Cutaneous squamous cell carcinoma (CSCC) is a common type of non-melanoma skin cancer, accounting for 20% of all skin neoplasms (Caudill et al., 2023). It originates from the gradual accumulation of genetic and epigenetic alterations and malignant proliferation in keratinocytes, ultimately facilitating the development of an invasive tumor (Hedberg et al., 2022). Invasive CSCC can recurrent and metastasize to adjacent lymph nodes or distant organs, leading to significant damage to local tissues if not promptly or appropriately treated (Lansbury et al., 2013). The principal therapies employed for the clinical management of CSCC are surgical intervention and radiotherapy; nevertheless, they present some disadvantages, including skin injury and difficulties in patient adherence and prognosis (Corchado-Cobos et al., 2020; Chabrillac et al., 2022). Thus, it is crucial to seek novel treatment agents to mitigate the malignant progression of CSCC, consequently improving patient overall well-being and mental health.

Artesunate (ART) is a water-soluble derivative of the antimalarial artemisinin, sourced from Artemisia annua; it is considered safe and well-tolerated via oral, intravenous, or intramuscular administration (Berkoz et al., 2021). ART demonstrates cytotoxic effects on various cancer cell lines, including those found in the colon, breast, leukemia, melanoma, central nervous system, ovaries, kidneys, and prostate (Augustin et al., 2020). Reportedly, ART effectively inhibits the malignant growth of human CSCC cells via the suppression of the PI3K/AKT pathway (Huang et al., 2022). Furthermore, Xu et al. have found that itraconazole suppresses the proliferation of CSCC by specifically targeting the HMGCS1/ACSL4 axis to induce ferroptosis (Xu et al., 2022a), implying the involvement of ferroptosis in CSCC. However, there are currently few reports on the regulation of CSCC ferroptosis by ART.

Notably, both glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) play crucial roles in the regulation of ferroptosis; SLC7A11 prevents ferroptosis by transporting cystine, promoting glutathione (GSH) production, and facilitating GPX4-mediated detoxification of lipid peroxides (Stockwell et al., 2017). SLC7A11 is frequently found to be overexpressed in many human malignancies, and a significant mechanism by which highly-expressed SLC7A11 contributes to tumor growth through the inhibition of ferroptosis (Koppula et al., 2021). Additionally, the growth of insulinoma cells is suppressed by ART via the SLC7A11-GPX4 ferroptosis pathway (Chen et al., 2024). Therefore, we hypothesized that ART might regulate the migration and proliferation of CSCC through the SLC7A11-GPX4 ferroptosis pathway.

p53 is a multifunctional tumor suppressor protein that is crucial in modulating cell cycle arrest, ferroptosis, senescence, and DNA damage repair (Xu et al., 2023). As previously described, SLC7A11 is recognized as a transcriptional target of p53, which promotes ferroptosis by inhibiting SLC7A11 expression (Koppula et al., 2021). Reportedly, ART up-regulates p53 expression in renal cell carcinoma cells (Markowitsch et al., 2020). p300 is a transcription-activating factor of p53; after DNA damage, p53 is bound with P300 and undergoes acetylation, and acetylated p53 exhibits enhanced stability and improved capacity for target protein recruitment (Adighibe and Pezzella, 2018). Importantly, the regulatory role of ART in acetylation has been documented (Zhang et al., 2022), and we also found that P300 served as a potential target for ART based on the SuperPred database. Based on the evidence, a hypothesis was postulated that ART may induce ferroptosis by regulating the SLC7A11-GPX4 pathway through the p300-p53 axis, thereby inhibiting the proliferation and migration of CSCC cells. Herein, the current study aims to investigate the molecular mechanism of ART in the proliferation and migration of CSCC through the p300-p53 axis, with the intention of providing a theoretical framework for new treatment approaches to CSCC.

Ethics statement

All animal experiments in this study were reviewed and approved by the Animal Ethics Committee of The Second Affiliated Hospital Zhejiang University School of Medicine (2024-296). We strictly adhered to the approved protocol, placing significant emphasis on minimizing the number of animals used and alleviating their suffering.

Cell culture

Human CSCC cells A431 were obtained from American Type Culture Collection (Manassas, VA, USA). A431 cells were plated at 2×10⁵ cells/well in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) comprising 10% fetal bovine serum (FBS) (Gibco). The medium was cultured in an incubator at 37°C with 74% N₂, 21% O₂ and 5% CO₂.

Cell grouping and treatment

A431 cells were subjected to 24-h treatment with 6.25, 12.5, 25, 50, 100, and 200 μM ART (HY-N0193, MedChem Express LLC, Monmouth Junction, NJ, USA) dissolved in dimethyl sulfoxide (DMSO), respectively. Subsequently, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the half maximal inhibitory concentration (IC₅₀) was 69.26 μM. Therefore, 70 μM ART was used for cell treatment in the subsequent experiments.

