Abstract
Background
Because of the high malignancy and subtle early symptoms of hepatocellular carcinoma (HCC), most patients lose the opportunity for surgery and opt for drug therapy. However, the current drugs for HCC treatment remain suboptimal, underscoring the urgent need to develop a novel anti-HCC agent. Curcumin is a natural chemical compound that has anti-cancer effects on various tumor cells, attracting sustained attention from researchers and clinicians. The present study aims to further elucidate curcumin’s anti-HCC mechanisms, thereby offering a promising therapeutic candidate for HCC patients.
Methods
The viability of HCC cells was evaluated by Cell Counting Kit-8 (CCK-8) assay. The levels of glutathione (GSH), malondialdehyde (MDA), and total iron in the cells were detected by biochemical kits. The levels of reactive oxygen species (ROS) and Fe2+ in the cells were detected by fluorescent probes. The protein expressions of protein kinase RNA-like endoplasmic reticulum kinase (PERK), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4) in the cells were detected by western blotting.
Results
Curcumin exerted a notably suppressive impact on HCC cells, which could be reversed by ferrostatin-1 (Fer-1) and desferrioxamine (DFO). By detecting cellular metabolic products, we observed an increase in total iron, Fe2+, MDA, ROS, and a reduction in GSH levels in HCC cells after treatment with curcumin, and these effects could be attenuated by Fer-1. Western blot analysis showed that curcumin significantly downregulated GPX4 levels in HCC cells while upregulating PERK, Nrf2 and HO-1 expression. Additionally, using PERK inhibitor, HO-1 inhibitor, and endoplasmic reticulum (ER) stress inhibitor could partially reverse the inhibition of HCC cells viability by curcumin and alleviate the reduction of GPX4 expression caused by curcumin.
Conclusions
Curcumin may induce ferroptosis in HCC cells through the PERK/Nrf2/HO-1 signaling pathway, thereby exerting its anti-cancer effects, suggesting that curcumin could potentially be used as a drug for treating HCC.
Keywords: Curcumin, ferroptosis, hepatocellular carcinoma (HCC), reactive oxygen species (ROS), nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1)
Highlight box.
Key findings
• Curcumin induces ferroptosis in hepatocellular carcinoma (HCC) by regulating protein kinase RNA-like endoplasmic reticulum kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway.
What is known and what is new?
• The majority of patients are first diagnosed with HCC in its middle or advanced stages. As a result, the search for low-toxicity and highly effective chemotherapeutic agents for HCC has emerged as a focal point in its treatment. Curcumin is a natural compound extracted from the rhizomes of plants in the ginger family. Previous studies have shown that curcumin has antioxidant, anti-inflammatory, antibacterial, and anti-cancer effects.
• This study provided evidence that curcumin could reduce the survival rate of HCC cells by inducing ferroptosis. The ferroptosis of HCC cells induced by curcumin may be mediated by the PERK/Nrf2/HO-1 signaling pathway.
What is the implication, and what should change now?
• This study has uncovered a previously unreported mechanism by which curcumin induces ferroptosis in HCC cells. The findings of this study may provide a basis for further research into the application of curcumin in the treatment of HCC. Although significant progress has been made in the study of the mechanisms of action of curcumin, more in vitro and in vivo experiments are still needed for further validation.
Introduction
As a frequently diagnosed malignant tumor, hepatocellular carcinoma (HCC) exhibits a high level of malignancy, ranking sixth in incidence rate and third in mortality rate among all malignant tumors (1). Most patients are diagnosed with HCC at mid-stage or late-stage, and the treatment regimen primarily relies on non-surgical methods. The current most commonly used targeted therapy drug, sorafenib, has a progression-free survival of only 4.3 months for liver cancer, and even the most recent immunotherapy regimen of atezolizumab combined with bevacizumab has a progression-free survival of only 6.8 months (2). HCC will rapidly develop resistance to current drugs, thus, there is an urgent need for a new drug to improve the prognosis of HCC patients.
