Astaxanthin inhibits hepatocellular carcinoma by targeting USP39-mediated β-catenin stabilization through deubiquitination

Abstract

Although studies suggest that astaxanthin exerts anticancer effects through Wnt/β-catenin pathway, its therapeutic target remains unclear. The deubiquitinase USP39 may be a potential target due to its ability to regulate this pathway in hepatocellular carcinoma (HCC). This study aims to identify the role and underlying mechanism of USP39 in the anti-HCC effects of astaxanthin via Wnt/β-catenin pathway. In vitro, HepG2 and HuH7 cell lines were treated with astaxanthin, and cell proliferation was assessed using CCK-8, colony formation, and EdU assays. Western blot and real-time qPCR were used to analyze the expression of USP39, β-catenin, and Cyclin D1. Co-immunoprecipitation and ubiquitination assays were performed to confirm the interaction between USP39 and β-catenin. In vivo, HuH7 xenografts in nude mice were established to evaluate tumor growth following astaxanthin treatment. Immunohistochemistry and western blot analyses were conducted to examine the expression of USP39, β-catenin, Cyclin D1, and Ki-67 in tumor tissues. Astaxanthin treatment significantly reduced cell viability and colony formation, accompanied by decreased levels of USP39, β-catenin, and Cyclin D1, and increased phosphorylated β-catenin. Astaxanthin-induced USP39 inhibition promoted β-catenin ubiquitination and degradation, thereby blocking Wnt/β-catenin pathway activation. β-catenin overexpression partially rescued the anti-tumor effects of USP39 knockdown. Astaxanthin significantly suppressed tumor growth, inhibited the expression of USP39, β-catenin, and Cyclin D1, and reduced the proportion of Ki-67-positive cells in tumor tissues. Astaxanthin inhibits HCC through USP39-mediated ubiquitination of β-catenin, providing novel insights into its anticancer mechanism. These findings highlight the potential of targeting USP39 for HCC therapy, especially in combination with astaxanthin.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-23891-2.

Introduction

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, largely due to limited therapeutic options and frequent resistance to molecular targeted therapies¹. The Wnt/β-catenin signaling pathway plays a pivotal role in liver regeneration and is widely implicated in HCC tumorigenesis, metastasis, and chemotherapy resistance². Despite advances in molecular targeted therapies such as sorafenib, clinical outcomes remain unsatisfactory due to pathway reactivation and off-target effects³. These challenges underscore the urgent need for novel agents that selectively inhibit Wnt/β-catenin signaling while minimizing toxicity.

Astaxanthin, a naturally occurring carotenoid with potent antioxidant and anti-inflammatory properties, has emerged as a promising anticancer agent. Preclinical studies have demonstrated that astaxanthin suppresses HCC progression by modulating key oncogenic pathways, including JAK2/STAT3 and NF-κB^4,5. Notably, astaxanthin inhibits Wnt/β-catenin signaling by downregulating β-catenin and its downstream targets⁶. However, the precise molecular targets underlying this regulation, particularly those associated with ubiquitination-dependent pathways, remain unidentified. Recent evidence identifies the deubiquitinating enzyme USP39 as a key regulator of β-catenin stability in HCC. USP39 promotes β-catenin accumulation through two distinct mechanisms: first, directly removing ubiquitin chains to prevent proteasomal degradation; second, inhibiting the maturation of TRIM26, an E3 ligase responsible for β-catenin degradation^7,8. This dual regulatory mechanism sustains Wnt/β-catenin hyperactivation, thereby promoting tumor proliferation and metastasis.

Despite these insights, the functional interplay between astaxanthin and USP39 in HCC remains unexplored. Given established anticancer properties of astaxanthin and the critical role of USP39 in β-catenin stabilization, we hypothesize that astaxanthin exerts its antitumor effects by targeting USP39-mediated β-catenin ubiquitination. This study aims to elucidate the role of USP39 in astaxanthin-mediated suppression of HCC via the Wnt/β-catenin pathway. Our findings will not only clarify the molecular mechanism underlying astaxanthin-induced Wnt/β-catenin inhibition but also establish USP39 as a potential therapeutic target for HCC.