Cell grouping was as below: (1) Blank group: normally cultured A431 cells; (2) ART group: A431 cells treated with 70 μM ART for 24 h; (3) Vehicle group: A431 cells were treated with equal amount of DMSO to the ART group for 24 h; (4) ART+Fer group: A431 cells underwent treatment with 70 μM ART and 2 μM of the ferroptosis inhibitor Ferrostatin-1 (HY-13823, MedChem Express LLC) dissolved in DMSO for 24 h (Zou et al., 2020); (5) ART+Vehicle I group: A431 cells underwent 24-h treatment with 70 μM ART and the same amount of DMSO as the ART+Fer group; (6) ART+oe-SLC7A11 group: A431 cells were transfected with overexpressing (oe)-SLC7A11 for 24 h and then treated with 70 μM ART for another 24 h; (7) ART+oe-NC group: A431 cells were transfected with oe-negative control (NC) for 24 h and then treated with 70 μM ART for 24 h; (8) ART+C646 group: A431 cells were subjected to 16-h treatment with 15 μM of a p300 inhibitor C646 (MedChem Express LLC) dissolved in DMSO, followed by 24-h treatment with 70 μM ART (van den Bosch et al., 2016); (9) ART+Vehicle II group: A431 cells were cultured in the presence of the equal amount of DMSO to the ART+C646 group for 16 h, followed by 24-h treatment with 70 μM ART.

Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) was utilized for cell transfection. oe-SLC7A11 and oe-NC were transfected into cells following the manuals at a transfection concentration of 50 nM. All transfected materials were purchased from GenePharma Corporation (Shanghai, China).

MTT assay

A431 cell viability was evaluated by MTT assay. A431 cells were cultured in 96-well plates at a density of 0.8×10⁴/well for 24 h, followed by treatment with 6.25, 12.5, 25, 50, 100 and 200 μM ART for 24 h. The medium was cultured in the presence of 20 μL MTT solution (5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) in an incubator for 3 h. The purple crystals that formed in the wells were dissolved with DMSO. The optical density (OD) value was measured at 490 nm using a microplate reader (Bio-Rad 680, Bio-Rad, Hercules, CA, USA). All experiments were repeated three times. Cell survival was computed following the formula: cell survival rate=[(OD490_{experimental well}–OD490_{blank well})/(OD490_{control well}–OD490_{blank well})]×100%.

Cell counting kit-8 (CCK-8)

CCK-8 assay was performed at 0, 6, 12, and 24 h after cell treatment. Cells were treated with the CCK-8 kit (96992, Sigma-Aldrich) as per the manufacturer’s protocols. The OD value at 450 nm was measured using a microplate reader (Bio-Rad 680, Bio-Rad). All experiments were repeated three times. The formula used for calculating the cell survival rate was: cell survival rate=[(OD450_{experimental well}–OD450_{blank well})/(OD450_{control well}–OD450_{blank well})]×100%.

Transwell assay

Cells were exposed to ART for 12 h and subjected to Transwell assay. Firstly, 100 μL A431 cell suspension was added to the apical chamber of Transwell, and 700 μL medium supplemented with 20% FBS (Sigma-Aldrich) was added to the basolateral chamber, followed by 24-h culture at 37°C with 5% CO₂ in the air. The Transwell chambers were taken out, rinsed with phosphate-buffered saline (PBS) thrice, and then fixed with 1% glutaraldehyde for 30 min. After being rinsed with PBS again and dried, cells were dyed with 0.1% crystal violet (Sigma-Aldrich) before photographing and observation under an optical microscope. Afterward, six visual fields were stochastically selected, and the number of positive cells was counted using the Image J software (National Institutes of Health, Bethesda, MD, USA).

Glutathione-S-transferase (GST) pull-down assay

The GST-p300 fusion protein was cloned into the expression vector pGEX-4T-1 (SHBCC, Shanghai, China), separated from a BL21 DE3 bacterial culture (Boster Biotechnology, Wuhan, Hubei, China), and fixed on Glutathione-Sepharose (C4-0531-01-03, Seplife, Xi’an, Shaanxi, China). Thereafter, the pre-rinsed streptavidin agarose beads (Yeasen Biotech, Shanghai, China) were added to the aforementioned system, before overnight incubation with free biotin (MedChem Express LCC) or 10 μM ART-biotin with rotation at 4°C. Following three elution buffer washes of the beads, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the denatured protein.

