www.elsevier.com/locate/ytaap

Toxicology and Applied Pharma

Broccoli and watercress suppress matrix metalloproteinase-9 activity and

invasiveness of human MDA-MB-231 breast cancer cells

Peter Rosea,*, Qing Huangb, Choon Nam Ongb, Matt Whitemana

aDepartment of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597, SingaporebDepartment of Community, Occupational and Family Medicine, National University of Singapore, 16 Medical Drive,

Singapore 117597, Singapore

Received 22 December 2004; accepted 6 April 2005

Available online 13 June 2005

Abstract

A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer.

In the current study, we examined the inhibitory effects of broccoli (Brassica oleracea var. italica) and watercress (Rorripa nasturtium

aquaticum) extracts on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity

using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is

associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed

TPA-induced MMP-9 activity and invasiveness in a concentration dependant manner as determined by zymographic analysis. Furthermore,

fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory

effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl

isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9

activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the

current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Cruciferous vegetables; Isothiocyanate; Matrix metalloproteinase; Invasion; Chemoprevention

Introduction

The process of metastasis consists of a series of complex

cascades involving cancer cell migration, adhesion, and

invasion. Indeed, cell invasion, the process of translocation

of neoplastic cells across extracellular matrix barriers, is

recognized as an essential biological event required for

tumor metastasis (Basset et al., 1997; Bogenrieder and

Herlyn, 2003). Although the mechanism(s) for the translo-

cation of tumor cells across matrix barriers are not fully

understood, it would appear that the secretion of proteolytic

enzymes is essential. The matrix metalloproteinases (MMP)

0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2005.04.010

* Corresponding author. Fax: +65 6779 1453.

E-mail address: bchpcr@nus.edu.sg (P. Rose).

represent a family of proteolytic enzymes that can degrade

extracellular matrix (ECM) components including collagen,

fibronectin, and laminin (Nabeshima et al., 2002). The

MMP family is represented by several sub-groups each

recognized for the type of substrates they degrade these

include the gelatinases, collagenases, stromelysins, strome-

lysin-like MMPs, matrilysins, and MMPs (Kerkela and

Saarialho-Kere, 2003). With regards to cell invasion both

matrix metalloproteinases 2 (MMP-2) and matrix metal-

loproteinases 9 (MMP-9) are known to be essential

(Bernardo and Fridman, 2003; Westermarck and Kahari,

1999). Indeed, elevated levels and activities of both MMP-

2 and MMP-9 are found in cancerous tissues and tumor

cells.

Due to the significant role that MMPs play in cancer as

well as additional human pathologies, considerable interest

cology 209 (2005) 105 – 113

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113106

has focused on identifying natural and synthetic com-

pounds that can inhibit MMP activities. To date, these have

included screening extracts derived from the traditional

Korean prescription drug Daesungki-Tang, the Chinese

medicinal herb Euonymus alatus, as well as polyphenolic

compounds isolated from green tea. The results obtained

from these studies indicate that extracts and compounds of

plant origin are potent sources of principles with inhibitory

effects against MMP activities (Cha et al., 2003; Chung et

al., 2004; Corps et al., 2004; Demeule et al., 2000; Garbisa

et al., 2001; Kaegi, 1998; Ha et al., 2004; Lin et al., 1998;

Mohan et al., 2000); however, the mechanism(s) for this

action remains largely unknown.

Considering the reported association of high cruciferous

vegetable consumption and reduced incidence of cancer,

little evidence exists showing the possible effects of

cruciferous vegetables on tumor invasion. An early report

by Scholar et al. (1989), demonstrated that a diet high in

crucifers (cabbage and collards) decreased the number of

pulmonary metastases after animals were intravenously

injected with mammary tumor cells. However, the potential

mechanism(s) and contributing active principles were not

determined. Therefore, in the current investigation, we

studied the inhibitory effects of extracts of broccoli and

Rorripa on the invasion potential of human MDA-MB-231

breast cancer cells and the activity of MMP-9. These data

could contribute to the cancer-inhibitory effects of diets high

in cruciferous vegetables.

Fig. 1. The effect of broccoli and Rorripa on MDA-MB-231 cell viability.

