Curcumin induces Ferroptosis in Hepatocellular Carcinoma by Regulating the P62-KEAP1-NRF2 signaling pathway

Curcumin induces Ferroptosis in Hepatocellular Carcinoma by Regulating the P62-KEAP1-NRF2 signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Curcumin induces Ferroptosis in Hepatocellular Carcinoma by Regulating the P62-KEAP1-NRF2 signaling pathway Jinlan Deng, Zhijuan Wu, Shangkun Ning, Xu Chang, Jibing Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7197765/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2025 Read the published version in BMC Cancer → Version 1 posted 11 You are reading this latest preprint version Abstract Context: Hepatocellular carcinoma (HCC) urgently requires new treatment strategies due to chemotherapy resistance and toxic side effects. Curcumin exhibits broad-spectrum anticancer activity against HCC and other malignant tumors. Ferroptosis, a form of cell death characterized by iron deposition, glutathione depletion, and lipid peroxidation, has emerged as a promising therapeutic target for cancer. The P62-KEAP1-NRF2 pathway is a key signaling pathway in ferroptosis. Objective: This study explored the mechanism by which curcumin induces ferroptosis in HCC cells by modulating the P62-KEAP1-NRF2 signaling pathway. Materials and methods: We constructed a Hepa1-6 xenograft mouse model to investigate changes in tumor growth, ferroptosis indicators, and the expression of P62, KEAP1, and NRF2. In vitro, HepG2 cell were treated with a ferroptosis inhibitor (Fer-1) or subjected to P62 overexpression, after which cell viability and key ferroptosis parameters Results: Curcumin (100 mg/kg, 15 days) significantly inhibited tumor growth, reduced glutathione (GSH) levels in tumor tissues, and increased reactive oxygen species (ROS), malondialdehyde (MDA), and Fe 2+ content. Additionally, in vitro experiments showed that curcumin inhibited HepG2 cell growth and proliferation, induced ferroptosis in HepG2 cells, decreased P62 and NRF2 levels, and increased KEAP1 levels. These effects were inhibited by the Fer-1. Overexpression of P62 reduced the impact of curcumin on the P62-KEAP1-NRF2 pathway and ferroptosis. Discussion and conclusion: Curcumin exerts its antitumor effects by inducing ferroptosis through the inhibition of the P62-KEAP1-NRF2 signaling pathway. This study provides important scientific basis for the application of curcumin and new potential targets for studying the pathological mechanisms of hepatocellular carcinoma. Curcumin HCC Ferroptosis P62-KEAP1-NRF2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and a leading cause of cancer-related deaths. Epidemiological data indicate that nearly 906,000 people are diagnosed with hepatocellular carcinoma annually, with HCC being the predominant form(Bray et al., 2024 ; Vogel, Meyer, Sapisochin, Salem, & Saborowski, 2022 ). Consequently, there is an urgent need to explore effective treatment strategies to combat this deadly disease. Ferroptosis, a novel form of regulated cell death distinct from apoptosis and necrosis, is characterized by the accumulation of iron and disruption of cellular redox homeostasis, leading to a decline in antioxidant capacity. Morphologically, ferroptosis is marked by intact cell nuclei, non-aggregated chromatin, unruptured cytoplasmic membranes, reduced or absent mitochondria with ruptured outer membranes, and increased inner membrane density(Gautheron, Gores, & Rodrigues, 2020 ). A key upstream process in ferroptosis involves the cystine/glutamate antiporter system (system xc⁻), which supplies cysteine for glutathione (GSH) synthesis(Hayano, Yang, Corn, Pagano, & Stockwell, 2016 ; Lee et al., 2017 ; Wang et al., 2018 ). Depletion of cysteine leads to GSH depletion, thereby triggering ferroptosis(Dixon et al., 2012 ). Glutathione peroxidase 4 (GPX4), a critical regulator of ferroptosis(W. S. Yang et al., 2014 ), converts GSH into oxidized glutathione (GSSG) and reduces cytotoxic lipid peroxides (PL-OOH) to non-toxic alcohols (PL-OH). Inhibition of GPX4 activity results in the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation products, ultimately leading to cell death(Friedmann Angeli, Krysko, & Conrad, 2019 ). P53, particularly the acetylation-deficient mutant p53 3KR, is responsible for suppressing the expression of XCT in the context of ferroptosis. The inhibition of XCT leads to the depletion of GSH and promotes ferroptosis(Jiang et al., 2015 ). Recent studies highlight that ceruloplasmin (CP) inhibits ferroptosis in HCC cells by regulating iron homeostasis, whereas CP inhibition increases Fe²⁺ and ROS levels, promoting erastin- and RSL3-induced ferroptosis(Shang et al., 2020 ). Notably, GPX4-associated ferroptotic hepatocyte death triggers a tumor-suppressive immune response, and combining ferroptosis inducers significantly improves survival in murine liver tumor models(Conche et al., 2023 ). These findings underscore the therapeutic potential of ferroptosis in treating primary liver tumors and metastases, positioning it as a promising avenue for HCC-specific therapy. The P62-KEAP1-NRF2 signaling pathway has been identified as a critical regulator of ferroptosis in HCC(Sun et al., 2016 ). Nuclear factor erythroid 2-related factor 2 (NRF2) plays a pivotal role in antioxidant responses and cellular defense mechanisms(Capelletti, Manceau, Puy, & Peoc'h, 2020 ). Under physiological conditions, Kelch-like ECH-associated protein 1 (KEAP1) maintains low NRF2 levels by promoting its ubiquitination and proteasomal degradation(Hirayama, Miki, & Nagasawa, 2019 ; R. Yang et al., 2020 ). During oxidative stress, NRF2 is stabilized and released from KEAP1-mediated inhibition, a process regulated by the autophagy receptor P62. Studies demonstrate that P62 knockdown in HCC cells enhances ferroptosis induced by erastin and sorafenib. Furthermore, NRF2 inhibition amplifies the anticancer effects of these agents in vitro and in xenograft models(Sun et al., 2016 ). In H22 cells, ATP2B3 mitigates erastin-induced ferroptosis by modulating the P62-KEAP1-NRF2-HO-1 pathway(Guo et al., 2023 ). Additionally, arenobufagin has been shown to induce autophagy-dependent ferroptosis in HepG2 cells via the P62-KEAP1-NRF2 axis(Y. Yang et al., 2024 ). Curcumin, a polyphenolic compound derived from turmeric roots and stems, is widely used as a food additive in Eastern cuisine. Preclinical studies highlight its pharmacological potential, particularly in cancer therapy(Prasad, Gupta, Tyagi, & Aggarwal, 2014 ). Curcumin exhibits antiproliferative and pro-oxidative effects in HCC cells, inhibiting tumor growth and metastasis by modulating transcription factors (e.g., growth factors, inflammatory cytokines), protein kinases, and other enzymes. It suppresses cancer cell proliferation by inducing cell cycle arrest, apoptosis, and ferroptosis(P. M. Li et al., 2014 ). Research confirms that curcumin-mediated NF-κB inhibition effectively curbs HCC cell growth(Marquardt et al., 2015 ). Moreover, curcumin has been shown to induce ferroptosis in cancer cells, exerting antitumor effects(R. Li et al., 2020 ). However, the precise mechanisms linking curcumin’s tumor-suppressive effects to ferroptosis in HCC remain unclear. This study elucidates that curcumin regulates ferroptosis via the P62/KEAP1/NRF2 signaling pathway, modulating the antioxidant capacity of HCC cells and inhibiting their growth. These findings reveal the protective effects, mechanisms, and molecular pathways of curcumin in HCC, providing a scientific basis for understanding the antitumor properties of traditional Chinese medicine in hepatocellular carcinoma treatment. Materials and Methods Chemicals and Reagents . Fetal bovine serum (cat. no. D1220D), DMEM medium (cat. no. MA0212), Cell Counting Kit-8 (cat. no. MA0218), MTT reagent (cat. no. 298-93-1), Annexin V-FITC/7-AAD cell apoptosis detection kit (cat. no. MA0428), EdU-555 cell proliferation detection kit (cat. no. CX003), PBS (cat. no. MA0015) and curcumin (cat. no. MB2147) were all purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). The malondialdehyde (MDA) detection kit (cat. no. S0131S) and Caspase inhibitor Z-VAD-FMK (cat. no. C1202) were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). Fer-1 (cat. no. 347174-05-4) was purchased from MedChemExpress (MCE, New Jersey, USA). The Reduced Glutathione (GSH) detection kit (cat. no. A006-2-1) was purchased from Nanjing Jiancheng Biotechnology (Nanjing, China). The ferrous ion (Fe 2+ ) detection kit (cat. no. BC5410) and DMSO dimethyl sulfoxide (cat. no. D8370) were both purchased from Solaibao Technology Co., Ltd. (Beijing, China). The DAPI staining solution kit (cat. no. EE0011) and Sorafenib (cat. no. SJ-MX0071A) were purchased from SparkJade Biotechnology Co., Ltd. (Shandong, China). In Western blot analysis, antibodies against P62 (species: rabbit, dilution: 1:5000, cat. no. ab109012) were purchased from Abcam (Cambridge, UK). Antibodies against KEAP1 (species: rabbit, dilution: 1:1000, cat. no. 8047), GPX4 (species: rabbit, dilution: 1:1000, cat. no. 59735), XCT (species: rabbit, dilution: 1:1000, cat. no. 12691) and P53 (species: mouse, dilution: 1:1000, cat. no. 2524) were purchased from CST (CST, USA). The antibody against β-actin (species: mouse, dilution: 1:4000,cat. no. GB15001) was purchased from Servicebio (Wuhan, China). The antibody against XCT (species: rabbit, dilution: 1:1000, cat. no. DF12509) was purchased from Affinity Biosciences (USA). The antibody against NRF2 (species: rabbit, dilution: 1:1000, cat. no. A0674) was purchased from Abclonal (Abclonal, USA). Goat anti-mouse IgG (cat. no. ZB-2305) and goat anti-rabbit IgG (cat. no. ZB-2301) were provided by Zhongshan Golden Bridge Biotechnology Co., Ltd. Animal model construction C57BL/6JNifdc mice [specific pathogen free (SPF) grade; Female; Weight, 17-19g; Age, 6–7 weeks provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in the SPF grade of Shandong University of Traditional Chinese Medicine (Jinan, China). The animal experimental protocol was conducted in accordance with the "National Institutes of Health Guidelines for the Care and Use of Laboratory Animals" and approved by the Institutional Animal Care and Research Advisory Committee of the Shandong University of Traditional Chinese Medicine (Approval No. SDUTCM20241113002; Jinan, China). A total of 30 mice were subjected to a 12-hour light/dark cycle at 25.0 ± 2.0°C and 55.0 ± 5.0% relative humidity, and were able to freely access food and water. After one week of adaptation, 1×10^6 Hepa1-6 cells in 0.2 ml of physiological saline were injected from the right axilla of mice to form subcutaneous xenograft tumors. When Hepa1-6 cell tumors were observed with the naked eye, mice were divided into the following three groups (n = 10/group): i) Model group (injected with cancer cells, intraperitoneal injection of an equal volume of physiological saline as a negative control); ii) Curcumin group (injection of cancer cells, daily intraperitoneal injection of 100mg/kg curcumin); iii) Chemotherapy group (injection of cancer cells, daily intraperitoneal injection of 60mg/kg Sorafenib; positive control). The regimens of Sorafenib and curcumin are based on their clinical applications and previous studies, respectively(Tian et al., 2021 ). Measure the weight and tumor volume of each mouse every 3 days within 15 days. The tumor volume is calculated using the following equation: tumor volume = length x width x width/2. When a tumor diameter of 1500mm 3 centimeters is recorded or severe disease symptoms (such as breathing difficulties or paralysis) are observed, euthanize the animal. On the 15th day, 30 mice (n = 10/group) were euthanized by intraperitoneal injection of excessive pentobarbital sodium (200mg/kg). The duration between injection and final tumor growth measurement is 20 days. Remove tumor tissue, weigh and store immediately at -80°C for further analysis. Cell Culture. The human hepatocellular