Figure 2.

COX2 inhibition with NS-398 blocks PGE2 production and restores natural killer (NK) cell activity reduced by thyroid cancer. (A) Concentrations of PGE2 in culture supernatants from thyroid cancer cells, as measured by enzyme-linked immunosorbent assay (ELISA). Bars represent mean ± SD of three independent experiments. Statistical analysis was performed using the paired two-tailed Student’s t-test. (B) mRNA expression of COX-2 in thyroid cancer cells, as determined by real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase served as an internal control. (C) Expression of COX-2 in thyroid cancer cells detected by Western blotting; β-actin served as a loading control. (D) PGE2 concentrations in supernatants of thyroid cancer cell cultures with or without NS398 for 72 h, as determined by ELISA. Bars represent mean ± SD of TPC-1 n = 6, FRO, and 850-5C n = 3. Statistical analysis was performed using the paired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001. (E) NK cells cultured with IL-2 in the control medium (circle) or in culture supernatant (triangle) or culture supernatant + NS398 for 48 h; cytotoxicity against K562 cells was evaluated as a control. NS398 was administered at 1 µM (square) or 5 µM (diamond). Bars represent mean ± SD of three independent experiments. (F) Expression of NK receptors NKp44, NKp30, and TRAIL (filled profiles) and isotype controls (white profiles) analyzed by flow cytometry. Numbers indicate mean fluorescence intensity. Y-axis shows cell counts.