Polyhydroxylated Fullerene C₆₀(OH)₄₀ Nanofilms Promote the Mesenchymal–Epithelial Transition of Human Liver Cancer Cells via the TGF-β1/Smad Pathway

Abstract

Background

The various growth factors change the phenotype of neoplastic cells from sedentary (epithelial) to invasive (mesenchymal), which weaken intercellular connections and promote chemotaxis. It can be assumed that the use of anti-inflammatory polyhydroxyfull nanofilms will restore the sedentary phenotype of neoplastic cells in the primary site of the tumor and, consequently, increase the effectiveness of the therapy.

Methods

The studies were carried out on liver cancer cells HepG2, C3A and SNU-449, and non-cancer hepatic cell line THLE-3. Transforming growth factor (TGF), epidermal growth factor and tumor necrosis factor were used to induce the epithelial–mesenchymal transition. C₆₀(OH)₄₀ nanofilm was used to induce the mesenchymal–epithelial transition. Obtaining an invasive phenotype was confirmed on the basis of changes in the morphology using inverted light microscopy. RT-PCR was used to confirm mesenchymal or epithelial phenotype based on e-cadherin, snail, vimentin expression or others. Water colloids at a concentration of 100 mg/L were used to create nanofilms of fullerene, fullerenol, diamond and graphene oxide. The ELISA test for the determination of TGF expression and growth factor antibody array were used to select the most anti-inflammatory carbon nanofilm. Mitochondrial activity and proliferation of cells were measured by XTT and BrdU tests.

Results

Cells lost their natural morphology of cells growing in clusters and resembled fibroblast cells after adding a cocktail of factors. Among the four allotropic forms of carbon tested, only the C₆₀(OH)₄₀ nanofilm inhibited the secretion of TGF in all the cell lines used and inhibited the secretion of other factors, including insulin-like growth factor system. Nanofilm C₆₀(OH)₄₀ was non-toxic to liver cells and inhibited the TGF-β1/Smad pathway of invasive cells treated with the growth factor cocktail.

Conclusion

The introduction of an anti-inflammatory, nontoxic component that can induce the mesenchymal–epithelial transition of cancer cells may represent a future adjuvant therapy after tumor resection.

Graphical Abstract

Keywords:

• carbon allotropes
• epithelial phenotype
• growth factors
• hepatocellular carcinoma
• mesenchymal phenotype
• metastasis

Acknowledgments

This report is a part of Malwina Sosnowska’s habilitation thesis.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This research was carried out in the framework of National Science Centre Poland project number 2019/33/N/NZ7/01392.