carcinoma cell line (HepG2, CVCL_0027, cat. no.CL-0103) and the mouse-derived hepatocellular carcinoma cell line (Hepa1-6, CVCL_0327, cat. no.HTX1770) were both purchased from MeilunBio (Dalian, China). The cells were cultured in DMEM medium. The medium was supplemented with 10% fetal bovine serum, 100 UI/mL penicillin, and 100 ug/mL streptomycin(cat. no. MA0110, Meilun, Dalian, China). These cells were cultured in a humidified incubator at 37°C and 5% carbon dioxide(Thermo Scientific, USA). H&E The tumor tissues were embedded in paraffin, sectioned, and stained according to standard operating procedures. After dehydration and transparency treatment, the sections were mounted. Whole-slide scanning was then performed using a digital slide scanning system for image acquisition (SQS-600P, China). Cell Viability Assay Cell viability was assessed using the CCK-8 and MTT assays. For the CCK-8 assay, cells were seeded in 96-well plates at a density of 5,000 cells per well. After treating HepG2 cells with curcumin (5, 10, 20, 30, 40, 50, 60, 70, 80, 90 µM) for 12 hours, 10 µL of CCK-8 reagent was added to each well. The plates were incubated at 37°C for 1 hour, and the absorbance (OD) of each well was measured at 450 nm according to the manufacturer's instructions. Similarly, for the MTT assay, cells were seeded in 96-well plates at a density of 5,000 cells per well. Hepatoma cells were treated with curcumin (0, 5, 20, 50 µM) for 12 hours, followed by the addition of MTT reagent (10% or 20 µL per well). After 4 hours, the culture was terminated, and the supernatant in each well was carefully aspirated. DMSO (150 µL per well) was added, and the plates were shaken on an orbital shaker (Qilinbeier, China) for approximately 10 minutes to dissolve the formed crystals. The absorbance was measured at a wavelength of 490 nm using a microplate reader (BioTek,USA). Migration assay HepG2 cells were added to the upper chamber for culture and then stained with H&E. HepG2 migrated to its lower face were counted in 3 views per membrane under a microscope (ZEISS, Germany). Flow cytometry Seed 1×10^5 cells per well in a 6-well plate, pretreat with Fer-1 and Z-VAD-FMK, then treat with curcumin for 12 hours. Wash the cells twice and incubate with fluorescent dye. Subsequently, allow flow cytometry (Beckman, USA) to scan and record the fluorescence signals emitted by FITC and 7-AAD. Cell proliferation assay The cells were seeded in 6-well plates at a density of 1×10^5 cells per well, treated with curcumin for 12 hours, and then incubated with EDU working solution, followed by the addition of fixative, permeabilization solution, and reaction solution. Finally, 1X Hoechst 33342 solution was added for nuclear staining. After washing, fluorescence detection was performed immediately under light-protected conditions. Transmission electron microscopy The processed cells were pre-embedded and fixed. After infiltration embedding, the mixture was polymerized into blocks, and ultrathin sections were cut at 60–80 nm. Negative staining was used to enhance contrast. Images were acquired using a transmission electron microscope (Hitachi, Japan). Cell transfection During transfection, cells were seeded in 6-well plates and cultured to 80%-90% confluency before plasmid transfection with the Homo sapiens sequestosome 1 (SQSTM1) (cat. no. HH20240517GX-PC02, Hanheng Biotechnology, Shanghai, China). When cells seeded in the 6-well plates reached 30%-50% confluency, siRNA transfection was performed using LipoFlyer 3.0 (cat. no. HB-LF3-1000, Hanheng Biotechnology, China) transfection reagent. Determination of intracellular reactive oxygen species . Detection of ROS production in cells using DCFH-DA. After oxidation in the cells, the fluorescence of the labeled probes significantly increased. HepG2 cells were cultured with curcumin for 12 hours, incubated with 10 µM DCFH-DA for 30 minutes, washed three times with PBS, and immediately observed and imaged under a fluorescence microscope (ZEISS, Germany).. Measurement of MDA, Total Iron, and GSH Content. Use commercial assay kits to measure the levels of MDA, total iron, and GSH in cells and animal serum. MDA levels are expressed in nmol/mg protein and are related to cellular protein concentration. GSH levels are expressed as µmol/gprot protein and are correlated with cellular protein concentration. Cellular immunofluorescence. Wash twice with PBS, fix with 4% paraformaldehyde (cat. no. EE0001, SparkJade, China) solution for 20 minutes, permeabilize with Triton X-100 solution (cat. no. MB2486, Meilunbio, China) for 30 minutes, then block with 5% BSA (cat. no. ED0017-B, SparkJade, China) solution at 37°C for 30 minutes. Finally, incubate with the primary antibody overnight at 4°C, followed by incubation with FITC-labeled secondary antibody (cat. no. P0186, Beyotime, China) for 1 hour at room temperature protected from light. The cells were washed three times with PBS, followed by counterstaining the nuclei with DAPI solution for 30 minutes. After another PBS wash, an anti-fade mounting medium was added, and the fluorescence intensity was observed under a fluorescence microscope (ZEISS, Germany). Western Blot Analysis . Collect and process HepG2 cells or tumor tissues, and place them in centrifuge tubes. Use cell lysis buffer containing a mixture of 10 µg/ml phosphatase inhibitors (cat. no. GRF102, Elegzyme, China) and 10 µg/ml protease inhibitors (cat. no. ST506, Beyotime, China) to lyse the cell membranes and release proteins. Centrifuge to remove cell debris and collect the supernatant. Next, measure the protein concentration in the supernatant using a BCA protein assay kit (cat. no. ZJ102, Elegzyme, China) to ensure the appropriate amount of protein sample is loaded. Load the protein (10–30 µg) and marker (cat. no. 26616,Thermo Scientific, USA) into the sample wells containing SDS-PAGE gel (cat. no. PG212, Elegzyme, China) electrophoresis buffer. Perform SDS-PAGE gel electrophoresis to separate the proteins, then transfer the separated proteins onto a PVDF (cat. no. IPVH00010, Merck KGaA, Germany) membrane using transfer buffer for blotting. Incubate the transferred membrane overnight at 60 rpm with anti-P62, anti-KEAP1, anti-NRF2, anti-GPX4, anti-XCT, and anti-P53 antibodies. After washing five times for 5 minutes each, incubate the membrane with a suitable horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour. Use an ECL detection kit (cat. no. ED0016-A, SparkJade China) for color development and exposure. Statistical Analysis . All experiments were performed independently at least three times, with data presented as mean ± standard error of the mean (SEM). Means were compared using either one-way ANOVA (for multiple groups) or t-tests (for two groups) via the Prism 8 software program. Differences were considered statistically significant when the p-value was less than 0.05. GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA) was used to visualize the data. Results Curcumin Suppresses Hepatocellular Carcinoma Growth To investigate the therapeutic potential of curcumin in hepatocellular carcinoma (HCC), we established Hepa1-6 cell-derived ectopic xenografts in mice. Following 15 days of treatment, curcumin administration significantly suppressed tumor growth, achieving comparable efficacy to the first-line agent sorafenib, as evidenced by reductions in tumor volume (54.3% vs. model group, p < 0.01) and weight (48.7% vs. model group, p < 0.01) (Fig. 1 A-C).Histopathological analysis further revealed pronounced tumor necrosis and decreased cellularity in both curcumin- and sorafenib-treated groups (Fig. 1 I), aligning with the observed growth inhibition. Additionally, compared to the model group, curcumin induced characteristic ferroptotic alterations, including glutathione (GSH) depletion (1.8-fold decrease vs. model, p < 0.05), malondialdehyde (MDA) accumulation (2.3-fold increase, p < 0.01), and elevated Fe²⁺ levels (1.6-fold increase, p < 0.05) (Fig. 1 D-F). Western blot analysis demonstrated that, compared to the model group, the expression of key ferroptosis-related proteins XCT and GPX4, as well as pathway proteins P62 and NRF2, was decreased in the curcumin- and sorafenib-treated groups (Fig. 1 G). Conversely, it upregulated KEAP1 and P53 (Fig. 1 H), suggesting activation of both KEAP1/NRF2 axis-mediated antioxidant suppression and P53-dependent ferroptotic signaling. Curcumin Suppresses HepG2 Hepatocellular Carcinoma Proliferation and Invasion To evaluate the anti-tumor activity of curcumin in hepatocellular carcinoma, we first assessed its effects on HepG2 cell proliferation.. HepG2 cells were treated with curcumin(0, 5, 20, 50 uM)for 12 hours, and MTT assays revealed a dose-dependent suppression of viability (Fig. 2 A). The calculated half-maximal inhibitory concentration (IC50) IC50 value was 50.75 µM (Fig. 2 B), which was selected for subsequent experiments EdU staining demonstrated a marked reduction in DNA synthesis activity in curcumin-treated cells compared to controls (Fig. 2 C).Furthermore, Transwell assays indicated that curcumin significantly attenuated the invasive capacity of HepG2 cells (Fig. 2 D). Flow cytometry analysis showed that curcumin treatment substantially increased non-apoptotic cell death(Chen, Eyupoglu, & Savaskan, 2017 ) (Fig. 2 G-H). Notably, this effect was not reversed by the pan-caspase inhibitor Z-VAD-FMK (curcumin + Z-VAD-FMK vs. curcumin alone: p > 0.05),but was significantly rescued by the ferroptosis inhibitor Fer-1 (curcumin + Fer-1 vs. curcumin alone: p < 0.01).These results collectively establish that curcumin exerts its anti-proliferative and anti-invasive effects on HepG2 cells primarily through ferroptosis induction. Curcumin induces Ferroptosis in HepG2 Cells To verify that curcumin induces ferroptosis in HepG2 cells, as shown in Fig. 3 A, we observed the morphology of HepG2 cells using transmission electron microscopy. The results revealed that, compared to the control group, the nuclei of the curcumin-treated group were irregular, The mitochondria were reduced in size, mostly round, with fewer and thicker cristae, increased membrane electron density, and continuous outer membranes.. These structural changes in HepG2 cells are consistent with the characteristics of ferroptosis. Next, we measured intracellular Fe 2+ accumulation, lipid peroxide levels (ROS, MDA), and antioxidant capacity (GSH), as shown in Figs. 3 B-F. The results indicated that, compared to the control group, the curcumin-treated HepG2 cells exhibited significantly increased Fe 2+ accumulation, elevated ROS and MDA levels, and a notable decrease in GSH content. Western blot and immunofluorescence results demonstrated that curcumin suppressed the expression of key ferroptosis-related proteins GPX4 and XCT, while inducing the expression of P53 (Figs. 3 G-I). Additionally, we found that curcumin inhibited the expression of pathway proteins P62 and NRF2, and induced the expression of KEAP1. These findings collectively indicate that curcumin induces ferroptosis in HepG2 cells. Ferrostatin-1 Suppress Curcumin-induced Ferroptosis in HepG2 Cells. To further verify that curcumin induces ferroptosis in HepG2 cells, we used Fer-1 for early intervention. As shown in Fig. 4 A, we found that the intracellular ROS levels in the group treated with curcumin combined with ferrostatin-1 were lower than those in the group treated with curcumin alone. Additionally, we observed that compared to the curcumin-only group, the intracellular levels of MDA and Fe 2+ decreased, while the intracellular GSH content significantly increased in the curcumin combined with Fer-1 group (Figs. 4 B-D). Furthermore, as shown in Figs. 4 E-G, immunofluorescence and Western blot results indicated that, compared to the curcumin group, the combination of curcumin and Fer-1 inhibited the expression of ferroptosis-related proteins GPX4, XCT, and P53. These results further confirm that curcumin