Assessment of Gelatinases (MMP-2 and MMP-9 by Gelatin Zymography

Abstract

The invasion and metastasis of tumor cells has been shown to require proteolytic activity in order to degrade components of the extracellular matrix (ECM). The hydrolysis of the ECM appears to facilitate tumor cell migration contributing to the metastatic dissemination of malignant cells (1). A major group of proteases that has been directly associated with tumor metastasis is the matrix metalloproteinases (MMPs), a family of endopeptidases known to cleave many ECM proteins (1). The MMPs are multidomain proteases that contain a zinc atom in the active site and are produced in a latent inactive form (zymogen) (2). Acquisition of enzymatic activity requires cleavage of the inhibitory N-terminal domain (3). Thus, generation of the active form usually occurs concomitantly with a decrease in molecular mass and exposure of the active site. Once activated, all the MMPs are specifically inhibited by a group of endogenous protease inhibitors known as the tissue inhibitors of metalloproteinases (TIMPs), which bind to the active site inhibiting catalytic activity (4).

Fig. 1

Gelatin zymogram of biological samples from different sources. Lanes 1–5, purified recombinant MMP-9 (lane 1, latent form; lane 2, activated form) and MMP-2 (lane 3, latent form; lane 4, active form; lane 5, mixture of active and latent forms). Note the presence of the MMP-9 dimer form (~200 kDa). Lane 6, serum-free conditioned media of HT1080 fibrosarcoma cells. Lanes 7–10, serum-free conditioned media of nonmalignant breast epithelial MCF10A cells untreated (lane 7) or treated with increasing concentrations (10, 25, and 50 ng/mL) of tumor necrosis factor-α (TNF-α) (lanes 8–10). Note the obvious induction of MMP-9 in response to TNF-α compared to untreated cells. Lane 11, cell lysate of TNF-α treated MCF10A cells. Note the presence of the intracellular, lower molecular mass (~83–85 kDa), precursor (partially glycosylated) form of latent MMP-9. Lanes 12 and 13, tissue extracts (30 μg of protein) from the benign (lane 12) and carcinoma (lane 13) section of a breast biopsy. Note the induction of MMP-9 and a 130–140-kDa band of unknown origin in the carcinoma. Also note the appearance of a approx 80–85 kDa form in the carcinoma. Note that it is difficult to determine whether this is active MMP-9 or the intracellular precursor form. Lane 14, unreduced molecular weight standard (Novex).