Increase of IDO expression and kynurenine production in fibroblasts co-cultured with COX-2-overexpressing breast cancer cells. (A) Metabolite profiling of the supernatant of RMF-EG fibroblasts co-cultured with MCF-7 (RMF-M) and COX-2-overexpressing MCF-7 (RMF-COX/M) cells and identified a peak with m/z ratio of 209 was increased. (B) UPLC/MS/MS fragmentation profile of the 209 (m/z) peak and the standard (L-kynurenine). (C) Increase of kynurenine in RMF-COX/M cells determined with an ELISA assay. The results from three independent assays were expressed as mean ± SEM. Statistical significance was evaluated with the Student t test. *P < 0.05. (D) Upregulation of IDO but not TDO in RMF-COX/M cells was assayed with quantitative RT-PCR. The results from three independent assays were expressed as mean ± SEM. Statistical significance was evaluated with Student t test (i). *P < 0.05. Protein level was also determined with Western blotting (ii). (E) MCF-7 cells were treated without (C) or with doxycycline (DOX, 1 μg/ml) for 72 hours to induce COX-2 expression. Protein level of COX-2 and IDO was studied with Western blotting. A 3.6-fold increase of COX-2 was found, whereas the expression of IDO was not detectable. (F) Protein level of COX-2 and IDO in MCF-7 and MDA-MB-231 cells was compared. In addition, IDO expression of RMF-EG cells co-cultured with MCF-7 or MDA-MB-231 cells was investigated.