Cycloheximide (CHX) assay

Cells were treated with 100 μg/mL CHX treatment (MedChem Express) for 2 h. Then, cells were lysed with PBS/Triton X-100 (Sigma-Aldrich) in the presence of proteinase K working solution (Sigma-Aldrich). The level of p53 was determined by western blot.

Western blot

The protein extraction kit (P0033, Beyotime, Shanghai, China) was utilized to extract the total proteins from the cells. The bicinchoninic acid protein concentration assay kit (P0009, Beyotime) was used to determine the protein concentration. Primary antibodies were added to the samples overnight at 4°C after electrophoresis, membrane transfer, and blockade. After the membrane was washed, the secondary antibody was added to the membrane for 1-h incubation at 37°C. The protein bands were assessed using a chemiluminescence kit (ECL Plus, Life Technology, Foster City, CA, USA). Image J was utilized for grayscale analysis, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. The antibody-related information is shown in Table 1.

Table 1 Antibody information

Antibodies	Cat No.	Dilution	Company
xCT(SLC7A11)	ab307601	1:1000
GPX4	ab41787	1 µg/mL
p300	ab275378	1:1000
p53	ab179477	1:2000	Abcam
Acetyl Lysine	ab190479	1:1000
GAPDH	ab9485	1:2500
Goat Anti-Rabbit IgG H&L (HRP)	ab205718	1:1000

Co-immunoprecipitation (Co-IP)

IP was conducted following the instructions provided by the manufacturers for the Pierce™ Classic Magnetic IP/Co-IP Kit (88804, Thermo Fisher Scientific). Briefly, cell lysis buffer was cultured with the anti-p53 antibody (1:2000, ab179477, Abcam, Cambridge, UK) overnight at 4°C, in a bid to form the immune complex. Afterward, the Pierce Protein A/G Magnetic Beads were mixed and cultivated with the antigen sample and antibody mixture for 1 h at room temperature. Finally, the binding proteins were eluted and then incubated with the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:1000, ab205718, Abcam) for 1 h for western blot analysis. The secondary antibody IgG (1:2000, ab172730, Abcam) was added to the proteins as a negative control. Cell complete lysate was detected as a positive control Input.

Experimental animal

Female athymic nude mice (n=30, weighing 18-20 g) were procured from Hubei Branch of Beijing Charles River Laboratory Animal Technologies Co. Ltd. [SCXK (e) 2022-0030]. Nude mice were reared at 26-28°C with 40-60% air humidity, provided with free access to food and water in a sterile environment, and maintained on a 12-h light/dark cycle.

Animal treatment and grouping

At first, 1×10⁶ A431 cells were injected subcutaneously into the axilla of the right forelimb of nude mice to induce tumorigenesis in vivo (Xu et al., 2022a; Li et al., 2023b). After 6 days, 30 nude mice were randomly classified into the following 5 groups (n=6): (1) the Model group: A431 cells induced tumorigenesis in vivo in nude mice; (2) the Model+ART group: after tumorigenesis in vivo, nude mice were administered with 30 mg/kg ART via oral gavage every other day (ART was sequentially dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% Saline) (Li et al., 2021); (3) the Model+Vehicle group: after tumorigenesis in vivo, nude mice were treated with equivalent dose of solvent to the Model+ART group via oral gavage every other day; (4) the Model+ART+C646 group: nude mice were injected intraperitoneally with 10 mg/kg C646 every other day following in vivo tumorigenesis (C646 was dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% Saline in turn) (Ono et al., 2021); (5) the Model+ART+Vehicle group: nude mice were injected intraperitoneally with the same amount of solvent as the Model+ART+C646 group every other day subsequent to in vivo tumorigenesis. After 22 days of treatment, mice were euthanized using an overdose of pentobarbital sodium (200 mg/kg), with their tumor tissues collected, photographed, and weighed. Subsequently, part of tumor tissues in each group were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 24 h, dehydrated with alcohol, cleaned with xylene, embedded in paraffin, sliced at 5 μm, and dewaxed to water for immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. The remaining tumor tissues were processed into tissue homogenate for western blot and kit detection.

Immunohistochemistry

The paraffin sections of tumor tissues were carefully rinsed with PBS and then subjected to boiling in 0.01 M sodium citrate buffer (Sigma-Aldrich) to facilitate antigen retrieval. Following this, the sections were cultured in 3% H₂O₂ for 15 min at room temperature, followed by closing with 5% goat serum (Solarbio) for 30 min. Sections were incubated with primary antibodies, including rabbit anti-p300 antibody (1:5000, ab275379, Abcam), rabbit anti-p53 antibody (1:50, ab32049, Abcam), rabbit anti-xCT (SLC7A11 antibody) (1:500, ab307601, Abcam), rabbit anti-GPX4 antibody (1:100, ab125066, Abcam), rabbit anti-Ki-67 antibody (0.5 μg/mL, ab15580, Abcam) at 4°C overnight. Then, the goat anti-rabbit immunoglobulin G H&L (HRP) (1:1000, ab6721, Abcam) was added to the sections for 2-h incubation at room temperature. Finally, the color was developed with 3,3-diaminobenzidine (Solarbio). After re-staining and mounting, sections were observed and photographed under an optical microscope. Image J software was used to calculate the percentage of positive cells.

