Time-dependent intracellular trafficking of FITC-conjugated epigallocatechin-3-O-gallate in L-929 cells

Many in vitro studies about green tea polyphenol, (–)-epigallocatechin-3-O-gallate (EGCG) focused on its pro-apoptotic and anti-proliferative effects on various types of cancer cells, while less attention has been paid to its incorporation into the cytoplasm and nuclear translocation. This study concentrated on the time-dependent intracellular trafficking of EGCG in L-929 cells. EGCG was conjugated with fluorescein-4-isothiocyanate (FITC) via the 3″-OH or 5″-OH group, as confirmed by NMR analysis, and then treated to either suspended or cultured cells. Confocal microscopic observations revealed that FITC-EGCG was clearly seen onto the membrane of suspended cells as well as into the cytoplasm and nucleus within 1 h. As an increase in treatment time, it concentrated on the nucleus and then was located at any places of the cells. The cellular uptake of FITC-EGCG in cultured cells was not observed until 1 h of culture, but started to be observed after at least 2 h. These results imply that although the cellular sensitivity and response to EGCG would be different from those of FITC-EGCG, it would be incorporated into the cytoplasm of cells and further be translocated into the nucleus in a time-dependent manner.

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.Figure optionsDownload as PowerPoint slide