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

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.


Introduction
Liver cancer is the fourth most common cause of cancer-related death worldwide, resulting in over 700000 deaths in 2018 [1,2]. Due to the development of medical science, the mortality rates of other major cancers, namely, prostate, breast, and colorectal cancer, have declined, but the mortality rate of liver cancer has rapidly increased [1,3]. Hepatocellular carcinoma (HCC) is derived from hepatocytes, which account for approximately 80% of primary liver cancers [4]. 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]. However, many HCCs are chemotherapy resistant [7]. Therefore, resensitizing cancer cells to chemotherapy drugs is a potential strategy for the development of chemotherapy.
Ceramide is a sphingolipid and structural molecule of the cell membrane that regulates fluidity [8]. Ceramide was first reported to induce cell apoptosis in leukemia 30 years ago [9]. The anticancer potential of ceramide has been found against many cancer types, such as nonsmall-cell lung carcinoma [10,11], head and neck squamous cell carcinoma [12], breast cancer [13], and multiple myeloma [14]. The metabolism of sphingolipids regulates the fate of the cell. Sphingolipids are roughly divided into prosurvival sphingolipids and apoptotic sphingolipids [15]. Sphingosine and ceramide are considered apoptotic sphingolipids, inducing cell death by regulating the apoptotic pathway, including extrinsic and intrinsic pathways [16]. The metabolites of ceramide and sphingosine, such as ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), or glucosylceramide, are considered prosurvival sphingolipids that prevent the cell from undergoing apoptosis [17][18][19]. Ceramides have been reported to play an essential role in the crosstalk of protective autophagy and apoptotic autophagy [15].

Cell Viability
The viability of HA22T cells was measured by trypan blue exclusion assay [23]. Briefly, the cells were resuspended in 0.05% trypsin (#TCL034, HIMEDIA, Mumbai, India) and exposed to 0.2% trypan blue reagent. Trypan blue dye did not stain the viable cells. Then, the viable cells were counted with a hemocytometer.

Measurement of Apoptotic Cells
Apoptotic HA22T cells were assessed by annexin V/7AAD double staining. An apoptosis detection kit (Strong Biotech Corporation, Taipei, Taiwan) and 7AAD (#11397, Cayman Chemicals, Ann Arbor, MI, USA) were used for annexin V/7AAD staining, and we replaced propidium iodide with 7AAD reagent. The procedure was performed according to the manufacturer's instructions. Briefly, the treated cells were harvested and stained with annexin V/7AAD, analyzed with an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) and visualized with FlowJo 7.6.1 software (TreeStar, Inc., Ashland, OR, USA).

Next-Generation Sequencing Analysis
RNA library construction and sequencing were commissioned by Tools Biotech (BIOTOOLS, Taipei, Taiwan). The mRNAs of HCC cells will be randomly fragmented in a fragmentation buffer, and then cDNA synthesis will be performed using random hexamers and reverse transcriptase. After the first strand synthesis, a custom second strand synthesis buffer (Illumina, San Diego, CA, USA) and dNTPs, RNase H, and E. coli polymerase I were added to form a second strand. After purification, repair of the terminal, A-tailing, sequence adaptor ligation, size selection, and PCR enrichment, the final cDNA library was prepared for completion. Next, the cDNA size will be checked in the library adaptors at both ends and quantified to higher accuracy (library activity >2 nM) by quantitative PCR (Q-PCR). RNA libraries were sequenced, and the sequencing data were processed. The NGS data were clustered using Expander 7 software [30], and GO analysis was performed with the Database for Annotation, Visualization, and Integrated Discovery (DAVID) website (v6.8) [31]. The PCA and heat map were generated with the ClustVis website [32]. Gene set enrichment analysis (GSEA) was utilized to analyze the biological process alterations in the C 2 -ceramide and C 2 -ceramide/diTFPP groups. The gene set was obtained based on false discovery rate (FDR) <0.25 and p < 0.05.

AVO Staining
The assessment of autophagy will use flow cytometry-based acidic vesicular organelle (AVO) staining. Briefly, the cells were grown in 6-well plates at a density of 5 × 10 4 cells per well, cultured for 24 h, and then treated with the indicated concentration of diTFPP (from 5 to 10 µM) combined with C 2 -ceramide for 24 h. The cells were collected and then stained with 1 µg/ml acridine orange (AO) at room temperature for 15 min. 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 (green) and 650 nm (red) barrier filters supported by the Center for Research Resources and Development of Kaohsiung Medical University, Taiwan.

ROS Detection
DHE was utilized to detect intracellular ROS formation. The cells were incubated with 1 µM DHE for 20 min and washed with PBS after incubation. The stained cells were visualized with an inverted fluorescence microscope.

