Pollonein-LAAO: uma nova L-aminoácido oxidase isolada de Bothrops moojeni induz estresse oxidativo em células tumorais de próstata e células endoteliais interferindo na progressão tumoral in vitro

Abstract

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to their progression. Due to the importance of ROS in promoting malignant phenotype, it has been widely accepted that augmenting intracellular ROS levels above threshold can trigger cancer cell death. In this framework, we hypothesized that the change in ROS concentration of prostate cancer cells (PC-3) and endothelial cells (HUVECs) could impair critical steps of turmor progression in vitro. Therefore, the purpose of this study was to evaluate the use of a new L-amino acid oxidase obtained from Bothrops moojeni venom (Pollonein-LAAO) as a ROS-based anti-tumorigenic and anti-angiogenic agent. Our results demonstrated that Pollonein-LAAO was cytotoxic to PC-3 cells in two-dimensional (IC50=2.4 µg/mL) and three-dimensional (tumor spheroid) in vitro assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and impaired cell cycle progression by causing G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited PC-3 cell migration, invasion and adhesion in collagen IV and fibronectin. Those results may be explained by the ability of Pollonein-LAAO to down-regulate genes that participate in critical steps of the cellular invasion process (SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3). We also observed that Pollonein-LAAO increased intracellular ROS levels, which culminate in reduction of endothelial capillary-tube formation in matrigel model and inhibition of critical angiogenesis steps such as migration, invasion and adhesion. Moreover, Pollonein-LAAO exhibited significant anti-angiogenic properties in 3D co-culture models. Those results may be explained to Pollonein-LAAO negatively modulates expression of genes that participate in critical steps of tumor angiogenesis (VEGFA, ANGPT-2, MMP-2 and MMP-9). Interestingly, Pollonein-LAAO decreased VEGF and α2β1 protein expression and reduced formation of actin fiber stress. Furthermore, the Pollonein-LAAO effects in PC-3 and HUVECs were abolished in the presence of catalase. Altogether, these results demonstrate that oxidative stress plays an intriguing role in the mechanism underlying tumor survival and invasion and tumor angiogenesis, since Pollonein-LAAO affected several malignant phenotypes. In this context, Pollonein-LAAO is an intriguing ROS-based agent that can be used as a biotechnological tool to enhance the current understanding of cancer treatment strategies.