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. 2008 Feb 1;103(2):509-19.
doi: 10.1002/jcb.21417.

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

Joel B Berletch  1 , Canhui Liu, William K Love, Lucy G Andrews, Santosh K Katiyar, Trygve O Tollefsbol
Affiliations

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

Joel B Berletch et al. J Cell Biochem. .

Abstract

The ends of human chromosomes are protected from the degradation associated with cell division by 15-20 kb long segments of hexameric repeats of 5'-TTAGGG-3' termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea catechins, in MCF-7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF-7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF-7 cells when treated with EGCG. Furthermore, down-regulation of hTERT gene expression in MCF-7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF-7 cells with EGCG resulted in a time-dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F-1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF-7 and HL60 cancer cell lines and may work through different pathways involving both anti-oxidant effects and epigenetic modulation.

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Figures

Fig. 1
Fig. 1
Growth analysis and morphological studies of MCF-7 breast cancer cells and HL60 promylocytic leukemia cells in response to EGCG treatment. Based on dose response analyses (data not shown), MCF-7 cells (A) were grown either in the presence or absence of 100 μM EGCG and HL60 cells (B) were grown in the presence or absence of 50 μM EGCG. Cells were counted using trypan blue staining. Morphological data were recorded at 3-day intervals for both MCF-7 (C) and HL60 cells (D). Cells were visualized at 100× magnification. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 2
Fig. 2
Time-dependent apoptosis induction in HL60 and MCF-7 cells by EGCG. HL60 and MCF-7 cells were treated with 50 and 100 μM EGCG, respectively for 9 days. Apoptosis was assayed by the Apoptag® Fluorescein Direct In Situ Apoptosis Detection Kit. A: HL60 nuclear DAPI stains (blue) and (B) HL60 TdT apoptosis stains (green) for untreated controls and cells treated through day 9. C: MCF-7 nuclear DAPI stains (blue) and (D) TdT apoptosis stains (green) for treated and untreated MCF-7 cells through day 9. Fluorescence activated cell sorting was used to further study apoptosis induced by EGCG (E). Levels of apoptosis were calculated using values for early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 3
Fig. 3
EGCG down-regulates hTERT mRNA expression in MCF-7 cells but not in HL60 cells. Both untreated and cells treated for specific time intervals were assayed for hTERT expression. A: hTERT mRNA expression in treated MCF-7 compared side-by-side with untreated controls and (B) treated HL60 cells presented in a similar fashion. Lane 1 contains 100 bp marker. (+) Denotes cells treated with either 50 μM (for HL60 cells) or 100 μM EGCG (for MCF-7 cells) based on dose response analysis (data not shown) and (-) indicates untreated controls. β-Actin was used as a loading control. For PCR amplification of hTERT, primers used were as follows: 5′-AGAACGTTCCGCAGAGAAAA-3′, sense and 5′-AAGCGTAGGAAGACGTCGAA-3′, anti-sense. Amplification of β-actin was accomplished using primers as follows: 5′-AGAAAATCTGGCACCACACC-3′, sense and 5′-AGCACTGTGTTGGCGTACAG-3′, antisense.
Fig. 4
Fig. 4
EGCG deacetylates histones at the hTERT promoter and the down-regulation of hTERT expression correlates with associated epigenetic modulation. ChIP analysis of histone H3 acetylation in EGCG treated and untreated MCF-7 cells. A: PCR amplification of ChIP-purified DNA using a rabbit anti-acetylated histone H3 antibody. One sample was incubated with only rabbit IgG to serve as a negative contamination control (Ø) and (B) 1% of the supernatant was removed prior to immunoprecipitation to serve as an input DNA control. Primers specific for the hTERT gene were used for PCR amplification: 5′-CTCCGTCCTCCCCTTCAC-3′, sense and 5′-CAGCGCTGCCTGAAACTC-3′, anti-sense. C: Graph representing the correlation between the epigenetic changes at the hTERT promoter and expression in MCF-7 cells. Percent methylation was obtained by dividing the number of methylated sites by the total number of sites (37) being assayed (see Fig. 5A for site-specific methylation of the hTERT promoter in response to EGCG). Relative hTERT expression was determined by the Kodak photo-imaging system after assigning the most intense band to be 100%.
Fig. 5
Fig. 5
EGCG induces demethylation at E2F-1 binding sites in the hTERT promoter and increases E2F-1 binding capacity. MCF-7 treated cells were assayed for hTERT promoter methylation. Sodium bisulfite conversion was used to study 37 CpG sites within the hTERT promoter (indicated by boxes). Nested PCR using primers specific for converted hTERT DNA were used: first round sense, 5′-ATTTGGAGGTAGTTTTGGGTT-3′; first round anti-sense, 5′-AACCACCAACTCCTTCAAA-3′. For second round sense, 5′-GTTTTTAGGGTTTTTATATTATGG-3′; second round anti-sense, 5′-CACACCAAACACTAAACCACCA-3′. A: As shown, EGCG caused hypomethylation at both the distal and proximal E2F-1 binding sites containing two and three CpG sites, respectively. The gray shaded boxes represent undetermined methylation status and possibly result from partial methylation at these sites. B: Gel electrophoresis showing an increase in E2F-1 binding at the hTERT promoter. C: Histogram illustrating the % binding of E2F-1.
Fig. 6
Fig. 6
Proposed mechanism of epigenetic gene control alterations by EGCG. A: Under normal growth conditions DNMT1 methylation capacity is not inhibited allowing methylation of the hTERT promoter in cancer cells which may block the binding of the Rb/E2F-1/DNMT1/HDAC1 repressor complex, the results of which include a hypermethylated promoter, acetylated histones and active gene transcription. B: Treatment of EGCG inhibits DNMT1 methylating ability leading to hypomethylation of the hTERT promoter [Fang et al., 2003] including key E2F-1 sites. This may allow the Rb/E2F-1/HDAC1 repressor complex to bind leading to hypoacetylation and reduced hTERT transcription. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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