β-elemene decreases cell invasion by upregulating E-cadherin expression in MCF-7 human breast cancer cells

  • Authors:
    • Xian Zhang
    • Yang Zhang
    • Yinghua Li
  • Corresponding author:
  • View Affiliations

  • Published online on: Tuesday, June 4, 2013
  • Pages: 745-750
  • DOI: 10.3892/or.2013.2519

Abstract

Inactivation of E-cadherin results in cell migration and invasion, hence leading to cancer aggressiveness and metastasis. Downregulation of E-cadherin is closely correlated with a poor prognosis in invasive breast cancer. Thus, re-introducing E-cadherin is a novel strategy for cancer therapy. The aim of the present study was to determine the effects of the traditional Chinese medicine, β-elemene (ELE), on E-cadherin expression, cell migration and invasion in the breast cancer cell line MCF-7. MCF-7 cells were treated with 50 and 100 µg/ml ELE. E-cadherin mRNA was analyzed by reverse transcription‑polymerase chain reaction. E-cadherin protein levels were determined by immunofluorescence and western blot assays. Cell motility was measured by a Transwell assay. ELE increased both the protein and mRNA levels of E-cadherin, accompanied by decreased cell migration and invasion. Further analysis demonstrated that ELE upregulated estrogen receptor‑α (ERα) and metastasis-associated protein 3 (MTA3), and decreased the nuclear transcription factor Snail. In conclusion, our results demonstrate that ELE decreases cell migration and invasion by upregulating E-cadherin expression via controlling the ERα/MTA3/Snail signaling pathway.

Introduction

E-cadherin is a calcium-regulated homophilic cell-cell adhesion molecule and is expressed in most normal epithelial tissues but is downregulated in most types of cancer cells (1). The absence of E-cadherin causes the dedifferentiation and invasiveness of human cancers (2), indicating that E-cadherin is a tumor suppressor (3). Either gene mutation or loss of the wild-type allele leads to inactivation of E-cadherin (3,4). Selective loss of E-cadherin is one of the hallmarks of invasive breast cancer phenotypes (5). Decreased levels of E-cadherin have been related to the distant metastasis and poor prognosis of breast cancer (6,7). Therefore, increasing the expression of functional E-cadherin is a novel cancer therapeutic strategy. However, the potential application of traditional Chinese medicine in inducing the expression of E-cadherin in breast cancers is largely unexplored.

Elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane) is an active anticancer component of the traditional Chinese medicine Curcuma wenyujin(8). The extract of elemene is a mixture of α-, β- and δ-elemene, with β-elemene (ELE) as the main component, which accounts for 60–72% of the three isoforms (9). ELE has shown anticancer activities in the clinical treatment of leukemia and carcinomas of the brain, breast and liver (8,1012). One formulation of ELE has been approved by the State Food and Drug Administration of China for the treatment of primary and secondary brain tumors.

ELE inhibits cell proliferation by inducing cell cycle arrest and apoptosis, thereby reducing the metastasis or tissue invasion of cancer cells (8,13,14). We previously found that ELE upregulated estrogen receptor-α (ERα) mRNA by downregulating the Ras/MAPK signaling pathway in the tamoxifen (TAM)-resistant cell line MCF-7/TAM (15). As ERα suppresses the expression of the nuclear transcription factor Snail, a negative transcription factor of E-cadherin gene expression (16,17), it is intriguing to propose that ELE may increase the expression of E-cadherin via activating the re-expression of ERα and hence inhibiting the gene transcription of Snail. In the present study, we analyzed the levels of E-cadherin expression and cell motility capacity of MCF-7 cells following ELE treatment.

Materials and methods

Chemicals and antibodies

ELE (98% purity) was purchased from Dalian Yuanda Pharmaceuticals (Liaoning, China). The primary antibodies against E-cadherin (ab1416), ERα (ab2746), metastasis-associated protein 3 (MTA3) (ab87275), Snail (ab53519) and β-actin were from Abcam (Cambridge, UK). The secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse-IgG and anti-rabbit-IgG antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cell line and drug treatment

The MCF-7 human breast cancer cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and propagated in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA). MCF-7 cells were cultured at 37°C in a humidified incubator (Heraeus, Germany) with 5% CO2 and seeded at 2.5×105 cells/ml in 6-well plates (Corning, Inc., Corning, NY, USA). The cells were divided into three treatment groups (0, 50 and 100 μg/ml ELE), observed and examined after 24 h.

