Phenethyl isothiocyanate inhibits the carcinogenic properties of hepatocellular carcinoma Huh7.5.1 cells by activating MAPK/PI3K-Akt/p53 signaling pathways

Cell culture and reagents

The human HCC Huh7.5.1 cells were procured from the China Typical Culture Collection Center (CCTCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (catalog no. C11995500BT; Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (catalog no. 04-001-1ACS; Biological Industries, Beit Haemek, Israel) and 1% v/v penicillin-streptomycin (catalog no. P1400; Solarbio, Beijing, P. R. China). The Huh7.5.1 cells used in this study were within the range of passages 5 to 10. Cells were incubated at 37 °C with a 5% CO₂ concentration.

Purified PEITC (>98%) was purchased from Aladdin (catalog no. 2257-09-2; Merck, Rahway, NJ, USA) and dissolved in dimethylsulphoxide (DMSO) (catalog no. D8371; Solarbio, Beijing, P. R. China) to prepare a 500 mmol/L stock solution, which was then stored at −80 °C.

MTT assay and colony formation assay

MTT assay was used to determine the anti-proliferative effect of PEITC on Huh7.5.1 cells. Huh7.5.1 cells were seeded in triplicate in 96-well plates (3 × 10³ cells per well) and incubated overnight. After treatment with varying concentrations of PEITC for 48 h, cells in each well were incubated with 30 µL of MTT (catalog no. 298-93-1; Solarbio) at 37 °C for 3.5 h. Once incubation was complete, the supernatant was discarded and 150 µL of DMSO (catalog no. D103272; Aladdin, Shanghai, China) was added into each well to fully dissolve the formazan crystal. To determine the absorbance at the wavelength of 490 nm in individual wells, a microplate spectrometer (SPARK, Tecan, Swiss) was used. The half maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 9.0 software. The cell survival rate was calculated using the following formula: cell survival rate (%) = (the average OD value of the treatment group/the average OD value of the control group) ×100%.

In the colony formation assay, Huh7.5.1 cells were introduced into a 6-well plate (5 × 10³ per well), incubated overnight, and then treated with either 15 or 30 µmol/L PEITC or 0.1% DMSO control for a duration of 12 h. Afterwards, cells were co-incubated within fresh medium and monitored for seven days. Then, the cell colonies were washed three times with phosphate buffer solution (PBS) (catalog no. C20012500BT; Gibco) and fixed with 4% paraformaldehyde (catalog no. BL539A; Biosharp, Heifei, P. R. China) for 15 min at room temperature. Following fixation, the cell colonies were further washed three times with PBS and stained with crystal violet (catalog no. C0121; Beyotime, Haimen, P. R. China) for 15 min. The stained colonies were counted and analyzed using ImageJ and GraphPad Prism 9.0.

Transwell invasion assay and wound healing migration assay

For the transwell invasion assay, 600 µL of serum-free medium was added to the lower chamber in a sterile 24-well plate. The transwell chamber was then carefully placed into the lower chamber with tweezers. Subsequently, Huh7.5.1 cells were inoculated into the transwell chamber (2 × 10⁴ cells per well) and incubated overnight. After co-culture with 15 and 30 µmol/L PEITC or 0.1% DMSO for 48 h, the cells adhering to the lower surface were washed with PBS and fixed for 15 min with 4% paraformaldehyde at room temperature followed by staining with crystal violet for 15 min. The migrated cells were photographed and counted with a bright field microscopy (TS2-S-SM; Nikon, Tokyo, Japan).

For the wound healing migration assay, Huh7.5.1 cells were inoculated into 6-well plates (1 × 10⁵cells per well). When the cells in each well reached 95%–100% confluence, a sterilized 10 µL pipette tip was used to create a straight scratch through the confluent monolayer. After rinsing with PBS, cells were treated with 15 µmol/L PEITC and 0.1% DMSO for 24 h, respectively, and photographed with a bright field microscope (TS2-S-SM; Nikon). At 0, 24, 48, 72, 96 and 120 h, the wound closure was analyzed using ImageJ software.

