EGCG Induce Iysosome-dependent,But Not Atuophagy-dependent Cell Death Under Serum-free Conditions

[Objective]EGCG is the most abundant and biologically active constituents in green tea, as well as the most extensive and in-depth researched tea polyphenols. Many in vitro studies indicated that EGCG will induce apoptosis in cancer cells. However, the effective concentration should reach at least200μM, or even higher. How to lower the work concentration of EGCG, but remain its cytotoxicity at the same time, is a pending problem. In our study, we found the EGCG cytotoxicity was enhanced by depriving the serum from the medium. Thus, the main objective of this study is to systematically investigate the underlying mechanism of the EGCG cytotoxicity in serum-free conditions.[Methods]1. Cell culture and treatment:the cells were precultred in full or serum-free medium for1hr, then added EGCG for different treatment time.2. Hoechst-PI staining to detect cell viability:the total cells were marked by Hoechst, while the dead were marked by PI, using the soft Image J to count the cell numbers; 3. western blotting:western blotting was applied to detect the expressions of different proteins in whole cell lysis or the digitonin extraction;4. cell staining:using Acridine Orange, oil red O, Lyso-Tracker Red, ROS probe and magic red;5. immunostaining:lysosomal membrane marker LAMP-1and ER marker Bip[Results]1. Serum starvation enhanced EGCG-induced cell death. Such kind of cell death with character of cytosolic vacuolization, which was obviously distinct from apoptosis, was a caspase-independent manner.2. Although we detected the increased numbers of autophagosomes, yet it was finally proved that the cell death is autophagy-independent either. In fact, EGCG leads to lysosomal membrane permeabilization(LMP) with a resulting leakage of lysosomal proteinase to the cytosol and cytoslic acidification, which were then involved to the cell death. The accumulation of autophagosomes was just a side effect of lysosome dysfunction caused by increased pH and decreased cathepsin activity in lysosomes.3. we also found the cytosolic vacuoles were derived from lysosome dilation.4. we identified that ROS, which trigged by EGCG in serum-free medium, is a potential factor caused lysosome damage.[Conclusions]The enhanced cytotoxicity of EGCG in serum-deprived conditions is associated with ROS-mediated lysosome destabilization.