Advanced glycation end products correlate with breast cancer metastasis by activating RAGE/TLR4 signaling

Abstract

Introduction: This study was aimed to investigate the mechanisms of advanced glycation end products (AGEs) in promoting invasion and metastasis of breast cancer.

Research design and methods: Patients with 131 breast cancer were enrolled in a cohort and followed up to investigate the association between AGEs and metastasis. Serum AGE concentrations were detected by ELISA. Breast cancer MDA-MB-231 cells were exposed to generated AGE-bovine serum albumin (BSA). CCK-8 assay was used to select the non-cytotoxic concentrations of AGE-BSA. Small interfering RNA was used to knock down Toll-like receptor 4 (TLR4). Migration and invasion were evaluated by wound healing and transwell assays. Real-time PCR and western blotting were used to detect the gene expressions.

Results: In the cohort study, metastasis incidence was significantly correlated with serum AGE concentrations in patients with breast cancer (adjusted OR=1.75, 95% CI=1.20 to 2.57, p=0.004). During follow-up, metastasis interval was significantly shorter in diabetic than non-diabetic subjects. In the in vitro study, AGE-BSA incubation significantly promoted migration and invasion of cancer cells in a concentration-dependent manner. AGE-BSA dramatically increased expressions of receptor for AGEs (RAGE), TLR4, myeloid differentiation factor (MyD88), matrix metalloproteinase 9 (MMP9), promoted nuclear translocation of nuclear factor κB (NFκB) p65, but decreased the expression of inhibitor of NFκB (IκBα). TLR4 silencing significantly suppressed migration and invasion of cancer cells exposed to AGE-BSA. TLR4 silencing reduced the expression of MyD88 and MMP9, as well as nuclear translocation of NFκB p65 but increased IκBα expression in AGE-BSA-incubated breast cancer cells.

Conclusions: AGEs are correlated with metastasis of breast cancer. AGEs' promoting effects on migration and invasion of breast cancer cells via activating RAGE/TLR4/MyD88 signaling were suggested as the involved mechanism.

Keywords: Breast Cancer; Diabetes Mellitus, Type 2.

Figure 1

(A) Columns indicated the cell viabilities calculated based on CCK-8 assays in MDA-MB-231 cells incubated with advanced glycation end product-bovine serum albumin (AGE-BSA) at 0, 10, 20, 40, 80 and 100 µg/mL for 8 hours, respectively. AGE-BSA began to show significant inhibitory effects on cell viabilities starting with the concentration at 40 µg/mL. (B) Immunoblots of Toll-like receptor 4 (TLR4) and GAPDH were demonstrated. Columns indicated relative expression of TLR4 in negative control (NC) cells, scrambled small interfering RNA (siRNA) transfected cells and TLR4-siRNA transfected cells, respectively (n=6, *p<0.05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

(A) Captured images of wound healing assay of MDA-MB-231 cells were demonstrated. (B) Columns indicated relative wound width of cells incubated with advanced glycation end product-bovine serum albumin (AGE-BSA) at 0, 5, 10 and 20 µg/mL for 8 hours, respectively. Several cells treated with AGE-BSA at 20 µg/mL were pretransfected with Toll-like receptor 4-small interfering RNA (TLR4-siRNA) (n=6, *p<0.05).

Figure 3

(A) Captured images of transwell assays were demonstrated. Columns indicated invaded cell number per field of MDA-MB-231 cells incubated with AGEs at 0, 5, 10 and 20 µg/mL for 8 hours, respectively. Several cells treated with AGEs at 20 µg/mL were pretransfected with TLR4-siRNA. (B) Columns indicated the relative expression levels of MMP9 mRNA expression levels in MDA-MB-231 cells. (C) Immunoblots of MMP9 and GAPDH were demonstrated. Columns indicated the relative expression levels of MMP9 in MDA-MB-231 cells (n=6, *p<0.05). AGE-BSA, advanced glycation end product-bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; TLR4-siRNA, Toll-like receptor 4-small interfering RNA.

Figure 4

(A) Columns indicated the relative mRNA expression levels of RAGE, TLR4 and MyD88 in MDA-MB-231 cells. (B) Immunoblots of RAGE, TLR4 and MyD88 were demonstrated. Columns indicated the relative expression levels of RAGE, TLR4 and MyD88 in MDA-MB-231 cells. (C) Left panel showed detected immunoblots of IκBα, GAPDH and Lamin B1 in cytoplasmic protein samples. Columns indicated the relative expression of IκBα in cytoplasm in MDA-MB-231 cells. (D) Left panel demonstrated the detected immunoblots of NFκB p65, GAPDH and Lamin B1 in nuclear protein in MDA-MB-231 cells. Columns indicated the relative expression of IκBα in nuclei. MDA-MB-321 cells were incubated with AGE-BSA at 0, 5, 10 and 20 µg/mL for 8 hours, respectively. Several cells treated with AGE-BSA at 20 µg/mL were pretransfected with TLR4-siRNA (n=6, p<0.05). AGE-BSA, advanced glycation end product-bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IκBα, inhibitor of nuclear factor κB; MyD88, myeloid differentiation factor; NFκB, nuclear factor κB; RAGE, receptor for advanced glycation end product; siRNA, small interfering RNA; TLR4, Toll-like receptor 4.