Materials and methods

Cell lines and culture

Human HCC cell lines (HepG2 and HuH7) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cell lines were characterized by cell vitality detection and tested for mycoplasma detection. The HepG2 cells were cultured in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS). HuH7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin according to standard conditions. Unless otherwise specified, all cell cultures were maintained in a humidified incubator with 5% CO₂ at 37 °C. For specific experiments, cells were treated with MG-132 (20 µM) for 4 h to inhibit proteasome activity (20 µM)⁹. Cells also were treated with astaxanthin (astaxanthin) (300 µM) for 24 h¹ or were treated with ICG-001 (35 µM) for 24 h¹⁰.

In vivo experiments

The nude mouse xenograft model: HuH7 cells (3 × 10⁶) stably transfected with either NC or shUSP39 were injected subcutaneously into the flanks of 6- to 8-week-old male BALB/c nude mice. Tumor growth was monitored by measuring the maximum length (a) and width (b) of the tumors using a vernier caliper every 3 days, and thereby the tumor volume (V) was calculated using the formula: V = ab²/2. After 30 days of cell injection, the mice were administered astaxanthin at a dose of 10 mg/kg body weight, given every other day. Following 21 days of astaxanthin treatment, the mice were euthanized, and the tumors were excised. Tumor weight was measured to assess the effect of astaxanthin on tumor growth.

The mice used in this study were obtained from the Shanghai SLAC Laboratory Animal Co Ltd (Shanghai, China). We used carbon dioxide (CO₂) inhalation equipment to carry out euthanasia. We placed the mice in a chamber during euthanasia, gradually supplying CO₂ to raise its concentration. The euthanasia personnel were required to observe the procedure and pause for a minimum of 1 min once they detected no movement, visible breathing, or heartbeat. The trials received approval from the Institutional Animal Care and Use Committee of Putian University (IACUC No. 2021-005). Animal experiments were complied with the ARRIVE guidelines and all methods were performed in accordance with the relevant guidelines and regulations.

Data processing and statistical analysis

Each experiment was performed independently at least three times to ensure reproducibility and reliability of the results. Categorical variables were expressed as frequencies and proportions (%). Continuous variables were reported as the mean ± standard deviation (SD), unless otherwise specified. To compare continuous variables between two groups, the unpaired two-tailed Student’s t-test was used. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was applied. All statistical analyses were conducted using GraphPad Prism 8 software (GraphPad Software, USA). A tests were two-sided where a P-value of < 0.05 was considered statistically significant. Asterisks were used to denote statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001, respectively.

Supplementary data file

The detailed protocols and other procedures are provided in the Supplementary Materials and Methods section. This file includes Supplementary Tables S1 and Supplementary Figures Fig. S1-5.

Results

Astaxanthin inhibits the HCC cell proliferation and decreases the expression levels of USP39 and β-catenin

The CCK8 kit was used to assess the effect of various concentrations of astaxanthin on HCC cell proliferation. HCC cell lines HuH7 and HepG2 were treated with DMSO and astaxanthin (50 µM, 100 µM, 150 µM, 200 µM, 250 µM and 300 µM) for 24 h. Cell growth was plotted based on optical density (OD) measurements. The data showed a gradual decrease in the growth of HCC cells (HuH7 and HepG2) with increasing astaxanthin concentration (Fig. 1A, B). Moreover, HCC cell growth was significantly inhibited at the maximum astaxanthin concentration of 300 µM. To facilitate observation, astaxanthin (300 µM) was selected as the optimal concentration for the subsequent experiments. A similar effect was observed in colony-forming assays, where astaxanthin significantly inhibited the colony-forming ability of HCC cells (Fig. 1C, D). The effect of astaxanthin (300 µM) on HCC cell proliferation was examined using the EdU assay, which showed a significant reduction in the number of EdU-positive HCC cells compared to the control group (Fig. 1E, F).

Fig. 1.

