Nucleoporin And Nuclophosmin--New Molecular Targets For Acute Leukemia With Natural Drugs

Part I. Effects of deguelin on the regulation of nucleoporins and nucleophosmin in Jurkat leukemia cells in vitroObjective: To study the antileukemia effects of deguelin on leukemia cells, and explore the possible mechanisms to provide more theories for clinical application.Methods: Human T cell leukemia cell line Jurkat was chosen as a model. The effect of deguelin on the growth of Jurkat cells was studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was detected through DNA fragmentation assay, Hoechst 33258 staining assay and Annexin V/PI double-labeled cytometry. The expression levels of nucleophosmin (NPM) and nucleoporins, including Nup88 and Nup214, were studied by flow cytometry, Western Blot and reverse transcription-polymerase chain reaction (RT-PCR). The subcellular distributions of nucleoporins and nucleophosmin were detected through confocal microscopy.Results: (1) The proliferation of Jurkat cells was inhibited by deguelin in a time- and dose-dependent manner, with IC50 value for 24 h being 46.03±0.13 nmol/L. (2) Deguelin induced DNA fragmentation and apoptotic cell death in a dose-dependent manner, which was accompanied by typical morphological changes associated with apoptosis. Nearly 20 percent of cells went apoptosis after being cultured with 40 nmol/L deguelin for 24 h. (3) The expression levels of Nup88, Nup214 and NPM were decreased in a dose-dependent manner both at protein level and transcription level when cultured with deguelin. Conclusion: Deguelin presents powerful anti-leukemia effects through both proliferation inhibition and apoptosis induction on Jurkat leukemia cells. The possible mechanism might be involved in the regulation of NPM and some nucleoporins, such as Nup88, Nup214.Part II The anti-tumor effects of gambogic acid through the regulation of cell proliferation, cell apoptosis and cell cycle in leukemia cellsObjective: To study the anti-tumor effects of gambogic acid on malignant leukemia cell lines in vitro.Methods: Acute myelocytic leukemia cell line (HL-60), acute monocytic leukemia cell line (U937) and human chronic myelocytic leukemia in blast crisis cell line K562 were chosen as models. The effects of gambogic acid on the growth of three leukemia cell lines were studied by MTT assay. Apoptosis was detected through Hoechst 33258 staining assay and Annexin V/PI double-labeled cytometry. The effects of gambogic acid on the cell cycle distribution of three leukemia cell lines were studied by propidium iodide method. Results: (1) Gambogic acid showed potent antileukemia effects on all three leukemia cell lines through proliferation inhibition. Among them, HL-60 was the most sensitive one to gambogic acid, while K562 was relatively insensitive to gambogic acid. (2) Gambogic acid induced apoptotic cell death in a dose-dependent manner in all three leukemia cell lines. Among them, HL-60 was still the most sensitive one, more than half of cells went early apoptosis when being cultured with 1.6μmol/L gambogic acid for 12 h, and the total apoptosis rate was (77.84±2.38)%. However, U937 and K562 were relatively less sensitive to gambogic acid. (3) The effects of gambogic acid on the cell cycle distribution of three leukemia cell lines were not the same. Both U937 and K562 cells were found arrested in G0/G1 phase when interfered by gambogic acid, while the percentage of cells in S phase was decreased accordingly. However, no obvious change was found in HL-60 cells, moreover, most of cells went apoptosis before being arrested in whatever phase. Conclusion: Gambogic acid presents powerful anti-leukemia effects through both proliferation inhibition and apoptosis induction. HL-60 cell line was the most sensitive one to gambogic acid of all three leukemia cell line. However, the exact mechanism may vary from one cell line to another.Part III Effects of gambogic acid on the regulation of nucleoporins and nucleophosmin in Jurkat leukemia cells in vitroObjective: To study the antileukemia effects of gambogic acid on leukemia cells, and explore the possible mechanisms to provide more theories for clinical application.Methods: Human T cell leukemia cell line Jurkat was chosen as a model. The effect of gambogic acid on the growth of Jurkat cells was studied by MTT assay. Apoptosis was detected through Hoechst 33258 staining assay and Annexin V/PI double-labeled cytometry. The expression levels of apoptosis regulatory proteins, such as Bcl-2 and Bcl-xL, and nucleoporins, including Nup88, Nup214 and Nup98, and nucleophosmin (NPM) were studied by flow cytometry, Western Blot and RT-PCR. The subcellular distributions of nucleoporins and nucleophosmin mentioned above were detected by both immunocytochemistry analysis and confocal microscopy.Results: (1) Gambogic acid showed potent antileukemia effects on Jurkat cells through the proliferation inhibition, with the IC50 value for 24 h being 1.822±0.148μmol/L. (2) Gambogic acid induced apoptotic cell death in a dose-dependent manner, which was accompanied by typical morphological changes associated with apoptosis and decreased expression of Bcl-2 and Bcl-xL. (3) The expression levels of Nup88, Nup214, Nup98 and NPM were also found decreased in a dose-dependent manner both at protein level and transcription level when cultured with gambogic acid. Moreover, the subcellular distributions of Nup88 and Nup214 in Jurkat cell were changed as well. Conclusion: Gambogic acid presents powerful antileukemia effects through both proliferation inhibition and apoptosis induction on Jurkat leukemia cells. The possible mechanism might be involved in the regulation of the expression levels of NPM and some nucleoporins, such as Nup88, Nup214 and Nup98, as well as the regulation of the subcellular distribution of nucleoporins.