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 C60nanofilm 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 C60nanofilm on restoring the epithelial phenotype at the protein level with protein membranes, Western blotting and mass spectrometry. C60nanofilm downregulated TGF and increased glutathione expression in SNU-449 cells. When grown on C60nanofilm, 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 C60nanofilm, 41 proteins are involved in EMT and EMT-modulating processes such as autophagy, inflammation and oxidative stress. The C60nanofilm 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 C60nanofilm induces a hybrid mesenchymal–epithelial phenotype and could be used in the prevention of postoperative recurrences.
上皮-间质转化（EMT）是指上皮细胞通过向间质表型转变获得主动迁移能力的过程。该机制常发生于细胞因子与活性氧富集而营养物质匮乏的微环境中。本研究旨在证明富勒烯C60纳米膜可通过恢复肝癌细胞非侵袭性上皮表型抑制其侵袭能力。实验采用上皮型与间质型HepG2、SNU-449肝癌细胞及非癌性间质HFF2细胞，通过酶联免疫吸附法测定细胞内谷胱甘肽和转化生长因子含量，采用商品化试剂盒检测总抗氧化能力，通过形态学变化、划痕实验和Boyden小室侵袭实验评估细胞侵袭行为。此外，运用蛋白膜技术、Western印迹和质谱分析在蛋白水平检测C60纳米膜对上皮表型恢复的影响。结果显示：C60纳米膜可下调SNU-449细胞中TGF表达并增强谷胱甘肽表达；侵袭性细胞在C60纳米膜上培养时表现出增强的细胞间连接性、降低的三维侵袭能力，以及关键侵袭标志物（MMP-1、MMP-9、TIMP-1、TIMP-2和TIMP-4）表达减少；质谱分析表明，在C60纳米膜上培养的HepG2细胞中发生变化的96种蛋白中，有41种蛋白参与EMT及其调控过程（如自噬、炎症和氧化应激）。C60纳米膜能抑制自噬、展现抗氧化与抗炎特性、促进葡萄糖转运，并调控β-连环蛋白/角蛋白/Smad4/snail+slug信号通路及MMP信号通路。综上所述，C60纳米膜可诱导间质-上皮混合表型的形成，有望应用于预防术后复发。
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