Nature products are precious resources for developing anti-cancer drugs, and it has been shown that 83% of the small molecule anti-cancer drugs that have gained approval for clinical use either originate from natural products or are inspired by them (3). Curcumin, a natural chemical compound, is extracted from the rhizomes of Zingiberaceae plants, and prior researches have revealed the antioxidant, anti-inflammatory, and antimicrobial capabilities of curcumin (4). In addition, researchers have found that curcumin shows anti-cancer effects against various types of cancer, including HCC (5). However, the mechanism of action of curcumin on HCC remains unclear.
Ferroptosis, a novel mode of cell death identified by Dixon et al., is iron-dependent in its mechanism (6). It differs from known cell death modes such as apoptosis, necrosis, and autophagy. In terms of morphology, ferroptosis exhibits characteristics such as shrinking mitochondria, increased membrane density, shrinking or disappearing cristae, and ruptured mitochondrial outer membrane. Mechanistically, ferroptosis is characterized by the generation of reactive oxygen species (ROS), which occurs mainly through the Fenton reaction between Fe2+ and hydrogen peroxide. This process promotes the buildup of lipid peroxides within cells and depletion of glutathione (GSH), eventually resulting in cellular demise (7-9). Furthermore, gene expression related to glutathione peroxidase 4 (GPX4), cysteine/glutamate antiporter, Cellular iron and lipid metabolism undergoes significant changes in cells undergoing ferroptosis (8-10). Recent researches have shown that inducing ferroptosis may serve as a novel therapeutic intervention for malignancies. It has been shown that induced ferroptosis may overcome the treatment resistance of breast cancer and ovarian carcinoma (11,12). Similarly, some chemicals such as polyunsaturated fatty acids in quinoa, Penexanthone A and andrographis can directly suppress the activity of cancer cells by inducing ferroptosis (13-15). These findings indicate a close relationship between ferroptosis and the occurrence or development of tumor. However, it remains unknown whether curcumin can induce HCC to undergo ferroptosis to exert anti-cancer effects.
The focus of this study was to delve into whether the anti-cancer effect of curcumin on HCC is associated with ferroptosis. Additionally, we found evidence suggesting that curcumin-inducing ferroptosis may be related to the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1208/rc).
Methods
Chemicals and reagents
The curcumin used in this study was sourced from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The ferrostatin-1 (Fer-1) used in this study was sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Deferoxamine mesylate (DFO), 4-phenylbutyric acid (4-PBA), GSK2606414, zinc protoporphyrin (ZnPP), 2’,7’-dichlorofluorescein diacetate (DCFH-DA) were sourced from MedChemexpress (New Jersey, USA). The fetal bovine serum (FBS) used in this study was provided by Lonsera (Shanghai, China). The Cell Counting Kit-8 (CCK-8), malondialdehyde (MDA) assay kit (Specifications: 96 T, Code: KTB1050), GSH assay kit (Specifications: 96 T, Code: KTB1600) were purchased at Abbkine Scientific Co., Ltd. (Wuhan, China). Iron content test kit (specifications: 48 T, code: BL898A) was sourced from Labgic Technology Co., Ltd. (Beijing, China). The ferroOrange used in this study was sourced from Dojindo (Kumamoto, Japan).
The antibodies against PERK, Nrf2, HO-1, GPX4 and Primary β-actin antibody were sourced from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). The goat anti-rabbit horseradish peroxidase-conjugated secondary antibody was sourced from Jiangsu Cowin Biotech Co., Ltd. (Taizhou, China).
Cell line and culture
The HCC cell lines HepG2 and PLC were purchased at Boster Biological Technology Co., Ltd. (Wuhan, China). HepG2 was cultured in DMEM high glucose medium (Gibco; Thermo Fisher Scientific, Waltham, USA) supplemented with 1% penicillin-streptomycin (Gibco) and 10% FBS, and maintained in an incubator with a 5% CO2 atmosphere at a temperature of 37 ℃. PLC was cultured in MEM medium (Boster Biological Technology Co., Ltd., Wuhan, China) containing 1% penicillin-streptomycin (Gibco) and 10% FBS, and maintained in an incubator with a 5% CO2 atmosphere at a temperature of 37 ℃.
