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Curcumin inhibits cell proliferation of MDA-MB-231 and BT-483 breast cancer cells mediated by down-regulation of NF[kappa]B, cyclinD and MMP-1 transcription.

Abstract

Curcumin, an active constituent of turmeric, has been shown to possess inhibitory effect of" cell proliferation and induction of apoptosis towards a board range of tumors. Cell inhibition activities of curcumin are behaved differently in various cell types. To investigate the mechanism basis for the cell inhibition of curcumin on breast cancer cell lines, we examine curcumin effect on NF[kappa]B, cell cycle regulatory proteins and matrix metalloproteinases (MMPs) in two breast cancer cell lines (MDA-MB-231 and BT-483). Cell proliferation was performed by water soluble tetrazolium WST-1 assay. The effect of curcumin's on the activity of matrix metalloproteinase-1, 3, 9 were analyzed by RT-PCR. Cell cycle regulatory protein including cyclin D1, CDK4 and p21 were examined by immunochemistry. The expressions of NF[kappa]B in breast cancer cells treated with curcumin were studied by immunochemistry and western blot. The results from WST-1 cell proliferation assay showed that curcumin exhibited the anti-proliferation effect on -MB-231 and BT-483 cells in a time- and dose-dependent manner. In response to the treatment, while, the expression of cyelin D1 had declined in MDA-MB-231 and the expression of **CDK4 in BT-483 had declined. MMP1 mRNA expression in BT-483 and MDA-MB-231 had significantly decreased in curcumin treatment group compared with control group. Our finding extrapolates the antitumor activity of curcumin in mediating the breast cancer cell proliferative rate and invasion by down-regulating the NF[kappa]B inducing genes.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Curcuma longa; Curcumin; NF[kappa]B; MMPs; CyclinD1/CDK4

Introduction

The first-line treatment of breast cancer is usually relied on surgery and radiotherapy, and is frequently supplemented by adjuvant chemo- or hormonotherapies. Unfortunately, breast cancer is highly resistant to chemotherapy, It has an urgent need in developing novel anticancer agents presenting low cytotoxic side-effects, but effective inhibit tumor cell proliferation pathway (Fisher 1994; Kaufmann 1989). Curcumin, a phenolic compound extract from rhizome of the plant Curcuma longa is found to have inhibitory effects towards a board range of tumors. in breast cancer, curcumin's anticancer effect has been anticipated in relating to induce apoptosis at G2 phase of cell cycle via a p53-dependent pathway (Choudhuri et al. 2005), but it's role in Gl/S transition of breast cancer has not been established well. Based on the evolving understanding of molecular basis of breast cancer, it has been explained that pathways activated by NF[kappa]B up-regulation are implicated not only in cell proliferation including cyclin Dl (Guttridge et al. 1999) but also regulate promoters that encode several MMPs. Therefore, in this study, we attempt to investigate the cell inhibition activity of curcumin on the NF[kappa]B and its regulatory genes including Gl cyclins and MMPs in BT-438 breast cancer cells and MDA-MB-231 breast cancer cells. Estrogen Receptor (ER) positive breast cancers are often beneficial from anti-hormonal drugs on either inhibiting interaction of oestradiol with ER or interrupting oestradiol synthesis. However, ER negative breast cancer, being lack of functional estrogen receptor, cannot be targeted by these therapies and are more aggressive with poorer prognoses (Rochefort et al. 2003). In our study, we used the MDA-MB-231 and BT-483 human breast cancer cell line as the model of ER-negative and ER-positive breast cancers respectively in comparing.

Materials and Methods

Curcumin (Molecular Weight 368.38) purchased from Sigma Chemicals (St. Louis, MO) was accurately weighed and dissolved in dimethyl sulphoxide (DMSO) (Sigma, St. Louis, MO) to a concentration of 500[micro]g/ml as a stock solution. It was diluted (50 [micro]g/ml) in DMEM and Leibovitz's L-15 supplemented with fetal bovine serum (10%) (Invitrogen, Carlsbad, California, US) respectively. The dilution (50 fig/ml) was further diluted with supplemented medium to a series of concentrations.

