The Phenoxyphenol Compound diTFPP Mediates Exogenous C2-Ceramide Metabolism, Inducing Cell Apoptosis Accompanied by ROS Formation and Autophagy in Hepatocellular Carcinoma Cells

Abstract

Hepatocellular carcinoma (HCC) is a severe disease that accounts for 80% of liver cancers. Chemotherapy is the primary therapeutic strategy for patients who cannot be treated with surgery or who have late-stage HCC. C2-ceramide is an effective reagent that has been found to inhibit the growth of many cancer types. The metabolism of C2-ceramide plays a vital role in the regulation of cell death/cell survival. The phenoxyphenol compound 4-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(4-hydroxyphenoxy)phenyl]phenoxy}phenol (diTFPP) was found to have a synergistic effect with C2-ceramide, resulting in considerable cell death in the HA22T HCC cell line. diTFPP/C2-ceramide cotreatment induced a two- to threefold increase in cell death compared to that with C2-ceramide alone and induced pyknosis. Annexin V/7-aminoactinomycin D (7AAD) double staining and Western blotting indicated that apoptosis was involved in diTFPP/C2-ceramide cotreatment-mediated cell death. We next analyzed transcriptome alterations in diTFPP/C2-ceramide-cotreated HA22T cells with next-generation sequencing (NGS). The data indicated that diTFPP treatment disrupted sphingolipid metabolism, inhibited cell cycle-associated gene expression, and induced autophagy and reactive oxygen species (ROS)-responsive changes in gene expression. Additionally, we assessed the activation of autophagy with acridine orange (AO) staining and observed alterations in the expression of the autophagic proteins LC3B-II and Beclin-1, which indicated autophagy activation after diTFPP/C2-ceramide cotreatment. Elevated levels of ROS were also reported in diTFPP/C2-ceramide-treated cells, and the expression of the ROS-associated proteins SOD1, SOD2, and catalase was upregulated after diTFPP/C2-ceramide treatment. This study revealed the potential regulatory mechanism of the novel compound diTFPP in sphingolipid metabolism by showing that it disrupts ceramide metabolism and apoptotic sphingolipid accumulation.

Antioxidant Effects of Irisin in Liver Diseases: Mechanistic Insights

“…What is more, Wang et al demonstrated that EPS364, a deep-sea bacterial exopolysaccharide, exhibited an antitumor trait by inducing hepatic cell death by upregulating ROS production, supporting the idea that oxidative stress could trigger cell death [45]. And the idea is likewise proofed by another research where diTFPP/C2-ceramide increased hepatic cell death by increasing ROS levels [39]. Evidence has shown that the inhibition of ROS production can alleviate abnormal cell death.…”

“…It is found that autophagy participated in the repair of ROS-induced oxidative proteins in LO2 cells [38]. Chang et al found that the production of ROS was elevated in diTFPP/C2-ceramide-treated cells with the activation of autophagy, proving autophagy is involved in the oxidative processes [39]. The induction of autophagy also helps to suppress oxidative stress.…”

Zebrafish Model-Based Assessment of Indoxyl Sulfate-Induced Oxidative Stress and Its Impact on Renal and Cardiac Development

“…Library construction and RNA sequencing (RNAseq) were performed by BIOTOOLS (Taipei, Taiwan) based on the manufacturer’s instructions [ 24 ]. In brief, the total RNA from zebrafish embryos treated with IS was analyzed by RNAseq using oligo(dT)-labeled magnetic beads.…”