Astragaloside IV represses hepatocellular carcinoma progression by modulating HMGB1-ferroptosis axis

Abstract

Astragaloside IV (AST-IV), as one of the main functional components of Astragalus membranaceus, has physiological functions such as regulating metabolism and anti-tumor. However, the role of AST-IV on hepatocellular carcinoma (HCC) was still poorly understood. In this study, our work explored whether AST-IV could induce ferroptosis and repress HCC tumorigenesis. Results indicated that AST-IV could repress the tumor progression (viability, migration) of HCC in vitro. Besides, AST-IV induced the ferroptosis (Fe²⁺, malondialdehyde, lipid peroxidation) in HCC cells. Molecular docking and microscale thermophoresis indicated that high mobility group protein B1 (HMGB1) acted as the target of AST-IV. AST-IV could repress the HMGB1 expression and HMGB1 reversed the role of AST-IV on HCC cells’ ferroptosis. In vivo, AST-IV administration repressed the tumor progression. In conclusion, AST-IV represses HCC progression by modulating HMGB1-ferroptosis axis, which provides a novel insight for HCC.

Introduction

Hepatocellular carcinoma (HCC) is the main type of liver cancer worldwide, accounting for 75–85% of liver cancer cases, and is one of the main causes of cancer-related deaths [1, 2]. Although there are currently various treatment methods such as surgical resection, ablation, local regional treatment, and targeted drug therapy targeting tumor characteristics and disease progression, the 5-year survival rate of HCC patients is still not ideal due to metastasis and drug resistance [3, 4]. The molecular basis of the pathogenesis, metastasis and treatment resistance of HCC has not been fully clarified [5]. Therefore, it is urgent to discover new biomarkers, analyze their molecular functions and develop targeted treatment strategies [6, 7].

Ferroptosis is a novel iron-dependent programmed cell death mode, and its mechanism is different from apoptosis, necrosis and autophagy [8, 9]. This process catalyzes the oxidation of abundant unsaturated fatty acids on the cell membrane by divalent iron or esterase, leading to lipid peroxidation and triggering cell death [10]. Ferroptosis plays a crucial yet complex role in HCC pathogenesis, progression, and recurrence. It can suppress tumor initiation by selectively eliminating malignant hepatocytes. However, cancer cells may escape this defense by altering glutathione metabolism (e.g., GPX4 downregulation) or iron regulation (e.g., TFRC upregulation), promoting HCC progression and therapy resistance [11]. HMGB1 inhibits the activity of system xc⁻ (SLC7A11), reduces cystine uptake and glutathione (GSH) synthesis, weakens the antioxidant function of GPX4, and leads to the accumulation of lipid peroxides. In addition, HMGB1 could up-regulate the intracellular iron (Fe²⁺) level and enhance the Fenton reaction.

Astragaloside IV (AST-IV) is a key bioactive saponin derived from Astragalus membranaceus (Huangqi), a traditional Chinese medicinal herb [12, 13]. AST-IV exhibits potent anti-inflammatory, antioxidant, and immunomodulatory properties [14]. AST-IV enhances the immune response capacity by regulating immune cells such as macrophages, T lymphocytes, regulatory T cells, dendritic cells and natural killer cells in the tumor immune microenvironment. AST-IV improves the function of immune cells and the secretion of related cytokines, can reverse the immunosuppressive state, thereby enhancing the killing and clearance ability of immune cells against tumor cells and exerting an anti-tumor effect [15, 16]. AST-IV has been reported to repress tumor progression, including nasopharyngeal carcinoma [17], prostate cancer [18], non-small cell lung cancer [19] and oral cancer [20] et al.

Here, this study found that AST-IV could repress the tumor progression of HCC. Therefore, a series of experiments were carried out to investigate whether and how AST-IV affected the progress of HCC. Overall, the findings about AST-IV provide a novel insight for traditional Chinese medicine on HCC.

Materials and methods

Cell culture and AST-IV treatment

The human HCC cell line (Huh7, MHCC97H) and normal human hepatic cells (THLE-3) were obtained from cell bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the DMEM culture medium (Gibco) with fetal bovine serum (FBS, 10%), streptomycin (100 mg/mL) and penicillin (100 U/mL, Invitrogen) in incubator at 37 ℃ (5% CO₂). AST-IV was purchased from Yuanye Biotech (CAS: 84687-43-4, Shanghai, China). AST-IV was mixed with DMSO and stored at − 20 ℃. The AST-IV (0 µmol/L, 25 µmol/L, 50 µmol/L) was administrated to cells in vitro.

