Molecular Medicine Reports Molecular Medicine Reports 1791-2997 1791-3004 D.A. Spandidos 10.3892/mmr.2025.13567 MMR-32-1-13567 Articles Co-treatment with triptolide and RSL3 induces hepatocellular carcinoma cell apoptosis and ferroptosis LiuWeixia WuGuodi WangJing WuShanshan ChenZhi The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, P.R. China Correspondence to: Dr Weixia Liu or Professor Zhi Chen, The State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Shangcheng, Hangzhou, Zhejiang 310003, P.R. China, E-mail: lwx10818036@zju.edu.cn, E-mail: zjuchenzhi@zju.edu.cn 072025 13052025 32 1 202 19022025 15042025 Copyright: © 2025 Liu et al. 2025 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Glutathione peroxidase 4 (GPx4; also known as phospholipid hydroperoxide glutathione peroxidase) inhibits cell death, including apoptosis and ferroptosis, by reducing lipid peroxidation. In addition, western blot assays showed that GPx4 protein levels were elevated in hepatocellular carcinoma (HCC) cells following triptolide (TPL) treatment. Therefore, it was hypothesized that HCC cells might develop partial resistance to TPL-induced cytotoxicity through upregulation of the GPx4 protein. To enhance anti-proliferative efficacy, the present study co-treated HCC cells with a combination of TPL and RAS-selective lethal 3 (RSL3), a well-characterized GPx4 activity inhibitor. Subsequent experimental data produced from Cell Counting Kit-8 and flow cytometric analyses demonstrated that co-administration of TPL and RSL3 promoted HCC cell apoptosis, elevated intracellular reactive oxygen species levels and induced ferroptosis. These collective findings suggested that co-treatment with TPL and RSL3 may induce both apoptotic and ferroptotic pathways in HCC cells.

 triptolide glutathione peroxidase 4 RAS-selective lethal 3 hepatocellular carcinoma apoptosis reactive oxygen species ferroptosis Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases zz202224 This work was supported by the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (grant no. zz202224). Introduction

Hepatocellular carcinoma (HCC) is the fourth leading cause of global cancer-related mortality (1), and is characterized by a poor prognosis due to its aggressive growth, propensity for metastasis and intrinsic resistance to current therapeutic approaches (2). Consequently, there is an urgent need to develop effective therapeutic strategies for the treatment of patients with HCC.

Triptolide (TPL), a structurally unique diterpene triepoxide isolated from Tripterygium wilfordii Hook F, is a traditional Chinese medicinal plant that has been used for centuries to treat a wide range of diseases. Notably, TPL has demonstrated potent anticancer activities, and exhibits cytotoxic activity against cancer cells originating from diverse tissues, including the colon, breast, stomach and liver (3–8). Emerging evidence has demonstrated that TPL exerts potent anti-HCC effects through multifaceted mechanisms. Studies have shown that TPL suppresses the invasion and tumorigenesis of MHCC-97H HCC cells by modulating the NF-κB signaling pathway (6). Furthermore, TPL can induce the apoptosis of HCC cells independent of p53 status (7), while its targeted delivery in BALB/c mice bearing HepG2 tumors effectively inhibits HCC progression through suppression of de novo lipogenesis (8). Furthermore, synergistic therapeutic outcomes have been observed when combining TPL with sorafenib for HCC treatment (9,10). These findings indicate that TPL may be a promising therapeutic candidate against HCC; however, its clinical translation remains constrained by dose-dependent toxicity (11).

Glutathione peroxidase 4 (GPx4), also known as phospholipid hydroperoxide glutathione peroxidase, serves a pivotal role in maintaining cellular redox homeostasis (12). Mechanistic studies have revealed functional differences between the GPx4 isoforms: The 20-kDa non-mitochondrial isoform suppresses ferroptosis through inhibition of lipid peroxidation (13), whereas the 23-kDa mitochondrial isoform prevents apoptosis by neutralizing cardiolipin hydroperoxide-mediated cytochrome c release (14,15). RAS-selective lethal 3 (RSL3) induces ferroptosis through direct GPx4 inactivation in RAS-mutated cancer cells, while sparing normal tissues (16). To potentiate the anti-HCC efficacy of TPL by pharmacological inhibition of GPx4 activity, the present study co-treated HCC cell lines in vitro with TPL and RSL3.

