Squamous cell carcinoma of the head and neck (SCCHN) accounts for 3% of all cancers in the United States, with approximately 45,660 new cases and more than 11,210 expected deaths in 2007.1 Despite advances in conventional therapies, including surgery, radiation and chemotherapy, the overall survival rate for SCCHN has not significantly improved in the past 3 decades.2 Overexpression of epidermal growth factor receptor (EGFR) and its ligands TGF-α or EGF has been observed in 80–90% of SCCHN specimens3–6 and correlates with poor disease-free and overall survival, and increased risk of disease recurrence and metastasis.6, 7 Erlotinib (OSI-774 or Tarceva), an EGFR tyrosine kinase inhibitor (TKI), has shown strong antitumor and chemopreventive efficacies in a variety of cancer types including SCCHN through blocking EGFR-related signal transduction pathways.5, 8–10 Our previous studies showed that EGFR-TKI could inhibit tumor growth through blocking EGFR-related pathways.3, 11 However, the efficacy of monotherapy is severely limited by heterogeneity of the tumor cell population and redundant growth and survival pathways.
The development of preventive approaches using specific natural or synthetic chemical compounds (chemoprevention) has become highly desirable to reduce the incidence of SCCHN. Currently, several chemopreventive regimens of either single agents or combinations are being actively investigated preclinically and clinically in patients with premalignant lesions of the head and neck or SCCHN.11–16 The chemopreventive or antitumor effect of (−)-epigallocatechin-3-gallate (EGCG), the most abundant and most active phenolic constituent of green tea, has been extensively studied in chemically induced rodent carcinogenesis models and in several types of cancer cells in culture.17–21 EGCG was reported to inhibit tumor cell growth through suppressing phosphorylation of EGFR and other signaling mediators, such as Akt and mitogen-activated protein kinase, in breast, colon, cervical and SCCHN cancers.22–24 In light of these studies, we have tested a hypothesis that combined treatment with erlotinib and EGCG could synergistically inhibit tumor growth by targeting EGFR/AKT signaling pathways, depleting total EGFR and thus ultimately undermining EGFR signaling. Our current studies suggest that the combination of EGCG and erlotinib is a promising strategy for chemoprevention of SCCHN that warrants further investigation.
Material and methods
Five cell lines (Tu177, Tu212, 886LN, SQCCY1 and 38) were selected for our study to represent different SCCHN sites. Tu177 and Tu212 cell lines established from a laryngeal and hypopharyngeal tumor, respectively, were provided by Dr. Gary L. Clayman (University of Texas M.D. Anderson Cancer Center, Houston, TX). The 886LN cell line was derived from a lymph node metastasis of squamous cell carcinoma of the larynx. SQCCY1 and 38 cell lines derived from oral cavity and tonsil fossa, respectively, were obtained from Dr. Shi-Yong Sun (Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA). All cells were maintained in DMEM/F12 (1:1) medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and antibiotics (streptomycin, penicillin G and amphotericin B) in a 37°C, 5% CO2 humidified incubator. For 3 to 5 days of treatment, medium was changed on day 3.
EGCG (Sigma Chemical, St. Louis, MO) and erlotinib (Genentech, South San Francisco, CA) were dissolved in autoclaved water and dimethyl sulphoxide (DMSO), respectively, as stock solutions for in vitro studies. Since there is substantial precipitate in DMSO-dissolved erlotinib, we harvested the supernatant after spinning down the precipitate for use as a stock solution. The reagents were further diluted in DMEM/F12 medium immediately before use. The final concentration of DMSO was less than 0.1%. For in vivo studies, EGCG was dissolved in autoclaved water, while erlotinib was dissolved in 1% Tween-80 (Sigma Chemical). Superoxide dismutase (SOD) was purchased from Sigma Chemical.