Migration assays

Cell migration assay was performed using the 24-well transwell chamber (Corning, cat no. 3422). The HCC cells were suspended in 200 µL of growth medium in the upper chamber, and 600 µL of growth medium with 10% fetal bovine serum (FBS) was added in lower chamber. After 24 h incubation in an incubator, the migrated cells on the upper filter surface were removed by using cotton swab. The filters were then fixed and stained by 0.4% crystal violet. The images were captured using a microscope (BZ-X810, Keyence).

EdU cell proliferation and CCK-8 assay

The proliferation of HCC cells were tested by the BeyoClick™ EdU Cell Proliferation Kit according to the manufacturer’s instructions. The fluorescence images were captured by fluorescence microscope (ZEISS, LSM900). The CCK-8 assay was performed by the cell counting kit-8 (CCK-8) reagent (CK04, Dojindo Laboratories, Kumamoto, Japan).

Fe²⁺, MDA, GSH analysis

The determination of Fe²⁺ level was tested by Iron Colorimetric Assay Kit (Elabscience, cat no. E-BC-K139-S). The quantitative detection of malondialdehyde (MDA) was performed by Lipid Peroxidation MDA Assay Kit (Beyotime Biotechnology, cat no. S0131S). The cellular GSH level was assessed using the GSH assay kit (Beyotime, cat no. S0053) following the manufacturer’s instructions.

Transmission electron microscopy (TEM)

HCC cells were trypsinized and fixed in glutaraldehyde overnight at 4 ◦C. Using 1% osmium tetroxide, the fixed cells was dehydrated in ethanol solutions, and embedded in Poly/Bed 812 resin. To examine mitochondrial ultrastructure, the samples were observed under a transmission electron microscope (HT7700, HITACHI).

Determination of lipid peroxidation

The cellular lipid peroxidation of HCC cells with erastin treatment was tested by the oxidation-sensitive probe C11-BODIPY581/591 (Thermo, cat no. D3861). HCC cells were treated with 5 µM of the oxidation-sensitive probe for 20 min. Besides, the cells were also stained with DAPI for cell nucleus.

RNA-Seq

Total RNA was extracted from HCC cells treated with AST-IV or blank using TRIzol reagent (Invitrogen). 150 bp paired-end sequencing was conducted by Illumina NovaSeq™ 6000 (LC-Bio Technology) according to the manufacturer’s protocol. Significant difference in differentially expressed genes (DEG) mRNAs with log2 (fold change) ≥1 and p < 0.05 was considered.

Molecular Docking

The 3D structure of AST-IV procured from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The target HMGB1 PDB file (PDB ID: 4qr9) garnered from the PDB (http://www.rcsb.org/). The calculate binding energy was calculated by AutoDock Vina software with a grid size of 20 × 20 × 20 Å. The results were visualized using PyMOL and LigPlot software.

MicroScale thermophoresis (MST) analysis

The purified recombinant HMGB1 protein was labeled using Protein labeling kit RED-NHS (NanoTemper, cat no. MO-L011). Under 100% excitation power and 40% MST power, the data were tested. The MST was performed by Nanotemper analysis software (NanoTemper Technologies) and the Kd was determined according to the protocol.

Quantitative reverse transcript-PCR (RT-qPCR)

The RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). The RNA was reversely transcribed by reverse transcription kit (Takara, Dalian, China). The level was detected by SYBR Green Taq Mix. GAPDH acted as the internal reference. Primers were as follows: HMGB1, F5’- TATGGCAAAAGCGGACAAGG-3’, and R5’- CTTCGCAACATCACCAATGGA-3’; GAPDH, F5’-CTGACATGCCGCCTGGAGA-3’, and R5’-ATGTAGGCCATGAGGTCCAC-3’.

Immunofluorescence

For immunofluorescence, HCC cells were plated in 12-well plates and exposed to AST-IV. The cells were fixed with 4% paraformaldehyde (Sigma) and permeabilized in 0.1% Triton X-100 and blocked in 3% BSA. Cells were incubated with HMGB1 antibodies overnight at 4 °C and then incubated with goat anti-mouse Alexa-549 antibody. The immunofluorescence image was captured by Zeiss LSM900 confocal microscope.

