Kynurenine induced E-cadherin ubiquitination and degradation and increased migration of breast cancer cells. (A) MCF-7 cells were treated with different concentrations of kynurenine for 48 hours, and cellular proliferation was investigated with MTT assay. (B) MCF-7- or COX-2-overexpressing MCF-7 cells were treated with 100 μM kynurenine, and cell migration was studied with transwell assays. The results from three independent assays were expressed as mean ± SEM. Statistical significance was evaluated with Student t test. *P < 0.05. (C) RMF-EG cells were cultured in the absence or presence of IDO inhibitor 1-methyl-L-tryptophan (L-1-MT) in the lower well of the transwell unit. MCF-7- or COX-2-overexpressing MCF-7 cells were seeded in the upper well. After 24 hours, migrated cell number was determined. *P < 0.05. (D) MCF-7 cells were incubated without (−) or with (+) 100 μM kynurenine for different times. Protein (i) and mRNA (ii) levels of E-cadherin were studied. (E) MCF-7 cells were incubated with kynurenine (100 μM) and MG132 (proteasome inhibitor, 10 μM) or chloroquine (CQ, lysosome inhibitor, 25 μM) for 24 hours. Protein level of E-cadherin was studied with Western blotting and normalized to actin. (F) Ubiquitination of E-cadherin was studied with immunoprecipitation of E-cadherin by specific antibody, and the ubiquitination status was detected with anti-ubiquitin antibody.