Influence of C60 Nanofilm on the Expression of Selected Markers of Mesenchymal–Epithelial Transition in Hepatocellular Carcinoma

Abstract

The epithelial–mesenchymal transition (EMT) is a process in which epithelial cells acquire the ability to actively migrate via a change to the mesenchymal phenotype. This mechanism occurs in an environment rich in cytokines and reactive oxygen species but poor in nutrients. The aim of this study was to demonstrate that the use of a fullerene C60 nanofilm can inhibit liver cancer cell invasion by restoring their non-aggressive, epithelial phenotype. We employed epithelial and mesenchymal HepG2 and SNU-449 liver cancer cells and non-cancerous mesenchymal HFF2 cells in this work. We used enzyme-linked immunosorbent assays (ELISAs) to determine the content of glutathione and transforming growth factor (TGF) in cells. We measured the total antioxidant capacity with a commercially available kit. We assessed cell invasion based on changes in morphology, the scratch test and the Boyden chamber invasion. In addition, we measured the effect of C60 nanofilm on restoring the epithelial phenotype at the protein level with protein membranes, Western blotting and mass spectrometry. C60 nanofilm downregulated TGF and increased glutathione expression in SNU-449 cells. When grown on C60 nanofilm, invasive cells showed enhanced intercellular connectivity; reduced three-dimensional invasion; and reduced the expression of key invasion markers, namely MMP-1, MMP-9, TIMP-1, TIMP-2 and TIMP-4. Mass spectrometry showed that among the 96 altered proteins in HepG2 cells grown on C60 nanofilm, 41 proteins are involved in EMT and EMT-modulating processes such as autophagy, inflammation and oxidative stress. The C60 nanofilm inhibited autophagy, showed antioxidant and anti-inflammatory properties, increased glucose transport and regulated the β-catenin/keratin/Smad4/snail+slug and MMP signalling pathways. In conclusion, the C60 nanofilm induces a hybrid mesenchymal–epithelial phenotype and could be used in the prevention of postoperative recurrences.