Objective: To investigate the role of human transcription factor NF-E2-related factor 2 (Nrf2) in the induction of the gene expression of uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A and its isoforms by epigallocatechin gallate (EGCG).
Methods: (1) Human colon carcinoma cells Caco-2 and HT-29 were cultured. Immunocytochemistry, western blotting and confocal laser microscopy were used to detect the protein expression of Nrf2. Twenty samples of colon carcinoma with surrounding normal tissues were collected during endoscopic course. (2) RNA interference expression vector pSilencer 3.1-H1 was used to construct four Nrf2-trageting plasmids: pSilence-Nrf2-A, B, C, and D and a control pSilence-CON. Cells were transfected with pSilence-Nrf2 for 48 hours to observe the effects of transient transfection. Cells were stably transfected with pSilence-Nrf2-B for 4 weeks and re-named as Caco-2-siNrf2 and HT-29-siNrf2 (siNrf2 cells), and others stably transfected with blank plasmid pSilencer 3.1-H1 were used as controls. (3) EGCG was added into the culture fluid of cells before and after the stably transfection. RT-PCR was used to detect the mRNA expression of Nrf2, UGT1A, UGT1A8 and UGT1A10 in cells and the samples of human colon cancer tissue.
Results: (1) The expressions of UGT1A8 and UGT1A10 mRNA were significantly lower than that in the surrounding healthy mucosa. (2) The mRNA expression of Nrf2, UGT1A8, and UGT1A10 increased by 1.8-9.2 times after the addition of EGCG (all P < 0.05). Immunocytochemistry, western blotting and immunofluorescence demonstrated a significant increase of Nrf2 protein expression in the nucleus after treatment with EGCG. (3) SalIenzyme digestion and DNA sequencing confirmed that pSilence-Nrf2-A, B, C, and D and pSilence-CON were all successfully constructed. The inhibition rate of Nrf2 gene expression was above 80% 48 h after transfection with pSilence-Nrf2-B, and that was no significant difference after transfection with pSilence-CON (P > 0.05). There was specific inhibition of Nrf2 in Caco-2-siNrf2, HT-29-siNrf2 cells (both P < 0.01). (4) The basal levels of UGT1A8 and UGT1A10 mRNA expression in the Caco-2-siNrf2 and HT-29-siNrf2 cells were lower by 15%-65% in comparison with those in control, and the induction of genes by EGCG was largely attenuated in them (all P > 0.05).
Conclusion: Nrf2 is localized in the cytoplasm of non-stimulated cells, and EGCG triggered its rapid nuclear accumulation. Suppression of Nrf2 gene expression results in down-regulation of the constructive expression of UGT genes and their induction by EGCG. EGCG induces the expression of UGT1A, UGT1A8 and UGT1A10 genes via a Nrf2-dependent mechanism.