Sumio Hayakawa

Apoptosis Induction by Lectin Isolated from the Mushroom Boletopsis leucomelas in U937 Cells

A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N′-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.

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

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.

Anti-Cancer Effects of Green Tea by Either Anti- or Pro-Oxidative Mechanisms

Tea derived from the leaves and buds of Camellia sinensis (Theaceae) is consumed worldwide. Green tea contains various components with specific health-promoting effects, and is believed to exert protective effects against diseases including cancer, diabetes and hepatitis, as well as obesity. Of the various tea components, the polyphenol catechins have been the subject of extensive investigation and among the catechins, (-)-epigallocatechin gallate has the strongest bioactivity in most cases. Our research group has postulated that hepatocyte nuclear factor-4α, sterol regulatory element-binding proteins, and tumor necrosis factor-α are targets of green tea constituents including (-)-epigallocatechin gallate for their anti-diabetes, anti-obesity, and anti-hepatitis effects, respectively. Published papers were reviewed to determine whether the observed changes in these factors can be correlated with anti-cancer effects of green tea. Two major action mechanisms of (-)-epigallocatechin gallate have been proposed; one associated with its anti-oxidative properties and the other with its pro-oxidative activity. When reactive oxygen species are assumed to be involved, our findings that (-)-epigallocatechin gallate down- regulated hepatocyte nuclear factor-4α, sterol regulatory element-binding proteins, and tumor necrosis factor-α may explain the anti-cancer effect of green tea as well. However, further studies are required to elucidate which determinant directs (-)-epigallocatechin gallate action as an anti-oxidant or a pro-oxidant for favorable activity.

In Vitro and In Silico Studies of the Molecular Interactions of Epigallocatechin-3-O-gallate (EGCG) with Proteins That Explain the Health Benefits of Green Tea

Green tea has been shown to have beneficial effects on many diseases such as cancer, obesity, inflammatory diseases, and neurodegenerative disorders. The major green tea component, epigallocatechin-3-O-gallate (EGCG), has been demonstrated to contribute to these effects through its anti-oxidative and pro-oxidative properties. Furthermore, several lines of evidence have indicated that the binding affinity of EGCG to specific proteins may explain its mechanism of action. This review article aims to reveal how EGCG-protein interactions can explain the mechanism by which green tea/EGCG can exhibit health beneficial effects. We conducted a literature search, using mainly the PubMed database. The results showed that several methods such as dot assays, affinity gel chromatography, surface plasmon resonance, computational docking analyses, and X-ray crystallography have been used for this purpose. These studies have provided evidence to show how EGCG can fit or occupy the position in or near functional sites and induce a conformational change, including a quaternary conformational change in some cases. Active site blocking, steric hindrance by binding of EGCG near an active site or induced conformational change appeared to cause inhibition of enzymatic activity and other biological activities of proteins, which are related to EGCG’s biological oligomer and formation of their toxic aggregates, leading to the prevention of neurodegenerative diseases and amyloidosis. In conclusion, these studies have provided useful information on the action of green tea/catechins and would lead to future studies that will provide further evidence for rational EGCG therapy and use EGCG as a lead compound for drug design.