induces ferroptosis in HepG2 cells. Moreover, we observed that after treatment with curcumin, the expression of the pathway proteins P62 and NRF2 decreased, while the expression of KEAP1 increased. However, after intervention with Fer-1, the expression of P62 and NRF2 increased, while the expression of KEAP1 decreased. Studies have confirmed that the P62-KEAP1-NRF2 signaling pathway is a key regulatory pathway for ferroptosis20. Therefore, we speculate that curcumin may induce ferroptosis in HepG2 cells by regulating the P62-KEAP1-NRF2 signaling pathway, thereby exerting its anti-tumor effects. Curcumin induces Ferroptosis by Regulating the P62-KEAP1-NRF2 Signaling Pathway To verify that curcumin induces ferroptosis in HepG2 cells by modulating the P62-KEAP1-NRF2 signaling pathway, we constructed a P62 overexpression plasmid (OE-P62) as shown in Fig. 5 A, and used it to increase the protein expression level of P62 (Fig. 5 B). We found that, compared to the group treated with curcumin alone, OE-P62 inhibited the increase in intracellular ROS levels (Fig. 5 C). Western blot results showed that, compared to the curcumin-only group, OE-P62 suppressed the downregulation of NRF2 and P62 proteins and the upregulation of KEAP1 protein in the pathway. Additionally, OE-P62 inhibited the reduction in expression of the ferroptosis key proteins GPX4 and XCT (Fig. 5 G). Furthermore, immunofluorescence results indicated that, compared to the control group, curcumin significantly reduced the levels of intracellular pathway proteins P62 (Fig. 5 D) and NRF2 (Fig. 5 E), and increased the content of KEAP1 (Fig. 5 F). However, OE-P62 reversed the changes in pathway proteins induced by curcumin. These results suggest that curcumin induces ferroptosis in HepG2 cells by regulating the P62-KEAP1-NRF2 signaling pathway. Discussion Although significant progress has been made in the treatment of HCC in recent years, chemotherapy resistance and toxic side effects remain important factors affecting the prognosis of HCC patients(Abou-Alfa et al., 2006 ; Llovet et al., 2008 ). There is an urgent need to develop new therapeutic strategies and drugs for HCC. Natural products from plants are well-known for their diverse targets and bioactive components, with minimal side effects, making them an important source for identifying drugs with anticancer properties(Atanasov et al., 2015 ; Aung, Qu, Kortschak, & Adelson, 2017 ). Ferroptosis, a newly identified form of cell death, is typically accompanied by lipid overload during each oxidative process(Conrad, Lorenz, & Proneth, 2021 ; Dixon & Stockwell, 2014 ). An increasing number of antitumor drugs and compounds have been reported to induce iron deposition. In other words, chemotherapeutic drugs not only induce apoptosis but also eliminate tumor cells by inducing iron deposition(R. Li et al., 2020 ). Ferroptosis has emerged as a promising approach in clinical cancer treatment strategies. Therefore, exploring natural active drug components that may induce ferroptosis in HCC is of great significance. In this study, we investigated the antitumor effects of curcumin by inducing ferroptosis in cancer cells. At the same time, we also confirmed the significant potential of the P62-KEAP1-NRF2 ferroptosis signaling pathway in combating tumors in hepatocellular carcinoma. Furthermore, our study elucidated that curcumin exerts its antitumor effects by inducing ferroptosis in HepG2 cells through the inhibition of the P62-KEAP1-NRF2 signaling pathway (Fig. 6 ). Curcumin, as an oxidizing agent, exhibits significant antitumor effects and is a promising anticancer drug. It has been reported that curcumin induces the production of ROS in cancers such as hepatocellular carcinoma and breast cancer, thereby exerting its antitumor effects(P. M. Li et al., 2014 ; R. Li et al., 2020 ). Numerous previous in vitro and in vivo studies have shown that curcumin can inhibit cancer growth in both animals and humans, including hepatocellular carcinoma(Cornblatt et al., 2007 ; Keum et al., 2006 ; Lanceta, Li, Choi, & Eaton, 2013 ; Zou et al., 2011 ). Some reports suggest that curcumin or its derivatives exhibit good anti-hepatocellular carcinoma activity when used alone or in combination with other drugs(Chiang, Chen, & Chang, 2018 ; Suttner & Dennery, 1999 ; W. S. Yang & Stockwell, 2008 ). In this study, data obtained from a xenograft mouse model further provided evidence that curcumin effectively inhibits hepatocellular carcinoma growth. We demonstrated that curcumin exerts its antitumor effects by inducing ferroptosis in HepG2 cells. Transmission electron microscopy and flow cytometry analysis revealed that the ferroptosis pathway is one of the most critical pathways through which curcumin induces HepG2 cell death. The accumulation of Fe 2+ , levels of lipid oxides (ROS, MDA), antioxidant levels (GSH), levels of ferroptosis-related proteins (GPX4, XCT, P53), and mitochondrial morphology in HepG2 cells were also regulated by curcumin, reflecting its pro-ferroptotic role in HepG2 cells. Further in vivo data were consistent with the in vitro findings, strengthening the evidence for curcumin-induced ferroptosis in HepG2 cells. Ferrostatin-1, a classical ferroptosis inhibitor, specifically targets lipid peroxidation-induced ferroptosis(Liu et al., 2020 ). We found that Ferrostatin-1 treatment effectively reversed curcumin-induced HepG2 cell death and alterations in ferroptosis-related parameters, further supporting the role of curcumin-induced ferroptosis as an anticancer mechanism. Studies have shown that the P62-KEAP1-NRF2 antioxidant signaling pathway, which transcriptionally activates ROS and iron metabolism-related genes, is a key negative regulator of ferroptosis in HCC cells. Inhibition of the P62-KEAP1-NRF2 pathway significantly enhances the anticancer activity of erastin and sorafenib in HCC cells both in vitro and in vivo. The expression of P62 prevents the degradation of NRF2 by inactivating KEAP1 and enhances subsequent nuclear accumulation of NRF2. As an important antioxidant transcription factor, NRF2 regulates the expression of antioxidant genes by binding to antioxidant response elements(Dixon et al., 2012 ; Sun et al., 2016 ). The main pathways of ferroptosis involve GSH metabolism imbalance, GPX4 inactivation, lipid peroxidation, and iron metabolism abnormalities. In this study, curcumin significantly inhibited GPX4 and GSH levels in HepG2 cells, promoted the accumulation of ROS and Fe 2+ , and induced ferroptosis in HepG2 cells. However, overexpression of P62 increased GPX4 levels and reduced intracellular ROS levels, similar to the results observed after Ferrostatin-1 treatment. These findings further indicate that curcumin-mediated ferroptosis is closely related to the P62-KEAP1-NRF2 signaling pathway. Conclusions In summary, our results indicate that curcumin induced ferroptosis plays an important role in anti-tumor activity. Fer-1 significantly inhibits curcumin induced ferroptosis in tumor cells by regulating ferroptosis, and overexpression of P62 can also alter the levels of key regulators of ferroptosis, alleviating curcumin induced ferroptosis in tumor cells. Therefore, our study suggests that curcumin promotes iron death in hepatocellular carcinoma cells by regulating the P62-KEAP1-NRF2 signaling pathway, and curcumin induced iron death is a promising potential therapeutic target for treating HCC. Declarations Funding This research was funded by Shandong Traditional Chinese Medicine Science and Technology Project (Q-2022110); Shandong Provincial Natural Science Foundation for Youth (ZR2023QH448); Shandong of The Outstanding Youth Innovation Team of Shandon Institutions of Higher Learning (2023KJ189). CRediT authorship contribution statement Jinlan Deng and Zhijuan Wu: Writing - original draft, Data curation. Shangkun Ning: Conceptualization. Xu Chang: Visualization, Formal analysis. Jibing Liu: Supervision. Yangli Yu: Validation. Min Zhang: Writing - review &editing, Data Curation, Resources. Lin Zhang: Writing - review &editing, Data Curation, Funding acquisition. All authors reviewed the manuscript. Availability of data and materials All data generated or analyzed during this study are included in this published article. Declaration of competing interest The authors have declared that they have no conflicts of interest. Acknowledgements Not applicable. References Abou-Alfa, G. K., Schwartz, L., Ricci, S., Amadori, D., Santoro, A., Figer, A., . . . Saltz, L. B. (2006). Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol, 24 (26), 4293-4300. doi:10.1200/jco.2005.01.3441 Atanasov, A. G., Waltenberger, B., Pferschy-Wenzig, E. M., Linder, T., Wawrosch, C., Uhrin, P., . . . Stuppner, H. (2015). Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv, 33 (8), 1582-1614. doi:10.1016/j.biotechadv.2015.08.001 Aung, T. N., Qu, Z., Kortschak, R. D., & Adelson, D. L. (2017). Understanding the Effectiveness of Natural Compound Mixtures in Cancer through Their Molecular Mode of Action. Int J Mol Sci, 18 (3). doi:10.3390/ijms18030656 Bray, F., Laversanne, M., Sung, H., Ferlay, J., Siegel, R. L., Soerjomataram, I., & Jemal, A. (2024). Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 74 (3), 229-263. doi:10.3322/caac.21834 Capelletti, M. M., Manceau, H., Puy, H., & Peoc'h, K. (2020). Ferroptosis in Liver Diseases: An Overview. Int J Mol Sci, 21 (14). doi:10.3390/ijms21144908 Chen, D., Eyupoglu, I. Y., & Savaskan, N. (2017). Ferroptosis and Cell Death Analysis by Flow Cytometry. Methods Mol Biol, 1601 , 71-77. doi:10.1007/978-1-4939-6960-9_6 Chiang, S. K., Chen, S. E., & Chang, L. C. (2018). A Dual Role of Heme Oxygenase-1 in Cancer Cells. Int J Mol Sci, 20 (1). doi:10.3390/ijms20010039 Conche, C., Finkelmeier, F., Pešić, M., Nicolas, A. M., Böttger, T. W., Kennel, K. B., . . . Greten, F. R. (2023). Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade. Gut, 72 (9), 1774-1782. doi:10.1136/gutjnl-2022-327909 Conrad, M., Lorenz, S. M., & Proneth, B. (2021). Targeting Ferroptosis: New Hope for As-Yet-Incurable Diseases. Trends Mol Med, 27 (2), 113-122. doi:10.1016/j.molmed.2020.08.010 Cornblatt, B. S., Ye, L., Dinkova-Kostova, A. T., Erb, M., Fahey, J. W., Singh, N. K., . . . Visvanathan, K. (2007). Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast. Carcinogenesis, 28 (7), 1485-1490. doi:10.1093/carcin/bgm049 Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, C. E., . . . Stockwell, B. R. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 149 (5), 1060-1072. doi:10.1016/j.cell.2012.03.042 Dixon, S. J., & Stockwell, B. R. (2014). The role of iron and reactive oxygen species in cell death. Nat Chem Biol, 10 (1), 9-17. doi:10.1038/nchembio.1416 Friedmann Angeli, J. P., Krysko, D. V., & Conrad, M. (2019). Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer, 19 (7), 405-414. doi:10.1038/s41568-019-0149-1 Gautheron, J., Gores, G. J., & Rodrigues, C. M. P. (2020). Lytic cell death in metabolic liver disease. J Hepatol, 73 (2), 394-408. doi:10.1016/j.jhep.2020.04.001 Guo, S., Zhong, A., Zhang, D., Gao, J., Ni, Y., Zhao, R., & Ma, W. (2023). ATP2B3 Inhibition Alleviates Erastin-Induced Ferroptosis in HT-22 Cells through the P62-KEAP1-NRF2-HO-1 Pathway. Int J Mol Sci, 24 (11). doi:10.3390/ijms24119199 Hayano, M., Yang, W. S., Corn, C. K., Pagano, N. C., & Stockwell, B. R. (2016). Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation. Cell Death Differ, 23 (2), 270-278. doi:10.1038/cdd.2015.93 Hirayama, T., Miki, A., & Nagasawa, H. (2019). Organelle-specific analysis of labile Fe(ii) during ferroptosis by using a cocktail of various colour organelle-targeted fluorescent probes. Metallomics, 11 (1), 111-117. doi:10.1039/c8mt00212f Jiang, L., Kon, N., Li, T., Wang, S. J., Su, T., Hibshoosh, H., . . . Gu, W. (2015). Ferroptosis as a p53-mediated activity during tumour suppression. Nature, 520 (7545), 57-62. doi:10.1038/nature14344 Keum, Y. S., Yu, S., Chang, P. P., Yuan, X., Kim, J. H., Xu, C., . . . Kong, A. N. (2006). Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells. Cancer Res, 66 (17), 8804-8813. doi:10.1158/0008-5472.Can-05-3513 Lanceta, L., Li, C., Choi, A. M., & Eaton, J. W. (2013). Haem oxygenase-1 overexpression alters intracellular iron distribution. Biochem J, 449 (1), 189-194. doi:10.1042/bj20120936 Lee, J., Kang, E. S., Kobayashi, S., Homma, T., Sato, H., Seo, H. G., & Fujii, J. (2017). The viability of primary hepatocytes is maintained under a low cysteine-glutathione redox state with a marked elevation in ophthalmic acid production. Exp Cell Res, 361 (1), 178-191. doi:10.1016/j.yexcr.2017.10.017 Li, P. M., Li, Y. L., Liu, B., Wang, W. J., Wang, Y. Z., & Li, Z. (2014). Curcumin inhibits MHCC97H liver cancer cells by activating ROS/TLR-4/caspase signaling pathway. Asian Pac J Cancer Prev, 15 (5), 2329-2334. doi:10.7314/apjcp.2014.15.5.2329 Li, R., Zhang, J., Zhou, Y., Gao, Q., Wang, R., Fu, Y., . . . Yu, H. (2020). Transcriptome Investigation and In Vitro Verification of Curcumin-Induced HO-1 as a Feature of Ferroptosis in Breast Cancer Cells. Oxid Med Cell Longev, 2020 , 3469840. doi:10.1155/2020/3469840 Liu, P., Feng, Y., Li, H., Chen, X., Wang, G., Xu, S., . . . Zhao, L. (2020). Ferrostatin-1 alleviates lipopolysaccharide-induced acute lung injury via inhibiting ferroptosis. Cell Mol Biol Lett, 25 , 10. doi:10.1186/s11658-020-00205-0 Llovet, J. M., Ricci, S., Mazzaferro, V., Hilgard, P., Gane, E., Blanc, J. F., . . . Bruix, J. (2008). Sorafenib in advanced hepatocellular carcinoma. N Engl J Med, 359 (4), 378-390. doi:10.1056/NEJMoa0708857 Marquardt, J. U., Gomez-Quiroz, L., Arreguin Camacho, L. O., Pinna, F., Lee, Y. H., Kitade, M., . . . Thorgeirsson, S. S. (2015). Curcumin effectively inhibits oncogenic NF-κB signaling and restrains stemness features in liver cancer. J Hepatol, 63 (3), 661-669. doi:10.1016/j.jhep.2015.04.018 Prasad, S., Gupta, S. C., Tyagi, A. K., & Aggarwal, B. B. (2014). Curcumin, a component of golden spice: from bedside to bench and back. Biotechnol Adv, 32 (6), 1053-1064. doi:10.1016/j.biotechadv.2014.04.004 Shang, Y., Luo, M., Yao, F., Wang, S., Yuan, Z., & Yang, Y. (2020). Ceruloplasmin suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma cells. Cell Signal, 72 , 109633. doi:10.1016/j.cellsig.2020.109633 Sun, X., Ou, Z., Chen, R., Niu, X., Chen, D., Kang, R., & Tang, D. (2016). Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology, 63 (1), 173-184. doi:10.1002/hep.28251 Suttner, D. M., & Dennery, P. A. (1999). Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. Faseb Journal, 13 (13), 1800-1809. doi:10.1096/fasebj.13.13.1800 Tian, S., Liao, L., Zhou, Q., Huang, X., Zheng, P., Guo, Y., . . . Tian, X. (2021). Curcumin inhibits the growth of liver cancer by impairing myeloid-derived suppressor cells in murine tumor tissues. Oncol Lett, 21 (4), 286. doi:10.3892/ol.2021.12547 Vogel, A., Meyer, T., Sapisochin, G., Salem, R., & Saborowski, A. (2022). Hepatocellular carcinoma. Lancet, 400 (10360), 1345-1362. doi:10.1016/s0140-6736(22)01200-4 Wang, L., Cai, H., Hu, Y., Liu, F., Huang, S., Zhou, Y., . . . Wu, F. (2018). A pharmacological probe identifies cystathionine β-synthase as a new negative regulator for ferroptosis. Cell Death Dis, 9 (10), 1005. doi:10.1038/s41419-018-1063-2 Yang, R., Song, C., Chen, J., Zhou, L., Jiang, X., Cao, X., . . . Zhang, Q. (2020). Limonin ameliorates acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1. Phytomedicine, 69 , 153211. doi:10.1016/j.phymed.2020.153211 Yang, W. S., SriRamaratnam, R., Welsch, M. E., Shimada, K., Skouta, R., Viswanathan, V. S., . . . Stockwell, B. R. (2014). Regulation of ferroptotic cancer cell death by GPX4. Cell, 156 (1-2), 317-331. doi:10.1016/j.cell.2013.12.010 Yang, W. S., & Stockwell, B. R. (2008). Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol, 15 (3), 234-245. doi:10.1016/j.chembiol.2008.02.010 Yang, Y., Liu, C., Wang, M., Cheng, H., Wu, H., Luo, S., . . . Li, Q. (2024). Arenobufagin regulates the p62-Keap1-Nrf2 pathway to induce autophagy-dependent ferroptosis in HepG2 cells. Naunyn Schmiedebergs Arch Pharmacol, 397 (7), 4895-4909. doi:10.1007/s00210-023-02916-5 Zou, C., Zhang, H., Li, Q., Xiao, H., Yu, L., Ke, S., . . . Gao, X. (2011). Heme oxygenase-1: a molecular brake on hepatocellular carcinoma cell migration. Carcinogenesis, 32 (12), 1840-1848. doi:10.1093/carcin/bgr225 Additional Declarations No competing interests reported. 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(A) Images of tumor xenograft mice and tumor resection mice from each group. The effects of curcumin on (B) body weight and (C) tumor volume were measured every 3 days. The levels of GSH (D), Fe\u003csup\u003e2+\u003c/sup\u003e (E), and MDA (F) in mouse serum were detected using Western blot (G), and the expression levels of pathway proteins P62, KEAP1, NRF2, and ferroptosis-related proteins GPX4, XCT, and P53 in mouse tumor tissues were analyzed (H) and quantified. (I) Hematoxylin and eosin staining of tumor tissues (magnification, ×100; scale bar, 200 µm). Data are presented as mean ± standard deviation. n=3, *P\u0026lt;0.05, **P\u0026lt;0.01, and ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/8c80481051d2b41d8c8110fe.png"},{"id":87840238,"identity":"1a2d8c92-a0e7-4439-997c-1f7f788deb5f","added_by":"auto","created_at":"2025-07-29 14:04:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":217524,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin can inhibit tumor activity. (A) MTT assay was used to detect cell proliferation ability; (B) CCK-8 method was used to screen concentrations; (D-E) Transwell assay was used to detect cell invasion ability (magnification ×20; scale bar, 50 µm); (C,F) EDU assay was used to detect the proliferation ability of HepG2 cells (magnification ×10; scale bar, 100 µm); (G-H) Flow cytometry was used to determine the type of cell death. Data are presented as the mean±SD, n=3, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/60c58bb83b3bbd7a9dd608b6.png"},{"id":87841990,"identity":"465a9112-b83c-4c65-bb06-a2334fe0078f","added_by":"auto","created_at":"2025-07-29 14:20:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156713,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin induced ferroptosis in HepG2 cells. (A) The morphology and structure of HepG2 cells were examined using transmission electron microscopy. (B-C) Intracellular ROS levels were detected using a cell detection kit. (D) Cellular GSH levels were measured using a GSH detection kit. (E) Changes in intracellular MDA content were assessed using an MDA detection kit. (F) Intracellular Fe\u003csup\u003e2+\u003c/sup\u003e levels were determined using an Fe\u003csup\u003e2+\u003c/sup\u003e detection kit. (G) Changes in the ferroptosis-related protein P53 were detected using cellular immunofluorescence (magnification, ×20; scale bar, 50 µm). (H-I) The expression levels of ferroptosis-related proteins XCT, GPX4, and pathway proteins P62, KEAP1, and NRF2 in HepG2 cells were analyzed. Data are presented as the mean±SD, n=3, *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/847a0430d36fdaf0f167da2e.png"},{"id":87840243,"identity":"1bdf6b05-cfc4-4f23-910f-b6e52d4234c4","added_by":"auto","created_at":"2025-07-29 14:04:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175987,"visible":true,"origin":"","legend":"\u003cp\u003eFerrostatin-1 suppress curcumin-induced ferroptosis in HepG2 cells. (A) Detection of intracellular ROS levels; (B) Changes in intracellular MDA content detected using an MDA detection kit; (C) Measurement of intracellular Fe\u003csup\u003e2+\u003c/sup\u003e levels using an Fe\u003csup\u003e2+\u003c/sup\u003e detection kit;(D) Measurement of intracellular GSH levels using a GSH detection kit; (E) Detection of P53 expression in cells using immunofluorescence (magnification, ×20; scale bar, 50 µm); (F) Immunoblot analysis and quantitative analysis; (G) Expression levels of pathway proteins P62, KEAP1, NRF2, and key ferroptosis proteins XCT and GPX4 in HepG2 cells.Data are presented as the mean±SD, n=3, *P \u0026lt;0.05, **P \u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/c36271140fd7fd16d7be75b3.png"},{"id":87840242,"identity":"22acc822-f2ed-4e39-86e5-37227dbdf96b","added_by":"auto","created_at":"2025-07-29 14:04:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":237166,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin induces ferroptosis by modulating the P62/KEAP1/NRF2 signaling pathway. (A) Western blot and quantitative analysis of P62 overexpression. (C) Detection kit for measuring intracellular ROS levels. Changes in intracellular pathway proteins P62 (D), KEAP1 (F), and NRF2 (E) were detected using cellular immunofluorescence. (G) Protein expression levels of pathway proteins P62, KEAP1, NRF2, and key ferroptosis proteins XCT and GPX4 were analyzed using immunoblotting and (H) quantitatively assessed. The data is expressed as mean ± standard deviation (n=3) *P\u0026lt;0.05 and ** P\u0026lt;0.01. *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/72def4c48ab6982be68d9f05.png"},{"id":87841438,"identity":"af60cbd1-1a36-4717-a8d7-2275107db3fa","added_by":"auto","created_at":"2025-07-29 14:12:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":121067,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphical representation of this study: An in-depth understanding of the mechanism by which curcumin promotes ferroptosis in hepatocytes by regulating the P62-KEAP1-NRF2 signaling pathway.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/ace3705c8a4db5cae64536c7.png"},{"id":97178994,"identity":"890b5afd-ec1a-4614-803d-0d2aa30d20f5","added_by":"auto","created_at":"2025-12-01 16:14:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1837261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/b80d0c7e-f7ee-4f0e-bcee-c3b51e66bceb.pdf"},{"id":87841432,"identity":"a87453f2-ae06-4e74-a32f-f3d5e8e92dae","added_by":"auto","created_at":"2025-07-29 14:12:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4409843,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7197765/v1/82f3235365b50b5b0c97c720.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Curcumin induces Ferroptosis in Hepatocellular Carcinoma by Regulating the P62-KEAP1-NRF2 signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and a leading cause of cancer-related deaths. Epidemiological data indicate that nearly 906,000 people are diagnosed with hepatocellular carcinoma annually, with HCC being the predominant form(Bray et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Vogel, Meyer, Sapisochin, Salem, \u0026amp; Saborowski, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, there is an urgent need to explore effective treatment strategies to combat this deadly disease.