Effects of escin on TNF-induced apoptosis. A, the chemical structure of escin. B, escin potentiates apoptotic effects of TNF. KBM-5 cells (5000 cells/well) were treated with the indicated amounts of escin for 2 h followed by the indicated amounts of TNF for 24 h at 37°C. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium uptake method. C, KBM-5 cells (left) and Jurkat cells (right) were pretreated with 30 μM escin for 2 h and then incubated with 1 nM TNF for 24 h. The cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope as described under Materials and Methods. Data indicate the percentage of proportions of apoptotic cells and are expressed as mean ± S.E. D, left, cells were pretreated with 30 μM escin for 2 h and then incubated with 1 nM TNF for 16 h. The cells were incubated with a fluorescein isothiocyanate-conjugated annexin V antibody and then analyzed by flow cytometry as described under Materials and Methods. Right, cells were pretreated with 30 μM escin for 2 h and then incubated with 1 nM TNF for 16 h. The cells were stained for TUNEL-positive cells and then analyzed by flow cytometry as described under Materials and Methods. Determinations were made in triplicate. Data represent the mean of two independent measurements ± S.E. **, p < 0.05. E, effect of escin on PARP cleavage. Cells were pretreated with 30 μM escin for 2 h and then incubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blotting using an anti-PARP antibody. F, escin suppresses TNF-induced invasion activity. H1299 cells (2.5 × 10⁴ cells) were seeded to the top chamber of a Matrigel invasion chamber system overnight in the absence of serum and then treated with 30 μM escin. After incubation, the cells were treated with TNF in the presence of 1% serum and then assayed for invasion as described under Materials and Methods. Results are expressed as the fold activity of the untreated control. All results shown are representative of two independent experiments.

Effects of escin on TNF-induced cell survival, cell-proliferative, and metastatic gene products. A, escin suppresses the expression of TNF-induced antiapoptotic proteins. B and C, escin inhibits the expression of TNF-induced cell proliferative and metastatic proteins. KBM-5 cells (2 × 10⁶ cells/ml) were incubated with 30 μM escin for 2 h and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blotting using the relevant antibodies. All results shown are representative of two independent experiments. Densitometric values of bands were corrected based on β-actin and were expressed relative to that of untreated cells, which was set as 1.0.

Effects of escin on constitutive and induced NF-κB activation. A, left, dose-dependent effect of escin on TNF-induced NF-κB activation. KBM-5 cells (2 × 10⁶ cells/ml) were incubated with the indicated concentrations of escin for 2 h and treated with 0.1 nM TNF for 30 min. The nuclear extracts were assayed for NF-κB activation by EMSA. Results are expressed as fold activity of the untreated control. Right, time-dependent effect of escin on TNF-induced NF-κB activation. KBM-5 (2 × 10⁶ cells/ml) cells were preincubated with 30 μM escin for the indicated time points and then treated with 0.1 nM TNF for 30 min. The nuclear extracts were prepared and assayed for NF-κB activation by EMSA. Results are expressed as fold activity of the untreated control. B, NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated cells or cells treated with 0.1 nM TNF were incubated with the indicated antibodies, an unlabeled NF-κB oligonucleotide probe, or a mutant oligonucleotide probe. They were then assayed for NF-κB activation by EMSA. C, effect of escin on the binding of NF-κB to DNA. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nM TNF and incubated for 30 min with the indicated concentrations of escin. They were then assayed for NF-κB activation by EMSA. All results shown are representative of two independent experiments. D, left, escin inhibits NF-κB activation induced by TNF in H1299 cells. The H1299 cells (1 × 10⁶ cells/ml) cells were preincubated with indicated concentration of escin for 2 h and then treated with 0.1 nM TNF for 30 min. The nuclear extracts were prepared and assayed for NF-κB activation by EMSA. Results are expressed as fold activity of the untreated control. Right, effect of escin on constitutive NF-κB activation. Multiple myeloma U266 cells (2 × 10⁶ cells/ml) were incubated with the indicated concentrations of escin for 2 h; the nuclear extracts were prepared and analyzed for NF-κB activation by EMSA. Results are expressed as fold activity of the untreated control. E, escin inhibits NF-κB activation induced by CSC, H₂O₂, PMA, LPS, OA, and TNF. KBM-5 (2 × 10⁶ cells/ml) cells were pre incubated with 30 μM escin for 2 h and then treated with 0.1 nM TNF for 30 min, 500 nM okadaic acid for 4 h, 250 μM H₂O₂ for 2 h, 25 ng/ml PMA for 2 h, 10 μg/ml LPS, and 10 μg/ml CSC for 1 h each. Nuclear extracts were analyzed for NF-κB activation by EMSA. Results are expressed as fold activity of the untreated control. All results shown are representative of two independent experiments. WT, wild type; MT, mutant; CV, cell viability.

