Effects Of Polyphyllin Ⅰ/Ⅱon Proliferation And Apoptosis Of Human Bladder Carcinoma Cells

Objective:1. To investigate the effects of Polyphyllin Ⅰ/Ⅱon proliferation of human bladder carcinoma cells.2. To investigate the effects of Polyphyllin Ⅰ/Ⅱon apoptosis of human bladder carcinoma cells.3. To study the mechanisms of Polyphyllin Ⅰ/Ⅱ on proliferation and apoptosis of human bladder carcinoma cells.4. To study the effects of f Polyphyllin Ⅱ on the Balb/c nude mice with human bladder carcinoma.Methods:1. Detection of cell proliferation about Polyphyllin Ⅰ/Ⅱon human bladder carcinoma cells lines EJ/BIU-87/T24:Cell proliferation were measured by MTT assay With mitomycin C (MMC) as positive control. The cell proliferation ratio were measured at 24h,48h, and 72 h with Polyphyllin Ⅰ/Ⅱ with different concentrations or not. The 50% inhibitory concentrations (IC50) of PP Ⅰ/Ⅱ were measured. Cell (EJ/BIU-87/T24) cycles were measured by flow cytometry with Polyphyllin Ⅰ/Ⅱ with different concentrations or not. The effects of Polyphyllin Ⅰ/Ⅱ on different bladder cancer cell line EJ, BIU-87and T24 were analysed.2. Detection of cell apoptosis about Polyphyllin Ⅰ/Ⅱ on human bladder carcinoma cells lines EJ/BIU-87/T24:The changes of cell morphology with polyphyllin Ⅰ/Ⅱ with different concentration were observed by microscope and transmission electron microscopy (SEM). Cell apoptosis were measured by flow cytometry.3. The preliminary study of the mechanisms of Polyphyllin Ⅰ/Ⅱ on proliferation and apoptosis of human bladder carcinoma cells:To explore the mechanism of cell apoptosis caused by polyphyllin Ⅰ/Ⅱ, the cell mitochondrial apoptotic pathways were chosed for further study. The total mRNA of each groups were extracted and cDNA were synthesized. Real-time polymerase chain reaction were used to quantify the relative quantification of cDNA of cytochrome c, caspse-3,caspase-9,Bax and Bcl-2. cytochrome c. The expression of protein of Bcl-2, Bax, caspase 3, caspase-9 were measured by western blot.4. To evaluate of the effects of polyphyllin II on the Balb/c nude mice with human bladder carcinoma:Transplanted subcutaneously tumor models of human bladder carcinoma were established in nude mice firstly. The effectiveness of polyphyllin Ⅱ on human bladder carcinoma was evaluated.Results:1. The abilities of proliferation of EJ/BIU-87/T24 cells were inhibited by polyphyllin Ⅰ/Ⅱ:No obviously inhibitory effects were seen when cells EJ/BIU-87/T24 were treated with PP I of the concentration below 1μg/ml, even prolonged the duration to 72h (P>0.05). The obviously inhibitory effects were found in these three cell lines with PP I of concentration more than or equals to 1μg/ml at 24h,48h and 72h. The results showed that treatment with PP I exhibited an anti-proliferation capacity in a dose-and time-dependent manner. The 50% inhibitory concentrations (IC50) of PP I were 8.099,4.931,3.933μg/ml to EJ cells at 24h,48h and 72h. It was 8.299,5.048, 3.999 μg/ml to BIU-87 cells and 8.751,5.297,4.012 μg/ml to T24 cells respectively. The inhibitory effects of PP I on these three cell lines were almost the same.The obviously inhibitory effects were found in these three cell lines with PP II of concentration more than or equals to 1 μg/ml at 24h,48h and 72h. The treatment with PP II exhibited an anti-proliferation capacity in a dose-and time-dependent manner. The 50% inhibitory concentrations (IC50) of PP I were 8.271,5.457,3.815μg/ml to EJ cells at 24h,48h and 72h. It was 8.133,5.387,3.636μg/ml to BIU-87 cells and 8.529,5.761,3.905μg/ml to T24 cells respectively. The inhibitory effects of PPⅡ on these three cell lines were almost the same.Furthermore, Analysis of cell cycles of EJ,BIU-87 and T24 cells treated with PP Ⅰ/Ⅱ was carried out. Cells were treated with PP Ⅰ/Ⅱ of different concentrations for 24h (1,2,4 and 8μg/ml respectively) and analyzed by flow cytometry. EJ, BIU-87 and T24 cells were arrested at G2/M phase to varying degrees with PP I of different concentration. EJ, BIU-87 and T24 cells were arrested at S phase with PP Ⅱ.With the increasing of concentration, this effect was more significant.The inhibitory effects of PP Ⅰ/Ⅱ on these three cell lines were almost the same.2. The effect of cell apoptosis about Polyphyllin Ⅰ/Ⅱ on human bladder carcinoma cells lines EJ/BIU-87/T24:EJ/BIU-87/T24 cells treated with PP Ⅰ/Ⅱ(concentration was 4μg/mg) displayed morphological changes after exposure. Control group of cells untreated with PP Ⅰ/Ⅱ showed that there were many mitochondria in cytoplasm, microvilli were seen on the cell surface,chromatin were clear in the nuclei, nucleoli and nuclear