Fig. 2.

Ectopic expression of exogenous paired-like homeodomain transcription factor 2 (PITX2) promotes letrozole resistance and cell invasiveness. (A) Western blotting analysis of PITX2 level in MCF7 cells stably transfected with pPM-His-PITX2 or pPM-His vector. After a 3-day culture and a following overnight starvation, MCF7 cells stably transfected with pPM-His-PITX2 or pPM-His vector were treated with 10^–5 M of letrozole, along with 25 nM of androstenedione, for another 4 days. Viable cell numbers were then determined using trypan blue staining (B) (fold change was determined for each treatment relative to the untreated control cells, ^*p < 0.05 and ^**p < 0.01) and cell apoptosis was determined using an apoptosis enzyme-linked immunosorbent assay kit (C). The results were presented as the mean±standard error of mean of the triplicate samples. (D) Each mouse received subcutaneous injections at one site on each flank with 0.1 mL of suspension of different transfected MCF7 cells (2×10⁷ cells/mL). Mice were then injected subcutaneously daily for 32 days with androstenedione (100 μg/day) plus letrozole (10 μg/day) from the day of inoculation. PBS, phosphate buffered saline. Tumor volumes were measured every 3 days. ^*p < 0.05 and ^**p < 0.01 when comparing letrozole (10 μg/day)+vector group to letrozole (10 μg/day)+His-PITX2 group. (E) Cell invasiveness assay: breast cancer cells (1×10⁴) were cultured in the upper chamber of Transwell with a membrane coated with Matrigel for 24 hours, followed by treatment with 10^–5 M of letrozole, along with 25 nM of Δ4A, or with dimethyl sulfoxide (Sigma-Aldrich) for 48 hours. The remaining cells on the membrane were then stained for 10 minutes with 0.1% crystal violet solution and subsequent spectrophotometry was developed at 570 nM.