Cell growth inhibition assay
To test the effects of single agent EGCG and erlotinib on cell growth of SCCHN, sulforhodamine B (SRB) cytotoxicity assays were adapted from Skehan et al.25 Cells maintained in medium with 5% FBS were seeded in 96-well plates at a density of 4,000 cells/well overnight prior to drug treatment. Afterwards, drugs were added as single agents in various concentrations (0–100 μM for EGCG, 0–40 μM for erlotinib), followed by incubation at 37°C and 5% CO2 for 72 hr. Cells were fixed for 1 hr with 10% cold trichloroacetic acid. Plates were washed 5 times in water, air-dried and then stained with 0.4% SRB for 10 min. After washing 4 times in 1% acetic acid and air-drying, bound SRB was dissolved in 10 mM unbuffered Tris base (pH 10.5). Plates were read in a microplate reader by measuring absorbance at 492 nm. The percent survival was then calculated based upon the absorbance values relative to untreated samples. The experiment was repeated 3 times.
Combination index assay
The interaction between EGCG and erlotinib was analyzed using the CalcuSyn software program (Biosoft, Ferguson, MO) which is based on the Chou and Talalay method.26 In brief, cells were seeded in 96-well plates at a density of 4,000 cells/well overnight prior to drug treatment. The next day, 1:2 serial dilutions of the drugs were prepared for EGCG, erlotinib and their combination based on their IC50, and added to the cells. After incubation for 72 hr at 37°C and 5% CO2, the plates were subjected to SRB assay. Data from the SRB assay were expressed as the fraction of cells with growth affected (FA) in drug-treated vs. untreated cells. The CI was calculated using CalcuSyn software. A CI value of >1 is defined as antagonism, equal to 1 as additivity and <1 as synergy. The experiment was repeated 3 times.
Flow cytometry analysis
Cell cycle arrest and apoptosis were analyzed in 2 cell lines, Tu212 and Tu177. Cells were treated with EGCG (30 μM), erlotinib (0.5 μM) or their combination (EGCG 30 μM + erlotinib 0.5 μM) for certain time points, then trypsinized and washed in cold 1× PBS. For cell cycle analysis, cells were fixed in 70% ethanol at 4°C for 2 hr, followed by a wash with 1× PBS. Cells were then stained with PI/RNase staining buffer (BD Pharmingen, San Diego, CA) for 15 min at room temperature. For Annexin V and 7-amino-actinomycin (7-AAD) binding assay, after trypsinizing and washing with cold PBS, the cells were resuspended in 1× Annexin binding buffer (BD PharMingen), and then stained with Annexin V-phycoerythrin (Annexin V-PE; BD PharMingen) and 7-AAD (BD PharMingen) for 15 min at room temperature. The stained samples for both cell cycle and apoptosis assays were measured using a fluorescence-activated cell sorting (FACS) caliber bench-top flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The data for cell cycle arrest were analyzed using Cell Quest software (Becton Dickinson, Franklin Lakes, NJ). FlowJo software (Tree Star, Ashland, OR) was used for apoptosis analysis. The experiments were repeated 3 times independently.
Tu212 cells were serum-starved overnight, then pre-exposed to 10 μM EGCG, 0.5 μM of erlotinib or the combination for 4 hr, followed by treatment with 100 ng/ml of EGF for 15 min, or cells were pre-exposed to EGCG for 7 hr and in the last 3 hr cells were treated with EGF for 3 hr.
Cells grown on coverslips were fixed with warm PHEMO buffer [68 mM PIPES, 25 mM HEPES (pH 6.9), 15 mM EGTA, 3 mM MgCl2, 10% (vol/vol) DMSO] containing 3.7% formaldehyde, 0.05% glutaraldehyde and 0.5% Triton X-100 for 10 min and incubated with primary antibody and fluor-conjugated secondary antibodies. Images were taken with Zeiss LSM510 META confocal microscope.
