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Introduction

An irreversible non-enzymatic reaction between carbohydrates and proteins produce advanced glycation end products (AGEs) (1,2). The accumulation of AGEs increases with age (3,4) and is typically higher in patients with diabetes mellitus (DM) (5). AGEs have been revealed to be a risk factor for complications of DM (6–9) and an important toxicity moiety for neuronal cells in Alzheimer's disease (10–13). Previous studies have demonstrated that cancer malignancy can be promoted by AGEs (14–16). Furthermore, the migration of oral cancer cells has been revealed to be increased by the receptor for AGEs (17). In a clinical setting, patients with DM exhibit a higher rate of metastasis of oral cancer and a lower cancer-associated survival rate (18). A strong association appears to exist between AGEs and oral cancer; however, the underlying mechanism of the involvement of AGEs in oral cancer remains to be elucidated.

Antioxidant responsive element is regulated by nuclear factor-erythroid 2-related factor 2 (Nrf-2), which in turn regulates the expression of antioxidant genes (19–22). Reactive oxygen species degradation (23,24), anti-inflammatory responses (25–28), and neuroprotection (29) are regulated by Nrf-2 through the downstream antioxidant genes heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (30–33). In addition, Nrf-2 regulates the apoptotic response via tumor protein p53 regulation (34). Furthermore, in oral cancer cells, Nrf-2 and HO-1 upregulation appear to be associated with apoptosis (35).

A previous study by our group demonstrated that AGEs regulate cell migration via extracellular signal-regulated kinase (ERK) phosphorylation (17). Therefore, it was hypothesized that AGEs regulate Nrf-2 and downstream signaling pathways through ERK phosphorylation. The expression of various apoptosis-associated proteins, including Nrf-2, HO-1, p53, Bcl-2 associated × apoptosis regulator (Bax) and apoptosis regulator Bcl-x (Bcl-xl), in SAS oral cancer cells following treatment with AGEs was analyzed through western blot analysis, in order to investigate the role and underlying mechanism of AGEs in oral cancer.

Materials and methods

Reagents

Phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), DL-glyceraldehyde, resveratrol and PD98059 were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and Hank's Balanced Salt Solution were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). GAPDH was purchased from Chemicon International, Inc. (Temecula, CA, USA). ERK, phosphorylated (p)-ERK, Nrf-2, HO-1, p53, Bax and Bcl-xl were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Nitrocellulose membranes were purchased from Pall Corporation (Port Washington, NY, USA). The enhanced chemiluminescence (ECL) Immobilon western chemiluminescent HRP substrate kit was purchased from EMD Millipore (Billerica, MA, USA).

Preparation of AGEs

AGEs were prepared by incubation with BSA (pH 7.4) in PBS with 20 mM DL-glyceraldehyde at 37°C for 1 week. The product was dialyzed using dialysis membranes (cat. no. MWCO 6000; Orange Scientific, Braine-l'Alleud, Belgium) in PBS at 4°C for 2 h, and the cycle was repeated five times. The product was then concentrated at 4°C using Amicon Ultra-15 centrifugal filter units (EMD Millipore) and centrifuged at 830 × g for 30 min prior to storage at −80°C as described in a previous study (36).

Cell culture and treatment

The oral cancer cell line SAS (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) was cultured in an atmosphere of 5% CO2 at 37°C. The culture was maintained in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin, 2 mM L-glutamine and 100 µg/ml streptomycin. Cells were cultured in serum-free DMEM for 24 h prior to treatment.

Western blot analysis

Total protein (30 µg) was resolved using SDS-PAGE on a 10% gel and transferred to nitrocellulose membranes (Pall Corporation). The membranes were blocked using non-fat milk and incubated overnight at 4°C with primary antibodies directed against the following proteins: p-ERK, ERK, Nrf-2, HO-1, p53, Bax, Bcl-xl (all dilution, 1:1,000) and GAPDH (dilution, 1:40,000). Primary antibodies were removed and the membranes were washed using PBS with Tween-20 (PBST) buffer three times for 30 min at room temperature. The membranes were subsequently incubated for 45 min at room temperature with the following secondary antibodies: Horseradish peroxidase-conjugated anti-mouse (cat. no. AP124P; Chemicon International, Inc.), anti-rabbit (cat. no. AP132P; Merck Millipore) and anti-goat (cat. no. 605-4313; Rockland Immunochemicals Inc., Limerick, PA, USA) (all dilutions, 1:4,000). The secondary antibodies were removed and the membranes were washed using PBST buffer twice for 30 min. Protein bands were detected using Millipore ECL. The density of the protein bands was quantified using Image J software version 1.4 (National Institutes of Health, Bethesda, MA, USA) following normalization with GAPDH. All data are presented as the mean ± standard deviation from experiments performed in triplicate.

