Figure 5
CAMP gene induction by Entinostat requires HIF-1α synthesis and activation.
(a) HIF-1α mRNA levels were analysed by qRT-PCR in HT-29 cells upon Entinostat (Entino) stimulation (2.5 μM, 24 h). Induced expression vs ctrl: *p < 0.05. (b,c) HEK293 cells transfected with the reporter vector pTRAF and stimulated for 24 h (b) with either 2 mM PBA or Entinostat (1–25 μM). HIF-1α activation was measured as Ypet intensity/cell. (c) Ypet fluorescence as seen in representative fields of unstimulated (ctrl) and 2.5 μM Entinostat (Entino) stimulated HEK293 cells. (d) ChIP assay for HIF-1α recruitment to the CAMP gene promoter was performed in HT-29 cells stimulated for 3 h with or without Entinostat. Significance vs ctrl: *p < 0.05. (e) HIF-1α knock-down was performed in HEK293 cells by transfecting with either a control vector (shSCR) or with shRNA specifically targeting HIF-1α (sh3_ HIF-1α and sh4_ HIF-1α) transcript. Cells were then stimulated with or without 2.5 μM Entinostat for 24 h, and analysed for the expression of the genes CAMP, HIF1A, STAT3, and DEFB1 (HBD1) expression by qRT-PCR. Significance vs each respective ctrl: *p < 0.05; significance vs untransfected (untrans) cells stimulated with Entino: §p < 0.05. (f) HEK293 cells were transfected with either a control vector (shSCR) or with shRNA specifically targeting STAT3 (sh3_STAT3 and sh4_STAT3) transcript. Cells were then stimulated with or without 2.5 μM Entinostat for 24 h, and then analysed for HIF-1α expression by qRT-PCR. Significance vs untransfected (untrans) control (ctrl) cells: *p < 0.05. (g) Representation of a proposed mechanism where Entinostat activates STAT3, possibly by inhibition of deacetylation of the transcription factor, which in turn increase expression of the HIF1A gene and stabilizes the protein product HIF-1α. Induced CAMP gene expression is activated by direct binding of HIF-1α.
