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Article

Cucurbitacin B Inhibits Hepatocellular Carcinoma by Inducing Ferroptosis and Activating the cGAS-STING Pathway

by
Huizhong Zhang
1,†,
Aqian Chang
1,†,
Xiaohan Xu
1,
Hulinyue Peng
1,
Ke Zhang
1,
Jingwen Yang
1,
Wenjing Li
1,
Xinzhu Wang
1,
Wenqi Wang
1,
Xingbin Yin
1,
Changhai Qu
1,
Xiaoxv Dong
1,* and
Jian Ni
1,2,*
1
School of Chinese Material Medica, Beijing University of Chinese Medicine, Beijing 102488, China
2
College of Traditional Chinese Medicine, Xinjiang Medical University, Urumqi 830017, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Curr. Issues Mol. Biol. 2026, 48(2), 138; https://doi.org/10.3390/cimb48020138
Submission received: 26 December 2025 / Revised: 21 January 2026 / Accepted: 26 January 2026 / Published: 27 January 2026
(This article belongs to the Special Issue Therapeutic Effects of Natural Bioactive Compounds in the Management of Human Diseases: 2nd Edition)
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Abstract

The incidence of primary liver cancer is increasing annually, with extremely high mortality and suboptimal therapeutic outcomes. The inefficient presentation of tumor antigens and low infiltration of specific cytotoxic T lymphocytes (CTLs) result in insufficient immunogenicity, which limits the efficacy of immunotherapy. Despite the popularity of immune checkpoint inhibitors (ICIs), insufficient immune activation means only a small subset of hepatocellular carcinoma (HCC) patients exhibit clinical responses to ICIs, showing significant inter-individual variability. The activation of the cyclic GMP-AMP synthase(cGAS)- stimulator of interferon genes(STING) pathway initiates the expression of type I interferons (IFNs) and inflammatory cytokines, promoting the formation of a pro-inflammatory environment at the tumor site. This pathway enhances anti-tumor immune responses by facilitating antigen processing and presentation, T cell priming and activation, and remodeling of the immunosuppressive microenvironment. Our research found that cucurbitacin B (CuB), a natural component derived from traditional Chinese medicine, had significant anti-hepatocellular carcinoma properties and exerted anti-tumor effects through the cGAS-STING pathway. Specifically, CuB regulated ferroptosis by down-regulating the expression of Solute Carrier Family 7 Member 11 (SLC7A11) and Glutathione Peroxidase 4 (GPX4) and upregulating the expression of Transferrin Receptor Protein 1 (TFR1) and Long-chain Acyl-CoA Synthetase 4 (ACSL4). These actions involved lipid substrates, iron ion homeostasis, and antioxidant defense systems. The release of mitochondrial DNA (mtDNA) triggered by ferroptosis activated the cGAS-STING immune signaling pathway, leading to the up-regulation of cGAS, phosphorylated STING (p-STING), phosphorylated TANK-binding kinase 1 (TBK1), phosphorylated Interferon regulatory factor3 (IRF3), and Interferon-β (IFN-β). This cascade activation pattern provides new insights into the drug treatment of tumors.