Cell viability analysis
Viability assessment of HepG2 and PLC cells was conducted through the CCK-8 assay. A density of 4×103 cells per well was used to seed the cells onto 96-well plates, which were then cultured for 24 hours, followed by treatment with specified concentrations of curcumin, Fer-1, DFO, 4-PBA, GSK2606414, or ZnPP alone or in combination for HCC cell lines. In the experiment of exploring the IC50 value of curcumin in two kinds of HCC cell lines, the concentrations of curcumin were 5, 10, 25, 50, 100 µm. For reliability and operational convenience, 50 µm was chosen as the curcumin concentration for all subsequent experiments because it approximates the 24 h half maximal inhibitory concentration (IC50) value determined in both HCC cell lines. According to the pre-experimental results, we selected 0.5 µm Fer-1, 100 µm DFO, 1 mm 4-PBA, 2.5 µm GSK2606414, 10 µm ZnPP to act with curcumin on two kinds of HCC cell lines, because these concentrations of drugs can better reduce the effect of curcumin on two kinds of HCC cell lines). Upon completion of the treatment, 10 µL of CCK-8 was dispensed into each well and incubated for 60 minutes, thereafter, the absorbance at 450 nm was recorded with a microplate reader (SPECTRA MAX 190).
Levels of total iron, GSH, and MDA assay
The HCC cell lines HepG2 and PLC were treated with curcumin and Fer-1 at a specified concentration alone or in combination for 24 hours. After the indicated treatment, the relative levels of total iron, GSH, and MDA were determined according to the methods provided in the respective reagent kit instructions from the manufacturers.
Levels of ROS assay
A density of 2×105 cells per well was used to seed the cells onto 6-well plates, and the cells were treated with specified concentrations of curcumin and Fer-1 alone or in combination for 24 hours. After treatment, the excess culture medium was removed, and then each well was incubated with 200 µL of 10 µm DCFH-DA working solution for 30 minutes, Subsequently, the samples were rinsed three times using phosphate-buffered saline (PBS) prior to examination with a fluorescence microscope.
Levels of Fe2+ assay
A density of 2×105 cells per well was used to seed the cells onto 6-well plates, and the cells were treated with specified concentrations of curcumin and Fer-1 alone or in combination for 24 hours. After treatment, we removed excess culture medium and washed the cells three times with PBS. Then added 200 µL of 1 µm concentration FerroOrange working solution to each well and incubated for 30 minutes before immediately observing under a fluorescence microscope.
Western blot analysis
HepG2 and PLC cells were processed with RIPA lysis buffer (Jiangsu Cowin Biotech Co., Ltd., Taizhou, China) containing 1% protease inhibitor (Jiangsu Cowin Biotech Co., Ltd.) to extract their total proteins. The lysates were spun at 12,000 g for 15 minutes at a chill temperature of 4 ℃ to precipitate the supernatant. The protein concentration of this supernatant was analyzed using the BCA assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). The protein samples were placed into 10% Bis-Tris gels for electrophoretic, followed by proteins transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). The PVDF membranes were first incubated with 5% milk for 1 hour to block non-specific binding. Subsequently, they were placed in a 1:2,000 dilution of primary antibodies and kept at 4 ℃ overnight in a cold chamber. Afterward, the membranes were incubated for 1 hour at room temperature with a 1:4,000 dilution of secondary antibodies. Finally, used the ECL chemiluminescence reagent (Abbkine Scientific Co., Ltd., Wuhan, China) to chemically luminescently visualize all the bands of proteins.
Statistical analysis
Results were illustrated with mean ± standard deviation (SD). Within-group and between-group comparisons were performed using t-tests or ANOVA. GraphPad Prism V9.5.0 was utilized for the statistical analysis, where P values below 0.05 were taken as statistically significant. All results were generated from at least three independent experiments.
Results
Curcumin decreased the cell viability of HCC cells and promoted HCC cells death
The structure of curcumin is shown in Figure 1A. To investigate the effect of curcumin on cell proliferation, we selected different concentrations of curcumin to treat HepG2 and PLC cells for 12, 24 and 48 hours. The CCK-8 assay was employed to determine cell viability following the treatment. After 12 hours of treatment, the IC50 values of curcumin in HepG2 and PLC cells were 75.01 µm (Figure 1B) and 193.9 µm (Figure 1C), respectively. After 24 hours of treatment, the IC50 values of curcumin in HepG2 and PLC cells were 64.55 µm (Figure 1B) and 42.51 µm (Figure 1C), respectively. After 48 hours of treatment, the IC50 values of curcumin in HepG2 and PLC cells were 30.86 µm (Figure 1B) and 25.08 µm (Figure 1C), respectively. Our results indicated that low-dose curcumin does not significantly influence the viability of the two HCC cell lines. while high concentrations of curcumin significantly and dose-dependently reduced the cell viability of HepG2 and PLC cells.
