The Tca8113 and TSCCa human tongue squamous cell carcinoma cell lines were purchased from the China Center for Type Culture Collection (Wuhan University) and cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics. For transfection experiments, the Lipofectamine™ 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer’s instructions.

EGCG was obtained from Sigma (St. Louis, MO, USA), and Myr-Akt1 was purchased from Addgene (Cambridge, MA, USA). Wortmannin, PD98059, anti-p-EGFR (Tyr1068), anti-Akt (pan), anti-Akt1, anti-p-Akt (S473), anti-ERK1/2, anti-p-ERK1/2 (Thr202/Tyr204), anti-HK2, anti-cleaved-PARP, anti-caspase3 and anti-α-tubulin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-β-actin, anti-rabbit IgG-HRP and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-VDAC was purchased from Abcam (Cambridge, UK).

Cells (5×10⁵) were seeded in 6-well plates. After incubation for 4 h, the culture medium was discarded and the cells were incubated in fresh medium for another 8 h. Glucose and lactate levels were measured using the Automatic biochemical analyzer (AU680; Beckman Coulter International, Brea, CA, USA) at the Clinical Biochemical Laboratory of Xiangya Hospital (Changsha, China). The relative glucose consumption and lactate production rates were normalized by the protein concentration of samples.

Cells were harvested by trypsinization and pelleted by centrifugation. Cell pellets were lysed in Nonidet P-40 cell lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40 and protease inhibitor mixture). Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Proteins were separated by SDS-PAGE and electrically transferred to a polyvinylidenedifluoride membrane (Millipore, Billerica, MA, USA). After blocking in 5% non-fat dry milk in TBS, the membranes were hybridized to specific primary antibodies overnight at 4°C, washed three times with TBS Tween-20, and then incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were then washed three times in TBS Tween-20 at room temperature. The protein bands were visualized using ECL chemiluminescence reagents (Pierce Chemical Co., Rockford, IL, USA) according to the manufacturer’s instructions.

Following EGCG treatments, ~5×10⁶ cells from a 10-cm plate were harvested by trypsinization and centrifuged at 800 rpm for 5 min at 4°C. The cell pellets were washed once with ice-cold PBS and then resuspended in three volumes of isolation buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM dithiothreitol, 10 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin and 10 mM aprotinin) in 250 mM sucrose. After chilling on ice for 3 min, the cells were disrupted by 60 strokes of a glass homogenizer. The homogenate was centrifuged once at 2,000 rpm at 4°C for 10 min to remove unbroken cells and nuclei. The mitochondria-enriched fraction (supernatant) was then pelleted by centrifugation at 13,000 rpm for 30 min. The pellets were lysed in RIPA buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl], and analyzed by western blotting.

To assess the anchorage-independent growth, tongue carcinoma cells were suspended (8×10³ cells/ml) in 1 ml of 0.3% agar with Eagle’s basal medium containing 10% FBS, 1% antibiotics, and different concentrations of EGCG (0, 20, 40 and 80 μM/l) overlaid in 6-well plates containing a 0.6% agar base. The cultures were maintained in a 37°C, 5% CO₂ incubator for 1–2 weeks, and then colonies were counted under a microscope using the Image-Pro Plus software program (Media Cybernetics, Silver Spring, MD, USA).

Statistical analyses were performed using the SPSS software (version 13.0). The experiments were performed in triplicate. Quantitative data are presented as means ± standard deviation. Significant differences between two groups were assessed by a two-tailed Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

Anchorage-independent growth is one of the malignant phenotypes of tumor cells and is considered to be one of the most accurate and stringent in vitro assays for detecting the malignant transformation of cells. Therefore, we first investigated the effect of EGCG on the anchorage-independent growth of human tongue carcinoma cells. Our results demonstrated that although EGCG had little effect on colony formation at 20 μM, the inhibition rate reached >60% after Tca8113 cells exposure to EGCG at the concentration of 40 μM. More importantly, 80 μM of EGCG almost blocked the colony growth in soft agar (Fig. 1A). We also found that EGCG had a similar effect on the suppression of the anchorage-independent growth of TSCCa cells in soft agar (Fig. 1B). These data indicated that EGCG inhibits anchorage-independent growth of human tongue carcinoma cells in a dose-dependent manner.