Immunofluorescence assay

For E-cadherin staining, cells were plated on glass coverslips in 6-well plates and treated with ELE for 24 h. The cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde solution containing 0.1% Triton X-100 for 20 min at room temperature (RT), incubated in 5% bovine serum albumin for 30 min at RT, and then treated with the anti-E-cadherin monoclonal antibody for 16 h at 4°C. After being washed three times in PBS, cells were further incubated with CY3 conjugated goat anti-mouse IgG (Beyotime Biotechnology, Haimeng, China) for 1 h at RT. After three washes with PBS, the cells were analyzed using a fluorescence inverted microscope (IX71; Olympus, Japan). The cells were counterstained with Hoechst 33258 (Beyotime Biotechnology) to label the cell nuclei.

Western blot analysis

Cells were washed twice with ice-cold PBS and lysed in 1% Triton buffer [1% Triton X-100, 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4 (Sigma, St. Louis, MO, USA), 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 μg/ml aprotinin] on ice. Total proteins were quantified using the Lowry method. Proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore, USA). The membranes were blocked with 5% bovine serum albumin (Sigma) at RT for 1 h and incubated overnight at 4°C with the following primary antibodies: E-cadherin (1:1,000), ERα (1:1,000), MTA3 (1:2,000) and β-actin (1:500). After washing with Tris-buffered saline with Tween-20 (TBST) buffer, the membranes were probed with HRP-conjugated secondary antibodies and developed with enhanced chemiluminescence reagent (Beyotime Biotechnology). To detect Snail, nuclear proteins were extracted with a nuclear extraction kit (Beyotime Biotechnology) and probed with the anti-Snail antibody (1:2,000). The images were analyzed by NIH ImageJ software.

Real-time RT-PCR

Total RNA was extracted by homogenization in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by chloroform re-extraction and isopropanol precipitation. RT-PCR was performed in a final volume of 20 μl containing 1.6 ml of cDNA template, 1 ml of primer (10 mM) and 10 ml of SYBR-Green Master Mix (Takara, Dalian, China). Primers used were 5′-TCCCATCAGCT GCCCAGAAA-3′ (sense) and 5′-TGACTCCTGTGTTCCTG TTA-3′ (antisense) for E-cadherin; 5′-GCACCGTCAAGG CTGAGAAC-3′ (sense) and 5′-TGGTGAAGACGCCAGT GGA-3′ (antisense) for human GAPDH. GAPDH was used as an endogenous housekeeping gene.

Transwell migration and invasion assay

Cell invasion was measured using a Transwell chamber. In brief, 2×105 cells were added per Transwell invasion chamber coated with 1–2 mg/ml Matrigel (reconstituted basement membrane; BD Biosciences, Mississauga, ON, Canada). MCF-7 cells were treated with ELE. Twenty-four hours later, the cells in the upper chamber were removed with a cotton swab. The remaining cells on the membrane were fixed for 10 min in methanol, stained with 1% crystal violet solution and washed with PBS. The number of invaded cells was counted for 5 fields/field of view at ×200 magnification.

Cell viability assay

Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded at 5×104 cells/well in 96-well plates, incubated overnight and then exposed to the indicated concentrations of ELE for the indicated times. Next, 20 μl of MTT (Sigma, St. Louis, MO, USA) solution (5 mg/ml) was added to each well, and the cells were incubated for another 4 h at 37°C. After removal of the culture medium, the cells were lysed in 200 μl of dimethyl sulfoxide (DMSO), and the optical density (OD) was measured at 570 nm with a microplate reader (Model 550; Bio-Rad Laboratories, USA). The following formula was used: Cell viability = (OD of the experimental sample/OD of the control group) × 100%.

Statistical analysis

The experiments were repeated at least three times. Data are expressed as the means ± standard deviation. Differences in the results between two groups were evaluated by the Student’s t-test. P<0.05 was considered to indicate a statistically significant result.