Both of ER-positive breast cancer cell line BT-483 and ER-negative breast cancer cell line MDA-MB-231 were bought from the American Type Culture Collection (Rockville, MD, USA). BT-483 were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), 1-glutamine (2mM) and antibiotics (penicillin 100 units/ml; streptomycin 100 mg/ml) at 37[degrees]C in an atmosphere of 5% [CO.sub.2] while MDA-MB-231 cells were grown in Leibovitz's L-15 medium with 2mM L-glutamine, supplemented with fetal bovine serum (10%), 1-glutamine (2mM) and antibiotics (penicillin 100 units/ml; streptomycin 100 mg/ml) at 37 [degrees]C without [CO.sub.2]. Cells were cultured at a density of 5 x [10.sub.3]) cells/well into 96-well plates. Cell growth was followed for 24 and 48 hr after stimulation in the presence or absence of cureumin (1, 1.25, 2.5, 5, 10, 20[micro]g/ml, namely 2.7, 4.1, 6.8, 13.6, 27, 54 [micro]M,). Medium alone was a negative control. Cell proliferation was assessed in vitro using the water soluble tetrazolium WST-1 (Roche, Indianapolis, IN).

Human breast cancer lines MDA-MB-231 and BT-483 were cultured with curcumin at the concentration of 2.5 [micro]g/ml and 5 [micro]g/ml respectively for 48 hours in standard humidified incubator. Total RNA was extracted by Geneaid[R] RNA Mini Kit (cultured cells) (Geneaid, Taiwan) according to the manufacture's instructions. The mRNA levels of the MMPs were determined by RT-PCR. Primer sequences for MMP-1, 3, 9 were previous described (Giambernardi et al. 1998). The relative target mRNA expression level was normalized by [alpha]-actin (forward 5'-CCTCTATGCCAACA-CAGTGC-3' reverse 5'-ATACTCCTGCTTGCTGAT-CC-3') in the same sample. PCR products were electrophoretic displayed on a 2% agrose gel with ethidium bromide staining and captured in gel documentation system (Bio-Rad, Hercules, CA, USA). Nuclear proteins were extracted from cultured cells as well. Protein concentrations were determined by using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Sample proteins were resolved by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis and then electrophoretically transferred to nitrocellulose. The primary antibody of NF[kappa]B were added and incubated at 4[degrees]C overnight. Then membranes were incubated with corresponding secondary antibodies for 1 hour. Protein bands were revealed by the ECL PLUS kit (GE. Healthcare Bio-Sciences, NJ, USA). The amount of NF[kappa]B protein was corrected by the signal of [Beta]-actin in the same sample. Quantification was achieved by quantifying the densities of the bands with the program of Quantity One.

The immunocytochemical studies for both cell lines were performed culturing in 24-well plate with glass-cover slides at a density of [10.sup.5] cells/well for 24 hours. The application of curcumin (5 [micro]g/ml) was followed by 48 hours incubation. The cells were fixed and immunostained with the following antibodies: Cyclin Dl, p21, CDK4 and NF[kappa]B. Staining was performed using 2% DAB substrate-chromogen solution. The intensity of DAB staining was examined under a standard light microscope using x 40 magnification.

Statistical analysis was performed using SPSS 16.0 (SPAA Inc., Chicago, IL, USA) including the ANOVA test for pairwise comparisons. The continuous variables were analyzed by independent-sample t test. All P values reported were two-sided with P <0.05 considered significant. Experimental results were presented as the mean [+ or -]SD.

Results and Disscussion

Effects of curcumin on breast cancer cell proliferation

Treatment of breast cancer cell BT-483 and MDA-MB-231 with curcumin exhibited inhibitory effect on cell growth (Figs. IB and C). Curcumin with the dose as low as 1.25[micro]g/ml was found to be effective on BT-483 and treatment with 5 [micro]g/ml for 48 h could result in a 60-70% inhibition. Curcumin with the dose as low as 2.5 [micro]g/ml was quite effective on MDA-MB-231 and treatment with 5 [micro]g/ml for 48 h resulted in a 50-60% inhibition. Thus we selected 5 [micro]g/ml concentration of curcumin as the optimal concentration for the subsequent experiments.

Curcumin decreased Matrix metalloproteinases-1 expression in MDA-MB231 and BT-483 breast cancer cells

[FIGURE 1 OMITTED]

To determine whether curcumin can down regulate MMPs expression in breast cancer cells, BT-483 and MD A-MB-231 were treated with 2.5[micro]g/ml, 5 [micro]g/ml curcumin for 48 hours, and then the mRNA was isolated and probed with MMP-1, MMP-3 and MMP-9 cDNA respectively. As shown in Fig. 2, curcumin at the concentration of 2.5 [micro]g/ml and 5[micro]g/m] can down-regulated MMP-1 mRNA in BT-483 breast cancer cell (Fig. 2B) and MDA-MB-231 breast cancer cell (Fig. 2A). In this study, 2.5 [micro]g/ml and 5 [micro]g/ml curcumin have not showed inhibitory effect on MMP-3 or MMP-9 mRNA expression.

