P27: Matrix metalloproteinase-9 and -13 as regulators of TGF-β-the signal transduction pathway in melanoma tumor cells

Abstract

Matrix metalloproteinases (MMPs) – zinc containing endopeptidases which cleave the different components of the extracellular matrix. However, not all biological mechanisms and the effects of these endopeptidases are well-established. Previously it was believed that MMP functions only as proteolytic enzymes, but it was found later that MMPs could act as regulators of protein kinase signal transduction pathways by modifying receptors and signaling molecules. One of the most important substrates for MMP is transforming growth factor-β (TGF- β ) , which is a receptor for the ligand TGF-β-signal transduction pathway comprises a network of protein kinase pathways responsible for many of the processes of tumor progression, including epithelial mesenchymal transformation (EMT). Despite the fact that melanoma cells are not of epithelial origin, they express the epithelial E-cadherin that is necessary for their interaction with the basal layers of the epidermis. The degradation of the extracellular matrix and loss of expression of E-cadherin are major changes needed to start the processes of melanoma invasion. One of the markers of EMT is a transcription factor twist1. Twist1 activates the processes of epithelial-mesenchymal transformation and increases the invasion of melanoma cells by increasing transcription of MMP. The aim of this study was to evaluate expression levels TGF-β, twist1 under selective inhibition of MMP-9 and combined inhibition of MMP-9 and-13 in melanoma model in vivo; to determine the role of MMPs as possible regulators of TGF-β-way signal transduction, as well as associated with TGF-β EMT process. The study was approved by the local Ethic Committee (protocol No. 144/ 2012 of 15.11.2012). Melanoma B16-bearing mice (control group consisted of 7 animals, MMP-9 inhibitor treatment group – 7, MMP-9/13 inhibitor treatment group – 7 animals) underwent experimental treatment by MMP inhibitor (Calbiochem, USA) by daily application within 7 days. Control group consisted of animals without treatment. Efficacy of MMP-9 inhibition was evaluated by gelatinize zymography. TGF-β1 and twist 1 transcription factor expression was determined by PCR real-time using StepOne System (Applied Biosystems, USA). Statistical analysis was done by Kruskal–Wallis test and Multiple Comparisons. The P values lower 0.05 were considered as significant. It was found that the selective inhibition of MMP-9 did not induce changes in expression levels of TGF-β and twist1, however, the combined inhibition of MMP-9 and MMP-13 significantly reduced the expression levels of the transcription factor twist1 and TGF-β. The results show that MMP-9 and MMP-13 act as activators of TGF-β-signal transduction pathway and could be considered as potential molecular targets for experimental therapy for cancer.