Results

Effect of ELE on the cell proliferation of MCF-7 cells

To examine the effect of ELE on the cell viability of MCF-7 cells, we treated the cells with 50 or 100 μg/ml ELE for 0, 12, 24, 36, 48, 60 and 72 h and determined the rate of cell survival with an MTT assay. MCF-7 cell proliferation was not inhibited at 50 or 100 μg/ml ELE for 24 h (P>0.05), whereas treatment of cells with both doses of ELE for 36 h significantly inhibited cell survival (Fig. 1) (P<0.05). Therefore, for our subsequent experiments, we treated cells with 50 and 100 μg/ml ELE for 24 h, respectively.

ELE transforms the phenotype of MCF-7 cells

We used a microscope to approximately evaluate the cell motility capacity with the change in cell morphology of MCF-7 cells treated with 50 and 100 μg/ml ELE for 24 h, respectively. ELE-treated cells demonstrated a loosely aggregated cell phenotype that switched gradually to a tight adherence, indicating increased cell-cell interactions and reduced capacity for motility (Fig. 2).

ELE increases the E-cadherin protein level in MCF-7 cells

To support the above-mentioned alteration in cell morphology, we next determined the E-cadherin protein levels in the MCF-7 cells by an immunofluorescence assay and found that E-cadherin protein was upregulated by ELE (Fig. 3A). Further western blot analysis showed that 50 and 100 μg/ml ELE increased E-cadherin in a concentration-dependent manner (Fig. 3B and C) (P<0.05 and <0.01, respectively). Thus, ELE upregulates E-cadherin protein levels.

ELE increases the E-cadherin mRNA level in MCF-7 cells

To investigate whether ELE enhances the gene transcription of E-cadherin, we determined the E-cadherin mRNA levels in MCF-7 cells using an RT-PCR assay. ELE at 50 and 100 μg/ml significantly increased the mRNA levels of E-cadherin in a concentration-dependent manner (Fig. 4) (P<0.05 and 0.01, respectively), which was consistent with the upregulation of E-cadherin protein (Fig. 3). These results showed that ELE increased E-cadherin gene transcription.

ELE regulates E-cadherin expression via ERα/MTA3/Snail signaling

To determine the underlying mechanism by which ELE increases the expression of E-cadherin, we determined the protein levels of ERα, MTA3 and Snail. ELE at 50 and 100 μg/ml increased both ERα and MTA3, whereas it decreased Snail (Fig. 5A and B) (P<0.05 and <0.01, respectively). In addition, increasing the concentration of ELE apparently enhanced these effects. Thus, ELE regulates E-cadherin expression via the ERα/MTA3/Snail signaling pathway.

ELE affects the cell migration and invasion of MCF-7 cells

An increase in E-cadherin may explain the increased cell-cell interactions and decreased capacity for motility of the MCF-7 cells following ELE treatment (Fig. 2). To further support this conclusion, we measured the cell migration and invasion of MCF-7 cells treated with ELE with a Transwell assay and found that cells successfully penetrated the basement membrane-coated chambers; however, the number of cells that penetrated the membrane was reduced significantly in the ELE-treated cells (Fig. 6) (P<0.05 and <0.01, respectively, for the 50 and 100 μg/ml treatment groups). These data demonstrated that ELE inhibits both cell migration and invasion.

Discussion

E-cadherin is a cell adhesion molecule that is expressed in normal breast tissue, and decreased E-cadherin expression is correlated with the poor clinical prognosis of breast cancer. The status of the E-cadherin gene, including promoter methylation and mutation, is related to the capacity for cell motility of breast cancers (18). Loss of E-cadherin expression results in increased cellular motility, invasiveness, and resistance to apoptosis, which leads to epithelial-mesenchymal transition (EMT) (19,20). Thus, reversing the functional protein levels of E-cadherin is an alternative strategy for cancer therapy. Reversing CpG hypermethylation in the promoter region of the E-cadherin gene could re-activate E-cadherin gene expression (21,22). It has been proposed that targeting signal transduction pathways such as EGFR and ER may be a more promising method with which to restore E-cadherin levels (2326).

