S100B Protein Stimulates Proliferation and Angiogenic Mediators Release through RAGE/pAkt/mTOR Pathway in Human Colon Adenocarcinoma Caco-2 Cells

Luisa Seguella,Laura Aurino,Barbara De Conno,Marcella Pesce,Giuseppe Annunziata,Giovanni Sarnelli,Giuseppe Esposito,Riccardo Capuano,Mirella Pesce

doi:10.3390/ijms20133240

Abstract

Chronic inflammation and angiogenesis are associated with colonic carcinogenesis. Enteric glia-derived S100B protein has been proposed as an “ideal bridge”, linking colonic inflammation and cancer, given its dual ability to up-regulate nuclear factor-kappaB (NF-κB) transcription via receptor for advanced glycation end products (RAGE) signaling and to sequestrate wild type pro-apoptotic wild type (wt)p53. However, its pro-angiogenic effects on cancer cells are still uninvestigated. To this aim, we evaluated the effect of exogenous S100B (0.05–5 µM) protein alone or in the presence of S100B blocking monoclonal antibody (mAb) (1:105–1:104 v/v diluted) on (1) cultured Caco-2 cells proliferation, migration and invasiveness in vitro, respectively by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-formazan, wound healing and matrigel invasion assays and (2) its effect on the release of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF) by ELISA and immunofluorescence analyses. The effect of S100B alone or in the presence of S100BmAb was then investigated on RAGE/pAkt/mammalian target of rapamycin (mTOR) signaling pathway by immunoblot analysis. Our results showed that S100B markedly increases proliferation and invasiveness of Caco-2 cells, through the release of pro-angiogenic VEGF and NO paralleled to a significant decrease of wtp53 expression mediated by RAGE-p38 mitogen-activated protein kinase (MAPK)/pAkt-mTOR and hypoxia-inducible factor 1-alpha (HIF1α) pathways. Such effects were counteracted by S100BmAb, indicating that S100B targeting is a potential approach to inhibit colon carcinoma proliferation and angiogenesis.

Highlights

In addition to genetic, environmental and dietetic influences [1,2], chronic inflammatory conditions are well-recognized risk factors for colonic carcinogenesis [3,4]
We evaluated the effect of exogenous S100B (0.05–5 μM) protein alone or in the presence of S100B blocking monoclonal antibody (1:105–1:104 v/v diluted) on (1) cultured Caco-2 cells proliferation, migration and invasiveness in vitro, respectively by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-formazan, wound healing and matrigel invasion assays and (2) its effect on the release of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF) by ELISA and immunofluorescence analyses
Our results showed that S100B markedly increases proliferation and invasiveness of Caco-2 cells, through the release of pro-angiogenic VEGF and NO paralleled to a significant decrease of wtp53 expression mediated by receptor for advanced glycation end products (RAGE)-p38 mitogen-activated protein kinase (MAPK)/pAkt-mammalian target of rapamycin (mTOR) and hypoxia-inducible factor 1-alpha (HIF1α) pathways

Summary

Introduction

Environmental and dietetic influences [1,2], chronic inflammatory conditions are well-recognized risk factors for colonic carcinogenesis [3,4]. The mammalian target of rapamycin (mTOR) is a protein kinase of the PI3K/Akt pathway that has been crucially involved in the activation and control of cell proliferation, survival, migration and angiogenesis. The identification of Akt/mTOR activators and their targeting, may be of potential interest to develop new drugs in CRC In this context, enteric glia-derived Ca+2/Zn+2-binding S100B protein emerges as a signaling molecule whose aberrant release plays a key role in both colonic inflammation [14] and carcinogenesis [15], given its downstream pro-inflammatory effects and mitogenic activity demonstrated in the gut, as well as in several highly malignant extra-intestinal cancers, such as melanoma and glioma [16,17,18]. We examined S100B effects (2) on Akt/mTOR pathway and VEGF signaling in the presence or absence of specific monoclonal anti-S100B antibody, in order to confirm/exclude its specific involvement in CRC cells-related malignancy and angiogenesis

Methods

Results

Conclusion