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Figure 1. Chemical structures of triterpene glycosides: cucumarioside A0-1 (a) and djakonovioside A (b) isolated from the sea cucumber Cucumaria djakonovi.
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Figure 2. Distribution of MDA-MB-231 cells according to the phases of the cell cycle after treatment with various concentrations of Cuc A0-1 and Dj A for 24 h.
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Figure 3. Visualization of cyclin B and A contents and cyclin-dependent kinases in MDA-MB-231 cells treated with triterpene glycosides Cuc A0-1 and Dj A at different concentrations. Representative Western blot membranes showing the effect of glycosides on cyclin and CDK protein expression levels (a). Processed data on cyclin B content in MDA-MB-231 cells treated with Cuc A0-1 (b). Processed data on cyclin A content in MDA-MB-231 cells treated with Dj A (c). Processed data on CDK-1 content in MDA-MB-231 cells treated with Cuc A0-1 (d). Processed data on CDK-2 content in MDA-MB-231 cells treated with Dj A (e). All data were normalized to the β-actin levels. Data are presented as means ± SEM. * p value < 0.05 was considered significant.
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Figure 4. Analysis of apoptosis induced by triterpene glycosides in MDA-MB-231 cells after 24 h of incubation. Flow cytometry assay for Annexin V-FITC/PI staining (a). Quantitative calculation of the data obtained via flow cytometry: Cuc A0-1 (0.5 and 1 μM)—(b) and Dj A (1 and 2 μM)—(c). Data are presented as means ± SEM. p value < 0.05 was considered significant. Apoptosis assay using Hoechst 33342 in a fluorescent microscopy analysis (d). Hoechst 33342 staining showed an increase in chromatin condensation and DNA fragmentation in apoptotic cells treated with Cuc A0-1 (1 μM) and Dj A (2 μM) compared with untreated control cells. Arrows indicate nuclei with condensed chromatin.
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Figure 5. Quantitative evaluation of ROS levels in MDA-MB-231 cells after incubation with Cuc A0-1 (a) and Dj A (b) for different times (6, 12, and 24 h) using the fluorescent dye H2DCF-DA. Glycosides Cuc A0-1 (c) and Dj A (d), at various concentrations, reduced the mitochondrial membrane potential (Δψm), as measured using the fluorescent dye TMRE. Data are presented as means ± SEM. * p value < 0.05 was considered significant. Staining of MDA-MB-231 cells with the fluorescent dye JC-1 showed a change in the mitochondrial membrane potential (e).
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Figure 6. Western blot analysis of cytoplasmic proteins: apoptosis promoter Bax (a,b) and apoptosis inhibitor Bcl-2 (a,c) with β-actin as a protein loading control under the treatment of MDA-MB-231 cells with different concentrations of Cuc A0-1 and Dj A. Cytoplasmic protein levels were normalized to the control group (untreated cells). * p < 0.05 compared with untreated MDA-MB-231 cells.
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Figure 7. Quantitative assessment of the contents of cytochrome C (a,b) and APAF-1 (c,d) in MDA-MB-231 cells after treatment with different concentrations of glycosides Cuc A0-1 (a,c) and Dj A (b,d) at different times (6, 12, and 24 h) using ELISA kits. * p < 0.05 compared with untreated MDA-MB-231 cells.
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Figure 8. Caspase-3/7 activity in the control cells and cells treated with triterpene glycosides for 12 and 24 h was measured using the Muse™ Caspase-3/7 Kit and flow cytometry in MDA-MB-231 cells.
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Figure 9. Western blot analysis of apoptotic markers (a) and quantitative analysis of the levels of cleaved caspase-9 (b), cleaved caspase-3 (c), and cleaved PARP-1 (d) in MDA-MB-231 cells treated with different concentrations of Cuc A0-1 and Dj A. β-actin was used as a protein loading control (a). The levels of apoptotic markers were normalized to those of the control group (untreated cells). * p < 0.05 compared to untreated MDA-MB-231 cells.
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Figure 10. Influence of Cuc A0-1 on the area (a) and integrated density (b) of the fluorescence zone detected by in vivo fluorescence imager, Fluor I. Effect of Cuc A0-1 (0.4 µg/mL) on tumor volume (c) and tumor growth index (d). The data are presented as a mean ± SEM (n = 7). Asterisks indicate the significance of the differences at p ≤ 0.05 * and p ≤ 0.01 ** according to one-factor analysis of variance (ANOVA) with Tukey’s correction.
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Figure 11. The visualization of tumor cells labeled with PKH800 NIR fluorescent dye using the fluorescence imager system, “Fluor I IN VIVO”, in untreated mice (a), mice treated with Cuc A0-1 in group II (b), mice treated with Cuc A0-1 in group III (c), and mice treated with doxorubicin in group IV (d). On day 12, the tumor area was visualized in live mice; afterward, the mice were euthanized, the skin was opened, and tumor cells were visualized again. Arrows indicate tumor metastasis in the abdominal cavity.
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Figure 12. Three-dimensional plot of cytotoxic activity (pIC50) dependence on the principal component values (PCA1–PCA3) calculated for 25 conformational forms of 20 glycosides tested against MDA-MB-231 cells. The glycosides demonstrating cytotoxic activity with IC50 ≤ 10 μM were outlined as active and are marked in red, while inactive glycosides are marked in violet.
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Figure 13. The PLS QSAR model correlation plot reflecting the relationship of predicted and experimental cytotoxicity of the glycosides against MDA-MB-231 cells. The cytotoxic action was expressed as pIC50.
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