Nude mouse xenograft model
The animal experiments were approved by the Animal Care and Use Committee of Emory University. Twenty-five nude mice (athymic nu/nu, Taconic, NY), aged 4 to 6 weeks (about 20 g weight), were randomly divided into 4 groups. Each group was orally gavaged for 7 days with vehicle control (1% Tween 80, n = 6), EGCG (125 mg/kg, n = 6), erlotinib (50 mg/kg, n = 6) or the combination (n = 7) of EGCG (125 mg/kg) and erlotinib (50 mg/kg) using a blunt tipped 20G 1.1/2 needle (Popper and Sons, New Hyde Park, NY) before inoculation of 2 × 106 Tu212 cells by s.c. injection into the right flank. The animals were continuously administered the agents 5 days a week. The tumor size was measured 3 times a week. The tumor volume was calculated using the formula: V = π/6 × larger diameter × (smaller diameter)2, as reported previously.27 Growth curves were plotted using average tumor size within each experimental group at the set time points. The median time from the tumor cell injection until the average tumor size (V) reached 500 mm3 in each group was evaluated by the Kaplan–Meier curve. The whole group of mice was sacrificed once the size of any tumor in that group reached 2 cm in diameter.
Western blot analysis
Whole cell lysates were extracted from drug-treated cells using lysis buffer. Fifty microgram protein was separated on 8–15% SDS-PAGE, transferred onto a PVDF membrane (Millipore, Bedford, MA) and immunostained with specific antibodies including rabbit anti-pEGFR and EGFR (Santa Cruz, CA), rabbit anti-pAKT and AKT (Cell Singling, Beverly, MA), mouse anti-caspase 3 (Imgenex, San Diego, CA), mouse anti-caspase 8, anti-caspase 9 and anti-PARP (Cell Singling, Beverly, MA). Rabbit anti-G3PDH or mouse anti-β-actin antibody (Trevigen, Gaithersburg, MD) was used as a sample loading control. Immunostained protein bands were detected with an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). The experiments were repeated 3 times.
Protein (100 μg) from each sample of nude mouse xenograft tissue was subjected to immunoblotting analysis using corresponding antibodies. The intensity of Western blotting was analyzed by densitometric image analysis system (UVP BioImaging System, Upland, CA).
For immunoprecipitation studies, about 300 μg of lysate was precleared with recombinant protein G-agarose (GIBCOBRL, Carlsbad, CA) for 4 hr at 4°C, then incubated with 4 μg of antibody against the N-terminus of EGFR (528, SC-120, Santa Cruz, CA) or nonimmunized mouse IgG precomplexed with protein G-agarose overnight. The membrane was probed with anti-ubiquitin antibody (Cell Signaling, Beverly, MA).
Immunohistochemistry and TUNEL assay
Immunohistochemistry analysis for Ki-67 staining on formalin-fixed, paraffin-embedded nude mouse xenograft tissue was performed using the R.T.U. Vectastain kit following the standard manufacture's protocol (Vector Laboratories, Burlingame, CA). Tissue sections were incubated with mouse anti-human Ki-67 (prediluted; Biomeda, Foster, CA) overnight at 4°C. The slides were stained with 3,3′-diaminobenzidine (DAB) (Sigma Chemical) and counterstained with hematoxylin (Vector Laboratories). TUNEL assay was performed by immunofluorescence using the same specimens as above following the procedure provided by the manufacturer (Promega, Madison, WI). The slides were counterstained with DAPI (Vector Laboratories).
Both Ki-67 staining and fluorescent signals from the TUNEL assay were visualized under Zeiss LSM 510 confocal microscopy. Their images were analyzed by MetaMorph software (Universal Imaging, Downingtown, PA) to measure the intensity or count the positive cells. Ten areas were randomly selected from each slide for measurement. For Ki-67 staining, we measured the intensity of positive cells by counting the absolute number of pixels. For TUNEL assay, we counted the total cell number and the positive cell number in the same area; the result was presented as the positive cell number per 1,000 total cells.
The two-tailed and paired t tests were used for the cell cycle and apoptosis assays. The two-tailed unpaired t test was used for analyzing the expression level of pEGFR and pAKT in xenograft tissue. The Wilcoxon rank-sum test was applied to evaluate the significance of differences in tissue necrosis, Ki-67 expression and TUNEL assay. The method of repeated measures ANOVA with between-subject factors was used to evaluate the significance of tumor cell growth inhibition among each group. Kaplan–Meier curves and log-rank test were employed for tumor progression analysis.