Apoptosis assay by flow cytometry (FCM)

The Annexin V-FITC/PI apoptosis detection kit (BD, the United States, catalog no. 556547) was used to detect cell apoptosis, according to the manufacturer’s instructions. Briefly, Huh7.5.1 cells were harvested and washed three times with PBS after incubation with 15 and 30 µmol/L PEITC or 0.1% DMSO for 12 h, respectively. Cells were then suspended with 400 µL of binding buffer followed by staining with 5 µL of fluorescein isothiocyanate-coupled annexin V (FITC) and 5 µL of propidium iodide (PI) solution. After incubation in the dark for 15 min at room temperature, samples were analyzed by FCM (NovoCyte, Agilent, the United States).

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay

A one step TUNEL apoptosis assay kit (catalog no. C1088; Beyotime) in combination with DAPI staining was used to detect PEITC-induced DNA fragmentation in Huh7.5.1 cells, according to the manufacturer’s instructions. Briefly, Huh7.5.1 cells were harvested and washed three times with PBS after incubation with 15 and 30 µmol/L PEITC or 0.1% DMSO for 12 h, respectively. Subsequently, cells were fixed with 4% paraformaldehyde for 10 min and then incubated with TUNEL reaction buffer for 30 min in the dark at 37 °C after infiltration with Triton X-100 (Beyotime, P. R. China, catalog no. P0096) for 5 min. After washing three times with PBS, 10 µL of 5 mg/L DAPI solution (catalog no. E607303; BBI, Shanghai, China) was added to stain the cells in the dark for 5 min at room temperature. Samples were observed under a fluorescence microscopy (TS2-S-SM, Nikon) to view the green fluorescence of apoptotic cells at 570 nm and blue DAPI-stained nuclei at 460 nm.

Detection of Caspase-3 activity

Huh7.5.1 cells were cultured overnight in 6-well plates and incubated with 15 and 30 µmol/L PEITC or 0.1% DMSO control for 12 h, respectively. The enzymatic activity level of Caspase-3 was assessed by identifying the cleavage of p-nitroaniline (pNA) through Caspase-3-specific substrates using a Caspase-3 assay kit (catalog no. C1116; Beyotime), according to the manufacturer’s instructions. Briefly, cells were harvested, centrifuged, and subsequently lysed in a 4 °C ice bath for a duration of 15 min. The Caspase-3 enzymatic reaction was conducted for 2 h at 37 °C, with 50 µL of cell lysate, 40 µL of reaction buffer, and 10 µL of Caspase-3 colorimetric substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide, Ac-DEVD-pNA) in each reaction. A microplate spectrometer (SPARK, Tecan, Swiss) was used to measure the colour intensity of pNA at the wavelength of 405 nm.

Determination of oxidative stress

Huh7.5.1 cells were cultured overnight in 6-well plates and incubated with 15 and 30 µmol/L PEITC or 0.1% DMSO control for 12 h, respectively. The intracellular ROS levels were determined using the oxidation-sensitive fluorescent probe DCFH-DA (Beyotime, P. R. China, catalog no. S0033S), according to the manufacturer’s instructions.

Mitochondrial transmembrane potential (MMP) measurement

According to the manufacturer’s instructions, MMP changes were measured using a Mitochondrial Membrane Potential Assay Kit with JC-1 (catalog no. C2006; Beyotime). Briefly, PEITC-treated cells were collected, washed twice with cold PBS, and then suspended with the mixture of culture medium and JC-1 staining solution (v/v =1:1). After incubation at 37 °C in the dark for 20 min, cells were centrifuged followed by tracking with 1 ×JC-1 dye. The stained cells were further detected by FCM and visualized under a fluorescence microscopy (OLYMPUS, IX73, Japan).

Cell cycle analysis

The cell cycle distribution was determined by FCM analysis. Briefly, Huh7.5.1 cells were cultured overnight in 6-well plates and incubated with 15 and 30 µmol/L PEITC or 0.1% DMSO control for 12 h, respectively. Both detached and adhered cells were collected into a centrifuge tube and washed twice with PBS. After fixation with 75% ethanol, cells were rinsed with PBS and then incubated in 0.5 mL of PI/RNaser (BD, the United States, catalog no. 550825) at room temperature for 15 min followed by FCM analysis.