 Materials and methods <sec> <title>Cell culture

HCC cell lines Hep3B, PLC/PRF/5 (PLC) and Huh7 were cultured in high-glucose Dulbecco's Modified Eagle Medium (cat. no. C11995500BT; DMEM; Hyclone; Cytiva) supplemented with 10% fetal bovine serum (cat. no. 35-015-CV; Corning, Inc.), streptomycin (100 µg/ml) and penicillin (100 U/ml) mix (cat. no. 15070063; Gibco; Thermo Fisher Scientific, Inc). All cell cultures were maintained in a humidified incubator with an atmosphere of 5% CO2 and a temperature of 37°C.

 Cell viability assay

Hep3B/PLC/Huh7 cells were seeded at a density of 2,000 cells/well into 96-well plates and allowed to adhere for 24 h. Subsequently, TPL (cat. no. E-0316; Shanghai Tauto Biotech Co. Ltd.) (0, 2.5, 5, 7.5, 10 and 12.5 ng/ml) or RSL3 (cat. no. HY-100218A; MedChemExpress) (0, 0.125, 0.25, 0.5, 1, 2 and 4 µM) was used for treating Hep3B/PLC cells for 48 h or Huh7 cells for 24 h at 37°C to determine the cytotoxicity of TPL or RSL3. To investigate the cytotoxicity of TPL combined with RSL3, Hep3B or PLC cells were incubated with TPL (5 and 7.5 ng/ml) plus RSL3 (0, 0.125, 0.25, 0.5 and 1 µM) for 48 h at 37°C, while Huh7 cells were treated with TPL (10 ng/ml) and RSL3 (0.5 and 1 µM) for 24 or 48 h at 37°C. In addition, Hep3B or PLC cells were treated with TPL (7.5 ng/ml) and RSL3 (1 µM) for 24 or 48 h with or without a 2-h pretreatment with a reactive oxygen species (ROS) inhibitor N-acetyl-cysteine (NAC) (12 mM) (cat. no. HY-B0215; MedChemExpress) at 37°C to assess whether co-treatment with TPL and RSL3 increases ROS levels. Likewise, Hep3B/PLC cell culture was subjected to a 2-h pretreatment with a specific ferroptosis inhibitor Ferrostatin-1 (Fer-1) (5 µM) (cat. no. HY-100579; MedChemExpress) or a potent free iron chelating agent Deferiprone (DEF) (100 µM) (cat. no. HY-B0568; MedChemExpress) to evaluate if co-treatment with TPL and RSL3 enhances ferroptosis. After that, Hep3B cells were treated with TPL (10 ng/ml) plus RSL3 (2 µM) and PLC cells were treated with TPL (7.5 ng/ml) plus RSL3 (1 or 2 µM) for 24 h at 37°C. Cell viabilities were measured using the Cell Counting Kit 8 (CCK-8; cat. no. HY-K0301; MedChemExpress) following the aforementioned various cell treatments. CCK-8 solution (10 µl/well) was added to the plate. The plates were then incubated for 2 h at 37°C. Subsequently, the absorbance was measured at 450 nm using a microplate reader (Biotek; Agilent Technologies, Inc.). All experiments were performed independently at least three times.

 Protein extraction and western blot analysis

Hep3B/PLC/Huh7 cells were inoculated into 6-cm dishes at a density of 1.7×105 cells/dish and cultured for 24 h in the incubator. To investigate GPx4 expression, Hep3B or PLC cells were treated with TPL at 0, 2.5, 5, 7.5, 10 and 12.5 ng/ml for 48 h or at 10 ng/ml for 0, 32, 48, 60 and 72 h. Huh7 cells were treated with TPL (0, 5, 10 and 15 ng/ml) for 24 or 48 h. To analyze apoptosis, Hep3B or PLC cells were treated with TPL (7.5 ng/ml)/RSL3 (1 µM)/TPL (7.5 ng/ml) combined with RSL3 (1 µM) for 30 h. After that, total cell lysates were extracted using RIPA lysis buffer containing PMSF (cat. no. C1055; Applygen Technologies, Inc.). Protein concentration was determined with the BCA Protein Assay Kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Proteins (40 µg/lane) were separated on SurePAGE, Bis-Tris, 10×8 cm gradient (4–20%) gels (cat. no. M00656; GenScript Biotech Corporation) and transferred onto PVDF membranes (cat. no. IPVH00010; Millipore; Merk Group), which were blocked in 5% BSA for 1 h at room temperature (RT). The membranes were then incubated with specific primary antibodies (1:1,000) at 4°C overnight. GAPDH (cat. no. BK7021-100 µl; Bioker Biotechnology Co.) was used as a loading control. Antibodies against GPx4 (cat. no. 52455), both cleaved and precursor proteins of PARP (cat. no. 9532), caspase-9 (cat. no. 9502) and caspase-3 (cat. no. 9662) were purchased from Cell Signaling Technology, Inc. After being washed, the membranes were incubated with HRP-conjugated Goat Anti-Rabbit-IgG (cat. no. SA00001-2; Proteintech Group, Inc.) (1:10,000) at RT for 1 h and detected using the FDbio-Femto ECL Kit (cat. no. FD8030; FDbio Science Biotech Co. Ltd.). Protein bands were semi-quantified and grayscale values were calculated using ImageJ 1.52a (National Institutes of Health).

