FIG. 3.
MitoTam efficiently suppresses Her2high breast carcinomas. (A) FVB/N c-neu mice s.c. injected with syngeneic NeuTL cells (2 × 106 cells per animal) were treated twice a week with tamoxifen (2.69 μmol/mouse/dose) or MitoTam (0.54 μmol/mouse/dose) dissolved in 4% EtOH in corn oil, 100 μl per dose, and tumor volume evaluated by USI. (B) Balb-c nu/nu mice were implanted with a slow-release estradiol pellet and injected s.c. with 2 × 106 MCF7 mock or MCF7 Her2high cells per animal. As soon as USI-detectable tumors appeared (∼50 mm3), the mice were treated with i.p. injection with 100 μl of tamoxifen (2 μmol/mouse/dose) or MitoTam (0.25 μmol/mouse/dose) dissolved in 4% EtOH in corn oil on days 3 and 7 of every week, and tumor volume was visualized and evaluated using USI. (C) Control and MitoTam-treated MCF7 mock and MCF7 Her2high cell-derived tumors, excised at the end of the experiment, were fixed, paraffin embedded, and stained using the TUNEL technique. The arrows indicate TUNEL-positive cells. Size bar = 50 μm. (D) Control and treated tumors as shown in (B) were lysed and evaluated for the level of procaspase-9, caspase-9, Bax, Bcl-2, and Her2 with actin as loading control using WB. (E) FVB/N c-neu mice with spontaneous tumors were treated twice a week with MitoTam (0.54 μmol/mouse/dose) or solvent control, and tumor volume was evaluated. (F) Balb/c mice were s.c. injected with syngeneic 4T1 cells (1 × 106 cells per animal) and treated with MitoTam (0.25 μmol/mouse/dose) or solvent control twice a week, and tumor volume was evaluated. (G) Blood, lung, and liver harvested from animals in (F) were homogenized and subjected to selection in the presence of 6-thioguanine for 10 days and colonies were counted. Data in (A) and (B) are mean values (n = 6) ± SEM and, in (E) and (F), are mean values (n ≥ 5) ± SEM. The symbols, *, **, and #, indicate statistically significant differences (p < 0.05). WB, Western blotting. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars