FBXO5 drives hepatocellular carcinoma progression and is a target for tea polyphenol-mediated inhibition

Introduction

The World Health Organization categorizes primary liver cancer into several histological types, with hepatocellular carcinoma (HCC) comprising 80–90% of cases (1). Although significant advances have been made in understanding HCC pathogenesis, several factors such as high recurrence rates, metastatic potential, and asymptomatic early-stage presentation contribute to unfavorable outcomes and reduced overall survival (2). Few patients are diagnosed at treatable early stages, with most cases identified only upon reaching advanced progression. While surgical techniques (resection and transplantation) and ablative therapies have improved recently, their effectiveness remains limited. Systemic therapies like radiotherapy and chemotherapy often cause significant adverse effects, collectively resulting in low overall treatment efficacy. These clinical challenges underscore the critical need for enhanced strategies in HCC prevention, early detection, therapeutic intervention, and outcome prediction.

The ubiquitin-proteasome system (UPS) is a critical protein degradation mechanism that maintains proteostasis and regulates gene expression (3), participating in diverse physiological processes including cell cycle regulation, signaling transduction, apoptosis, and DNA repair (4). Within this system, E3 ubiquitin ligases serve as key regulatory components. FBXO5 (F-box only protein 5), as a constituent of the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, mediates ubiquitination-dependent protein degradation and plays significant roles in tumorigenesis and cancer progression (5). Emerging evidence implicates FBXO5 in the development and prognosis of multiple malignancies. Elevated FBXO5 expression promotes proliferation in esophageal squamous cell carcinoma and correlates with poorer ovarian cancer outcomes and higher histologic grades (6). Conversely, FBXO5 downregulation induces apoptosis in lung cancer cells (7). These observations suggest FBXO5’s oncogenic potential across tumor types (8). In HCC, FBXO5 overexpression correlates with advanced disease stage and unfavorable prognosis (9), FBXO5 promotes HCC cell proliferation by inhibiting the anaphase-promoting complex/cyclosome (APC/C) inhibitor complex, stabilizing Skp2 (S-phase kinase-associated protein 2), and promoting the degradation of p27 (Kip1, Cyclin-dependent kinase inhibitor 1B) (10), while ribavirin treatment response may involve FBXO5 messenger RNA (mRNA) modulation (11). Although FBXO5 is frequently overexpressed in primary tumors and drives cancer progression, its precise functions and prognostic value in HCC remain poorly characterized.

Tea ranks as the world’s second most consumed beverage after water, containing numerous bioactive compounds, most notably tea polyphenols (TPs) (12). TPs, the predominant polyphenolic phytochemicals in tea, exhibit diverse pharmacological properties including antitumor, antioxidant, antibacterial, anti-inflammatory, lipid-lowering, and hepatoprotective effects (13,14). These compounds demonstrate therapeutic potential against various diseases, particularly heart disease, diabetes, cardiovascular diseases, and cancers (15,16). Mechanistic studies reveal that TPs modulate multiple signaling pathways to regulate tumor cell apoptosis. They effectively inhibit proliferation and induce apoptosis in various cancer cell lines, including MCF-7 breast cancer cells, SW780 bladder cancer cells, HepG2 HCC cells, and lung carcinoma cells, while sparing normal cells (17-19). Notably, murine experiments demonstrate that green TP pretreatment significantly reduces DEN-induced hepatocellular tumor incidence (20). Despite these findings, the molecular mechanisms underlying TPs’ anti-HCC activity remain unclear. This study therefore aims to investigate the regulatory relationship between FBXO5 and TPs in HCC progression through integrated bioinformatics and in vitro experiments, thereby elucidating TPs’ antitumor mechanisms. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1462/rc).

Methods

Reagents and antibodies

Human HCC cell lines HEP-3B and SNU-449 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, while Huh-7 was acquired from Wuhan Procell (Wuhan, China). Cells were cultured in DMEM or RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, USA) supplemented with 10% fetal bovine serum (FBS) (Clark, Williamsburg, USA). The FBXO5 small interfering RNA (siRNA) fragment was purchased from Shanghai GenePharma (Shanghai, China), and sh-FBXO5 lentiviral vectors were constructed by Wuhan Hanheng Biological (Wuhan, China). The following reagents were used Cell Counting Kit-8 (CCK-8) (Beyotime Biotechnology, Shanghai, China), Transwell chambers (Corning Inc., Corning, USA), TPs (MedChemExpress, Monmouth Junction, USA), FBXO5 antibody (Proteintech, Wuhan, China), and β-actin antibody (Abclonal, Wuhan, China).

