Biochemical and Biophysical Research Communications
Epigallocatechin gallate inhibits HIF-1α degradation in prostate cancer cells
Section snippets
Materials and methods
Materials. (−)-Epigallocatechin gallate (EGCG), ferrous sulfate (FeSO4), desferrioxamine (DFX), and protease inhibitor cocktail were obtained from Sigma. 2-Oxoglutarate (2-OG) was obtained from Fisher Scientific. Primary antibodies to HIF-1α and HIF-1β were from Santa Cruz Biotechnology and anti-β-actin antibody was from Sigma. Secondary antibodies, horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG, M-PER mammalian protein extraction reagent, and neutravidin beads were from
EGCG upregulates HRE-mediated transcription under normoxia
To determine the effect of EGCG on HIF-1 transcription factor, we used reporter gene assay in which PC-3 and PC-3ML cells were transiently co-transfected with pGL3-6xHRE-Luc to test HIF-1 transcriptional activity, and pRL-TK as a control. PC-3 and PC-3ML cells were treated with EGCG at 0, 20, or 40 μg/ml for 24 h, followed by dual luciferase assay. As shown in Figs. 1A and B, EGCG significantly and specifically increased HRE-mediated luciferase activity from pGL3-6xHRE-Luc in a dose-dependent
Discussion
Herein, we determined that EGCG upregulated HRE-mediated promoter activity in hormone-refractory prostate cancer cells, PC-3 and PC-3ML cells. Since pGL3-6xHRE-Luc and pRL-TK plasmids have the same thymidine kinase basal promoter, this result suggested that EGCG specifically upregulated HRE-mediated promoter activity without affecting basal transcriptional activity. Furthermore, this study provides the first evidence of EGCG, a major green tea polyphenol, inhibiting HIF-1α hydroxylation and
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