Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating bax expression

Delta-lactoferrin (a dagger Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that a dagger Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell viability assay and DNA fragmentation. Western blot analyses showed that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of the endonuclease PARP was significantly increased. The levels of expression of Bcl family members were detected by immunochemistry and showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased. We determined that the pro-apoptotic effects of a dagger Lf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by a dagger Lf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a a dagger Lf response element located at -155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that a dagger Lf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished a dagger Lf transcriptional activity, identifying it as a a dagger Lf-responsive element. These results indicate that the Bax gene is a novel a dagger Lf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls a dagger Lf transcriptional activity, modulates Bax transactivation.