Bioinformatics analysis

FBXO5 mRNA expression data across various tumors and in HCC tissues were obtained from The Cancer Genome Atlas (TCGA) database (https://ualcan.path.uab.edu/analysis.html). The downloaded mRNA data underwent identifier conversion and log2 transformation to compare FBXO5 expression between paired tumor and adjacent normal tissues. Differential expression analysis was performed using R software with the ggplot2, limma, and beeswarm packages. Additionally, patient survival analysis based on FBXO5 expression levels was conducted through GEPIA (http://gepia.cancer-pku.cn/).

Cell culture

Human HCC cell lines HEP-3B and SNU-449 were maintained in RPMI-1640 (1) or DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Clark) and 1% penicillin-streptomycin (Beyotime Biotechnology). Huh-7 cells are human HCC cells (Wuhan Procell), cultured in Procell-specific medium. All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The culture medium was refreshed every 1–2 days, and cells were passaged or harvested for experiments upon reaching 90% confluence.

Cell transfection

Three FBXO5-specific siRNAs (Shanghai GenePharma) were transfected into HEP-3B and Huh-7 HCC cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). The experimental setup included four groups: (I) control siRNA; (II) FBXO5 siRNA1; (III) FBXO5 siRNA2; and (IV) FBXO5 siRNA3. For the SNU-449 cell line, an FBXO5 overexpression cell line was established by infecting cells with a recombinant lentiviral vector (Hankio Biotechnology, Shanghai, China) targeting the human FBXO5 gene (NM_012177.5). Cells infected with a negative control virus served as the control group. After infection, cells are screened using puromycin. These cells were divided into two groups: (I) control; (II) FBXO5 Overexpression. The siRNA sequences targeting FBXO5 were: siRNA1: sense 5'-CCG GGA CUU AAA CUG GUA AAT T-3', antisense 5'-UUU ACC AGU UUU UAA GUC CGG GTT-3'; siRNA2: sense 5'-GCC CUA GGA UUG UAC AAC UTT-3', antisense 5'-AGU UGU ACA AUC CUA GGG CTT-3'; siRNA3: sense 5'-GGG AGA UGC UGA AGG AAA UTT-3', antisense 5'-AUU UCC UUC AGC AUC UCC CTT-3'.

CCK-8 analysis

Following transfection or drug treatment, HCC cells were collected and seeded into 96-well plates (LABSELECT, Tianjin, China) at a density of 3,000 cells per well, with triplicate wells for each experimental group. After incubation for 24, 48, and 72 hours, 10 µL of CCK-8 reagent (Beyotime Biotechnology) was added to each well, followed by additional incubation at 37 °C for 4 hours. Absorbance at 450 nm was subsequently measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The assay was repeated thrice.

Clone formation experiment

HCC cells post-transfection or drug treatment were trypsinized and seeded into 6-well plates (LABSELECT) at a density of 1,000 cells per well. After 5–7 days of culture until visible colonies formed, cells were processed as follows: (I) medium removal; (II) phosphate buffered saline (PBS) (Servicebio, Wuhan, China) washing; (III) fixation with 4% paraformaldehyde (Biosharp, Anhui, China); and (IV) staining with 0.1% crystal violet (Beyotime Biotechnology) for 10 minutes at room temperature. Stained plates were PBS-washed, air-dried, and imaged for colony counting (ImageJ software).

Wound healing assays

Transfected or drug-treated cells were cultured in 6-well plates (LABSELECT) to 80% confluence. Uniform scratches were created across each well using a pipette tip, followed by PBS (Servicebio) washing to remove dislodged cells. Serum-free medium was added, and wound closure was monitored at 0, 24, and 48 hours. Scratch areas were quantified using ImageJ software (triplicate experiments).

Transwell migration and invasion assays

HCC cells transfected or treated with drugs were collected by trypsin (Biosharp) digestion and counted, and cell suspensions were prepared with serum-free medium at a density of about 5×10⁴/well. For cell invasion, Matrigel (Corning Inc.) was thawed and added to the upper chambers after 10-fold dilution with basal medium and placed in a 37 °C incubator for 1–2 hours. After that, the cells were inoculated into the Matrigel-coated upper chamber, and 800 µL of complete medium was added to the lower chamber and placed in the cell culture incubator to continue incubation for 24 hours. For cell migration, Matrigel-free chambers (Corning Inc.) were used and cultured in the same way for 24 hours. After that, the cells were fixed with 4% paraformaldehyde (Biosharp) for 30 min and stained with 0.1% crystal violet (Beyotime Biotechnology) for 20 min, and then the floating color was washed with pure water, and the un-crossed cells were swabbed and photographed by observing them under a light microscope (Olympus, Tokyo, Japan).

