Nucleoporin And Protein Acetylation

AIM: To investigate anticancer effects and molecular mechanism of deguelin on human Burkitt's lymphoma Raji and Daudi cells in vitro and compare the cytotoxicities of deguelin on Raji and Daudi cells and human peripheral blood monocular cells (PBMC).METHODS: The effects of deguelin on the growth of Daudi cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium (MTT) assay. Apoptosis were studied through Hoechst 33258 staining and Annexin V/PI double-labeled cytometry. The effect of deguelin on the cell cycle of Daudi cells were studied by a propidium iodide method. The expressions of cyclin D1 and pRb were checked by Western blot.RESULTS: The proliferation of Daudi cells were decreased in deguelin-treated group with with a 24 h IC₅₀ value of 51.55 nmol/L and Raji cells with a 24 h IC₅₀ value of 55.98 nmol/L. Deguelin induced Raji and Daudi cells apoptosis in a time- and dose-dependent manner. Daudi cells treated with deguelin showed G₀/G₁ phase increase and S phase decrease. 0, 5, 10, 20, and 40 nmol/L deguelin-treated for 24 h, G₀/G₁ phase increased from 37.34% to 56.56%, whereas S phase decreased from 31.12% to 21.36%. Deguelin arrested G₂/M phase in Raji, correspondingly reducing and increasing cells in G1 and an S phase. Treated with 0, 10, 20, and 40 nmol/L deguelin for 24 h, the rate of S phase cells were 28.63%, 24.91%, 22.94%, and 17.29%, and G₂/M phase cells were 13.04%, 13.97%, 36.32%, and 54.67%. PBMC was less sensitive to the cytotoxic effect of deguelin than Daudi cells. The expression of cyclin D1 and pRb protein were decreased sharply in Daudi cells treated with deguelin.CONCLUSION: Deguelin is able to inhibit the proliferation of Raji and Daudi cells by regulating the cell cycle that arrested cells at G₂/M phase and G₀/G₁ phase respectively and inducing the cell apoptosis. Moreover, deguelin demonstrated low toxicity in PBMC but selectively induced apoptosis of Daudi cells. The antitumor effects of deguelin were related to down-regulating the expression of cyclin D1 and pRb protein.PART IIDeguelin regulates the nuclear pore complex protein Nup98 and Nup88 in U937 cells in vitroAIM: To investigate anticancer effects and molecular mechanism of deguelin on the human U937 leukaemia cells and to explore the underlying mechanism regulating the nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro.METHODS: The effects of deguelin on the growth of U937 cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium (MTT) assay. The effect of deguelin on the cell cycle of U937 cells was studied by a propidium iodide method. The localization of the nuclear pore complex protein Nup98 and Nup88 was checked by immunoflurescence and immunoelectron microscopy. The expressions of Nup98 and Nup88 in U937 cells were checked by flow cytometry (FCM) and by Western blot.RESULTS: The proliferation of U937 cells were inhibited in deguelin-treated group with a 24 h IC₅₀ value of 21.61 nmol/L and 36 h IC₅₀ value of 17.07 nmol/L. U937 cells treated with deguelin showed reduction in the percentages of cells in G₀/G₁, whereas accumulation of cells in S and G₂/M phase. Nup88 and Nup98 were found on both the nuclear and cytoplasmic side of the U937 cells by immunoflurescence and immunoelectron microscopy. The expression of Nup98 was up-regulated and Nup88 protein was down-regulated in U937 cells treated with deguelin.CONCLUSION: Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle that arrested cells at S and G2/M phase, whereas G0/G1 phase decreased. The antitumor effects of deguelin were related to up-regulating the expression of Nup98 and down-regulating the expression of Nup88 protein in U937 cells.PART IIICurcumin, a potent anti-tumor reagent, is a novel histone deacetylase inhibitor regulating B-NHL cell line Raji proliferation effectAIM: To investigate curcumin (diferuloylmethane) induced apoptosis and its molecular mechanism of action in B-NHL cell line Raji cells.METHODS: Raji cells were cultured in RPMI-1640 medium and treated with curcumin in different concentrations. 3-(4, 5-dimethyl -2- thiazolyl) -2, 5diphenyl -2 H- tetrazolium (MTT) assay was used to detect growth inhibition and Hoechst 33258 staining was used to detect apoptosis. Immunocytochemistry and Western blot were used to detect the expressions of histone deacetylase 1, 3, and 8 (HDAC1, HDAC3, HDAC8) and acetylated histone H4 (Ac-histone H4) protein.RESULTS: Curcumin inhibited the proliferation of B-NHL cell line Raji cells with a 36 h IC₅₀ value of 24.l±2.01 μmol/L. Hoechst 33258 staining showed that curcumin can induce Raji cell apoptosis. The expression levels of HDAC1, HDAC3, and HDAC8 proteins were downregulated following curcumin treatment in Raji cells, whereas Ac-histone H4 protein expression was upregulated after treatment with curcumin.CONCLUSION: Curcumin, as a new member of the histone deacetylase inhibitors, can inhibit the expression of class I HDACs (HDAC1, HDAC3, and HDAC8), and can increase the expression of Ac-histone H4 in Raji cells. Curcumin plays an important role in regulating B-NHL cell line Raji cell proliferation and apoptosis.