Takeshi Minami

Inhibition of Matrix Metalloproteinases by Tea Catechins and Related Polyphenols

We have reported that green tea infusion inhibited in vitro invasion and in vivo metastasis of murine tumor cells. 1 Since matrix metalloproteinases (MMPs) play roles in these processes, we examined whether green tea catechins inhibit MMPs from the murine tumor cells. 2 Mouse Lewis lung carcinoma LL2-Lu3 cells were cultured in serum-free medium Cosmedium 001, and MMPs were partially purified from the medium by affinity chromatography with gelatin-agarose. Gelatin zymography showed that the carcinoma cells produced MMP-2 and MMP-9 as judged by their molecular masses (F IG . 1). ( − )-Epicatechin gallate and ( − )-epigallocatechin gallate (EGCG) inhibited in a dose-dependent manner these MMP activities (F IG . 2B), while no inhibition was observed for ( + )-catechin and ( − )-epicatechin at least up to 100 μ M. The black tea components theaflavin and its digallate also exhibited the inhibitory activity (F IG . 2B) and bound LL2-Lu3 MMPs as EGCG did (F IG . 1). The results of affinity chromatography with EGCG, theaflavin, or theaflavin digallate immobilized on agarose (F IG . 1) indicated the direct binding of MMPs to these compounds, which is likely to cause the inhibition. Since MMPs have been linked to tumor cell invasion, we examined if these catechin-related compounds inhibit the invasion by using a Matrigel (reconstituted basement membrane) invasion system. 1,2 The results indicated that the inhibitors of MMPs inhibited the invasion as well (F IG . 2A). In order to examine the importance of galloyl groups, galloyl monosaccharides were tested for these activities. 3 The degrees of inhibition of LL2-Lu3 MMPs were 78.9%, 0%, and 0% by tetragalloyl glucose, digalloyl hamamelose, and (mono)galloyl α -glucoside each at 50 μ M, respectively. The degrees of inhibition of Matrigel invasion of LL2-Lu3 cells were 99.2%, 72.4%, and 0% each at 50 μ M, respectively. The inhibition of invasion by digalloyl hamamelose with no MMP inhibitory activity may be explained by its inhibition (by 39% at 50 μ M) of cell adhesion of LL2-Lu3 cells to Matrigel. These results suggest that previously observed inhibition of metastasis by green tea infusion 1 can be explained at least partly by inhibition by tea catechins of MMPs the activities of which are necessary for tumor cell invasion. In a subsequent experiment, we examined the effects of EGCG on mRNA expression of MMPs. Expression of mRNAs for MMP-2, MMP-9, and MT1-MMP were assessed by the reverse transcriptase–polymerase chain reaction method, using total