Zymography-Based Assay for Screening Potential Anti-Gelatinase Agents using Serratia marcescens Serralysin

Abstract

Matrix metalloproteinases (MMPs) are a class of over 20 zinc-dependent proteolytic enzymes that have been implicated in the physiologic process of aging and in the pathogenesis of cancer. Specifically, the gelatinases MMP-2 and MMP-9 have been intensively studied in connection with tumor invasion and metastasis, and have been considered as promising targets for cancer treatment. Herein, we introduce a fast, reliable and cost-effective zymography-based assay that can be used routinely for pre-screening agents with potential anti-gelatinolytic activity. Agents which exhibit antigelatinolytic activity in this assay can then be tested using M conventional screening assays for anticancer agents. The gelatinase serralysin from culture supernatants of the Gramnegative bacterium Serratia marcescens was employed. Several anti-aging creams and plant extracts were tested. Vitamin E- and tretinoin-containing creams showed remarkable antigelatinolytic activity. For the natural products, aqueous extract of papaya gave the most remarkable anti-gelatinolytic activity as compared to aqueous extracts of guava and tomato. To verify these results, culture supernatants of normal human keratinocytes and HaCaT cells were treated with the same agents. Keratinocyte-secreted gelatinases (MMP-2, MMP-9) and Serratia marcescens–secreted gelatinase (serralysin) were inhibited in a similar manner by the tested anti-aging creams and by papaya extract. Taken together, zymography using gelatinases from microorganisms can serve as a useful tool for pre-screening synthetic and natural products with potential anti-MMP-2/-9 activities.