Enhancing hepatocellular carcinoma treatment: synergistic cytotoxicity and mechanistic insights of Ramucirumab and 5-Azacytidine combination therapy.

Abstract

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with limited therapeutic options and poor prognosis for advanced stages. This study investigates the synergistic anticancer effects of Ramucirumab (RAM), a VEGFR-2 inhibitor, and 5-Azacytidine (5-Aza), a hypomethylating agent, on HCC cells, focusing on mechanisms of cytotoxicity, DNA damage, apoptosis, and cell cycle modulation. HuH-7 cells were treated with RAM and 5-Aza, alone and in combination, across varying concentrations. Cell viability was assessed using the Neutral Red Uptake assay, while DNA damage and apoptosis were evaluated through the TUNEL assay and protein array analysis. The expression of cell cycle and inflammatory genes was analyzed using quantitative real-time PCR (qRT-PCR). Result shows combination treatment significantly enhanced cytotoxicity compared to individual dose-dependent therapies. DNA damage was markedly increased in RAM-treated cells, with upregulation of apoptotic proteins CAS3, BID, BAD, p53, and FAS observed. In contrast, apoptotic proteins were markedly decreased in combination-treated cells. Cell cycle arrest was evident through the downregulation of key regulatory genes, including MCM2, MCM3, cyclin B1, and CDK2. Inflammatory cytokines IL-1β and IL-6 were repressed, while COX2 expression was elevated, suggesting oxidative stress as a possible mechanism. In conclusion, the synergistic effects of RAM and 5-Aza in HCC cells are mediated through increased damage to DNA, apoptosis, and arrest of cell cycle, offering potential treatment strategy for advanced HCC. Further experiments conducted in vivo are warranted to validate these findings and optimize treatment regimens.