Growth of SCCHN cells
To study the sensitivity of SCCHN to EGCG and erlotinib, we initially examined 5 SCCHN cell lines (Tu212, Tu177, 886LN, SQCCY1 and 38), of which Tu212, Tu177, 886LN express comparable amounts of total EGFR protein and activated EGFR at basal level. Tu212 and Tu177 are sensitive to EGFR-TKI inhibition3; SQCCY1 is sensitive to apoptosis induced by inhibition of the AKT pathway28; and 38 is a chemoresistant cell line.29 Cells were treated with EGCG (0–100 μM) or erlotinib (0–40 μM) for 72 hr. A cell growth inhibition assay (SRB) showed that both single agent EGCG and erlotinib inhibited growth of the 5 SCCHN cell lines in a dose-dependent manner (Figs. 1a and 1b). The effect of combined treatment with EGCG and erlotinib on growth inhibition of SCCHN cells was determined by combination index (CI) assay. The concentrations were based on IC50 derived from the growth inhibition assay for single agents with a series of 2-fold reduction or escalation. The same concentration used for the single drug was also applied for the combination. The CI indicated that the two-drug combination of EGCG and erlotinib synergistically inhibited cell growth in 4 of 5 tested SCCHN cell lines (Fig. 1c). A synergistic effect on cell growth inhibition was also observed at relatively high concentrations in 886LN cells. Therefore, single doses of erlotinib and EGCG at their IC50s were applied for the remaining in vitro studies.
Cell cycle progression and induction of apoptosis
The effects of the two-drug combination on cell cycle progression were investigated in 2 representative SCCHN cell lines, Tu212 and Tu177. Cells were treated for 24 and 48 hr with 30 μM EGCG and 0.5 μM erlotinib (Fig. 2a). Compared to the control group, erlotinib alone induced significant cell cycle arrest at G0/G1 phase in both Tu212 and Tu177 cells as early as 24 hr after treatment. EGCG alone exerted no effect on cell cycle arrest. The combined treatment induced additive cell cycle arrest compared to that induced by EGCG or erlotinib alone in either cell line. The two-drug combination did not affect cell cycle changes at either S or G2/M phase. The apoptotic effect of the combination treatment was examined by treating Tu212 and Tu177 cells for 72 and 96 hr. Single agent EGCG or erlotinib induced moderate apoptosis, but the two-drug combination resulted in time-dependent significantly greater apoptotic cell death (Fig. 2b, p < 0.05).
Targeting phosphorylation of EGFR and AKT pathways
To elucidate the molecular mechanisms of EGCG and erlotinib treatment in SCCHN, we examined whether the drugs could modulate pEGFR and pAKT levels. Tu212 cells were treated with EGCG (30 μM), erlotinib (0.5 μM) or their combination for 30 min, 6, 24 and 72 hr. Erlotinib alone inhibited the phosphorylation of both pEGFR and pAKT after 30 min and retained this effect until 72 hr. EGCG alone clearly suppressed pAKT expression but did not substantially alter pEGFR levels until 72 hr. However, the combination of EGCG and erlotinib further downregulated pAKT after 30 min and pEGFR after 72 hr (Fig. 3a).
A study by Hou et al. suggested that SOD can stabilize EGCG in vitro by blocking auto-oxidation, resulting in greater inhibition of cell growth by EGCG.30 Therefore, we examined the effect of SOD on the efficacy of EGCG and its synergy with erlotinib in inducing apoptosis. Tu212 cells were treated with EGCG, erlotinib or their combination for 3 and 5 days in the presence or absence of SOD (5 U/ml). Figure 3b shows that (i) inhibition of pEGFR, pERK and pAKT by erlotinib was associated with marked increases of cleaved caspase 9, PARP and caspase 3; (ii) in the presence of SOD, EGCG induced greater reduction of pEGFR on day 3 and total EGFR on day 5 than that without SOD; (iii) the combination treatment induced substantially greater reduction of pEGFR, pERK, total EGFR and AKT levels associated with greater increases in activation of caspase 3 than the treatment with the single agents. This effect was greater in the presence vs. the absence of SOD and became more pronounced by day 5 relative to day 3. We did not observe noticeable changes in the activation of caspase 8. Similar results were obtained with other SCCHN cell lines (data not shown).
