Figure 1.
Cryptopleurine inhibits NF-κB activation by different stimuli.
(A) Structure of cryptopleurine. (B) MDA-MB231 cells (upper panel) and Hep3B cells (lower panel) were preincubated with indicated concentrations of cryptopleurine for 12 h and then treated with TNF-α (20 ng/ml) for 15 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. In lane AP-1, a 100-fold excess of unlabeled AP-1 consensus oligonucleotide was added to the reaction mixture. In lane κB, a 100-fold excess of unlabeled κB consensus oligonucleotide was added to the reaction mixture. The arrow indicates the location of the DNA-NF-κB complex. (C) MDA-MB435, and MCF-7 cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for 15 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. (D) Hep3B, and RAW264.7 cells were preincubated with 30 nM cryptopleurine for 12 h and then treated with 25 ng/ml PMA or 1 μg/ml LPS for 90 min. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA.
Figure 2.
Effect of cryptopleurine on the TNF-α-induced phosphorylation and degradation of IκBα.
(A) MDA-MB231 cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. (B) MDA-MB231 cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Cytoplasmic extracts were analyzed by Western blot using indicated antibodies for p-IκBα, IκBα, and tubulin. (C) MDA-MB231 cells were preincubated with indicated concentrations of cryptopleurine for 12 h and then treated with TNF-α (20 ng/ml) for 15 min. Cytoplasmic extracts were analyzed by Western blot using indicated antibodies for p-IκBα, IκBα, and tubulin. (D) MDA-MB231 cells were preincubated with 30 nM cryptopleurine for 12 h, incubated with 50 μg/ml ALLN for 30 min, and then treated with 20 ng/ml TNF-α for 15 min. Cytoplasmic extracts were analyzed by Western blot using indicated antibodies for p-IκBα, IκBα, and tubulin.
Figure 3.
Effect of cryptopleurine on the TNF-α-induced activation of IκBα kinase, p65 phosphorylation, and p65 nuclear translocation.
(A) HEK293 cells transfected with IKKα or IKKβ stimulated with TNF-α for the indicated times in the presence or absence of 30 nM cryptopleurine. Whole-cell extracts were immunoprecipitated with antibody for IKKα or IKKβ, and In vitro kinase assays were performed with GST-IκBα and [γ-32p]ATP. To show the equal amount of immunocomplex in each reaction, the bottom gels represent IKKα or IKKβ detected with Western blot. (B) HEK293 cells transfected with IKKα or IKKβ stimulated with TNF-α for 15 min, and whole-cell extracts were immunoprecipitated with antibody for IKKα. Indicated concentrations of cryptopleurine was added to the immunoprecipitated IKK complex, incubated for 30 min, and analyzed by an immune complex kinase assay. MDA-MB231 cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Nuclear extracts (C) and cytoplasmic extracts (D) were analyzed by Western blot using indicated antibodies for Ser536 phosphorylation in p65, p65, tubulin, and Topo-I.
Figure 4.
Effect of cryptopleurine on the TNF-α-induced NF-κB-dependent reporter gene expression.
(A) MDA-MB231 cells were transiently transfected with a NF-κB reporter construct pNF-κB-Luc for 24 h. After transfection, cells were incubated with indicated concentrations of cryptopleurine for 12 h and then treated with 20 ng/ml TNF-α for an additional 12 h. The lysates of MDA-MB231 cells were subject to the measurement of dual luciferase activity. Data represented as mean ± standard deviation of three independent experiments. **p<0.01, ***p<0.001, significantly different when compared with TNF-α-stimulated normal cells. (B) MDA-MB231 cells were transiently transfected with a NF-κB reporter construct pNF-κB-Luc alone or with plasmids expressing the indicated proteins. After transfection, cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with the relevant plasmid for an additional 12 h. TNF-α-treated cells were incubated with 30 nM cryptopleurine for 12 h and then treated with 20 ng/ml TNF-α for an additional 12 h. The lysates of MDA-MB231 cells were subject to the measurement of dual luciferase activity. Data represented as mean ± standard deviation of three independent experiments. ***p<0.001, significant with respect to control.
Figure 5.
Effect of cryptopleurine on the TNF-α-induced NF-κB-dependent inflammatory cytokines, antiapoptotic, and proliferation genes expression.
(A) MDA-MB231 cells were preincubated with indicated concentrations of cryptopleurine for 12 h and then treated with TNF-α (20 ng/ml) for an additional 12 h. RNA was isolated from cells, reverse-transcribed, and analyzed by real-time PCR for IL-6, IL-8, and IL-1β. Data represented as mean ± standard deviation of three independent experiments. *p<0.05, ***p<0.001, significantly different when compared with TNF-α-stimulated normal cells. (B) MDA-MB231 cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Whole cell extracts were analyzed by Western blot using indicated antibodies for TRAF2, Bcl2, cIAP1, FLIP, COX-2, cyclinD1, and tubulin. (C) MDA-MB231, MDA-MB435, RAW264.7, and Hep3B cells were plated in triplicate, treated with 0, 10 or 30 nM cryptopleurine, and subjected to MTT assay on days 1, 2, 3 to analyze cell proliferation. Absorbance was measured at 570 nm.
Figure 6.
Effect of cryptopleurin on the TNF-α-induced apoptosis.
(A) MDA-MB231 cells were pretreated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for 24 h, and subsequently stained with Annexin V-FITC and propidium iodide, followed by analysis using a flow cytometer. Representative plots of one set of triplicate experiments. Early apoptotic cell (Annexin-V+ and PI) were displayed in the lower right quadrant and late apoptotic cells (Annexin-V+ and PI+) were shown in the upper right quadrant. (B) MDA-MB231 cells were pretreated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for 24 h. Whole cell extracts were analyzed by Western blot using indicated antibodies for cleaved capase-8, cleaved capase-3, cleaved PARP, and tubulin.
Figure 7.
Effect of cryptopleurine on the TNF-α-induced invasion activity.
(A) MDA-MB231 cells were incubated with 30 nM cryptopleurine for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Whole cell extracts were analyzed by Western blot using indicated antibodies for ICAM-1, MMP-9, VEGF, and tubulin. (B) MDA-MB231 cells invaded through the pores in the Matrigel-coated filters were fixed, stained and counted in five random field visualized by microscopy (100×). Data represented as mean ± standard deviation of three independent experiments. ***p<0.001, significant with respect to control.