 Real-time cellular analysis (RTCA)

The inhibitory effect produced by TPL and RSL3 on cell proliferation was evaluated by RTCA. Hep3B or PLC cells (2,000 cells/well) were seeded into E-Plate 16 (cat. no. 5469813001; Agilent Technologies, Inc.) and cultured in medium containing TPL (7.5 and 12.5 ng/ml) with or without RSL3 (1 µM) at 37°C for 80 h. xCELLigence RTCA S16 Analyzer (ACEA Biosciences, Inc.) was used to monitor cell proliferation in real-time. Cell proliferation signals were transformed into cell indexes, which were displayed in RTCA S16 software (version 1.0.1; ACEA Biosciences, Inc.).

 Cell apoptosis assay

Hep3B or PLC cells were seeded into 6-cm dishes at the density of 1.7×105 cells/dish, cultured for 24 h in the incubator, and then treated with TPL (7.5 ng/ml) or RSL3 (1 µM) or TPL (7.5 ng/ml) combined with RSL3 (1 µM) for 24 h. Thereafter, cells were harvested, stained using BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I (cat. no. 556547; BD Pharmingen; BD Biosciences) according to the manufacturer's protocol and then detected with flow cytometry (LSRFortessa SORP; BD Biosciences). Data were recorded using BD FACSDiva™ software and analyzed with FlowJo software (version V10; BD Biosciences). Annexin V+/PI− stained cells represented apoptotic cells.

 Detection of ROS

Hep3B or PLC cells were seeded into 10-cm dishes at a density of 5×105 cells/dish, allowed to adhere for 24 h in the incubator, then treated with TPL (7.5 and 10 ng/ml)/RSL3 (1 µM)/TPL (7.5, 10 ng/ml) combined with RSL3 (1 µM) for another 24 h at 37°C. Subsequently, intracellular ROS levels were determined by detecting the fluorescence intensity of DCF as previously described (17) with minor modifications. That is, the cells were harvested and incubated with DMEM containing 2.5 µM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (cat. no. S0033S; Beyotime Institute of Biotechnology) for 1 h at 37°C, with mixing performed every 5 min. The cells were then washed three times with DMEM to remove DCFH-DA that had not entered the cells. Finally, DCF emission was recorded on the FITC channel of a flow cytometer (LSRFortessa SORP; BD Biosciences) using the BD FACSDiva software. Data were analyzed with FlowJo software (version V10; BD Biosciences).

 Measurement of lipid peroxidation levels

BODIPY® 581/591 C11 (cat. no. D3861; Molecular Probes; Thermo Fisher Scientific, Inc.) is a fluorescent probe used to determine lipid peroxidation in live cells. PLC cells were seeded into 10-cm dishes at a density of 5×105 cells/dish, cultured for 24 h in the incubator, then treated with TPL (7.5 ng/ml)/RSL3 (1 and 2 µM)/TPL (7.5 ng/ml) combined with RSL3 (1 and 2 µM) for 24 h at 37°C. Cell pellets were obtained by 0.25% trypsin digestion and centrifugation at 200 × g for 3 min at RT. After washing with PBS, the cells were resuspended in PBS containing 10 µM BODIPY 581/591 C11 and incubated for 30 min at RT. Subsequently, the excess dye was removed by rinsing with PBS and centrifugation at 200 × g for 3 min at RT. Finally, the cells were resuspended in PBS and detected by flow cytometry (LSRFortessa SORP; BD Biosciences) and the fluorescence signals were recorded using BD FACSDiva software. FlowJo software (version V10; BD Biosciences) was used for data analysis.

