Doxorubicin‐Induced Cytotoxicity in Adipose‐Derived Stem Cells Is Associated With Altered SAPK / JNK Signaling

Figures

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  FIGURE 1. Effect of different Dox concentrations (0.1–100 μM) on mitochondrial activity, ATP content, and total protein content in ADSCs, expressed as a percentage of the control cell values (0 μM); means ± SD, *p < 0.05 versus control cells (0 μM); c neg—death cells treated with 1% of Triton X, N = 8. The circles indicate the threshold concentrations above which cellular activity changes significantly.

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  FIGURE 2. Flow cytometry analysis of the cell cycle in ADSCs after exposure to Dox at 5, 25, or 50 μM for 24 h, *p < 0.05 versus control (0 μM), # p < 0.05 versus 5 μM. (A) Mean percentage of ADSCs representing different phases of the cell cycle; mean ± SD, N = 3. (B) Histograms showing the distributions of ADSCs in different phases of the cell cycle.

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  FIGURE 3. Morphological and structural changes in ADSCs. (A) Morphology of ADSCs after 24 h exposure to Dox (5, 25, or 50 μM) compared to control cells (0 μM), (magn. 100×; magn. in the inset: 200×, scale bar = 50 μm). (B) F‐Actin filament arrangement ADSCs stained with Alexa Fluor 555 coupled phalloidin (red) after 24 h exposure to Dox at 5, 25, or 50 μM compared to control cells (0 μM). Cell nuclei were visualized with DAPI (blue), magnification 400×, scale bar = 20 μm. (C) Quantitative analysis of ADSCs size (μm) after Dox treatment. Data were derived from 6 independent photos. Mean ± SD, *p < 0.05 versus control (0 μM), N = 6.

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  FIGURE 4. Assessment of apoptosis in ADSCs (A) Kinetic profiles of phosphatidylserine translocation related to apoptosis in ADSCs after exposure to 5, 25, or 50 μM Dox for 0, 3, 6, 24, and 48 h, *p < 0.05 versus control (0 μM), N = 6. (B) Comparison of phosphatidylserine translocation related to apoptosis in ADSCs after 24 h and 48 h of exposure to Dox. Means ± SD, *p < 0.05 versus control (0 μM). The circles indicate the threshold concentrations above which cellular activity changes significantly. (C) Expression of Bax, Bcl‐2, TP53, CASP3, and CASP7 genes in ADSCs after exposure to Dox in comparison to control (0 μM). Means ± SD, *p < 0.05 versus control (0 μM), N = 6. (D) The Bax/Bcl‐2 ratio. (E) The expression of cleaved Caspase 3, cleaved Caspase 9, BAX, and BCL‐2 proteins in ADSCs after exposure to Dox versus control cells. Means ± SD, *p < 0.05 versus control (0 μM), N = 4. (F) Representative Western blot results for the analyzed proteins.

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  FIGURE 5. Assessment of senescence and oxidative stress in ADSCs (A) β‐galactosidase staining in ADSCs after exposure to 5, 25, and 50 μM Dox; Representative images. Magn. 200×, scale bar = 50 μm. (B) Quantification of SA‐β‐gal‐positive cells expressed as percentage of total number of cells, mean ± SD, *p < 0.05 versus control (0 μM), N = 8. (C) Senescence‐associated β‐galactosidase activity after exposure to 5, 25, and 50 μM Dox analyzed with flow cytometry. (D) Expression of RB1 gene in ADSCs after exposure to Dox, median and quartiles; *p < 0.05 versus control (0 μM), N = 6. (E) ROS generation in ADSCs after exposure to 5, 25, and 50 μM Dox, means ± SD, *p < 0.05 versus control cells (0 μM), # p < 0.05 versus 5 μM, N = 8. (F) Expression of CAT, SOD1, SOD2, and GPX4 genes in ADSCs after exposure to Dox, medians and quartiles; *p < 0.05 versus control cells (0 μM), N = 8. (G) GSH/GSSG ratio in ADSCs after exposure to 5, 25, and 50 μM Dox. Means ± SD, *p < 0.05 versus control cells (0 μM), N = 3. (H) Mitochondrial superoxide identification in ADSCs after exposure to Dox. Representative images. Magn. 100×, scale bar = 20 μm.

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  FIGURE 6. Effects of Dox on SAPK/JNK pathway (A) Expression of the MAP3K2, MAP2K4, MAP2K7, JUN, and MAP3K1 genes in ADSCs exposed to Dox (5, 25, and 50 μM) compared to control cells (0 μM). Means ± SD, *p < 0.05 versus control (0 μM), # p < 0.05 versus 5 μM, N = 6. (B) Comparison of the expression of JNK2, c‐JUN, p‐JNK in ADSCs after exposure to Dox versus control cells. Means ± SD; *p < 0.05 versus control (0 μM), # p < 0.05 versus 5 μM, ^p < 0.05 versus 25 μM, N = 4. (C) Representative Western blot results for the analyzed proteins.