Antiproliferative and proapoptotic effects of epigallocatechin gallate on human leiomyoma cells

Abstract

Objective: To investigate the effects of epigallocatechin gallate (EGCG), an extract of green tea on cultured human leiomyoma cells (HuLM).

Design: Laboratory study.

Setting: University hospitals.

Patient(s): Not applicable.

Intervention(s): Not applicable.

Main outcome measure(s): The HuLM cells were treated with various EGCG concentrations. Cell proliferation was assayed using Hoechst 33258 dye, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Total RNA was isolated, and gene expression profiling was performed on 84 key genes related to 18 different signal transduction pathways. The protein levels of PCNA, CDK4, BCL2, and BAX were examined by Western blot analysis.

Result(s): The HuLM cells treated with EGCG showed a dose-dependent and time-dependent inhibition of cell proliferation. The TUNEL staining indicated a significant increase in apoptosis in HuLM cells treated with 100 μM of EGCG compared with untreated control. Gene expression profiling indicated that EGCG treatment up-regulated representative genes from the transforming growth factor β (TGF-β) and stress pathways, while inhibiting the survival pathway and NFκB-dependent inflammatory pathway. Western blot analysis confirmed that EGCG at ≥50 μM significantly decreased the expression of PCNA, CDK4, and BCL2 as well as increased the expression of the proapoptotic BAX in a dose-dependent manner.

Conclusion(s): Epigallocatechin gallate inhibits the proliferation of HuLM cells and induces apoptosis. These results suggest that EGCG may be a potential anti-uterine fibroid agent acting through multiple signal transduction pathways.

Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Effect of EGCG on morphology of HuLM cells. HuLM cells were treated with EGCG concentrations at 0µM(a), 10 µM(b), 50µM(c), 100µM(d) and 200µM(e, f) for 48h. Pictures were taken using a phase-contrast microscope at 100× (a–e) or 200 × (f) magnifications. The data shown here are representative of experiments repeated twice.

Figure 2

EGCG inhibits the proliferation of HuLM cells. HuLM cells were treated with EGCG (0, 0.1, 1, 10, 50, 100 and 200 uM) for up to 7 days. The cells were lysed and DNA content was measured with Hoechst 33258 dye solution. The results are presented as the percent changes from control (0µM EGCG). The inhibitory effect of ≥ 50µM EGCG and above was statistically significant (p

Figure 3

EGCG-mediated apoptosis in HuLM cells assessed by TUNEL staining (green fluorescence- panel A). Arrows point to representative apoptotic HuLM cells treated with 100µM EGCG (A). Nuclei were stained with propidium iodide (B-red fluorescence). Images in A and B are super imposed in panel C (note the apoptotic nuclei appear yellow). Magnification 200×.

Figure 4

Effect of EGCG treatment on the expression of genes related to multiple signal transduction pathways in HuLM cells. Expression levels in HuLM cells treated with 100µM EGCG were normalized against untreated cells. Only genes with expression fold change of ≥2 (green bars) or ≤2 (red bars) are depicted.

Figure 5

Western blot analysis detecting the effects of various EGCG concentrations on pro-apoptosis and proliferation-associated genes in HuLM cells. EGCG, at 50µM or higher concentration significantly decreased the expression of PCNA (A), Cdk4 (B), and Bcl-2 (C), in comparison to untreated control culture (p