1. Introduction

Primary liver cancer ranks as the 6th most common malignant tumor and the third leading cause of cancer-related death worldwide, exhibiting an extremely high mortality rate, with its incidence on the rise annually. HCC accounts for the highest proportion of primary liver cancer cases, ranging from 85% to 90%. Notably, HCC is difficult to detect in its early stages and has a poor prognosis. Due to complex pathogenic factors, high recurrence rates, high metastasis rates, and drug resistance, therapeutic modalities for primary liver cancer—including surgical resection, liver transplantation, minimally invasive interventional therapy, and immunotherapy—exhibit suboptimal efficacy and are associated with numerous adverse reactions [1,2]. Unfortunately, it remains impossible to observe the progression or recurrence of hepatocellular carcinoma based on existing molecular studies. It has created a significant gap in the pursuit of precision medicine [3]. In addition, its significant heterogeneity and immunosuppressive microenvironment pose challenges for the treatment of HCC, which depends on the strong proliferative capacity, phenotypic plasticity, epithelial–mesenchymal transition (EMT), and immune evasion capabilities of hepatocellular carcinoma cells [4,5]. It seems that most therapeutic approaches ultimately focus on activating and amplifying the anti-tumor immune strategy. The immune system plays a crucial role in tumor prevention, progression, and metastasis. Immunotherapy activates the immune system and leverages its robust anti-tumor responses to eliminate cancer cells [6,7]. Currently, the main types of immunotherapies for liver cancer include ICIs [8,9,10], liver cancer vaccines, and cellular therapies [11,12,13,14]. However, the overall clinical response rate of tumor immunotherapy remains unmet. Despite being at the forefront of immunotherapy, ICIs only elicit clinical responses in less than 30% of HCC patients, primarily due to insufficient immune activation and intrinsic and extrinsic resistance of the tumor microenvironment in hepatocellular carcinoma [15,16,17]. Immune cells within the tumor microenvironment are “educated” by tumor cells, enabling tumor cells to evade immune surveillance. Additionally, the efficacy of the naturally occurring adaptive immune response in tumors is relatively low, which severely limits the response to immunotherapy [18,19,20].
Novel strategies for immunotherapy include reversing the immunosuppression mediated by regulatory T cells (Tregs) and activating the innate immune system [21,22,23]. Given that the innate immune system serves as the primary protective mechanism against pathogen infections and constitutes the foundation of adaptive immunity, activating the innate immune system has emerged as a new approach to exploring cancer therapy. The lesion sites of solid tumors often exhibit insufficient innate immune responses. This deficiency severely hinders tumor recognition and may further promote tumor progression. Therefore, finding ways to activate the innate immune response within tumors has become a primary concern in immunotherapy [24,25,26,27]. mtDNA contains hypomethylated CpG sequences similar to those in bacterial DNA, enabling it to act as an activator of the innate immune system [28,29,30,31]. mtDNA in the cytoplasm binds to the DNA sensor cGAS, leading to the production of cyclic GMP-AMP (cGAMP) [32,33,34,35,36,37]. cGAMP binds to the stimulator of STING on the endoplasmic reticulum, recruits and activates TBK1, and thereby induces the phosphorylation of IRF3 [38]. Following phosphorylation, IRF3 translocates into the nucleus, thereby triggering the secretion of pro-inflammatory cytokines and type I interferon responses and ultimately enhancing innate immune responses. These effectors collectively promote the cytotoxic activity of natural killer cells, guide the expansion of cytotoxic CD8+ T cells, activate pro-inflammatory cellular programs, and regulate the expression of immunomodulatory genes [39,40].
CuB is an oxidized tetracyclic triterpenoid primarily found in plants of the Cucurbitaceae and Brassicaceae families [41]. Modern pharmacological studies have confirmed that CuB exhibited a broad range of pharmacological activities, including anti-inflammatory, antioxidant, antiviral, hypoglycemic, hepatoprotective, neuroprotective, and anti-cancer effects [42,43,44,45,46,47,48]. To a certain extent, it contributes to the prevention and treatment of various diseases such as inflammatory disorders, neurodegenerative diseases, diabetes, and cancer. Notably, CuB has demonstrated significant anti-tumor activity in both in vitro and in vivo studies [49]. It exerted its effects on the growth and progression of various tumors—including liver cancer, breast cancer, colon cancer, non-small cell lung cancer [50], prostate cancer [51], pancreatic cancer [52], nasopharyngeal carcinoma, tongue cancer, cutaneous squamous cell carcinoma, cholangiocarcinoma, and myeloid leukemia [53]—primarily through mechanisms such as inhibiting cell growth and proliferation [54], arresting the cell cycle [55,56,57], disrupting the cytoskeleton [58], inhibiting tumor angiogenesis [59], and inducing cell apoptosis and autophagy [60,61]. The anti-tumor activity of CuB might be closely associated with the ferroptosis mechanism. Studies have shown that CuB promoted iron accumulation and glutathione (GSH) depletion, leading to excessive lipid peroxide production and down-regulation of GPX4 expression, thereby inhibiting nasopharyngeal carcinoma cells both in vitro and in vivo [62]. CuB also exerted strong inhibitory effects on five non-small cell lung cancer cell lines, and traditional ferroptosis inhibitors (e.g., deferoxamine (DFO), liproxstatin-1 (Lip-1), ferrostatin-1 (Fer-1)) could counteract CuB-induced cell death. Specifically, CuB induced ferroptosis in H358 cells by promoting the accumulation of lipid ROS, malondialdehyde (MDA), and ferrous ions, increasing the expression of ferroptosis-related proteins, and reducing the level of GSH and mitochondrial membrane potential [63]. Thus, CuB-induced ferroptosis in tumor cells to inhibit tumor cell proliferation may be one of its potential anti-tumor pathways. It is worth noting that ferroptosis does not occur selectively in a specific cancer cell line, and the molecular mechanism network of ferroptosis remains valid in hepatocellular carcinoma [64]. Therefore, we boldly hypothesize that CuB can exert an inhibitory effect on hepatocellular carcinoma cells through ferroptosis, which triggers a cascade of mitochondrial DNA oxidative damage to activate the STING pathway. In this context, ferroptosis acts as a trigger for immunotherapy. We conducted experiments at the cellular level to validate this hypothesis.
Mitochondria, composed of a double-membrane structure, are widely distributed in various tissues and organs [65,66]. There is a closely interrelated connection between mitochondria and ferroptosis. In the ferroptosis state, mitochondria exhibit reduced volume, outer membrane rupture, and decreased or even complete loss of cristae. Lipid peroxides, the key drivers of ferroptosis, are primarily produced by mitochondria [67]. Ferroptosis-induced mitochondrial damage disrupts the activation of the cGAS-STING pathway. Arsenic trioxide (ATO)-induced ferroptosis in vitro in hepatocellular carcinoma cells, effectively eliminating tumors. Furthermore, ATO served as an immunostimulant, activating the cGAS/STING/IFN cascade by promoting mitochondrial damage and the release of mitochondrial DNA [68]. Thus, we hypothesized that the anti-tumor effect of CuB might exhibit a hierarchical activation characteristic, where the release of mtDNA triggered by ferroptosis activated the cGAS-STING pathway, thereby exerting potent anti-tumor activity.

