Molecular and Cellular Pharmacology
Curcumin induces down-regulation of EZH2 expression through the MAPK pathway in MDA-MB-435 human breast cancer cells

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Abstract

Curcumin, a natural compound isolated from turmeric, may inhibit cell proliferation in various tumor cells through a mechanism that is not fully understood. The enhancer of zeste homolog 2 (EZH2) gene is overexpressed in human breast cancers with poor prognosis. In this study, we observed a dose- and time-dependent down-regulation of expression of EZH2 by curcumin that correlates with decreased proliferation in the MDA-MB-435 breast cancer cell line. The curcumin treatment resulted in an accumulation of cells in the G1 phase of the cell cycle. Further investigation revealed that curcumin-induced down-regulation of EZH2 through stimulation of three major members of the mitogen-activated protein kinase (MAPK) pathway: c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. These data suggest that an underlying mechanism of the MAPK pathway mediates the down-regulation of EZH2, thus contributing to the anti-proliferative effects of curcumin against breast cancer.

Introduction

Curcumin, a polyphenol isolated from the plant turmeric (Curcuma longa), has been found to inhibit cell proliferation in a variety of human cancer cell lines in vitro and has been used both to prevent and to treat various cancers in vivo (Kunnumakkara et al., 2008, Lopez-Lazaro, 2008). In breast cancer, it has been reported that curcumin was able to inhibit the expression of HER2, cyclin E, cyclin D1 and others (Hong et al., 1999, Mukhopadhyay et al., 2002, Aggarwal et al., 2007, Kim et al., 2008, Liu et al., 2009). These data provided evidence that the anticancer properties of curcumin in breast cancer might be attributable to the modulation of various molecular targets.

Recently, Margarita and colleagues reported that curcumin could down-regulate the protein expression of MEL18, a member of the polycomb group (PcG), in Hodgkin's lymphoma HD-MY-Z cells (Sanchez-Beato et al., 2004), suggesting that curcumin might be involved in the regulation of PcG proteins and related signaling pathways. It is known that the enhancer of zeste homolog 2 (EZH2) is a critical component of PcG proteins, and that EZH2 plays an important role in cell proliferation and cell cycle regulation (Tonini et al., 2008). Overexpression of EZH2 protein has been reported to be linked to invasive growth, aggressive clinical behavior and poor prognoses of breast cancers, and it has been suggested as a new molecular target for breast cancer treatment (Kleer et al., 2003, Raaphorst et al., 2003, Collett et al., 2006).

The aims of this study were therefore (a) to determine whether curcumin could inhibit the proliferation of breast cancer cells by regulating the expression and/or activity of EZH2; and if so, (b) to investigate its potential mechanism.

Section snippets

Materials

Curcumin (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored in a dark-colored bottle at − 20 °C as a stock solution. The stock was diluted to the required concentration with serum-free medium immediately before use. The final DMSO concentration did not exceed 0.1% DMSO throughout the study. Antibodies against ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JNK1/2, phospho-JNK1/2(Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182) and β-actin were obtained

Curcumin inhibits the proliferation of human breast cancer cell line MDA-MB-435.

MDA-MB-435 cells were exposed to various concentrations of curcumin for 12, 24 or 48 h, and their cellular proliferation was monitored. The results demonstrated that curcumin inhibited the proliferation of MDA-MB-435 cells. The anti-proliferative effects of curcumin were both dose- and time-dependent (Fig. 1A). A marked inhibition in cellular proliferation was observed in cells treated for 48 h with 50 μM curcumin compared with untreated control cells. Flow cytometric analysis was used to detect

Discussion

Breast cancer is one the most common human cancers and the second leading cause of cancer deaths in women in the United States (Jemal et al., 2008). It is therefore highly desirable that an effective chemotherapy agent against this form of cancer is developed as soon as possible. Curcumin, a natural polyphenol product isolated from the C. longa, has emerged in recent years as a promising anticancer therapeutic agent (Pari et al., 2008), and its anti-tumor properties have been reliably

Acknowledgments

This work was supported by grants from the Major State Basic Research Program of China (2006CB910104) and the 863 Project of China (2007AA021901).

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    These authors contributed equally to this article.

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