Investigating the role of SIRT 1/Autophagy/NF-kB regulatory axis in chemoresistance in Hepatocellular Carcinoma (HCC)

Abstract

Liver cancer is the fifth most common cancer in the world and the third leading cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) which originates from the main liver cells (hepatocytes), accounting for about 90% of total liver cancer cases. HCC remains a major challenge in health as patients are often detected in the late stage with high mortality. HCC therapy becomes even more challenging in patients with drug resistance. Sirtuin 1 (SIRT 1), a member of the sirtuin family is known to regulate various oncogenic events such as cell survival, apoptosis, autophagy, tumourigenesis, metastasis and drug resistance in various cancers, but its role in HCC, particularly in chemoresistance is underexplored. Besides SIRT 1, the event of autophagy and nuclear factor kappa B (NF-κB) signaling are also involved in cancer chemoresistance. However, the regulatory role of the SIRT 1/autophagy/NF-κB signaling in HCC chemoresistance remains unclear. In the present study, a multikinase inhibitor sorafenib, was selected as a chemotherapeutic agent to generate chemoresistance cell lines. Sorafenib stands out as the first-line systemic treatment for advanced unresectable HCC and has demonstrated a notable improvement in patient survival. Cell viability was measured using MTS assay. Clonogenic assays were performed to determine the colony-forming ability of the parental and its respective chemoresistant cells. The expression of ATP binding cassette (ABC) transporters and the expression of SIRT 1, autophagy, and NF-κB were examined by western blot. Sorafeni resistant HCC cells demonstrated increased IC50 values of sorafenib, a greater number of clones were formed, and elevated expression of ATP binding cassette (ABC) transporters includes ABCB1, ABCC1 and ABCG2, which confirmed the acquired sorafenib. resistance. Next, a high expression of SIRT 1 was seen in chemoresistant HepG2 and Huh-7 cells when compared to its respective parental cells. Activation of autophagy was seen in chemoresistant Huh-7 cells, evidenced by reduction LC3A-I and LC3B-I expression, and increased LC3A-II and LC3B-II expression were observed, suggesting the conversion of LC3-I to LC3-II and reflecting the progression of autophagy. However, the upregulation of autophagy was not seen in chemoresistant HepG2 cells, suggesting that this upregulation could be cell-dependent, which requires further investigations. Activation of autophagy was significantly inhibited by silencing SIRT 1 expression, while NF-κB remains active following SIRT 1 -knockdown in both parental and resistance cell. Overall, the results of the present study suggest that autophagy is highly regulated by SIRT 1, while regulation of NF-κB may involve other signaling pathways. SIRT/ autophagy / NF-κB signaling provides a novel perspective on regulation of chemoresistance in HCC