2. Materials and Methods

2.1. Materials

Cucurbitacin B (CuB) (Yuanye, Shanghai, China, CAS#: 6199-67-3, No: JB245504, 98% purity); Ferrostatin-1 (Fer-1) (Abcam, Cambridge, UK, No: GR3378683-6); HepG2-specific medium (Procell, Wuhan, China); 0.25% Trypsin-EDTA (Thermo, Waltham, MA, USA); MTT (LabLead, Beijing, China); Dimethyl Sulfoxide(DMSO) (Sigma, St. Louis, MO, USA); Reactive Oxygen Species (ROS) Detection Kit, DAPI Stain, Mitochondrial Membrane Potential Assay Kit (JC-1), Total GSH Detection Kit, Lipid Peroxidation Detection Kit (BODIPY 581/591 C11) (Beyotime, Shanghai, China); Z-VAD-FMK, Necrostatin-1, Disulfiram, Ferrostatin-1, Liproxstatin-1 (MCE, Monmouth Junction, NJ, USA); FeRhoNox-1 (Abmole, Houston, Texas, USA); Malondialdehyde (MDA) Content Assay Kit (AIDISHENG, Yancheng, China); Tris-Glycine-SDS Electrophoresis Buffer, Tris-Glycine Transfer Buffer, SDS-PAGE Gel Preparation Kit (Boster, Wuhan, China); Bicinchoninic Acid (BCA) Protein Assay Kit (UtiBody, Tianjin, China); Mitochondria Isolation Kit, Animal Tissue/Cell Genomic DNA Isolation Kit (Solarbio, Beijing, China); ACSL4/FACL4 Polyclonal antibody, BAX Polyclonal antibody, Caspase 3/P17/P19 Polyclonal antibody, SLC7A11/xCT Polyclonal antibody, GPX4 Polyclonal antibody, BCL2 Polyclonal antibody, TMEM173/STING Polyclonal antibody, CD71 Polyclonal antibody, IRF3 Polyclonal antibody, Phospho-IRF3 (Ser396) Recombinant antibody, TBK1 Polyclonal antibody, Phospho-TBK1 (Ser172) Recombinant antibody, cGAS Polyclonal antibody, IFN-beta Polyclonal antibody (Proteintech, Wuhan, China).

2.2. Cell Culture

Human hepatoma cell line HepG2 (CL-0103), mouse-derived hepatoma cell line Hepa1-6 (CL-0105), and human immortalized liver cell line THLE-2 (CL-0833) were obtained from Procell and cultured in accordance with guidance from Procell. In brief, HepG2 cells were seeded in a 25 cm2 cell culture flask (Corning, Corning, NY, USA) and incubated in 5 mL of cell-specific medium (Procell, Wuhan, China). The HepG2 cell line was maintained in a constant-temperature cell culture incubator (Nuaire, Plymouth, MN, USA) at 37 °C and 5% CO2 with 95% humidity. The media was changed every two days until the cells reached 90% confluency. At this stage, cells were harvested using 0.25% Trypsin/EDTA (Gibco, Grand lsland, NY, USA) and subcultured for subsequent experiments.

2.3. Cytotoxicity Assay

HepG2 cells were seeded in a 96-well plate at a density of 7 × 103 cells/well and treated with different concentrations of CuB for 24 h, following the manufacturer’s instructions. The absorbance was measured at 490 nm using a microplate reader (Thermo, Waltham, MA, USA).
HepG2 cells were seeded in 96-well plate. Following the treatment of cells with inhibitors for 4 h, cells were treated with CuB for 24 h. The detection was carried out with a microplate reader.

2.4. Lipid Peroxidation Measurement

Cells were collected with trypsin and treated with C11-BODIPY (10 μM) for 20 min. The oxidation of the polyunsaturated butadienyl portion of C11-BODIPY resulted in a fluorescence emission peak between ~590 nm and ~510 nm. Cells were analyzed using flow cytometry (Exc = 488 nm, Em = 510 nm) after washing 3 times with PBS. The results were analyzed using CytExpert 2.6 software. Observe each group of cells with a fluorescence microscope and collect images. Measure relative fluorescence intensity using the ImageJ 1.54g software.

2.5. Total ROS and MMP Measurement

For total ROS detection, cells were treated with 10 μM DCFH-DA for 20 min. After washing cells three times with PBS, cells were collected and observed under a fluorescence microscope, with images captured (Exc = 488 nm, Em = 525 nm). For MMP measurement, cells were stained with JC-1 (1×) for 20 min, then washed three times with PBS before analysis. Both JC-1 monomers (Exc = 490 nm, Em = 530 nm) and aggregates were observed (Exc = 525 nm, Em = 590 nm) under a fluorescence microscope, with images captured. Measure relative fluorescence intensity using the ImageJ 1.54g software.

2.6. Measurement of Fe2+ Level

Drug-treated and untreated cells were collected and loaded with FeRhonox-1 (5 μM) for 30 min. After washing 3 times with PBS, each group of cells was observed with a fluorescence microscope (Exc = 540 nm, Em = 575 nm), with images captured. Relative fluorescence intensity was measured using ImageJ 1.54g software.

2.7. Morphological Changes

Firstly, different concentrations of CuB were added for 24 h, and a control group was prepared. The cells were collected and fixed with 2.5% glutaraldehyde for 12 h and 1% osmium tetroxide for 2 h. The samples were dehydrated by a series of gradient ethanol solutions, embedded in epoxy resin, and sectioned by a microtome. Double staining was performed with uranyl acetate and lead citrate, and the mitochondrial structure was observed under a transmission electron microscope, with images captured.

