Mechanism of Action of (−)-Epigallocatechin-3-Gallate: Auto-oxidation–Dependent Inactivation of Epidermal Growth Factor Receptor and Direct Effects on Growth Inhibition in Human Esophageal Cancer KYSE 150 Cells

Abstract

Abstract(À)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the phosphorylation of epidermal growth factor receptor (EGFR). We observed similar effects of EGCG in esophageal squamous cell carcinoma KYSE 150 cells and epidermoid squamous cell carcinoma A431 cells. Pretreatment of KYSE 150 cells with EGCG (20 Mmol/L) for 0.5 to 24 hours in HAM's F12 and RPMI 1640 mixed medium at 37°C, before the addition of EGF, resulted in a decreased level of phosphorylated EGFR (by 32-85%). Prolonged treatment with EGCG (8 or 24 hours) also decreased EGFR protein level (both by 80%). EGCG treatment for 24 hours also caused decreased signals of HER-2/neu in esophageal adenocarcinoma OE19 cells. These effects of EGCG were prevented or diminished by the addition of superoxide dismutase (SOD, 5 units/mL), or SOD plus catalase (30 units/mL), to the cell culture medium. A similar phenomenon on inactivation of EGFR was observed in A431 cells as well. Under culture conditions for KYSE 150 cells, EGCG was unstable, with a half-life of f30 minutes; EGCG dimers and other oxidative products were formed. The presence of SOD in the culture medium stabilized EGCG and increased its half-life to longer than 24 hours and some EGCG epimerized to (+)-gallocatechin-3-gallate. A mechanism of superoxide radical-mediated dimerization of EGCG and H 2 O 2 formation is proposed. The stabilization of EGCG by SOD in the culture medium potentiated the activity of EGCG in inhibiting KYSE 150 cell growth. The results suggest that in cell culture conditions, the auto-oxidation of EGCG leads to EGFR inactivation, but the inhibition of cell growth is due to other mechanisms. It remains to be determined whether the presently observed auto-oxidation of EGCG occurs in vivo. In future studies of EGCG and other polyphenolic compounds in cell culture, SOD may be added to stabilize EGCG and to avoid possible artifacts. (Cancer Res 2005; 65(17): 8049-56)

Autoxidative quinone formation in vitro and metabolite formation in vivo from tea polyphenol (-)-epigallocatechin-3-gallate: Studied by real-time mass spectrometry combined with tandem mass ion mapping

“…We and other investigators have shown that under cell culture conditions, EGCG is subject to oxidation, resulting in the formation of reactive oxygen species [9][10][11][12]. Our recent results further indicated that under cell culture conditions, EGCG (20 μM) underwent autoxidation and EGCG dimers were formed [9].…”

“…This can explain why it takes longer for P2 to reach a peak level than for theasinensin A and its quinone ( Figure 2B). This is the alternative mechanism that we proposed previously [11]. It is preferred over the mechanism in which EGCG • react with EGCG to form EGCG dimer radical and the EGCG dimer [11], in that case the formation of EGCG quinone would not far precede the formation of EGCG dimer.…”

“…The O 2 •− can then react with another molecule of EGCG for the propagation of the chain reaction of EGCG autoxidation. The involvement of O 2 •− in these reactions is supported by the observation that these reactions are inhibited by the presence of superoxide dismutase (SOD) [11]. EGCG quinone can react with EGCG to form EGCG dimer, such as theasinensin A.…”

N-Acetylcysteine enhances the lung cancer inhibitory effect of epigallocatechin-3-gallate and forms a new adduct

“…In the absence of SOD and catalase, M24.7 formed rapidly and then disappeared as a function of time, whereas in the presence of SOD (5 U/mL) and catalase (30 U/mL), M24.7 formed more slowly and was stable. This behavior was similar to what we and others have previously observed for EGCG under cell culture conditions [5,10].…”

“…These effects are diminished by inclusion of superoxide dismutase (SOD) which stablizes EGCG, and apparently prevents oxidative damage of EGFR [10]. Similarly, inclusion of SOD and/or catalase reduces the growth inhibitory and pro-apoptotic activity of EGCG in many in vitro systems.…”