Figure 1.
Curcumin decreased the cell viability of HCC cells and promoted HCC cells death. (A) Chemical structure of curcumin. (B) Effect of curcumin on the growth of HepG2 cells following treatment with various concentrations for 12, 24, and 48 hours. (C) Effect of curcumin on the growth of PLC cells following treatment with various concentrations for 12, 24, and 48 hours. Compared to the control group, ns, P>0.05; **, P<0.01. HCC, hepatocellular carcinoma.
Curcumin-induced ferroptosis in HCC cells
To determine if curcumin is capable of inducing ferroptosis in the HCC cell lines HepG2 and PLC, we treated these cells with curcumin alone or in combination with two inhibitors that prevent ferroptosis, Fer-1 and DFO. The results showed that both ferroptosis inhibitors partially reversed the decrease in cell viability induced by curcumin on HepG2 and PLC cells (Figure 2A). Additionally, we also examined the variations in the levels of GPX4 and HO-1 proteins in these cells after treatment with curcumin alone or in combination with the ferroptosis inhibitor Fer-1, as these two proteins have been shown to be closely associated with ferroptosis in previous studies. The outcomes demonstrated that curcumin reduced the expression of GPX4 while increasing HO-1 expression in both cell lines. Furthermore, the ferroptosis inhibitor Fer-1 attenuated the impact of curcumin on GPX4 and HO-1 protein levels in both cell lines (Figure 2B-2D).
Figure 2.
Curcumin induced ferroptosis in HCC cells. (A) Effects of curcumin (50 µm) alone or in combination with Fer-1 (0.5 µm) or DFO (100 µm) on the growth of HepG2 and PLC cells after the treatment for 24 hours. (B) Effects of curcumin (50 µm) and Fer-1 (0.5 µm) alone or in combination on the expression of protein levels of HO-1 and GPX4 in HepG2 and PLC cells after the treatment for 24 hours. (C,D) Relative levels of HO-1 and GPX4, normalized to levels of β-actin, as determined by Western blotting. (E) Levels of total iron in HepG2 and PLC cells treated with curcumin (50 µm) and Fer-1 (0.5 µm) alone or in combination for 24 hours. (F,G) Determination of Fe2+ levels via fluorescence intensity measurements in HepG2 and PLC cells after treatment with curcumin (50 µm) and Fer-1 (0.5 µm) alone or in combination. ns, P>0.05; *, P<0.05; **, P<0.01. DFO, desferrioxamine; Fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; HCC, hepatocellular carcinoma; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2.
To further validate the effect of curcumin on two types of HCC cells and its association with ferroptosis, we investigated the changes in ferroptosis-related metabolites in two types of cells after treatment with curcumin alone or in combination with the ferroptosis inhibitor Fer-1. The results showed that the total iron, Fe2+, MDA, and ROS levels in HepG2 and PLC cells were significantly increased due to the curcumin treatment, at the same time, GSH levels underwent a significant reduction. Furthermore, the ferroptosis inhibitor Fer-1 was able to attenuate the effects of curcumin on two cell lines (Figure 2E-2G and Figure 3A-3D).
Figure 3.
Curcumin induced ferroptosis in HCC cells. (A) Levels of MDA in HepG2 and PLC cells treated with curcumin (50 µm) and Fer-1 (0.5 µm) alone or in combination for 24 hours. (B,C) Determination of ROS levels via fluorescence intensity measurements in HepG2 and PLC cells after treatment with curcumin (50 µm) and Fer-1 (0.5 µm) alone or in combination. (D) Levels of GSH in HepG2 and PLC cells treated with curcumin (50 µm) and Fer-1 (0.5 µm) alone or in combination for 24 hours. ns, P>0.05; **, P<0.01. a.u., arbitrary units; Fer-1, ferrostatin-1; GSH, glutathione; HCC, hepatocellular carcinoma; MDA, malondialdehyde; ROS, reactive oxygen species.