RNA sequencing and data analysis

A total of 1 µg of RNA was extracted using Trizol (Invitrogen, Waltham, MA, USA) from Huh7.5.1 cells subjected to treatment with 15 µmol/L PEITC or 0.1% DMSO for 10 h, respectively, each with three biological replicates. Total RNA was enriched with polyA+ using Dynabeads Oligo (dT) magnetic beads (catalog no. 25-61005; Thermo Fisher Scientific, Waltham, MA, USA) and served as the input for preparing libraries with the NEBNext^® Magnesium RNA Fragmentation Module (catalog no. E6150S; NEB, Ipswich, MA, USA). Subsequently, Illumina NovaseqTM 6000 was used by LC-Bio Technology Co., Ltd. (Hangzhou, China) for paired-end sequencing. Fragments per kilobase of transcript per million mapped (FPKM) reads indicated the abundance of gene expression. The statistical power of RNA sequencing was calculated in RnaSeqSampleSize (https://cqs-vumc.shinyapps.io/rnaseqsamplesizeweb/). To annotate gene function, gene set enrichment analysis (GSEA) was performed using hallmark gene sets and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets from MSigDB.

RNA extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)

Huh7.5.1 cells were seeded into 6-well plates (1 ×10⁵ cells per well). After overnight culture, Huh7.5.1 cells were treated with 15 µmol/L PEITC or 0.1% DMSO for 10 h, respectively, each with three biological replicates. A total of 1 µg of RNA was extracted using Trizol and then treated with DNase I. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The values of A260/A280 of all RNA samples were between 1.8 and 2.0. The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) with RIN number > 7.0, and confirmed by electrophoresis with denaturing agarose gel. RNA was reverse-transcribed to cDNA using the MonScript™ RTIII Super Mix with dsDNase (Two-Step) (catalog no. MR05201M; Monad, Wuhan, China). The cDNA synthesis reaction was carried out at 50 °C for 15 min, followed by thermal inactivation at 85 °C for 5 min. Subsequently, the produced cDNA was utilized for qRT-PCR.

The qRT-PCR reaction was set up with a total volume of 20 µL, consisting of 10 µL SYBR Green Master Mix (catalog no. MQ10101; Monad), 0.4 µL each of forward and reverse primers, 1.5 µL cDNA template, and 7.7 µL ddH₂O. qRT-PCR was conducted on a BioRad CFX Connet qRT-PCR Dectction system (Bio-Rad, Hercules, CA, USA). Briefly, the reaction conditions comprised an initial pre-denaturation at 95 °C for 30 s, succeeded by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. The specificity of the PCR products was confirmed through melt curve analysis, with amplification lengths ranging from 110 to 300 bp. For normalization purposes, expression of β-actin was analyzed. Three biological replicates and three technical replicates were performed. Data were collected and analyzed as previously described in Guan et al. (2024). Specifically, the relative gene expression levels were determined by using the 2^−ΔΔCt method, wherein the PCR efficiencies were >95% and the R2 was >0.990. All primers used are listed in Table S1.

Statistical analysis

All experiments were performed in three biological replicates in the present study. GraphPad Prism version 9.0 was used to assess statistical differences between groups using t-test or one-way ANOVA. P-values <0.05 were considered statistically significant.

PEITC suppresses the activity and proliferation of Huh7.5.1 cells

To determine the anti-proliferative effects of PEITC (Fig. 1A) against HCC, Huh7.5.1 cells were treated with various concentrations (0∼100 µmol/L) of PEITC for 48 h and then subjected to the MTT assay. We found that the anti-proliferative effects on Huh7.5.1 cells were positively correlated to the dose of PEITC and the IC₅₀ value of PEITC was 29.6 µmol/L (Fig. 1B). Based on the results of the MTT assay, the 2D-clonony-formation assay was further conducted. The results showed that the number of colonies significantly decreased in the PEITC-treated Huh7.5.1 cells in a dose-dependent manner relative to the control (DMSO-treated) cells (Fig. 1C). Taken together, these results suggest that PEITC exerts anti-cancer effects on human HCC Huh7.5.1 cells.