 Statistical analysis

SPSS 17.0 (SPSS, Inc.) was used for analyzing data. All experiments were repeated three or more times. Quantitative data from the experiments were analyzed and presented in graphs using GraphPad Prism 8 (Dotmatics). Data are presented as the mean ± standard deviation. The statistical significance of differences among three or more groups was determined by one-way ANOVA, followed by Tukey's multiple comparisons test for post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>TPL treatment induces accumulation of GPx4

It has been reported that TPL inhibits the proliferation of HCC cells in a concentration-dependent manner (17). Consistent results were obtained in the present study. TPL significantly inhibited both Hep3B and PLC cell viability in a concentration-dependent manner (Fig. 1A and B). Furthermore, the protein expression levels of GPx4 were elevated in Hep3B and PLC cells 48 h after treatment with ≥7.5 ng/ml TPL (Fig. 1C and D). Furthermore, upregulation of GPx4 was detected in cells 32, 48, 60 and 72 h after TPL (10 ng/ml) treatment (Fig. 1E and F). GPx4 levels were also evidently increased in Huh7 cells after 48 h of 10 or 15 ng/ml TPL treatment (Fig. S1).

 Co-treatment with TPL and RSL3 promotes the inhibition of cell viability

As an antioxidant enzyme, GPx4 protects cells from oxidative stress-induced death (12); however, RSL3 can decrease the activity of GPx4 (16). Therefore, TPL in combination with RSL3 was used with the aim of improving the efficiency of TPL in inducing cell death. As shown in Fig. 2A and B, treatment of Hep3B or PLC cells with RSL3 (≤1 µM) for 48 h did not result in reduced cell viability. However, the inhibitory effect of TPL (7.5 ng/ml) together with RSL3 (1 µM) on Hep3B or PLC cell viability was greater than that produced by TPL (7.5 ng/ml) alone, and was positively related to the concentration of RSL3 (Fig. 2C and D). In addition, the viability of Huh7 cells was reduced by RSL3 (2 µM) (Fig. S2A), but not by TPL (≤10 ng/ml) (Fig. S2B) after 24 h of treatment duration. Furthermore, Huh7 cell viability was also significantly reduced by co-treatment with TPL (10 ng/ml) and RSL3 (0.5 or 1 µM) for 24 or 48 h (Fig. S2C). However, subsequent experiments used Hep3B and PLC cells, and not Huh7 cells, considering that Huh7 cells were sensitive to RSL3-induced cytotoxicity and might not necessitate co-treatment with TPL and RSL3.

Additionally, cell indexes at the 80-h timepoint monitored by RTCA indicated that TPL (7.5 ng/ml) together with RSL3 (1 µM) inhibited the proliferation of Hep3B and PLC cells more effectively than TPL (12.5 ng/ml) alone (Table I). The mechanism underlying the reduction in cell viability induced by the combination of TPL and RSL3 was subsequently elucidated through analysis of Hep3B and PLC cell death.

 TPL combined with RSL3 increases apoptosis

Since TPL induces mitochondrial pathway-mediated apoptosis (18) and mitochondrial GPx4 suppresses apoptosis mediated by the mitochondrial death pathway (14), the present study examined whether representative hallmarks of classic apoptosis were at higher levels in TPL and RSL3 co-treated Hep3B and PLC cells. As shown in Fig. 3A, the proportion of Annexin V+/PI− Hep3B or PLC cells was higher in the group co-treated with TPL and RSL3 than that in the group treated with TPL or RSL3 alone after 24 h. Furthermore, the expression levels of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 were increased in Hep3B and PLC cells co-treated with TPL and RSL3 compared with those treated with TPL or RSL3 alone (Fig. 3B). Thus, an increase in apoptosis was confirmed in HCC cells treated with TPL combined with RSL3.