In the present study, we discovered that ELE positively regulated the expression of E-cadherin in MCF-7 cells. Consistently, ELE induced a switch in cell morphology and reduced cell migration and invasion. Estrogen and its receptors regulate the expression of E-cadherin and EMT in breast cancer cells (27). The ERα maintains the epithelial morphology of breast cancer cells by activating CDH-1, the E-cadherin encoding gene (28). It has been reported that ER signaling upregulates MTA3 levels to negatively modulate Snail-mediated repression of E-cadherin (29). Further studies have demonstrated that activation of the ER-MTA3-Snail-E-cadherin pathway in breast cancer is generally associated with a more favorable clinical outcome (3033).

We previously found that ELE upregulates ERα mRNA and promotes the re-expression of ERα through downregulating the Ras/MAPK/ERK signaling pathway in MCF-7/TAM cells (15). In the present study, we showed that ELE increased ERα and MTA3, while reducing Snail. Thus, we demonstrated that ELE enhances the E-cadherin system and decreases the cell motility capacity by mediating the ER/MTA3/Snail/E-cadherin pathway in the breast cancer cell line MCF-7. These results suggest that the traditional Chinese medicine β-elemene is a promising agent for the treatment of breast cancers.

Acknowledgements

We thank Luping Zheng (The Research Institute of Integrated Traditional and Western Medicine, Dalian Medical University) for the technical assistance. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University.

References

1 

Hazan RB and Norton L: The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton. J Biol Chem. 273:9078–9084. 1998.

2 

Cheng CW, Wu PE, Yu JC, et al: Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two-hit hypothesis of tumor suppressor gene. Oncogene. 20:3814–3823. 2001.

3 

Berx G, Cleton-Jansen AM, Nollet F, et al: E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J. 14:6107–6115. 1995.

4 

Vos CB, Cleton-Jansen AM, Berx G, et al: E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis. Br J Cancer. 76:1131–1133. 1997.

5 

Yoshida R, Kimura N, Harada Y and Ohuchi N: The loss of E-cadherin, α- and β-catenin expression is associated with metastasis and poor prognosis in invasive breast cancer. Int J Oncol. 18:513–520. 2001.

6 

Parker C, Rampaul RS, Pinder SE, et al: E-cadherin as a prognostic indicator in primary breast cancer. Br J Cancer. 85:1958–1963. 2001.

7 

Kowalski PJ, Rubin MA and Kleer CG: E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res. 5:R217–R222. 2003.

8 

Lu JJ, Dang YY, Huang M, et al: Anti-cancer properties of terpenoids isolated from Rhizoma Curcumae - a review. J Ethnopharmacol. 143:406–411. 2012.

9 

Wang G, Li X, Huang F, et al: Antitumor effect of β-elemene in non-small-cell lung cancer cells is mediated via induction of cell cycle arrest and apoptotic cell death. Cell Mol Life Sci. 62:881–893. 2005.

10 

Yao YQ, Ding X, Jia YC, et al: Anti-tumor effect of β-elemene in glioblastoma cells depends on p38 MAPK activation. Cancer Lett. 264:127–134. 2008.

11 

Liu J, Zhang Y, Qu J, et al: β-Elemene-induced autophagy protects human gastric cancer cells from undergoing apoptosis. BMC Cancer. 11:1832011.

12 

Li X, Wang G, Zhao J, et al: Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase. Cell Mol Life Sci. 62:894–904. 2005.

13 

Chen W, Lu Y, Wu J, et al: Beta-elemene inhibits melanoma growth and metastasis via suppressing vascular endothelial growth factor-mediated angiogenesis. Cancer Chemother Pharmacol. 67:799–808. 2011.

14 

Yu Z, Wang R, Xu L, et al: β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS. PLoS One. 6:e158432011.

15 

Zhang B, Zhang X, Tang B, et al: Investigation of elemene-induced reversal of tamoxifen resistance in MCF-7 cells through oestrogen receptor α (ERα) re-expression. Breast Cancer Res Treat. 136:399–406. 2012.

16 

Oesterreich S, Deng W, Jiang S, et al: Estrogen-mediated down-regulation of E-cadherin in breast cancer cells. Cancer Res. 63:5203–5208. 2003.

