Fig 1.
1A and 1B. Western blotting analysis comparing doxorubicin (DOX) samples with control (CTRL) samples. (A) Human cardiac fibroblasts (HCFs) were exposed to 0.1 to 1.0 μM DOX for 24 h. DOX increased the expression of p53 protein in a dose-dependent manner (one-way ANOVA followed by Tukey’s test, n = 4, *** p < 0.001, ** p < 0.01, NS: no significant difference). (B) HCFs were exposed to 0.5 μM DOX for 1 to 24 h. DOX increased the expression of p53 protein in a time-dependent manner (one-way ANOVA followed by Tukey’s test, n = 4, * p < 0.05, NS: no significant difference). 1C. Apoptosis assay comparing DOX samples with CTRL samples. Flow cytometry analysis showed that DOX significantly increased early apoptosis (Q3) (unpaired t-test, n = 4, ** p < 0.01). 1D and 1E. Cell cycle analysis comparing DOX samples with CTRL samples. (D) Western blotting analysis. HCFs were exposed to 0.5 μM DOX for 1 to 24 h. DOX increased the expression of p21 protein in a time-dependent manner (one-way ANOVA followed by Tukey’s test, n = 4, *** p < 0.001, NS: no significant difference). (E) Cell cycle assay by FACS showed that DOX significantly decreased the proportion of cells in the G1 phase and increased those in the G2 phase of the cell cycle (unpaired t-test, n = 6, *** p < 0.001, ** p < 0.01, NS: no significant difference). 1F. ROS production measured by fluorescence 24 h after administration of DOX. DOX increased ROS production significantly in the samples with 5.0 μM DOX (one-way ANOVA followed by Tukey’s test, n = 6, * p < 0.05, NS: no significant difference).
Fig 2.
Western blotting analysis for four groups to evaluate the effects of store-operated Ca2+ entry (SOCE) inhibition; CTRL group, DOX group, YM group, and YM+DOX group.
(A) YM-58483 significantly attenuated the DOX-induced upregulation of p53 protein. (B) YM-58483 significantly attenuated the DOX-induced upregulation of p21 protein. (one-way ANOVA followed by Tukey’s test, n = 6, *** p < 0.001, NS: no significant difference).
Fig 3.
Western blotting analysis to evaluate the effects of ORAI1 gene knockdown.
(A) Expression of ORAI1 mRNA by qPCR. The expression level of ORAI1 mRNA was reduced in samples with siRNA against ORAI1. In samples with control siRNA, DOX significantly upregulated the expression level of ORAI1 mRNA (two-way ANOVA followed by Bonferroni’s post hoc test, n = 6, *** p < 0.001, ** p < 0.01, NS: no significant difference). (B) Western blotting of four groups; CTRL siRNA group without DOX, CTRL siRNA group with DOX, ORAI1 gene knockdown (KD) group without DOX, and ORAI1 gene KD group with DOX. (C) There were no significant differences in the expression of STIM1 protein (two-way ANOVA followed by Bonferroni’s post hoc test, n = 7, NS: no significant difference). (D) Knockdown of the ORAI1 gene eliminated the expression of ORAI1 protein. DOX upregulated the expression level of ORAI1, but there was no significant difference in the samples with ORAI1 KD siRNA (two-way ANOVA followed by Bonferroni’s post hoc test, n = 7, *** p < 0.001, * p < 0.05, NS: no significant difference). (E) Knockdown of ORAI1 gene negated DOX-induced expression of p53 protein (two-way ANOVA followed by Bonferroni’s post hoc test, n = 8, *** p < 0.001, ** p < 0.01, * p < 0.05, NS: no significant difference). (F) In the samples with control siRNA, DOX upregulated the expression level of p21 protein. No significant difference was identified in samples with ORAI1 KD siRNA (two-way ANOVA followed by Bonferroni’s post hoc test, n = 8, ** p < 0.01, NS: no significant difference).
Fig 4.
Apoptosis assay to evaluate the effects of SOCE inhibition.
(A) Apoptosis assay of four groups: CTRL group, DOX group, YM group, and YM+DOX group. Flow cytometry showed that YM-58483 significantly attenuated DOX-induced early apoptosis (one-way ANOVA followed by Tukey’s test, n = 6, *** p < 0.001, * p < 0.05, NS: no significant difference). (B) Apoptosis assay of four groups: CTRL siRNA group without DOX, CTRL siRNA group with DOX, ORAI1 KD siRNA group without DOX, and ORAI1 KD siRNA group with DOX. Flow cytometry showed that the knockdown of the ORAI1 gene significantly attenuated DOX-induced early apoptosis (two-way ANOVA followed by Bonferroni’s post hoc test, n = 6, *** p < 0.001, ** p < 0.01, NS: no significant difference).
Fig 5.
ROS production measured by fluorescence to evaluate the effects of SOCE inhibition.
(A) Four groups were analyzed: the CTRL group, DOX group, YM group, and YM+DOX group. DOX significantly increased ROS production. There were no significant differences among the ROS productions of YM-58483-treated groups. (one-way ANOVA followed by Tukey’s test, n = 6, *** p < 0.001, * p < 0.05, NS: no significant difference). (B) Four groups were analyzed: CTRL siRNA group without DOX, CTRL siRNA group with DOX, ORAI1 KD siRNA group without DOX, and ORAI1 KD siRNA with DOX. In the CTRL siRNA groups, DOX significantly increased ROS production in both conditions. In the ORAI1 knockdown siRNA groups, there was no significant difference in ROS production between the with DOX and without DOX groups (two-way ANOVA followed by Bonferroni’s post hoc test, n = 6, *** p < 0.001, NS: no significant difference).
Fig 6.
Assessment of DOX-induced cardiotoxicity in mice.
Saline or 40 mg/kg DOX was administered intraperitoneally. In addition, 0.2% DMSO or 10 mg/kg YM-58483 was administered intraperitoneally prior to saline or DOX. (A) TUNEL apoptosis staining in heart sections. Apoptotic nuclei were quantitatively assessed in five random fields. Scale bar, 100 μm. (B) YM-58483 significantly attenuated DOX-induced apoptosis (one-way ANOVA, n = 7, *** p < 0.001, NS: no significant difference).