Statistical analysis

Student's t-tests were conducted using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Pathways associated with apoptosis are regulated by resveratrol

SAS cells were treated with resveratrol (1.25 and 2.5 µM) for 24 h, and Nrf-2, HO-1, p53 and Bax protein expression was detected using western blot analysis. The results demonstrated that, compared with the untreated control group, resveratrol significantly increased Nrf-2 (1.25 µM, 1.42±0.06, P=0.002; 2.5 µM, 1.35±0.1, P=0.03), HO-1 (1.25 µM, 1.24±0.04, P=0.003; 2.5 µM, 1.37±0.11, P=0.03), p53 (1.25 µM, 1.32±0.1, P=0.04; 2.5 µM, 1.79±0.24, P=0.03) and Bax (2.5 µM, 1.49±0.03, P<0.0001) expression (Fig. 1).

Figure 1. Regulation of apoptosis-associated signaling pathways by resveratrol. SAS cells were treated with resveratrol (1.25 and 2.5 µM) for 24 h. Nrf-2, HO-1, p53 and Bax were subsequently detected by western blot analysis. Resveratrol resulted in a significant increase in the expression level of Nrf-2, HO-1, p53 and Bax compared with the untreated control group. Results are presented as the mean ± standard deviation. *P<0.05; **P<0.001; ***P<0.0001 all in comparison to the control. Nrf-2, nuclear factor-erythroid 2-related factor 2; p53, tumor protein p53; HO-1, heme oxygenase 1; Bax, Bcl-2 associated × apoptosis regulator; Res, resveratrol.

Regulation of apoptosis-associated pathways by AGEs

SAS cells were treated with AGEs (200 and 400 µg/ml) or BSA (400 µg/ml; negative control) for 24 h. Western blot analysis was used to detect Nrf-2, HO-1, p53 and Bax protein expression. The results revealed that compared with the untreated control group, treatment with 400 µg/ml AGEs significantly decreased Nrf-2 (0.64±0.05; P=0.002), p53 (0.72±0.06; P=0.008) and Bax (0.7±0.03; P=0.0005) expression (Fig. 2).

Figure 2. Regulation of apoptosis-associated pathways by AGEs. SAS cells were treated with AGEs (200 and 400 µg/ml) or BSA (400 µg/ml; negative control) for 24 h. Western blot analysis was then performed to detect Nrf-2, HO-1, p53 and Bax protein expression. Treatment with AGEs resulted in a significant decrease in Nrf-2, p53 and Bax expression compared with the untreated control group. Results are presented as the mean ± standard deviation. **P<0.001; ***P<0.0001 all in comparison to the control. AGEs, advanced glycation end products; BSA, bovine serum albumin; Nrf-2, nuclear factor-erythroid 2-related factor 2; p53, tumor protein p53; HO-1, heme oxygenase 1; Bax, Bcl-2 associated × apoptosis regulator; Res, resveratrol; conc., concentration.

AGEs regulation of apoptosis-associated signaling pathways via ERK phosphorylation

Following the treatment of SAS cells with AGEs or BSA for 24 h, Bcl-xl and p-ERK were detected using western blot analysis. Treatment with AGEs was associated with a significant increase in Bcl-xl (200 µg/ml, 1.23±0.07, P=0.03; 400 µg/ml, 1.36±0.04; P=0.0006; Fig. 3A) and ERK phosphorylation (200 µg/ml, 1.2±0.01, P<0.00001; 400 µg/ml, 1.26±0.02, P=0.0004; Fig. 3B) compared with the untreated control groups. However, treatment with 400 µg/ml BSA also significantly increased p-ERK compared with the control group (1.25±0.04; P=0.003; Fig. 3B). A pretreatment of 10 µM PD98059 for 1 h was used on the cells to inhibit the phosphorylation of ERK (Fig. 4A). Pretreatment with PD98059 blocked the effect of AGEs on HO-1, Bcl-xl and Bax expression (Fig. 4B). Furthermore, compared with the untreated control group, pretreatment with PD98059 prior to treatment with AGEs significantly increased the expression of Nrf-2 (200 µg/ml, 1.27±0.04, P=0.003; 400 µg/ml, 1.24±0.03, P=0.001) and p53 (200 µg/ml, 1.43±0.06, P=0.002; 400 µg/ml, 1.39±0.08, P=0.01) (Fig. 4B). The mechanism by which AGEs were observed to influence the survival rate of oral cancer cells in the present study is presented in Fig. 5.

Figure 3. Regulation of Bcl-xl and p-ERK expression by AGEs. SAS cells were treated with AGEs or BSA for 24 h. Bcl-xl and p-ERK were detected using western blot analysis. These results demonstrated that treatment with AGEs significantly increased (A) Bcl-xl and (B) p-ERK expression compared with the untreated control group. Results are presented as the mean ± standard deviation. *P<0.05; **P<0.001; ***P<0.0001 all in comparison to the control. AGEs, advanced glycation end products; BSA, bovine serum albumin; Bcl-xl, apoptosis regulator Bcl-x; ERK, extracellular signal-regulated kinase; p-, phosphorylated; Res, resveratrol; conc., concentration.