17 

Scherbakov AM, Andreeva OE, Shatskaya VA and Krasil’nikov MA: The relationships between snail1 and estrogen receptor signaling in breast cancer cells. J Cell Biochem. 113:2147–2155. 2012.

18 

van Horssen R, Hollestelle A, Rens JA, et al: E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells. Breast Cancer Res Treat. 136:365–377. 2012.

19 

Foroni C, Broggini M, Generali D and Damia G: Epithelial-mesenchymal transition and breast cancer: role, molecular mechanisms and clinical impact. Cancer Treat Rev. 38:689–697. 2012.

20 

Korpal M, Lee ES, Hu G and Kang Y: The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem. 283:14910–14914. 2008.

21 

Nam JS, Ino Y, Kanai Y, et al: 5-aza-2′-deoxycytidine restores the E-cadherin system in E-cadherin-silenced cancer cells and reduces cancer metastasis. Clin Exp Metastasis. 21:49–56. 2004.

22 

Peng G, Wargovich MJ and Dixon DA: Anti-proliferative effects of green tea polyphenol EGCG on Ha-Ras-induced transformation of intestinal epithelial cells. Cancer Lett. 238:260–270. 2006.

23 

Lu Z, Ghosh S, Wang Z and Hunter T: Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion. Cancer Cell. 4:499–515. 2003.

24 

Mauro L, Pellegrino M, Lappano R, et al: E-cadherin mediates the aggregation of breast cancer cells induced by tamoxifen and epidermal growth factor. Breast Cancer Res Treat. 121:79–89. 2010.

25 

Belguise K, Guo S and Sonenshein GE: Activation of FOXO3a by the green tea polyphenol epigallocatechin-3-gallate induces estrogen receptor α expression reversing invasive phenotype of breast cancer cells. Cancer Res. 67:5763–5770. 2007.

26 

Helguero LA, Lindberg K, Gardmo C, et al: Different roles of estrogen receptors α and β in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line. Cancer Res. 68:8695–8704. 2008.

27 

Planas-Silva MD and Waltz PK: Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells. J Steroid Biochem Mol Biol. 104:11–21. 2007.

28 

Cardamone MD, Bardella C, Gutierrez A, et al: ERα as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells. Proc Natl Acad Sci USA. 106:7420–7425. 2009.

29 

Kumar R: Another tie that binds the MTA family to breast cancer. Cell. 113:142–143. 2003.

30 

Fujita N, Kajita M, Taysavang P and Wade PA: Hormonal regulation of metastasis-associated protein 3 transcription in breast cancer cells. Mol Endocrinol. 18:2937–2949. 2004.

31 

Mishra SK, Talukder AH, Gururaj AE, et al: Upstream determinants of estrogen receptor-α regulation of metastatic tumor antigen 3 pathway. J Biol Chem. 279:32709–32715. 2004.

32 

Yu JC, Hsu HM, Chen ST, et al: Breast cancer risk associated with genotypic polymorphism of the genes involved in the estrogen-receptor-signaling pathway: a multigenic study on cancer susceptibility. J Biomed Sci. 13:419–432. 2006.

33 

Toh Y and Nicolson GL: The role of the MTA family and their encoded proteins in human cancers: molecular functions and clinical implications. Clin Exp Metastasis. 26:215–227. 2009.

Journal Cover

August 2013
Volume 30 Issue 2

Print ISSN: 1021-335X
Online ISSN:1791-2431

2013 Impact Factor: 2.191
Ranked #33/202 Oncology
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APA
Zhang, X., Zhang, Y., & Li, Y. (2013). β-elemene decreases cell invasion by upregulating E-cadherin expression in MCF-7 human breast cancer cells. Oncology Reports, 30, 745-750. http://dx.doi.org/10.3892/or.2013.2519
MLA
Zhang, X., Zhang, Y., Li, Y."β-elemene decreases cell invasion by upregulating E-cadherin expression in MCF-7 human breast cancer cells". Oncology Reports 30.2 (2013): 745-750.
Chicago
Zhang, X., Zhang, Y., Li, Y."β-elemene decreases cell invasion by upregulating E-cadherin expression in MCF-7 human breast cancer cells". Oncology Reports 30, no. 2 (2013): 745-750. http://dx.doi.org/10.3892/or.2013.2519