[FIGURE 2 OMITTED]

Immunochemical detection of cyclin Dl, CDK4 and p21 in MDA-MB231 and BT-483 breast cancer cells

The D-type cyclins with their catalytic partners, CDK4 and CDK6, are rate limiting for the progression from [G.sub.0] into S phase of cell cycle (Sherr 1996). [p21.sup.cip1/waf1]wan is known to inhibit the kinase activity of the CDK4-Cyclin Dl. To determine whether curcumin modulate its anti-proliferative effects through the regulation of cyclin Dl and its catalytic partners, CDK, Immunochemistry were performed after MDA-MB-231 AND BT-483 treated with curcumin at 5 [micro]g/ml. As shown in Fig. 3.1, Curcumin decreased the cyclin D1 expression and induced the level of p21 expression in MDA-MB231 while the decrease of CDK4 expression has not been found. Down-regulated level of CDK4 expression has been found in BT-483 treated with curcumin, while no significant differences were observed in the level of cyclin Dl or p21 expression in ER-positive breast cancer BT-483 (Fig. 3.2).

[FIGURE 3 OMITTED]

Effect of curcumin on NF[kappa]Bprotein of MDA-MB-231 and BT-483 breast cancer cells.

To investigate whether curcumin inhibit NF-kB in BT-483 and MDA-MB-231 human breast cancer, we perform immunostaining and western blot of NF-kB p65 protein. As shown in Fig. 4. Treatment of BT-483 and MDA-MB-231 cell lines with curcumin at the concentration of 5[micro]g/ml for 12, 24, 48 hours respectively shown a reduced expression of nuclear NF-kB.

Treatment of breast cancer cell BT-483 and MDA-MB-231 with curcumin exhibited inhibitory effect on cell growth (Figs. IB and C). In mammalian cells, the D cyclins with their catalytic partners, CDK4 and CDK6, function as the cells leave [G.sub.0]and progress through [G.sub.1] into S phase (Buckley et al. 1993; Hall and Peters 1996; Sherr 1996). Three D-type cyclins, designated Dl, D2, and D3, have been identified to date. D-cyclin isoforms have implicated a tissue and cell specificity and may play a different role during normal and tumor development. In majority of human breast cancers, deregulated overexpression of cyclin Dl is found (Buckley et al. 1993). The presence of specific CDK inhibitory proteins regulates the activities of cyclins and CDKs. Whether curcumin modulate its anti-proliferative effects through the regulation of cyclin Dl was examined. Immuno-chemistry was carried out after treatment with 5 [micro]g/ml. The result showed that curcumin decreased cyclin Dl expression (Fig. 3.1B) in MDA-MB-231 while the decrease of cyclin Dl has not been observed in BT-483. Recently, the role of cyclin D3 in breast cancer progression has been suggested (Wong et al. 2001). Cyclin D3 share a similar function as well as cyclin Dl in regulating the Gl check-point of the cell cycle. Though cyclin D3 share a similar function of cycline Dl inregulating the Gl check-pint of the cell cycle, the role of cyclin D3 in breast cancer progression had just been recently suggested. Garcia-Morales (Garcia-Morales et al. 2006) have identified that ER-positive MCF-7 breast cancer cells growth inhibition by rapamycin is due to a Gl arrest that specifically caused by cyclin D3 down-regulation but not cyclin Dl. In our study, down-regulated cyclin Dl expression has not been found in ER-positive breast cancer BT-483, wherease a lower level of CDK4 expression has been found in BT-483 treated with curcumin. It straightly indicates the decrease of CDK4 occurred in BT-483 could be interpreted as a consequence of the cyclin D3 loss but not the cyclin Dl.

Proteins termed CDK inhibitory proteins including INK4 proteins and the Kip/Cip proteins can also bind to cyclin-Cdk complexes. Kip/Cip proteins, comprised of p27, p21, and p57, can inhibit each of the cyclin-CDK complexes being essential for Gl progression and S phase entry (Sherr and Roberts 1995). [p21.sup.cipl/wafl] is known to inhibit the kinase activity of the CDK4-Cyclin Dl. Inhibition by p27 is another generally proposed mechanism to explain the inactivity of D-type cyclin-CDK4 complexes (Sherr and Roberts 1995). p27 concentration allows the nuclear import and activation of cyclin D3-CDK4 complexes (Coulonval et al. 2003). We investigated curcumin's regulatory effect on [p2l.sup.cipl/wafl] by immunochemistry. As results shown in Fig. 3F, we observed curcumin induced high level of [p21.sup.cip1/wafl] protein expression in MDA-MB-231 but failed to see such difference in case of between ER-positive BT-483 cells no matter it treated with curcumin or not. The possibility that curcumin inhibits cyclin D3-CDK4 complex to bind p27 in breast caner related to ER status should be tested in the future study.