membrane were clear, cytoplasmic staining were well-distributed. After being treated with PP Ⅰ/Ⅱ, the cells were shrinked, microvilli reduced or disappeared, cytoplasm condensed, mitochondria were swelling and a handful of endoplasmic reticulum were expansion, part of the nucleus fragmentated and dissolved. Cells have more significant change afer 48h with PP Ⅰ/Ⅱ treatment. The morphology of cells were inconformity, chromatin blocked at the edge of nuclear membrane, nuclear fragmentated and dissolved, the number of organelles decreased and the number of lysosome increased, membrane blebbing phenomenon and apoptotic body were seen. As the extension of time, the cell apoptosis phenomenon was more obvious.EJ,BIU-87 and T24 cells were treated with PP I of different concentrations for 24h (1,2,4 and 8μg/ml respectively). Flow cytometry testing results showed that apoptosis rates of the experimental cells were more higher than that of control group’s. It was in a time-and dose-dependence manner.3. The mechanisms of Polyphyllin Ⅰ/Ⅱ on proliferation and apoptosis of human bladder carcinoma cells:The gene of cytochrome c, the Bcl-2, caspase 3, caspase-9 and Bax were amplified. With the increasing of concentration of PP I, the RQ of cytochrome c, caspase-3 increased and Bcl-2, caspase-9 decreased significantly. The RQ of Bax decreased to some extent. The expressions of protein showed that cytochrome c, caspase 3 were increased and Bcl-2, caspase-9 were decreased. The change of Bax is not obvious.With the increasing of concentration of PP II, the RQ of cytochrome c, caspase-3 and caspase-9 increased and Bcl-2 decreased significantly. The RQ of Bax decreased to some extent. The expressions of protein showed that cytochrome c, caspase-3 and caspase-9 were increased and Bcl-2 decreased distinctly. The change of Bax is not obvious.4. The evaluation of the effects of polyphyllin II on the Balb/c nude mice with human bladder carcinoma:The growth of control subcutaneous transplantation tumor is rapid and the diameter of tumor was increased to 17 mm in 30 days. The tumor volume increased slowly with polyphyllin II in the first 2 weeks, and then tumor volume began to shrink, but didn’t completely disappear. The spirit of experimental animals was good and the weight were good. With Kaplan-Meier survival analysis, survival curve are obtained. The Survival rates of treatment group with MMC or polyphyllin II were significantly higher than the control group. The median survival and averaged survival time was 33 and 33.7 days in negative control group. It was 44 and 50.3 days in treatment group with MMC and 47 and 48.7 days in polyphyllin II group.Conclusion1. The abilities of proliferation of EJ/BIU-87/T24 cells were inhibited by polyphyllin I. The inhibitory effects of PP I on these three cell lines were almost the same. EJ, BIU-87 and T24 cells were arrested at G2/M phase to varying degrees with PP I of different concentration and apopotosis were induced. That maybe associated with the etiopathogenesis.2. The abilities of proliferation of EJ/BIU-87/T24 cells were inhibited by polyphyllin Ⅱ. The inhibitory effects of PP Ⅱ on these three cell lines were almost the same. EJ, BIU-87 and T24 cells were arrested at S phase to varying degrees with PP Ⅱ of different concentration and apopotosis were induced. That maybe associated with the etiopathogenesis.3. The gene of cytochrome c, the Bcl-2, caspase 3, caspase-9 and Bax were amplified. With the increasing of concentration of PP Ⅰ, the RQ of cytochrome c, caspase-3 increased and Bcl-2, caspase-9 decreased significantly. The RQ of Bax decreased to some extent. The expressions of protein showed that cytochrome c, caspase 3 were increased and Bcl-2, caspase-9 were decreased. The change of Bax is not obvious.With the increasing of concentration of PP Ⅱ, the RQ of cytochrome c, caspase-3 and caspase-9 increased and Bcl-2 decreased significantly. The RQ of Bax decreased to some extent. The expressions of protein showed that cytochrome c, caspase-3 and caspase-9 were increased and Bcl-2 decreased distinctly. The change of Bax is not obvious. PP Ⅰ/PP Ⅱ play the role of resistance to bladder cancer not only through affecting the copy of apoptosis related gene,but also influencing the expression links of apoptosis related protein.4. Polyphyllin Ⅱ have the effects of inhibition the proliferation of bladder cancer cell in vivo. It can inhibit tumor growth and prolong the survival time, well be tolerated in nude mice.5. Polyphyllin Ⅰ/Ⅱ may become one of the alternative medicine of bladder cancer patients postoperative perfusion treatment.