Determination of lactate dehydrogenase (LDH), oxidized glutathione (GSSG), GSH, reactive oxygen species (ROS), malonaldehyde (MDA), and Fe²⁺ levels

According to the manufacturer’s instructions, the levels of LDH in cell culture supernatant and the levels of GSSG, GSH, ROS, MDA and Fe²⁺ in cells and tumor tissues were measured using GSH and GSSG assay kit (S0053, Beyotime), LDH activity assay kit (BC0685, Solarbio), Lipid Peroxidation MDA assay kit (S0131S, Beyotime), ROS assay kit (S0033S), and Fe²⁺ content assay kit (MAK025, Sigma-Aldrich). The ratio of GSSG/GSH was calculated.

TUNEL staining

Tumor tissue sections were incubated with protease K working solution (Sigma-Aldrich) at 37°C for 22 min, prior to three washes with PBS. Next, tissue paraffin sections were incubated for 20 min with 0.1% triton. After being rinsed with PBS, sections were cultured at room temperature for 10 min with buffer dropwise. Proper amounts of dUTP, TDTase, and buffer were mixed together at a 1:5:50 ratio as per the TUNEL staining kit guidelines (T2196, Solarbio). Additionally, sections were incubated for 2 h at 37°C, followed by PBS rinsing again. Afterward, sections were supplemented with 4’,6-diamidino-2-phenylindole staining solution dropwise, incubated at room temperature for 10 min, shielded from light, and sealed using an anti-fluorescence quenching sealer. The observation of sections and photo shoots were performed under a confocal fluorescence microscope (DMI8, Leica, Solms, Germany). Images were quantified using Image J software.

Statistical analysis

Statistical analyses and data visualizations were performed using GraphPad Prism 9.5 software (GraphPad Software Inc, San Diego, CA, USA). Normally distributed measurement data were expressed as mean ± standard deviation (SD). The independent sample t-test was employed for comparisons between the two groups. One-way analysis of variance (ANOVA) was used to compare multiple groups, with Tukey’s test conducted for post hoc analysis. The p-value was acquired from a two-sided test, with p<0.05 considered statistically significant.

ART promoted ferroptosis and inhibited CSCC cell proliferation and migration

We first evaluated the IC₅₀ of ART for CSCC cell line A431. A431 cells were cultivated for 24 h, thereafter exposed to ART at doses of 6.25, 12.5, 25, 50, 100, and 200 μM for 24 h, and cell viability was evaluated by the MTT assay. The results elicited that ART showed concentration-dependent growth inhibition on A431 cells within the concentration range of 6.25-200 μM, and IC₅₀ was 69.26 μM (Fig. 1A). In the follow-up experiment, we used 70 μM ART to treat A431 cells. As reflected by CCK-8 and Transwell assays, A431 cell proliferation (p<0.01, Fig. 1B) and migration (p<0.01, Fig. 1C) were abated after ART treatment. Upon cellular injury or death, LDH may be released into the extracellular environment (Segal and Levi, 1975). We determined the LDH level in the cell culture supernatant by kits. The results showed that the cellular LDH level was increased after ART treatment (p<0.01) (Fig. 1D). When ferroptosis occurs, the cycle of GSSG and reduced GSH is dysregulated, which leads to GSSG buildup (Rochette et al., 2022). We further assessed the cell GSSG/GSH ratio by kits and found that the cellular GSSG/GSH ratio was elevated after ART treatment (p<0.01) (Fig. 1E). It’s commonly acknowledged that oxidative damage appears during the occurrence of cell ferroptosis (Li et al., 2023a). Also, kit detection elicited that the levels of ROS and MDA were increased following ART treatment (all p<0.01, Fig. 1F, 1G). There is evidence that accumulation of Fe²⁺ occurs when cells undergo ferroptosis (Stockwell et al., 2020). We assayed the cellular active Fe²⁺ level by kits and observed that the level was augmented after ART treatment (p<0.01) (Fig. 1H). The results demonstrated that ART accelerated ferroptosis and delayed the malignant progression of CSCC cells.