Total RNA extraction and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA from transfected or drug-treated HCC cells was extracted with the Rapid Cell/Tissue Total RNA Extraction Kit (Norevizan, Nanjing, China), and RNA was reverse transcribed with the Reverse Transcription Kit (Norevizan) to obtain the complementary DNA (cDNA) real-time PCR reactions were performed on a Roche 9600, and the primers were supplied by Shanghai Sangyo Biotech. The primer sequences were as follows: FBXO5, forward primer 5'-GCC AGA GGA AAT TTT AGA CTG C-3', reverse primer 5'-CCA AGT TGT GCT CAC TTT AGA C-3'. GAPDH was used as an internal reference: GAPDH, forward primer 5'-CAG CCT CAA GAT CAT CAG CA-3', reverse primer 5'-TGT GGT CAT GAG TCC TTC CA-3'. PCR parameters included: initial denaturation at 95 °C for 10 min, 40 cycles of initial denaturation at 95 °C for 5 s; incubation at 63 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min (21).

Western blotting analyses

Transfected or drug-treated HCC cells were collected by digestion with trypsin, and the protease inhibitor (Beyotime Biotechnology) RIPA lysis buffer (strong) (Beyotime Biotechnology) and phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology) were prepared as a lysate at a ratio of 100:1, and the cells were lysed on ice after adding the lysate to the cells to obtain proteins. The protein concentration was determined using BCA kit (Beyotime Biotechnology), and after quantification, the proteins were denatured by placing the proteins in a metal bath (Thermo Fisher Scientific) at 100 °C for 5 min to obtain the up-sampled proteins. The proteins were separated by polyacrylamide gel electrophoresis, and the proteins on the separation gel were transferred to polyvinylidene fluoride (PVDF) membranes (TransGen Biotech, Beijing, China). The PVDF membranes containing the proteins were closed by placing them in 5% skimmed milk in a rapid closure solution for 15 min, and then the membrane strips were placed in the primary antibody, and the membrane strips were incubated in a shaking bed at 4 °C overnight. On the following day, the protein bands were detected by incubation with the secondary antibody for 2 hours and enhanced chemiluminescence (ECL) development after washing the membrane three times with Tris-Buffered Saline with Tween 20 (TBST) (22). Immunoblotting was performed using anti-FBXO5 (1:1,000; Cat No.10872-1-AP, Proteintech), anti-β-actin (1:2,000; Cat No. GB11001-100, Servicebio), and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:5,000; Cat No. GAR0072, Lianke Biotech, Hangzhou, China).

Statistical analyses

The experimental data were statistically analyzed using Statistical Package for the Social Sciences (SPSS v16.0) and GraphPad Prism software (v6.0 Inc., San Diego, CA, USA). Independent samples Student’s t-test was taken for comparison between two groups, and analysis of variance (ANOVA) was used for comparison between multiple groups. P<0.05 was taken as the difference was statistically significant.

Ethics statement

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Results

Expression of FBXO5 in HCC and its effect on the prognosis of HCC patients

To investigate the role of FBXO5 in HCC, we analyzed its expression patterns across multiple tumor types using TCGA data. mRNA expression and clinical follow-up data from 371 HCC patients and 50 normal liver tissue samples revealed significantly upregulated FBXO5 expression in tumor tissues compared to non-tumor controls (Figure 1A). This upregulation was consistently observed when comparing tumor tissues with their paired adjacent normal tissues (Figure 1B). Kaplan-Meier analysis demonstrated that HCC patients with high FBXO5 expression had significantly shorter overall survival (Figure 1C), suggesting FBXO5 may function as an oncogene in HCC.

Figure 1 Bioinformatics analysis of FBXO5 expression levels in HCC. (A) Expression of FBXO5 in a variety of tumors; (B) expression of FBXO5 mRNA in normal and HCC tissues; (C) analysis of FBXO5 expression and survival of patients with HCC. HCC, hepatocellular carcinoma; HR, hazard ratio; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

Screening of FBXO5 interfering fragments and verification of FBXO5 lentiviral vector overexpression effect

To study the effect of FBXO5 expression on HCC cells, we transfected FBXO5 siRNAs into HEP-3B and Huh-7 cells to construct FBXO5 overexpressing HCC cell lines. Lentiviral vectors were constructed and HCC SNU-449 cell lines were transfected with lentiviral vectors carrying FBXO5 and empty loads, respectively, to establish overexpression of FBXO5 cell lines. RT-qPCR and Western blot analyses confirmed successful FBXO5 knockdown in HEP-3B and Huh-7 cells (Figure 2A,2B) and effective FBXO5 overexpression in SNU-449 cells (Figure 2C,2D). It indicated that the interference effect of FBXO5 interference fragment was better and the overexpression FBXO5 lentiviral vector was successfully constructed.

