Synergistic inhibition of head and neck tumor growth by green tea (-)-epigallocatechin-3-gallate and EGFR tyrosine kinase inhibitor

Abstract

One of the mechanisms of the antitumor activity of green tea (-)-epigallocatechin-3-gallate (EGCG) is associated with its effect on epidermal growth factor receptor (EGFR)-mediated signaling transduction pathways. We investigated whether combining EGCG with the EGFR-tyrosine kinase inhibitor (EGFR-TKI) erlotinib may augment erlotinib-induced cell growth inhibition of squamous cell carcinoma of the head and neck (SCCHN) in a mouse xenograft model. In vitro studies with 5 head and neck cancer cell lines revealed that synergistic cell growth inhibition by the combination of EGCG and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, increased activation of caspases 9, 3 and PARP compared to the inhibition induced by EGCG or erlotinib alone. Erlotinib inhibited phosphorylation of EGFR, stabilizing EGFR at the plasma membrane, whereas EGCG induced EGFR internalization and ubiquitin-degradation, ultimately undermining EGFR signaling. The efficacy of the combination treatment was investigated with nude mice (n = 25) orally gavaged with vehicle control, EGCG, erlotinib or the combination at the same doses for 7 days, followed by subcutaneous injection with Tu212 cells. Animals were continuously administered the agents 5 days weekly for 7 weeks. The combined treatment resulted in significantly greater inhibition of tumor growth and delayed tumor progression as a result of increased apoptosis, decreased cell proliferation and reduced pEGFR and pAKT compared to the single agent treatment groups. Our results suggest a synergistic antitumor effect of a combined treatment with EGCG and erlotinib, and provide a promising regimen for future chemoprevention and treatment of SCCHN.

Figure 1

Effect of EGCG and erlotinib on growth of SCCHN cell lines. SCCHN cell lines Tu177, Tu212, 886LN, 38, SQCCY1 were treated with (a) EGCG (0–100 μM), or (b) erlotinib (0–40 μM) as single agents for 72 hr, as described in the “Materials and methods” section. SRB assay was performed to determine cell growth inhibition. (c) Combination Index derived by CalcuSyn software (see the “Materials and methods” section). Data from SRB assay are illustrated as fraction of affected cells (FA), i.e. drug-treated vs. untreated cells. A CI value of >1 is antagonism, equal to 1 is additivity and <1 is synergy.

Figure 2

Effect of EGCG and erlotinib on cell cycle and apoptosis. Tu177 and Tu212 cells were treated with EGCG (30 μM), erlotinib (0.5 μM) or their combination for 24 and 48 hr (for cell cycle), or 72 and 96 hr (for apoptosis). (a) Statistical analyses of effects on cell cycle in Tu177 and Tu212 cells. Left panel, symbols * and ** indicate a comparison of the erlotinib group with the control group (p = 0.002 for 24 hr, p = 0.005 for 48 hr) and the combination group with the control group (p = 0.03 for 24 hr, p = 0.009 for 48 hr) in Tu177 cells. Right panel, symbols * and ** indicate a comparison of the erlotinib group with the control group (p = 0.03 for 24 hr, p = 0.02 for 48 hr) and the combination group with the control group (p = 0.05 for 24 hr and p = 0.01 for 48 hr) in Tu212 cells. (b) Quantification of apoptosis in Tu177 and Tu212 cells. Left panel, symbols * and ** indicate a comparison of the combination group with all other groups at 72 and 96 hr, respectively, with p < 0.05 in Tu177 cells. Right panel, symbols * and ** indicate a comparison of the combination group with all other groups at 72 and 96 hr, respectively, with p < 0.05 in Tu212 cells.

Figure 3

Effect of EGCG and erlotinib on EGFR signaling. (a) Tu212 cells maintained in medium with 5% serum were treated with EGCG (30 μM), erlotinib (0.5 μM) or their combination for different time points. (b) Effect of SOD on the efficacy of EGCG. Tu212 cells were treated with EGCG (10 μM) with or without SOD (5 U/ml), erlotinib (0.5 μM) or their combination for 3 and 5 days. G3PDH or β-actin served as a loading control. Western blotting analysis was the representative of 3 independent experiments.

