Amiodarone promotes cancer cell death through elevated truncated SRSF3 and downregulation of miR-224

Figure 1. The effects of amiodarone on target gene and protein expression in HeLa cells HeLa cells were treated with indicated amount of amiodarone for indicated time. The cells were collected and subjected to (A) RT-PCR analysis of SRSF3, Slu7, p53, cyclin D1, p21, ATF3, COX-2, and GAPDH (loading control) mRNA expression; (B) immunoblot analysis for the detection of SLU7, SRSF3, p53, cyclin D1, p21, ATF3, COX-2, PARP, and ACTN (loading control) protein expression. PCR bands (A) were quantified through pixel density scanning and evaluated by ImageJ software, version 1.44a (http://imagej.nih.gov/ij/). The results are representative of two independent experiments.

Figure 2. Amiodarone caused cellular apoptosis HeLa cells were treated with (A) lower concentration of amiodarone for 20 h and (B) higher concentration of amiodarone for 6 h. The cells were stained with PI, and the DNA content was determined by FACS analysis. The results are representative of three independent experiments.

Figure 3. Amiodarone triggered intrinsic apoptosis HeLa cells were treated indicated amount amiodarone for 20 h. Cells were subject to the flow cytometry analysis using (A) the PE Annexin V Apoptosis Detection Kit (BD Biosciences) (B) the JC-1 staining kit. The results (A and B) are representative of three independent experiments. (C) The data from (B) were further measured the values of FL1-H and FL2-H.

Figure 4. The effects of amiodarone on the cell colony and anchorage-independent growth (A) HeLa cells were treated indicated amount amiodarone for 7 days and cells were subject to the colony formation. After 7 days incubation, colonies were fixed with methanol and stained with 0.005% crystal violet solution for 1 h and photographed. Colonies were counted and quantified using ImageJ software. (B) HeLa cells were treated indicated amount amiodarone for 19 days and cells were subject to the colony formation in soft agar. After 19 days incubation, colonies were stained with 0.005% Crystal Violet solution for 30∼60 min and photographed. Colonies were counted and quantified using ImageJ software. All assays were done in triplicate.

Figure 5. The regulatory mechanism of amiodarone on the SRSF3 expression HeLa cells were treated indicated amount amiodarone with pre-treated 2 h of vehicle, CHX (50 mg/ml), Act D (1 µM), and calcium chloride (1 mM) for 6 h. Cells were subject to the (A) Western blotting analysis and (B) RT-PCR analysis. The numerical data below each band is to indicate the ratio of specific mRNA (A) or protein (B) with control mRNA (GAPDH) or control protein (HuR) using the ImageJ software. The results (A and B) are representative of three independent experiments. (C and D) HeLa, GBM8401, U118MG cells were transiently transfected with 0.5 mg pGL3.SRSF3 (–1650/+171)-LUC and cells were treated with indicated amount of amiodarone for 20 h. Cells were harvested for luciferase reporter assay with the Promega Luciferase Assay Kit. The numbers (C and D) above the columns indicate the luciferase activity relative to an index of 1 for the reporter alone with vehicle.

Figure 6. Amiodarone synergistically enhanced caffeine- or digoxin-induced SRSF3 alternative splicing and p53-independent apoptosis in HeLa cells HeLa cells were treated with 5 mM caffeine or 0.1 µM digoxin and along with indicated amount of amiodarone for 16 h. The cells were collected and subjected to (A) RT-PCR analysis of SRSF3, Slu7, p53, cyclin D1, p21, ATF3, COX-2, and GAPDH (loading control) mRNA expression; (B) immunoblot analysis for the detection of p53, cyclin D1, LC3B, PARP, γ−H2AX, and ACTN (loading control) protein expression. PCR bands (A) were quantified through pixel density scanning and evaluated by ImageJ software, version 1.44a (http://imagej.nih.gov/ij/). The results are representative of two independent experiments.

Figure 7. Amiodarone augmented the effects on the increase of sub-G1 population in HeLa cells by caffeine or digoxin HeLa cells were treated with 5 mM caffeine or 0.1 µM digoxin and along with indicated amount of amiodarone for 16 h. The cells were stained with PI, and the DNA content was determined by FACS analysis. The results are representative of three independent experiments.

Figure 8. Amiodarone induced the ROS generation in HeLa cell HeLa cells were treated with (A) lower concentration of amiodarone for 20 h and (B) higher concentration of amiodarone for 16 h. After incubated, we stained live cells with 10 µM DCFH-DA for 30∼60 min at 37° C, harvested cells were then subjected to FACS analysis. The results are representative of three independent experiments.

Figure 9. Caffeine or digoxin increased the effects of amiodarone for the elevation of ROS generation in HeLa cell HeLa cells were treated with (A) 10 mM caffeine or (B) 0.3 µM digoxin and lower concentration of amiodarone for 16 h. After incubation, we stained live cells with 10 µM DCFH-DA for 30∼60 min at 37° C, harvested cells were then subjected to FACS analysis. The results are representative of three independent experiments.

Figure 10. The comparison of the effects of caffeine, amiodarone, and digoxin on the miRs regulation in HeLa cell HeLa cells were treated with 10 mM caffeine, 0.3 µM digoxin, and 30 mM amiodarone for 16 h. The cells were collected and subjected to the miR analysis for (A) miR-34a; (B) miR-145; (C) miR-125b; (D) miR-200c; (E) miR-504; and (F) miR-224 expression. The results are representative of three independent experiments.

Figure 11. The effects of amiodarone on miR-224 and its target genes in HeLa cell HeLa cells were treated with lower concentration of amiodarone for 20 h. The cells were collected and subjected to (A) the miR analysis for miR-224 expression; (B) the RT-PCR analysis for caspase3, TNFAIP1, COX-2, and GAPDH (loading control); and (C) the Western blotting analysis using antibodies against COX-2 and ACTN (loading control). The results are representative of three independent experiments.