 Co-treatment with TPL and RSL3 promotes the production of soluble ROS

A comparison analysis of CCK-8 assay results revealed that NAC inhibited the reduction in Hep3B and PLC cell viability caused by TPL combined with RSL3, although this was not significant in Hep3B cells at 24 h (Fig. 4A and B). To verify whether the amount of soluble ROS was increased in TPL and RSL3 co-treated cells, soluble ROS levels were measured by flow cytometry. As shown in Fig. 4C, in the TPL (7.5 ng/ml) and RSL3 (1 µM) co-treated PLC cells, though not the TPL (7.5 and 10 ng/ml) and RSL3 (1 µM) co-treated Hep3B cells, a significant increase in soluble ROS levels was detected 24 h after treatment.

 Co-treatment with TPL and RSL3 induces ferroptosis

It has been reported that non-mitochondrial GPx4 inhibition induces ferroptosis (13). The present study investigated whether ferroptosis of Hep3B and PLC cells was induced following treatment with TPL in the presence of RSL3. Fer-1 was added to the cell culture medium and cell viability was assessed 24 h after co-treatment with TPL and RSL3. The results demonstrated that PLC cell viability was enhanced by Fer-1, compared with the cell viability in the TPL and RSL3 co-treatment group (Fig. 5B). In addition, the inhibition of cell viability induced in PLC cells by TPL + RSL3 was reduced in the presence of DEF when compared with that in TPL + RSL3 group (Fig. 5D). In comparison to the inhibition of Hep3B cell viability reduction by Fer-1 (P>0.05) or DEF (P>0.05), the suppression of PLC cell viability reduction by Fer-1 (P<0.05) or DEF (P<0.001) was significant. Therefore, PLC cells were selected to undergo lipid peroxidation level evaluation after co-treatment with TPL and RSL3. Consistent with our prediction, lipid peroxidation levels were significantly increased in PLC cells after co-treatment with TPL (7.5 ng/ml) and RSL3 (2 µM) for 24 h, compared with that in only TPL (7.5 ng/ml) or RSL3 (2 µM) group or TPL (7.5 ng/ml) and RSL3 (1 µM) co-treatment group (Fig. 6). These results suggested that ferroptosis was induced by the combination of TPL and RSL3 in HCC cells.

 Discussion

Current therapeutic strategies for advanced HCC, encompassing both surgical interventions and non-surgical approaches, remain suboptimal regarding clinical efficacy. Sorafenib, a multi-kinase inhibitor, is the first-line systemic therapy for unresectable HCC, which received approval from the U.S. Food and Drug Administration in 2007, and demonstrates survival benefits in this patient population (19,20). However, the clinical use of sorafenib is constrained by its modest survival extension and the critical challenge of treatment resistance (21). Emerging evidence has indicated that both intrinsic and acquired resistance mechanisms can undermine its therapeutic potential, necessitating the urgent development of novel therapeutic approaches.

T. wilfordii Hook F (thunder god vine) is a traditional medicinal plant with a long clinical history in the treatment of autoimmune diseases. In recent years, the antitumor effects of its active compound TPL have emerged as a new research focus for this traditional therapy, demonstrating broad-spectrum anticancer properties that make it a highly promising candidate as a novel anticancer drug (22–25). Notably, TPL demonstrates regulatory effects on lipid metabolism, a mechanism that may antagonize HCC progression by altering cancer cell metabolic dependencies (26,27). However, the clinical application of TPL is limited by its narrow therapeutic window and cumulative toxicity during prolonged use. Researchers have explored various strategies for reducing toxicity while enhancing efficacy, among which drug combination therapy has shown particularly promising results (28). The present study investigated the combination of TPL with RSL3, a compound known for its ferroptosis-inducing activity, to observe their combined effects on human HCC cell lines. The results revealed a cooperative effect between the two agents, allowing dose reduction of both drugs while maintaining inhibitory effects on cell viability, thereby potentially mitigating their adverse effects.

Notably, the current study observed an elevation of GPx4 protein levels in TPL-treated HCC cells. This finding contrasts with the results of a recent report demonstrating TPL-mediated GPx4 suppression in leukemia models, wherein GPx4 downregulation sensitized malignant cells to doxorubicin (29). The observed discrepancy suggests a cell lineage-dependent dichotomy in the regulatory effects of TPL on GPx4 expression, potentially reflecting tissue-specific redox adaptation mechanisms across malignancies of distinct origins. This tissue-specific regulation may originate from differential basal Nrf2 activity between epithelial-derived HCC and hematopoietic malignancies, as GPx4 expression is known to be transcriptionally controlled by the Nrf2-Keap1-ARE pathway (30,31). These findings highlight the necessity of context-specific therapeutic strategies when targeting ferroptosis pathways in different cancer types.

