Suberoylanilide hydroxamic acid upregulates reticulophagy receptor expression and promotes cell death in hepatocellular carcinoma cells

Abstract

BACKGROUND

Hepatocellular carcinoma (HCC) is a common clinical condition with a poor prognosis and few effective treatment options. Potent anticancer agents for treating HCC must be identified. Epigenetics plays an essential role in HCC tumorigenesis. Suberoylanilide hydroxamic acid (SAHA), the most common histone deacetylase inhibitor agent, triggers many forms of cell death in HCC. However, the underlying mechanism of action remains unclear. Family with sequence similarity 134 member B (FAM134B)-induced reticulophagy, a selective autophagic pathway, participates in the decision of cell fate and exhibits anticancer activity. This study focused on the relationship between FAM134B-induced reticulophagy and SAHA-mediated cell death.

AIM

To elucidate potential roles and underlying molecular mechanisms of reticulophagy in SAHA-induced HCC cell death.

METHODS

The viability, apoptosis, cell cycle, migration, and invasion of SAHA-treated Huh7 and MHCC97L cells were measured. Proteins related to the reticulophagy pathway, mitochondria-endoplasmic reticulum (ER) contact sites, intrinsic mitochondrial apoptosis, and histone acetylation were quantified using western blotting. ER and lysosome colocalization, and mitochondrial Ca²⁺ levels were characterized via confocal microscopy. The level of cell death was evaluated through Hoechst 33342 staining and propidium iodide colocalization. Chromatin immunoprecipitation was used to verify histone H4 lysine-16 acetylation in the FAM134B promoter region.

RESULTS

After SAHA treatment, the proliferation of Huh7 and MHCC97L cells was significantly inhibited, and the migration and invasion abilities were greatly blocked in vitro. This promoted apoptosis and caused G1 phase cells to increase in a concentration-dependent manner. Following treatment with SAHA, ER-phagy was activated, thereby triggering autophagy-mediated cell death of HCC cells in vitro. Western blotting and chromatin immunoprecipitation assays confirmed that SAHA regulated FAM134B expression by enhancing the histone H4 lysine-16 acetylation in the FAM134B promoter region. Further, SAHA disturbed the Ca²⁺ homeostasis and upregulated the level of autocrine motility factor receptor and proteins related to mitochondria-endoplasmic reticulum contact sites in HCC cells. Additionally, SAHA decreased the mitochondrial membrane potential levels, thereby accelerating the activation of the reticulophagy-mediated mitochondrial apoptosis pathway and promoting HCC cell death in vitro.

CONCLUSION

SAHA stimulates FAM134B-mediated ER-phagy to synergistically enhance the mitochondrial apoptotic pathway, thereby enhancing HCC cell death.

Keywords: Suberoylanilide hydroxamic acid; Histone H4 lysine-16; Reticulophagy; Apoptosis; Autophagic cell death; Hepatocellular carcinoma

Core Tip: Family with sequence similarity 134 member B (FAM134B) is considered to be a tumor suppressor protein that can play a pivotal role in inhibiting hepatocellular carcinoma (HCC) cells. In addition, FAM134B acts as a putative reticulophagy receptor in the regulation of the reticulophagy process. Furthermore, suberoylanilide hydroxamic acid (SAHA) upregulates FAM134B expression in HCC cells and promotes apoptosis and autophagy-mediated cell death. Thus, FAM134B-mediated reticulophagy synergizes with SAHA to induce HCC cell death. Our findings offer novel insights into the mechanism underlying SAHA-induced HCC cell death.