Figure 1. ART boosted ferroptosis and suppressed CSCC cell proliferation and migration. A431 cells were treated with ART. (A) MTT method to determine the IC₅₀ of ART on A431; (B) CCK-8 assay to evaluate cell proliferation; (C) Transwell method to assess cell migration; (D-H) Assay kits to measure the LDH level in cell culture supernatant and cellular GSSG/GSH ratio and ROS, MDA and active Fe²⁺ levels (n=3). Data were expressed as mean ± SD. One-way ANOVA analysis was used for inter-group comparisons. The post hoc test was performed using Tukey’s multiple comparison test. *p<0.05, **p<0.01.

Inhibition of ferroptosis partially reversed the inhibitory effect of ART on CSCC cell proliferation and migration

To investigate whether ART inhibited the proliferation and migration of CSCC cells by inducing ferroptosis, we concurrently administered Ferrostatin-1 and ART to treat A431 cells. In contrast to the ART+Vehicle I group, the ART+Fer group displayed a lessened LDH level in cell culture supernatant (Fig. 2A), a decreased cellular GSSG/GSH ratio (Fig. 2B), reduced levels of cellular ROS (Fig. 2C), MDA (Fig. 2D) and cellular active Fe²⁺ (Fig. 2E) (all p<0.05), enhanced cell proliferation (p<0.05, Fig. 2F), as well as increased number of migratory cells (p<0.05, Fig. 2G). These findings collectively suggested that inhibition of ferroptosis partly counteracted the inhibitory effect of ART on CSCC cell proliferation and migration.

Figure 2. Inhibition of ferroptosis partially abolished the hindrance function of ART on CSCC cell proliferation and migration. ART and Ferrostatin-1 were used to treat A431 cells. (A-E) Assay kits to measure the LDH level in cell culture supernatant, as well as cellular GSSG/GSH ratio and ROS, MDA, and active Fe²⁺ levels; (F) CCK-8 assay to evaluate cell proliferation; (G) Transwell assay to assess cell migration (n=3). Data were presented as mean ± SD. One-way ANOVA analysis was implemented for inter-group comparisons, followed by Tukey’s multiple comparison test. *p<0.05.

ART promoted ferroptosis of CSCC cells by suppressing the SLC7A11-GPX4 ferroptosis pathway, thereby inhibiting cell proliferation and migration

We further used reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot to detect the levels of cellular SLC7A11 and GPX4. The results elicited that ART treatment brought about markedly decreased SLC7A11 and GPX4 levels in A431 cells (all p<0.01, Fig. 3A, 3B). Subsequently, we treated A431 cells with oe-SLC7A11 and ART concurrently. Compared to the ART+oe-NC group, the ART+oe-SLC7A11 group exhibited augmented SLC7A11 and GPX4 levels (all p<0.05, Fig. 3A, 3B), a diminished LDH level in the cell culture supernatant (Fig. 3C), a reduced cellular GSSG/GSH ratio (Fig. 3D), decreased levels of cellular ROS (Fig. 3E), MDA (Fig. 3F), and cellular active Fe²⁺ (Fig. 3G), along with enhanced cell proliferation (Fig. 3H) and migration (Fig. 3I) (all p<0.05). Overall, these results revealed that ART strengthened CSCC cell ferroptosis by dampening the SLC7A11-GPX4 ferroptosis pathway, hence impeding cell proliferation and migration.

Figure 3. ART facilitated ferroptosis of CSCC cells by repressing the SLC7A11-GPX4 ferroptosis pathway, thereby inhibiting cell migration and proliferation. ART and oe-SLC7A11 were utilized to treat A431 cells. (A) RT-qPCR to determine the levels of SLC7A11 and GPX4; (B) Western blot to measure the levels of SLC7A11 and GPX4 in cells; (C-G) Assay kits to measure the LDH level in cell culture supernatant, as well as cellular GSSG/GSH ratio and ROS, MDA and active Fe²⁺ levels; (H) CCK-8 assay to assess cell proliferation; (I) Transwell method to evaluate cell migration (n=3). Data were exhibited as mean ± SD. One-way ANOVA analysis was conducted for inter-group comparisons, and Tukey’s multiple comparison test was performed for the post hoc test. *p<0.05, **p<0.01.