GPx4 exerts cytoprotective effects against oxidative stress-mediated cell death. Based on this premise, it was hypothesized that suppressing GPx4 upregulation with RSL3 in combination with TPL would enhance cell death. As expected, co-treatment with RSL3 (1 µM) and TPL (7.5 ng/ml) significantly reduced HCC cell viability. However, RSL3 monotherapy (1 µM) for 48 h exhibited negligible cytotoxicity in both Hep3B and PLC cell lines, a phenomenon consistent with the findings of a previous study, which demonstrated that GPx4 ablation alone fails to induce ferroptosis in glioblastoma models (32). This evidence suggested that GPx4 inhibition may require concomitant disruption of compensatory antioxidant pathways to achieve therapeutic efficacy.

Co-treatment with TPL (7.5 ng/ml) and RSL3 (1 µM) induced apoptosis in Hep3B and PLC cells in the present study. Notably, the percentage of apoptotic cells in all groups was low in Hep3B cells, with the maximum % being <10%. It is possible that low treatment dose (7.5 ng/ml TPL and 1 µM RSL3) and short duration (24 h) could not induce a high proportion of apoptosis in Hep3B cells. In addition, Hep3B cells lack p53. They may rely on alternative death mechanisms (e.g. necrosis), with the exception of canonical apoptosis pathways. Soluble ROS levels were not markedly increased in Hep3B cells after co-treatment with TPL (7.5 and 10 ng/ml) and RSL3 (1 µM) for 24 h, which may be reversed by prolonging treatment duration, for NAC significantly increased Hep3B cell viability after co-treatment with TPL (7.5 ng/ml) and RSL3 (1 µM) for 48 h. Fer-1 is a synthetic antioxidant and prevents damage to membrane lipids by a reductive mechanism and thereby inhibits ferroptosis (33). Ample iron ions engage in producing ROS by oxidizing lipid and thus induce ferroptosis (34,35). DEF is a potent free iron chelating agent and has antioxidant activity (36). In the present study, the reduction in PLC but not Hep3B cell viability resulting from co-treatment with TPL and RSL3 was inhibited by both Fer-1 and DEF, suggesting that co-treatment with TPL and RSL3 at 1 or 2 µM induced ferroptosis in PLC rather than Hep3B cells. This result might be associated with the findings that RSL3 (2 µM) induced PLC (Fig. 2B) instead of Hep3B (Fig. 2A) cell death after 48 h of treatment duration. Both results indicated that 2 µM of RSL3 might be not high enough to induce Hep3B cell ferroptosis. Hence, co-treatment with TPL and increased dose of RSL3 might induce Hep3B cell ferroptosis.

In conclusion, building on the observation that TPL upregulated GPx4 levels in HCC cells, the current study co-treated HCC cells with TPL and RSL3 in vitro. The findings demonstrated that these agents cooperated in inducing the apoptosis and ferroptosis of HCC cells, thereby providing novel mechanistic insights into TPL-mediated anti-HCC effects.

 Supplementary Material Supporting Data Acknowledgements

The authors would like to thank Dr Xue Liang (The First Affiliated Hospital, Zhejiang University School of Medicine) for maintaining cell lines.

 Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

 Authors' contributions

WXL and ZC confirm the authenticity of all the raw data. WXL designed and performed most of the experiments, analyzed the data and wrote the manuscript. GDW and JW performed the flow cytometry experiments. SSW participated in interpreting the flow cytometry data. ZC contributed to the conception and secured funding. All authors have read and approved the final manuscript.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

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TPL treatment induces the accumulation of GPx4. Cytotoxicity of TPL at the indicated concentrations in (A) Hep3B and (B) PLC cells was measured using the Cell Counting Kit 8 assay. Elevation of GPx4 in (C) Hep3B and (D) PLC cells after 48 h of treatment with various concentrations of TPL and elevations of GPx4 in (E) Hep3B and (F) PLC cells 32, 48, 60 and 72 h after TPL (10 ng/ml) treatment were assessed by western blot analyses. Quantitative data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. 0 ng/ml TPL group in (A). **P<0.01 and ***P<0.001 vs. 0 ng/ml TPL group in (B, C and D). **P<0.01 vs. 0 h of treatment duration in (E). **P<0.01 and ***P<0.001 vs. 0 h of treatment duration in (F). GPx4, glutathione peroxidase 4; RSL3, RAS-selective lethal 3; TPL, triptolide.