\u003c/p\u003e\u003cp\u003eFerroptosis, a novel form of regulated cell death distinct from apoptosis and necrosis, is characterized by the accumulation of iron and disruption of cellular redox homeostasis, leading to a decline in antioxidant capacity. Morphologically, ferroptosis is marked by intact cell nuclei, non-aggregated chromatin, unruptured cytoplasmic membranes, reduced or absent mitochondria with ruptured outer membranes, and increased inner membrane density(Gautheron, Gores, \u0026amp; Rodrigues, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A key upstream process in ferroptosis involves the cystine/glutamate antiporter system (system xc⁻), which supplies cysteine for glutathione (GSH) synthesis(Hayano, Yang, Corn, Pagano, \u0026amp; Stockwell, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Depletion of cysteine leads to GSH depletion, thereby triggering ferroptosis(Dixon et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Glutathione peroxidase 4 (GPX4), a critical regulator of ferroptosis(W. S. Yang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), converts GSH into oxidized glutathione (GSSG) and reduces cytotoxic lipid peroxides (PL-OOH) to non-toxic alcohols (PL-OH). Inhibition of GPX4 activity results in the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation products, ultimately leading to cell death(Friedmann Angeli, Krysko, \u0026amp; Conrad, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). P53, particularly the acetylation-deficient mutant p53 3KR, is responsible for suppressing the expression of XCT in the context of ferroptosis. The inhibition of XCT leads to the depletion of GSH and promotes ferroptosis(Jiang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Recent studies highlight that ceruloplasmin (CP) inhibits ferroptosis in HCC cells by regulating iron homeostasis, whereas CP inhibition increases Fe\u0026sup2;⁺ and ROS levels, promoting erastin- and RSL3-induced ferroptosis(Shang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, GPX4-associated ferroptotic hepatocyte death triggers a tumor-suppressive immune response, and combining ferroptosis inducers significantly improves survival in murine liver tumor models(Conche et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings underscore the therapeutic potential of ferroptosis in treating primary liver tumors and metastases, positioning it as a promising avenue for HCC-specific therapy.\u003c/p\u003e\u003cp\u003eThe P62-KEAP1-NRF2 signaling pathway has been identified as a critical regulator of ferroptosis in HCC(Sun et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Nuclear factor erythroid 2-related factor 2 (NRF2) plays a pivotal role in antioxidant responses and cellular defense mechanisms(Capelletti, Manceau, Puy, \u0026amp; Peoc'h, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Under physiological conditions, Kelch-like ECH-associated protein 1 (KEAP1) maintains low NRF2 levels by promoting its ubiquitination and proteasomal degradation(Hirayama, Miki, \u0026amp; Nagasawa, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; R. Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). During oxidative stress, NRF2 is stabilized and released from KEAP1-mediated inhibition, a process regulated by the autophagy receptor P62. Studies demonstrate that P62 knockdown in HCC cells enhances ferroptosis induced by erastin and sorafenib. Furthermore, NRF2 inhibition amplifies the anticancer effects of these agents in vitro and in xenograft models(Sun et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In H22 cells, ATP2B3 mitigates erastin-induced ferroptosis by modulating the P62-KEAP1-NRF2-HO-1 pathway(Guo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, arenobufagin has been shown to induce autophagy-dependent ferroptosis in HepG2 cells via the P62-KEAP1-NRF2 axis(Y. Yang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurcumin, a polyphenolic compound derived from turmeric roots and stems, is widely used as a food additive in Eastern cuisine. Preclinical studies highlight its pharmacological potential, particularly in cancer therapy(Prasad, Gupta, Tyagi, \u0026amp; Aggarwal, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Curcumin exhibits antiproliferative and pro-oxidative effects in HCC cells, inhibiting tumor growth and metastasis by modulating transcription factors (e.g., growth factors, inflammatory cytokines), protein kinases, and other enzymes. It suppresses cancer cell proliferation by inducing cell cycle arrest, apoptosis, and ferroptosis(P. M. Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Research confirms that curcumin-mediated NF-κB inhibition effectively curbs HCC cell growth(Marquardt et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, curcumin has been shown to induce ferroptosis in cancer cells, exerting antitumor effects(R. Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the precise mechanisms linking curcumin\u0026rsquo;s tumor-suppressive effects to ferroptosis in HCC remain unclear.\u003c/p\u003e\u003cp\u003eThis study elucidates that curcumin regulates ferroptosis via the P62/KEAP1/NRF2 signaling pathway, modulating the antioxidant capacity of HCC cells and inhibiting their growth. These findings reveal the protective effects, mechanisms, and molecular pathways of curcumin in HCC, providing a scientific basis for understanding the antitumor properties of traditional Chinese medicine in hepatocellular carcinoma treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eChemicals and Reagents\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eFetal bovine serum (cat. no. D1220D), DMEM medium (cat. no. MA0212), Cell Counting Kit-8 (cat. no. MA0218), MTT reagent (cat. no. 298-93-1), Annexin V-FITC/7-AAD cell apoptosis detection kit (cat. no. MA0428), EdU-555 cell proliferation detection kit (cat. no. CX003), PBS (cat. no. MA0015) and curcumin (cat. no. MB2147) were all purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). The malondialdehyde (MDA) detection kit (cat. no. S0131S) and Caspase inhibitor Z-VAD-FMK (cat. no. C1202) were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). Fer-1 (cat. no. 347174-05-4) was purchased from MedChemExpress (MCE, New Jersey, USA). The Reduced Glutathione (GSH) detection kit (cat. no. A006-2-1) was purchased from Nanjing Jiancheng Biotechnology (Nanjing, China). The ferrous ion (Fe\u003csup\u003e2+\u003c/sup\u003e) detection kit (cat. no. BC5410) and DMSO dimethyl sulfoxide (cat. no. D8370) were both purchased from Solaibao Technology Co., Ltd. (Beijing, China). The DAPI staining solution kit (cat. no. EE0011) and Sorafenib (cat. no. SJ-MX0071A) were purchased from SparkJade Biotechnology Co., Ltd. (Shandong, China). In Western blot analysis, antibodies against P62 (species: rabbit, dilution: 1:5000, cat. no. ab109012) were purchased from Abcam (Cambridge, UK). Antibodies against KEAP1 (species: rabbit, dilution: 1:1000, cat. no. 8047), GPX4 (species: rabbit, dilution: 1:1000, cat. no. 59735), XCT (species: rabbit, dilution: 1:1000, cat. no. 12691) and P53 (species: mouse, dilution: 1:1000, cat. no. 2524) were purchased from CST (CST, USA). The antibody against β-actin (species: mouse, dilution: 1:4000,cat. no. GB15001) was purchased from Servicebio (Wuhan, China). The antibody against XCT (species: rabbit, dilution: 1:1000, cat. no. DF12509) was purchased from Affinity Biosciences (USA). The antibody against NRF2 (species: rabbit, dilution: 1:1000, cat. no. A0674) was purchased from Abclonal (Abclonal, USA). Goat anti-mouse IgG (cat. no. ZB-2305) and goat anti-rabbit IgG (cat. no. ZB-2301) were provided by Zhongshan Golden Bridge Biotechnology Co., Ltd.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal model construction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eC57BL/6JNifdc mice [specific pathogen free (SPF) grade; Female; Weight, 17-19g; Age, 6\u0026ndash;7 weeks provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in the SPF grade of Shandong University of Traditional Chinese Medicine (Jinan, China). The animal experimental protocol was conducted in accordance with the \"National Institutes of Health Guidelines for the Care and Use of Laboratory Animals\" and approved by the Institutional Animal Care and Research Advisory Committee of the Shandong University of Traditional Chinese Medicine (Approval No. SDUTCM20241113002; Jinan, China). A total of 30 mice were subjected to a 12-hour light/dark cycle at 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u0026deg;C and 55.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0% relative humidity, and were able to freely access food and water. After one week of adaptation, 1\u0026times;10^6 Hepa1-6 cells in 0.2 ml of physiological saline were injected from the right axilla of mice to form subcutaneous xenograft tumors. When Hepa1-6 cell tumors were observed with the naked eye, mice were divided into the following three groups (n\u0026thinsp;=\u0026thinsp;10/group): i) Model group (injected with cancer cells, intraperitoneal injection of an equal volume of physiological saline as a negative control); ii) Curcumin group (injection of cancer cells, daily intraperitoneal injection of 100mg/kg curcumin); iii) Chemotherapy group (injection of cancer cells, daily intraperitoneal injection of 60mg/kg Sorafenib; positive control). The regimens of Sorafenib and curcumin are based on their clinical applications and previous studies, respectively(Tian et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Measure the weight and tumor volume of each mouse every 3 days within 15 days. The tumor volume is calculated using the following equation: tumor volume\u0026thinsp;=\u0026thinsp;length x width x width/2. When a tumor diameter of 1500mm\u003csup\u003e3\u003c/sup\u003e centimeters is recorded or severe disease symptoms (such as breathing difficulties or paralysis) are observed, euthanize the animal. On the 15th day, 30 mice (n\u0026thinsp;=\u0026thinsp;10/group) were euthanized by intraperitoneal injection of excessive pentobarbital sodium (200mg/kg). The duration between injection and final tumor growth measurement is 20 days. Remove tumor tissue, weigh and store immediately at -80\u0026deg;C for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Culture.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe human hepatocellular carcinoma cell line (HepG2, CVCL_0027, cat. no.CL-0103) and the mouse-derived hepatocellular carcinoma cell line (Hepa1-6, CVCL_0327, cat. no.HTX1770) were both purchased from MeilunBio (Dalian, China). The cells were cultured in DMEM medium. The medium was supplemented with 10% fetal bovine serum, 100 UI/mL penicillin, and 100 ug/mL streptomycin(cat. no. MA0110, Meilun, Dalian, China). These cells were cultured in a humidified incubator at 37\u0026deg;C and 5% carbon dioxide(Thermo Scientific, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eH\u0026amp;E\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe tumor tissues were embedded in paraffin, sectioned, and stained according to standard operating procedures. After dehydration and transparency treatment, the sections were mounted. Whole-slide scanning was then performed using a digital slide scanning system for image acquisition (SQS-600P, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Viability Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCell viability was assessed using the CCK-8 and MTT assays. For the CCK-8 assay, cells were seeded in 96-well plates at a density of 5,000 cells per well. After treating HepG2 cells with curcumin (5, 10, 20, 30, 40, 50, 60, 70, 80, 90 \u0026micro;M) for 12 hours, 10 \u0026micro;L of CCK-8 reagent was added to each well. The plates were incubated at 37\u0026deg;C for 1 hour, and the absorbance (OD) of each well was measured at 450 nm according to the manufacturer's instructions. Similarly, for the MTT assay, cells were seeded in 96-well plates at a density of 5,000 cells per well. Hepatoma cells were treated with curcumin (0, 5, 20, 50 \u0026micro;M) for 12 hours, followed by the addition of MTT reagent (10% or 20 \u0026micro;L per well). After 4 hours, the culture was terminated, and the supernatant in each well was carefully aspirated. DMSO (150 \u0026micro;L per well) was added, and the plates were shaken on an orbital shaker (Qilinbeier, China) for approximately 10 minutes to dissolve the formed crystals. The absorbance was measured at a wavelength of 490 nm using a microplate reader (BioTek,USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMigration assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHepG2 cells were added to the upper chamber for culture and then stained with H\u0026amp;E. HepG2 migrated to its lower face were counted in 3 views per membrane under a microscope (ZEISS, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow cytometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeed 1\u0026times;10^5 cells per well in a 6-well plate, pretreat with Fer-1 and Z-VAD-FMK, then treat with curcumin for 12 hours. Wash the cells twice and incubate with fluorescent dye. Subsequently, allow flow cytometry (Beckman, USA) to scan and record the fluorescence signals emitted by FITC and 7-AAD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell proliferation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cells were seeded in 6-well plates at a density of 1\u0026times;10^5 cells per well, treated with curcumin for 12 hours, and then incubated with EDU working solution, followed by the addition of fixative, permeabilization solution, and reaction solution. Finally, 1X Hoechst 33342 solution was added for nuclear staining. After washing, fluorescence detection was performed immediately under light-protected conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransmission electron microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe processed cells were pre-embedded and fixed. After infiltration embedding, the mixture was polymerized into blocks, and ultrathin sections were cut at 60\u0026ndash;80 nm. Negative staining was used to enhance contrast. Images were acquired using a transmission electron microscope (Hitachi, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell transfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring transfection, cells were seeded in 6-well plates and cultured to 80%-90% confluency before plasmid transfection with the Homo sapiens sequestosome 1 (SQSTM1) (cat. no. HH20240517GX-PC02, Hanheng Biotechnology, Shanghai, China). When cells seeded in the 6-well plates reached 30%-50% confluency, siRNA transfection was performed using LipoFlyer 3.0 (cat. no. HB-LF3-1000, Hanheng Biotechnology, China) transfection reagent.