In this study, we demonstrated that EGCG induced human mesothelioma cell death in a dose-dependent manner. We further clarified the mechanism responsible for such cell killing. EGCG induced reactive oxygen species (ROS) and impaired the mitochondrial membrane potential. The use of ROS scavengers, catalase and tempol, significantly inhibited the EGCG-induced apoptosis. Furthermore, we found that EGCG induced autophagy, and that the suppression of autophagy enhanced the EGCG-induced cell death.
There are many reports about the effects of EGCG on cancer cell growth
[7, 29, 30]. However, there are only two papers concerning EGCG-induced mesothelioma cell death
[8, 9]. In the former paper it was reported that the EGCG-induced cell death occurred via H2O2-dependent T-type Ca2+ channel opening. The data are not inconsistent with our present data showing that EGCG-induced mesothelioma cell death occurs via the production of ROS (H2O2 and superoxide). As we did not analyze the H2O2-dependent T-type Ca2+ channel opening, it is unclear whether H2O2-dependent T-type Ca2+ channel opening is involved in our case.
As both apoptosis and autophagy are triggered by common upstream signals
[27, 28], we tested whether EGCG induced autophagy, and found that it did induce autophagy, and that treatment of cells with CQ, an autophagy inhibitor, augmented the EGCG-induced cell death. Autophagy is known to play dual roles in cancer, acting as both a tumor inhibitor and as a tumor growth promoter
. In our present study, autophagy protected the mesothelioma cells from death. These data are consistent with several reports in other cancer cells demonstrated that the inhibition of autophagy restored chemosensitivity and augmented tumor cell death
[13–19]. CQ is a well-known drug that is widely used for the prophylaxis treatment of malaria because of both its efficacy and low toxicity to humans
[31–33]. It is also widely used as an anti-rheumatoid agent
, and our data suggests that it may be useful for treating mesothelioma patients if used in combination with EGCG.
The cell death induced by EGCG was prevented by treatment with catalase, thus suggesting that the effects of EGCG were largely due to the production of hydrogen peroxide by the cells. Because the catalase was added extracellularly, it could decrease the hydrogen peroxide that was extracellularly induced by EGCG. In contrast, tempol, a membrane-permeable radical scavenger, also prevented the EGCG-induced cell death. This reagent reduced the formation of the hydroxyl radical by scavenging superoxide anions. These results suggest that the superoxide anion produced in the cells could lead to cell death either directly or indirectly. Therefore, EGCG treatment may induce the disruption of the mitochondrial membrane potential inside cells. In fact, as shown in Figure
3B, EGCG did decrease the mitochondrial membrane potential.
Several studies report that EGCG has dual function of anti-oxidant and pro-oxidant potential
[34, 35]. Low concentrations (i.e. 10 μM) of EGCG scavenged free radicals thereby inhibiting oxidative damage to cellular DNA. In contrast, higher concentrations (i.e. 100 μM) of EGCG induced cellular DNA damage
. Dual function of EGCG in normal human lymphocytes is reported in
. In our present study we have shown similar results as shown in Figure
1. In most mesothelioma cell lines higher concentrations (i.e. 100 μM or 200 μM) of EGCG induced cell death and low concentrations (i.e. 10 μM) of EGCG failed to induce cell death.
The accumulating experimental evidence that cancer cells are more susceptible to hydrogen peroxide and to hydrogen peroxide-induced cell death than normal cells was discussed in a mini-review paper
. However, it is unclear what specific concentrations of hydrogen peroxide are required to kill cancer cells. It has been speculated that hydrogen peroxide may be present at low levels in normal cells because there are higher levels of catalase activity.