Role of advanced glycation endproducts in breast cancer

Sharaf, Hanaa (2014) Role of advanced glycation endproducts in breast cancer. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Diabetes and cancer are major health problems because of their high incidences worldwide. A growing body of epidemiological evidence indicats a molecular link between diabetes and breast cancer. Patients with diabetes mellitus have an increased likelihood of developing various types of cancers including breast cancer through the formation of advanced glycation endproducts (AGEs) which lead to cellular and bio-molecular dysfunction. However, the effects of AGEs have been poorly investigated on breast cancer cells. This current study examined for the first time the biological effects of various concentrations of BSA-derived AGEs on the invasive and hormone–independent breast cancer cell line MDA-MB-231 and on non- invasive hormone-dependent human breast cancer cell line MCF-7. Bovine serum albumin was glycated-using methylglyoxal for three days. Crosslinked AGEs were assessed using sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining. Different assays including cell proliferation, migration and invasion through the MatrigelTM with assessment of matrix metalloproteinase (MMP) activity by using Zymogaraphy were performed to investigate the effect of different concentrations of BSA-AGE on both cell lines in vitro. Furthermore, signalling pathways were investigated by Western blotting and using kinexusTM phspho-protein microarray. The expression of the main receptor for AGEs (RAGE) involved on the MDA-MB231 and MCF-7 breast cancer cell lines was assessed by Western blotting and Calibur Flow Cytometer System. The results of this study demonstrated that BSA-AGEs increased MDA-MB-cell, proliferation, migration and invasion through the MatrigelTM associated with an enhancement of matrix metalloproteinase (MMP)-9 activities, in a dose-dependent manner, up-regulated the expression of the receptor for AGEs (RAGE) and of the key signalling protein, phospho-extracellular-signal regulated kinase (p-ERK)-1/2. In addition, the blockade of BSA-AGE/RAGE interactions using anti-neutralizing RAGE antibody reduced the expression of p-ERK1/2. Furthermore, in BSA-AGE-treated cells, phospho-protein micro-array analysis revealed the main enhancement of the over-phosphorylation of (ERK1/2), (p70S6K1), (STAT)-3 and (MAPK) p38, involved in cell survival, cell growth cell cycle and protein synthesis. In contrast, MCF-7 showed stimulatory effects of BSA-AGEs, on cell proliferation and migration, as compared to non-modified BSA. However, BSA-AGEs did not change the weak invasive capacity of MCF-7 cells to cross a reconstituted basement membrane. In addition, BSA-AGEs induced over-phosphorylation of RAGE in MCF-7 cells. The investigation of signalling pathways suggests that BSA-AGEs might contribute to breast cancer development through activation of MAPK pathway and activation of CREB1 transcription factor in MCF-7 cells. Throughout the study, the non-modified BSA had a negligible effect. In conclusion, BSA-AGEs might contribute to breast cancer development and progression of breast cancer. In addition, the up-regulation of RAGE and key phosphor-protein signalling expression induced by BSA-AGEs might be a promising target for therapy to prevent the development of breast cancer in diabetic patients.