fig5
Figure 5. E2 effects on tumoral osteolysis and secretion of osteolytic PTHrP. (A) Osteoclast number at tumor-bone interface (N.Oc/mm) in tibiae from tumor cell-inoculated mice supplemented with the lowest (0.05 mg) and highest (0.72 mg) E2 doses (n = 8-11 tibiae/group), with representative images of TRAP-positive multinucleated osteoclasts (see arrow for example of red-stained multinucleated osteoclast). *P < 0.01 by t-test. (B) Osteolytic PTHrP secretion from inoculated tumor cells vs. ER+ tumor cells isolated from BMETs of 2 different mice. Cells were treated with 10-7 M E2 or media control for 48 h after 4 days in E2-free media (n = 4/group). Cell number, as assessed by MTT assay, was not different between cell lines or altered by E2 treatment (data not shown). *P ≤ 0.05, **P ≤ 0.001 E2 vs. control; ^P ≤ 0.05, ^^P ≤ 0.0001 inoculated vs. BMET-derived control cells; ***P ≤ 0.001 inoculated vs. BMET-derived E2-treated cells, by 2-way ANOVA with Tukey post-test. (C) E2 dose-dependency of PTHrP secretion in MCF-7 maintained in E2-deplete media for 4 days prior to treatment with various concentrations of E2 (M), as indicated, for 72 h (n = 3-4/group). (D) PTHrP secretion from ER+ BMET-derived cells treated with E2 (10-8 M), E2 and MPP (10-6 M, ERα antagonist), PPT (10-8 M, ERα agonist), or media control for 52 h after 4 days in E2-deplete media (n = 3-4/group). Cell numbers (by MTT assay) were not altered by treatments (data not shown). *P ≤ 0.05 vs. control; **P ≤ 0.01 E2 vs. MPP + E2; not significant (n.s.) vs. control or as shown, by 1-way ANOVA with Sidak post-test. MPP: Methyl-piperidino-pyrazole; PPT: propyl pyrazole triol; PTHrP: parathyroid hormone-related protein.