Figure 4. Effects of AGEs following PD98059 pretreatment. Pretreating cells with 10 µM PD98059 for 1 h resulted in (A) a reduction in the expression of p-ERK and (B) inhibition of the effects of AGEs on HO-1, Bcl-xl and Bax expression, and significant increase in Nrf-2 and p53 expression compared with the untreated control group. Results are presented as the mean ± standard deviation. **P<0.001 all in comparison to control. AGEs, advanced glycation end products; Bcl-xl, apoptosis regulator Bcl-x; Nrf-2, nuclear factor-erythroid 2-related factor 2; p53, tumor protein p53; HO-1, heme oxygenase 1; Bax, Bcl-2 associated × apoptosis regulator; ERK, extracellular signal-regulated kinase; p-, phosphorylated; Res, resveratrol; conc., concentration.

Figure 5. AGEs influence signaling pathways associated with oral cancer cell survival. AGEs increase ERK phosphorylation, which stimulates downstream Nrf-2 inhibition and Bcl-xl upregulation. This subsequently suppresses p53 and Bax expression, which manifests as a change in the survival rate of oral cancer cells. AGEs, advanced glycation end products; Bcl-xl, apoptosis regulator Bcl-x; Nrf-2, nuclear factor-erythroid 2-related factor 2; p53, tumor protein p53; HO-1, heme oxygenase 1; Bax, Bcl-2 associated × apoptosis regulator; p-ERK, phosphorylated-extracellular signal-regulated kinase.

Discussion

An association has been demonstrated between oral cancer and DM (37–39); however, the mechanism underlying this association remains to be elucidated. A previous study by our group revealed that AGEs regulate oral cancer cell migration via the ERK signaling pathway (17). The results demonstrated the mechanism by which AGEs regulate p53 via ERK and downstream Nrf-2 and Bcl-xl. To the best of our knowledge, this is the first study to elucidate the mechanism of action of AGEs in oral cancer cells.

Previous studies have demonstrated the antitumorigenic effects of resveratrol (40–42). In oral cancer, resveratrol suppresses cell growth, DNA synthesis, migration and invasion, and increases cell apoptosis (43–45). A previous study reported that Nrf-2 regulates the p53 signaling pathway, leading to an apoptotic response (34). A study by Lee et al (35) suggested that the apoptosis of oral cancer cells is regulated by Nrf-2 and downstream HO-1 and p53. These results support the contention that Nrf-2 enhances cell apoptosis via the p53 signaling pathway. The results of the present study demonstrated that resveratrol significantly increased Nrf-2, HO-1, p53 and Bax protein expression, suggesting that resveratrol induces apoptosis through Nrf-2, HO-1, p53 and Bax signaling pathways. The results of the current study demonstrated that, in contrast to resveratrol, AGEs significantly decrease Nrf-2, HO-1, p53 and Bax, and increase Bcl-xl protein expression. This indicates that AGEs modulate oral cancer survival via regulation of the expression of Nrf-2 and Bcl-xl.

In the present study, ERK phosphorylation was significantly upregulated by AGEs and the pretreatment of SAS cells with PD98059 to suppress ERK activation inhibited the effects of AGEs on p-ERK, HO-1, Bcl-xl and Bax expression, whereas Nrf-2 and p53 expression significantly increased. Treatment with AGEs significantly increased Bcl-xl, and decreased p53 protein expression. A previous study by Chipuk et al (46) suggested that an interaction between Bcl-xl and p53 inhibits the activation of Bax. Li et al (47) reported that Bcl-xl inhibits p53 resulting in an anti-apoptotic effect. These results suggest that AGEs regulate p53 via ERK phosphorylation to inhibit Nrf-2 and activate Bcl-xl. In addition, AGEs and BSA increased ERK phosphorylation in the current study. However, AGEs decreased Nrf-2 and p53 protein expression. The pretreatment of SAS cells with PD98059 increased Nrf-2 and p53 expression. The results of the present study suggest that treatment with AGEs or BSA differ regarding their effects on oral cancer cells (17).

The results of the current study suggest that AGEs decrease Nrf-2 and p53 expression and increase Bcl-xl expression via ERK phosphorylation. To the best of our knowledge, this is the first study to demonstrate that AGEs regulate the expression of Nrf-2 and Bcl-xl, which subsequently influences p53 expression via ERK phosphorylation. In conclusion, the results of the present study suggest a mechanism by which AGEs influence the survival rate of oral cancer cells. This mechanism involves AGEs increasing ERK phosphorylation, which stimulates downstream Nrf-2 inhibition and Bcl-xl upregulation. This subsequently suppresses p53 and Bax expression, the effects of which manifest as a change in the survival rate of oral cancer cells. These findings explain the increase in oral cancer invasiveness and decrease in the survival rate of patients with DM, who typically have higher levels of AGEs. In addition, the results of the current study indicate that the accumulation of AGEs due to aging or DM promotes the progression of oral cancer.

Acknowledgements

The present study was supported by the National Science Council of Taiwan (grant no. 100-2314-B-309-002-MY3).

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References