Effect of gemcitabine-ALK-014 combination on MMP9 expression in primary pancreatic adenocarcinoma CELL cultures (PCCs).

Abstract

332 Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth highest cause of death in the western world. The adjuvant chemotherapy can increase the overall survivor (OS) of PDAC patients. The Gemcitabine (GEM) and alkaloids mixture (ALK-014) combination shows the increase two time the OS non resectable in PDAC patients with respect to gemcitabine treatment alone. Indeed the Alkaoids compound modulated the Matrix MetalloProteinase 9 (MMP9) in PDAC cell clines. AIM: Our goal was to investigate the MMP 9 protein modulation in primary cell cultures (PCCs) of PDAC by Gem and ALK-014 combination treatment. Methods: Two PCCs (LPc006 and LPc028) were seeded in 4 multi-well chamber slids (8,000 each/well) for cytotoxicity study by Gem [10 Nm], ALk-014 [1µm]and their combination. After 48 h of treatment the cells were stained Immunocytochemistry (IHC) using polyclonal antibody for MMP9. We used no-treated cells to evaluate basic level of MMP9 protein. No–stained cells were used as negative control. All experiments were done in triplicates. We evaluated protein expression and cellular morphology by computer for imaging analysis (D-Sight, Menarini, Italy). The expression differences was evaluated by ANOVA and t-test analyses. Results: We found a significant reduction of MMP9 expression in both cell lines treated with Gem/ALK-014 combination with respect to their controls ( p<0.01). The same drug combination reduced significantly the number of proliferating cells and showed the modification of the structure of nuclei with respect to untreated cells (p<0.001). Conclusions: New therapeutic approaches are warranted to reduce the metastatic behavior of PDAC. The present drug combination seems to be a promising tool for PDAC treatment in vitro. The new computerized approach for MMP9 evaluation could be a first quantitative attempt for protein quantification in preclinical model.