PGC-1α Increases The Sensitivity Of 5′-DFUR To Cancer Cells By Inducing UPase Sirt3 Acts As A Novel Target Of PGC-1α In ROS And Mitochondrial Generation

Peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α) is capable of coactivating several nuclear receptors and transcription factors that participate in the regulation of multiple metabolic processes, including gluconeogenesis, mitochondrial biogenesis, and adaptive thermogenesis. Uridine phosphorylase (UPase) catalyzes the reversible conversion of uridine into uracil and contributes to the antineoplastic activity of 5’-deoxy-5-fluorouridine (5’-DFUR) and homeostasis of uridine levels in plasma and tissues. This study demonstrates uridine phosphorylase as a novel target gene of PGC-1α, which induces the transcription and enzymatic activity of UPase in various cancer cells and thus augments their susceptibility to 5’-DFUR. PGC-1α-induced activation of UPase expression occurs at its transcription level that is mediated by an estrogen-related receptor (ERR) binding site (-1078 to-1070 base pairs) mapped in the promoter region of UPase gene. Our mutational studies using luciferase reporter construct together with electrophoretic mobility shift assays confirm the binding of ERR to PGC-1α-responsive element. Moreover, the inhibition of PGC-1α/ERRα-dependent signaling by 3-[4-(2,4-bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluo romethyl-1,3,4-thiadiazol-2-yl) acrylamide (XCT790) compromises the ability of PGC-1αto induce the transcript of UPase, indicating PGC-1α-dependent and ERRa-mediated upregulation of UPase. Finally, the overexpression of PGC-1a sensitizes breast and colon cancer cells to growth inhibition by 5’-DFUR presumably by inducing apoptosis in tumor cells and XCT790 can inhibit the process. Taken together, our results corroborate the regulatory function of PGC-1αin uridine homeostasis and imply its links with the energy metabolism. The mechanistic elucidation of this association between both cellular pathways should advance the clinical use of 5-fluorouracil-based chemotherapy. Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which can decrease membrane potential while increasing cellular respiration. In keeping with the role of peroxisome proliferator-activated receptor-y coactivator-la (PGC-1α) on mitochondrial biogenesis and respiration, we demonstrate a stimulatory effect of PGC-la on SIRT3 mRNA and protein expression in muscle cells and hepatocytes. PGC-1αalso stimulated the activity of the mouse SIRT3 promoter, which is mediated by an estrogen-related receptor (ERR) binding site (-407/-399). Our mutational studies using luciferase reporter constructs and chromatin immunoprecipitation assay together with electrophoretic mobility shift assays confirm the binding of ERR to PGC-la responsive element. Moreover, PGC-1αis coinduced with several key reactive oxygen species (ROS)-detoxifying enzymes and increases the number of mitochondria; but the molecular mechanisms underlying that are incompletely understood. Studies with SIRT3 knockdown indicate that SIRT3 is required for the induction of PGC-la-mediated ROS-detoxifying enzymes and the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2 and ATP synthase 5c, cytochrome c. Functionally, overexpression of PGC-la decreases the ROS level while coinfected with SIRT3 shRNA recovers the ROS products level, which suggests SIRT3 mediated PGC-la effect on ROS production in cells. Furthermore, SIRT3 loss of function reduced the stimulatory effect of PGC-1αon mitochodrial number in C2C12 myotubes. In summary, our results indicated the existence of a regulatory pathway involving PGC-la, ERRa, SIRT3 and ROS. Alterations in this regulatory pathway may participate in the therapeutic manipulation of degenerative disease, diabetes and aging.