Curcumin regulated the expression of PERK, Nrf2, and HO-1 in HCC cells
We investigated the changes in the protein concentrations of PERK, Nrf2, HO-1, and GPX4 in HepG2 and PLC cells following curcumin treatment at varying concentrations. The results showed that curcumin dose-dependently increased the expression of PERK, Nrf2, and HO-1 proteins in both HCC cells, while dose-dependently down-regulating the expression of GPX4 in both cell types (Figure 4A-4C). It suggested that curcumin-induced ferroptosis in HCC cells was likely to be correlated with the PERK/Nrf2/HO-1 signaling pathway.
Figure 4.
Curcumin regulated the expression of PERK, Nrf2, and HO-1 in HCC cells. (A) Effects of curcumin (10, 25, 50 µm) on the expression of protein levels of PERK, Nrf2, HO-1 and GPX4 in HepG2 and PLC cells after the treatment for 24 hours. (B,C) Relative levels of PERK, Nrf2, HO-1 and GPX4, normalized to levels of β-actin, as determined by Western blotting. ns, P>0.05; *, P<0.05; **, P<0.01. GPX4, glutathione peroxidase 4; HCC, hepatocellular carcinoma; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; PERK, protein kinase RNA-like endoplasmic reticulum kinase.
Inhibition of PERK, Nrf2, and HO-1 expression could alleviate the sensitivity of HCC cells to curcumin-induced ferroptosis
To investigate whether the changes in PERK/Nrf2/HO-1 expression are linked to curcumin-induced ferroptosis in HCC cells, we treated HepG2 and PLC cells with the PERK inhibitor GSK2606414, the HO-1 inhibitor ZnPP, and curcumin alone or in combination. The results showed that the impact of curcumin on HCC cells was counteracted by GSK2606414, a PERK inhibitor, and ZnPP, an HO-1 inhibitor (Figure 5A,5B). GSK2606414 successfully inhibited PERK expression in both HCC cells, while downregulating the expression of Nrf2 and HO-1 proteins upregulated by curcumin, reversing the downregulation of GPX4 expression by curcumin. ZnPP successfully inhibited HO-1 expression in both cell lines, while reversing the downregulation of GPX4 expression by curcumin (Figure 5C-5H).
Figure 5.
Inhibition of PERK, Nrf2, and HO-1 expression could alleviate the sensitivity of HCC cells to curcumin-induced ferroptosis. (A,B) Effects of curcumin (50 µm) alone or in combination with GSK2606414 (2.5 µm) or ZnPP (10 µm) on the growth of HepG2 and PLC cells after the treatment for 24 hours. (C,D) Effects of curcumin (50 µm) alone or in combination with GSK2606414 (2.5 µm) or ZnPP (10 µm) on the expression of protein levels of PERK, Nrf2, HO-1 and GPX4 in HepG2 and PLC cells after the treatment for 24 hours. (E-H) Relative levels of PERK, Nrf2, HO-1 and GPX4, normalized to levels of β-actin, as determined by Western blotting. ns, P>0.05; *, P<0.05; **, P<0.01. GPX4, glutathione peroxidase 4; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; PERK, protein kinase RNA-like endoplasmic reticulum kinase.
PREK is an important protein in endoplasmic reticulum (ER) stress, and previous studies have also shown that curcumin can induce ER stress in cells (16-18). With the aim of investigating the potential relationship between ER stress and curcumin-induced ferroptosis, we exposed HepG2 and PLC cells to curcumin, with or without 4-PBA, an inhibitor of ER stress. The results showed that co-treatment with 4-PBA partially reversed the effect of curcumin on the viability of two types of HCC cells (Figure 6A). In addition, 4-PBA reversed the up-regulated expression of PERK, Nrf2, and HO-1 by curcumin in both cell lines, and reversed the down-regulated expression of GPX4 by curcumin (Figure 6B-6D).
Figure 6.
