RAGE antibody blocked AGE regulation.

<p>The use of RAGE antibody (10 ng/ml pretreatment for 1 hour) to block AGE conjugation resulted in a significant increase in the ERK phosphorylation, MMP2, and MMP9 due to AGEs. Compared with AGEs treatment (#), RAGE antibody blocked ERK phosphorylation, MMP2, and MMP9 (A and B) as well as cell migration (C).</p

Cell Migration Is Regulated by AGE-RAGE Interaction in Human Oral Cancer Cells <i>In Vitro</i>

<div><p>Advanced glycation end products (AGEs) are produced in an irreversible non-enzymatic reaction of carbohydrates and proteins. Patients with diabetes mellitus (DM) are known to have elevated AGE levels, which is viewed as a risk factor of diabetes-related complications. In a clinical setting, it has been shown that patients with oral cancer in conjunction with DM have a higher likelihood of cancer metastasis and lower cancer survival rates. AGE-RAGE (a receptor of AGEs) is also correlated with metastasis and angiogenesis. Recent studies have suggested that the malignancy of cancer may be enhanced by glyceraldehyde-derived AGEs; however, the underlying mechanism remains unclear. This study examined the apparently close correlation between AGE-RAGE and the malignancy of SAS oral cancer cell line. In this study, AGEs increased ERK phosphorylation, enhanced cell migration, and promoted the expression of RAGE, MMP2, and MMP9. Using PD98059, RAGE antibody, and RAGE RNAi to block RAGE pathway resulted in the inhibition of ERK phosphorylation. Cell migration, MMP2 and MMP9 expression were also reduced by this treatment. Our findings demonstrate the importance of AGE-RAGE with regard to the malignancy of oral cancer, and help to explain the poor prognosis of DM subjects with oral cancer.</p></div

RAGE RNAi mediated influence of AGEs.

<p>RAGE RNAi (20 nM for 48 hours) was used to silence protein expression. Compared with the RNAi negative control (N), RAGE RNAi significantly reduced RAGE expression (A). The suppression of cell migration by RAGE RNAi occurred independently from treatment with AGEs (B). RAGE RNAi also inhibited ERK phosphorylation, MMP2, and MMP9. Compared with N+AGEs (#), RNAi +AGEs presented a significant reduction (C and D) and the negative control had no effect.</p

Inhibition of cell proliferation by AGEs.

<p>SAS cells were treated with AGEs (0–400 µg/ml) or BSA (0–400 µg/ml) for 24 to 48 hours. The number of cells and proliferation were detected using trypan blue dye exclusion (A) and a WST-1 assay (B). AGEs significantly reduced the number of cells; BSA (as a negative control) increased viability (A). AGEs also inhibited cell proliferation (B). Treatment with AGEs (400 µg/ml; 0–4 hours) enhanced cell migration, while treatment with BSA did not (C).</p

AGEs increased ERK phosphorylation.

<p>Western blot analysis showing that treating SAS cells with AGEs for 24 hours resulted in increased ERK phosphorylation (A). Pretreatment with PD98059 for 1 hour reduced ERK phosphorylation, MMP2 and MMP9 (B and C) as well as cell migration (D); however RAGE levels still increased (E).</p

AGEs regulated RAGE, MMP2, and MMP9.

<p>After treating cells with AGEs (200 and 400 µg/ml) or BSA (400 µg/ml) for 24 hours, RAGE, MMP2, and MMP9 were detected by western blot analysis (A). AGEs significantly increased the expression of RAGE, MMP2, and MMP9 (B).</p

Mechanism by which AGEs-RAGE influences cancer malignancy.

<p>AGEs increase RAGE expression and stimulate the downstream pathway of ERK phosphorylation. This has the effect of up-regulating MMP2 and MMP9 expression and enhancing cell migration, which manifests as malignancy.</p

Resveratrol regulated the effects of AGEs via ROS.

<p>Detection of ROS by DCFH-DA fluorescence: (A) After treating cells with AGEs and resveratrol (Res; 20μM) for 24 hours, the level of ROS significantly increased; (B) After treating cells with AGEs the levels of APP, BACE, and PS1 were increased. (C) Treatment with resveratrol alone had no changes of APP expression. (D) H₂O₂ enhanced APP, BACE, and PS1 expression. (B and D) Resveratrol significantly inhibited the effects of AGEs and H₂O₂. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 vs. control. ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 vs. AGEs.</p

Cell death related pathway was regulated by AGEs.

<p>Detection of the death related pathway, GRP78, p53, bcl-2, and bax: (A) AGEs increased GRP78 and p53 significantly, but decreased the bcl-2/bax ratio; (B) after pretreating cells with NAC for 2 hours, the effects of AGEs were inhibited. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 vs. control. ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 vs. AGEs.</p

Regulation of APP processing by AGEs via ROS.

<p>Cells were treated with AGEs (0.5 mg/ml), BSA (0.5 mg/ml; as a negative control) or H₂O₂ (100 μM; as an ROS positive control) for 24 hours. AGEs increased the levels of APP, BACE, and PS1, and H₂O₂ enhanced the expression of APP, BACE, and PS1; some cells were also pretreated with ROS scavenger NAC for 2 hours before being treated with AGEs, which reduced the effects of AGEs. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 vs. control.</p