2.8. Measurement of GSH and MDA

The supernatant was collected after cell lysis and disruption. A sample was mixed with 150 µL of reaction liquid and incubated at 25 °C for 20 min. The level of GSH was determined by a microplate reader (Thermo, Waltham, MA, USA) at 412 nm.
A sample was mixed with 300 µL of reaction liquid and incubated at 95 °C for 30 min. The level of GSH was determined by a microplate reader (Thermo, Waltham, MA, USA) at 532 nm and 600 nm.

2.9. Detection of mtDNA Content

Cells were harvested by trypsinization, and an appropriate amount of ice-cold Lysis Buffer was added. The mixture was homogenized under ice bath conditions, and the cytoplasmic components and mitochondria were collected from the cell homogenate via differential centrifugation, followed by DNA extraction. The reaction solution was added, and the thermal cycling conditions were set as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s.
Primer sequences:
mt-F: 5′-CCTCCCATTCATTATCGCCGCCCTTGC-3′
mt-R: 5′-GTCTGGGTCTCCTAGTAGGTCTGGGAA-3′

2.10. Expression of Ferroptosis-Related Proteins

Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature, then subjected to 10% SDS-PAGE gel electrophoresis. The separated proteins were transferred onto PVDF membranes, which were then blocked with Blotto at room temperature with shaking for 1.5 h. Primary antibodies against ACSL4, GPX4, SLC7A11, and TFR1 (1:750 dilution) were added, followed by overnight incubation. Membranes were rinsed 3 times with TBST. Corresponding secondary antibodies (1:8000 dilution) were added and incubated for 1.5 h, after which membranes were rinsed 3 times with TBST again.The membranes were developed in a chromogenic reagent for 30 s, then visualized using an ECL chemiluminescence imaging system (Clinx, Shanghai, China), and images were captured.

2.11. Expression of cGAS-STING Pathway-Related Proteins

Cells were collected for total protein quantification. Protein samples were boiled for 5 min to denature and then subjected to 10% SDS-PAGE gel electrophoresis. The separated proteins were transferred onto PVDF membranes, which were blocked with Blotto at room temperature with shaking for 1.5 h. Primary antibodies against cGAS, STING, p-STING, IRF3, p-IRF3, TBK1, p-TBK1, and IFN-β (1:750 dilution) were added, followed by overnight incubation. Membranes were rinsed 3 times with TBST. Corresponding secondary antibodies (1:8000 dilution) were added and incubated for 1.5 h, after which membranes were rinsed 3 times with TBST again. The membranes were developed in a chromogenic reagent for 30 s, visualized using an ECL chemiluminescence imaging system, and images were captured.

2.12. Statistical Analyses

Statistical analysis and chart drawing were conducted using GraphPad Prism 8.0 software. One-way analysis of variance (ANOVA) was employed to analyze statistical significance.

3. Results

3.1. Ferroptosis Caused by CuB Is Related to Oxidative Stress

CuB significantly inhibits the proliferation of liver cancer cells in vitro. As the concentration of CuB increases, the cell viability in the CuB treatment group gradually decreases, showing a dose-dependent manner (Figure 1A). By calculation and curve fitting, the half-maximal inhibitory concentrations (IC50) of CuB for HepG2 and Hepa1-6 cells were determined to be 38.35 μM and 21.38 μM, respectively. CuB exhibits significant cytotoxicity against both liver cancer cell lines, while its toxicity to the hepatocyte THLE-2 is relatively weak. Collectively, these results demonstrated that CuB exerted a potent cytotoxic effect on HepG2 hepatocellular carcinoma cells and effectively inhibited the in vitro proliferation of HepG2 cells. Subsequently, to elucidate the mechanism underlying CuB-induced tumor cell death, HepG2 cells were preconditioned with inhibitors Z-VAD-FMK, Nec-1, Dis, Fer-1, and Lip-1 prior to exposure to CuB for 24 h. Our data demonstrated that compared with other inhibitors, ferroptosis inhibitors Fer-1 and Lip-1 could have minimized CuB-mediated cytotoxicity in HepG2 cells and partially rescued cell viability (Figure 1B). These findings suggested that the initiation of CuB-triggered cell death was linked to ferroptosis.
Ferroptosis is induced by the lipid peroxidation of iron-dependent unsaturated fatty acids highly expressed on the plasma membrane [69,70,71]. The accumulation of iron-dependent lipid reactive oxygen species is a common feature of all ferroptosis pathways. It is undeniable that lipid metabolism influences ferroptosis [72]. Changes in lipid peroxidation levels are the primary indicator for determining whether ferroptosis has occurred. Polyunsaturated fatty acids in the cell membrane are esterified into membrane phospholipids and then oxidized to form lipid ROS, which transmit ferroptotic signals and promote the occurrence of ferroptosis [73]. Subsequently, we evaluated the changes in intracellular ROS and LPO levels in the drug groups using DCFH-DA and BODIPY 581/591 C11 probes. Under the indication of DCFH-DA, cells in different drug dose groups showed gradually enhanced green fluorescence under the microscope (Figure 2C). Flow cytometry results revealed that the relative fluorescence intensity of intracellular ROS in the drug-treated groups was significantly higher than that in the normal group (Figure 2A), indicating that CuB upregulated intracellular ROS levels. BODIPY 581/591 C11 converted from red fluorescence to green fluorescence upon exposure to lipid peroxides, and the ratio of red to green fluorescence intensity was negatively correlated with the degree of cellular LPO. With the increase in drug concentration, weakened red fluorescence and enhanced green fluorescence were observed in cells under the microscope (Figure 3C), and the ratio of red to green relative fluorescence intensity decreased (Figure 3A). These data demonstrated an increase in the degree of intracellular lipid peroxidation.

