Low dose amiodarone reduces tumor growth and angiogenesis

Figure 1. Amiodarone affects U-87 MG cell function. (A) Viability assay of U-87 MG cells using WST8, after 24 or 72 h of incubation with ranging concentrations of Amiodarone (0, 1, 10 and 50 μM), investigating cytotoxic and proliferation-rate effects, respectively. n = 9–10. ***p < 0.001, compared with non-treated control cells with the same incubation time. (B) Transwell migration assay of U-87 MG cells after 18 h of incubation with ranging concentrations of Amiodarone (0, 1, 10, and 50 μM). Inverted microscope used to detect and count cells on the lower side of inserts' filter (five different fields per well) and average number of migrated cells calculated, n = 6. ***p < 0.001, compared with non-treated control cells. Results are presented as mean ± SEM. Figure supplement 1. Amiodarone’s effect on U-87 MG cell viability.

Figure 2. Amiodarone affects U-87 MG cell function in 3D. (A) Anoikis assay with U87-MG cells, using p-HEMA coated plates, incubated with ranging concentrations of Amiodarone (0, 1, 10 and 50 μM) for 72 h. Cell viability measured using WST8 assay, n = 6. ***p < 0.001, compared with non-treated control cells. (B, C) Soft agar colony formation assay of U-87 MG cells cultured for 15 days under ranging concentrations of Amiodarone (0, 1, 5, 7, 10, and 50 μM). Colonies visualized using inverted microscope on day 15. Images processed, analyzed, then average number of colonies per well using ImageJ calculated. n = 3. *p < 0.05, ***p < 0.001, compared with non-treated control cells. Scale bar: 100 µm. (D, E) Spheroid assembly and growth assay of U-87 MG cell spheroids. Relative spheroid size grown in rounded-bottom 96-well plate after 1, 5 and 7 days under ranging concentrations of Amiodarone (0, 1, 10 and 50 μM). Calculated using MATLAB code analysis (as shown in Fig. 2–figure supplement 1). Further normalization done in the plots—spheroid growth calculated as the ratio between the current size and the initial size, scaling the initial size values of day 1 as 100%, n = 3. *p < 0.05, **p < 0.01,…

Figure 3. Amiodarone affects HUVEC function. (A) Viability assay of HUVECs, using WST8, after 24 and 72 h of incubation with ranging concentrations of Amiodarone (0, 0.1, 1, 3, 5, 7, and 10 μM), investigating cytotoxic and proliferation-rate effects, respectively, n = 8. ***p < 0.001, compared with untreated control cells with the same incubation time. (B, C) Scratch assay of HUVECs after 36 h of incubation using the IncuCyte S3 Live-Cell Analysis System, with ranging concentrations of Amiodarone (0, 1, 5, 7, and 10 μM), n = 8. Scale bar: 400 µm. *p < 0.05, ***p < 0.001, compared with non-treated control cells. Results are presented as mean ± SEM. Figure supplement 1. Amiodarone reduces cell viability of endothelial, fibroblast and melanoma cells.

Figure 4. Amiodarone affects HUVEC function in 3D. (A, B) Angiogenesis tube formation assay of HUVECs seeded on Matrigel after 6 h of incubation, using IncuCyte S3 Live-Cell Analysis System with ranging concentrations of Amiodarone (0, 0.1, 1 and 5 μM). Statistical analysis of tube formation hallmark (number of meshes formed) and images processed using ImageJ software; representative images are presented, n = 4. Scale bar: 1.9 mm. *p < 0.05, compared with non-treated control cells. Results are presented as mean ± SEM.

Figure 5. Amiodarone inhibits corneal neovascularization after I.P. treatment. Amiodarone’s anti-angiogenic activity evaluated following systemic administration in the mouse corneal micropocket assay using bFGF-induced neovascularization. C57BL/6 mice were injected I.P. with Amiodarone 0.05 mg/kg or 0.1 mg/kg q.d. for 5 days. (A) Representative images taken from treated (0.1 mg/kg q.d.) and untreated mice on day 5. bFGF pellets detected as a white spot in the center of the cornea. Blood vessels growing from limbal periphery are reduced in the Amiodarone-treated group compared with the untreated group. (B) Normalization of vessel area (%) for untreated and Amiodarone treated mice (0.05 mg/kg and 0.1 mg/kg q.d.). Vessel area was calculated using the following formula: [Clock hours × Vessel length (mm) × π × 0.2 mm]. Graphs indicate significant inhibition of vessel formation after 5 days of treatment. Objective lens 400 × , (in each group n = 10). *p < 0.05, ***p < 0.001, compared with non-treated control mice. Results are presented as mean ± SEM.

Figure 6. Amiodarone inhibits angiogenesis in Matrigel plugs. Matrigel containing VEGF and bFGF (control) or Matrigel containing VEGF and bFGF mixed with Amiodarone 0.05 mg/plug was injected S.C. into C57BL/6 mice to determine the drug’s effect on angiogenesis. (A, B) Representative plugs removed from Amiodarone-treated and untreated mice after 8 days. Untreated mice presented bloody plugs surrounded by massive blood vessels, compared with Amiodarone-treated mice which had poor vasculature. (C) Representative histologic sections of untreated plug (left) and Amiodarone-treated plug (right) of mice post anti-CD31 antibody (DAB-brown) and Gill’s Hematoxylin for nuclei (blue) staining. Arrows point to large vessels with open lumens. Scale bar: 50 µm. (D) Quantification of infiltrating endothelial cells in Matrigel plugs employing the flow cytometry assay. FACS dot plots of endothelial cells (CD31 + /CD45, marked in red squares) from single-cell suspension originated from untreated (left) and treated (right) Matrigel plugs. Data are presented as percent of specific cell population out of the total cell population (p < 0.05). Infiltrating endothelial cells were reduced by 60% in the…

Figure 7. Amiodarone inhibits GBM tumor xenograft growth. Amiodarone’s anti-tumor activity following systemic administration (I.P.) evaluated in a S.C. tumor model. Foxn1 nu mice were inoculated S.C. with 5 × 106 U-87 MG cells. When tumors reached an average volume of ~ 200 mm3, mice were divided into two groups of treated (Amiodarone 0.1 mg/kg I.P. injection q.d.) and untreated (given saline) mice over a course of 14 days. (A, B) Tumors were surgically resected and collected when an average volume of ~ 1000 mm3 (day 14) was reached. (C, D) Weight and volume of tumors from treated and untreated mice were measured. Tumor volume was calculated using the standard equation (length × width2 × 0.52). n = 3–5. *p < 0.05, **p < 0.01, compared with non-treated control mice. Results are presented as mean ± SEM. (E) Representative histologic sections of untreated tumor (left) and Amiodarone-treated tumor (right) of mice post-H&E staining. Scale bar: 100 µm. (F) Representative images of tumor sections stained with anti-CD31 (DAB-brown) and Gill’s Hematoxylin for nuclei staining (blue). Untreated tumor (top) and Amiodarone-treated tumor (bottom). Scale bar: 100 µm. Objective lens 10 × . The data of…