ART promoted p53 acetylation, improved p53 protein stability, and limited the SLC7A11-GPX4 pathway by targeting p300

As reported, ART possesses the ability to modulate acetylation (Zhang et al., 2022). Besides, we analyzed potential targets of ART through the prediction of the SuperPred database (https://prediction.charite.de/index.php) (Supplementary Table 1). It was found that p300 was a potential target for ART through the prediction analysis of the SuperPred database (https://prediction.charite.de/index.php) (Fig. 4A). GST pull-down assay revealed that biotin-labeled ART could pull down GST-labeled purified p300 protein (Fig. 4B). Western blot experiment validated that the levels of p300, total acetylation, and p53 were elevated following ART treatment (all p<0.01, Fig. 4C). Also, ART treatment brought about increased p53 acetylation levels (p<0.01, Fig. 4D). Subsequently, we treated cells with CHX (75 μg/mL) for 2 h and discovered that cell treatment with ART resulted in an increase in the stability of the p53 protein (Vehicle group vs ART group, p<0.01, Fig. 4E). To further validate the role of ART targeting p300 in the SLC7A11-GPX4 pathway, we used the p300 inhibitor C646 and ART to treat A431 cells. Based on the results, compared with the ART+Vehicle II group, cells in the ART+C646 group exhibited diminished total acetylation and p53 levels (all p<0.05, Fig. 4F), diminished p53 acetylation levels (p<0.01, Fig. 4D), suppressed p53 protein stability (p<0.05, Fig. 4E), and elevated levels of SLC7A11 and GPX4 (all p<0.05, Fig. 4F). In short, these results indicated that ART targeted p300 to stimulate p53 acetylation, encouraged p53 protein stability, and hindered the SLC7A11-GPX4 pathway.

Figure 4. ART stimulated p53 acetylation, increased p53 protein stability, and limited the SLC7A11-GPX4 pathway by targeting p300. A431 cells were treated with ART and p300 inhibitor C646. (A) Predictive analysis of the SuperPred database (https://prediction.charite.de/index.php) revealed that p300 was a potential target for ART. Red percentages indicated the likelihood of ART binding to p300, and green percentages indicated the accuracy of the predictive modeling; (B) GST pull-down assay to detect the binding of ART and p300. B-ART is biotin-labeled ART. Cell complete lysis buffer was used as a positive control Input, and Biotin was used as a biotin negative control; (C, F) Assessment of levels of p300, total acetylation, p53, SLC7A11, and GPX4 using western blot; (D) Determination of the p53 acetylation level by Co-IP assay. IgG was used as the negative control, and cell complete lysis buffer was used as the positive control Input; (E) Evaluation of p53 protein stability by CHX test (n=3). Data were exhibited as mean ± SD, and the inter-group comparisons were made by one-way ANOVA, followed by the post hoc test using Tukey’s test. *p<0.05, **p<0.01.

Suppression of p300 partially annulled the regulatory effects of ART on ferroptosis, proliferation, and migration of CSCC cells

We further examined the changes in ferroptosis, proliferation, and migration of A431 cells treated with ART and C646. Compared to the ART+Vehicle II group, the ART+C646 group showed decreased LDH level in cell culture supernatant (Fig. 5A), cellular GSSG/GSH ratio (Fig. 5B), cellular ROS (Fig. 5C), MDA level (Fig. 5D), and cellular active Fe²⁺ levels (Fig. 5E) (all p<0.05), together with heightened cell proliferation (Fig. 5F) and migration (Fig. 5G) (p<0.01). In summary, these aforementioned findings suggested that the inhibition of p300 partly invalidated the modulation mediated by ART on migration, proliferation, and ferroptosis of CSCC cells.

Figure 5. Suppressing p300 partly averted the regulatory effects of ART on proliferation, ferroptosis, and migration of CSCC cells. A431 cells were administered ART and C646. (A-E) Assay kits to measure the LDH level in cell culture supernatant, as well as cellular GSSG/GSH ratio and ROS, MDA, and active Fe²⁺ levels; (F) Cell proliferation was assessed by CCK-8; (G) Cell migration was evaluated by Transwell method (n=3). Data were expressed as mean ± SD. One-way ANOVA analysis was conducted for inter-group comparisons, with Tukey’s multiple comparison test performed afterward. *p<0.05, **p<0.01.

ART regulated the SLC7A11-GPX4 ferroptosis pathway by up-regulating the p300-p53 axis, thereby preventing tumor growth in vivo

