Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells

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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.

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 translocation 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) 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, stromelysin-like MMPs, matrilysins, and MMPs (Kerkela and Saarialho-Kere, 2003). With regards to cell invasion both matrix metalloproteinases 2 (MMP-2) and matrix metalloproteinases 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 has focused on identifying natural and synthetic compounds 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.

Section snippets

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.

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

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

Acknowledgment

The authors would like to thank Bee-Lan Lee.

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