Astaxanthin inhibits HCC proliferation and decreases the expression levels of USP39 and β-catenin. (A,B) The growth trend of HCC cells (HepG2 and HuH7) treated with astaxanthin at varying concentrations (0-300 µM) for 24 h was assessed using CCK8 assays. (C,D) astaxanthin (300 µM) inhibited the colony-forming ability of HCC cells. (E,F) The EdU assay revealed a reduced number of EdU-positive HCC cells treated with astaxanthin. (G–I) astaxanthin influences the expression levels of USP39, β-catenin, Cyclin D1, and p-β-catenin in HCC cells (HepG2 and HuH7). Student’s t-test: * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are representative of at least three independent experiments and are presented as mean ± SD.

HCC cells (HepG2 and HuH7) were treated with astaxanthin (300 µM), then RNA and proteins were extracted after 24 h to measure the expression levels of target molecules. The results demonstrated a decrease in the RNA and protein expression levels of USP39, β-catenin, and Cyclin D1, while astaxanthin inhibited HCC proliferation. Conversely, the level of phosphorylated β-catenin (p-β-catenin) protein increased (Fig. 1G-I and Fig. S1 A, B). The in vitro results confirm that astaxanthin inhibits HCC cell proliferation and possesses anticancer effects. Additionally, these results suggest that astaxanthin-mediated inhibition of HCC cell proliferation is associated with alterations in levels of USP39, β-catenin, and Cyclin D1.

Down-regulated USP39 inhibits the HCC cell proliferation and decreases the expression levels of β-catenin and Cyclin D1

Before elucidating the role and mechanism of astaxanthin-mediated HCC inhibition, we established that USP39 regulates the Wnt/β-catenin pathway to suppress cell proliferation. We generated cells with stable knockdown of USP39 protein using a lentiviral shRNA technique in HepG2 and HuH7 cells. The effects of USP39 on the HCC cell proliferation were evaluated using MTT and colony-forming assays. MTT results demonstrated that the down-regulated USP39 significantly inhibited the HCC cell proliferation (Fig. 2A, B). Similarly, colony-forming assays demonstrated that down-regulated USP39 significantly inhibited the colony-forming ability of HCC cells (Fig. 2C, D). The effect of down-regulated USP39 on HCC cell proliferation was assessed using the EdU assay, which revealed a significant reduction in the number of EdU-positive HCC cells compared to the NC group (Fig. 2E, F). These results confirm that USP39 h significantly affects HCC cell proliferation.

Fig. 2.

Down-regulation of USP39 inhibits the HCC proliferation and decreases the expression levels of β-catenin and Cyclin D1. (A,B) The effect of USP39 on HCC cells (HepG2 and HuH7) proliferation was assessed using MTT assays at different time points. (C,D) Colony-forming unit assays demonstrated that down-regulated USP39 significantly inhibited the colony-forming ability of HCC cells. (E,F) The effect of USP39 on HCC cells (HepG2 and HuH7) proliferation was evaluated using EdU assays. (G–I) The effect of USP39 on the expression levels of β-catenin, Cyclin D1, and p-β-catenin in HCC cells (HepG2 and HuH7) was confirmed by Western blotting. Student’s t-test: * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are representative of at least three independent experiments and are presented as mean ± SD.

To determine whether the Wnt/β-catenin pathway is involved in the inhibition of HCC proliferation by USP39, we assessed the expression levels of key molecules, β-catenin and Cyclin D1, in the HCC cells with down-regulated USP39. The results demonstrated that down-regulated USP39 significantly decreased the protein levels of β-catenin and Cyclin D1, while increasing the protein level of p-β-catenin in HCC cells (Fig. 2G-I). Subsequently, β-catenin RNA level was analyzed by RT-PCR. Interestingly, β-catenin mRNA levels showed no significant difference in HCC cells transduced with shUSP39 (Fig. S2 A, B), suggesting that USP39 may regulate β-catenin expression through posttranslational regulation, such as protein ubiquitination.