Inhibition of PERK, Nrf2, and HO-1 expression could alleviate the sensitivity of HCC cells to curcumin-induced ferroptosis. (A) Effects of curcumin (50 µm) alone or in combination with 4-PBA (1 mm) on the growth of HepG2 and PLC cells after the treatment for 24 hours. (B) Effects of curcumin (50 µm) alone or in combination with 4-PBA (1 mm) on the expression of protein levels of PERK, Nrf2, HO-1 and GPX4 in HepG2 and PLC cells after the treatment for 24 hours. (C,D) Relative levels of PERK, Nrf2, HO-1 and GPX4, normalized to levels of β-actin, as determined by Western blotting. ns, P>0.05; *, P<0.05; **, P<0.01. 4-PBA, 4-phenylbutyric acid; GPX4, glutathione peroxidase 4; HCC, hepatocellular carcinoma; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; PERK, protein kinase RNA-like endoplasmic reticulum kinase.
Discussion
HCC is highly malignant, and its early symptoms are subtle. Most patients are diagnosed with HCC in the middle or late stage for the first time. Therefore, finding low-toxicity and highly effective chemotherapy drugs for HCC has become a hot topic in its treatment. Curcumin is classified as a polyphenol and is obtained from the rhizomes of Zingiberaceae plants. Curcumin is recognized for its anti-inflammatory and antioxidant characteristics, as indicated by earlier studies, and it also has anti-cancer effect in tumors such as HCC, breast cancer, pancreatic cancer, and colorectal cancer (4,5). Therefore, curcumin is considered a promising natural product for development as an anticancer agent. The present study provided evidence that curcumin could reduce the viability of HCC cells by inducing ferroptosis. Further cellular experiments suggested that curcumin-induced ferroptosis in HCC cells may be mediated through the PERK/Nrf2/HO-1 signaling pathway. Therefore, this study revealed a previously unreported mechanism by which curcumin induces ferroptosis in HCC cells. The findings of this study may provide a basis for further research on the application of curcumin in the treatment of HCC.
Our results suggested that curcumin had an anti-tumor effect on HCC cells, and this effect could be partially reversed by two different ferroptosis inhibitors, Fer-1 and DFO. GPX4 is associated with the intracellular GSH homeostasis and is a key protein in ferroptosis, while HO-1 is associated with the concentration of iron in the cells (9,19,20). We found that curcumin significantly down-regulated GPX4 expression while up-regulating HO-1 expression in HCC cells, suggesting that its ability to reduce HCC cell survival may be linked to modulation of GPX4 and HO-1 protein levels. Furthermore, our findings showed that curcumin elevated the concentrations of total iron, Fe2+, MDA, ROS within HCC cells and decreases the GSH content, all of which could be attenuated by the ferroptosis inhibitor Fer-1. These findings provide evidence that curcumin can act as an inducer of ferroptosis in HCC cells to exert its anti-cancer effects.
We next investigated the downstream effectors of curcumin-induced ferroptosis in HCC cells. Our data showed that the expression levels of PERK, Nrf2, as well as HO-1 were upregulated in HCC cells after curcumin treatment, accompanied by a downregulation of GPX4 expression. The effects of curcumin on HCC cells were reversed by PERK inhibitor GSK2606414, HO-1 inhibitor ZnPP, and ER stress inhibitor 4-PBA. PERK is a central regulatory factor of ER stress, and the phosphorylation mediated by PERK can facilitate the detachment of Nrf2 from its cytosolic anchor, Kelch-like ECH-associated protein 1 (Keap1), enabling Nrf2 to translocate into the nucleus and initiate the transcription of its downstream target genes. The upregulation of NRF2 further leads to the upregulation of HO-1 expression (21,22). To sum up, curcumin may induce ferroptosis in HCC cells by upregulating PERK through ER stress, resulting in the upregulation of Nrf2, which in turn promotes the further enhancement of HO-1 expression.
