STUDIO DELL'EFFETTO DI UKRAIN SUI MECCANISMI DI TRANSIZIONE EPITELIO-MESENCHIMALE E INVASIVITA' TUMORALE IN CELLULE DI CARCINOMA RENALE

Renal cell carcinoma (RCC) is the most common malignancy of the kidney, arising from the cortical tubular epithelium and accounting for 85% of renal cancers. Recent clinical investigations suggested that Ukrain (UK), a semisynthetic derivative of the greater celandine alkaloids, exerts beneficial effects in the treatment of several solid tumors, including the RCC. In this study we investigated whether UK was able to modulate the malignant phenotype of RCC cells, by analyzing the expression of proteins involved in tumor progression. For this purpose, we investigated whether Ukrain modulates the malignant phenotype of clear cell renal carcinoma (ccRCC) cells Caki-1, Caki-2, ACHN treated with four doses (5, 10, 20, 40 µM) for 24 and 48h. The epithelial-to-mesenchymal transition (EMT) markers E-cadherin, β-catenin, vimentin were analyzed by immunofluorescence, as well as actin and tubulin; matrix metalloproteinase-2 and -9 activity was analyzed by SDS-zymography, intracellular and secreted SPARC protein levels by Western blot, cell cycle by flow cytometry. Ukrain did not induce E-cadherin/β-catenin immunoreactivity at cell-cell boundary, although determined actin cortical expression in Caki-2 and ACHN, and did not affect vimentin organization; however, in some Caki-1 and ACHN cells the perinuclear concentration of vimentin was consistent with its down-regulation. Matrix metalloproteinase-2 and -9 activity was significantly down-regulated 48h after 20 µM Ukrain administration. At this time point Ukrain significantly decreased migration and invasion, and down-regulated SPARC levels in cell supernatants at all doses in Caki-2, and at 20 µM in Caki-1 and ACHN cells, possibly rendering tumor microenvironment less permissive for tumor invasion. Concomitantly, SPARC was up-regulated in all ccRCC cells, suggesting that Ukrain could also affect cell proliferation by cell cycle inhibition, as supported by the cell cycle analysis, since SPARC acts also as cell cycle inhibitor. Our results suggest that Ukrain may switch the EMT-related phenotype of ccRCC cells, and targets the two major aspects involved in RCC progression, such as tumor invasion/microenvironment remodeling and cell proliferation.