USP39 promotes the HCC cell proliferation by regulating the expression level of β-catenin

The above results indicated that down-regulation of USP39 inhibited β-catenin protein expression. We further investigated whether USP39 influences HCC cell proliferation by regulating the expression levels of β-catenin. β-catenin expression in HuH7 cells from both the NC group and shUSP39 group was elevated by plasmid transfection. MTT results demonstrated that elevated β-catenin significantly promoted HCC cell proliferation in both the NC and shUSP39 groups (Fig. 3A). Additionally, the impact of elevated β-catenin on HCC cell proliferation was assessed using the EdU assay, which revealed an increased number of EdU-positive HCC cells in both the NC and shUSP39 groups. These results confirmed that elevated β-catenin promotes the HCC cell proliferation. Furthermore, we observed that this inhibition of proliferation could be reversed by β-catenin supplementation in the shUSP39 group (Fig. 3B, C).

Fig. 3.

USP39 promotes the HCC proliferation by regulating β-catenin expression level. (A) The effect of USP39 on the proliferation of HuH7 cells with up-regulated β-catenin was assessed using MTT assays after 24 h. (B,C) The effect of USP39 on HuH7 cell proliferation with up-regulated β-catenin was evaluated using EdU assays. (D,E) The effect of β-catenin on USP39 expression levels in HuH7 cells was verified by Western blotting. (F,G) The effect of β-catenin on Cyclin D1 expression levels in HuH7 cells was verified by Western blotting. (H-I) The effect of USP39 on β-catenin expression levels in HuH7 cells was verified by Western blotting. The anti-β-catenin antibody was used to detect β-catenin levels. Student’s t-test: * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are representative of at least three independent experiments and are presented as mean ± SD.

RNA and proteins were extracted to assess the expression levels of target molecules in HCC cells. The results showed that elevated β-catenin had no significant effect on USP39 expression, whereas USP39 overexpression significantly increased β-catenin protein levels (Fig. 3D-E, H-I). Together with the knockdown and rescue experiments, these findings confirm that USP39 positively regulates β-catenin stability, while β-catenin does not reciprocally regulate USP39. Additionally, elevated β-catenin increase the expression of Cyclin D1 protein (Fig. 3F, G and Fig. S3). These findings suggest that USP39 promotes HCC cell proliferation by regulating β-catenin protein expression.

HCC cell lines (HepG2 and HuH7) were treated with ICG-001, a Wnt/β-catenin pathway inhibitor, and was analyzed using the MTT and EdU assays. MTT results showed that ICG-001 significantly inhibited HCC cell proliferation (Fig. 4A). The EdU assay indicated that ICG-001 reduced the number of EdU-positive HCC cells (Fig. 4B, C). These results confirm that blocking the Wnt/β-catenin pathway can inhibit the HCC cell proliferation. Western blot results showed that ICG-001 down-regulated β-catenin and Cyclin D1 protein levels (Fig. 4D-F). These results demonstrate that down-regulation of β-catenin significantly inhibits HCC cell proliferation.

Fig. 4.

Regulation of the Wnt/β-catenin pathway inhibits HCC proliferation and reduces Cyclin D1 expression levels. (A) The effect of the Wnt/β-catenin pathway inhibitor (ICG-001) on the proliferation of HCC cells (HepG2 and HuH7) proliferation was assessed using MTT assays after 24 h. (B,C) The effect of the Wnt/β-catenin pathway inhibitor (ICG-001) on HCC cells (HepG2 and HuH7) proliferation was evaluated using EdU assays. (D–F) The effect of the Wnt/β-catenin pathway inhibitor (ICG-001) on Cyclin D1 expression levels in HCC cells (HepG2 and HuH7) was verified by Western blotting. Student’s t-test: * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are representative of at least three independent experiments and are presented as mean ± SD.