Cells were treated with either broccoli or Rorripa extracts (0.1–1 mg/ml)

and cell viability determined using the MTT and LDH leakage assays as

described in Materials and methods. Data are means T SD of three or more

independent experiments. Significance testing was performed using an

independent t test P < 0.01 compared to the control.

Materials and methods

Cell culture

The human MDA-MB-231 breast cancer cell line was

purchased from ATCC. Cells were cultured in monolayers

at 37 -C, 5% CO2 in Dulbecco’s modified Eagle’s

medium (DMEM) supplemented with 10% fetal bovine

serum (FBS), 100 mg/ml streptomycin, and 100 U/ml

penicillin.

Cell viability testing. Cell viability was determined as

previously described (Hansen et al., 1989). In brief, MDA-

MB-231 cells were seeded at a density of 1 � 104 cells/well

in a 96-well microtitre plate and incubated for 24 h.

Following cell adhesion the cells were washed twice in

PBS and fresh media (DMEM FBS free) were replaced

containing the designated concentrations of vegetable

extract. Plates were incubated for a further 24 h prior to

the determination of cell viability, as measured by the

calorimetric change using a plate reader at 595 nm (Bio-

Rad, model 3550, Biorad, Hercules, CA, USA).

Activity of lactate dehydrogenase (LDH) in the medium

was measured using an Abbott VP Biochemical Analyser

with the test kit (Abbott Laboratories, Irving, TX, USA).

The total LDH activity was determined by ultrasonication

and assessed by expressing as percentage LDH leakage

(LDH in medium/total LDH activity �100).

Plant material and liquid chromatography mass

spectroscopy (LC-MS) analysis. Individual cruciferous

vegetables, broccoli (Brassica oleracea var. italica) and

watercress (Rorripa nasturtium aquaticum), were collected

over a 3-month period from local supermarkets. Both

species were placed on dry ice and freeze dried immediately

to preserve freshness. All individual vegetable samples were

then pooled, this being conducted to eliminate variation.

Extracts were made as previously described (Mithen et al.,

2003; Rose et al., 2000). In brief, 100 mg of freeze-dried

tissue was weighed into a 50-ml polypropylene tube,

hydrated with 2.0 ml of deionized water, and homogenized

for 15 s (Ultraturrax homogenizer) and left at room

temperature for 1 h with occasional vortexing. Boiling

70% methanol (3.0 ml) was added to the mix and incubated

for a further 15 min at +70 -C. The mixture was cooled to

room temperature, and centrifuged at 3000 rpm for 5 min.

After centrifugation 1 ml aliquots were removed and

vacuum condensed to 200 Al volumes. The resultant

concentrates were filtered through sterile non-pyrogenic

filters (0.2 Am; Millipore) and stored at �70 -C prior to

testing. Extracts gave an equivalent concentration of 100 mg

ml�1 for each sample. All extracts were analyzed for their

respective ITC composition using a Finnigan-LCQ LC-MS

system.

Gelatin zymography

Gelatin zymography was performed essentially as

reported earlier with modifications (Albini et al., 2003; Ha

et al., 2004; Huang et al., 2004). Cells were seeded onto six-

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113 107

well plates in DMEM with 10% FBS and cultured to 80%

confluence. Cells were subsequently washed in PBS twice

and cultured in serum-free medium for an additional 24 h.

Following serum starvation the cells were then treated with

TPA (80 nM) and/or vegetable extract (0.1–1 mg/ml) for 24

h. The conditioned media for each designated treatment was

then collected and standardized to cell number (5 � 105

cells), mixed with non-reducing sample buffer (62.5 mM

Tris–HCl, pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01%

bromophenol blue), and subjected to electrophoresis on a

10% SDS–PAGE gel containing 0.1% (w/v) gelatin

(Sigma). The resulting gels were washed twice for 60 min

in wash buffer (10 mM Tris (pH 8.0) containing 2.5% (v/v)

Triton X-100) to remove residual SDS and incubated for a

further 16 h at 37 -C h in developing buffer (50 mM Tris–

HCl, pH 7.5, 0.2 M NaCl, 10 mM CaCl2, and 1 mM ZnCl2).