The Nrf2/HO-1 signaling axis is a repeatedly validated ferroptosis-inducing pathway. For example, Levistilide A can induce ferroptosis in breast cancer through the activation of the Nrf2/HO-1 signaling cascade (23). Metformin can trigger ferroptosis in lung cancer through the Nrf2/HO-1 signaling axis (24). However, there are also reports suggesting that the stimulation of the Nrf2/HO-1 signaling axis can help cells resist oxidative stress and play a protective role for cells, such as Loganin mitigates septic acute kidney injury via the Nrf2/HO-1 signaling axis, and Astaxanthin can provide neuroprotective effects for traumatic brain injury via the Nrf2/HO-1 signaling axis (25,26). These studies imply that a slight upregulation of HO-1 expression can exert a protective effect on cells against oxidation. However, a significant upregulation of HO-1 can lead to the disruption of cellular redox equilibrium and result in ferroptosis. Curcumin demonstrated anti-tumor effects in this study, and it has also shown antioxidant protective effects in other experiments. Whether the dual effects of curcumin are related to the level of HO-1 protein expression is worthy of further study.
Conclusions
In conclusion, our results suggest that curcumin may activate the PERK/Nrf2/HO-1 pathway through ER stress, leading to significant upregulation of HO-1 expression. This in turn results in an increase in intracellular Fe2+ levels, disruption of redox homeostasis, and ultimately inducing HCC cells ferroptosis. Our study also indicates that curcumin as a natural compound possesses the potential to serve as a therapeutic intervention for HCC. However, there are still limitations in this study. The main limitations are the insufficient experiments conducted on ER stress and cell viability, as well as the absence of in vivo experiments. We will conduct additional experiments for validation in future studies.
Supplementary
The article’s supplementary files as
Acknowledgments
The authors thank the editor and the reviewers for their useful feedback that improved this paper.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Footnotes
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1208/rc
Funding: This study was supported by grants from the Suzhou medical key discipline (No. szzdxk1901) and the Hospital Internal Scientific Research Foundation of Kunshan Hospital Affiliated to Jiangsu University (No. CXTD21-C06).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1208/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1208/dss
References
- 1.Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. 10.3322/caac.21834 [DOI] [PubMed] [Google Scholar]
- 2.Finn RS, Qin S, Ikeda M, et al. Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med 2020;382:1894-905. 10.1056/NEJMoa1915745 [DOI] [PubMed] [Google Scholar]
- 3.Man S, Luo C, Yan M, et al. Treatment for liver cancer: From sorafenib to natural products. Eur J Med Chem 2021;224:113690. 10.1016/j.ejmech.2021.113690 [DOI] [PubMed] [Google Scholar]
- 4.Wahnou H, El Kebbaj R, Liagre B, et al. Curcumin-Based Nanoparticles: Advancements and Challenges in Tumor Therapy. Pharmaceutics 2025;17:114. 10.3390/pharmaceutics17010114 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Gutsche LC, Dörfler J, Hübner J. Curcumin as a complementary treatment in oncological therapy: a systematic review. Eur J Clin Pharmacol 2025;81:1-33. 10.1007/s00228-024-03764-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012;149:1060-72. 10.1016/j.cell.2012.03.042 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bipasha M, Deepali V, Prabal D, et al. Ferroptosis: A Mechanism of Cell Death With Potential Scope in Cancer Therapy. Asia Pac J Clin Oncol 2025;21:465-73. 10.1111/ajco.14172 [DOI] [PubMed] [Google Scholar]
