Effect And Mechanism Of Triptolide On Mice Prostatic Carcinoma

Objective:Based on the application of the experienced prescriptions against tutor in the treatment of prostate carcinoma which has achieved preferable clinical effectiveness, this experimentation, through in vivo and in vitro experiments, will further study the functions and possible mechanism of Triptolide's (TL) effective anti-cancer ingredient in suppressing the proliferation of mice prostate carcinoma , so as to provide a safe and effective anti-prostate cancer medicine for clinical purposes.Method:The RM-1 passage cells of mice prostate carcinoma are used in this experimentation, and TL's suppression on the proliferation of RM-1 cells is tested through MTT experiment; the influences of TL on the apoptosis RM-1 cell is tested through acridine orange fluorescent staining; the function of TL on the cycle and apoptosis peak of RM-1 cells is analyzed through flow cytometry; the function of TL on the DNA apoptosis of RM-1 cells is tested through "Trapezoid" electrophoresis; and the caspase-3 , bcl-2 mRNA expression levels of treated cells are tested through RT-PCR testing agent. The hypodermic injection of RM-1 cells in C57BL/6 mice is carried out in the in vivo experiment to establish the animal prostate carcinoma model, and then the volume and weight of the tumor is measured to calculate the tumor growth suppression rates; with the help of TUNEL technology, the apoptosis cells in the prostate carcinoma organism are tested; and through immunization group measurement method, the influences of the medicine on the tumor caspase-3 gene and its protein bcl-2 expression level of the experimented animals are observed. Result:1. The growth suppression rates of TL (5, 10, 20, 40 and 80ng/ml) treated RM-1 cells are 9.8%, 25.1%, 39.2%, 48.8% and 53.2%, and 12, 24, 36 and 48h after the treatment of TL ( 10 and 20ng/ml) on RM-1 cells, the cell growth suppression rates are 8.4%, 25.1%, 36.1%, 42.4% and 10.2%, 39.2%, 50.2% and 58.5%, and MTT tests show that Triptolide is able to suppress the growth of RM-1 cells depending on dosages and time durations.2. 14 days after prostate carcinoma mice models are established, the growth of tumors of the TL (0.2mg/kg), TL(0.4mg/kg) and TL (0.8g/kg) treated mice groups is obviously slower than that of the model reference group, and the weight differences of tumors between the several TL treated groups and the model reference group are obvious ( P<0.01 ) . The in vivo experiment results show that TL at a certain concentration is able to suppress the growth of prostate carcinoma cells in mice.3. Acridine orange fluorescent staining observation shows that, after the TL treatment on RM-1 cells, nucleus condensation, cell membrane invagination, irregular orange particles in the cells, and apoptosis morphological changes are found.4. After the analysis of cell cycles and apoptosis peaks through flow cytometry, the results show that, 48h after the TL (10 and 20ng/ml) treatment on RM-2 cell, obvious apoptosis peak appears before the G₁ stage, and apoptosis rates are 41.1% and 57.8%.5. The DNA electrophoresis analysis shows that, 24h, 36h and 48h after the TL treatment, DNA "trapezoid" spectrum can be seen, and DNA "trapezoid" strips in RM-1 cells 48h after the TL treatment are especially obvious.6. TUNEL staining results show that, in prostate carcinoma animals after the TL treatment, positive TUNEL staining cells increase, and there are light brown particles inside the nucleus, and small apoptosis roundlets, nucleus condensation and large amount of tiny nucleuses form. Experiments have shown that TL has the function of inducing the apoptosis of prostate carcinoma cells.7. RP-PCR tests show that, the caspase-3 mRNA expressions of TL treated RM-1 cells are higher than those untreated reference cells, indicating that the medicine is able to induce the apoptosis of RM-1 cells through the heightening of their caspase-3 expressions. And the bcl-2 mRNA expressions are lower than those untreated reference cells, indicating that the medicine is able to induce the apoptosis of RM-1 cells through the lowering of their bcl-2 expressions.8. The immunization group results show that, in the TL treated mice prostate carcinoma organism, the positive cell caspase-3 protein expressions are obviously higher than that of the model reference group, indicating that the medicine is able to induce the apoptosis of RM-1 cells through the heightening of their caspase-3 expressions. And positive cell bcl-2 protein expressions are obviously lower than that of model reference group , further indicating that the medicine is able to lower the bcl-2 expression of RM-1 cells, and the bcl-2 protein is related to the division degrees of the prostate carcinoma cells. The above experiments show that TL is able to induce the apoptosis of RM-1 prostate carcinoma cell strains through the heightening of caspase-3 expressions and lowering of bcl-2 expressions.Conclusion:1. TL suppresses the proliferation of mice prostate carcinoma RM-1 cell strains, and is dependent on time duration and dosages. 2. TL suppresses the growth of prostate carcinoma cells in mice.3. TL induces the apoptosis of RM-1 cells.4. TL induces the apoptosis of prostate carcinoma cells in mice.5. The function of TL suppressing the growth of tumor is realized through the induction of cell apoptosis.6. TL raises the caspase-3 expression and lowers bcl-2 mRNA expression of RM-1 cells.7. TL raises the caspase-3 expression and lowers the bcl-2 expression of prostate carcinoma cells in mice.8. TL induces the apoptosis of prostate carcinoma cells through the heightening of their caspase-3 expressions and lowering of their bcl-2 expressions.