Fig 1.
Effects of doxazosin and Lapatinib on non-tumor cells stained with Annexin V (AnV) and Propidium Iodide (PI).
(A) Doxazosin’s molecular structure. (B) Dot plot and graph (D) of primary astrocytes cultures after treatment with doxazosin and Lapatinib for 48h. (C) Dot plot and graph (E) of organotypic hippocampal cultures treated with doxazosin and Lapatinib for 48h. Data are represented as means (n = 4).
Fig 2.
Citotoxicity of doxazosin on C6 (A,C,E,G) and U138-MG (B,D,F,H) cells after treatment for 48 h.
MTT assay of C6 (A) and U138-MG (B). LDH activity of C6 (C) and U138-MG (D). SRB assay of C6 (E) and U138-MG (F). Data are represented by means±SEM (n = 4). ***p<0.001 compared to respective control, ANOVA followed by Tukey’s test. Confocal microscopy of C6 (G) and U138-MG (H). Photomicrographs are on top and SRB staining in the bottom. Magnification: 10X + 2,5.
Fig 3.
Effects of doxazosin and Lapatinib on glioma cells stained with Annexin V (AnV) and Propidium Iodide (PI).
(A) Dot plot and graph (C) of C6 cells after treatment with doxazosin and Lapatinib for 48h. (B) Dot plot and graph (D) of U138-MG cells treated with doxazosin and Lapatinib for 48 h. Data are represented as means (n = 4).
Fig 4.
Flow cytometry of cleaved caspase-3 of C6 (A) and U138-MG (B) glioma cells.
Fig 5.
Cell cycle analysis (A,B) and mitotic index (C,D) of glioma cells treated with doxazosin for 48 h.
(A) Cell cycle of C6 cells and (B) U138-MG cells. (C) Mitotic index of C6 cells and (D) U138-MG cells. Data are represented by means±SEM (n = 4). *p<0.05, **p<0.01, ***p<0.001 compared to respective control, ANOVA followed by Tukey’s test.
Fig 6.
Western blotting of C6 (A) and U138-MG (B) cells after treatment with doxazosin for 48h (n = 4).
β-actin was used as a loading control. (C) Effect of doxazosin on cell death and GSK-3β Ser9 phosphorylation compared with LY294002 (LY) on glioma cells. Magnification: 40X. C–control; V–vehicle; DZ–doxazosin. Data are represented as means (n = 4).
Fig 7.
Suggested model of the effects of doxazosin on C6 and U138-MG glioma cells.
Doxazosin promotes inhibition of PI3K/Akt pathway, which activates GSK-3β and p53. GSK-3β inhibits cell cycle progression and cell proliferation and p53 induce apoptosis via caspase-3 activation, and also inhibits cell cycle progression.