Animal assay

For the in vivo tumor study, BALB/c mice (male, 18–22 g, 4–5 weeks) were provided by Guangdong Provincial People’s Hospital. The animal studies were carried out and approved by the Laboratory Animal Welfare and Ethics Committee of the Guangdong Provincial People’s Hospital. BALB/c mice with Huh7 subcutaneous injection were randomly divided into two groups (n = 5), and each group was administrated with AST-IV intraperitoneal injection. Tumor size volume was detected by caliper each 3 days following: Volume = a×b²/2 (a and b are the major and minor axes of the tumor). The maximal tumor sizeburden was not exceeded, which was permitted by the ethics committee. The euthanasia for mice was CO₂. The animal studies were performed in accordance with the Ethics Committee of Guangdong Provincial People’s Hospital.

Statistical analysis

All the statistical analysis was performed using GraphPad Prism 8.0. Student’s t tests with two-tailed were utilized for two groups comparisons. One-way ANOVA was utilized for multiple comparisons. P < 0.05 was utilized for statistical significance.

Results

AST-IV repressed the tumor progression of HCC in vitro

Astragaloside IV (AST-IV) is a key bioactive saponin derived from Astragalus membranaceus (Huangqi). Here, the chemical formula of AST-IV was C₄₁H₆₈O₁₄ with formula weight 784.97 (Fig. 1A). To examine the effect of AST-IV on HCC cell phenotype, the assay conducted the following series of experiments to check its effects. As shown in Fig. 1B, AST-IV inhibited the proliferation of HCC cells in the concentration range of 0 to 200 µmol/L, but did not significantly inhibit the normal cell. Secondly, the assay also observed the inhibitory effect of AST-IV on the migration of HCC cells at various concentrations (0 µmol/L, 25 µmol/L, 50 µmol/L). In the case of HCC proliferation, EdU analysis showed that AST-IV inhibited proliferative ability at concentration-dependent concentration (Fig. 1D, E and F). Briefly, this data suggested that AST-IV inhibited proliferation and migration of HCC cells in vitro.

Fig. 1.

AST-IV repressed the tumor progression of HCC in vitro. (A) The chemical structural formula of AST-IV. (B) The human HCC cell line (Huh7, MHCC97H) and normal human hepatic cells (THLE-3) were administrated by continuous concentration of AST-IV (0-200 µmol/L). The viability was tested by CCK-8. (C) Transwell assay was performed to test the migration of HCC cells. AST-IV (0 µmol/L, 25 µmol/L, 50 µmol/L) was administrated to cells in vitro. (D, E, F) EdU analysis was performed to test the proliferative ability of HCC cells with AST-IV concentration-dependent treatment. ** p < 0.01; * p < 0.05

AST-IV accelerated the ferroptosis of HCC cells

To investigate the role of AST-IV on HCC cells’ malignant characteristics, the functional assays about ferroptosis was performed. The functional assays revealed that AST-IV up-regulated the iron (Fe²⁺) concentration accumulation (Fig. 2A). Malondialdehyde (MDA) analysis revealed that AST-IV accelerated the MDA level (Fig. 2B). GSH analysis showed that AST-IV reduced the GSH expression (Fig. 2C). The transmission electron microscopy (TEM) indicated that AST-IV triggered the mitochondria structural alterations, e.g. reduced size, increased membrane density, and loss of cristae (Fig. 2D). In addition to the characteristics iron, MDA and GSH, ROS was also closely related to ferroptosis of tumor cells. Lipid peroxidation analysis showed that AST-IV up-regulated the ROS accumulation (Fig. 2E and F). Overall, the findings indicated that AST-IV accelerated the ferroptosis of HCC cells.

Fig. 2.

AST-IV accelerated the ferroptosis of HCC cells. (A) The iron (Fe²⁺) concentration accumulation was tested in HCC cells (Huh7, MHCC97H) treated with AST-IV (0 µmol/L, 25 µmol/L, 50 µmol/L). (B) The content of MDA was determined in HCC cells (Huh7, MHCC97H) treated with AST-IV. (C) The GSH was determined by the commercial kits. (D) The TEM was determined for the ultrastructure of the mitochondria in HCC cells. (E, F) The cellular lipid peroxidation level was tested by BODIPYTM 581/591 C11 on flow cytometer. ** p < 0.01; * p < 0.05