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of intracellular reactive oxygen species\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eDetection of ROS production in cells using DCFH-DA. After oxidation in the cells, the fluorescence of the labeled probes significantly increased. HepG2 cells were cultured with curcumin for 12 hours, incubated with 10 \u0026micro;M DCFH-DA for 30 minutes, washed three times with PBS, and immediately observed and imaged under a fluorescence microscope (ZEISS, Germany)..\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of MDA, Total Iron, and GSH Content.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUse commercial assay kits to measure the levels of MDA, total iron, and GSH in cells and animal serum. MDA levels are expressed in nmol/mg protein and are related to cellular protein concentration. GSH levels are expressed as \u0026micro;mol/gprot protein and are correlated with cellular protein concentration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCellular immunofluorescence.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWash twice with PBS, fix with 4% paraformaldehyde (cat. no. EE0001, SparkJade, China) solution for 20 minutes, permeabilize with Triton X-100 solution (cat. no. MB2486, Meilunbio, China) for 30 minutes, then block with 5% BSA (cat. no. ED0017-B, SparkJade, China) solution at 37\u0026deg;C for 30 minutes. Finally, incubate with the primary antibody overnight at 4\u0026deg;C, followed by incubation with FITC-labeled secondary antibody (cat. no. P0186, Beyotime, China) for 1 hour at room temperature protected from light. The cells were washed three times with PBS, followed by counterstaining the nuclei with DAPI solution for 30 minutes. After another PBS wash, an anti-fade mounting medium was added, and the fluorescence intensity was observed under a fluorescence microscope (ZEISS, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blot Analysis\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eCollect and process HepG2 cells or tumor tissues, and place them in centrifuge tubes. Use cell lysis buffer containing a mixture of 10 \u0026micro;g/ml phosphatase inhibitors (cat. no. GRF102, Elegzyme, China) and 10 \u0026micro;g/ml protease inhibitors (cat. no. ST506, Beyotime, China) to lyse the cell membranes and release proteins. Centrifuge to remove cell debris and collect the supernatant. Next, measure the protein concentration in the supernatant using a BCA protein assay kit (cat. no. ZJ102, Elegzyme, China) to ensure the appropriate amount of protein sample is loaded. Load the protein (10\u0026ndash;30 \u0026micro;g) and marker (cat. no. 26616,Thermo Scientific, USA) into the sample wells containing SDS-PAGE gel (cat. no. PG212, Elegzyme, China) electrophoresis buffer. Perform SDS-PAGE gel electrophoresis to separate the proteins, then transfer the separated proteins onto a PVDF (cat. no. IPVH00010, Merck KGaA, Germany) membrane using transfer buffer for blotting. Incubate the transferred membrane overnight at 60 rpm with anti-P62, anti-KEAP1, anti-NRF2, anti-GPX4, anti-XCT, and anti-P53 antibodies. After washing five times for 5 minutes each, incubate the membrane with a suitable horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour. Use an ECL detection kit (cat. no. ED0016-A, SparkJade China) for color development and exposure.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eStatistical Analysis\u003c/b\u003e.\u003c/h2\u003e\u003cp\u003eAll experiments were performed independently at least three times, with data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Means were compared using either one-way ANOVA (for multiple groups) or t-tests (for two groups) via the Prism 8 software program. Differences were considered statistically significant when the p-value was less than 0.05. GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA) was used to visualize the data.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCurcumin Suppresses Hepatocellular Carcinoma Growth\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the therapeutic potential of curcumin in hepatocellular carcinoma (HCC), we established Hepa1-6 cell-derived ectopic xenografts in mice. Following 15 days of treatment, curcumin administration significantly suppressed tumor growth, achieving comparable efficacy to the first-line agent sorafenib, as evidenced by reductions in tumor volume (54.3% vs. model group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and weight (48.7% vs. model group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C).Histopathological analysis further revealed pronounced tumor necrosis and decreased cellularity in both curcumin- and sorafenib-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), aligning with the observed growth inhibition. Additionally, compared to the model group, curcumin induced characteristic ferroptotic alterations, including glutathione (GSH) depletion (1.8-fold decrease vs. model, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), malondialdehyde (MDA) accumulation (2.3-fold increase, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and elevated Fe\u0026sup2;⁺ levels (1.6-fold increase, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). Western blot analysis demonstrated that, compared to the model group, the expression of key ferroptosis-related proteins XCT and GPX4, as well as pathway proteins P62 and NRF2, was decreased in the curcumin- and sorafenib-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Conversely, it upregulated KEAP1 and P53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), suggesting activation of both KEAP1/NRF2 axis-mediated antioxidant suppression and P53-dependent ferroptotic signaling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCurcumin Suppresses HepG2 Hepatocellular Carcinoma Proliferation and Invasion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the anti-tumor activity of curcumin in hepatocellular carcinoma, we first assessed its effects on HepG2 cell proliferation.. HepG2 cells were treated with curcumin(0, 5, 20, 50 uM)for 12 hours, and MTT assays revealed a dose-dependent suppression of viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The calculated half-maximal inhibitory concentration (IC50) IC50 value was 50.75 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which was selected for subsequent experiments EdU staining demonstrated a marked reduction in DNA synthesis activity in curcumin-treated cells compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).Furthermore, Transwell assays indicated that curcumin significantly attenuated the invasive capacity of HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Flow cytometry analysis showed that curcumin treatment substantially increased non-apoptotic cell death(Chen, Eyupoglu, \u0026amp; Savaskan, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). Notably, this effect was not reversed by the pan-caspase inhibitor Z-VAD-FMK (curcumin\u0026thinsp;+\u0026thinsp;Z-VAD-FMK vs. curcumin alone: p\u0026thinsp;\u0026gt;\u0026thinsp;0.05),but was significantly rescued by the ferroptosis inhibitor Fer-1 (curcumin\u0026thinsp;+\u0026thinsp;Fer-1 vs. curcumin alone: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).These results collectively establish that curcumin exerts its anti-proliferative and anti-invasive effects on HepG2 cells primarily through ferroptosis induction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCurcumin induces Ferroptosis in HepG2 Cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify that curcumin induces ferroptosis in HepG2 cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, we observed the morphology of HepG2 cells using transmission electron microscopy. The results revealed that, compared to the control group, the nuclei of the curcumin-treated group were irregular, The mitochondria were reduced in size, mostly round, with fewer and thicker cristae, increased membrane electron density, and continuous outer membranes.. These structural changes in HepG2 cells are consistent with the characteristics of ferroptosis. Next, we measured intracellular Fe\u003csup\u003e2+\u003c/sup\u003e accumulation, lipid peroxide levels (ROS, MDA), and antioxidant capacity (GSH), as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-F. The results indicated that, compared to the control group, the curcumin-treated HepG2 cells exhibited significantly increased Fe\u003csup\u003e2+\u003c/sup\u003e accumulation, elevated ROS and MDA levels, and a notable decrease in GSH content. Western blot and immunofluorescence results demonstrated that curcumin suppressed the expression of key ferroptosis-related proteins GPX4 and XCT, while inducing the expression of P53 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I). Additionally, we found that curcumin inhibited the expression of pathway proteins P62 and NRF2, and induced the expression of KEAP1. These findings collectively indicate that curcumin induces ferroptosis in HepG2 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFerrostatin-1 Suppress Curcumin-induced Ferroptosis in HepG2 Cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further verify that curcumin induces ferroptosis in HepG2 cells, we used Fer-1 for early intervention. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, we found that the intracellular ROS levels in the group treated with curcumin combined with ferrostatin-1 were lower than those in the group treated with curcumin alone. Additionally, we observed that compared to the curcumin-only group, the intracellular levels of MDA and Fe\u003csup\u003e2+\u003c/sup\u003e decreased, while the intracellular GSH content significantly increased in the curcumin combined with Fer-1 group (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Furthermore, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G, immunofluorescence and Western blot results indicated that, compared to the curcumin group, the combination of curcumin and Fer-1 inhibited the expression of ferroptosis-related proteins GPX4, XCT, and P53. These results further confirm that curcumin induces ferroptosis in HepG2 cells. Moreover, we observed that after treatment with curcumin, the expression of the pathway proteins P62 and NRF2 decreased, while the expression of KEAP1 increased. However, after intervention with Fer-1, the expression of P62 and NRF2 increased, while the expression of KEAP1 decreased. Studies have confirmed that the P62-KEAP1-NRF2 signaling pathway is a key regulatory pathway for ferroptosis20. Therefore, we speculate that curcumin may induce ferroptosis in HepG2 cells by regulating the P62-KEAP1-NRF2 signaling pathway, thereby exerting its anti-tumor effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCurcumin induces Ferroptosis by Regulating the P62-KEAP1-NRF2 Signaling Pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify that curcumin induces ferroptosis in HepG2 cells by modulating the P62-KEAP1-NRF2 signaling pathway, we constructed a P62 overexpression plasmid (OE-P62) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, and used it to increase the protein expression level of P62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We found that, compared to the group treated with curcumin alone, OE-P62 inhibited the increase in intracellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Western blot results showed that, compared to the curcumin-only group, OE-P62 suppressed the downregulation of NRF2 and P62 proteins and the upregulation of KEAP1 protein in the pathway. Additionally, OE-P62 inhibited the reduction in expression of the ferroptosis key proteins GPX4 and XCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Furthermore, immunofluorescence results indicated that, compared to the control group, curcumin significantly reduced the levels of intracellular pathway proteins P62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and NRF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), and increased the content of KEAP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). However, OE-P62 reversed the changes in pathway proteins induced by curcumin. These results suggest that curcumin induces ferroptosis in HepG2 cells by regulating the P62-KEAP1-NRF2 signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough significant progress has been made in the treatment of HCC in recent years, chemotherapy resistance and toxic side effects remain important factors affecting the prognosis of HCC patients(Abou-Alfa et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Llovet et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). There is an urgent need to develop new therapeutic strategies and drugs for HCC. Natural products from plants are well-known for their diverse targets and bioactive components, with minimal side effects, making them an important source for identifying drugs with anticancer properties(Atanasov et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Aung, Qu, Kortschak, \u0026amp; Adelson, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ferroptosis, a newly identified form of cell death, is typically accompanied by lipid overload during each oxidative process(Conrad, Lorenz, \u0026amp; Proneth, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dixon \u0026amp; Stockwell, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). An increasing number of antitumor drugs and compounds have been reported to induce iron deposition. In other words, chemotherapeutic drugs not only induce apoptosis but also eliminate tumor cells by inducing iron deposition(R. Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ferroptosis has emerged as a promising approach in clinical cancer treatment strategies. Therefore, exploring natural active drug components that may induce ferroptosis in HCC is of great significance. In this study, we investigated the antitumor effects of curcumin by inducing ferroptosis in cancer cells. At the same time, we also confirmed the significant potential of the P62-KEAP1-NRF2 ferroptosis signaling pathway in combating tumors in hepatocellular carcinoma. Furthermore, our study elucidated that curcumin exerts its antitumor effects by inducing ferroptosis in HepG2 cells through the inhibition of the P62-KEAP1-NRF2 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurcumin, as an oxidizing agent, exhibits significant antitumor effects and is a promising anticancer drug. It has been reported that curcumin induces the production of ROS in cancers such as hepatocellular carcinoma and breast cancer, thereby exerting its antitumor effects(P. M. Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; R. Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Numerous previous in vitro and in vivo studies have shown that curcumin can inhibit cancer growth in both animals and humans, including hepatocellular carcinoma(Cornblatt et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Keum et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lanceta, Li, Choi, \u0026amp; Eaton, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zou et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Some reports suggest that curcumin or its derivatives exhibit good anti-hepatocellular carcinoma activity when used alone or in combination with other drugs(Chiang, Chen, \u0026amp; Chang, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Suttner \u0026amp; Dennery, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; W. S. Yang \u0026amp; Stockwell, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this study, data obtained from a xenograft mouse model further provided evidence that curcumin effectively inhibits hepatocellular carcinoma growth. We demonstrated that curcumin exerts its antitumor effects by inducing ferroptosis in HepG2 cells. Transmission electron microscopy and flow cytometry analysis revealed that the ferroptosis pathway is one of the most critical pathways through which curcumin induces HepG2 cell death. The accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e, levels of lipid oxides (ROS, MDA), antioxidant levels (GSH), levels of ferroptosis-related proteins (GPX4, XCT, P53), and mitochondrial morphology in HepG2 cells were also regulated by curcumin, reflecting its pro-ferroptotic role in HepG2 cells. Further in vivo data were consistent with the in vitro findings, strengthening the evidence for curcumin-induced ferroptosis in HepG2 cells. Ferrostatin-1, a classical ferroptosis inhibitor, specifically targets lipid peroxidation-induced ferroptosis(Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We found that Ferrostatin-1 treatment effectively reversed curcumin-induced HepG2 cell death and alterations in ferroptosis-related parameters, further supporting the role of curcumin-induced ferroptosis as an anticancer mechanism.\u003c/p\u003e\u003cp\u003eStudies have shown that the P62-KEAP1-NRF2 antioxidant signaling pathway, which transcriptionally activates ROS and iron metabolism-related genes, is a key negative regulator of ferroptosis in HCC cells. Inhibition of the P62-KEAP1-NRF2 pathway significantly enhances the anticancer activity of erastin and sorafenib in HCC cells both in vitro and in vivo. The expression of P62 prevents the degradation of NRF2 by inactivating KEAP1 and enhances subsequent nuclear accumulation of NRF2. As an important antioxidant transcription factor, NRF2 regulates the expression of antioxidant genes by binding to antioxidant response elements(Dixon et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The main pathways of ferroptosis involve GSH metabolism imbalance, GPX4 inactivation, lipid peroxidation, and iron metabolism abnormalities. In this study, curcumin significantly inhibited GPX4 and GSH levels in HepG2 cells, promoted the accumulation of ROS and Fe\u003csup\u003e2+\u003c/sup\u003e, and induced ferroptosis in HepG2 cells. However, overexpression of P62 increased GPX4 levels and reduced intracellular ROS levels, similar to the results observed after Ferrostatin-1 treatment. These findings further indicate that curcumin-mediated ferroptosis is closely related to the P62-KEAP1-NRF2 signaling pathway.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our results indicate that curcumin induced ferroptosis plays an important role in anti-tumor activity. Fer-1 significantly inhibits curcumin induced ferroptosis in tumor cells by regulating ferroptosis, and overexpression of P62 can also alter the levels of key regulators of ferroptosis, alleviating curcumin induced ferroptosis in tumor cells. Therefore, our study suggests that curcumin promotes iron death in hepatocellular carcinoma cells by regulating the P62-KEAP1-NRF2 signaling pathway, and curcumin induced iron death is a promising potential therapeutic target for treating HCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Shandong Traditional Chinese Medicine Science and Technology Project (Q-2022110); Shandong Provincial Natural Science Foundation for Youth (ZR2023QH448); Shandong of The Outstanding Youth Innovation Team of Shandon Institutions of Higher Learning (2023KJ189).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinlan Deng and Zhijuan Wu: Writing - original draft, Data curation. Shangkun Ning: Conceptualization. Xu Chang: Visualization, Formal analysis. Jibing Liu: Supervision. Yangli Yu: Validation. Min Zhang: Writing - review \u0026amp;editing, Data Curation, Resources. Lin Zhang: Writing - review \u0026amp;editing, Data Curation, Funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that they have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbou-Alfa, G. K., Schwartz, L., Ricci, S., Amadori, D., Santoro, A., Figer, A., . . . Saltz, L. B. (2006). Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. \u003cem\u003eJ Clin Oncol, 24\u003c/em\u003e(26), 4293-4300. doi:10.1200/jco.2005.01.3441\u003c/li\u003e\n \u003cli\u003eAtanasov, A. G., Waltenberger, B., Pferschy-Wenzig, E. M., Linder, T., Wawrosch, C., Uhrin, P., . . . Stuppner, H. (2015). Discovery and resupply of pharmacologically active plant-derived natural products: A review. \u003cem\u003eBiotechnol Adv, 33\u003c/em\u003e(8), 1582-1614. doi:10.1016/j.biotechadv.2015.08.001\u003c/li\u003e\n \u003cli\u003eAung, T. N., Qu, Z., Kortschak, R. D., \u0026amp; Adelson, D. L. (2017). Understanding the Effectiveness of Natural Compound Mixtures in Cancer through Their Molecular Mode of Action. \u003cem\u003eInt J Mol Sci, 18\u003c/em\u003e(3). doi:10.3390/ijms18030656\u003c/li\u003e\n \u003cli\u003eBray, F., Laversanne, M., Sung, H., Ferlay, J., Siegel, R. L., Soerjomataram, I., \u0026amp; Jemal, A. (2024). Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J Clin, 74\u003c/em\u003e(3), 229-263. doi:10.3322/caac.21834\u003c/li\u003e\n \u003cli\u003eCapelletti, M. M., Manceau, H., Puy, H., \u0026amp; Peoc\u0026apos;h, K. (2020). Ferroptosis in Liver Diseases: An Overview. \u003cem\u003eInt J Mol Sci, 21\u003c/em\u003e(14). doi:10.3390/ijms21144908\u003c/li\u003e\n \u003cli\u003eChen, D., Eyupoglu, I. Y., \u0026amp; Savaskan, N. (2017). Ferroptosis and Cell Death Analysis by Flow Cytometry. \u003cem\u003eMethods Mol Biol, 1601\u003c/em\u003e, 71-77. doi:10.1007/978-1-4939-6960-9_6\u003c/li\u003e\n \u003cli\u003eChiang, S. K., Chen, S. E., \u0026amp; Chang, L. C. (2018). A Dual Role of Heme Oxygenase-1 in Cancer Cells. \u003cem\u003eInt J Mol Sci, 20\u003c/em\u003e(1). doi:10.3390/ijms20010039\u003c/li\u003e\n \u003cli\u003eConche, C., Finkelmeier, F., Pe\u0026scaron;ić, M., Nicolas, A. M., B\u0026ouml;ttger, T. W., Kennel, K. B., . . . Greten, F. R. (2023). Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade. \u003cem\u003eGut, 72\u003c/em\u003e(9), 1774-1782. doi:10.1136/gutjnl-2022-327909\u003c/li\u003e\n \u003cli\u003eConrad, M., Lorenz, S. M., \u0026amp; Proneth, B. (2021). Targeting Ferroptosis: New Hope for As-Yet-Incurable Diseases. \u003cem\u003eTrends Mol Med, 27\u003c/em\u003e(2), 113-122. doi:10.1016/j.molmed.2020.08.010\u003c/li\u003e\n \u003cli\u003eCornblatt, B. S., Ye, L., Dinkova-Kostova, A. T., Erb, M., Fahey, J. W., Singh, N. K., . . . Visvanathan, K. (2007). Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast. \u003cem\u003eCarcinogenesis, 28\u003c/em\u003e(7), 1485-1490. doi:10.1093/carcin/bgm049\u003c/li\u003e\n \u003cli\u003eDixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, C. E., . . . Stockwell, B. R. