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.
Highlights
Liver cancer is the fourth most common cause of cancer-related death worldwide, resulting in over 700000 deaths in 2018 [1,2]
After the staining solution was removed, the cells were washed with phosphate-buffered saline (PBS) and immediately analyzed in an LSR II flow cytometer using 488-nm bandpass blue excitation filters and 515 nm and 650 nm barrier filters supported by the Center for Research Resources and Development of Kaohsiung Medical University, Taiwan
C2 -Ceramide is a ceramide with a methyl group on the R chain, and it contributes to apoptosis in cancer cells [10,11,12,33]
Summary
Liver cancer is the fourth most common cause of cancer-related death worldwide, resulting in over 700000 deaths in 2018 [1,2]. Chemotherapy is one of the most critical therapeutic procedures against advanced HCC, and it acts by inducing programmed cell death (PCD), especially apoptosis [5,6]. Sphingosine and ceramide are considered apoptotic sphingolipids, inducing cell death by regulating the apoptotic pathway, including extrinsic and intrinsic pathways [16]. As indicated in our previous studies, autophagy plays a role in many anticancer drug treatments and leads to cell death or cell survival [10,21,22,23]. ROS are involved in autophagy, apoptosis, and apoptotic cell death regulation. ROS were shown to be associated with chemotherapeutic drug treatment and apoptosis. In this study, we revealed the role of diTFPP in sensitizing HCC to C2 -ceramide by activating the ROS/autophagy pathway.
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