The green tea polyphenol EGCG potentiates the antiproliferative activity of c-Met and epidermal growth factor receptor inhibitors in non-small cell lung cancer cells

Abstract

Purpose: Activation of the c-Met and epidermal growth factor receptors (EGFR) promotes the growth and survival of non-small cell lung cancer (NSCLC). Specific receptor antagonists have shown efficacy in the clinic, but tumors often become resistant to these therapies. We investigated the ability of (-)-epigallocatechin-3-gallate (EGCG) to inhibit cell proliferation, and c-Met receptor and EGFR kinase activation in several NSCLC cell lines.

Experimental design: NSCLC cell lines with variable sensitivity to the EGFR antagonist erlotinib were studied. Cell growth was evaluated using proliferation and colony formation assays. Kinase activation was assessed via Western blot analysis. Experiments were conducted with EGCG, the EGFR antagonist erlotinib, and the c-Met inhibitor SU11274. The antagonists were also tested in a xenograft model using SCID mice.

Results: EGCG inhibited cell proliferation in erlotinib-sensitive and -resistant cell lines, including those with c-Met overexpression, and acquired resistance to erlotinib. The combination of erlotinib and EGCG resulted in greater inhibition of cell proliferation and colony formation than either agent alone. EGCG also completely inhibited ligand-induced c-Met phosphorylation and partially inhibited EGFR phosphorylation. The triple combination of EGCG/erlotinib/SU11274 resulted in a greater inhibition of proliferation than EGCG with erlotinib. Finally, the combination of EGCG and erlotinib significantly slowed the growth rate of H460 xenografts.

Conclusion: EGCG is a potent inhibitor of cell proliferation, independent of EGFR inhibition, in several NSCLC cell lines, including those resistant to both EGFR kinase inhibitors and those overexpressing c-Met. Therefore, EGCG might be a useful agent to study as an adjunct to other anticancer agents.

Fig 1

EGCG effects cell growth of NSCLC cells, and increases their sensitivity to erlotinib. Cell viability of H2122 (A), H358 (B) and H460 (C) non-small cell cancer cells in vitro in response to EGCG and erlotinib. Cells were cultured in 96-well plates in the presence of increasing concentrations of EGCG, erlotinib at 2 μM or combinations of EGCG plus erlotinib. Viability was assessed using a tetrazolium assay after allowing growth of cells for 72 h, and the change in the number of cells in treated wells were expressed as a percent relative to the changes in the number of cells in the control wells over 72 hours; normalized to 100. Negative numbers in the graph for the treated wells mean the number of cells decreased from the number of cells that were found in the control wells at the initiation of treatment (T=0). * P < 0.05 compared with the control. + P < 0.05 EGCG alone vs. EGCG as same dose plus erlotinib.

Fig 2

EGCG and erlotinib are more effective than either agent alone at inhibiting growth of colonies. Cells were cultured at low density and treated with EGCG, erlotinib or combinations every 48 h for 7 days. (A) Colonies were stained with crystal violet and then photographed. In (B), the crystal violet was extracted and assayed by spectrophotometry. EGCG was used at the concentrations noted in the figure and erlotinib was used at 2 μM. * P < 0.05 compared with FBS control. + P < 0.05 EGCG alone vs. EGCG as same dose plus erlotinib.

Fig 3

EGCG effects signaling in growth factor receptor signaling in H2111, H358 and H460 NSCLC cells. Cells were stimulated with either HGF at 33 ng/ml (A) or EGF at 100 ng/ ml (B). As noted, cells were pretreated with EGCG 10 μM to 40 μM for 4 hours (A and B) or 2 mM erlotinib for 1 hour (C). Cell lysates were collected and subjected to SDS-PAGE, western blotting and analyzed with phospho-specific antibodies to the c-Met receptor (pMet), Akt (p-Akt), Erk1/2 (pErk) and the EGFR (pEGFR). Tubulin was used as a control for loading.

Fig 4

EGCG effects cell proliferation and kinase activity in the H1993 c-Met overexpressing cell line and the H1975 T790M EGFR TKI resistant cell line. In (A) (H1993) & (C) (H1975), cells were cultured in the presence of EGCG 2.5, 5.0, 10, 20 and 40 μM, or erlotinib 2 μM or combinations as noted. Viability was assessed after 72 h using a tetrazolium based assay and the data were expressed as described in the legend to Figure 1. In (B), H1993 cells were pretreated with EGCG 10-40 μM and then left unstimulated or EGF or HGF was added. In (D), H1975 cells were pretreated with EGCG 10-40 μM and left unstimulated or stimulated with HGF at 33 ng/ml or EGF at 100 ng/ml as noted. Cells were also treated with 2 μM erlotinib prior to exposure to EGF. In (B), and (D), cell lysates were subjected to SDS-PAGE and western blotting with phospho-specific antibodies to the c-Met receptor (p-Met), Akt (p-Akt), Erk1/2 (p-Erk) and the EGFR (pEGFR). Tubulin was used as a control for loading. * P < 0.05 compared with FBS control. + P < 0.05 EGCG alone vs. EGCG as same dose plus erlotinib.

Fig 5

Combinations of EGCG, a c-Met inhibitor and erlotinib synergistically block proliferation of H460 cells. In (A), H460 cells were pretreated with SU11274 at varying doses as indicated prior to stimulation with HGF 33 ng/ml. Lysates were subjected to PAGE and Western blotting with a phosphospecific antibody to the c-Met receptor. Tubulin was uses as a control for loading. In (B), H460 cells were cultured in the presence of EGCG at 10 or 20 μM, erlotinib at 2μM, or SU11274 at 5 μM alone or in combination. Viability was assessed after 72 h using a tetrazolium based assay and the data were expressed as described in the legend to Figure 1. In (C), H1975 cells were cultured in the presence of erlotinib at 2 μM, SU11274 at 5 μM, EGCG at 20 μM or combinations as noted. Viability was again assessed as described in the legend to Figure 1. * P < 0.05 compared with FBS control.

Fig 6

Combinations of EGCG and erlotinib are effective a slowing the growth of H460 tumors. Two million H460 cells were injected subcutaneously in male SCID mice. After 3 days, the mice were gavaged with EGCG (15/mg/kg), erlotinib (10/mg/kg), both EGCG and erlotinib or with 2% Tween-80 control as indicated. In (A), tumor volumes were measured. Data is expressed as cubic mm volume. In (B), animals were sacrificed at day 22 and tumors were dissected and weighed. * P < 0.05 EGCG + erlotinib vs. control.