Estudo do efeito antitumoral da associação da metformina com a nanoparticula de Poli(ácido lático-co-glicólico) de metotrexato em células de adenocarcinoma ductal pancreático

Abstract

Pancreatic ductal adenocarcinoma (PDAC) corresponds to 90% of malignant pancreatic tumors and one of the main risk factors for this cancer is type 2 diabetes mellitus, as well as smoking and obesity. Metformin is an oral anti-glycemic widely used in the treatment of diabetes mellitus. In addition to this property of use, metformin has recently been reported to have antitumor activity. The aim of this study was to perform the in vitro evaluation of the pró-apoptotic effect by combining metformin and methotrexate-associated PLGA nanoparticle, a standard chemotherapy used in antineoplastic treatment. To this end, Alamar Blue viability tests, annexin / PI flow cytometry and immunofluorescence with anti-caspase-3, anti-bcl-2, and anti-FADD antibodies in pancreatic ductal adenocarcinoma (PANC-1) cells were performed. and in human kidney embryonic cells (HEK-293). The internalization of FITC-labeled nanoparticles by immunofluorescence was also evaluated. Evaluation of drug resistance and cell survival pathway were done by analyzing mRNA expression of the MDR1, survivin, EGFR, Akt, and mTOR genes by RT-PCR. The results showed that isolated doses of metformin and nanoparticle do not induce significant apoptosis, however, the combined use induced up to 55.3% of apoptosis. The high expression of FADD and caspase-3 was statistically significant with p <0.01, as well as the low expression of bcl-2, with p <0.05, in the 48 hours period when combined with metformin and PLGA- MTX, showing activation of apoptosis by both extrinsic and intrinsic pathways. Moreover, the increase of GSH and reduction of MDA reveal antioxidant effect of treatment in both strains. Nanoparticles reduced MDR1, survivin, EGFR drug resistance gene expression with p <0.0001 in all combination treatments and also reduced Akt and mTOR survival life gene expression with p <0.0001 in all treatments. all treatments combined. The use of metformin prior to PLGA-MTX increases tumor cell apoptosis induction, reduces resistance to antitumor treatment and inhibits cell survival pathways. In this context, it is concluded that metformin enhances the antitumor activity of PLGA-MTX, suggesting that it is a promising integrated system in the future when used in combination within a nanoparticle system.