Figure 2 Validation of interference and overexpression efficiency of FBXO5. (A,B) FBXO5 mRNA (A) and protein (B) levels in HEP-3B and Huh-7 cells following siRNA-mediated knockdown. (C,D) FBXO5 mRNA (C) and protein (D) levels in SNU-449 cells following plasmid-mediated overexpression. Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. siRNA, small interfering RNA.

Effect of FBXO5 on the activity and proliferation of HCC cells

CCK-8 assays demonstrated that FBXO5 knockdown significantly inhibited viability of both HEP-3B and Huh-7 cells (Figure 3A), while FBXO5 overexpression enhanced SNU-449 cell proliferation (Figure 3B). Colony formation assays revealed that FBXO5 silencing markedly reduced clonogenic capacity in HEP-3B and Huh-7 cells (Figure 3C), whereas FBXO5-overexpressing SNU-449 cells showed significantly increased colony formation compared to empty vector controls (Figure 3D). These findings indicate that FBXO5 promotes HCC cell proliferation and survival.

Figure 3 Effect of FBXO5 on the activity and proliferation of HCC cells. (A) CCK-8 assay to detect the effect of HEP-3B and Huh-7 knockdown of FBXO5 on cell activity; (B) CCK-8 assay to detect the effect of SNU-449 overexpression of FBXO5 on cell activity; (C) clonal spots to detect the effect of HEP-3B and Huh-7 knockdown of FBXO5 on cell proliferation (stained with crystal violet; magnification, 2×); (D) clonal spots to detect the effect of SNU-449 overexpression of FBXO5 on cell proliferation (stained with crystal violet; magnification, 2×). Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; HCC, hepatocellular carcinoma.

Effect of FBXO5 on wound healing, migration and invasion ability of HCC cells

To investigate the role of FBXO5 in HCC cell motility, we performed wound healing and Transwell assays. Wound healing assays revealed that FBXO5 knockdown significantly impaired migration capacity in both HEP-3B and Huh-7 cells (Figure 4A), with prolonged wound closure time compared to controls. Conversely, FBXO5 overexpression enhanced SNU-449 cell migration, resulting in faster wound closure (Figure 4B). Transwell assays demonstrated that FBXO5 silencing inhibited cell migration and invasion (Figure 4C), while FBXO5 overexpression promoted these processes (Figure 4D). These findings collectively demonstrate that FBXO5 enhances motility in HCC cells.

Figure 4 Effect of FBXO5 on wound healing, migration and invasion ability of HCC cells. (A) Scratch assay to detect the effect of HEP-3B and Huh-7 knockdown of FBXO5 on cellular trauma healing ability (magnification, 100×); (B) scratch assay to detect the effect of SNU-449 overexpression of FBXO5 on cellular trauma healing ability (magnification, 100×); (C) Transwell assay of the effect of HEP-3B and Huh-7 knockdown of FBXO5 on cell migration, invasion ability (stained with crystal violet; magnification, 200×); (D) Transwell detection of the effect of SNU-449 overexpression of FBXO5 on cell migration, invasion ability (stained with crystal violet; magnification, 200×). Compared with control: **, P<0.01; ***, P<0.001; ****, P<0.0001. HCC, hepatocellular carcinoma.

Effect of TPs on the activity of HCC cells

In order to investigate the effects of TPs on HCC cells, we utilized CCK-8 assay to detect the changes in cellular activity after different doses of TPs acted on HCC cells for 24 hours. We found that the cellular activity of TPs decreased with the increase of drug concentration. We found that the activity of the three liver cancer cell lines weakened with the increase of drug concentration (Figure 5A-5C). Whereas the sensitivity of TPs to the three cell lines was not the same, 100 µg/mL TPs inhibited SNU-449 cells by about 50%, whereas the inhibitory effect on HEP3B cells were inhibited by only about 10%. In order to study the effects of different concentrations of TPs on HCC cells more comprehensively, we chose the concentrations of action of TPs that inhibited HCC cells by about 50% and 75%, respectively, for the subsequent experiments, and chose 92 and 120 µg/mL TPs for treating SNU-449 cells, the HEP-3B cells were treated with the concentrations of 250 and 350 µg/mL of TPs, respectively, while Huh-7 cells were treated with 143 and 196 µg/mL TPs.

