Pro-oxidative activities and dose-response relationship of (-)-epigallocatechin-3-gallate in the inhibition of lung cancer cell growth: a comparative study in vivo and in vitro

Abstract

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. Nevertheless, the dose-response relationship of the inhibitory activity in vivo has not been systematically characterized. The present studies were conducted to address these issues, as well as the involvement of reactive oxygen species (ROS), in the inhibitory action of EGCG in vivo and in vitro. We characterized the inhibitory actions of EGCG against human lung cancer H1299 cells in culture and in xenograft tumors. The growth of tumors was dose dependently inhibited by EGCG at doses of 0.1, 0.3 and 0.5% in the diet. Tumor cell apoptosis and oxidative DNA damage, assessed by the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and phosphorylated histone 2A variant X (gamma-H2AX), were dose dependently increased by EGCG treatment. However, the levels of 8-OHdG and gamma-H2AX were not changed by the EGCG treatment in host organs. In culture, the growth of viable H1299 cells was dose dependently reduced by EGCG; the estimated concentration that causes 50% inhibition (IC(50)) (20 microM) was much higher than the IC(50) (0.15 microM) observed in vivo. The action of EGCG was mostly abolished by the presence of superoxide dismutase (SOD) and catalase, which decompose the ROS formed in the culture medium. Treatment with EGCG also caused the generation of intracellular ROS and mitochondrial ROS. Although EGCG is generally considered to be an antioxidant, the present study demonstrates the pro-oxidative activities of EGCG in vivo and in vitro in the described experimental system.

Fig. 1.

Inhibitory effects of EGCG treatments on the growth of H1299 lung cancer cell xenografts. (A) Mean tumor volume as a function of time. (B) Final tumor weight. The values shown are mean ± SE of 10 mice (20 tumors) per group. Different superscripts (a and b) indicate statistical difference among groups (by one-way analysis of variance; P < 0.05).

Fig. 2.

EGCG levels in the plasma (A), tumor (B), liver (C) and lung (D). The values are shown as mean ± SE of 10 mice. Statistical significance from control group by two-tailed t-test: *P < 0.05 and **P < 0.01.

Fig. 3.

Effects of EGCG treatments on oxidative stress, DNA damage repair and apoptosis in the xenograft tumors. Representative immunohistochemisty microphotographs and percentage for 8-OHdG-positive-stained cells (A) and γ-H2AX-positive-stained cells (B) and apoptotic index (C). Values in the bar graphs represent mean ± SE (n = 6 mice). Different superscripts (a, b, c and d) indicate statistical difference among groups (by one-way analysis of variance; P < 0.05).

Fig. 4.

Inhibition of cancer cells growth and induction of apoptosis by EGCG. (A) Morphological change of H1299 cells and percentage of viable cells for H1299, CL13, A549, H460 and HT29 (n = 3) after treatment with EGCG for 24 h in serum-free medium with (closed circles) or without (open circles) added SOD/catalase (CAT) (5 U/ml, 30 U/ml). (B) Annexin-V/propidium iodide (PI) costained cells after treatment with 50 μM EGCG for different time periods and (C) with 25 or 50 μM EGCG for 24 h in the presence or absence of SOD/CAT in serum-free medium. The values shown are mean ± SD.

Fig. 5.

Induction of intracellular ROS, mitochondrial ROS and mitochondrial membrane potential change by EGCG in H1299 cells. (A) Representative fluorescence images of intracellular ROS after induction for 6 h. (B) Time-dependent generation of intracellular ROS. (C) Intracellular ROS formation in the presence or absence of SOD/catalase (CAT) (5 U/ml, 30 U/ml) or NAC (2 mM) for 12 and 24 h. (D) Flow cytometry representative histogram and time-dependent generation of mitochondrial ROS with 25 or 50 μM of EGCG in the presence or absence of SOD/CAT. (E) Comparison of mitochondrial membrane potential change and mitochondrial ROS production with 25 or 50 μM of EGCG in the presence or absence of SOD/CAT for 24 h. The values shown are mean ± SD.

Fig. 6.

Effects of EGCG treatments on oxidative stress and DNA damage repair in the H1299 cell lines. The cells were treated with 5–50 μM EGCG for different time periods. (A) Representative microphotographs of 8-OHdG- and γ-H2AX-stained cells in the presence of 50 μM EGCG for 12 and 24 h. (B) Western blots for γ-H2AX with 5–50 μM EGCG in the presence or absence of SOD/catalase (CAT) (5 U/ml, 30 U/ml) for different time periods. The values shown are mean ± SD.