Doxorubicin-Induced Modulation of TGF-β Signaling Cascade in Mouse Fibroblasts: Insights into Cardiotoxicity Mechanisms

Figure 1. Cell viability remains high at DOX concentrations less than 1 μM. Cell viability analysis was performed using the alamarBlue assay on NIH3T3 cells and CFs exposed to specific concentrations of DOX for 24 hours. Error bars represent ± standard error represented a sample size of n=8 for NIH3T3 and n=6 for CFs.

Figure 2. DOX inhibited TGF-β1 induced expression of CCN2 in a dose-dependent manner. The effect of DOX on CCN2 protein expression in NIH3T3 cells (A) and CFs (B) with and without 10 ng TGF-β1 stimulation was assessed. The cells were tested with increasing concentrations of DOX (0, 0,25, 1, or 2.5 μM), and 100 μg/mL ascorbic acid for 24 hours. Values represent mean ± standard error (n=4). Statistical analysis performed with two-way ANOVA. * P-value < 0.05 is compared between the control group and the treatment group stimulated with TGF-β1 without DOX, and # P-value < 0.05 are treatments compared to the treatment group stimulated with TGF-β1 without DOX.

Figure 3. DOX inhibited the TGF-β1 induced expression of BMP-1 in NIH3T3 cells, while BMP-1 levels remained high in CFs. The effect of DOX on BMP1 protein expression in NIH3T3 cells (A) and cardiac fibroblasts (CFs) (B), with and without TGF-β1 stimulation was assessed. Cells were treated with 1 μM DOX and 100 μg/mL ascorbic acid for 24 hours, and protein extracts were collected for western blot analysis. Expression levels of BMP1 and the housekeeping protein β-actin were determined, and densitometry was performed for BMP1, normalized to β-actin. Error bars represent ± standard error (n=4). Statistical analysis was completed with Student’s t-test assuming unequal variance. # P-value < 0.05.

Figure 4. TGF-β1 induced the phosphorylation of SMAD2 in both NIH3T3 cells and CFs. DOX treatment diminished the extent of phosphorylation; however, the reduction did not reach statistical significance. The effect of DOX on SMAD2 and phosphorylated SMAD2 (pSMAD2) in NIH3T3 cells (a) and CFs (B) was assessed. Cells were treated with 1 mM DOX and 100 mg/mL ascorbic acid and stimulated with or without TGF-β1for one hour. Densitometry was performed on SMAD2, pSMAD2, and normalized to housekeeping protein β-actin. Values represent mean ± standard error (n=4). * P-value < 0.05 when compared to treatments without DOX and # P-value < 0.05 compared to treatments with DOX. & P-value < 0.05 are treatments with and without DOX without any TGF-β1 stimulation.

Figure 5. DOX inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells and CFs. Effect of DOX on COL1 protein expression in NIH3T3 cells (A) and CFs (B), with and without TGF-β1 stimulation was assessed. Cells were treated with 1 μM DOX and 100 μg/mL ascorbic acid for 24 hours, with or without 10 ng TGF-β1 stimulation, after which protein extracts were collected and subjected to western blotting to determine the expression levels of COL1 and the housekeeping protein β-actin. Densitometry was performed for COL1 and normalized to β-actin. Values represent mean ± standard error (n=4). P-values were determined using Student t-test assuming unequal variance * P-value < 0.05 control compared to treatment stimulated with TGF-β1 without DOX, and # P-value < 0.05 compared to both treatment groups treated with TGF-β1.

Figure 6. DOX inhibited the TGF-β1 induced expression of Atf4 and increased the expression of Cdkn1a, Id1, Id2, Runx1, Thbs1, Tgfb1, and Stat1 in NIH3T3 cells. Effects of DOX on the expression of selected genes in the TGFβ/BMP Signaling Pathways in NIH3T3 cells was assessed. Cells were treated with 10 ng TGF-β1 with or without 1 μM DOX for 24 hours.