HMGB1 acted as the target of AST-IV

In the AST-IV treated HCC cells, the differentially expressed genes (DEGs) were detected by the RNA-Seq (Fig. 3A). The GSEA pathway enrichment analysis indicated that the FERROPTOSIS pathway was remarkable (Fig. 3B). Among these DEGs, HMGB1 was a significantly expressed gene. To confirm the potential target for AST-IV, the molecule docking was performed to test possible binding between HMGB1 and the AST-IV bioactive compound (Fig. 3C). The results indicated that the binding energy was − 7.2 kcal/mol. The interaction of HMGB1-AST-IV was further verified by microscale thermophoresis (MST) assay. The MST results indicated that the equilibrium dissociation constant (Kd) value was 1.03 µM (Fig. 3D). overall, these data concluded that AST-IV could interact with HMGB1, and HMGB1 acted as the downstream target of AST-IV in HCC.

Fig. 3.

HMGB1 acted as the target of AST-IV. (A) RNA-Seq was performed in the AST-IV treated HCC cells (Huh-7). (B) Gene set enrichment analysis (GSEA) shows the ferroptosis characteristic in AST-IV treated HCC cells (Huh-7) compared with control. (C) The molecule docking was performed to test the possible interaction between HMGB1 and the AST-IV bioactive compound. (D) Microscale thermophoresis (MST) assay was performed to test the interaction of HMGB1-AST-IV

AST-IV targeted HMGB1 to trigger HCC ferroptosis

To test the role of AST-IV on HMGB1, the following assays were performed in HCC cells. Firstly, the immunofluorescence analysis found that AST-IV administration reduced the HMGB1 level in Huh7 cells (Fig. 4A). Moreover, to investigate the role of AST-IV on ferroptosis characteristic of HCC cells, the overexpression of HMGB1 was performed in the Huh7 cells, as well as the co-administration of AST-IV. Results indicated that HMGB1 overexpression administration inhibited the iron (Fe²⁺) concentration accumulation (Fig. 4B) and malondialdehyde (MDA) (Fig. 4C), and co-administration of AST-IV up-regulated them. Besides, HMGB1 overexpression administration increased the GSH expression, and co-administration of AST-IV repressed it (Fig. 4D). Lipid peroxidation analysis showed that HMGB1 overexpression administration repressed the ROS accumulation, and co-administration of AST-IV increased it (Fig. 4E and F). Given these findings, the data demonstrated that AST-IV targeted HMGB1 to trigger HCC ferroptosis.

Fig. 4.

AST-IV targeted HMGB1 to trigger HCC ferroptosis. (A) Immunofluorescence analysis was performed to test the HMGB1 level in Huh7 cells with overexpression of HMGB1 (HMGB1) and AST-IV administration (50 µmol/L). (B) The iron (Fe²⁺) concentration accumulation was tested in HCC cells (Huh7) treated with overexpression of HMGB1 (HMGB1) and AST-IV (50 µmol/L). (C) The content of MDA was determined in HCC cells (Huh7) treated with HMGB1 and AST-IV. (D) The GSH was determined by the commercial kits. (E, F) The cellular lipid peroxidation level was tested by BODIPYTM 581/591 C11 on flow cytometer. ** p < 0.01; * p < 0.05

AST-IV repressed the HCC tumor growth in vivo

To determine the role of AST-IV on HCC tumor growth, the in vivo mice assay was performed with Huh-7 cells subcutaneous tumor (Fig. 5A). Mice were treated with AST-IV intraperitoneal injection. Results indicated that AST-IV repressed the HCC tumor growth in vivo, including the volume (Fig. 5B) and weight (Fig. 5C). Immumohistochemical (IHC) staining revealed that AST-IV repressed the expression of HMGB1, as well as the tumor growth (Fig. 5D). Given these findings, the data demonstrated that AST-IV repressed the HCC tumor growth in vivo.

Fig. 5.

AST-IV repressed the HCC tumor growth in vivo. (A) The in vivo mice assay was performed with Huh-7 cells subcutaneous tumor. Mice were treated with AST-IV intraperitoneal injection. (B) The volume and (C) weight were calculated in the AST-IV treated mice or control group. (D) Immumohistochemical (IHC) staining revealed the expression of HMGB1, ki67, and GPX4. * p < 0.05

Discussion

Astragaloside IV (AST-IV) is a key bioactive saponin derived from Astragalus membranaceus (Huangqi) which is one of the traditional tonifying herbs in China. In modern clinical practice, it is often combined with other traditional Chinese medicines herbs for anti-tumor treatment, and the therapeutic effect is definite. Modern pharmacology shows that the active components of Astragalus membranaceus mainly include three categories: polysaccharides, saponins and flavonoids. AST-IV is the main indicator for evaluating the quality of Astragalus membranaceus and an important material basis for exerting its efficacy, which has been proven to exert pharmacological immunity and anti-tumor effects Fig 6.

