Phenylmethylsulfonyl fluorides (PMSF), bovine serum albumin (BSA), DL-Glyceraldehyde, and PD98059 were purchased from Sigma (St. Louis, MO, USA). DMEM media, fetal bovine serum (FBS), penicillin, streptomycin, Hanks Balanced Salt Solution (HBSS), trypan blue, and Lipofectamine RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). GAPDH (Cat No.: MAB374) was purchased from Chemicon (Temecula, CA, USA). ERK (Cat No.: SC-94), p-ERK (Cat No.: SC-7383), RAGE (Cat No.: SC-94), and RAGE siRNA (Cat No.: SC-36374) were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). MMP2 (Cat No.: 2763-S) and MMP9 (Cat No.: 2551-S) were purchased from Epitomics (Burlingame, CA, USA). Nitrocellulose membranes were purchased from PALL corp. (Ann Arbor, MI, USA). Enhanced chemiluminescence (ECL) was purchased from Millipore (Billerica, MA, USA). Wst-1 kit was purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA). Culture-Insert was purchased from ibidi (Verona, WI, USA).

AGEs were prepared by incubating BSA (pH = 7.4) in PBS with 20 mM DL-Glyceraldehyde at 37°C for 1 week. The product was dialyzed in PBS at 4°C for 2 hours, and this cycle was repeated 5 times. The product was then concentrated at 4°C using an Amicon Ultra protein concentration tube (Millipore) and centrifuged at 3,000 rpm for 30 minutes before being stored at −80°C [28].

The oral cancer cell line SAS (Japanese Collection of Research Bioresources Cell Bank [JCRB], Japan) was grown at 37°C under a 5% CO₂ atmosphere. The culture was maintained in DMEM media (Invitrogen) routinely supplemented with 10% FBS, 100 units/ml of penicillin, 2mM L-glutamine, and 100 µg/ml streptomycin. Cells were incubated serum-free for 24 hours prior to treatment.

Cells were seeded on 6-well plates at a concentration of 10⁵ per well and cultured for 24 hours. Cell viability was determined according to their ability to exclude 0.5% trypan blue (Invitrogen) following treatment with 100–400 µg/ml AGEs for 24 and 48 hours, respectively. An equal volume of trypan blue dye solution (0.1%, w/v), HBSS, and cell slurry were combined and maintained at room temperature for five minutes. Samples were then loaded onto a hemocytometer to categorize cells as living or dead according to the uptake of dye. All figures presented in this study are the mean values of triplicate experiments.

Cells were seeded on 96-well plates at a concentration of 5×10³ per well and cultured for 24 hours. Cell proliferation was detected by the WST-1 kit (Clontech) in conditioned medium. Samples were prepared according to the protocol outlined by the manufacturer. Briefly, following treatment with AGEs (0–400 µg/ml), the conditioned medium was centrifuged at 2,000 rpm for 10 minutes. Supernatant was then transferred into 96 well plates (100 µl/well). Reaction Mixture (100 µl) was added to each well, followed by incubation for 30 minutes. During the incubation period, samples were maintained at room temperature (RT) and protected from light. Absorbance of the samples was measured at 490 nm. Reference wavelength should be greater than 600 nm. All figures presented in this study are the mean values from experiments performed in triplicate.

Cells were seeded on 6-well plates at a concentration of 2×10⁵ cells per well and cultured for 24 hours. The cells were then transfected with RAGE siRNA (a pool of 3 target-specific 19–25 nt siRNA; Santa Cruz) or the sense strand sequence of the RNAi as a negative control (5′-UUCUCCGCCCGUGUCACGU-3′) [29]. Transfections were conducted using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer's protocol. Specifically, dilute 40 pmol of RNAi duplex in 250 µl Opti-MEM I reduced serum medium without serum. We then gently mix the Lipofectamine RNAiMAX and diluted 4 µl of the mixture into 50 µl Opti-MEM I reduced serum medium. The dilute RNAi duplex was combined with dilute Lipofectamine RNAiMAX and incubated for 20 minutes at room temperature. Finally, the RNAi duplex- Lipofectamine RNAiMAX complexes were added to each well. This resulted in a final volume of 600 µl and a final siRNA concentration of 20 nM. Cells were incubated for 48 hours prior to being used in experiments.

Proteins (30 µg) were resolved using 10% SDS-PAGE gel and then transferred onto nitrocellulose membranes (PALL Corp.). The membranes were blocked using non-fat milk and incubated overnight at 4°C with the following primary antibodies: anti-p-ERK (dilution of 1∶1,000); anti-ERK (dilution of 1∶1,000); anti-MMP2 (dilution of 1∶1,000); anti-MMP9 (dilution of 1∶1,000); anti-RAGE (dilution of 1∶1,000; anti-GAPDH (dilution of 1∶40,000). Primary antibodies were then removed and membranes were washed with PBST buffer for 30 minutes. The membranes were subsequently incubated for 45 minutes at RT with the following secondary antibodies: horseradish peroxidase conjugated anti-mouse (dilution of 1∶4,000) and anti-rabbit (dilution of 1∶4,000) (Chemicon). Finally, secondary antibodies were removed and the membranes were washed with PBST buffer twice for 30 minutes. Signals were detected using the Western Lighting Chemiluminescence Reagent Plus kit (Millipore). The densities of the signals were measured using an imaging system, and the signals were quantified after being normalized against those of GAPDH. All figures presented in this paper are the mean values from experiments performed in triplicate.

Cells were seeded on 6-well plates using Culture-Insert (ibidi) at a concentration of 6×10⁵ cells per well and then cultured for 24 hours. After a period of 24 hours in serum-free conditions, the Culture-Inserts were removed and cells were washed using HBSS, before being treated with AGEs for analysis of cell migration.