PEITC induces apoptosis-related cell death in Huh7.5.1 cells

In order to evaluate whether PEITC could inhibit the growth of Huh7.5.1 cells by inducing the apoptosis-related cell death, an Annexin V-FITC/PI double staining assay by FCM was conducted. The apoptotic rates of Huh7.5.1 cells significantly increased from 2.15% to 37.84% and 74.05% after treatment with 15 and 30 µmol/L PEITC for 12 h, respectively, compared with that of control cells (Fig. 2A), suggesting a significant induction of apoptosis in Huh7.5.1 cells by PEITC. As the decline of MMP is one of the landmark events in the early stage of apoptosis (Gottlieb et al., 2003), the change of MMP was assessed using the JC-1 probe. The results of fluorescence assay showed a increase in the green JC-1 monomer fluorescence intensity and simultaneously a progressive decline of the red JC-1 aggregate signal in the PEITC-treated Huh7.5.1 cells, indicating that PEITC markedly decreased the MMP in Huh7.5.1 cells in a dose-dependent manner (Figs. 2B and 2C). To further verify the apoptosis induced by PEITC treatment in Huh7.5.1 cells, the detection of Caspase-3 activity was also performed. The result revealed that the activity of Caspase-3 in the PEITC-treated Huh7.5.1 cells increased significantly in a dose-dependent manner, compared with that in control cells (Fig. 2D). Overall, all these results suggest that PEITC induces mitochondrial-related apoptosis in Huh7.5.1 cells.

PEITC enhances oxidative stress and induces DNA damage in Huh7.5.1 cells

Mitochondria-related apoptotic responses, such as the decrease of MMP that promotes the enhancement of mitochondrial out membrane permeabilization (MOMP), activate the generation of ROS (Estaquier et al., 2012; Liu et al., 2018). The result of FCM analysis showed that treatment with PEITC for 12 h could significantly enhance the intracellular ROS level in Huh7.5.1 cells compared with that in control cells (Fig. 3A).

DNA damage is frequently observed in apoptotic cells (Wu & Crowe, 2020). When apoptosis occurs, DNA breakage, nuclear chromatin agglutination, and nuclear membrane rupture are usually induced (CortésGutiérrez et al., 2020). ROS accumulation has been implicated in causing DNA damage and apoptosis in diverse cancers (Barzilai & Yamamoto, 2004; Guachalla & Rudolph, 2010; Guo et al., 2014; Sadiq, 2023). To investigate whether PEITC could induce DNA damage in Huh7.5.1 cells, a DAPI staining and TUNEL assay was conducted. The results showed that PEITC induced a significant increase of green fluorescence in Huh7.5.1 cells in a dose-dependent manner (Figs. 3B and 3C), indicating that DNA damage occurred in the nuclei. Moreover, DAPI staining also showed a large number of nuclear ruptures (Figs. 3B and 3C). The merged images revealed the DNA-damaged nuclei in Huh7.5.1 cells (Figs. 3B and 3C).

PEITC induces cell cycle arrest in S phase in Huh7.5.1 cells

As DNA damage usually arrests or blocks the cell cycle, we further evaluated the effect of PEITC on cell cycle distribution in Huh7.5.1 cells by FCM analysis. The results demonstrated that PEITC treatment significantly increased the population of Huh7.5.1 cells in S phase, but markedly reduced the population of cells in G2/M and G0/G1 phases, indicating that PEITC could induce S-phase arrest to play its inhibitory roles in Huh7.5.1 cells (Fig. 4).