Gels were stained using 0.5% Coomassie blue R-250 in 5%

Fig. 2. Inhibitory effect of broccoli and Rorripa extracts against MMP-9 activi

exposed to increased concentrations of vegetable extract. Conditioned media were

gel co-polymerized with 0.1% gelatin. Following electrophoresis, gelatin zymogr

unstained band. (B) Densitometric intensity of the MMP-9 band quantified using a

separate days using freshly prepared reagents.

(v/v) methanol and 10% (v/v) acetic acid for 1 h and

destained in 10% (v/v) methanol, 5% (v/v) acetic acid until

the bands could be visualized. The presence of MMP-9 was

indicated as an opaque band, the density of which was

quantified using a Kodak Scientific Imaging system (Kodak,

CT, USA).

Cell invasion assay

The ability of cells to invade through the BD Matrigel

invasion chambers were conducted as previously described

(Baba et al., 2000). In brief, cells (2 � 105/ml) were

inoculated into the culture inserts and pretreated with

different concentrations of broccoli and watercress extracts

(0.1–1 mg/ml) for 1 h prior to stimulation with 80 nM

TPA. After 12 h incubation, the lower surfaces of the

membrane were fixed with 100% methanol and stained

ty. (A) Conditioned media derived from TPA-treated MDA-MB-231 cells

normalized to cell number prior to loading on a 10% SDS–polyacrylamide

aphy was performed and the presence of MMP-9 determined as an opaque

Kodak Scientific Imaging system. All experiments were repeated 3 times on

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113108

Fig. 3. (A) Chromatogram and respective mass spectrum showing the presence of 7-methylsulfinylheptyl isothiocyanate in Rorripa and 4-methylsulfinylbutyl

nitrile and 4-methylsulfinylbutyl isothiocyanate in broccoli extracts. (B) MDA-MB-231 cell viability treated with the respective fractions of broccoli and

watercress. Cells were treated with individual fractions of broccoli or watercress extract (1 mg/ml) and cell viability determined using the MTT and LDH

leakage assays as described in Materials and methods. (C) Conditioned media from TPA-treated MDA-MB-231 cells were subject to zymographic analysis in

the presence of individual fractions of broccoli and watercress extract. (D) Densitometric intensity of the MMP-9 band was quantified using a Kodak Scientific

Imaging system. All experiments were repeated 3 times on separate days using freshly prepared reagents. Data are means T SD of three or more independent

experiments.

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113 109

with Giemsa solution. Cells that had invaded to the lower

surface of the membranes were counted using a high-power

field microscope.

Statistical analysis

Data are presented as means T SD and analyzed by

Student’s t test.

Results

Cruciferous vegetable extract cytotoxicity on MDA-MB-231

cells

The cytotoxicity of each vegetable extract towards

MDA-MB-231 breast cancer cells was evaluated using the

MTT and LDH leakage assays. The results showed that no

appreciable loss in cell viability was observed in cells

incubated with increased concentrations of broccoli or

Rorripa extract (0, 0.1, 0.2, 0.5, and 1 mg/ml) (Fig. 1).

Effect of broccoli and Rorripa extracts on MMP-9 activities

To examine the inhibitory effect of cruciferous vegetable

extracts against MMP-9 enzymatic activity, cultured condi-

tioned media of MDA-MB-231 cells were subjected to

zymographic analysis. In the presence of increased con-

centrations of broccoli or Rorripa extract (0, 0.1, 0.2, 0.5,

and 1 mg/ml), MMP-9 activity was shown to be reduced in

a concentration-dependent manner (Figs. 2A–B).

4-Methylsulfinylbutyl and 7-methylsulfinylheptyl

isothiocyanates contribute to the inhibitory effects of

broccoli and Rorripa on MMP-9

Identification of candidate MMP-9 inhibitor(s), in

extracts of broccoli and Rorripa, was determined by LC-

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113110

MS analysis. Because both species are members of the

family Cruciferae we focused our attention towards

isothiocyanate (ITCs) constituents. ITCs have previously

been shown to have numerous biological effects in

mammalian cells, including phase I enzyme inhibition

and phase II enzymatic induction properties. The major

ITC products found in the vegetable extracts were the non-

volatile ITCs, 4-methylsulfinylbutyl (broccoli; 4-MSB

ITC), and 7-methylsulfinylheptyl isothiocyanates (Rorripa;

7-MShep ITC). In addition, we also found 4-methylsulfi-

nylbutyl nitrile (4-MSB nitrile) in the broccoli extract (Fig.