Figure 4

Short-term effect of EGCG and erlotinib on EGF-induced EGFR degradation. (a) Tu212 cells were serum-free starved overnight and then pre-exposed to 10 μM EGCG, 0.5 μM of erlotinib or the combination in the presence of SOD (5 U/ml) for 4 hr, followed by treatment with 100 ng/ml of EGF for 15 min, or pre-exposed to the indicated treatments for 7 hr and treated with EGF for the last 3 hr cells. Parallel studies of immunofluorescence staining of EGFR (a, ×1000) and Western blot analysis (b) were performed. Western blotting analysis and images were the representative of 3 independent experiments. For comparison, 2 membranes from each Western blot were exposed at the same time. (c) EGCG-induced ubiquitination of EGFR. Tu212 cells were serum-free starved overnight, then treated with 10 or 20 μM EGCG alone for 3 hr, 10 μM MG132 alone for 6 hr, EGCG combined with MG132 during the second 3 hr of MG132 treatment, 100 ng/ml of EGF alone for 15 min or 20 μM of EGCG followed with EGF for 15 min (left panel); EGF alone for 15 min, 0.5 μM erlotinib alone for 3 hr, erlotinib followed with EGF for 15 min or 20 μM of EGCG plus erlotinib followed with EGF for 15 min (right panel). All treatments were performed in the presence of SOD (5 U/ml). Ubiquitinated EGFR was detected by immunoprecipitating lysates (300 μg protein) with 4 μg of antibody against the N-terminus of EGFR (528), and then probing the membrane with an anti-ubiquitin monoclonal antibody. Mouse IgG was used as a control. The level of β-actin in lysate before immunoprecipitation indicates equal amount of protein. Experiments were performed independently 4 times.

Figure 4

Short-term effect of EGCG and erlotinib on EGF-induced EGFR degradation. (a) Tu212 cells were serum-free starved overnight and then pre-exposed to 10 μM EGCG, 0.5 μM of erlotinib or the combination in the presence of SOD (5 U/ml) for 4 hr, followed by treatment with 100 ng/ml of EGF for 15 min, or pre-exposed to the indicated treatments for 7 hr and treated with EGF for the last 3 hr cells. Parallel studies of immunofluorescence staining of EGFR (a, ×1000) and Western blot analysis (b) were performed. Western blotting analysis and images were the representative of 3 independent experiments. For comparison, 2 membranes from each Western blot were exposed at the same time. (c) EGCG-induced ubiquitination of EGFR. Tu212 cells were serum-free starved overnight, then treated with 10 or 20 μM EGCG alone for 3 hr, 10 μM MG132 alone for 6 hr, EGCG combined with MG132 during the second 3 hr of MG132 treatment, 100 ng/ml of EGF alone for 15 min or 20 μM of EGCG followed with EGF for 15 min (left panel); EGF alone for 15 min, 0.5 μM erlotinib alone for 3 hr, erlotinib followed with EGF for 15 min or 20 μM of EGCG plus erlotinib followed with EGF for 15 min (right panel). All treatments were performed in the presence of SOD (5 U/ml). Ubiquitinated EGFR was detected by immunoprecipitating lysates (300 μg protein) with 4 μg of antibody against the N-terminus of EGFR (528), and then probing the membrane with an anti-ubiquitin monoclonal antibody. Mouse IgG was used as a control. The level of β-actin in lysate before immunoprecipitation indicates equal amount of protein. Experiments were performed independently 4 times.

Figure 5

Effect of treatment with EGCG, erlotinib and their combination on SCCHN xenograft tumors. Four groups of animals were orally gavaged with control (1% Tween-80, n = 6), EGCG (125 mg/kg, n = 6), erlotinib (50 mg/kg, n = 6) or their combination (same doses, n = 7) for 7 days prior to a subcutaneous inoculation of 2 × 10⁶ Tu212 cells. The animals were continuously gavaged with the agents 5 days a week for a total of 7 weeks as described in the “Materials and methods” section. (a) Tumor volume was measured 3 times per week at indicated time points. Tumor growth was significantly inhibited in the combination group as compared to the control (p = 0.006), EGCG (p = 0.02) or erlotinib (p = 0.01) groups. (b) The Kaplan–Meier curves were recorded for the 4 treatment groups. The median times to reach a tumor size of 500 mm³ were 19.5 (control), 25 (EGCG) or 27 (erlotinib) days, and not reached at the time of termination (combined treatment). (c) Representative image of immunohistochemical staining of Ki-67 (×200) and quantification of the signal. Symbols * and ** illustrate comparisons between the combination group and the erlotinib group (p = 0.05) and between the combination group and the control group (p = 0.03), respectively. (d) Representative image of TUNEL assay (×400) and quantification of the signal. Symbol * illustrates comparison between the combination group and the control group (p = 0.04).

Figure 6

Effect of EGCG and erlotinib on the levels of phosphorylated EGFR and AKT in xenograft tumors. Proteins were extracted from each mouse xenograft. Protein (100 μg) for each sample was used for Western blot as described in the “Materials and methods” section. (a) Representative Western blot analysis of the levels of pEGFR and total EGFR protein in tumor tissues. (b) Quantification of pEGFR in all tumor tissues using densitometric image analyses. Symbols * and ** illustrate comparisons between the erlotinib group and the control group (p = 0.001) and between the combination group and the control group (p = 0.002), respectively. (c) Representative Western blot analysis of the levels of pAKT and total AKT protein in tumor tissues. (d) Quantification of pAKT in all tumor tissues using densitometric image analyses.