Co-immunoprecipitation assays revealed a direct interaction between USP39 and β-catenin in HCC cells (HepG2 and HuH7) (Fig. 5A, B). Immunofluorescence staining assays also revealed that USP39 was predominantly localized in the nucleus, while β-catenin was mainly cytoplasmic but also exhibited nuclear localization. Higher-magnification images confirmed partial nuclear co-localization of USP39 and β-catenin in HepG2 and HuH7 cells (Fig. 5C). Additional experiments in SK-Hep-1 cells further supported the nuclear overlap between USP39 and β-catenin (Supplementary Fig. S3B). These observations suggested that deubiquitinase USP39 interacts with β-catenin to inhibit the HCC cell proliferation. Subsequently, β-catenin ubiquitination increased in HCC cells with down-regulated USP39 (Fig. 5D). In addition, the level of β-catenin ubiquitination was downregulated in USP39 rescued HepG2 cells co-transfected with HA-β-catenin and Myc-Ub (Fig. 5E). We further treated cells with cycloheximide (CHX, an inhibitor of protein synthesis) and monitored β-catenin expression by western blotting at different time points. The results indicated that USP39 overexpression largely increased the half-life of the β-catenin protein in HepG2 cells (Fig. 5F-G). In addition, downregulation of β-catenin was dramatically reversed in USP39 knockdown HCC cells (HepG2) treated with MG132 (an inhibitor of the ubiquitin–proteasome pathway) (Fig. 5H-I). Together, these results identified that USP39 has a direct deubiquitinating activity towards the regulation of β-catenin protein.

Fig. 5.

USP39 interacts with β-catenin through ubiquitination regulation. (A,B) Co-immunoprecipitation assays revealed an interaction between endogenous USP39 and β-catenin in HCC cells (HepG2 and HuH7). USP39 and β-catenin immunoprecipitates were normalized to immunoglobulin (Ig) G. Whole-cell lysates were subjected directly to immunoblotting (IB) using specific antibodies. (C) Immunofluorescence staining assays for USP39 and β-catenin in HCC cells (HepG2 and HuH7) were visualized using confocal microscopy. (D) The ubiquitination level of β-catenin is increased in shUSP39 HCC cells (HepG2 and HuH7) co-transfected with HA-β-catenin and Flag-Ub. (E) β-catenin ubiquitination in USP39 rescued HepG2 cells co-transfected with HA-β-catenin and Myc-Ub. (F) Quantitation of β-catenin in HepG2 cells transfected with USP39 overexpressing plasmid was monitored by western blotting at the indicated times after cyclohexamide (CHX, 0.2 mg/ml) addition. (G) Signal for β-catenin was quantified densitometrically and relative aboundance of β-catenin protein at the time of CHX addition (0 h) was set to 1. (H,I) The protein level of β-catenin in HepG2 cells transfected with shUSP39 and treated with MG132 as indicated.

Astaxanthin inhibits HCC proliferation via the USP39/β-catenin/cyclin D1 axis in vitro

The above experiments confirmed the mechanism that down-regulated USP39 inhibits HCC cell proliferation through the deubiquitination regulation of β-catenin. We further explored whether astaxanthin’s inhibition of HCC proliferation involves this mechanism. HCC cell lines (HepG2 and HuH7) were treated with astaxanthin (300 µM) for 24 h in both the NC and shUSP39 groups. Proliferation was then assessed using the CCK8 kit. The results showed that astaxanthin (300 µM) significantly inhibited HCC cell proliferation in the NC group. In contrast, proliferation inhibition was more pronounced in the shUSP39 group (Fig. 6A, B). Similarly, astaxanthin (300 µM) significantly inhibited the colony formation ability of HCC cells in the NC group, with a more pronounced effect in the shUSP39 group (Fig. 6C, D). Additionally, the EdU assay revealed a significant reduction in the number of EdU-positive HCC cells in both the NC group and the shUSP39 group treated with astaxanthin (300 µM), with a more pronounced effect in the latter (Fig. 6E-H).

Fig. 6.