Figure 5 Effect of TPs on the activity of HCC cells. (A) CCK-8 assay to detect the effect of TPs on the activity of HCC cells HEP-3B; (B) CCK-8 assay to detect the effect of TPs on the activity of HCC cells SNU-449; (C) CCK-8 assay to detect the effect of TPs on the activity of HCC cells Huh-7. Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; HCC, hepatocellular carcinoma; TPs, tea polyphenols.

Effects of TPs on the malignant biological behavior of HCC cells

To elucidate the effects of TPs on the proliferation of HCC cells, we performed clone formation experiments. We found that with the increase of TPs concentration, the clone formation ability was significantly weakened and the cell colony formation ability was obviously inhibited (Figure 6A). Then, to clarify the effect of TPs on the wound healing ability of HCC cells, we examined the wound healing of HEP-3B, SNU-449, and Huh-7 cells after 24 hours of the effect of different concentrations of TPs, and the results showed that the exposure of TPs decreased the wound healing ability of HEP-3B, SNU-449, and Huh-7 cells (Figure 6B). Next, we utilized Transwell assays to investigate whether TPs affected the migration and invasion of HCC cells. The results showed that TPs exposure impaired the migration and invasion abilities of HEP-3B, SNU-449, and Huh-7 cells (Figure 6C). This showed that TPs inhibited the proliferation of HCC cells and decreased the trauma healing ability, migration and invasion of HCC cells.

Figure 6 Effect of FBXO5 on wound healing, migration and invasion ability of HCC cells. (A) Colony formation test was carried out in HEP-3B, SNU-449, and Huh-7 cells treated with TPs at different concentrations (stained with crystal violet; magnification, 2×); (B) the wound healing test was used to measure the motility of HEP-3B, SNU-449, and Huh-7 cells treated with different concentrations of TPs (magnification, 100×); (C) Transwell migration and invasion assays were performed to measure the migratory and invasive capacity in HEP-3B, SNU-449, and Huh-7 cells after treatment with different concentrations of TPs (stained with crystal violet; magnification, 200×). Compared with control, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. HCC, hepatocellular carcinoma; TPs, tea polyphenols.

TPs regulate FBXO5 expression in HCC cells

FBXO5 may be a key oncogenic protein in HCC development and progression, and we hypothesized whether TPs inhibit the malignant biological functions of HCC cells by down-regulating the expression of FBXO5 in HCC cells. To answer this speculation, we transfected FBXO5 lentiviral vector into HCC cells to increase the expression level of FBXO5. RT-qPCR and Western blotting techniques were used to analyze whether TPs could affect the expression of FBXO5 in HCC cells. The results showed that after the action of TPs, the expression of FBXO5 in HCC cells was significantly reduced at both mRNA and protein levels (Figure 7A,7B); Western blotting assay of overexpression of FBXO5 in combination with TPs showed that compared with the group of overexpression of FBXO5 alone, the protein level of FBXO5 in the group of overexpression of FBXO5 in conjunction with TPs was reduced and was close to the control group (Figure 7C). It indicated that TPs could down-regulate the expression of FBXO5 in HCC cells.

Figure 7 TPs modulate FBXO5 expression in HCC cells. (A) Real-time PCR to detect the mRNA expression of FBXO5 after the effect of TPs; (B) Western blot detection of protein expression of FBXO5 after TPs action; (C) Western blot detection of protein expression of FBXO5 after overexpression of FBXO5 and TPs. Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Compared with TPs treatment only or transfection of FBXO5 plasmid only: ^#, P<0.05. HCC, hepatocellular carcinoma; PCR, polymerase chain reaction; TPs, tea polyphenols.

Overexpression of FBXO5 affects the antitumor activity of TPs in HCC cells

Next, to verify whether the anti-tumor activity of TPs was related to FBXO5, we overexpressed FBXO5 and then combined it with TPs in HCC cells, and detected whether up-regulation of FBXO5 could affect the inhibitory effect of TPs on cancer cells by functional assays. Clone formation assays showed that FBXO5 overexpression promoted colony formation in HEP-3B and SNU-449 cells, whereas FBXO5 overexpression neutralized the reduction of colony formation in HCC cells caused by TPs exposure (Figure 8A). Wound healing assay showed that FBXO5 overexpression promoted the wound healing ability of HEP-3B and SNU-449 cells, and sustained up-regulation of FBXO5 eliminated the attenuation of wound healing ability induced by TPs exposure (Figure 8B). Transwell results showed that FBXO5 up-regulation promoted the migratory and invasive ability of HEP-3B and SNU-449 cells, whereas FBXO5 overexpression reversed the attenuation of HCC cell migration and invasion ability caused by TPs exposure (Figure 8C). These results demonstrated that the upregulation of FBXO5 significantly attenuated the effects of TPs, suggesting that TPs might be used to inhibit the growth of HCC cells by downregulating the expression of FBXO5.

