhibition to bone marrow, liver damage are not obvious in a short period.