Study On The Anti-tumor Effect Of Gambogic Acid Upon Lymphoid Neoplasm Cell Lines And Its Underline Molecular Mechanism

PART I Regulation of gambogic acid on cell proliferation and cell cycle of lymphoid neoplasm cell lines【Objective】To explore the Regulation of gambogic acid on cell proliferation and cell cycle of Jurkat cells and Raji cells.【Methods】Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyl- tetrazolium bromide assay. Cell cycle was exmined by PI staining thorough flow cytometry. Expressions of Cyclin D3 and Cyclin E were detected by western blotting.【Results】GA inhibited the proliferation of Jurkat cells and Raji cells with 50% inhibitory concentration values of 1.69±0.09 (24 h), 0.98±0.13 (48 h), and 0.67±0.12 mmol/L (72 h) and 1.34±0.06 (24 h),0.39±0.03 (48 h),0.30±0.02μmol/L (72 h), respecitvely. In a dose-dependent manner, Gambogic acid arrested cell cycle of Jurkat and Raji cells at G0/G1 phase, the percentage of which increased from 41.78% to 62.72% and from 40.23% to 67.30%, respectively. Gambogic acid downregulated the expression of Cyclin D3 and Cyclin E dose and time dependently. 【Conclusion】Gambogic acid suppresses the proliferation of Jurkat cells and Raji cells and arrested cell cycle of them at G0/G1 phase. Cell cycle arrest may be through downregulation of Cyclin D3 and Cyclin E.Part II Regulation of gambogic acid on anti-apoptotic protein of Jurkat cells and Raji cells【Objective】To study the Regulation of gambogic acid on anti-apoptotic protein of Jurkat cells and Raji cells.【Methods】Annexin V-fluorescein-isothiocyanate/propidium iodide were used to detect apoptosis. Expressions of Bcl-xL and NF-κB were detected by western blotting.【Results】Treated with 0, 1.0, 2.0, and 3.0μmol/L GA for 24 h, percentages of the early apoptotic cells in the whole Jurkat population was from 0.95% and 2.09% to 34.03% and 38.30%, and that of Raja cells from 4.21% and 10.76% to 16.13% and 24.38%. Treated with 0, 0.5, 1.0, 2.0, and 4.0μmol/L GA for 24 h, expression of Bcl-xL and NF-κB was downregulated dose-dependently except Bcl-xL in Jurkat cells by 0.5μmol/L GA and NF-κB in Raji cells by 0.5μmol/L GA. There were no difference in those groups.【Conclusion】Gambogic acid is able to induce the apoptosis of Jurkat cells and Raji cells, which is partly through the downregulation of the expression of antiapoptotic protein, Bcl-xL and NF-κB. Part III Gambogic acid induces death inducer-obliterator 1-mediated apoptosis【Objective】To explore the apoptosis inducing effects of GA on Jurkat cells and Raji cells and its underlying mechanism.【Methods】Western blotting was used to study the expression of DIO-1 and pro-caspase 3, as well as 2 activated subunits: p17 and p20. The subcellular localization of DIO-1 was examined by immunofluorescence and Hoechst33258 staining.【Results】Treated with 0, 0.5, 1.0, 2.0, and 4.0μmol/L GA for 24 h, the expression of DIO-1 increased dose-dependently and then declined, and pro-caspase 3 was cleavage into 2 subunits: p17 and p20. Treated with 2.0μmol/L GA for 0, 2, and 24h, the expression of DIO-1 increased time-dependently. GA induced the translocation of DIO-1 from cytoplasm into the nucleus in Jurkat and Raji cells.【Conclusion】Gambogic acid upregulates the DIO-1 expression and was related to DIO-1 translocation, leading to the apoptosis of Jurkat and Raji cells. The DIO-1 expression is associated with the activation of pro-caspase 3 accompanied with the downregulation of Bcl-xL and NF-kB. DIO-1 triggered early-stage cell death in GA-treated Jurkat cells.