Figure 8 Overexpression of FBXO5 affects the antitumor activity of TPs in HCC cells. (A) Clonogenic spot assay to detect the effect of overexpression of FBXO5 in combination with TPs on cell proliferation (stained with crystal violet; magnification, 2×); (B) scratch assay to detect the effect of overexpression of FBXO5 in combination with TPs on cellular wound healing ability (magnification, 100×); (C) Transwell assay to detect the effect of overexpression of FBXO5 in combination with TPs on cell migration and invasion ability (stained with crystal violet; magnification, 200×). Compared with control: *, P<0.05; ***, P<0.001; ****, P<0.0001. Compared with TPs treatment only or transfection of FBXO5 plasmid only: ^#, P<0.05; ^###, P<0.001; ^####, P<0.0001. HCC, hepatocellular carcinoma; TPs, tea polyphenols.

Discussion

FBXO5, also known as Early Mitotic Inhibitor 1 (Emil1), prevents premature exit from mitosis by inhibiting the activity of the APC/C. The degradation of FBXO5 itself is an essential step for initiating the anaphase stage and ensuring the successful completion of cell division (23). Cell cycle regulation is closely related to tumor proliferation, and polyploidy generation and chromosomal instability are important factors in carcinogenesis and important hallmarks of malignancy. Previous studies have shown that high FBXO5 expression leads to chromosomal instability and mitotic disorders (24), and that aberrant FBXO5 expression affects chromosomal stability and normal cell division (5), whereas abnormalities in the cell cycle and genomic instability can promote tumor growth (25). FBXO5 is overexpressed as an oncogene in a variety of human tumors (26), statistical analysis indicates that the expression level of FBXO5 is significantly correlated with tumor prognosis, staging, and lymph node metastasis in patients (27). A study has found that FBXO5 is highly expressed in gastric cancer tissues and plays an important role in tumor cell proliferation, differentiation and cell cycle regulation (28). And our in vitro results showed that knockdown of FBXO5 reduced the activity of HCC cells and inhibited cell proliferation, migration and invasive ability, while overexpression of FBXO5 played a promotional role.

TPs demonstrate broad biological potential in cancer prevention, cardiovascular protection, metabolic regulation, neuroprotection, and gut health through their antioxidant, anti-inflammatory, and multi-pathway regulatory effects (29). Reactive oxygen species (ROS) have a dual role in promoting and inhibiting carcinogenesis in human cancers (30). In cellular experiments, TPs demonstrate exceptionally potent antioxidant, anti-inflammatory, and anticancer activities (16). With potent antioxidant properties, TPs effectively scavenge excess ROS and regulate key signaling pathways, thereby significantly mitigating oxidative stress, inflammatory responses, and apoptosis triggered by ROS in multiple organs such as the stomach and lungs. Ultimately, they exert broad-spectrum protective effects (31,32). In addition, TPs can regulate the composition of the intestinal flora, improve immunity, modulate immune cells, and reduce inflammatory responses by increasing beneficial flora and decreasing harmful flora (33). TPs hold potential for disease prevention and adjunctive intervention in various conditions. At reasonable doses, their risk of side effects is generally lower than that of traditional chemical drugs, making them promising candidates for novel disease prevention and adjunctive therapeutic agents (34). In addition, the combination of chemotherapeutic agents and TPs can synergistically improve therapeutic efficacy and reduce the adverse side effects of anticancer drugs (35). This suggests that TPs have a promising future in anticancer therapy and are ideal drugs for the treatment of cancer or as adjuvants in combination with other anticancer drugs. Our results indicated that TPs could reduce the activity of HCC cells and inhibit cell proliferation, migration and invasion ability; while overexpression of FBXO5 could weaken the inhibitory effect of TPs on HCC cells after the combination of FBXO5 and TPs.