Depletion of EGFR protein
To further explore the mechanism by which combination treatment affects EGFR and other cellular signaling pathways, we tested a hypothesis that erlotinib inhibits phosphorylation of EGFR, immobilizing it at the plasma membrane, as seen in SCCHN,31 while EGCG enhances EGFR internalization and degradation; thus the combination of EGCG and erlotinib leads to the ultimate depletion of total EGFR protein. We performed parallel immunofluorescence (Fig. 4a) and Western blot analyses (Fig. 4b) using Tu212 cells in the presence or absence of EGF to examine whether EGCG affects EGF-induced EGFR activation, internalization and degradation.32, 33
Immunofluorescence staining of EGFR (Fig. 4a) shows that at the basal level, EGCG alone induced EGFR internalization, shown as diffusive cytoplasmic staining which became more pronounced by 7 hr of treatment. While erlotinib alone stabilized EGFR at the membrane, it did not prevent EGFR internalization induced by EGCG when in combination treatment for 7 hr. EGFR was internalized when cells were briefly exposed to EGF; clusters of weak staining in the cytoplasm were detected after 3 hr. In the presence of EGF, internalization of EGFR was detected in cells treated with EGCG, whereas erlotinib consistently prevented EGFR internalization even after 3 hr of exposure, as shown by the membranous staining of EGFR. However, when erlotinib was combined with EGCG, most EGFR remained in the cytoplasm with a markedly reduced intensity by 3 hr of treatment compared to the level at 15 min, suggesting that EGFR fails to recycle back to plasma membrane and may undergo degradation.
Western blot analysis (Fig. 4b) revealed that at basal level, EGCG alone or in combination with erlotinib reduced pEGFR, pERK, pAKT and total EGFR protein levels. Similarly, erlotinib inhibited pEGFR, pERK and pAKT but not total EGFR levels. EGF induced intense activation of pEGFR (tyr1173, tyr 1045) and pERK. EGF also induced significant downregulation of EGFR to undetectable levels after 3 hr of treatment. EGCG had no effect on activation of EGFR (tyr 1173 and tyr1045), pERK and pAKT by EGF, but specifically augmented ligand-induced downregulation of total EGFR protein, whereas erlotinib substantially abrogated the activation of pEGFR, pERK and pAKT by EGF without altering the total levels of EGFR protein. Erlotinib in combination with EGCG induced greater inhibition of pEGFR (tyr1173 and tyr1045), pERK and pAKT and greater downregulation of EGFR protein than erlotinib alone.
Ubiquitin-dependent degradation of EGFR
We consistently observed that EGCG induced downregulation of EGFR protein expression, either in short-term serum-free conditions (Fig. 4b) or in long-term culture with serum (Fig. 3b) in Tu212 and other cell lines (data not shown). The presence of SOD in all treatments should prevent loss of EGFR by auto-oxidation. Hence, we asked whether EGCG could induce ubiquitin-dependent degradation of EGFR. Immunoprecipitation revealed that with equal amounts of EGFR precipitated from the lysate, cells treated with EGCG displayed a dose-dependent increase in ubiquitinated EGFR compared to the control (Fig. 4c). When proteasomal activity was inhibited by MG132, a specific proteasome inhibitor, a dose-dependent increase in ubiquitinated EGFR was detected in cells treated with EGCG. Furthermore, ubiquitinated EGFR induced by EGF was augmented in the presence of EGCG, or EGCG with erlotinib compared to the level suppressed by erlotinib alone. Western blot analysis of the lysates for immunoprecipitation revealed that, consistent with the observations shown in Figure 4b, EGCG alone indeed downregulated EGFR and reduced protein levels even further when combined with EGF. In the presence of MG132, total EGFR levels appeared to be increased by EGCG compared to that by MG132 alone.
