Malyarenko OS et al. (2026), Anticancer Effect of Pacificusoside D from the ...

ECB-ART-54805

Mar Drugs 2026 Feb 11;242:. doi: 10.3390/md24020075.

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Anticancer Effect of Pacificusoside D from the Starfish Solaster pacificus in Combination with 2-Deoxy-D-glucose on Oxidative Phosphorylation in Triple-Negative Breast Cancer Cells MDA-MB-231.

Malyarenko OS, Malyarenko TV, Kicha AA, Ermakova SP, Ivanchina NV.

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Triple-negative breast cancer (TNBC) represents significant therapeutic challenges due to its aggressive behavior, metabolic plasticity, and lack of targeted treatments, prompting investigation of biologically active triterpene glycosides from the starfish Solaster pacificus. This study evaluated the ability of pacificusoside D (SpD) to synergistically enhance the anticancer efficacy of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) in TNBC MDA-MB-231 cells by targeting mitochondrial oxidative phosphorylation (OXPHOS). Methods included metabolic profiling via glucose uptake, lactate, and glutamate Glo assays; IC50 determination by MTS and trypan blue assays; colony formation evaluation using a soft agar assay; and molecular mechanism elucidation by Western blot, fluorescence microscopy and spectrometry, and flow cytometry analyses. Results demonstrated that MDA-MB-231 cells predominantly utilized glycolysis under basal conditions, shifting to OXPHOS with 2-DG (0.5 mM). IC50 values were 8.0/8.4 mM for 2-DG and 0.3/0.25 μM for SpD after 24 h of cell treatment. SpD exhibited a significant decrease in the number of colonies in MDA-MB-231 cells and possessed synergistic anticancer effects with 2-DG. Mechanistically, SpD increased tumor suppressor VHL expression level, down-regulated expression level of electron transport chain enzymes, generated reactive oxygen species, induced mitochondrial dysfunction, and triggered Bax/Bak-mediated apoptosis. These findings highlighted the synergistic anticancer potential of SpD in combination with 2-DG in aggressive breast cancer, offering insights into improved clinical outcomes in the future.

