Time-dependent intracellular trafficking of FITC-conjugated epigallocatechin-3-O-gallate in L-929 cells
Graphical abstract
In the present study, epigallocatechin-3-O-gallate (EGCG) was conjugated with fluorescein-4-isothiocyanate (FITC) via the 3″-OH or 5″-OH group on the gallate ring of EGCG. We demonstrated the binding of FITC-conjugated EGCG onto membranes, its incorporation into cytoplasm and subsequent translocation into nucleus in suspended (left) and cultured (right) L-929 cells after 4 h and 8 h of treatment, respectively.
Introduction
Conventional studies have shown that green tea catechins have potent suppressive effects on cellular responses of various cancer cells and their intracellular signaling cascades.1, 2, 3 and references therein Most of this anti-cancer activity of green tea are believed to be mediated by its major polyphenolic antioxidant constituent, (−)-epigallocatechin-3-O-gallate (EGCG).4, 5, 6 Furthermore, some previous reports have demonstrated the incorporation of EGCG into the cytosol and/or even the nucleus of cells as well as its tissue distribution and metabolism in animals by using radioisotope-labeled EGCG, for example, [3H]EGCG.7, 8, 9, 10, 11, 12 This cellular internalization of EGCG is considered to be involved in the mechanism of the anti-cancer activity of EGCG as a possible chemopreventive agent, since catechins exert pro-apoptotic and anti-proliferative activities on cancer cells.
Although the biological effects of EGCG have been extensively investigated and believed to be mainly due to its potent antioxidant activities, its time-dependent intracellular trafficking is still unclear. In these previous studies, EGCG was added to cell culture media, and the effects were examined 24, 48 or even 72 h later. It is not known whether the action of EGCG was exerted in the first few hours or throughout the experimental period. Our earlier study also demonstrated the suppressive effects of EGCG on vascular smooth muscle cells after 48 h.13 In the present study, the binding of EGCG onto membranes, its incorporation into cytoplasm and subsequent translocation into nucleus in suspended and cultured L-929 cells for 4 or 8 h were demonstrated by FITC-conjugation, which would be exploited to give a clue to identify the primary targets and action mechanisms of EGCG.
Section snippets
Characterization of EGCG conjugated with FITC
Figure 1 shows representative HPLC profiles of free FITC, EGCG and their conjugate, FITC-EGCG, submitted to HPLC analysis with UV detection at 280 nm.14 One major peak and other minor were shown on the chromatogram of FITC-EGCG, which were named as peak 1 and 2, respectively (Fig. 1C). Peak 1 was identified as EGCG by comparison with the chromatogram of EGCG and its authentic standard.15 Since the column used in HPLC was not suitable to differentiate free FITC from FITC-EGCG, it was not clear
Discussion
As a great deal of attention has been focused on various beneficial activities of EGCG, exact information about the time-dependent intracellular trafficking of this compound is required in order to elucidate its primary targets of action and precise mechanisms of its actions. For this purpose, EGCG was conjugated with FITC via 3″-OH or 5″-OH group in the gallate ring of EGCG, which was confirmed by NMR analysis (Fig. 2).
The binding of FITC-EGCG onto membranes, its incorporation into cytoplasm
Conjugation of EGCG with FITC
EGCG, the major polyphenolic component of green tea, was purchased from DSM Nutritional Products Ltd. (Teavigo™, Basel, Switzerland).29 In order to visualize the intracellular trafficking of EGCG, it was conjugated with fluorescein-4-isothiocyanate (FITC, Dojindo Lab., Kumamoto, Japan) by using a modified labeling method as reported previously.30 In brief, EGCG (1 g) was dissolved in 100 ml of 0.2 M K2HPO4 (pH 9.2) and incubated for 72 h at 37 °C with FITC (60 mg). After incubation, the conjugate was
Acknowledgments
This work was supported by the Japan Society for the Promotion of Science Grant funded by the Japanese Government (FY2006 JSPS Postdoctoral Fellowship for Foreign researchers, P 06451). We appreciate Dr. Naoki Nakajima and Mr. Hajime Sugai (Institute for Frontier Medical Sciences, Kyoto University) for technical assistance.
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