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. \u003cem\u003eCell, 149\u003c/em\u003e(5), 1060-1072. doi:10.1016/j.cell.2012.03.042\u003c/li\u003e\n \u003cli\u003eDixon, S. J., \u0026amp; Stockwell, B. R. (2014). The role of iron and reactive oxygen species in cell death. \u003cem\u003eNat Chem Biol, 10\u003c/em\u003e(1), 9-17. doi:10.1038/nchembio.1416\u003c/li\u003e\n \u003cli\u003eFriedmann Angeli, J. P., Krysko, D. V., \u0026amp; Conrad, M. (2019). Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. \u003cem\u003eNat Rev Cancer, 19\u003c/em\u003e(7), 405-414. doi:10.1038/s41568-019-0149-1\u003c/li\u003e\n \u003cli\u003eGautheron, J., Gores, G. J., \u0026amp; Rodrigues, C. M. P. (2020). Lytic cell death in metabolic liver disease. \u003cem\u003eJ Hepatol, 73\u003c/em\u003e(2), 394-408. doi:10.1016/j.jhep.2020.04.001\u003c/li\u003e\n \u003cli\u003eGuo, S., Zhong, A., Zhang, D., Gao, J., Ni, Y., Zhao, R., \u0026amp; Ma, W. (2023). ATP2B3 Inhibition Alleviates Erastin-Induced Ferroptosis in HT-22 Cells through the P62-KEAP1-NRF2-HO-1 Pathway. \u003cem\u003eInt J Mol Sci, 24\u003c/em\u003e(11). doi:10.3390/ijms24119199\u003c/li\u003e\n \u003cli\u003eHayano, M., Yang, W. S., Corn, C. K., Pagano, N. C., \u0026amp; Stockwell, B. R. (2016). Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation. \u003cem\u003eCell Death Differ, 23\u003c/em\u003e(2), 270-278. doi:10.1038/cdd.2015.93\u003c/li\u003e\n \u003cli\u003eHirayama, T., Miki, A., \u0026amp; Nagasawa, H. (2019). Organelle-specific analysis of labile Fe(ii) during ferroptosis by using a cocktail of various colour organelle-targeted fluorescent probes. \u003cem\u003eMetallomics, 11\u003c/em\u003e(1), 111-117. doi:10.1039/c8mt00212f\u003c/li\u003e\n \u003cli\u003eJiang, L., Kon, N., Li, T., Wang, S. J., Su, T., Hibshoosh, H., . . . Gu, W. (2015). Ferroptosis as a p53-mediated activity during tumour suppression. \u003cem\u003eNature, 520\u003c/em\u003e(7545), 57-62. doi:10.1038/nature14344\u003c/li\u003e\n \u003cli\u003eKeum, Y. S., Yu, S., Chang, P. P., Yuan, X., Kim, J. H., Xu, C., . . . Kong, A. N. (2006). Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells. \u003cem\u003eCancer Res, 66\u003c/em\u003e(17), 8804-8813. doi:10.1158/0008-5472.Can-05-3513\u003c/li\u003e\n \u003cli\u003eLanceta, L., Li, C., Choi, A. M., \u0026amp; Eaton, J. W. (2013). Haem oxygenase-1 overexpression alters intracellular iron distribution. \u003cem\u003eBiochem J, 449\u003c/em\u003e(1), 189-194. doi:10.1042/bj20120936\u003c/li\u003e\n \u003cli\u003eLee, J., Kang, E. S., Kobayashi, S., Homma, T., Sato, H., Seo, H. G., \u0026amp; Fujii, J. (2017). The viability of primary hepatocytes is maintained under a low cysteine-glutathione redox state with a marked elevation in ophthalmic acid production. \u003cem\u003eExp Cell Res, 361\u003c/em\u003e(1), 178-191. doi:10.1016/j.yexcr.2017.10.017\u003c/li\u003e\n \u003cli\u003eLi, P. M., Li, Y. L., Liu, B., Wang, W. J., Wang, Y. Z., \u0026amp; Li, Z. (2014). Curcumin inhibits MHCC97H liver cancer cells by activating ROS/TLR-4/caspase signaling pathway. \u003cem\u003eAsian Pac J Cancer Prev, 15\u003c/em\u003e(5), 2329-2334. doi:10.7314/apjcp.2014.15.5.2329\u003c/li\u003e\n \u003cli\u003eLi, R., Zhang, J., Zhou, Y., Gao, Q., Wang, R., Fu, Y., . . . Yu, H. (2020). Transcriptome Investigation and In Vitro Verification of Curcumin-Induced HO-1 as a Feature of Ferroptosis in Breast Cancer Cells. \u003cem\u003eOxid Med Cell Longev, 2020\u003c/em\u003e, 3469840. doi:10.1155/2020/3469840\u003c/li\u003e\n \u003cli\u003eLiu, P., Feng, Y., Li, H., Chen, X., Wang, G., Xu, S., . . . Zhao, L. (2020). Ferrostatin-1 alleviates lipopolysaccharide-induced acute lung injury via inhibiting ferroptosis. \u003cem\u003eCell Mol Biol Lett, 25\u003c/em\u003e, 10. doi:10.1186/s11658-020-00205-0\u003c/li\u003e\n \u003cli\u003eLlovet, J. M., Ricci, S., Mazzaferro, V., Hilgard, P., Gane, E., Blanc, J. F., . . . Bruix, J. (2008). Sorafenib in advanced hepatocellular carcinoma. \u003cem\u003eN Engl J Med, 359\u003c/em\u003e(4), 378-390. doi:10.1056/NEJMoa0708857\u003c/li\u003e\n \u003cli\u003eMarquardt, J. U., Gomez-Quiroz, L., Arreguin Camacho, L. O., Pinna, F., Lee, Y. H., Kitade, M., . . . Thorgeirsson, S. S. (2015). Curcumin effectively inhibits oncogenic NF-\u0026kappa;B signaling and restrains stemness features in liver cancer. \u003cem\u003eJ Hepatol, 63\u003c/em\u003e(3), 661-669. doi:10.1016/j.jhep.2015.04.018\u003c/li\u003e\n \u003cli\u003ePrasad, S., Gupta, S. C., Tyagi, A. K., \u0026amp; Aggarwal, B. B. (2014). Curcumin, a component of golden spice: from bedside to bench and back. \u003cem\u003eBiotechnol Adv, 32\u003c/em\u003e(6), 1053-1064. doi:10.1016/j.biotechadv.2014.04.004\u003c/li\u003e\n \u003cli\u003eShang, Y., Luo, M., Yao, F., Wang, S., Yuan, Z., \u0026amp; Yang, Y. (2020). Ceruloplasmin suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma cells. \u003cem\u003eCell Signal, 72\u003c/em\u003e, 109633. doi:10.1016/j.cellsig.2020.109633\u003c/li\u003e\n \u003cli\u003eSun, X., Ou, Z., Chen, R., Niu, X., Chen, D., Kang, R., \u0026amp; Tang, D. (2016). Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. \u003cem\u003eHepatology, 63\u003c/em\u003e(1), 173-184. doi:10.1002/hep.28251\u003c/li\u003e\n \u003cli\u003eSuttner, D. M., \u0026amp; Dennery, P. A. (1999). Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. \u003cem\u003eFaseb Journal, 13\u003c/em\u003e(13), 1800-1809. doi:10.1096/fasebj.13.13.1800\u003c/li\u003e\n \u003cli\u003eTian, S., Liao, L., Zhou, Q., Huang, X., Zheng, P., Guo, Y., . . . Tian, X. (2021). Curcumin inhibits the growth of liver cancer by impairing myeloid-derived suppressor cells in murine tumor tissues. \u003cem\u003eOncol Lett, 21\u003c/em\u003e(4), 286. doi:10.3892/ol.2021.12547\u003c/li\u003e\n \u003cli\u003eVogel, A., Meyer, T., Sapisochin, G., Salem, R., \u0026amp; Saborowski, A. (2022). Hepatocellular carcinoma. \u003cem\u003eLancet, 400\u003c/em\u003e(10360), 1345-1362. doi:10.1016/s0140-6736(22)01200-4\u003c/li\u003e\n \u003cli\u003eWang, L., Cai, H., Hu, Y., Liu, F., Huang, S., Zhou, Y., . . . Wu, F. (2018). A pharmacological probe identifies cystathionine \u0026beta;-synthase as a new negative regulator for ferroptosis. \u003cem\u003eCell Death Dis, 9\u003c/em\u003e(10), 1005. doi:10.1038/s41419-018-1063-2\u003c/li\u003e\n \u003cli\u003eYang, R., Song, C., Chen, J., Zhou, L., Jiang, X., Cao, X., . . . Zhang, Q. (2020). Limonin ameliorates acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-\u0026kappa;B inflammatory response via upregulating Sirt1. \u003cem\u003ePhytomedicine, 69\u003c/em\u003e, 153211. doi:10.1016/j.phymed.2020.153211\u003c/li\u003e\n \u003cli\u003eYang, W. S., SriRamaratnam, R., Welsch, M. E., Shimada, K., Skouta, R., Viswanathan, V. S., . . . Stockwell, B. R. (2014). Regulation of ferroptotic cancer cell death by GPX4. \u003cem\u003eCell, 156\u003c/em\u003e(1-2), 317-331. doi:10.1016/j.cell.2013.12.010\u003c/li\u003e\n \u003cli\u003eYang, W. S., \u0026amp; Stockwell, B. R. (2008). Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. \u003cem\u003eChem Biol, 15\u003c/em\u003e(3), 234-245. doi:10.1016/j.chembiol.2008.02.010\u003c/li\u003e\n \u003cli\u003eYang, Y., Liu, C., Wang, M., Cheng, H., Wu, H., Luo, S., . . . Li, Q. (2024). Arenobufagin regulates the p62-Keap1-Nrf2 pathway to induce autophagy-dependent ferroptosis in HepG2 cells. \u003cem\u003eNaunyn Schmiedebergs Arch Pharmacol, 397\u003c/em\u003e(7), 4895-4909. doi:10.1007/s00210-023-02916-5\u003c/li\u003e\n \u003cli\u003eZou, C., Zhang, H., Li, Q., Xiao, H., Yu, L., Ke, S., . . . Gao, X. (2011). Heme oxygenase-1: a molecular brake on hepatocellular carcinoma cell migration. \u003cem\u003eCarcinogenesis, 32\u003c/em\u003e(12), 1840-1848. doi:10.1093/carcin/bgr225\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Curcumin, HCC, Ferroptosis, P62-KEAP1-NRF2","lastPublishedDoi":"10.21203/rs.3.rs-7197765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7197765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eContext:\u003c/strong\u003e Hepatocellular carcinoma (HCC) urgently requires new treatment strategies due to chemotherapy resistance and toxic side effects. Curcumin exhibits broad-spectrum anticancer activity against HCC and other malignant tumors. Ferroptosis, a form of cell death characterized by iron deposition, glutathione depletion, and lipid peroxidation, has emerged as a promising therapeutic target for cancer. The P62-KEAP1-NRF2 pathway is a key signaling pathway in ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e This study explored the mechanism by which curcumin induces ferroptosis in HCC cells by modulating the P62-KEAP1-NRF2 signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods:\u003c/strong\u003e We constructed a Hepa1-6 xenograft mouse model to investigate changes in tumor growth, ferroptosis indicators, and the expression of P62, KEAP1, and NRF2. In vitro, HepG2 cell were treated with a ferroptosis inhibitor (Fer-1) or subjected to P62 overexpression, after which cell viability and key ferroptosis parameters\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Curcumin (100 mg/kg, 15 days) significantly inhibited tumor growth, reduced glutathione (GSH) levels in tumor tissues, and increased reactive oxygen species (ROS), malondialdehyde (MDA), and Fe\u003csup\u003e2+\u003c/sup\u003e content. Additionally, in vitro experiments showed that curcumin inhibited HepG2 cell growth and proliferation, induced ferroptosis in HepG2 cells, decreased P62 and NRF2 levels, and increased KEAP1 levels. These effects were inhibited by the Fer-1. Overexpression of P62 reduced the impact of curcumin on the P62-KEAP1-NRF2 pathway and ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion and conclusion:\u003c/strong\u003e Curcumin exerts its antitumor effects by inducing ferroptosis through the inhibition of the P62-KEAP1-NRF2 signaling pathway. This study provides important scientific basis for the application of curcumin and new potential targets for studying the pathological mechanisms of hepatocellular carcinoma.\u003c/p\u003e","manuscriptTitle":"Curcumin induces Ferroptosis in Hepatocellular Carcinoma by Regulating the P62-KEAP1-NRF2 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 14:04:19","doi":"10.21203/rs.3.rs-7197765/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-01T05:06:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T20:24:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117828434841870616819780550359723047757","date":"2025-08-27T11:00:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T07:37:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238069154178348097699664600920239901937","date":"2025-08-13T11:58:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258535043816588110288931783706009129241","date":"2025-08-07T03:48:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T02:27:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-25T10:47:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T07:36:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-25T07:35:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-07-23T14:52:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"821d9a37-f868-4179-91b4-4b3f42be760d","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:07:55+00:00","versionOfRecord":{"articleIdentity":"rs-7197765","link":"https://doi.org/10.1186/s12885-025-15307-1","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2025-11-29 15:58:23","publishedOnDateReadable":"November 29th, 2025"},"versionCreatedAt":"2025-07-29 14:04:19","video":"","vorDoi":"10.1186/s12885-025-15307-1","vorDoiUrl":"https://doi.org/10.1186/s12885-025-15307-1","workflowStages":[]},"version":"v1","identity":"rs-7197765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7197765","identity":"rs-7197765","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}