Fig. 6.

AST-IV represses HCC progression by modulating HMGB1-ferroptosis axis, which provides a novel insight for HCC

Here, these findings of our research revealed that AST-IV could repress the tumor progression (viability, migration) of HCC in vitro. Besides, AST-IV induced the ferroptosis (Fe²⁺, malondialdehyde, lipid peroxidation) in HCC cells. Therefore, the data concluded that AST-IV could exert the anti-tumor for HCC by inducing ferroptosis.

In HCC tumorigenesis, the ferroptosis plays a critical role with dual functions [21]. On the one hand, ferroptosis can inhibit the progression of HCC because it leads to the accumulation of ROS through the inactivation of GPX4 or the obstruction of cystine uptake (such as the down-regulation of SLC7A11), selectively killing tumor cells [22]. On the other hand, chronic inflammation and iron overload in the HCC microenvironment may accelerate tumorigenesis by promoting lipid peroxidation. Furthermore, HCC cells may resist ferroptosis by up-regulating FTH1 (iron storage protein) or activating the NRF2 antioxidant pathway, leading to treatment resistance [23, 24].

The effective active components of traditional Chinese medicine exert anti-cancer effects through multi-target mechanisms [25]. For example, Curcumin down-regulates the Wnt/β-catenin pathway and inhibits the proliferation of HCC tumors [26]. Paclitaxel blocks the cell cycle of HCC cells [27]. Baicalein inhibits the growth of HCC through the PI3K/AKT pathway. Artemisinin promotes the accumulation of ROS and triggers ferroptosis in HCC tumors. Tanshinone IIA enhances lipid peroxidation by down-regulating GPX4, inducing apoptosis and ferroptosis. Ginsenoside Rg3 inhibits MMP-9 to reduce metastasis and resists the metastasis and invasion of HCC. Quercetin enhances the sensitivity of HCC to sorafenib. Oridonin enhances the radiotherapy effect of HCC.

For the downstream target of AST-IV, this study found that high mobility group protein B1 (HMGB1) was regulated by it. Molecular docking and microscale thermophoresis indicated that HMGB1 was bound with AST-IV, and AST-IV could repress the expression level of HMGB1. Overall, HMGB1 acted as the target of AST-IV to regulate the HCC cells’ ferroptosis. HMGB1 plays multiple regulatory roles in ferroptosis, and its mechanism involves the regulation of inflammatory responses, oxidative stress and cell death signaling pathways [28, 29]. For example, HMGB1 promotes the accumulation of intracellular iron ions and intensifies iron-dependent lipid peroxidation by interacting with transferrin receptor (e.g. TfR1) [30].

In summary, this research revealed that AST-IV administration repressed the HCC tumor progression. Accurately, AST-IV could repress the HMGB1 expression and HMGB1 reversed the role of AST-IV on HCC cells’ ferroptosis (Fig. 6). Here, AST-IV represses HCC progression by modulating HMGB1-ferroptosis axis, which provides a novel insight for HCC.

Author contributions

Xingyang Zhao and Ruizhe Liu performed the assays. Xingyang Zhao and Ruizhe Liu wrote the main manuscript and prepared Figs. 1, 2, 3, 4, 5 and 6. Deqing Wu and Haiyu Zhou were responsible for the funding. All authors reviewed the manuscript.

Funding

This study was supported by the Natural Science Foundation of Jiangxi (S2024ZRMSL0165), the National Natural Science Foundation of China (82472064), the International Science and Technology Cooperation Program of Guangdong (2022A0505050048), the Natural Science Foundation of Guangdong (2024A1515012369), the Beijing Xisike Clinical Oncology Research Foundation (Y-HS202102-0038), and the GDPH Postdoctoral Start-up Funding (BY012024022).

Data availability

The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal protocol and details in present study had been conducted in accordance with the ethical principles and guidelines of the International Council for Laboratory Animal Science (ICLAS). The study had been approved by the Ethical Committee of Guangdong Provincial People’s Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

Not applicable.