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	Figure 1. Structures of triterpene glycosides CucD, SpD, SpF, SpH, and SpI from the starfish S. pacificus. Xyl: β-D-xylopuranose, Qui: β-D-quinovopuranose, Glc: β-D-glucopuranose, 3-OMe-Glc: 3-O-methyl-β-D-glucopuranose, 3-OMe-Xyl: 3-O-methyl-β-D-xylopuranose, 6-O-SO3-Glc: 6-O-sulfoxy-β-D-glucopuranose.
	Figure 2. The effect of 2-DG on the metabolic profile, cell viability, and proliferation of TNBC cells MDA-MB-231. (a) MDA-MB-231 cells (5 × 103 cells/200 µL) were treated with either DMSO (vehicle control) or 2-DG at 0.1, 0.5, 5 mM and incubated for 24 h. The ability of MDA-MB-231 cells to take up glucose, excrete the lactate and glutamate, as determined by Glucose uptake or Lactate/Glutamate Glo assays. (b) MDA-MB-231 cells (1 × 104 cells/200 µL) were treated with either DMSO (vehicle control) or 2-DG at 0.1, 0.5, 5, and 10 mM and incubated for a time point of 24, 48, or 72 h as compared to DMSO-treated cells (control). Cell viability was estimated by the MTS and trypan blue exclusion assays. Data show the mean of three independent experiments ± SD. The asterisks indicate (a) a significant changes in the content of 2-deoxy-D-glucose-6-phosphate (2-DG6P), lactate and glutamate or (b) a significant decrease in cell viability of MDA-MB-231 cells treated with different concentrations of 2-DG at one time point of 24 or 48 or 72 h as compared to DMSO-treated cells (control) (* p < 0.05; p < 0.01; * p < 0.001).
	Figure 3. The effect of 2-DG, CucD, SpD, SpF, SpH, and SpI from the starfish S. pacificus on the colony formation in TNBC cells MDA-MB-231. MDA-MB-231 cells were treated with (a) 2-DG at concentrations of 0.1, 0.5, and 2.5 mM; (b) CucD; (c) SpD; (d) SpF; (e) SpH; (f) SpI at concentrations of 0.05, 0.1, and 0.2 µM in soft agar. The number of colonies was counted under a Motic AE 20 microscope (total magnification of 10×) using ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. Results are presented as mean ± standard deviation (SD). The asterisks indicate a significant decrease in the number of colonies in MDA-MB-231 cells treated with different concentrations of 2-DG and triterpene glycosides from the starfish S. pacificus as compared to DMSO-treated cells (control) (* p < 0.05; p < 0.01; * p < 0.001).
	Figure 4. The effect of SpD from the starfish S. pacificus in combination with 2-DG on the colony formation in TNBC cells MDA-MB-231. (a,b) MDA-MB-231 cells were pre-treated with 2-DG at concentrations of 0.5 mM and then treated with SpD at concentrations of 0.025, 0.05, and 0.1 µM in soft agar. The number of colonies was counted under a Motic AE 20 microscope (total magnification of 10×) using ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. Results are presented as mean ± standard deviation (SD). The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells ( p < 0.01; * p < 0.001).
	Figure 5. The effect of SpD from the starfish S. pacificus in combination with 2-DG on the expression level of enzymes of mitochondrial complexes and ROS generation in TNBC cells MDA-MB-231. MDA-MB-231 cells were treated by either 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM) and subjected to Western Blot or fluorescence microscopy and spectrometry assays. (a) The regulation of the expression level of VHL tumor suppressor, UQCRC2 (complex III), COX1 (complex IV), ATP5a1 (complex V), and β-actin was quantified by Western Blot assay. (b) Relative expression level was measured using the Quantity One 1D analysis version 4.6.7 software. Expression level was normalized to the total level of β-actin. (c) Fluorescence microscopic images were taken under CELENA® X High Content Imaging System at a total magnification of 10×, aided with cell-integrated CELENA® X Cell Analyzer software version 1.6.0. (d) Fluorescence intensity of H2DCFDA in MDA-MB-231 cells was determined by ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. (e) Fluorometric quantification of ROS level by Synergy H1 multimode microplate reader. Results are expressed as mean ± SD. The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells (* p < 0.05; p < 0.01; * p < 0.001).
	Figure 6. The effect of SpD from the starfish S. pacificus in combination with 2-DG on the alteration of mitochondrial mass in TNBC cells MDA-MB-231. MDA-MB-231 cells were treated with either 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM) and subjected to fluorescence microscopy and flow cytometry assays. (a) Fluorescence microscopic images were taken under CELENA® X High Content Imaging System (at a total magnification of 10×), aided with cell-integrated CELENA® X Cell Analyzer software version 1.6.0. (b) Fluorescence intensity of MitoTracker Green in MDA-MB-231 cells was determined by ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. (c) Flow cytometry analysis of MDA-MB-231 cells stained with MitoTracker Green dye. (d) Quantitative calculation of the data obtained by NovoExpress flow cytometry software (version 1.6.1). Results are expressed as mean ± SD. The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells (* p < 0.05; *** p < 0.001).
	Figure 7. The effect of SpD from the starfish S. pacificus in combination with 2-DG on apoptosis induction in TNBC cells MDA-MB-231. (a) MDA-MB-231 cells were treated by either 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM). The regulation of the expression level of Bax, Bak, caspase 9, caspase 7, caspase 3, cleaved caspase 3, and β-actin were quantified by Western Blot assay. (b) Relative expression level was measured using the Quantity One 1D analysis version 4.6.7 software. Expression level was normalized to the total level of β-actin. (c) Dot plots show a representative experiment of treatments. (d) Bar charts show percentage values of live, early apoptotic, late apoptotic/dead, and dead MDA-MB-231 cells treated by 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM). Results are presented as mean ± standard deviation (SD). The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells (* p < 0.05; p < 0.01; * p < 0.001).