- 8.Koppula P, Zhuang L, Gan B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 2021;12:599-620. 10.1007/s13238-020-00789-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Yang WS, SriRamaratnam R, Welsch ME, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 2014;156:317-31. 10.1016/j.cell.2013.12.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Li M, Wang Y, Li X, et al. Pharmacological targeting of the mitochondrial phosphatase PTPMT1 sensitizes hepatocellular carcinoma to ferroptosis. Cell Death Dis 2025;16:257. 10.1038/s41419-025-07581-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Pottier C, Montero-Ruiz L, Jehay R, et al. Targeting ferroptosis resistance resensitizes metastatic HR+HER2- breast cancer cells to palbociclib-hormone therapy. Cancer Commun (Lond) 2025;45:460-4. 10.1002/cac2.12646 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kapper C, Oppelt P, Arbeithuber B, et al. Targeting ferroptosis in ovarian cancer: Novel strategies to overcome chemotherapy resistance. Life Sci 2024;349:122720. [DOI] [PubMed] [Google Scholar]
- 13.Li S, Ding M, Feng M, et al. Polyunsaturated Fatty Acids in Quinoa Induce Ferroptosis of Colon Cancer by Suppressing Stemness. J Agric Food Chem 2024;72:16152-62. [DOI] [PubMed] [Google Scholar]
- 14.Zhao G, Liu Y, Wei X, et al. Identification of Penexanthone A as a Novel Chemosensitizer to Induce Ferroptosis by Targeting Nrf2 in Human Colorectal Cancer Cells. Mar Drugs 2024;22:357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Miyazaki K, Xu C, Shimada M, et al. Curcumin and Andrographis Exhibit Anti-Tumor Effects in Colorectal Cancer via Activation of Ferroptosis and Dual Suppression of Glutathione Peroxidase-4 and Ferroptosis Suppressor Protein-1. Pharmaceuticals (Basel) 2023;16:383. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Zhang MJ, Shi M, Yu Y, et al. Curcuminoid PBPD induces cuproptosis and endoplasmic reticulum stress in cervical cancer via the Notch1/RBP-J/NRF2/FDX1 pathway. Mol Carcinog 2024;63:1449-66. [DOI] [PubMed] [Google Scholar]
- 17.Cheng CY, Lin YH, Su CC. Curcumin inhibits the proliferation of human hepatocellular carcinoma J5 cells by inducing endoplasmic reticulum stress and mitochondrial dysfunction. Int J Mol Med 2010;26:673-8. [DOI] [PubMed] [Google Scholar]
- 18.Al Azzani M, Nizami ZN, Magramane R, et al. Phytochemical-mediated modulation of autophagy and endoplasmic reticulum stress as a cancer therapeutic approach. Phytother Res 2024;38:4353-85. [DOI] [PubMed] [Google Scholar]
- 19.Du HF, Wu JW, Zhu YS, et al. Fucoxanthin Induces Ferroptosis in Cancer Cells via Downregulation of the Nrf2/HO-1/GPX4 Pathway. Molecules 2024;29:2832. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Lv YT, Liu TB, Li Y, et al. HO-1 activation contributes to cadmium-induced ferroptosis in renal tubular epithelial cells via increasing the labile iron pool and promoting mitochondrial ROS generation. Chem Biol Interact 2024;399:111152. [DOI] [PubMed] [Google Scholar]
- 21.Wei R, Zhao Y, Wang J, et al. Tagitinin C induces ferroptosis through PERK-Nrf2-HO-1 signaling pathway in colorectal cancer cells. Int J Biol Sci 2021;17:2703-17. 10.7150/ijbs.59404 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Ji X, Chen Z, Lin W, et al. Esculin induces endoplasmic reticulum stress and drives apoptosis and ferroptosis in colorectal cancer via PERK regulating eIF2α/CHOP and Nrf2/HO-1 cascades. J Ethnopharmacol 2024;328:118139. 10.1016/j.jep.2024.118139 [DOI] [PubMed] [Google Scholar]
- 23.Jing S, Lu Y, Zhang J, et al. Levistilide a Induces Ferroptosis by Activating the Nrf2/HO-1 Signaling Pathway in Breast Cancer Cells. Drug Des Devel Ther 2022;16:2981-93. 10.2147/DDDT.S374328 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Deng C, Xiong L, Chen Y, et al. Metformin induces ferroptosis through the Nrf2/HO-1 signaling in lung cancer. BMC Pulm Med 2023;23:360. 10.1186/s12890-023-02655-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Zhang J, Wang C, Kang K, et al. Loganin Attenuates Septic Acute Renal Injury with the Participation of AKT and Nrf2/HO-1 Signaling Pathways. Drug Des Devel Ther 2021;15:501-13. 10.2147/DDDT.S294266 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Cai X, Hua S, Deng J, et al. Astaxanthin Activated the Nrf2/HO-1 Pathway to Enhance Autophagy and Inhibit Ferroptosis, Ameliorating Acetaminophen-Induced Liver Injury. ACS Appl Mater Interfaces 2022;14:42887-903. 10.1021/acsami.2c10506 [DOI] [PubMed] [Google Scholar]