Growth of mouse xenograft tumors
The antitumor efficacy of combined treatment with EGCG and erlotinib was determined in xenograft mice bearing Tu212 cells, a known tumorigenic cell line. We pretreated mice with vehicle control, EGCG alone, erlotinib alone or their combination for 7 days to imitate an intrinsically preventive environment in the mouse body before injection of Tu212 tumor cells. The animals were continuously given the agents after the tumor cell injection. The tumor growth data indicated that, although EGCG and erlotinib as single agents moderately inhibited tumor growth, no statistical significance was reached compared to the control (p = 0.14, p= 0.07, respectively). However, the combination of EGCG and erlotinib significantly suppressed tumor growth compared to the control (p = 0.006), the EGCG alone treated (p = 0.02) or the erlotinib alone treated group (p = 0.01) (Fig. 5a). Mice treated with EGCG and two-drug combination were observed to be more energetic and active than those in the other 2 groups.
Kaplan–Meier curves revealed that the median times to reach a tumor size of 500 mm3 were 19.5, 25 and 27 days, and not reached at the time of termination for the control, EGCG alone, erlotinib alone and the combined treatment, respectively (Fig. 5b). Log-rank tests showed that this time delay induced by the combined treatment was statistically significant compared to control (p = 0.005), EGCG alone (p = 0.001) or erlotinib alone (p = 0.03). There is a pattern of time delay for the tumor volume to reach 500 mm3 in both the EGCG and erlotinib treatment groups relative to control, but this is not statistically significant, p = 0.5 and p = 0.1, respectively.
Ki-67 expression and apoptosis in xenograft tumors
Expression of Ki-67 and TUNEL assays revealed that the combination treatment resulted in greater inhibition of proliferation and in induction of apoptosis in the xenograft tumors (Fig. 5c). There were statistically significant differences in mean Ki-67 expression between the combination and the control groups (p = 0.03) and between the combination group and the erlotinib group (p = 0.05). Induction of apoptosis by the combination treatment was significantly greater than that by the control group (p = 0.04).
Phosphorylated EGFR and AKT in xenograft tumors
Protein extracted from the xenograft tumor tissues was examined to evaluate whether the inhibition of pEGFR and pAKT by treatment with EGCG and erlotinib in vitro is also seen in vivo. We found that erlotinib alone, but not EGCG alone, significantly suppressed pEGFR levels compared to the control treatment (p = 0.001), and the combined treatment yielded significantly lower mean pEGFR levels than that in the control group (p = .002, Figs. 6a and 6b). Both EGCG and erlotinib moderately inhibited pAKT, the combined treatment further reduced its levels but this was not statistically significant (Figs. 6c and 6d).
Overexpression of EGFR is observed in 80–90% of SCCHN cases studied.3–6 Thus, EGFR-TKIs such as erlotinib offer promising therapies. The specific antitumor activity of EGCG on EGFR signaling pathways22–24 suggested its potential as a less-toxic, inexpensive therapy when combined with EGFR-TKIs, based on the premise that the combination treatment could generate synergistic antitumor potency compared to the limited therapeutic value of many monotherapies. Indeed, we have demonstrated that EGCG synergizes with erlotinib in suppressing tumor growth by targeting the common EGFR/AKT signaling pathways, internalizing EGFR and subsequently inducing EGFR degradation. To the best of our knowledge, this is the first report that combined treatment with EGCG and erlotinib synergistically inhibits SCCHN tumor growth both in vitro and in vivo.
