Study On Ursolic Acid Induced Apoptosis In Jurkat Cells

ObjectiveUrsolic acid ( UA, C₃₀ H₄₈ O₃ ) , belongs to triterpenoids, the molecular weight of which is 456. 68. Its melting point is 285 291℃ ,and it can be dissolved in methanol, ethanol, butanol and butanone. It is sparingly soluble to acetone, benzene, chloroform, ethylether and unsoluble to water and ligroin. UA exists in many kinds of plants. According to present data, UA can be isolated from 34 families 108 species of plants in nature, such as the leaves and the fruits of arbutus uva - ursi, the leaves of paulownia. For its liposolubility, UA can pass through the cell membrane to produce its extensive biological effects, the most prominent of which is to prevent the development of tumors. It usually has great inhibiting effects to agents with carcinogenesis, and also has cytotoxic action to many kinds of malignant tumors, by inducing their differentiation and preventing their vasifaction. All their effects above have been verified recently. In 1990, UA was classified as one of the most promosing chemoprophylactic drugs in Japan. In addition, UA can improve liver function, treat dyslipidemia, and also has anti - bacteria and anti - virus effects.UA has remarkable effect on treatment of malignant tumor of hematological system, and can inhibit the proliferation of P3HR1 cells of Burkitt lymphoma and K562 cells of chronic myelogenous leukemia. Some researches have found that UA can decrease the activity of Daudi cells of human lymphoma, and meanwhile it can induce changes of their cellular morphology, DNA disruption and nucleus condensation.Study in the past few years, found that the mechanisms of ursolic acid for inhibiting tumors are;â‘  resisting carcinogenous agents;â‘¡ antioxidation;â‘¢blocking cell cycle;(4) inducing differentiation and apoptosis of tumor cells;(5) inhibiting angiogenesis;?preventing invasion of tumor cells;(7) enhancing im-munological function;? protecting liver function. However, the study on the molecular mechanism of the antineoplastic action of ursolic acid, is not clear. It has impeded the exploitation and application of the new drug. Apoptosis plays an important role in the antineoplastic action, so we will investigate the apoptosis action induced by ursolic acid and its molecular mechanism in Jurkat cells in this study. It may be useful for the treatment of hematological malignant tumors in application of ursolic acid.Materials1. Jurkat Cells2. Ursolic Acid3. Reagents for FACS4. Reagents for Western BlottingMethods1. Cell CultureJurkat cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, lOOunits/ml penicillin, and 100|xg/ml streptomycin. They were maintained in a 37^ , 5% CO2 , fully humidified incubator.2. Cytotoxic Action AssayThe toxic action of ursolic acid in Jurkat cells was assessed using WST kit - 8. Cells in logarithm growth stage were transferred in 96 - well plates. Ursolic acid was added at various concentrations, and lOul WST kit -8 was added after different hours. One hour later, cell survival rate were assayed by a Spectra max microplate at 450 nm. Complete medium and DMSO were used as blank control and solventia control respectively. Repeated five wells were set up for each concentration in each time.3. Cell Apoptosis AnalysisJurkat cells, treated by 20 or 40jxmol/L ursolic acid for 2 or 4 hours, were incubated with FITC - conjugated Annexin V and counterstained with propidium iodide (PI) in order to allow exclusion of necrotic cells. The cells were subsequently analyzed using a flow cytometer. 10 000 cells were analyzed in every sample.4. Protein ExtractionCells, treated by ursolic acid for 4 to 8 hours or treated by ursolic acid for 1 to 2 hours after cultured with medium without fetal bovine serum for 24 hours, were collected and washed twice with phosphate buffered saline, than solved by cell lysis buffer. Cell protein extracts were obtained with centrifugation at 15, 000 rpm for 15 minutes after three freeze - thaw cycles. Then the concentration of protein was determined.5. Western Blotting analysisProtein, extracted from ursolic aicd treated Jurkat cells, were electrotrans-fered to PVC membrane after separated by SDS - PAGE electrophoresis. Pro-caspase - 3 , cleaved caspase - 3 , Procaspase - 9, cleaved caspase - 9 , Akt and Akt phosphorylation were analyzed by Western Blotting.Results1. The cytotoxic action of ursolic acid is significant on Jurkat cells.2. Ursolic acid induced apoptosis in Jurkat cells.3. Caspase -9 and caspase -3 were activated, and the phosphorylation of Akt was inhibited in the apoptosis process induced by ursolic acid in Jurkat cells.ConclusionUrsolic acid could induce apoptosis in Jurkat cells through the mitochondria pathway. The mechanism is associated with the inhibition of Akt phosphorylation.