Figure 1.
The chemical structure of cucurbitacin IIb (CuIIb) (A) and its effect on the proliferation of Con A-stimulated lymphocytes.
Mouse lymphocytes were incubated with different concentrations of CuIIb for 4 h, 24 h or 48 h, respectively and analyzed with WST-1 assay (B, C and D). Cell division was also measured by CFSE staining assay (E) and the proliferation indexes of CFSE-labeled cells are presented in (F). Data are analyzed with ModFit software. One representative data of three independent experiments with similar results are shown as mean ± SD (n = 3). ##P<0.01 versus control group; **P<0.01 versus Con A group.
Figure 2.
CuIIb induced cell cycle arrest by modulating the expression of cell cycle-related proteins in activated lymphocytes.
(A and B) Flow cytometry analysis showing cell cycle distribution of Con A-stimulated lymphocytes upon CuIIb treatment for 48 h. #P<0.05 versus control group; *P<0.05 and **P<0.01 versus Con A group. (C and D) Cells were pretreated with CuIIb (10 µM) for 1 h, then exposed to Con A (5 µg/ml) for 0, 6, 24, and 48 h, respectively. The expression of p27Kip1 and cyclin proteins at various time points was determined by Western blotting. β-Tubulin was used as a loading control. Representative blots of three independent experiments are shown in (C) and the relative densitometric ratios of each protein to β-tubulin are shown in (D). Values are shown as mean ± SD of three experiments. Arrow indicates a nonspecific band. **P<0.01.
Figure 3.
Inhibitory effect of CuIIb on CD69 and CD25 expression in activated lymphocytes.
Mouse lymphocytes were stimulated with Con A in the presence or absence of CuIIb for 24 h. CD69 and CD25 expression was evaluated by flow cytometry. (A and C) Representative flow cytometric dot plots from one of three independent experiments. (B and D) Quantitative histograms of (A) and (C), respectively. Values are shown as mean ± SD of three experiments. ##P<0.01 versus control group; *P<0.05 and **P<0.01 versus Con A group.
Figure 4.
CuIIb suppressed inflammatory cytokine expression in activated lymphocytes.
(A, D and G) Cells were pretreated with 2.5, 5 and 10 µM CuIIb for 1 h followed by PDB plus ionomycin (Ion) stimulation for 4 h, TNF-α, IFN-γ, and IL-6 were determined by intracellular cytokine staining. Both dot plots (A, D and G) and quantitative histograms (B, E and H) are presented to show the percentages of CD3+ T lymphocytes expressing TNF-α, IFN-γ or IL-6, respectively. (C, F and I) Lymphocytes were pretreated with 10 µM CuIIb for 1 h followed by Con A (5 µg/mL) stimulation for 0, 6, 24 and 48 h, TNF-α, IFN-γ, and IL-6 protein expression in culture medium were determined by cytometric bead array. Values are shown as mean ± SD (n = 3). ##P<0.01 versus control group; *P<0.05 and **P<0.01 versus PDB+Ion or Con A-activated group.
Figure 5.
Effect of CuIIb on the activation of MAPKs in Con A-activated lymphocytes.
Cells were pretreated with CuIIb (10 µM) for 1 h, and then exposed to Con A (5 µg/mL) for 0, 15, 30, and 60 min, respectively. The phosphorylation of Erk1/2, JNK and p38 MAPKs at various time points was determined by Western blotting. β-Tubulin was used as a loading control. Representative western blots are shown in (A) and the relative densitometric ratios of each protein to β-tubulin are shown in (B). Arrow indicates a nonspecific band. Values are shown as mean ± SD of three experiments. *P<0.05; **P<0.01.
Figure 6.
CuIIb modulated the NF-κB and STAT3 pathways in activated lymphocytes.
Cells were pretreated with CuIIb (10 µM) for 1 h, then exposed to Con A for indicated time periods. The phosphorylation of NF-κB/p65, IκB (A) and STAT3 (E) at various time points was determined by Western blotting. β-Tubulin was used as a loading control. Representative western blots are shown in (A and E) and the relative densitometric ratios of each phosphorylated protein to its total protein are shown in (B and F). (C) Immunofluorescence analysis of the distribution of NF-κB/p65 in lymphocytes. After co-treatment with CuIIb and Con A for 1 h, cells were fixed and then immunostained with anti-p65 antibody (red) followed by CF568-labeled second antibody. Nuclei (blue) were revealed by Hochest 33342 staining. Fluorescent images were obtained by fluorescence microscopy with a 100× oil objective lens. Representative of at least 10 images for each group were shown. Scale bar, 5 µm. (D) Quantitative PCR analysis of the mRNA levels of IκBα and TNF-α. CuIIb were pretreated for 1 h prior to stimulation with Con A. Levels of IκBα and TNF-α mRNA were determined 3 h after Con A stimulation. The mRNA levels normalized to β-actin are expressed as mean ± SD with controls defined as 1 (n = 3). *P<0.05; **P<0.01.
Figure 7.
CuIIb disrupted actin dynamics in activated lymphocytes.
Cells were pretreated with CuIIb (10 µM) for 1 h, then exposed to Con A for indicated time periods. The phosphorylation of cofilin at various time points was determined by Western blotting. β-Tubulin was used as a loading control. Representative blots are shown in (A) and the relative densitometric ratios of P-cofilin to cofilin are shown in (B). **P<0.01. (C) Immunofluorescence analysis of the distribution of β-actin in lymphocytes. After co-treatment with CuIIb and Con A for 1 h, cells were fixed and then immunostained with anti-β-actin antibody followed by appropriate second antibody. Nuclei (blue) were revealed by Hochest 33342 staining. Fluorescent images were obtained by fluorescence microscopy with a 100× oil objective lens. Representative of at least 10 images for each group were shown. Arrow indicates aggregated actin. Scale bar, 5 µm.
Figure 8.
CuIIb-induced cell apoptosis in Con A-stimulated mouse lymphocytes.
(A) Cells were stimulated with Con A in the absence or presence of CuIIb for 24 h. Apoptosis was determined by annexin V-PE/7-AAD staining. The percentages of annexin V+ cells (sum of 7-AAD− annexin V+ cells and 7-AAD+annexin V+ cells) within total cells are shown. Values are shown as mean ± SD (n = 3). (B and C) Cells were pretreated with CuIIb (10 µM) for 1 h, then exposed to Con A for indicated time periods. The cleaved caspase-3 and PARP levels were determined by Western blotting. β-Tubulin was used as a loading control. Representative blots of three independent experiments are shown in (B) and the relative densitometric ratios of each protein to β-tubulin are shown in (C). *P<0.05; **P<0.01.