In vitro studies show that the combination of EGCG with erlotinib inhibited cell growth in a dose-dependent manner, exerting greater potency than either single agent alone in 4 of 5 SCCHN cell lines. The combination treatment not only synergistically inhibited cell growth of erlotinib-sensitive cell lines such as Tu212, Tu177, but also markedly inhibited growth of EGFR-TKI-insensitive 886LN cells3 and a chemoresistant cell line 38.29 SQCCY1, an AKT pathway inhibition-sensitive cell line exhibited a similar pattern of growth inhibition to that in EGFR-TKI-sensitive cell lines such as Tu212 and Tu177. Numerous studies suggest that both EGCG and erlotinib as single agents can induce cell cycle arrest at G0/G1 phase.24, 34–38 In the current studies, during the early time period of treatment (24–48 hr), cell cycle arrest induced by erlotinib was accompanied by the marked suppression of pEGFR, pERK and pAKT. Even though EGCG alone did not induce significant cell cycle arrest, the moderate inhibition of EGFR downstream signaling pathways when combined with erlotinib did yield a significant additive effect on cell cycle arrest at G0/G1 phase. Sequential treatment with EGCG alone followed by erlotinib or vice versa failed to induce significant growth inhibition compared to the combination treatment (data not shown). These results suggest that the synergistic effect of the combined treatment on cell growth inhibition in SCCHN is at least partially attributed to induction of cell cycle arrest, and is dependent upon the concomitant presence of both agents.
EGCG and EGFR-TKIs as single agents can induce apoptosis in a variety of cancer types, including SCCHN.3, 21, 38–40 In the current study, when cells were treated with erlotinib for more than 3 days, the inhibition failed to provoke significant apoptosis, in spite of greater inhibition of pEGFR, pERK and pAKT than that by EGCG (Figs. 2b and 3b). However, the additive effect on inhibition of pEGFR, pERK and pAKT signaling generated by the combined treatment was accompanied by drastically augmented induction of apoptosis by 72 and 96 hr (Fig. 2b). The further activation of caspases 9, 3 and PARP by the combination treatment, with no appreciable changes in caspase 8, suggests that the synergy in induction of apoptosis could be mediated mainly through intrinsic apoptosis pathways. It has been reported that EGCG exhibits greater inhibition of protein tyrosine kinase activities of EGFR, platelet derived growth factor receptor and fibroblast growth factor receptor than the nonreceptor type protein tyrosine kinases, with the strongest inhibition on EGFR.41 In addition, EGCG inhibits the EGFR downstream signaling cascade by directly inhibiting ERK and AKT kinase activities.23 Consistent with these observations, treatment with EGCG alone inhibited pAKT even before EGCG-induced downregulation of total EGFR protein was observed by 5 days of treatment, indicating that EGCG may directly affect AKT kinase activity. Compared to the effect of EGCG, erlotinib substantially inhibited EGF-induced pEGFR, pERK and pAKT. The combination treatment yielded synergistic inhibition of pEGFR, pERK and pAKT, associated with marked increases in apoptosis.
The significant downregulation of EGFR by EGCG alone and in combination with erlotinib suggests that a reduction in total EGFR protein levels could be one mechanism underlying the synergistic inhibition of EGFR signaling by the combined treatment. By performing parallel immunofluorescence and Western blot analyses, we demonstrated that erlotinib inhibited the EGF-induced signaling cascade, thereby immobilizing EGFR at the membrane, in agreement with the findings by Thelemann et al.31 This inhibition was also effective under basal nonstimulating conditions. On the other hand, EGCG induced internalization of EGFR in the absence of activated EGFR (tyr 1173) and pEGFR (tyr1045), a critical tyrosine phosphorylation site for EGF-induced EGFR internalization.42 The rapid degradation of EGFR induced by EGCG either alone or in the presence of EGF suggests that EGFR downregulation is unlikely a consequence of transcriptional regulation. Further studies revealed that the EGFR internalized by EGCG underwent ubiquitin-dependent degradation. Treatment with the proteasomal inhibitor MG132 and EGCG induced dose-dependent increases in ubiquitinated EGFR along with accumulated total EGFR protein (Fig. 4c), indicating that EGCG may not affect proteasomal activity but rather enhance ubiquitin-conjugating enzyme activity. Such novel findings remain to be investigated. A longer treatment time with the combined agents may be required to deplete EGFR under serum culture conditions compared to serum-free conditions, as observed by day 5. Most importantly, the markedly diminished total EGFR level induced by the combined treatment was associated with abrogated pEGFR, pERK and pAKT signaling and greater increases in apoptosis by day 5 than those observed by day 3.