Combination of TPL and RSL3 promotes the inhibition of hepatocellular carcinoma cell viability. The cytotoxic effects of RSL3 at various concentrations on (A) Hep3B and (B) PLC cells were determined by cell viability analysis. Quantitative data are presented as the mean ± standard deviation (n=3). *P<0.05 and ***P<0.001 vs. 0 µΜ RSL3 group. (C) Hep3B and (D) PLC cells were treated with TPL (5 or 7.5 ng/ml) combined with or without RSL3 (0.125, 0.25, 0.5 and 1 µΜ) for 48 h, followed by cell viability analysis. *P<0.05, **P<0.01 and ***P<0.001 vs. TPL (5, 7.5 ng/ml) + RSL3 (0 µΜ) group. RSL3, RAS-selective lethal 3; TPL, triptolide.

TPL combined with RSL3 increases apoptosis. Hep3B and PLC cells were treated with RSL3 (1 µM), TPL (7.5 ng/ml) or TPL (7.5 ng/ml) combined with RSL3 (1 µM) for (A) 24 h or (B) 30 h. (A) Apoptosis was detected by flow cytometry. A total of 1×104 cells were recorded to analyze Annexin V+/PI− cells in each experiment. Quantitative data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. TPL (7.5 ng/ml) or RSL3 (1 µM) group. (B) Western blot analysis of classic apoptotic markers; these proteins were effectively activated in Hep3B and PLC cells treated with 7.5 ng/ml TPL combined with 1 µM RSL3. Grayscale was analyzed using ImageJ 1.52a software. Quantitative data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. TPL (7.5 ng/ml) or RSL3 (1 µM) group. FITC, fluorescein isothiocyanate; PI, propidium iodide; RSL3, RAS-selective lethal 3; TPL, triptolide.

Co-treatment with TPL and RSL3 promotes the production of soluble ROS. (A) Hep3B and (B) PLC cells were treated with 1 µM RSL3, 7.5 ng/ml TPL or both, combined with or without NAC at 12 mM for 24 or 48 h, followed by cell viability assay. Quantitative data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. TPL (7.5 ng/ml) and RSL3 (1 µM) co-treatment group. (C) ROS levels were detected in Hep3B and PLC cells by flow cytometry. ***P<0.001 vs. RSL3 (1 µM) or TPL (7.5 ng/ml) group. NAC, N-acetyl-cysteine; ns, not significant; ROS, reactive oxygen species; RSL3, RAS-selective lethal 3; TPL, triptolide.

TPL in combination with RSL3 induces the ferroptosis of hepatocellular carcinoma cells. Cell viability was measured in (A and C) Hep3B and (B and D) PLC cells treated with RSL3, TPL or both combined with or without (A and B) Fer-1 or (C and D) DEF at the indicated concentration for 24 h. Quantitative data are presented as the mean ± standard deviation (n=3). *P<0.05 and ***P<0.001 vs. TPL and RSL3 co-treatment group. DEF, deferiprone; Fer-1, ferrostatin-1; ns, not significant; RSL3, RAS-selective lethal 3; TPL, triptolide.

Ratios of green-to-red fluorescence of BODIPY® 581/591 C11 staining in PLC cells were detected by flow cytometry after 24 h of treatment with RSL3, TPL or both. Quantitative data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. RSL3 (2 µM), TPL (7.5 ng/ml) or RSL3 (1 µM) + TPL (7.5 ng/ml) group. R1, RSL3 (1 µM); R2, RSL3 (2 µM); RSL3, RAS-selective lethal 3; TPL, triptolide.

Effects of TPL and RSL3 on cell proliferation.

Cell index at 80 h
Treatment Hep3B cells PLC cells
Control 3.59±0.04 2.16±0.14
RSL3 (1 µM) 3.34±0.04 2.03±0.14
TPL (7.5 ng/ml) 2.17±0.00 2.88±0.02
TPL (12.5 ng/ml) 2.29±0.00 2.31±0.02
TPL (7.5 ng/ml) + RSL3 (1 µM) 1.69±0.01 1.78±0.02
TPL (12.5 ng/ml) + RSL3 (1 µM) 0.22±0.00 0.83±0.01

RSL3, RAS-selective lethal 3; TPL, triptolide.