Following treatment with 200–400 µg/ml AGEs for 4 or 24 hours, cells were seeded on 7 cm dishes at a concentration of 1×10⁶ cells. Zymography was used to detect the function of MMP2 and MMP9 in conditional medium. To achieve this, we used 7.5% SDS-PAGE gel containing 0.1% gelatin (J.T.Baker, Center Valley, PA, USA). The gel was then washed with 2.5% Triton X 100 for 30 minutes at RT. Triton X 100 was removed and gel was washed with RO H₂O. We then added renaturing buffer (50mM Tris-HCl pH 7.2,20%glycerol) for 30 minutes at RT. Renaturing buffer was removed and gel was washed with RO H₂O. We then added developing buffer (50mM Tris-HCl pH 8.8, 5mM CaCl2, 1mM ZnCl2) for 24 hours at 37°C. Developing buffer was removed and gel was washed with RO H₂O. Gel staining was conducted for 1 hour at RT using PageBlue Protein Staining (Fermentas, Lafayette, CO, USA). Gel destaining was conducted using RO H₂O at RT. (Fig S1).

We conducted student t-tests, one-way ANOVA, and p values of <0.05 were considered statistically significant.

We first assessed the influence of AGEs on cell viability. SAS cells were treated with AGEs (0–400 µg/ml) or BSA (0–400 µg/ml) for 24 or 48 hours, whereupon the number of cells and proliferation rate were determined according to trypan blue dye exclusion (Fig. 1A) and WST-1 assays (Fig. 1B). Compared with control cells, AGE treated cells showed a significant reduction in number of cells (24 hours AGEs 100: 2.88±0.16, P = 0.006; AGEs 200: 2.6±0.25, P = 0.008; AGEs 400: 1.85±0.05, P <0.0001; 48 hours AGEs 100: 3.1±0.18, P = 0.05; AGEs 200: 2.57±0.17, P = 0.01; AGEs 400: 2.03±0.08, P = 0.003). In contrast, BSA was shown to increase the number of cells (as a negative control, 24 hours: 4.77±0.32, NS; 48 hours: 9.25±0.35, P = 0.0004) (Fig. 1A). In addition, cell proliferation was shown to be inhibited by AGEs (Fig. 1B). Finally, treating cells with AGEs (400 µg/ml; 0–4 hours) enhanced migration; however, BSA did not have any effect on migration (Fig. 1C).

Cells were treated with AGEs (200 and 400 µg/ml) or BSA (400 µg/ml) for 24 hours. We then used western blot analysis to detect RAGE, MMP2, and MMP9. Compared with control cells, the result showed that AGEs treated cells presented a significant increase in RAGE (AGEs 400: 1.3±0.03, P = 0.0007), MMP2 (AGEs 200: 1.28±0.04, P = 0.002; AGEs 400: 1.47±0.04, P = 0.004), and MMP9 (AGEs 400: 1.24±0.03, P = 0.0008) (Fig. 2). Moreover, the functionality of MMP2 and MMP9 was increased after treatment with AGEs for 4 or 24 hours (Fig S2).

In order to identify the pathway of AGEs, SAS cells were treated with AGEs or BSA for 24 hours. ERK phosphorylation was then detected using western blot analysis. Our results show that treatment with AGEs significantly increased ERK phosphorylation (AGEs 400: 1.26±0.06, P = 0.01) (Fig. 3A). ERK phosphorylation was enhanced following 4 hours AGEs treatment (Fig S2); however, pretreating cells with PD98059 for 1 hour resulted in a reduction in the expression of MMP2 and MMP9 (Fig. 3B and C). It appears that PD98059 pretreatment blocked the effects of AGEs on cell migration (Fig. 3D); however, RAGE expression was not affected (AGEs 400: 1.27±0.04, P = 0.003) (Fig. 3E).

RAGE antibodies (10 ng/ml pretreatment for 1 hour) were used to block AGE conjugation. Treatment with AGEs resulted in a significant increase in ERK phosphorylation and the expression of MMP2, and MMP9 (ERK: 1.31±0.03, P = 0.0008; MMP2: 1.38±0.04, P = 0.0005; MMP9: 1.32±0.09, P = 0.02). Compared to cells treated with AGEs alone, cells treated with both AGEs and RAGE antibody demonstrated significantly inhibited ERK phosphorylation (P = 0.009) as well as reduced expression of MMP2 (P = 0.05), and MMP9 (P = 0.0003) (Fig. 4 A and B). RAGE antibodies were also shown to suppress cell migration (Fig. 4C).

RAGE RNAi (20 nM for 48 hours) was then used to silence protein expression. Compared with the RNAi negative control (N), RAGE RNAi was shown to have a significantly suppress RAGE expression (0.64±0.07, P = 0.006) (Fig. 5A). The suppression of cell migration by RAGE RNAi was found to occur independently from the effects of AGEs (Fig. 5B). Furthermore, RAGE RNAi inhibited ERK phosphorylation (RNAi: 0.64±0.08, P = 0.01; N+AGEs: 1.26±0.03, P = 0.001) MMP2 (N+AGEs: 1.28±0.04, P = 0.003), and the secretion of MMP9 (RNAi: 0.58±0.06, P = 0.002; N+AGEs: 1.36±0.05, P = 0.003). Compared with N+AGEs, treatment with RNAi +AGEshad a significant effect on all three of these processes (ERK: P = 0.02; MMP2: P <0.003; MMP9: P = 0.04) (Fig. 5C and D). The negative control did not show significant effects.