Finally, we subcutaneously inoculated 1×10⁶ A431 cells into the right forelimb axilla of athymic nude mice to establish a CSCC in vivo tumorigenesis model and treated nude mice with ART. Compared with the Model+Vehicle group, mice in the Model+ART group showed decreased tumor tissue weight (p<0.01, Fig. 6A). In comparison to the Model+Vehicle group, the Model+ART group exhibited rising levels of p300 and p53, as well as reduced levels of SLC7A11 and GPX4 (all p<0.01, Fig. 6B, 6C). Furthermore, western blot assay manifested that the acetylation level of mouse tumor tissues was increased in the Model+ART group versus the Model+Vehicle group (p<0.01, Fig. 6D). Besides, the GSSG/GSH ratio (Fig. 6E), ROS (Fig. 6F), MDA (Fig. 6G), and active Fe²⁺ (Fig. 6H) levels were up-regulated in the Model+ART group relative to the Model+Vehicle group (all p<0.01). Nude mice in the Model+ART group had more TUNEL-positive cells than mice in the Model+Vehicle group (p<0.01, Fig. 6I). The Ki-67 level in the Model+ART group was lower than that of the Model+Vehicle group (p<0.01, Fig. 6C). Lastly, we treated CSCC nude mice with C646 and ART. Compared with the Model+ART+Vehicle group, the Model+ART+C646 group displayed lessened acetylation levels (p<0.05, Fig. 6D), augmented nude mouse tumor weight (p<0.05, Fig. 6A), decreased p53 levels, increased GPX4 and SLC7A11 levels (all p<0.05, Fig. 6C), diminished GSSG/GSH ratio (Fig. 6E), reduced levels of ROS (Fig. 6F), MDA (Fig. 6G) and active Fe²⁺ (Fig. 6H) (all p<0.05), decreased TUNEL positive cells (p<0.05, Fig. 6I), and increased Ki-67 positive cells (p<0.05, Fig. 6C). Based on these findings, we concluded that ART modulated the SLC7A11-GPX4 ferroptosis pathway by stimulating the p300-p53 axis, thereby preventing in vivo tumor growth.

Figure 6. ART modulated the SLC7A11-GPX4 ferroptosis pathway by up-regulating the p300-p53 axis, thus hindering tumor in vivo growth. The CSCC in vivo model was developed by subcutaneous inoculation of 1×10⁶ A431 cells in the armpit of the right forelimb of athymic nude mice, and CSCC nude mice were treated with ART and C646. (A) Tumor weight; (B, C) IHC to determine SLC7A11, p300, p53, GPX4 and Ki67 levels in nude mouse tumor tissues; (D) Western blot to measure acetylation levels in tumor tissues of nude mice; (E-H) Assay kits to measure GSSG/GSH ratio and ROS, MDA and active Fe²⁺ levels; (I) TUNEL staining to assess apoptosis in nude mouse tumor tissues. n=6. The data were expressed as mean ± SD. One-way ANOVA was implemented for inter-group comparisons, and Tukey’s multiple comparison test was conducted afterward. *p<0.05, **p<0.01.

CSCC is the second most prevalent cancer in humans, with its prevalence persistently increasing (Corchado-Cobos et al., 2020). Natural herbal remedies have shown considerable efficacy in inhibiting the malignant progression of cancer (Xiong et al., 2021). ART, a Chinese patent medicine, distinguishes itself from other treatments due to its exceptional efficacy, minimal toxicity, and low tolerance (Wang et al., 2017). ART has been confirmed to selectively inhibit the proliferation of head and neck cancer cells by enhancing iron-dependent and ROS-induced ferroptosis (Roh et al., 2017). Accumulating studies have indicated that ferroptosis is strongly associated with the advancement of CSCC (Li et al., 2022b; Su et al., 2022), but little is known about the specific mechanism of ART in CSCC. In the present study, we found that ART could inhibit the malignant behaviors of CSCC cells by regulating ferroptosis. More importantly, ART could target the p300-p53 axis to modulate SLC7A11-GPX4-mediated ferroptosis, thereby affecting CSCC cell proliferation and migration. The specific mechanism diagram is shown in Fig. 7.

Figure 7. A graphical mechanism diagram. ART: artesunate, Ac: acetylation.

ART, a derivative of artemisinin, exhibits enhanced bioactivity and water solubility, rendering it very efficacious and safe for the treatment of different malignancies (Sun et al., 2019). Research has demonstrated that ART therapy increases autophagy-dependent apoptosis in human bladder cancer cells by elevating ROS levels and activating the AMPK-mTOR-ULK1 axis (Zhou et al., 2020). Pirali et al. have revealed that ART contributes to caspase-dependent apoptosis by declining HSP70 levels in breast cancer cells (Pirali et al., 2020). On the other hand, numerous studies have demonstrated that ART inhibits cancer malignant progression. For instance, ART prevents thyroid cancer cells from proliferating, migrating, and invading via modulation of the PI3K/AKT/FKHR pathway (Xu et al., 2022b). Other research has shown that ART can induce apoptosis and repress the migration and viability of ER-positive endometrial cancer cells in a dose-dependent manner (Yin et al., 2020). In a similar light, our findings also suggested that ART facilitated ferroptosis and repressed CSCC migration and proliferation. Additionally, it has been documented that the ferroptosis-specific inhibitor ferrostatin-1 can counteract the inhibition of ART on myeloma cell proliferation (Liang et al., 2023). Our findings aligned with the previous report, indicating that the suppressive effects of ART on the proliferation and migration of CSCC cells could be partly rescued by suppression of ferroptosis.

