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Figure 1. The structure of pacificusoside D (SpD) from the starfish S. pacificus.
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Figure 2. The effect of fucoidan from the brown alga S. cichorioides (ScF), its derivative ScF_AH (AH), pacificusoside D from starfish S. pacificus (SpD), and X-ray irradiation on viability of 3D mouse epidermal cells JB6 Cl41 and human melanoma cells SK-MEL-2. JB6 Cl41 and SK-MEL-2 spheroids were treated by (a) ScF (F) (100, 200, 400, and 800 µg/mL); (b) ScF_AH (AH) (100, 200, 400, and 800 µg/mL); (c) SpD (S) (0.5, 1, 5, 25, 50, and 100 µM); (d) X-ray (2, 4, 8, and 16 Gy) and incubated for 24 h. The cell viability was estimated using the MTS assay. Data are represented as the means ± SD as determined from triplicate experiments.
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Figure 3. Two-component combined effect of fucoidan from S. cichorioides (ScF) with pacificusoside D from S. pacificus (SpD) or X-ray and the product of autohydrolysis of fucoidan ScF_AH (AH) with SpD (D) or X-ray on the viability of 3D human melanoma cells SK-MEL-2. SK-MEL-2 spheroids were treated by (a) ScF (50, 100, and 200 µg/mL) in combination with SpD (1 µM) (FS combination); (b) ScF (50, 100, and 200 µg/mL) in combination with X-ray (FX combination); (d) ScF_AH (50, 100, and 200 µg/mL) in combination with SpD (1 µM) (AHS combination); (e) ScF_AH (50, 100, and 200 µg/mL) in combination with X-ray (AHX combination) for total 72 h. The cell viability was estimated using MTS assay. Photographs (n = 6, where n = number of photographs) of each spheroid were taken using the ZOE™ Fluorescent Cell Imager. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey’s HSD test for multiple comparisons indicated the statistical significance (* p < 0.05 and ** p < 0.01). (c,f) Type of combined action of FS, FX, AHS, and AHX combinations calculated using Compusyn 1.0.1 software (ComboSyn, Inc., USA). The combined index (CI) is a quantitative measure of the degree of interaction between test compounds. CI equal to 0.9–1.1 is considered additive; a CI value greater than 1.1 represents antagonism; and CI values less than 0.7 indicate synergism.
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Figure 4. Two-component combined effect of pacificusoside D from S. pacificus (SpD) with fucoidan from S. cichorioides (ScF) or the product of autohydrolysis (ScF_AH) or with an X-ray, as well as an X-ray with SpD or with ScF or ScF_AH, on the viability of 3D human melanoma cells SK-MEL-2. SK-MEL-2 spheroids were treated by (a) SpD (1 µM) in combination with ScF (50, 100, and 200 µg/mL) (SF combination); (b) SpD (1 µM) in combination with ScF_AH (50, 100, and 200 µg/mL) (SAH combination); (c) SpD (1 µM) in combination with X-ray (2 Gy) (SX combination); (e) X-ray (2 Gy) in combination with ScF (50, 100, and 200 µg/mL) (XF combination); (f) X-ray (2 Gy) in combination with ScF_AH (50, 100, and 200 µg/mL) (XAH combination); (g) X-ray (2 Gy) in combination with SpD (1 µM) (XS combination) for total 72 h. The cell viability was estimated using the MTS assay. The cell viability was estimated using the MTS assay. Photographs (n = 6, where n = number of photographs) of each spheroid were taken using the ZOE™ Fluorescent Cell Imager. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey’s HSD test for multiple comparisons indicated the statistical significance (* p < 0.05 and ** p < 0.01). (d,h) Type of combined action of SF, SAH, SX, XF, XAH, and XS combinations calculated using Compusyn 1.0.1 software (ComboSyn, Inc., USA).
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Figure 5. Three-component combined effect of fucoidan from S. cichorioides (ScF) with pacificusoside D from S. pacificus (SpD) and X-ray on the viability of 3D human melanoma cells SK-MEL-2. SK-MEL-2 spheroids were treated by (a) ScF (50, 100, and 200 µg/mL) in combination with X-ray (2 Gy) and SpD (1 µM) (FXS combination); (b) SpD (1 µM) in combination with X-ray (2 Gy) and ScF (50, 100, and 200 µg/mL) (SXF combination); (d) X-ray (2 Gy) in combination with ScF (50, 100, and 200 µg/mL) and SpD (1 µM) (XFS combination) (e) X-ray (2 Gy) in combination with SpD (1 µM) and ScF (50, 100, and 200 µg/mL) (XSF combination) for total 72 h. The cell viability was estimated using the MTS assay. The cell viability was estimated using the MTS assay. Photographs (n = 6, where n = number of photographs) of each spheroid were taken using the ZOE™ Fluorescent Cell Imager. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey’s HSD test for multiple comparisons indicated the statistical significance (* p < 0.05 and ** p < 0.01). (c,f) Type of combined action of FXS, SXF, XFS, and XSF combinations calculated using Compusyn 1.0.1 software (ComboSyn, Inc., USA).
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Figure 6. The combined radiomodifying effect of fucoidan from S. cichorioides (ScF) with X-ray and pacificusoside D from S. pacificus (SpD) on the invasion of 3D human melanoma cells SK-MEL-2. (a) Photographs of SK-MEL-2 spheroids (40×, scale 500 µm) after invasion into the gelatin matrix for 72 h treated with ScF (50, 100, and 200 µg/mL) in combination with X-ray (2 Gy) and SpD (1 μM). (b) Quantification of invasion of spheroids treated with the combination of investigated compounds was performed using the ImageJ program and was determined as the difference between the area of invasion of spheroid cells and the area of the spheroid. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey’s HSD test for multiple comparisons indicated the statistical significance (* p < 0.05 and ** p < 0.01). (c) The Combination Index (CI) for the interaction between ScF, X-ray, and SpD was calculated using the Compusyn software (ComboSyn, Inc.).
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Figure 7. The combined radiomodifying effect of fucoidan from S. cichorioides (ScF) with X-ray and pacificusoside D from S. pacificus (SpD) on the induction of apoptosis and DNA degradation of 3D human melanoma SK-MEL-2. (a) Western blot analyses of caspase 9, 7, and 3 activation, and cleaved caspase 3 expression in spheroids treated by ScF in combination with X-ray and SpD for 72 h. β-actin was used as an internal control. Relative band density was measured using Image Lab™ Software 4.1. (b) DNA degradation of 3D SK-MEL-2 cells treated ScF in combination with X-ray and SpD for 72 h determined by the DNA comet method. Photographs (n = 3, where n = number of photographs, scale 100 µm) of each spheroid were taken using the ZOE™ Fluorescent Cell Imager.
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