Relationship between hypoxia and proteolytic activity in two clear cell renal car-cinoma cell lines: caki-1 and caki-2 cells

PALOMAR, LUCAS S.; OLEA, GABRIELA; RODRÍGUEZ, JUAN P.; BARNES, TAMARA; GAY CLAUDIA CAROLINA; MELANA COLAVITA, JUAN PABLO; AGUIRRE, MARÍA VICTORIA

Jornada; X Jornadas anuales de la Sociedad Argentina de Biología; 2018

Metastasis is the main mortality cause in cancer patients (90%) and particularly this is important in clear cell renal carcinoma (CCRC). Likewise, extracellular proteolysis is one of the main mechanisms that allow the degradation and remodeling of the extracellular matrix and the basement membrane, thus favoring proliferation and tumor metastasis. Recent studies have established a link between this factor and intratumoral hypoxia conditions. Proteolytic enzymes, such as metalloproteinases (MMPs), belonging to a broad family of Zn2+-dependent enzymatic proteins, regulate this microenvironment by controlling angiogenesis and tissue remodeling. Up to date, these studies have not yet been addressed on CCRC. For that reason, in the present work the characterization of the extracellular proteolytic activity was initiated, using the zymography technique for its detection, in "in vitro" cultures of two CCRC cell lines: Caki-1 and Caki-2 cells. Both cell lines were exposed to chemical hypoxia, generated by CoCl2 addition (0-300 M) for 24 h. Then, cellular supernatants were collected for protein and zymographic profile analysis. Additionally, the enzyme activity inhibition assay was performed, using EDTA (50 mM). The preliminary results found, show differential proteolytic activity between both cell lines, being higher in Caki-1 cells, as well as variations among the different treatments. Also, the inhibition experiments demonstrate that the proteolytic activity can be attributed to metallo-dependent enzymes. Currently, using immunochemical assays, we are looking for the action of different MMP isoforms in the process.