3A). The presence of each individual ITC was confirmed

by LC-MS; the ITCs having a [M + H+] ion and a major

[M + H+ � 64] fragment (corresponding to the loss of

the terminal methylsulfinyl groups). These data are con-

sistent with the fragmentation patterns of synthetic

standards, sulforaphane, and 8-methylsulfinyloctyl ITCs.

Moreover, the relative amount of the [M + H+]+1 and [M +

H+]+2 were consistent with the predicted isotopic occur-

rence of 13C, 15N, and 33S. Thus, in each extract we had

identified putative candidate compounds that may inhibit

MMP-9induction 4-MSB nitrile, 4-MSB ITC, and 7-

MShep ITC.

We next fractionated the broccoli and Rorripa extracts

into 6 individual fractions. The candidate compounds 4-

MSB ITC and 7-MShep ITC were separated into fraction

4, while 4-MSB nitrile was present in fraction 3. Cells

were incubated for 24 h in the presence of each fraction

at 1 mg/ml equivalents and cell viability determined.

None of the fractions tested were cytotoxic in either of

the assays used (Fig. 3B). Next we analyzed each

vegetable fraction (1–6) to determine the inhibitor effects

of towards MMP-9 activity using the zymography assay.

Using the conditioned media obtained from the MDA-

MB-231 cells treated with each fraction (1 mg/ml) we

found that only fraction 4 exhibited any strong inhibitory

effect against MMP-9, as shown in Figs. 3C–D. None of

the other components showed any inhibition action

towards MMP-9 activity. Therefore, ITCs present within

broccoli and Rorripa can be regarded as the main active

components with respect to the inhibition of MMP-9

activity.

Suppressive effect of broccoli and Rorripa extracts on

TPA-induced invasion of MDA-MB-231 cells

To determine whether inhibition of MMP-9 activity

could prevent the invasive properties of MDA-MB-231

cells. Using transwell plates we measured the cell invasion

property following TPA stimulation. As shown in Figs. 4A

and B, crude extracts of broccoli and Rorripa and their

respective ITC fractions could inhibited the invasiveness of

TPA-stimulated MDA-MB-231 cells through the Matrigel-

coated membrane in a concentration-dependent manner. We

can rule out the possibility that such inhibition is due to

cytotoxicity, as the cell viability was unaffected by the

vegetable extracts, as determined using the MTT assay and

LDH leakage assay. These results show that the broccoli

and Rorripa extracts could inhibit MDA-MB-231 cells

invasion, a breast cancer cell line with a high invasive

capacity.

Discussion

The process of invasion requires the active degradation

of environmental barriers including components of the

basement membrane (BM) and extracellular matrix

(ECM). Essential in this step are several proteolytic

enzymes including the MMPs. To date, MMP-9 (gelatinase

B) and -2 (gelatinase A) have received considerable

attention due to their ability to degrades type IV collagen,

this being a major structural component of the BM and

ECM (Nabeshima et al., 2002). Moreover, increased

expression and activities of MMP-9 and -2 are often found

in tumor tissues and malignant cells (Woessner, 1991) and

are known to be crucial in the invasion process (Aimes and

Quigley, 1995; Woessner, 1991). Indeed, elevated protein

levels and activities of MMP-9 and -2 in invasive breast

cancers are associated with poor survival rates in patients. It

is therefore not surprising that current research seeks to

identify mechanism(s) to inhibit or reduce the invasive

process.

