Inhibition of Specificity Protein 1 Is Involved in Phloretin-Induced Suppression of Prostate Cancer
Phloretin decreased the expression of Sp1-targeted genes by downregulating Sp1 levels in PCa cells. (a, b) LNCaP and PC-3 cells were treated with phloretin (0, 20, 50, and 100 μM) for 24 h and then harvested for western blot (a) and RT-PCR (b) assays to check the expressions of Sp1 and its target genes (including Sp3/4, XIAP, VEGF, Survivin, Cyclin D1, and AR) in both mRNA and protein levels (β-actin as control). (c) LNCaP and PC-3 cells were cultured in 12-well plates with cover slips and then treated with phloretin (0, 20, 50, and 100 μM) for 24 h. Cells grown on cover slips were treated for immunofluorescent confocal analysis with Sp1 and Sp3/4 antibodies (the cell nucleus was stained with DAPI). (d) PC-3 cells were transfected with pSp1-751/-20-Luc, pSp3-417/-38-Luc, pVEGF-2018/+50-Luc, pSurvivin-269/-39-Luc, and pGL3-basic and treated with phloretin (0, 20, 50, and 100 μM), and then luciferase activities were measured by using the Dual-Luciferase Reporter Assay Kit. The relative luciferase units (RLU) were calculated. .
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