Elevated glycolysis is considered to be an emerging hallmark of human cancer. HK2, a rate-limiting enzyme of glycolysis, is upregulated in various cancers (31). Thus, in the present study, we investigated whether EGCG-mediated antitumor activity is associated with tongue carcinoma cell glycometabolism. Immunoblot analysis indicated that the HK2 protein level decreased in response to EGCG treatment in Tca8113 cells (Fig. 2A, left) and a similar result was also observed in TSCCa cells (Fig. 2B, left). To assess the effect of EGCG on glycolysis, we examined the level of glucose consumption and lactate production in the two human tongue carcinoma cells. Consistent with the results of the immunoblot analysis, EGCG dose-dependently inhibited the consumption of glucose (Fig. 2A and B, middle) and production of lactate (Fig. 2A and B, right) in the Tca8113 and TSCCa cells. These results suggested that EGCG-mediated tumor suppression is partially dependent on glycolysis inhibition, and HK2 is involved in this process.

Findings of recent studies demonstrated that the Akt signaling pathway is a hub in the regulation of cancer metabolism (36,37). As an Akt upstream receptor tyrosine kinase, the oncoprotein EGFR is always overexpressed in human tongue carcinoma (38). The results showed that EGCG strongly inhibited EGF-induced EGFR activation and EGCG also significantly inhibited the phosphorylation of EGFR downstream kinases Akt and ERK1/2. Furthermore, we found that the protein levels of HK2 were inhibited following EGCG treatment (Fig. 3A). To determine the connection between ERKs/Akt and HK2 suppression in the presence of EGCG, we used PD98059, a specific inhibitor of the ERKs pathway, to treat Tca8113 and TSCCa cells and examined whether HK2 expression would be affected by the inhibition of the ERKs signaling pathway in these cells. Results indicated that treatment with PD98059 markedly decreased the phosphorylation level of ERKs, however, there was no obvious effect on the expression of HK2 (Fig. 3B). By contrast, wortmannin, a specific inhibitor of the Akt pathway, not only substantially inhibited Akt activity, but also strongly suppressed HK2 expression in these cells (Fig. 3C). These results indicated that Akt, but not ERKs, was a key kinase of the EGCG-mediated downregulation of HK2.

Based on the previous data, in order to confirm that the regulation of glycolysis by EGCG in human tongue carcinoma cells is dependent on Akt activity, we transfected constitutively activated Akt1 (Myr-Akt1) into Tca883 and TSCCa cells. The results indicated that 80 μM EGCG significantly decreased the expression of HK2 in Tca8113 and TSCCa cells. However, Myr-Akt1 transfection markedly upregulated HK2 expression in these EGCG-treated cells as expected (Fig. 4A and B, left). Moreover, Myr-Akt1 rescued over 70 and 60% of the deficient glucose uptake (Fig. 4A and B, middle) and lactate production (Fig. 4A and B, right) in EGCG-treated Tca8113 and TSCCa cells, respectively. These results suggested that EGCG regulates glycolysis in human tongue carcinoma cells, and these biochemical processes are partly mediated by Akt activation.

The HK2 protein is crucial in cell proliferation as well as apoptosis resistance. In cancer cells, HK2 overexpression is associated with hyperactivated glycolysis and upregulated cell growth. More importantly, for HK2 to exert its anti-apoptotic function, HK2 translocates to the mitochondrial outer membrane and interacts with the voltage-dependent anion channel (VDAC) to block the release of cytochrome c, eventually inhibiting the apoptotic pathway (39–42). In order to assess the influence of EGCG on the subcellular localization of HK2, mitochondrial fractions from Tca8113 and TSCCa cells, with or without 80 μM EGCG treatment, were extracted and analyzed by immunoblotting with antibodies to detect HK2, α-tubulin and VDAC. The results indicated that the translocation of HK2 in the mitochondrial outer membrane fraction was decreased in the Tca8113 and TSCCa cells following EGCG treatment (Fig. 5A). Furthermore, we evaluated the apoptotic signaling pathway by immunoblotting to examine the cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) expression after EGCG treatment. The results demonstrated that the levels of cleaved-caspase-3 and cleaved-PARP in Tca8113 and TSCCa cells were markedly increased in response to EGCG treatment (Fig. 5B). Based on these data, we suggested that the EGCG-mediated downregulation of HK2 inhibits anti-apoptotic effects of human tongue carcinoma cells.