Statistics
The comparison of two different groups was analyzed at least in triplicate by oneway analysis of variance (ANOVA), and the comparison between pairs was analyzed by Student's t-test. A p-value < 0.05 was considered statistically significant.

diTFPP Sensitizes Hepatocellular Carcinoma Cells to C 2 -Ceramide
C 2 -Ceramide is a ceramide with a methyl group on the R chain, and it contributes to apoptosis in cancer cells [10][11][12]33]. The human hepatocellular carcinoma cell line HA22T was observed to have resistance to C 2 -ceramide, which led to a cell death rate of less than 35% at a 20 µM dose ( Figure 1B). Treatment with the phenoxyphenol compound diTFPP and 20 µM C 2 -ceramide led to a 70% cell death rate with no toxicity when diTFPP was used alone ( Figure 1B). Cell morphology was also observed; treatment with diTFPP induced considerable cell death when coadministered with 20 µM C 2 -ceramide ( Figure 1C). Next, we stained the cells with hematoxylin and observed pyknosis. Interestingly, the number of pyknotic cells increased after cotreatment with diTFPP/C 2 -ceramide ( Figure S1).

The diTFPP/C 2 -Ceramide Cotreatment Triggers Apoptosis
Apoptosis is a well-known intercellular process associated with homeostasis, autophagy, and anticancer mechanisms, resulting in programmed cell death (PCD) [34,35]. Apoptosis is related to many chemotherapy drugs, such as irinotecan and fluorouracil (5-FU) [36][37][38]. To determine whether apoptosis is associated with the role of diTFPP/C 2ceramide-induced cell death, we analyzed the cells by annexin V/7-aminoactinomycin D (7AAD) double staining by flow cytometry. The cells in quadrants I (Q I ) and IV (Q IV ) were considered early-stage and late-stage apoptotic cells, respectively. The data indicated that cotreatment with diTFPP and C 2 -ceramide increased the percentage of annexin V-positive cells, suggesting that the synergism of diTFPP and C 2 -ceramide amplified apoptotic activity in HA22T cells (Figure 2A,B). On the other hand, we also measured the expression alteration of apoptotic proteins by Western blot analysis. The extrinsic apoptotic protein caspase-8 was cleaved after diTFPP/C 2 -ceramide treatment but not in the other treatment groups. (Figure 2C). In addition, diTFPP/C 2 -ceramide cotreatment also induced the expression of caspase 8 downstream and the pro-apoptotic protein Bax as well as the cleavage of caspase-9 ( Figure 2C). Poly (ADP-ribose) polymerase-1 (PARP-1) is associated with DNA repair, rescuing cells from cell damage. During the activation of apoptosis, PARP-1 is cleaved and deactivated, resulting in DNA double strain breakage and apoptosis [39]. Cleaved PARP-1 was upregulated significantly after diTFPP/C 2 -ceramide cotreatment ( Figure 2C). Alterations in the percentage of annexin V-positive cells and the levels of proapoptotic protein markers indicated that the potential synergism of diTFPP and C 2 -ceramide plays a vital role in the activation of apoptosis.

Transcriptomic Analysis Reveals the Role of diTFPP/C 2 -Ceramide Treatment in Hepatocellular Carcinoma
Apoptosis was found to play a critical role in diTFPP/C 2 -ceramide-induced cell death, but the apoptotic mechanism of diTFPP/C 2 -ceramide is still unknown. Thus, we investigated alterations in the transcriptome after diTFPP/C 2 -ceramide treatment by next-generation sequencing (NGS). The results showed that the expression of over 20,000 genes was altered after 24 h of treatment with diTFPP, C 2 -ceramide, or diTFPP/C 2ceramide. Principal component analysis (PCA) was utilized to analyze the similarity of the transcriptome after the treatments. The cotreatment with the diTFPP/C 2 -ceramide group was found to be an immense distance from the other groups, suggesting that the cotreatment resulted in considerable alterations to the transcriptome in the other groups ( Figure 3A). The expression of ceramide metabolism genes was investigated from NGS data. C 2 -ceramide treatment upregulated most of the ceramide metabolic genes, but cells cotreated with diTFPP with C 2 -ceramide showed an expression map distinct from that of those treated with C 2 -ceramide only. The results revealed alterations in sphingolipid metabolic genes ( Figure 3B,C). Ceramides can be categorized as prosurvival or proapoptotic ceramides. Ceramide and sphingosine are associated with the proapoptotic function that induces endoplasmic reticulum stress, which activates the unfolded protein response and ultimately results in apoptosis [40,41]. On the other hand, the prosurvival class of ceramides usually comprises metabolites of ceramide and sphingosine, such as glucosylceramide (GlcCer), ceramide-1-phosphate (C1P), or sphingosine-1-phosphate (S1P), all of which inhibit caspase activation and induce autophagy to prevent apoptosis and induce cell proliferation [8,18,19,42]. Therefore, the balance of ceramides is critical in the initiation of apoptosis and regulation of cell fate [8]. The expression of genes that catalyze proapoptotic ceramides into prosurvival ceramides, such as SPHK1, SPHK2, CERK, and UGCG, was upregulated after C 2 -ceramide treatment ( Figure 3B). However, the changes in response to C 2 -ceramide treatment were subverted by cotreatment with diTFPP. In contrast, the expression of the genes ASAH1 and ASAH2, which mediate sphingosine catalysis into ceramide, was upregulated ( Figure 3B). These alterations in the expression of ceramide metabolic proteins suggested that the potential mechanism of diTFPP/C 2ceramide treatment is apoptosis activation. To investigate the potential role of diTFPP in this process, we analyzed the NGS data of C 2 -ceramide and C 2 -ceramide/diTFPP treatment with gene set enrichment analysis (GSEA) and found that the combined treatment of diTFPP and C 2 -ceramide decreased the gene sets enriched in DNA replication and cell cycle transition pathways ( Figure 3D,E), suggesting that diTFPP treatment induces cell cycle arrest.
Additionally, we clustered the NGS data by gene expression level and found 1986 downregulated genes and 2240 upregulated genes ( Figure 3F,H). Gene ontology (GO) analysis was utilized to analyze the upregulated and downregulated genes. Downregulated genes were found to be primarily associated with the cell cycle and mitosis, suggesting that cotreatment with diTFPP/C 2 -ceramide results in cell cycle and mitosis arrest ( Figure 3G). On the other hand, upregulated genes were found to be associated with protein transport, ROS homeostasis, and autophagy ( Figure 3I).