Astaxanthin inhibits HCC proliferation via the regulation of the USP39/β-catenin/Cyclin D1 axis in vitro. (A,B) The effect of astaxanthin (300 µM) on the proliferation of shUSP39 HCC cells (HepG2 and HuH7) was assessed by CCK8 assays after 24 h. (C,D) The effect of astaxanthin (300 µM) on the colony-forming ability of shUSP39 HCC cells (HepG2 and HuH7) was evaluated by colony-forming assays. (E–H) The effect of astaxanthin (300 µM) on the proliferation of shUSP39 HCC cells (HepG2 and HuH7) was evaluated by EdU assays. (I–K) The effect of astaxanthin (300 µM) on the expression levels of USP39, β-catenin, Cyclin D1, and p-β-catenin in shUSP39 HCC cells was determined by Western blotting. Student’s t-test: * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are representative of at least three independent experiments and are presented as mean ± SD.

RNA and proteins were subsequently extracted to examine the expression levels of target molecules in the HCC cells. The results showed that astaxanthin (300 µM) decreased the protein levels of USP39, β-catenin, and Cyclin D1, while increasing the expression level of p-β-catenin protein both in the NC group and the shUSP39 group (Fig. 6I-K). Moreover, these results were more pronounced in the shUSP39 + astaxanthin (300 µM) group when compared with shUSP39 alone group. mRNA levels of the target molecules were detected by RT-PCR, and the results indicated that astaxanthin (300 µM) also down-regulate mRNA expression levels of USP39 and β-catenin (Fig. S4 A, B). These results suggest that astaxanthin inhibits the HCC cell proliferation by regulating the USP39/β-catenin/Cyclin D1 axis.

USP39 involved in the molecular mechanism by which astaxanthin inhibits the growth of HCC in vivo

To further demonstrate that astaxanthin inhibits HCC progression by down-regulating USP39 and consequently decreasing the expression of β-catenin in vivo, we generated shUSP39 HuH7 cells and transplanted them subcutaneously into the flanks of BALB/c nude mice. Astaxanthin (10 mg/kg) was administered every other day for 21 days following the establishment of xenografts, and tumor weight was measured after this period. During this period, the subcutaneous tumor volume was measured every 3 days. The results showed that down-regulated USP39 in HuH7 cells significantly reduced the volume and weight of subcutaneous tumors in mice compared to the NC group (mean tumor size: 1.0 cm * 1.1 cm vs. 1.4 cm * 1.5 cm), thus inhibiting HCC growth (Fig. 7A-C). Notably, tumors in the shUSP39 group were smaller than those in the NC group. Furthermore, these effects were more pronounced in the shUSP39 + astaxanthin group when compared with shUSP39 alone group (mean tumor size: 0.6 cm * 0.6 cm vs. 1.0 cm * 1.1 cm) (Fig. 7A-C).

Fig. 7.

Astaxanthin inhibits the HCC growth through regulation of the USP39/β-catenin/Cyclin D1 axis in vivo. (A) The impact of astaxanthin and USP39 on HCC growth in vivo was evaluated using xenograft assays. (B) Tumor volumes were measured every 3 days. (C) Tumor weights were recorded after 21 days of astaxanthin treatment. (D,E) The expression levels of USP39, β-catenin, and Cyclin D1 in tumors were analyzed by Western blotting. (F,G) Immunohistochemical (IHC) staining was used to assess the expression levels of USP39, β-catenin, Cyclin D1, and Ki67 in tumors. Student’s t-test: * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are representative of at least three independent experiments and are presented as mean ± SD.

Furthermore, RNA and proteins were extracted from these tumors to examine the expression levels of the target molecules. The results indicated that astaxanthin significantly reduced the protein expression levels of USP39, β-catenin, and Cyclin D1 both in the NC and shUSP39 groups (Fig. 7D-E). Furthermore, these effects were more pronounced in the shUSP39 + astaxanthin group when compared with shUSP39 alone group (Fig. 7D-E). mRNA levels of the target molecules were measured by RT-PCR, and the results demonstrated that astaxanthin also down-regulated the mRNA expression levels of USP39 and β-catenin in vivo (Fig. S5). Additionally, IHC assays revealed that the protein levels of USP39, β-catenin, Cyclin D1, and Ki67 were reduced in shUSP39 HuH7 cells, with the most pronounced reductions observed in the shUSP39 + astaxanthin group (Fig. 7F-G). Therefore, in vivo experiments confirmed that USP39/β-catenin/Cyclin D1 axis is involved in the molecular mechanism by which astaxanthin inhibits the HCC growth.

