Figure 1.
(A) MCF7 cells were transfected with an ER luciferase reporter gene along with the internal transfection control Renilla Luciferase and treated with increasing concentrations (logarithmic scale) of E2 and 25HC. Moreover, cells were treated simultaneously with similar doses of each compound. The normalized luciferase activity values of cells treated with vehicle (-) were set as 1-fold induction, upon which the activity induced by treatments was calculated. (B) MCF7 cells transfected with the ER reporter gene were treated with 10nM E2 or 1µM 25HC alone and in combination with increasing concentration of the ER antagonist ICI, as indicated. Each data point represents the mean ± SD of three experiments performed in triplicate. (C–F) Hek293 cells were transfected with ER luciferase reporter gene (EREluc) and ERα (C) or ERβ (D) expression plasmids, with Gal4 reporter gene GK1 and the Gal4 fusion proteins encoding the Ligand Binding Domain (LBD) of ERα (GalERα) (E) or ERβ (GalERβ) (F) and treated with 10nM E2 or 1µM 25HC alone and in combination with 10µM ER antagonist ICI, as indicated. Each data point represents the mean ± SD of three experiments performed in triplicate.
Figure 2.
The graphs show residual binding of the radioactive tracer in the presence of increasing concentrations of unlabelled E2 and 25HC in MCF7 cells (A), in Hek293 cell lysates in the presence (B) or absence of recombinant ERα protein (C). Each data point represents the mean ± SD of triplicate samples of three separate experiments. Note that the amount of tracer bound in the absence of competitor was arbitrarily set to 100% and that the underlying absolute values differ between the three panels.
Figure 3.
25HC up-regulates the expression of estrogen target genes.
(A) E2 and 25HC induce the recruitment of ERα to the ERE site located in the pS2 promoter sequence in MCF7 cells. Cells were treated for 1 h with vehicle, E2 (10nM) or 25HC (1µM) and submitted to the chromatin immunoprecipitation procedure using anti-ERα or nonspecific anti-IgG antibodies. For Re-ChIP assays, anti-SRC-1, SRC-3 and CBP antibodies were used. The amplified sequences were evaluated by real-time PCR. (B–C) evaluation of mRNA expression of pS2, Progesterone Receptor (PR), Cathepsin D, Cyclin A and Cyclin D1 by real-time PCR in MCF7 and BG-1 cells. Cells were treated for 24h with 10nM E2 and 1µM 25HC. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean ± SD of three experiments performed in triplicate. (•), (°), (▪), (□) (♦) indicate p<0.05 for cells receiving vehicle (–) versus treatments.
Figure 4.
25HC induces cancer cell proliferation.
Immunoblots of ERα (A, B) and Cyclin D1 (C, D) from MCF7 and BG-1 cells. Cells were treated for 24h with vehicle (-), 10nM E2 or 1µM 25HC and in presence of 10µM ER-antagonist ICI, as indicated. β-actin serves as loading control. 25HC induces proliferative effects in MCF7 and BG-1 cells (E–H). Cells were treated for 5 days with increasing concentrations (logarithmic scale) of E2 and 25HC and counted on day 6 (E,G). Cells were treated with 10nM E2 or 1µM 25HC and in combination with 10µM ER-antagonist ICI and counted on day 6 (F,H). Proliferation of cells receiving vehicle was set as 100% upon which cell growth induced by treatments was calculated. Each data point is the average ± SD of three independent experiments. (•), (°) indicate p<0.05 for cells receiving vehicle (–) versus treatments.
Figure 5.
E2 and 25HC prevent CoCl2-induced apoptosis in HL-1 cells.
Apoptotic changes were detected using TUNEL (in green) and nuclei were stained by propidium iodide (PI, in red), as indicated. Representative photographs after 18h treatment with vehicle and 100µM CoCl2, 10nM E2, 1µM 25HC alone, or a combination of 100µM CoCl2 with 10nM E2 in presence or absence of 10µM ICI, 100µM CoCl2 with 1µM 25HC in presence or absence of 10µM ICI.
Figure 6.
Evaluation of HIF-1α and CTGF expression in HL-1 cells.
HIF-1α and CTGF mRNA expression were evaluated by real-time PCR after 100µM CoCl2 treatment (A) and incubating HL-1 cells under hypoxia (2%O2) (B). Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle (A) or cultured under normoxia (B). (C) immunoblots of HIF-1α and CTGF from HL-1 cells after treatment with vehicle (-) or 100µM CoCl2, as indicated. β-tubulin serves as a loading control. Side panel shows densitometric analysis of the blots, normalized to β-tubulin. (D) immunoblots of HIF-1α and CTGF from HL-1 cells cultured under normoxia or in presence of low oxygen tension (2% O2), as indicated. β-tubulin serves as a loading control. Side panel shows densitometric analysis of the blots, normalized to β-tubulin. Cells were transfected with a control shRNA or shHIF-1α and exposed for 8h to 100µM CoCl2 (E) or hypoxic conditions (2% O2) (F). Side panels show densitometric analysis of the blots, normalized to β-tubulin. (G) efficacy of HIF-1α silencing obtained using shHIF-1α. Each data point is the average ± SD of three independent experiments. (•), (°) indicate p<0.05 for cells receiving vehicle (–) versus treatments or for cells cultured under normoxia versus cells exposed to hypoxia.
Figure 7.
Immunoblots of HIF-1α and CTGF from HL-1 cells.
(A) cells were treated for 8h with 100µM CoCl2 or (B) exposed to hypoxia (2% O2), in combination with 10nM E2, 1µM 25HC and 10µM ICI, as indicated. β-tubulin serves as loading control. Data (mean ± SD) are representative of three independent experiments. Side panels show densitometric analysis of the blots, normalized to β-tubulin. (•), (°) indicate p<0.05 for cells receiving vehicle (–) versus treatments or for cells cultured under normoxia versus cells exposed to hypoxia.
Figure 8.
25HC prevents the hypoxia-induced expression of HIF-1α and CTGF through kinase-mediated signalling.
(A) immunoblots of p-ERK1/2 and p-p38 from HL-1 cells treated for 10 min with vehicle (-), 10nM E2 and 1µM 25HC. Total ERK/2 and p38 serve as loading control. (B) immunoblots of HIF-1α and CTGF from HL-1 cells exposed for 8h to 100µM CoCl2 or hypoxia (2% O2) (C), in combination with 10nM E2, 1µM 25HC and 10µM PD or 10µM SB, as indicated. β-tubulin serves as loading control. Data (mean ± SD) are representative of three independent experiments. Side panels show densitometric analysis of the blots, normalized to β-tubulin. (•), (°) indicate p<0.05 for cells receiving vehicle (–) versus treatments or for cells cultured under normoxia versus cells exposed to hypoxia.
Figure 9.
Evaluation of HIF-1α and CTGF mRNA expression in hypoxic rat heart preparations.
HIF-1α and CTGF mRNA expression was evaluated by real time PCR after 1h exposure to low pO2 levels (40%) and in presence of 1nM E2 or 100nM 25HC treatment, as indicated. mRNA values obtained from control and hypoxic rat hearts were normalized for 18S expression and shown as fold change in triplicate experiments. (°), (•) indicate p<0.05 for control versus hypoxic rat hearts.