Effects of escin on TNF-induced IKK activation and IκBα degradation. A, escin inhibits TNF-induced NF-κB activation and IκBα degradation. Top, KBM-5 cells (2 × 10⁶ cells/ml) were incubated with 30 μM escin for 2 h and treated with 0.1 nM TNF for the indicated times. Nuclear extracts prepared from treated cells were analyzed for NF-κB activation by EMSA. Results are expressed as fold activity of the untreated control. Bottom, cytoplasmic extracts prepared from treated cells were analyzed by Western blotting using antibody against anti-IκBα. Equal protein loading was evaluated by β-actin. B, effect of escin on phosphorylation of IκBα induced by TNF. Cells were preincubated with 30 μM escin for 2 h, incubated with 50 μg/ml ALLN for 30 min, and then treated with 0.1 nM TNF for 10 min. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using phosphospecific IκBα antibody. C, escin inhibits ubiquitination of IKK-γ induced by TNF. KBM-5 (4 × 10⁶ cells/ml) cells were pre incubated with 30 μM escin for 2 h, incubated with 50 μg/ml ALLN for 30 min, and then treated with 0.1 nM TNF for 10 min. Whole-cell extracts were prepared and immunoprecipitated with IKK-γ antibody. Immunoblot was performed against ubiquitin antibody, and loading was confirmed by IKK-γ antibody. D, escin inhibits IKK activation induced by TNF. KBM-5 (4 × 10⁶ cells/ml) cells were pretreated with 30 μM escin for 2 h and then challenged with 1 nM TNF for indicated time points. Whole-cell extracts were immunoprecipitated with antibody against IKK-β and analyzed by an immune complex kinase assay. To examine the effect of escin on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies. E, direct effect of escin on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nM TNF and immunoprecipitated with anti-IKK-β antibody. The immune complex kinase assay was performed in the absence or presence of the indicated concentration of escin. All results shown are representative of two independent experiments. F, role of ROS on escin suppressed IKK activation by TNF. KBM-5 (4 × 10⁶ cells/ml) cells were preincubated with 10 mM NAC for 1 h, 30 μM escin for 2 h, and then challenged with 1 nM TNF for 10 min. Whole-cell extracts were immunoprecipitated with antibody against IKK-β and analyzed by an immune complex kinase assay. G, effect of escin on TNF-induced MAPK activation. Cells were preincubated with 30 μM escin for 2 h and then treated with 0.1 nM TNF for 10 min. Whole-cell extracts were fractionated and then subjected to Western blot analysis using the relevant antibodies. For JNK activation, whole-cell extracts were immunoprecipitated with antibody against JNK1 and analyzed by an immune complex kinase assay. To examine the effect of escin on the level of expression of JNK protein, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-JNK1 antibody.

Effects of escin on phosphorylation and nuclear translocation of p65 induced by TNF. A, escin inhibits TNF-induced nuclear translocation of p65. KBM-5 cells were first treated with 30 μM escin for 2 h at 37°C and then exposed to 0.1 nM TNF for 15 min. After cytospin, immunocytochemical analysis was done as described under Materials and Methods. B, escin inhibits TNF-induced translocation and phosphorylation of p65. KBM-5 cells were either untreated or pretreated with 30 μM escin for 2 h at 37°C and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic and nuclear extracts were prepared and analyzed by Western blotting using p65 and phosphospecific p65 antibodies. For loading control of nuclear protein, the membrane was reblotted with either β-actin or anti-PARP antibody.

Effects of escin on NF-κB-dependent reporter gene expression. A, escin inhibits TNF-induced NF-κB-dependent reporter gene (SEAP) expression. A293 cells (2.5 × 10⁵ cells/ml) were transiently cotransfected with an NF-κB-containing plasmid linked to the SEAP gene; after 24 h of transfection, cells were treated with the indicated concentrations of escin for 2 h followed by 1 nM TNF for an additional 24 h. Cell supernatants were collected and assayed for SEAP activity as described under Materials and Methods. Results are expressed as fold activity over the activity of the vector control. B, escin inhibited NF-κB-dependent reporter gene expression induced by TNF, TNFR1, TRADD, TRAF2, NIK, IKK, and p65. A293 cells were transiently transfected with the indicated plasmids along with an NF-κB-containing plasmid linked to the SEAP gene and after 24 h cells were either treated or untreated with 30 μM escin for 2 h. Where indicated, cells were exposed to 1 nM TNF for 12 h. Cell supernatants were assayed for SEAP activity as described under Materials and Methods. Results are expressed as fold activity over the activity of the vector control. C, whole-cell extracts from transfected cells with various plasmids were prepared and analyzed by Western blotting using antibodies against TNFR1, TRADD, TRAF2, NIK, IKK-β, and p65. DN, dominant-negative. All results shown are representative of two independent experiments.