Discussion

HCC represents a significant global health challenge, characterized by its high incidence and poor prognosis¹¹. Despite advances in therapeutic strategies, effective treatment options remain limited. Thus, new approaches need to be explored¹². Astaxanthin is a xanthophyll ketocarotenoid pigment with diverse applications across various industries. The eco-friendly extraction methods of astaxanthin from natural sources, such as crawfish exoskeletons, offer opportunities for sustainable production of this compound¹³. Originally known for imparting a red-pink color to organisms, astaxanthin is now gaining attention for its potential health benefits and research interest^14,15. Accumulating evidence has demonstrated that astaxanthin is a promising candidate agent in the field of HCC prevention and therapy^1,4,5.

Astaxanthin has been studied for its potential effects on Wnt/β-catenin pathway^1,6,16,17, which plays a crucial role in HCC development and progression. However, the precise mechanisms by which astaxanthin affects Wnt/β-catenin signalling in HCC remains to be elucidated. Recently, astaxanthin has also been linked to the ubiquitin-proteasome system in protein degradation¹⁸, providing new hypotheses and mechanisms for how astaxanthin regulates Wnt/β-catenin signalling. USP39 is a deubiquitinating enzyme, and we have previously reported that it affects cell proliferation through the deubiquitination of β-catenin in HCC^7,8. Therefore, this study focuses on the hypothesis that USP39-mediated β-catenin ubiquitination is a potential mechanism by which astaxanthin inhibits HCC proliferation.

In this study, astaxanthin was confirmed to have an inhibitory effect on HCC both in vitro and in vivo. The study utilized multiple experimental approaches to evaluate the efficacy of astaxanthin in inhibiting the proliferation of HCC cell lines (HuH7 and HepG2) in vitro. The results indicated that astaxanthin significantly inhibited HCC cell proliferation-related phenotypes. Additionally, astaxanthin significantly reduced the growth of subcutaneous tumors in nude mice. The anti-HCC effects of astaxanthin involve various well-documented cellular biological processes¹⁹. For instance, astaxanthin reduces hypoxia within the tumor microenvironment⁴. Additionally, astaxanthin mitigates oxidative stress, DNA damage, and cell death, and it reduces the induction of early hepatocarcinogenesis in rats induced by cyclophosphamide^20,21. The restoration of glucose metabolism through the inhibition of aerobic glycolysis and promotion of the tricarboxylic acid cycle also plays a crucial role in the inhibition of hepatoma cell proliferation induced by astaxanthin²². Astaxanthin exhibits anticancer effects by inducing cell apoptosis through mitochondrial-dependent mechanisms in rat HCC CBRH-7919 cells²³.

However, the specific mechanisms by which astaxanthin regulates the Wnt/β-catenin signaling pathway in HCC remain unknown²⁴. In light of this issue, Zhu et al. reported that astaxanthin prevents osteoarthritis by inhibiting Rspo2-mediated Wnt/β-catenin signaling in chondrocytes and reducing Rspo2-related inflammatory factors in macrophages²⁵. Furthermore, Kavitha et al. demonstrated that astaxanthin inhibits Wnt/β-catenin signaling pathways through the inactivation of Erk/MAPK and PI3K/Akt, thereby inducing intrinsic apoptosis in a hamster model of oral cancer¹⁶. The results of the present study indicate that astaxanthin inhibits HCC proliferation by downregulating β-catenin expression and enhancing β-catenin phosphorylation. Astaxanthin treatment increases p-β-catenin expression, which may be associated with enhanced ubiquitination and degradation of β-catenin. Our findings indicate that the deubiquitinase USP39 influences HCC cell proliferation by modulating β-catenin ubiquitination. USP39 is known to impact the stability of several oncoproteins, and its inhibition has been associated with reduced tumor growth in various cancer types²⁶. Our findings enhance this understanding by elucidating the specific role of USP39 in HCC and its modulation by astaxanthin.

