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Bovine papillomavirus: from mutation to metastasis

Araldi RP1, Magnelli RF2, Frare EO2,2, Módolo DG2, Mazzuchelli-de-Souza J3, Carvalho MACR3,2, Spadacci-Morena DD4, De-Sá-Junior PL2, Costa AS2, Souza DM3, Carvalho RF2, Beçak W2, Stocco RC2,

1 Biotecnologia, Universidade de São Paulo ; 2 Laboratório de Genética, 4 Laboratório de Fisiopatologia, Instituto Butantan, Brasil; 3 Programa de Pós-Graduação Interunidades em Biotecnologia, Universidade de São Paulo, Instituto Butantan, Instituto de Pesquisas Tecnológicas Universidade de

Introduction: Bovine papillomavirus (BPV) is the aetiological agent of bovine papillomatosis, infectious disease characterized by the presence of multiple papillomas, which can regress spontaneously or progress to malignancy. Moreover, BPV is considered a useful model for HPV oncogenic process studies. Evidences have been suggested that BPV promotes cancer initiation, progression and also induces epithelial-mesenchymal transition (EMT), contributing to cell migration and metastatic process. Reactive oxygen species (ROS) are considered the major promoter of EMT.  Objectives: This study evaluated the role of BPV in ROS production and its association to EMT in different cell lineages: normal skin uninfected by BPV (control), three lines of cutaneous papilloma (P01, P02 and P03) obtained from three afflicted bovines and one line obtained from bovine esophagus carcinoma (EC) (Ethic Committee process 1319/14). Methods: All samples were subjected to molecular BPV diagnosis, confirming the absence of viral sequences in control and the co-infection in papillomas and EC lineages (BPV-1, 2 and 4). DNA damages were analyzed by comet assay, considered that they are always occur in cancer initiation.  Cell morphological changes were verified by inverted microscopy using phase contrast. Mitochondrial membrane potential (ΔΨm) was analyzed using the MitoTracker probe. Disruption of ΔΨm indicates cell metabolic changes which are associated to ATP supply and apoptosis. ROS production was verified using the DCFH-DA assay.  ΔΨm and ROS production were analyzed qualitatively by immunofluorescence and quantitatively by flow cytometer. Results and Discussion: Comet assay indicated clastogenesis in both papilloma and EC. These lines also revealed morphological changes, including the filopodia and lamelipodia formation, which suggest cell migration and EMT. Mitochondrial membrane potential analysis pointed out increase ΔΨm in papillomas in relation to control. This data suggest a BPV anti-apoptotic effect. However, EC’s cells showed a reduction of ΔΨm. This data suggest a reduction of ATP production by oxidative phosphorilation, resulting in anaerobic glycolysis (Warburg effect). DCFH-DA assay pointed out a reduction of ROS production in EC’s cell, reinforcing the results. This is the first study that evaluated the role of BPV in metabolic change inducement. These data emphasize that BPV participate of all stages of carcinogenesis, from mutation to metastasis.

Supported by FAPESP

7 - CELLULAR BIOLOGY AND GENETICS 21th Annual Scientific Meeting of Instituto Butantan.