MMPs are a family of proteolytic enzymes that can degrade extracellular matrix (ECM) components including collagen, fibronectin, and laminin. Due to the significant role that MMPs play in cancer as well as additional human pathologies, interest has focused on identifying natural and synthetic compounds that can inhibit MMP activities. Curcumin has been reported that it can inhibit the activity of MMP2 in MDA-MB-231 cell line by western blot (Shao et al. 2002). As shown in Fig. 2A and B, our RT-PCR result showed that curcumin can inhibit the mRNA expression of MMP-1 in MDA-MB-231 and BT-483 while having no effect on the MMP-3, 9.

Constantly activated NF-kB directly induce genes that promote cell proliferation including cyclin Dl and the cyclin-dependent kinase inhibitors p21 (Guttridge et al. 1999). The transcription factor NF[kappa]B has also been shown to be critically involved in the regulation of MMP-1, MMP-3 (Mengshol et al. 2000). Curcumin has been reported to have a synergistic effect against the growth of both ER-positive and ER-negative cells (Shao et al. 2002). We proposed that curcumin may suppress NF-kB which in turn suppressing MMPs, cyclin Dl. Under the normal conditions, NF-kB as an inactive heterotrimer present in the cytoplasm. Cytokines such as TNF-[alpha] and environmental hazards like ionizing radiation or toxins trigger the translocation of NF-kB from the cytoplasm to the nucleus. Activation of this transcription factor protects a variety of cell types from the damage that induced by pro-apoptotic stimuli (Haefner 2002). It is therefore, inhibition of NF-kB activity is not only desirable in the treatment of inflammation but also in cancer therapy. Curcumin decreased nuclear and cytoplasmic p65 immunopositiv-ity of both BT-483 and MDA-MB-231 cells (Fig. 4A). Curcumin inhibited intranuclear expression of NF-kB p65 in MDA-MB-231 and BT-483 cells were also confirmed by western blot (Fig. 4B).

Breast cancer often progresses from the estrogen-dependent, nonmetastatic phenotype to the estrogen-independent, metastatic phenotypes. This progression is usually accompanied by altered function of the estrogen receptor or outgrowth of ER-negative cancer cells with generally high constitutive activity of NF-kB (Nakshatri et al. 1997). Although many estrogen receptor-positive breast cancers initially respond to antihormones drugs, responses are commonly incomplete when resistance ultimately emerging. Preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NF-kB in cell-line models of ER-positive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen. On the other hand, activated ER can inhibit both NF-kB activation and the ability of NF-kB to regulate gene expression (Agresti and Bianchi 2003). Further studies are necessary to investigagte how curcumin suppress the activation of NF-kB via inhibition of osteoclastogenesis in ER-positive breast cancer cell. Conceivably, our results show that curcumin can treat both ER-negative breast cancer (with generally high constitutive activity of NF-kB) and perhaps hormone-resistant ER-positive breast cancer via inhibiting the activation of NF-kB.

In summary, our finding extrapolates the antitumor activity of curcumin in mediating the breast cancer cell proliferative rate and invasion by down-regulating the NF-kB inducing genes. Our findings suggest curcumin could be a potentially therapeutic agent for both ER-positive and ER-negative breast cancer.

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Q.Liu, Wings T.Y. Loo, S.C.W. Sze, Y. Tong*

School of Chinese Medicine, The University of Hong Kong, G/F Estates Building, 10 Sassoon Road, Pokfulam, Hong Kong

* This research was supported in part by grant from Seed Funding Programme for Basic Research, the University of Hong Kong (No. 200611159207).

* Corresponding author.

E-mail address: tongyao@hku.hk (Y. Tong).

0944-7113/$-see front matter [c] 2009 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2009.04.008
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Title Annotation:nuclear factor kappa-light-chain-enhancer of activated B cells; matrix metalloproteinase
Author:Liu, Q.; Loo, Wings T.Y.; Sze, S.C.W.; Tong, Y.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9HONG
Date:Oct 1, 2009
Words:3281
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