In addition to post-translational regulation via USP39, our data revealed a reduction in β-catenin mRNA levels after astaxanthin treatment (Fig. S1), suggesting that transcriptional suppression may also contribute to the observed effects. Although not explored in the present study, previous work has shown that astaxanthin can modulate upstream pathways such as PI3K/Akt and Erk/MAPK¹⁶, which regulate β-catenin transcription. Future studies using promoter activity assays will be required to delineate this additional mechanism.

It is also worth noting that we observed that combined treatment with astaxanthin and USP39 knockdown exerted a stronger inhibitory effect than either intervention alone (Figs. 6 and 7). This synergistic effect suggests that astaxanthin not only acts through USP39-mediated deubiquitination but may also engage USP39-independent pathways of β-catenin suppression. Consistent with this, our data indicate a reduction in β-catenin mRNA levels following astaxanthin treatment, implying potential transcriptional regulation in addition to post-translational control. Further studies will be required to dissect these complementary mechanisms.

This study has several limitations. We did not perform in vivo ubiquitination assays after astaxanthin treatment, nor reciprocal rescue experiments with USP39 overexpression, which would further strengthen the mechanistic link. Our analyses mainly focused on β-catenin and Cyclin D1, without evaluating additional Wnt targets or pathway activity reporters. While USP39 was shown to regulate β-catenin stability, whether this occurs through direct deubiquitination remains unclear, and the specificity of astaxanthin for USP39 requires confirmation through binding or structure–activity studies. The in vitro concentration of astaxanthin (300 µM) exceeds physiologically achievable levels, and pharmacokinetics or toxicity analyses were not conducted in vivo, highlighting the need for optimized formulations and dosing strategies. Moreover, only two HCC cell lines, with supplementary validation in SK-HEP-1, were examined, which does not fully capture tumor heterogeneity. Finally, although previous reports support the clinical relevance of USP39 and β-catenin, direct validation in patient tissues was not included. Future work will expand mechanistic studies, include additional models, and incorporate clinical samples to enhance translational significance.

Conclusion

In summary, we have detailed the mechanism by which astaxanthin inhibits HCC proliferation through USP39-regulated β-catenin ubiquitination. Additionally, the study highlights the role of USP39 in β-catenin stabilization and its potential as a therapeutic target for HCC treatment in combination with astaxanthin. This novel mechanism offers a new strategy for HCC treatment through astaxanthin supplementation or USP39 depletion in cases with elevated USP39/β-catenin protein levels.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conception and design: C. Song; Data acquisition: X. Li and H. Xu; Draft and revise: C. Song and X. Li. Animal experiments: R. Chen, X. Huang, Z. Lin, L. Lin, and Y. Qi; Analysis and interpretation: S. Wang, L. Han, J. Li, and G. Song.

Funding

This study was supported by the National Natural Science Foundation of China (grant no. 82200676), the Natural Science Foundation of Fujian Province of China (grant nos. 2020J01920), the Open Project of Putian University Key Laboratory of Translational Tumor Medicine in Fujian Province (grant no. 2022KF005), the Science and Technology Planning Projects of Putian of Fujian Province (grant nos. 2023SZ3001PTXY01 and grant nos. 2020SP004), College Student Innovation and Entrepreneurship Training Program of Putian University (grant no. X202411498008), the Collaborative Project Between the Hospital and College of Putian University (grant no. 2024106), the Joint Fund Project for Scientific and Technological Innovation in the Field of Medical and Health Care in Putian (grant no. 2024SJYL079), and the Fujian Provincial Health Technology Project (grant no. 2024GGA089).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. Raw data are available from the corresponding author upon reasonable request.

Declarations

Ethical statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Putian University (IACUC No. 2021-005). All methods were performed in accordance with the relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines 2.0. for animal research.

Competing interests

The authors declare no competing interests.