The SLC7A11-GPX4 pathway is the principal mechanism that protects against ferroptosis by promoting intracellular GSH synthesis and declining lipid peroxidation (Zheng and Conrad, 2020). The SLC7A11-GPX4 axis, as the principal inhibitor of ferroptosis, has attracted interest as a prospective therapy for illnesses associated with ferroptosis. Specifically, inhibition of SLC7A11 leads to the depletion of GPX4 and GSH, resulting in damage to cellular and subcellular membranes by the accumulation of iron-dependent lipid peroxides (Wu et al., 2021). Moreover, SLC7A11 overexpression in cancer cells accelerates tumor proliferation and migration via suppressing ferroptosis. (Lei et al., 2022). Similar to the evidence, we uncovered that ART treatment caused decreased SLC7A11 and GPX4 levels in A431 cells, whereas overexpressed SLC7A11 in ART-exposed CSCC cells increased SLC7A11 and GPX4 levels and cell growth and migration, as well as lessened GSSG/GSH ratio and levels of LDH, active Fe²⁺, MAD and ROS. In contrast, Li et al. have reported that the knockdown of SLC7A11/GPX4 induces ferroptosis and curtails cell malignant phenotypes in lung cancer cells (Li et al., 2022a). Strikingly, ART can decelerate insulinoma cell growth and viability via SLC7A11/GPX4-mediated ferroptosis (Chen et al., 2024). Altogether, these data highlighted that ART suppressed CSCC cell proliferation and migration via the promotion of SLC7A11/GPX4-mediated ferroptosis.

Our subsequent analysis demonstrated that p300 was a potential target for ART and verified the binding of ART to p300. Acetylation is a common modification associated with the activation of p53 in response to various p53-activating agents, and p300 functions as a p53 acetylase, enhancing the stability of p53 acetylation (Ito et al., 2001). The depletion of p53 has been reported to restore the protein levels of SLC7A11 and GPX4 (Kan et al., 2021). Therefore, it is plausible that ART might target the p300-p53 axis to regulate the SLC7A11-GPX4 pathway. We also noticed that p300 knockdown partially abrogated the regulatory effects of ART on ferroptosis and malignant phenotypes in CSCC cells. Likewise, the p300 inhibitor C646 reportedly represses RSL3-induced cell death and ferroptosis in human pancreatic ductal adenocarcinoma cells, as evidenced by decreased levels of lipid ROS, iron, and MDA (Wang et al., 2023). Rotte et al. have also found that the reduced expression of nuclear p300 protein is associated with the malignant progression of disease and poor outcomes for melanoma patients (Rotte et al., 2013). Despite multiple findings emphasizing the importance of p300’s intracellular localization with regard to its activity, the exact regulatory mechanisms regulating its activity are still unknown (Mackeh et al., 2014). Subsequent experiments using the CSCC in vivo tumor model further validated the effect of p300-p53 axis-mediated SLC7A11-GPX4 on tumor growth. The present study is, to our knowledge, the first to demonstrate that ART could up-regulate the p300-p53 axis to inhibit the SLC7A11-GPX pathway, hence limiting the in vivo growth of CSCC tumors.

In conclusion, in this study, we presented the findings for the first time to elucidate the targeting effect of ART on p300. Our findings indicated that ART suppressed the proliferation and migration of CSCC through the modulation of the SLC7A11-GPX4 ferroptosis pathway mediated by the p300-p53 axis. p300 repression partially counteracted the modulatory impacts of ART on CSCC cell malignant phenotypes and ferroptosis. Nonetheless, the majority of our research on ferroptosis and its correlation with tumors was performed using cellular and animal models, and there was a lack of validated clinical evidence. Besides, further investigation is required to fully understand the treatment effect of ART on CSCC. Future efforts should focus on conducting clinical verification and further investigating the mechanisms of ART against CSCC.

Not Applicable.

The authors have no conflicts of interest to declare.

Conceptualization, Xinyan Huang and Wenxi Wang; Methodology, Xinyan Huang; Software, Songzhao Zhang and Jihui Huang; Validation, Xinyan Huang, Songzhao Zhang and Lili Li; Formal Analysis, Xinyan Huang; Investigation, Wenxi Wang and Lili Li; Resources, Songzhao Zhang; Data Curation, Xinyan Huang; Writing – Original Draft Preparation, Xinyan Huang; Writing – Review & Editing, Wenxi Wang; Visualization, Songzhao Zhang; Supervision, Wenxi Wang; Project Administration, Wenxi Wang.