Investigation of the effect of tazemetostat on hepatocellular carcinoma cells through the NF-κB pathway using in vitro and in silico methods

Primary liver cancer is considered the seventh most common and second leading cause of death worldwide; globally, hepatocellular carcinoma (HCC) accounts for approximately 75% of these cases. Several genetic and epigenetic alterations are involved in the process of carcinogenesis by affecting signaling pathways together. Tazemetostat is a drug developed for the inhibition of the histone methyltransferase EZH2, which can cause epigenetic changes when abnormal expression is involved. Another pathway implicated in cancer is NF-κB/IKK, which is associated with and plays a central role in regulating many biological processes such as cell growth, proliferation, differentiation, viability, apoptosis. Inhibition of NF-kB prevents cancer development, while its activation is associated with epithelial-mesenchymal transition (EMT) leading to invasion and metastasis of tissues. In this thesis, it was aimed to investigate the effect of tazemetostat on the viability and proliferation of HEPG2 (hepatocellular carcinoma) cells, the effects on the expression of MMP2 and MMP9 genes and the relationship between the drug and the NF-kB pathway. Immunocytochemistry was used to measure the levels of MMP2 and MMP9 proteins, which play an active role in the EMT process. Molecular docking was used to measure the interaction between NF-kB and Tazemetostat, ELISA was used to measure NF-kB activity, and qRT-PCR was used to analyze MMP2 and MMP9 gene expression. Molecular docking results showed that Tazemetostat exhibited affinity for the NF-kB p65 subunit with negative binding energy (kcal/mol). Different doses of Tazemetostat (5 µM, 12 µM, 30 µM, 60 µM, and 100 µM) reduced the proliferation and viability of HEPG2 and HUVEC cells and NF-κB p65 activity in a dose-dependent manner, with a more significant and pronounced reduction observed in HEPG2 cells. Doses of 60 µM and 100 µM of tazemetostat reduced the expression of MMP2 and MMP9 genes and MMP2 and MMP9 proteins in HEPG2 cells, while the 100 µM dose reduced the expression of MMP2 and MMP9 genes and MMP2 and MMP9 proteins in HUVEC cells.