3.2. CuB Induced Ferroptosis in HepG2 Cells via the SLC7A11/GPX4 Pathway

As endogenous cellular metabolites, ROS maintain a delicate balance between their production and scavenging [74]. When this balance between oxidative and antioxidant mechanisms is disrupted, cells undergo oxidative stress [75]. The essence of inducing cellular oxidative stress lies in disrupting the balance of the endogenous oxidation regulatory system, which manifests as an accelerated dynamic process of oxidative damage and a breakdown of the antioxidant defense system [76]. The glutathione system is a crucial antioxidant defense mechanism against ROS: it maintains cellular redox homeostasis by regulating the balance of redox-modulating molecules such as NAD/NADH, NADP/NADPH, and GSH/GSSG [77,78]. GSH, the most abundant intracellular antioxidant, can scavenge cellular ROS under the catalysis of GPX4 and serves as a key regulator of ferroptosis. Compared with the control group, the content of the intracellular antioxidant GSH was significantly decreased in the drug-treated group (Figure 4B).
Ferroptosis relies on the involvement of iron. Iron exists intracellularly in the forms of Fe2+ and Fe3+, which interconvert under the action of a series of proteins such as transferrin (TF) and ferroportin (FPN). Cellular iron homeostasis is tightly regulated through a rigorous internal iron recycling process [79,80]. Excessive accumulation of Fe2+ triggers ferroptosis. We detected labile Fe2+ in cells using FeRhoNox-1 and observed that the red fluorescence inside the cells increased and intensified (Figure 5B), showing an upward trend with increasing drug concentration (Figure 5A). Mitochondria are the core of iron metabolism and play a crucial role in ferroptosis induction [81,82,83]. Mitochondria contain approximately 20–50% of the total cellular iron. Iron-containing proteins in mitochondria act as essential cofactors to participate in electron transfer during enzymatic redox reactions. Once mitochondrial free iron is overloaded, it can increase mtROS levels via the Fenton reaction, activate mitochondrial NOX4 and ALOX15, and ultimately lead to lipid peroxidation and ferroptosis. We observed that drug treatment induced significant changes in mitochondrial structure, a marked decrease in mitochondrial membrane potential (Figure 6A,B), as well as mitochondrial shrinkage with reduced or even vanished cristae (Figure 7A). We detected a large amount of mtDNA spillover in the cytoplasm (Figure 7B), indicating that CuB stimulated mitochondria to release mtDNA. These findings indicate that CuB-induced ferroptosis severely damaged the mitochondria of HepG2 cells, rendering them unable to maintain normal function and morphology.
In summary, CuB increased intracellular ROS, LPO, and MDA levels, decreased GSH content, upregulated Fe2+ levels, and disrupted mitochondrial structure in HepG2 cells. These findings further confirmed that CuB might exert its effects by inducing ferroptosis in HepG2 cells. Subsequently, we detected the expression of proteins related to the ferroptosis pathway in cells and found that the expression levels of ACSL4 and TFR1 were significantly upregulated, while those of SLC7A11 and GPX4 were significantly downregulated, with a clear dose-dependent pattern (Figure 8A–E). ACSL4, TFR1, SLC7A11, and GPX4 are all core molecules in the ferroptosis regulatory pathway. They participate in determining whether ferroptosis occurs by regulating lipid substrates, iron ion homeostasis, and antioxidant defense systems, respectively. Free polyunsaturated fatty acids are esterified into membrane-bound phospholipid hydroperoxides (PLOOHs), which exhibit lethality after peroxidation. ACSL4 can preferentially bind long-chain polyunsaturated fatty acids such as arachidonic acid (AA) and adrenic acid (AdA) to coenzyme A to form intermediates, which are then esterified into phosphatidylethanolamine (PE) by various LPCAT enzymes, directly driving ferroptosis [84,85]. Iron primarily exists in the body in the form of Fe3+, which tends to bind with transferrin. With the assistance of the membrane protein transferrin receptor (TFR1), it enters the cell, where it is reduced by six-transmembrane epithelial antigen 3 (STEAP3) to the less stable Fe2+ form [86]. The Fe2+ released into the cytoplasm via solute carrier family 11 member 2 (SLC11A2) catalyzes the generation of hydroxyl radicals and triggers iron-mediated phagocytosis [87,88,89]. GPX4 uses two electrons provided by GSH to directly reduce PLOOH to the corresponding phospholipid alcohol (PLOH). In this process, if the generated PLOOH is not rapidly reduced by GPX4, it will interact with Fe2+ to produce alkoxy and peroxy radicals (Fenton reaction), thereby initiating the production of PLOOHs [90,91]. SLC7A11 is involved in the synthesis of the reducing agent GSH.

