br Hematoxylin and eosin H E staining and immunohistochemica
2.16. Hematoxylin and eosin (H&E) staining and immunohistochemical analysis of xenograft tumors
The MDA-MB-231 xenograft tumors were removed and fixed in formalin solution (10% neutral buffered formaldehyde) at room tem-perature for 48 h. Slides containing 4 μm section of formalin-fixed and paraffin-embedded specimens of xenograft tumors were prepared for histopathological and immunohistochemical analyses. H&E staining was carried out as described previously . The tumor sections were used to determine the expression of P-STAT3Y705 by
immunohistochemical analysis. Slides were incubated with a primary antibodies for P-STAT3Y705 and visualized using the anti-rabbit horse-radish peroxidase Envision System (DAKO). The counterstaining was done using Mayer's hematoxylin.
2.17. Statistical analysis
Statistical analysis for single comparison was performed using the Student's t-test. Data were expressed as means of ± SD from at least three independent experiments. p < 0.05 was considered a statistically significant difference.
3.1. Comparison of enzymes involved in PGE2 metabolism in MCF10A-ras cells and non-oncogenic MCF10A cells
COX-2 converts arachidonic SB 203580 into PGE2, whereas 15-PGDH oxidizes the 15(S)-hydroxyl group of PGE2 to generate 15-keto PGE2 (Fig. 1A). In various tumors and transformed cells, down-regulation of
E.J. Lee, et al. Redox Biology xxx (xxxx) xxxx
Fig. 3. Inhibitory effects of 15-keto PGE2 on STAT3 target gene expression and neoplastic transformation of MCF10A-ras cells. A. MCF10A-ras cells were treated with 15-keto PGE2 (20 μM) or vehicle for 24 h. The mRNA levels of two re-presentative STAT3 target genes Cyclin D1 and Bcl2 were determined by real-time PCR. β-Actin was measured to ensure equal amount of cDNA loaded. N.S.: not significant. *p < 0.05, **P < 0.01, N.S, not significant. B. MCF10A-ras cells were seeded in 6 well plates and treated with two different con-centrations of 15-keto PGE2 (10 or 20 μM) or vehicle (DMSO) in DMEM/F12 medium containing 5% heat-inactivated horse serum and supplementary agents. The incubation conditions and other experimental details are described in Materials and methods. The colony size > 100 μm was counted under a light microscope. Results are the means ± S.D. *p < 0.05, ***P < 0.001. C. MCF10A-ras cells were plated on a 60 mm dish containing 0.5% (down) and 0.33% (up) double layer agar. The cells were treated every day with DMEM/F-12 containing DMSO or 15-keto PGE2. After 3-week of incubation, the colonies were stained with crystal violet, and the number of colonies was counted as described in Materials and methods. Results are the means ± S.D. *p < 0.05, ***P < 0.001.
15-PGDH is frequently observed with concomitant overexpression of COX-2. Therefore, 15-PGDH has been speculated as an endogenous COX-2 antagonist . In human breast cancer cells, PGE2 production has been shown to be markedly elevated compared to normal cells . We analyzed total lysates of MCF10A and MCF10A-ras cells for the basal levels of COX-2 and 15-PGDH. The protein level of COX was higher in MCF10A-ras cells compared to normal breast MCF10A epi-thelial cells (Fig. 1B). However, the basal level of 15-PGDH was higher in MCF10A cells. Moreover, the migration of MCF10A-ras cells in terms of their wound healing ability was much higher than that in non-on-cogenic MCF10A cells (Fig. 1C).
Ras gene mutation is found in certain types of human breast cancers, which associates with metastatic dissemination and poor prognosis . STAT3 is constitutively activated in ras-transfected rat intestinal epithelial cells . Phosphorylation of STAT3 at tyrosine 705 is a pivotal event for the activation of this transcription factor for cancer progression . We investigated whether 15-keto PGE2 could affect the STAT3 signaling in MCF10A-ras cells. 15-Keto PGE2 significantly decreased STAT3 phosphorylation in a dose- (Fig. 2A) and time-de-pendent (Fig. 2B) manners. Phosphorylated STAT3 forms a homodimer and translocates to the nucleus where it binds to specific DNA