Unraveling the Effects of Epigallocatechin-3-gallate on Hepatocellular Carcinoma Cells: A Comparative Analysis of Monolayer vs Multicellular Tumor Spheroids

The tumor microenvironment is a complex milieu that has not been properly studied in cells cultured in conventional monolayer. Studies have demonstrated the antitumor activity of epigallocatechin-3-gallate (EGCG), present in green tea, using monolayer cultures without considering the three-dimensional microenvironment of a tumor. Furthermore, many studies have shown the effect of EGCG on the transcriptional profile of cancer cells, but each study has been limited to only one or a few cell types. Using the LINCS database, we characterized the gene signatures produced by EGCG treatment in different cell types and reported a variation in EGCG-induced gene signatures depending on the cell type analyzed. GSEA analysis revealed that EGCG influenced multiple biological pathways related to cell signaling, proliferation, epigenetic modifications, and the tumor microenvironment. Then, we cultured hepatocellular carcinoma cells (HepG2) as multicellular tumor spheroids (MTS) to evaluate the effects of EGCG on growth, morphological integrity, cell migration, and cell viability in MTS. We also evaluated the expression of genes related to cell survival and proliferation (IL6, TNF, RELA, BAX, BCL2), chromatin modification and DNA methylation (EZH2, KDM1A, HAT1, DNMT3A), and cell adhesion (CDH1, CD44, ITGB2, MMP2). The cell culture condition influenced EGCG effects on gene expression and cell viability, with more significant effects in monolayer than in MTS. After 15 days, control MTS showed cellular dissociation, whereas EGCG-treated MTS showed decreased cell viability and no growth. ECGG blocked the migration of MTS cells into Matrigel and decreased the expression of matrix metalloproteinase MMP2. These results suggest that EGCG could prevent cell migration from small nonirrigated tumors in vitro by affecting cell adhesion molecules such as MMP2, decreasing the catalytic activity of enzymes associated with metastasis.