Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H₂O₂.

It is commonly recognized that diabetic complications involve increased oxidative stress directly triggered by hyperglycemia. The most important cellular protective systems against such oxidative stress have yet remained unclear. Here we show that the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the Txnrd1 gene, is an essential enzyme for such protection. Individually grown Txnrd1 knockout (Txnrd1(-/-)) mouse embryonic fibroblasts (MEFs) underwent massive cell death directly linked to glucose-induced H2O2 production. This death and excessive H2O2 levels could be reverted by reconstituted expression of selenocysteine (Sec)-containing TrxR1, but not by expression of Sec-devoid variants of the enzyme. Our results show that Sec-containing TrxR1 is absolutely required for self-sufficient growth of MEFs under high-glucose conditions, owing to an essential importance of this enzyme for elimination of glucose-derived H2O2. To our knowledge, this is the first time a strict Sec-dependent function of TrxR1 has been identified as being essential for mammalian cells

Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide gene expression profiling on doxorubicin-treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel protein target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells