Characterization of Matrix Metalloproteinases From Breast Cancer Cells

Abstract

Matrix metalloproteinases (MMPs) are enzymes in the extracellular matrix that are involved in a variety of functions including differentiation of cells, cell adhesion, and cell-to-cell interactions. A subclass of MMPs known as gelatinases (MMP-2 and -9) are associated with breast cancer metastasis due to their ability to degrade type-IV collagen, a constituent of basement membranes. Therefore, significant interest exists in developing therapeutic agents, which might prevent or decrease cancer metastasis. Breast cancer is the most prevalent invasive cancer in women worldwide. Genistein, a soy isoflavone has anti-breast cancer properties. This project seeks to measure the effect that genistein on the activity of the gelatinases MMP-2 and MMP-9. Here we describe our experiments to (i) establish assays for the gelatinases, (ii) detection of gelatinases in human breast cancer cells and (iii) analyze effects of genistein on gelatinase activity. Mammary carcinoma cells HTB-19 and HTB-20 are grown to log phase, harvested by trypsinization and whole cell extracts were prepared using well-established protocols. Activity of MMP-2 and MMP-9 is detected by gelatin zymography using 10% acrylamide gel electrophoresis followed by reactivation, incubation of the gel in presence of specific metal ions and coomassie blue staining. Presence of MMP-2 and MMP-9 in human breast cancer cells is confirmed by western blot analysis using specific antibodies against these proteins. Results obtained in these studies and their significance for growth of human mammary cancer cells will be discussed.