3.3. Amplified Activation of cGAS-STING Pathway by CuB-Induced DNA Release

cGAS can recognize double-stranded DNA (dsDNA) derived from bacteria, viruses, micronuclei, and mitochondria; catalyze the production of second messenger molecules; and then bind to and activate the STING molecule. CuB could induce mitochondrial shrinkage and structural disruption in HepG2 cells. We detected a large amount of mtDNA spillover in the cytoplasm, indicating that CuB stimulated mitochondria to release mtDNA, which might trigger conformational changes in cGAS and further activate the cGAS-STING pathway to release signaling factors. The protein expression levels of cGAS, phosphorylated STING (p-STING), phosphorylated TBK1 (p-TBK1), phosphorylated IRF3 (p-IRF3), and IFN-β were significantly increased in HepG2 cells after drug treatment (Figure 9A–F), demonstrating the activation of the cGAS-STING signaling pathway.
We investigated the expression levels of GPX4, cGAS, and p-STING in HepG2 cells treated with CuB and the ferroptosis inhibitor Fer-1, aiming to further elucidate the important role of ferroptosis in activating the cGAS-STING signaling pathway(Figure 10A–D). The results indicated that CuB decreased the expression of GPX4 while increasing the levels of cGAS and p-STING. The ferroptosis inhibitor Fer-1 reversed this trend. Under the influence of Fer-1, the expression levels of the key proteins cGAS and p-STING in the cGAS-STING signaling pathway were reduced, further demonstrating that the activation of the cGAS-STING signaling pathway by CuB was dependent on ferroptosis.

4. Discussion

Natural small-molecule compounds and their derivatives exhibit unique advantages in the prevention and treatment of various diseases, particularly in cancer and chronic conditions [92,93,94]. Firstly, natural small-molecule drugs typically exert their potent therapeutic effects by targeting multiple signaling pathways, which significantly reduces the likelihood of drug resistance [95,96]. In addition to their excellent pharmacological activity, natural small molecule compounds exhibit low toxicity, favorable pharmacokinetic properties, and outstanding biocompatibility [97,98]. In our study, the natural small-molecule compound CuB, derived from Cucurbitaceae and Brassicaceae plants, demonstrated strong anti-tumor activity. Our findings in the present study indicated that CuB induced ferroptosis in HepG2 cells through dual regulatory effects: it downregulated the expression of SLC7A11 and GPX4, leading to a significant increase in intracellular LPO and ROS levels, reduced GSH content, obvious mitochondrial shrinkage, and the reduction in or even disappearance of mitochondrial cristae, while simultaneously upregulating TFR1 expression to promote cellular Fe3+ uptake and ACSL4 expression to accelerate the production of lipid peroxidation substrates. By synergizing with its action on the SLC7A11/GPX4 signaling pathway axis, CuB collectively enhanced intracellular PL-PUFA-OOH accumulation, disrupted antioxidant system homeostasis, and thereby triggered ferroptosis.
The occurrence of ferroptosis drove the release of mtDNA from cells, and cGAS recognized this cytoplasmic mtDNA to activate the cGAS-STING signaling pathway. Excitingly, CuB can induce ferroptosis in HepG2 cells and thereby trigger an anti-tumor immune response. The bidirectional regulatory role between ferroptosis and the cGAS-STING signaling pathway is based on redox imbalance, iron homeostasis disruption, and metabolic reprogramming. Specifically, ROS produced by oxidative stress leads to DNA damage and the activation of damage-associated molecular patterns (DAMPs) in the cytoplasm, which further activate the cGAS-STING signaling pathway [99]. Research has shown that key regulatory targets associated with ferroptosis, such as GPX4, NCOA4, and LOX-1, have become potential targets in diseases involving STING signaling dysregulation. Multilayered regulatory networks collectively have viewed ferroptosis as a metabolic-immunity intersection [100]. An increasing number of studies have confirmed the interaction between ferroptosis and the cGAS-STING signaling pathway. The release of mtDNA caused by ferroptosis is considered one of the upstream events that activate the tumor cGAS-STING pathway. The telomerase inhibitor BIBR1532 synergistically enhanced anti-tumor immunity in conjunction with radiotherapy, relying on the inhibition of DNA damage repair and the occurrence of ferroptosis. Ferroptosis-induced mitochondrial oxidative stress stimulated the release of mtDNA into cytoplasm, which subsequently affected the translocation of STING from the endoplasmic reticulum to the Golgi apparatus, thereby activating the cGAS-STING pathway [101]. In hepatocytes, the depletion of Transforming Growth Factor β-Activated Kinase 1 (TAK1) induced ferroptosis through oxidative stress and iron overload. The oxidative DNA damage caused by ferroptosis promoted the activation of macrophage cGAS-STING signaling, intervening in liver injury, fibrosis, and tumor development [102]. Moreover, ferroptosis led to the production of oxidative DNA damage products, such as 8-hydroxy-2′-deoxyguanosine (8-OHG). 8-OHG acted as a direct ligand for cGAS, activating the STING-dependent DNA sensing pathway and contributing to the development of pancreatic tumors [103]. In summary, the natural small-molecule compound CuB could activate the cGAS-STING signaling pathway through ferroptosis, regulating downstream anti-tumor signal transduction and cytokine secretion. This avoids the need for repeated administration of chemotherapeutic drugs and immune activators, reduces the toxicity risk associated with multi-drug combination, and provides a novel development strategy for future hepatocellular carcinoma therapy.