Natural products including green tea polyphenols, iso-

thiocyanates (ITC), resveratrol, limonene, and Allium-

derived organosulfur compounds can all inhibit chemically

induced tumor formation in rodent models (Park and

Pezzuto, 2002; Surh, 2003). Attributed to the anticancer

effects of these dietary agents is their ability to inhibit cancer

cell proliferation, invasion, and metastatic potential. For

example, with regards to invasion, green tea polyphenols are

strong inhibitors of the gelatinolytic activity of MMP-9 and

the secretion of MMP-2 (Annabi et al., 2002). Surprisingly,

little is known of the role of cruciferous vegetable species or

their phytochemical constituents in the prevention and or

inhibition of cancer cell invasion. Recent epidemiological

evidence has shown that a diet high in cruciferous

vegetables can reduce the incidence of breast cancer risk

in females (Ambrosone et al., 2004; Fowke et al., 2003).

These data are further supported by the findings from

experimental rodent models in which the principal bioactive

components of cruciferous vegetables, the ITCs, can induce

phase II detoxification enzymes, induce cell cycle arrest,

and promote of apoptosis in breast cancer cells in vitro

(Jackson and Singletary, 2004; Tseng et al., 2004; Zhang et

al., 1992). Sulforaphane, derived from broccoli, has also

been demonstrated to inhibit chemically induced breast

cancer in female rats (Fahey et al., 1994). Moreover, early

work by Wattenberg (1981), showed that benzyl isothio-

cyanate could inhibit 7, 12-dimethylbenz(a)anthracene-

induced neoplasia of the breast of Sprague–Dawley rats.

To date, no studies have addressed whether phytochem-

Fig. 4. Inhibition of TPA-induced cell invasion by broccoli and Rorripa (A) Photographs of MDA-MB-231 cells after invasion through Matrigel pre-treated for

1 h with individual plant extracts and fractions. (B) Inhibitory effect of broccoli and Rorripa on the invasiveness of MDA-MB-231 cells. Cells (2 � 105/ml)

were seeded onto Matrigel\ coated membranes of transwell plates and then pretreated with 1 mg/ml of each extract for 1 h, followed by incubation with 80 nM

TPA for additional 16 h. The lower surfaces of the membranes from the transwell units were fixed with 100% methanol and stained with Giemsa solution. Cells

that had invaded to the lower surface of the membranes were counted under a light microscope. Data are presented as means T SD. All experiments were done

in triplicate. (*P < 0.05 compared to the TPA-treated group).

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113 111

icals derived from cruciferous vegetables are inhibitory

against cancer cell invasion in vitro. Therefore, in the

current investigation we evaluated extracts of broccoli,

B. oleracea var. italica, and watercress, R. nasturtium

aquaticum, both major dietary sources of ITCs, against the

invasive potential of a human breast cancer cell line.

We show that extracts of broccoli and Rorripa can

inhibit MMP-9 activities and the invasive potential of the

human breast cancer cell line MDA-MB-231. Analysis of

individual plant extracts using LC-MS revealed that two

major ITC were present, these being 4-methylsulfinylbutyl

and 7-methylsulfinylheptyl ITCs, respectively. This find-

ing corresponded with previous reports (Mithen et al.,

2003; Rose et al., 2000). Fractionation of each plant

extract showed that the anti-invasive properties of broccoli

and Rorripa were mediated by the ITC constituents.

Previous investigations have shown that ITCs can inhibit

the NF-KB signaling pathway in murine Raw 264.7

macrophages, human pancreatic cancer BxPC-3, and

human colon HT29 cells (Heiss et al., 2001; Jeong et

al., 2004; Murakami et al., 2003; Srivastava and Singh,

2004). Moreover, recent evidence has also demonstrated

that the ITC sulforaphane can inhibit AP-1 DNA binding

in human keratinocyte exposed to UV-B irradiation (Zhu

et al., 2004). Considering the role of NF-KB and AP-1

signaling pathways in the expression of pro-inflammatory

genes, including MMPs, perhaps detailed characterization

of the AP-1 and NF-KB pathways in the anti-invasive

properties of other cruciferous vegetable species and ITCs

is warranted.

In conclusion, the findings herein suggest that broccoli

and Rorripa have anti-invasive and anti-metalloproteinase

activities, and that the phytochemical constituents, the ITCs,

are a new class of invasion inhibitors. The results provide

P. Rose et al. / Toxicology and Applied Pharmacology 209 (2005) 105–113112

further insight as to how cruciferous vegetable consumption

perhaps contributes to human chemoprevention.

Acknowledgment

The authors would like to thank Bee-Lan Lee.

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