PEITC inhibits the migration and invasion of Huh7.5.1 cells

In order to further investigate whether PEITC could impact the migration of Huh7.5.1 cells, wound healing migration assay was conducted. The percentages of the open wound area at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h after the treatment with 15 µmol/L PEITC were calculated. Our result indicated that the migration of Huh7.5.1 cells was markedly delayed, compared with that of control cells (Fig. 5A). Specifically, at 24 h, the wound closure in the control group reached 21.7%, whereas that in the PEITC-treated group was only 2%; by 120 h, the would closure in the control group achieved 73.6%, while that in the PEITC-treated group was at 33.5%. This data clearly demonstrated the significant effect of PEITC in inhibiting cell migration (Fig. 5A). Consistent with the result of wound healing migration assay, the transwell invasion assay also confirmed that PEITC treatment had a negative influence on the invasion ability of Huh7.5.1 cells in a dose-dependent manner (Fig. 5B). Compared with the control group (2,058 invading cells), the number of invading cells in the 15 µmol/L PEITC-treated group dropped to 900, and further reduced to 130 in the 30 µmol/L PEITC-treated group (Fig. 5B). These results not only confirmed the inhibitory effect of PEITC on the invasive capacity of cells, but also presented a distinct dose-dependent pattern.

Transcriptome analysis suggests that PEITC activates MAPK, PI3K-Akt and p53 signaling pathways in Huh7.5.1 cells

In order to explore the potential molecular mechanisms of the inhibitory effects of PEITC on HCC, transcriptome sequencing was conducted in Huh7.5.1 cells treated with or without PEITC for 10 h. The statistical power of this experimental design calculated in RnaSeqSampleSize is 0.80. Pairwise comparisons showed that a total of 2889 genes were significantly differentially expressed with a —log₂ fold change (FC)— ≥ 1 and a p-value <0.05, among which 2339 genes were significantly up-regulated and 550 down-regulated in the PEITC-treated Huh7.5.1 cells relative to control cells (Fig. 6A and Table S2). To cross-check the accuracy of the RNA-seq results, qRT-PCR analysis was conducted on eight differentially expressed genes (DEGs), including four genes involved in cell proliferation, invasion and migration (FOS, PDE6G, RGS16 and HBEGF) (MildeLangosch, 2005; Nikolova et al., 2010; Carper et al., 2014; Bakiri et al., 2017; Hu et al., 2020; Ji & Wang, 2023; Zhang et al., 2023) and four genes related to oxidative stress, DNA damage and apoptosis (ATF3, DDIT4, SOCS1 and MXD1) (Hai et al., 1999; Kim, Kim & Lee, 2020; Ding et al., 2021; Ryu et al., 2022; Xiong et al., 2022; Shao et al., 2023) (Fig. 6D). The strong consistency between the qRT-PCR validation and RNA-seq results indicated high reliability of the transcriptomic profiling data.

The KEGG pathway analysis of DEGs manifested that the top 20 significantly enriched pathways involved two typical signaling pathways in cancer pathogenesis, namely MAPK and PI3K-Akt signaling pathways (Fig. 6B). There were 27 and 25 DEGs enriched in the MAPK and PI3K-Akt signaling pathways, respectively (Table S3). In addition, GSEA was also applied to analyze the global changes of the signaling pathways regulated by PEITC. It was found that 21 activated gene sets (NES >1.5, p-val <0.05, and false discovery rate (FDR) < 0.25) were identified in Huh7.5.1 cells after PEITC treatment (Table 1), including hallmarks E2F targets, PI3K-Akt-mTOR signaling and p53 pathway (Fig. 6C). The core enrichment genes for these three hallmark gene sets were further investigated (Table S4). Among these, one gene encoding cell cycle-related targets of E2F transcription factors, one gene up-regulated by activation of the PI3K-Akt-mTOR pathway and nine genes involved in the p53 pathway were up-regulated in the PEITC-treated Huh7.5.1 cells (Table 2). All these results showed that MAPK, PI3K-Akt and p53 signaling pathways could be activated by PEITC, suggesting the potential regulatory mechanisms of PEITC in inhibiting proliferation, migration and invasion, stimulating oxidative stress and inducing cell cycle arrest and apoptosis in Huh7.5.1 cells.