Author Contributions

Conceptualization, H.Z. and A.C.; methodology, H.Z. and A.C.; validation, H.Z. and X.X.; formal analysis, H.Z. and H.P.; investigation, H.Z. and K.Z.; data curation, H.Z. and A.C.; writing—original draft preparation, H.Z. and A.C.; writing—review and editing, X.Y. and C.Q.; visualization, W.L. and J.Y.; supervision, X.W. and W.W.; project administration, X.D.; funding acquisition, X.D. and J.N. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded supported by the fundamental Research Funds for the Central Universities (No. 2022-JYB-XJSJJ015) and National Natural Science Foundation of China (No. 82405037).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CTLsCytotoxic T lymphocytes
ICIsImmune checkpoint inhibitors
HCCHepatocellular carcinoma
IFNstype I interferons
cGAS-STINGGMP-AMP synthase-stimulator of interferon genes
cGASCyclic GMP-AMP synthase
CuBCucurbitacin B
mtDNAMitochondrial DNA
TBK1TANK-binding kinase 1
IRF3Interferon regulatory factor3
EMTepithelial–mesenchymal transition
TregsRegulatory T cells
cGAMPcyclic GMP-AMP
STINGStimulator of interferon genes
DFODeferoxamine
Lip-1Liproxstatin-1
Fer-1Ferrostatin-1
MDAMalondialdehyde
ROSReactive oxygen species
GSHglutathione
DRP1Dynamin-related protein 1
ATOarsenic trioxide
ACSL4Long-chain Acyl-CoA Synthetase 4
GPX4Glutathione Peroxidase 4
SLC7A11Solute Carrier Family 7 Member 11
TFR1Transferrin Receptor Protein 1
IFN-βInterferon-β
Z-VAD-FMKBenzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone
Nec-1Necrostatin-1
DisDisulfiram
DMSODimethyl sulfoxide
BCABicinchoninic acid
TFTransferrin
FPNFerroportin
LPOlipid peroxidation
PLOOHsphospholipid hydroperoxides
AAarachidonic acid
AdAadrenic acid
PEphosphatidylethanolamine
LIPlabile iron pool
STEAP3Six-transmembrane Epithelial Antigen of Prostate 3
SLC11A2Solute Carrier Family 11 Member 2
FTH1Ferritin Heavy Chain 1
FTLFerritin Light Chain
NCOA4Nuclear Receptor Coactivator 4
SLC40A1Solute Carrier Family 40 Member 1
dsDNAdouble-stranded DNA

