Electric cell substrate impedance sensing (ecis) for the study of adrenomedul- lin on human renal clear caki-2 cells in hypoxic conditions

STOYANOFF, TANIA; MARTINEZ RAMIREZ, ALFREDO; RODRÍGUEZ, JUAN PABLO; MELANA COLAVITA, JUAN PABLO; TODARO, JUAN; AGUIRRE, MARÍA VICTORIA

Congreso; XLIX Reunión de Anual de la Asociación Argentina de Farmacología Experimental (SAFE); 2017

Angiogenesis is a crucial process in tumor development and metastasis. The hypoxic feature of solid tumors contributes locally and systemically to tumor progression. Clear cell renal carcinoma (CRCC) is the most common histological subtype of renal cancer (80-90% of cases). It is characterized by an intense vasculariza- tion implying a close relationship among angiogenesis, cellular re- sponse to hypoxic stress and tumor progression. There is limited evidence about the use of electric cell?substrate impedance sensing (ECIS) assay to evaluate angiogenesis and / or metastatic potential in CRCC. In addition, the function of adrenomedullin (AM) and the effects of its inhibition on solid tumors (such as CRCC) are not fully known. The aim of this study was to assess the effects of AM, a pro-angiogenic molecule, and its inhibition in CRCC using the ECIS assay. Briefly, Caki-2 cells were cultured on ECIS 8W10E electrode plates (Applied Biophysics, Troy, NY). Cellular impedance variations were measured against different concentrations of cobalt chloride (1-100 μM) for simulating hypoxic conditions and with/ without the inhibitory agents: 16311(10 to 1000 nM) and 22-52 (0,01 to 1 μM). Monolayers were summited to 1400μA current at 60Hz for 20 sec for migration assay.Results showed that 100 μM CoCl2 was the most effective dose for mimicking hypoxia in Caki-2 cultures. Expression of AM was evaluated by Western Blotting (WB), RT-PCR and qPCR at 0,12, 24 and 36 h post cobalt chloride. AM expression increased significative- ly between 12 and 24 h in hypoxic conditions by WB and RT-PCR (p≤0.001). qPCR revealed a significant increment of AM RNAm at 24 h of hypoxia (p≤0.01). Finally, AM inhibitors caused the decrease in cell growth and migration by ECIS assays. This preliminary study contributes to a better understanding of CCRC tumor biology and encourages us to propose AM as a potential anti-tumor target in relation to angiogenesis.