The results of this experiment have largely aligned with our anticipated expectations and are consistent with bioinformatics database findings. This study has established FBXO5 as a key oncogenic driver and prognostic biomarker in HCC, demonstrating for the first time that TPs exert antitumor effects by directly inhibiting it. These findings position FBXO5 as a new diagnostic marker and reveal TPs as a promising targeted therapy to suppress HCC progression. Consequently, clinical research should now prioritize developing TPs or their derivatives for precision oncology treatments and validating FBXO5’s utility in patient prognosis. However, our study still lacks support from in vivo experiments. The therapeutic role of TPs needs to be verified in mouse models, and the molecular mechanisms of FBXO5 in TPs-mediated hepatocarcinogenesis require further in-depth exploration.

Conclusions

We found that FBXO5 could enhance the activity of HCC cells and promote cell proliferation, migration and invasion through in vitro cellular experiments, and it may be a pro-carcinogenic gene in HCC, and FBXO5 might serve as a biomarker to guide the early diagnosis and treatment of HCC patients. We found that TPs could inhibit the expression of FBXO5 in HCC cells in vitro, and might exert anti-HCC effects by down-regulating the expression of FBXO5, which could be used as an effective drug to target FBXO5 for the treatment of HCC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1462/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1462/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1462/prf

Funding: This work was supported by the Anhui Provincial Department of Education Outstanding Young Teachers Development Program in Universities (No. YQZD2024028) and the Major Program of Anhui Province Education Department (No. KJ2021ZD0087).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1462/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Satriano L, Lewinska M, Rodrigues PM, et al. Metabolic rearrangements in primary liver cancers: cause and consequences. Nat Rev Gastroenterol Hepatol 2019;16:748-66. [Crossref] [PubMed]
2. Oura K, Morishita A, Tani J, et al. Tumor Immune Microenvironment and Immunosuppressive Therapy in Hepatocellular Carcinoma: A Review. Int J Mol Sci 2021;22:5801. [Crossref] [PubMed]
3. Park J, Cho J, Song EJ. Ubiquitin-proteasome system (UPS) as a target for anticancer treatment. Arch Pharm Res 2020;43:1144-61. [Crossref] [PubMed]
4. Dagar G, Kumar R, Yadav KK, et al. Ubiquitination and deubiquitination: Implications on cancer therapy. Biochim Biophys Acta Gene Regul Mech 2023;1866:194979. [Crossref] [PubMed]
5. Ji J, Jing A, Ding Y, et al. FBXO5-mediated RNF183 degradation prevents endoplasmic reticulum stress-induced apoptosis and promotes colon cancer progression. Cell Death Dis 2024;15:33. [Crossref] [PubMed]
6. Guan C, Zhang J, Zhang J, et al. Enhanced expression of early mitotic inhibitor-1 predicts a poor prognosis in esophageal squamous cell carcinoma patients. Oncol Lett 2016;12:114-20. [Crossref] [PubMed]
7. Wang K, Qu X, Liu S, et al. Identification of aberrantly expressed F-box proteins in squamous-cell lung carcinoma. J Cancer Res Clin Oncol 2018;144:1509-21. [Crossref] [PubMed]
8. Liu P, Wang X, Pan L, et al. Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis. Front Immunol 2022;13:901784. [Crossref] [PubMed]
9. Gong Q, Zhang L, Guo J, et al. FBXO family genes promotes hepatocellular carcinoma via ubiquitination of p53. J Cancer Res Clin Oncol 2024;150:458. [Crossref] [PubMed]
10. Zhao Y, Tang Q, Ni R, et al. Early mitotic inhibitor-1, an anaphase-promoting complex/cyclosome inhibitor, can control tumor cell proliferation in hepatocellular carcinoma: correlation with Skp2 stability and degradation of p27(Kip1). Hum Pathol 2013;44:365-73. [Crossref] [PubMed]
11. Xu C, Luo L, Yu Y, et al. Screening therapeutic targets of ribavirin in hepatocellular carcinoma. Oncol Lett 2018;15:9625-32. [Crossref] [PubMed]
12. Mao X, Xiao X, Chen D, et al. Tea and Its Components Prevent Cancer: A Review of the Redox-Related Mechanism. Int J Mol Sci 2019;20:5249. [Crossref] [PubMed]
