The green tea component EGCG inhibits RNA polymerase III transcription

Abstract

RNA polymerase III (RNA pol III) transcribes many small structural RNA molecules involved in RNA processing and translation, and thus regulates the growth rate of a cell. Accurate initiation by RNA pol III requires the initiation factor TFIIIB. TFIIIB has been demonstrated to be regulated by tumor suppressors, including ARF, p53, RB, and the RB-related pocket proteins, and is a target of the oncogene c-myc and the mitogen-activated protein kinase ERK. EGCG has been demonstrated to inhibit the growth of a variety of cancer cells, induce apoptosis and regulate the expression of p53, myc, and ERK. Thus, we hypothesized that EGCG may regulate RNA pol III transcription in cells. Here, we report that EGCG (1) inhibits RNA pol III transcription from gene internal and gene external promoters (2) EGCG inhibits protein expression of the TFIIIB subunits Brf1 and Brf2, and (3) EGCG inhibits Brf2 promoter activity in cervical carcinoma cells.

Figure 1. EGCG inhibits HeLa cell proliferation

(A) Structure of (−) - epigallocatechin gallate (EGCG). (B) Hela cells treated with increasing concentrations of EGCG as indicated and counted using a hemocytometer at the time points indicated. Each dose and time point was performed in triplicate.

Figure 2. EGCG inhibits RNA pol III transcription in HeLa cells

(A) HeLa cells transiently transfected with pGL3-U6 (100ng), or empty pGL3 vector (100ng), treated with increasing concentrations of EGCG (0uM, 5uM, 10uM, 25uM, 50uM). Lower panel corresponds to total cellular protein concentrations used in luciferase assay as determined by Lowry assay. (B) HeLa cells transiently transfected with pGL3-VAI (100ng), or empty pGL3 vector (100ng), treated with increasing concentrations of EGCG. Lower panel corresponds to total cellular protein concentrations used in luciferase assay as determined by Lowry assay. All luciferase assay results are expressed as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three times, and representative experiments are depicted. Statistical analysis was performed using one-way ANOVA with a Tukey post-test with a 95% confidence interval (Graphpad Prism 3.03); * = p <0.05; ** = p < 0.01; *** = p < 0.001.

Figure 3. EGCG inhibits expression of the TFIIIB subunits Brf1 and Brf2

(A) Nuclear extract prepared from HeLa cells transiently transfected with FlagMaf1 and untransfected HeLa cells treated with 0uM or 25uM EGCG and immunoblotted with an anti-flag and CSH1043 antibodies. Arrows depict migration of Brf1, Brf2 and Maf1. (B) Same immunoblot from 2A reprobed using anti-actin antibody as loading control.

Figure 4. EGCG inhibits activity from the human Brf2 promoter

(A) Schematic representation of the human Brf2 promoter as determined by Gene2Promoter software of Genomatix Suite. Putative transcription factor binding sites are depicted as identified by MapInspector [38]. Bolded ATG indicates start of translation, and italicized sequence denotes 5' untranslated sequence. (B) HeLa cells transiently transfected with pGL3 (200ng), pGL3-U6 (200ng), Brf2-pGL3 (50ng, 100ng, 200ng). (3) HeLa cells transiently transfected with pGL3 (100ng), Brf2-pGL3 (100ng) and treated with increasing concentrations of EGCG. Statistical analysis was performed using one-way ANOVA with a Tukey post-test with a 95% confidence interval (GraphpadPrism3.03);**=p<0.01.