Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation

Riyo Morimoto-Kamata,Satoru Yui,Takeo Ichisugi,Sei-Ichiro Mizoguchi

doi:10.1155/2012/456462

Abstract

Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG) is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering) CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

Highlights

Cathepsin G (CG) is a serine protease that is secreted from activated neutrophils and a subset of monocytes, and belongs to the chymotrypsin superfamily [1,2,3,4]
phenylmethylsulfonyl fluoride (PMSF)-treated CG was prepared by incubating CG with PMSF (4 mM) for 2 h at 37◦C followed by dialysis to remove unbound PMSF that would cause cytotoxicity in MCF-7 cells in cell aggregation assays
We have previously demonstrated that CG induces homotypic cell aggregation and the formation of multicellular 3D spheroids of MCF7 cells [23, 24]

Summary

Introduction

Cathepsin G (CG) is a serine protease that is secreted from activated neutrophils and a subset of monocytes, and belongs to the chymotrypsin superfamily [1,2,3,4]. CG plays important roles in the hydrolysis of the extracellular matrix and microbicidal system and in immune response, apoptosis, chemotaxis, and blood coagulation [1, 3,4,5,6,7]. CG and other serine proteases, such as neutrophil elastase and proteinase 3, act in conjunction with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes [1, 3, 8]. In human leukemic NB4 cells, CG cleaves the protein highly homologous to the Drosophila protein “brahma” (brm), which regulates chromatin conformation and the nuclear matrix during apoptosis [9]. CG is reported to facilitate and impede blood coagulation [6], and it can be considered a regulatory factor in inflammatory and apoptotic reactions

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Conclusion