13. Bakun P, Mlynarczyk DT, Koczorowski T, et al. Tea-break with epigallocatechin gallate derivatives - Powerful polyphenols of great potential for medicine. Eur J Med Chem 2023;261:115820. [Crossref] [PubMed]
14. Chen Y, Cheng S, Dai J, et al. Molecular mechanisms and applications of tea polyphenols: A narrative review. J Food Biochem 2021;45:e13910. [Crossref] [PubMed]
15. Khan N, Mukhtar H. Tea Polyphenols in Promotion of Human Health. Nutrients 2018;11:39. [Crossref] [PubMed]
16. Yang M, Zhang X, Yang CS. Bioavailability of Tea Polyphenols: A Key Factor in Understanding Their Mechanisms of Action In vivo and Health Effects. J Agric Food Chem 2025;73:3816-25. [Crossref] [PubMed]
17. Hessien M, El-Gendy S, Donia T, et al. Growth inhibition of human non-small lung cancer cells h460 by green tea and ginger polyphenols. Anticancer Agents Med Chem 2012;12:383-90. [Crossref] [PubMed]
18. Liang J, Li F, Fang Y, et al. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells. Mater Sci Eng C Mater Biol Appl 2014;36:7-13. [Crossref] [PubMed]
19. Luo KW, Chen Wei, Lung WY, et al. EGCG inhibited bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via down-regulation of NF-κB and MMP-9. J Nutr Biochem 2017;41:56-64. [Crossref] [PubMed]
20. Umemura T, Kai S, Hasegawa R, et al. Prevention of dual promoting effects of pentachlorophenol, an environmental pollutant, on diethylnitrosamine-induced hepato- and cholangiocarcinogenesis in mice by green tea infusion. Carcinogenesis 2003;24:1105-9. [Crossref] [PubMed]
21. Sunaga F, Tsuchiaka S, Kishimoto M, et al. Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases. J Vet Med Sci 2020;82:217-23. [Crossref] [PubMed]
22. de Almeida VH, de Melo AC, Meira DD, et al. Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer. Braz J Med Biol Res 2017;51:e6822. [Crossref] [PubMed]
23. Margottin-Goguet F, Hsu JY, Loktev A, et al. Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase. Dev Cell 2003;4:813-26. [Crossref] [PubMed]
24. Fan X, Guan G, Wang J, et al. Licochalcone A induces cell cycle arrest and apoptosis via suppressing MAPK signaling pathway and the expression of FBXO5 in lung squamous cell cancer. Oncol Rep 2023;50:214. [Crossref] [PubMed]
25. Chen D, Lu S, Huang K, et al. Cell cycle duration determines oncogenic transformation capacity. Nature 2025;641:1309-18. [Crossref] [PubMed]
26. Wang R, Gao X, Xie L, et al. METTL16 regulates the mRNA stability of FBXO5 via m6A modification to facilitate the malignant behavior of breast cancer. Cancer Metab 2024;12:22. [Crossref] [PubMed]
27. Li H, Sun X, Lv Y, et al. Downregulation of Splicing Factor PTBP1 Curtails FBXO5 Expression to Promote Cellular Senescence in Lung Adenocarcinoma. Curr Issues Mol Biol 2024;46:7730-44. [Crossref] [PubMed]
28. Zhang J, Zhang G, Wang K, et al. Exploring the role of FBXO5 in gastric cancer. Mol Cell Probes 2023;69:101915. [Crossref] [PubMed]
29. Luo Q, Luo L, Zhao J, et al. Biological potential and mechanisms of Tea's bioactive compounds: An Updated review. J Adv Res 2024;65:345-63. [Crossref] [PubMed]
30. Yan Y, Zhou S, Chen X, et al. Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis. Signal Transduct Target Ther 2024;9:58. [Crossref] [PubMed]
31. Yang J, Geng Y, Zhao B, et al. Green tea polyphenols alleviate TBBPA-induced gastric inflammation and apoptosis by modulating the ROS-PERK/IRE-1/ATF6 pathway in mouse models. Food Funct 2024;15:10179-89. [Crossref] [PubMed]
32. Lv H, Wang J, Geng Y, et al. Green tea polyphenols inhibit TBBPA-induced lung injury via enhancing antioxidant capacity and modulating the NF-κB pathway in mice. Food Funct 2024;15:3411-9. [Crossref] [PubMed]
33. Wang ST, Cui WQ, Pan D, et al. Tea polyphenols and their chemopreventive and therapeutic effects on colorectal cancer. World J Gastroenterol 2020;26:562-97. [Crossref] [PubMed]
34. Yan Z, Zhong Y, Duan Y, et al. Antioxidant mechanism of tea polyphenols and its impact on health benefits. Anim Nutr 2020;6:115-23. [Crossref] [PubMed]
35. Cao J, Han J, Xiao H, et al. Effect of Tea Polyphenol Compounds on Anticancer Drugs in Terms of Anti-Tumor Activity, Toxicology, and Pharmacokinetics. Nutrients 2016;8:762. [Crossref] [PubMed]