Green tea polyphenols affect invasiveness of human gastric MKN-28 cells by inhibition of LPS or TNF-α induced Matrix Metalloproteinase-9/2

Abstract

Several studies demonstrated a correlation between green tea consumption and a reduced cancer risk. Among different components, green tea polyphenols have been identified as molecules responsible for the beneficial effects showed by the green tea against oxidative stress and cell invasiveness. In this study, we investigated the effects of green tea polyphenol extracts (GTPs) in human gastric MKN-28 cell line. To this aim, we have first evaluated the effect of GTPs on oxidative stress induced cell injury. The pre-treatment with 10^-4 M catechin equivalents of GTPs exerts a protective effect on xanthine-xanthine oxidase induced cell cytotoxicity, thus confirming the anti-oxidant properties of GTPs. The effect of GTPs was also extended to the invasive ability of MKN-28 cells stimulated with TNF-α or LPS, as pro-inflammatory factors. Migration and matrigel invasion assays demonstrated that GTPs exposure (10^-6 M) prevents the increase in cell invasiveness induced by TNF-α or LPS. Finally, we have analyzed the effect of GTPs on the levels of Matrix Metalloproteinases (MMP)-9/2, whose expression is up-regulated by TNF-α or LPS. Our results indicated that the pre-treatment with GTPs was able to reduce MMP-9/2 expression at both protein and enzyme activity levels in the conditioned media of TNF-α or LPS stimulated MKN-28 cells. In conclusion, our results demonstrated that green tea polyphenol extract reduces the invasiveness of gastric MKN-28 cancer cells through the reduction of TNF-α or LPS induced MMP-9/2 up-regulation. Therefore, these data support the hypothesis that GTPs could exert a protective role against the metastatic process in gastric cancer.

Keywords: Cell migration; Cell invasion; DMEM, Dulbecco's Modified Eagles's Medium; DMSO, Dimethyl sulfoxide; ECM, Extracellular matrix; FBS, fetal bovine serum; GTPs, Green tea polyphenols extract; Green tea polyphenols; LPS, Lipopolysaccharide; MKN-28 gastric cancer cells; MMP-, Matrix metalloproteinase; Matrix Metalloproteinase-2 (MMP-2); Matrix Metalloproteinase-9 (MMP-9); PBS, Phosphate-buffer saline; ROS, Reactive Oxygen Species; TNF-α, Tumor necrosis factor α.

Graphical abstract

Fig. 1

Effect of the pretreatment with GTPs on Xanthine–Xanthine Oxidase induced cell cytotoxicity in MKN-28 cells. MKN-28 cells were pre-incubated with the indicated GTPs concentration in serum-free medium containing 0.1% (v/v) DMSO (vehicle) for three hours; the cells were then exposed to DMEM containing Xanthine (1 mM) and Xanthine oxidase (75 mU/ml) or with serum-free medium containing the vehicle alone for additional two hours. Control cells were incubated with DMEM in the presence (10%) or in the absence of FBS, as well as in the presence of 10⁻⁴ M GTPs. Cell viability was determined as reported in Section 2.3. The values represent the mean of three separate experiments performed in triplicate.

Fig. 2

Effect of GTPs on cell migration or invasion in TNF-α or LPS stimulated MKN-28 cells. For migration (Panel A) or invasion assay (Panel C) MKN-28 cells were incubated in Boyden chambers or Matrigel coated membranes, respectively. Cells were treated with 10⁻⁶ M GTPs (images b, d, f) or vehicle alone (0.1% DMSO, images a) for six hours and then stimulated for 18 h with 10 ng/ml TNF-α (images c, d) or LPS (images e, f). Cells were then fixed and stained as detailed in Section 2.7. Each image shows a representative of five fields of three different experiment, visualized by a phase contrast microscope (40× objective). The quantification of the migrating (Panel B) or invading (Panel D) cells was calculated as the ratio versus the control unstimulated cells (representative images a, set as 1).

Fig. 3

Concentration-dependent effect of GTPs on MMP-9/2 protein levels induced by TNF-α stimulation in MKN-28 cells. Cells were pre-incubated with the indicated concentration of GTPs for six hours and then exposed to 10 ng/ml TNF-α for additional 18 h. The concentrated conditioned media were analyzed by Western Blotting for MMP-9 (Panel A) and MMP-2 (Panel B), respectively for a representative experiment (Panel C). Densitometric analysis of MMP-9/2 protein expression levels. The data obtained from three different experiments were averaged and reported as MMP-9/2 fold change vs control cells (set as 1) cultured in the absence of GTPs and TNF-α.

Fig. 4

Concentration-dependent effect of GTPs on MMP-9/2 protein levels induced by LPS stimulation in MKN-28 cells. Cells were pre-incubated with the indicated concentration of GTPs for six hours and then exposed to 10 ng/ml LPS for additional 18 h. The concentrated conditioned media were analyzed by Western Blotting for MMP-9 (Panel A) and MMP-2 (Panel B), respectively for a representative experiment. (Panel C) Densitometric analysis of MMP-9/2 protein expression levels. The data obtained from three different experiments were averaged and reported as MMP-9/2 fold change vs control cells (set as 1) cultured in the absence of GTPs and LPS.

Fig. 5

Concentration-dependent effect of GTPs on MMP-9/2 gelatinolytic activity levels induced by TNF-α or LPS stimulation in MKN-28 cells. Cells were pre-incubated with the indicated concentration of GTPs for six hours and then exposed to 10 ng/ml TNF-α (Panel A) or LPS (Panel B) for additional 18 h. The concentrated conditioned media were analyzed by gelatin zymography as detailed in Section 2.6. The position of the MMP-9/2 migrating band is indicated by an arrow on the right. The images show a representative experiment. (Panel C) Densitometric analysis of MMP-9/2 gelatinolytic activity levels. The data obtained from three different experiments were averaged and reported as MMP-9/2 fold change gelatinase activity vs control cells (set as 1) cultured in the absence of GTPs and TNF-α or LPS.