	Figure 1. Structures of triterpene glycosides CucD, SpD, SpF, SpH, and SpI from the starfish S. pacificus. Xyl: β-D-xylopuranose, Qui: β-D-quinovopuranose, Glc: β-D-glucopuranose, 3-OMe-Glc: 3-O-methyl-β-D-glucopuranose, 3-OMe-Xyl: 3-O-methyl-β-D-xylopuranose, 6-O-SO3-Glc: 6-O-sulfoxy-β-D-glucopuranose.
	Figure 2. The effect of 2-DG on the metabolic profile, cell viability, and proliferation of TNBC cells MDA-MB-231. (a) MDA-MB-231 cells (5 × 103 cells/200 µL) were treated with either DMSO (vehicle control) or 2-DG at 0.1, 0.5, 5 mM and incubated for 24 h. The ability of MDA-MB-231 cells to take up glucose, excrete the lactate and glutamate, as determined by Glucose uptake or Lactate/Glutamate Glo assays. (b) MDA-MB-231 cells (1 × 104 cells/200 µL) were treated with either DMSO (vehicle control) or 2-DG at 0.1, 0.5, 5, and 10 mM and incubated for a time point of 24, 48, or 72 h as compared to DMSO-treated cells (control). Cell viability was estimated by the MTS and trypan blue exclusion assays. Data show the mean of three independent experiments ± SD. The asterisks indicate (a) a significant changes in the content of 2-deoxy-D-glucose-6-phosphate (2-DG6P), lactate and glutamate or (b) a significant decrease in cell viability of MDA-MB-231 cells treated with different concentrations of 2-DG at one time point of 24 or 48 or 72 h as compared to DMSO-treated cells (control) (* p < 0.05; p < 0.01; * p < 0.001).
	Figure 3. The effect of 2-DG, CucD, SpD, SpF, SpH, and SpI from the starfish S. pacificus on the colony formation in TNBC cells MDA-MB-231. MDA-MB-231 cells were treated with (a) 2-DG at concentrations of 0.1, 0.5, and 2.5 mM; (b) CucD; (c) SpD; (d) SpF; (e) SpH; (f) SpI at concentrations of 0.05, 0.1, and 0.2 µM in soft agar. The number of colonies was counted under a Motic AE 20 microscope (total magnification of 10×) using ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. Results are presented as mean ± standard deviation (SD). The asterisks indicate a significant decrease in the number of colonies in MDA-MB-231 cells treated with different concentrations of 2-DG and triterpene glycosides from the starfish S. pacificus as compared to DMSO-treated cells (control) (* p < 0.05; p < 0.01; * p < 0.001).
	Figure 4. The effect of SpD from the starfish S. pacificus in combination with 2-DG on the colony formation in TNBC cells MDA-MB-231. (a,b) MDA-MB-231 cells were pre-treated with 2-DG at concentrations of 0.5 mM and then treated with SpD at concentrations of 0.025, 0.05, and 0.1 µM in soft agar. The number of colonies was counted under a Motic AE 20 microscope (total magnification of 10×) using ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. Results are presented as mean ± standard deviation (SD). The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells ( p < 0.01; * p < 0.001).
	Figure 5. The effect of SpD from the starfish S. pacificus in combination with 2-DG on the expression level of enzymes of mitochondrial complexes and ROS generation in TNBC cells MDA-MB-231. MDA-MB-231 cells were treated by either 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM) and subjected to Western Blot or fluorescence microscopy and spectrometry assays. (a) The regulation of the expression level of VHL tumor suppressor, UQCRC2 (complex III), COX1 (complex IV), ATP5a1 (complex V), and β-actin was quantified by Western Blot assay. (b) Relative expression level was measured using the Quantity One 1D analysis version 4.6.7 software. Expression level was normalized to the total level of β-actin. (c) Fluorescence microscopic images were taken under CELENA® X High Content Imaging System at a total magnification of 10×, aided with cell-integrated CELENA® X Cell Analyzer software version 1.6.0. (d) Fluorescence intensity of H2DCFDA in MDA-MB-231 cells was determined by ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. (e) Fluorometric quantification of ROS level by Synergy H1 multimode microplate reader. Results are expressed as mean ± SD. The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells (* p < 0.05; p < 0.01; * p < 0.001).
	Figure 6. The effect of SpD from the starfish S. pacificus in combination with 2-DG on the alteration of mitochondrial mass in TNBC cells MDA-MB-231. MDA-MB-231 cells were treated with either 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM) and subjected to fluorescence microscopy and flow cytometry assays. (a) Fluorescence microscopic images were taken under CELENA® X High Content Imaging System (at a total magnification of 10×), aided with cell-integrated CELENA® X Cell Analyzer software version 1.6.0. (b) Fluorescence intensity of MitoTracker Green in MDA-MB-231 cells was determined by ImageJ version 1.50i software bundled with 64-bit Java 1.6.0_24. (c) Flow cytometry analysis of MDA-MB-231 cells stained with MitoTracker Green dye. (d) Quantitative calculation of the data obtained by NovoExpress flow cytometry software (version 1.6.1). Results are expressed as mean ± SD. The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells (* p < 0.05; *** p < 0.001).
	Figure 7. The effect of SpD from the starfish S. pacificus in combination with 2-DG on apoptosis induction in TNBC cells MDA-MB-231. (a) MDA-MB-231 cells were treated by either 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM). The regulation of the expression level of Bax, Bak, caspase 9, caspase 7, caspase 3, cleaved caspase 3, and β-actin were quantified by Western Blot assay. (b) Relative expression level was measured using the Quantity One 1D analysis version 4.6.7 software. Expression level was normalized to the total level of β-actin. (c) Dot plots show a representative experiment of treatments. (d) Bar charts show percentage values of live, early apoptotic, late apoptotic/dead, and dead MDA-MB-231 cells treated by 2-DG (0.5 mM) or SpD (0.1 µM) alone or by 2-DG (0.5 mM) in combination with SpD (0.025, 0.05, and 0.1 µM). Results are presented as mean ± standard deviation (SD). The one-way ANOVA and the Tukey’s HSD test for multiple comparisons show statistical significance as compared to DMSO-treated cells (control) or 2-DG-treated cells (* p < 0.05; p < 0.01; * p < 0.001).