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Figure 1. (A) Cell viability of HepG2, Hepa1-6, THLE-2 cells assessed under different conditions (n = 6). (B) Cell viability of HepG2 cells pre-protected with Z-VAD-FMK, Nec-1, Dis, Fer-1 and Lip-1 (n = 6) (** p < 0.01).
Figure 1. (A) Cell viability of HepG2, Hepa1-6, THLE-2 cells assessed under different conditions (n = 6). (B) Cell viability of HepG2 cells pre-protected with Z-VAD-FMK, Nec-1, Dis, Fer-1 and Lip-1 (n = 6) (** p < 0.01).
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Figure 2. (A) Analysis of the flow cytometry results of ROS among different groups (n = 3). (B) Semi-quantitative analysis of ROS levels in different groups (n = 3). (C) Fluorescence images of HepG2 cells for ROS under different conditions (scale bar: 30 μm, 50 μm) (A: ×200, B: ×400) (*** p < 0.001).
Figure 2. (A) Analysis of the flow cytometry results of ROS among different groups (n = 3). (B) Semi-quantitative analysis of ROS levels in different groups (n = 3). (C) Fluorescence images of HepG2 cells for ROS under different conditions (scale bar: 30 μm, 50 μm) (A: ×200, B: ×400) (*** p < 0.001).
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Figure 3. (A) Analysis of the flow cytometry results of LPO among different groups (n = 3). (B) Semi-quantitative analysis of LPO levels in different groups (n = 3). (C) Fluorescence images of HepG2 cells for LPO under different conditions (scale bar: 30 μm) (*** p < 0.001).
Figure 3. (A) Analysis of the flow cytometry results of LPO among different groups (n = 3). (B) Semi-quantitative analysis of LPO levels in different groups (n = 3). (C) Fluorescence images of HepG2 cells for LPO under different conditions (scale bar: 30 μm) (*** p < 0.001).
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Figure 4. (A) Determination of intracellular MDA levels in different groups (n = 3). (B) Determination of intracellular GSH levels in different groups (n = 3) (*** p < 0.001).
Figure 4. (A) Determination of intracellular MDA levels in different groups (n = 3). (B) Determination of intracellular GSH levels in different groups (n = 3) (*** p < 0.001).
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Figure 5. (A) Semi-quantitative analysis of relevant Fe2+ levels in different groups (n = 6). (B) Fluorescence images of HepG2 cells for Fe2+ under different conditions (scale bar: 10 μm) (* p < 0.05, ** p < 0.01).
Figure 5. (A) Semi-quantitative analysis of relevant Fe2+ levels in different groups (n = 6). (B) Fluorescence images of HepG2 cells for Fe2+ under different conditions (scale bar: 10 μm) (* p < 0.05, ** p < 0.01).
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Figure 6. (A) Analysis of the flow cytometry results of MMP among different groups (n = 3). (B) Semi-quantitative analysis of MMP levels in different groups (n = 3). (C) Fluorescence images of HepG2 cells for MMP under different conditions (scale bar: 50 μm) (*** p < 0.001).
Figure 6. (A) Analysis of the flow cytometry results of MMP among different groups (n = 3). (B) Semi-quantitative analysis of MMP levels in different groups (n = 3). (C) Fluorescence images of HepG2 cells for MMP under different conditions (scale bar: 50 μm) (*** p < 0.001).
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Figure 7. (A) Observation of cellular mitochondria before and after drug treatment. Red arrows indicate mitochondria. (A. Control Group, B. Drug-treatment group, scale bar: 5 μm). (B) PCR detection of the effect on mtDNA expression levels in different groups (n = 3) (** p < 0.01, *** p < 0.001).
Figure 7. (A) Observation of cellular mitochondria before and after drug treatment. Red arrows indicate mitochondria. (A. Control Group, B. Drug-treatment group, scale bar: 5 μm). (B) PCR detection of the effect on mtDNA expression levels in different groups (n = 3) (** p < 0.01, *** p < 0.001).
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Figure 8. (A–D) Western blotting detection of the expression levels of ACSL4, GPX4, SLC7A11, and TFR1 in cells under different treatment conditions (n = 3). (E) Band diagrams showing the expression of ACSL4, GPX4, SLC7A11, and TFR1 in cells under different treatment conditions (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 8. (A–D) Western blotting detection of the expression levels of ACSL4, GPX4, SLC7A11, and TFR1 in cells under different treatment conditions (n = 3). (E) Band diagrams showing the expression of ACSL4, GPX4, SLC7A11, and TFR1 in cells under different treatment conditions (* p < 0.05, ** p < 0.01, *** p < 0.001).
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Figure 9. (A) Band diagrams showing the expression of cGAS, STING, p-STING, IRF3, p-IRF3, TBK1, p-TBK1, IFN-β, p-IFN-β in cells under different treatment conditions. (B–F) Western blotting detection of the expression levels of cGAS, p-STING, p-IRF3, p-TBK1, and p-IFN-β in cells under different treatment conditions (n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 9. (A) Band diagrams showing the expression of cGAS, STING, p-STING, IRF3, p-IRF3, TBK1, p-TBK1, IFN-β, p-IFN-β in cells under different treatment conditions. (B–F) Western blotting detection of the expression levels of cGAS, p-STING, p-IRF3, p-TBK1, and p-IFN-β in cells under different treatment conditions (n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001).
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Figure 10. (A) Band diagrams showing the expression of cGAS, STING, p-STING, GPX4 in cells under different treatment conditions. (B–D) Western blotting detection of the expression levels of cGAS, p-STING, and GPX4 in cells under different treatment conditions (n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05).
Figure 10. (A) Band diagrams showing the expression of cGAS, STING, p-STING, GPX4 in cells under different treatment conditions. (B–D) Western blotting detection of the expression levels of cGAS, p-STING, and GPX4 in cells under different treatment conditions (n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05).
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MDPI and ACS Style

Zhang, H.; Chang, A.; Xu, X.; Peng, H.; Zhang, K.; Yang, J.; Li, W.; Wang, X.; Wang, W.; Yin, X.; et al. Cucurbitacin B Inhibits Hepatocellular Carcinoma by Inducing Ferroptosis and Activating the cGAS-STING Pathway. Curr. Issues Mol. Biol. 2026, 48, 138. https://doi.org/10.3390/cimb48020138

AMA Style

Zhang H, Chang A, Xu X, Peng H, Zhang K, Yang J, Li W, Wang X, Wang W, Yin X, et al. Cucurbitacin B Inhibits Hepatocellular Carcinoma by Inducing Ferroptosis and Activating the cGAS-STING Pathway. Current Issues in Molecular Biology. 2026; 48(2):138. https://doi.org/10.3390/cimb48020138

Chicago/Turabian Style

Zhang, Huizhong, Aqian Chang, Xiaohan Xu, Hulinyue Peng, Ke Zhang, Jingwen Yang, Wenjing Li, Xinzhu Wang, Wenqi Wang, Xingbin Yin, and et al. 2026. "Cucurbitacin B Inhibits Hepatocellular Carcinoma by Inducing Ferroptosis and Activating the cGAS-STING Pathway" Current Issues in Molecular Biology 48, no. 2: 138. https://doi.org/10.3390/cimb48020138

APA Style

Zhang, H., Chang, A., Xu, X., Peng, H., Zhang, K., Yang, J., Li, W., Wang, X., Wang, W., Yin, X., Qu, C., Dong, X., & Ni, J. (2026). Cucurbitacin B Inhibits Hepatocellular Carcinoma by Inducing Ferroptosis and Activating the cGAS-STING Pathway. Current Issues in Molecular Biology, 48(2), 138. https://doi.org/10.3390/cimb48020138

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