The Autophagy Regulatory Mechanism Plays a Critical Role in diTFPP/C 2 -Ceramide-Induced Apoptosis
Autophagy plays a role in the apoptotic pathway, and the fate of cells-either cell survival or cell death-depends on the regulatory mechanism [43]. From the NGS data, autophagic genes were found to be upregulated, suggesting that autophagy plays an essential role in diTFPP/C 2 -ceramide-induced cell apoptosis. Acidic vesicular organelle (AVO) staining was utilized to detect the generation of autophagosomes to verify the activation of autophagy. The results indicated an increase in AVO signaling after diTFPP/C 2 -ceramide cotreatment ( Figure 4A,B). Nevertheless, microtubule-associated protein 1A/1B light chain 3B (LC3B) was modified to LC3B-I and LC3B-II and showed an additive effect after diTFPP or C 2 -ceramide treatment. Additionally, the expression of Beclin-1, a protein associated with autophagosome formation, increased. The alterations in protein expression reflect autophagy activation ( Figure 4C). Additionally, we treated the cells with the autophagy inhibitor 3-MA before treatment with C 2 -ceramide and diTFPP and observed that the levels of the apoptotic marker cleaved PARP-1 were significantly decreased in the 3-MA-treated group ( Figure S2). (B) Quantification of (A). ns, no significant p>0.05, ** p < 0.01, *** p < 0.001 compared with the control groups. #### p < 0.0001 compared with the C 2 -ceramide alone group; all data are presented as the means ±SD of four independent experiments. (C) Western blot analysis of autophagic proteins after 24 h of diTFPP/C 2-ceramide treatment. GAPDH as an internal control.

ROS Formation Is Involved in diTFPP/C 2 -Ceramide-Induced Cell Death
The NGS data also demonstrated that genes related to the response to H 2 O 2 and cell redox homeostasis were upregulated, suggesting that ROS play a role in diTFPP/C 2ceramide treatment. ROS formation in the cell was measured with dihydroethidium (DHE), which is a superoxide-targeting red fluorescence reagent. The results indicated that combined treatment with diTFPP and C 2 -ceramide significantly induced the formation of ROS ( Figure 5A,B). Additionally, ROS-associated proteins such as catalase were found to increase after diTFPP/C 2 -ceramide treatment. The superoxide dismutase 1 (SOD1)-to-superoxide dismutase 2 (SOD2) "switch" phenomenon occurred, which was also observed in our previous study and was associated with autophagic apoptosis ( Figure 5C). Mitochondria are the major source of ROS production [44]. We observed the generation of mitochondrial ROS with MitoSOX red and found that the C 2 -ceramide/diTFPP combined treatment induced considerable mitochondrial ROS production in HA22T cells ( Figure S3). In addition, the ROS inhibitor NAC was utilized to inhibit the formation of ROS to observe the effect of ROS on C 2 -ceramide/diTFPP-induced apoptosis. The results indicated that pretreatment with NAC blocked the formation of cleaved PARP-1 and consequently inhibited apoptosis ( Figure S2).

