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

Delta-lactoferrin (δLf) 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 δLf 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 δLf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by δLf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a δLf response element located at -155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that δLf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished δLf transcriptional activity, identifying it as a δLf-responsive element. These results indicate that the Bax gene is a novel δLf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls δLf transcriptional activity, modulates Bax transactivation.