There are several implications for the specific action of EGCG on EGFR protein. First, regardless of EGFR activation, internalization of membrane EGFR may disturb specific EGFR signaling destined to various cellular compartments, such as the plasma membrane, mitochondria, endoplasmic reticulum and nucleus.43 Second, consistent with findings using black tea extract,44 upon EGFR internalization by EGCG, its subsequent ubiquitin-dependent degradation may limit the amount of EGFR recycling back to the membrane, thus diminishing EGFR signaling strength and duration. Studies of EGF binding have shown that pretreatment with EGCG induced a marked inhibition of EGF binding to A431 cells41 or to HT29 cells.45 We have observed by flow cytometry that EGCG reduced both the level of cell surface EGFR and the amount recycling back to the membrane (data not shown). Clinical studies have indicated that dependence on EGFR signaling for cell growth and survival, increased expression of mutant and/or wild-type EGFR or gene copies of EGFR could be molecular determinants for the efficacy of EGFR-targeting therapy.46 The current study indicates that endocytosis and turnover rate of EGFR could also be molecular determinants. Third, EGFR-TKI inhibits phosphorylation by competing with ATP. Thus a certain fraction of phosphorylated EGFR escaped from TKI would undergo endocytosis and eventually recycle back to the plasma membrane. Stabilization of EGFR at the membrane by erlotinib may allow EGCG to better capture either phosphorylated or inactivated EGFR, thus resulting in suppressed EGFR signaling. A recent report that inhibition of EGFR activation by EGCG is mediated by altering lipid order, thus preventing EGF-induced EGFR from dimerization in HT29 colon cancer cells, further strengthens our findings.45 Finally, depletion of plasma membrane EGFR is an important mechanism underlying the antitumor activity of antibodies against EGFR such as cetuximab and for gemcitabine-mediated cytotoxicity in SCCHN cell lines.47 The mechanisms by which EGCG indirectly or directly interacts with EGFR and whether ubiquitin-conjugating enzymes are affected by EGCG demand further investigation.
The stability of EGCG is always a major concern in an in vitro study.21, 30 EGCG dimers and other oxidative products such as hydrogen peroxide are formed, making EGCG unstable with a half-life of less than 30 min in some culture conditions,30 thus demanding pretreatment. Consistent with the study by Hou et al.,30 the presence of SOD enhanced efficacy of EGCG in inducing apoptosis and we did not observe marked degradation of EGFR by EGCG alone or in combination with erlotinib until day 5 (Fig. 3b). In the presence of SOD, degradation of EGFR induced by EGCG was mediated through ubiquitin-dependent degradation and unlikely due to auto-oxidative effect of EGCG.
The significance of the combined EGCG and erlotinib treatment on tumor growth was examined using a chemoprevention xenograft mouse model. Nude mice were pretreated with control, erlotinib, EGCG or the combination 1 week before the injection of Tu212 cells. Our in vitro studies indicated the importance of pretreatment in creating a unique environment in the body of nude mice to prevent tumor cell initial intake and progression. Consistent with the in vitro studies, compared to the control or single agent treatment groups, substantially diminished tumor growth in the combined treatment group resulted from greater growth inhibition (Ki67) and apoptosis (TUNEL) mediated by significant suppression of pEGFR and pAKT pathways.
EGCG exhibited a synergistic antitumor activity when combined with an EGFR-TKI, erlotinib, with no significant side effects in our animal experiment. In addition to targeting the common EGFR downstream signaling pathways, the current study has suggested a novel mechanism by which the combination of erlotinib and EGCG results in depletion of membrane EGFR and ultimately decreases both total and activated EGFR levels. In conclusion, our studies suggest that EGCG represents a novel chemopreventive therapeutic agent for SCCHN in synergizing with EGFR-TKI.