Discussion
HCC is a severe disease that was responsible for over 700,000 deaths in 2018 [1,2]. The results presented in this study indicated the apoptotic role of C 2 -ceramide and diTFPP cotreatment in the HA22T HCC cell line. The cells treated with C 2 -ceramide exhibited pyknosis, which is a cellular process that reduces cellular and nuclear volume and is considered a characteristic of apoptosis or necrosis [45,46]. Although both cell death processes can involve pyknosis, annexin V/PI staining and the expression of apoptotic proteins revealed the apoptotic role of C 2 -ceramide/diTFPP treatment.
The assessment of sphingolipid metabolism provided evidence that the disruption of ceramide metabolism induces cell apoptosis or survival. Sphingolipid metabolites show two different mechanisms: some induce cell apoptosis, while others promote cell survival [40]. Treatment with C 2 -ceramide upregulated most of the ceramide metabolic genes, catalyzing apoptotic ceramide into glycosylceramide, ceramide-1-phosphate, and sphingosine-1-phosphate, which are known for cell survival ( Figure 3B), to maintain healthy homeostasis from exogenous ceramide administration. UGCG is a glucosylceramide synthase that catalyzes the formation of glucosylceramide from ceramide [47]. UGCG has been reported to be a regulator of Akt activation and a promoter of cell proliferation [48]. Salustiano and Previato also discovered that UGCG is involved in multidrug resistance [49]. The synthesis of ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) is associated with ceramide kinase (CERK) and sphingosine kinase (SPHK), which phosphorylate ceramide and sphingosine, respectively [50]. C1P and S1P play a role in cell survival by regulating members of the Bcl family to inhibit apoptosis [51]. Sphingosine and ceramide are sphingolipids with pro-apoptotic functions [40]. Akt, c-Myc, and Bcr-Abl are oncogenes that have been reported to be suppressed by the regulatory mechanisms of ceramide and sphingosine synthesis [52][53][54]. Ceramide has also been reported as a necessary mediator of caspase-3 cleavage in response to radiation [55]. The disruption of C 2 -ceramide signaling indicated the potential pathway by which diTFPP induces apoptosis.
In this study, ROS played a vital role in diTFPP/C 2 -ceramide-induced cell death. According to the alteration of sphingolipid metabolism, we assumed that ROS production is associated with sphingolipid disturbance. In our previous study, C 8 -ceramide induced lung cancer apoptosis by producing ROS and disrupting superoxide dismutase (SOD) expression [11]. Knupp and Chang indicated that mutant-induced sphingolipid accumulation led to mitochondrial dysfunction and ROS production [56]. The study provided a view of sphingolipid-induced ROS production associated with the dysregulation of mitochondria. In an early study, exogenous ceramide was found to inhibit the activity of mitochondrial respiratory chain (MRC) proteins and induce the production of ROS [57][58][59]. The NGS data also showed that mitochondrial (mt)-proteins were all upregulated after diTFPP/C 2 -ceramide treatment (data not shown). The alteration of mt-protein expression might compensate for MRC inhibition by ceramide accumulation.
Autophagy also plays a vital role in apoptosis, regulating the fate of cells from survival to death [60]. The crosstalk of apoptosis and autophagy has been reported to be associated with sphingolipid metabolism [15]. As mentioned before, ceramide inhibits Akt protein kinase via pyrophosphatase protein 2A (PP2A), resulting in the activation of mammalian target of rapamycin (mTOR)-mediated autophagy [61,62]. Ceramide is also associated with the transcription and lipidation of LC3, an autophagic protein associated with autophagosome generation during lipidation [63]. Sun and Zhu reported that ceramide treatment mediated JNK activation and the phosphorylation of c-Jun and was associated with the transcription and lipidation of LC3 [63]. We also observed the upregulation of endoplasmic reticulum (ER) stress response proteins, indicating that ceramide-mediated ER stress regulates the activation of autophagy [15] (data not shown). These results indicate that diTFPP/C 2 -ceramide-mediated cell death is multifactorial and is associated with autophagy, mitochondrial dysfunction, ROS production, and ER stress-mediated responses.

Conclusions
The results indicated that diTFPP disrupts C 2 -ceramide metabolism, leading to the activation of autophagy, which results in the formation of ROS and ultimately induces cell apoptosis in HA22T hepatocellular carcinoma ( Figure 6). Figure 6. The potential regulatory mechanism of diTFPP in C 2 -ceramide-mediated cell death. Endogenous sphingolipid metabolism favors the conversion of exogenous C 2 -ceramide to prosurvival sphingolipids and causes the attenuation of C 2ceramide-induced anti-HCC effects, including anti-proliferation and apoptosis induction in HCC cells. In contrast, cotreatment with C 2 -ceramide and diTFPP promotes the switching of sphingolipid metabolism from prosurvival to proapoptosis, enhances ROS production and autophagic stress, and eventually sensitizes HCC cells to C 2 -ceramide-induced apoptosis.

Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study are available within the article.