METTL16 antagonizes astaxanthin-induced ferroptosis in colorectal cancer cells

Introduction

Colorectal cancer (CRC) ranks as the fourth most commonly diagnosed human malignancy and the third leading cause of cancer-related mortality worldwide (1). As a gastrointestinal malignant tumor, CRC involves genetic, environmental, and lifestyle risk factors. Over the past decades, despite significant improvements in early screening methods and rapid advancements in therapeutic approaches, CRC remains one of the most challenging cancers to treat due to the lack of targeted therapies. Additionally, common treatment approaches for CRC include multimodal combination therapy involving surgery, chemotherapy, and radiation therapy. However, recurrence and metastasis rates remain high (2). Therefore, discovering novel biomarkers, identifying natural compounds with potent tumor-killing activity, elucidating their mechanisms of action, and improving drug targeting while reducing toxicity have become key focuses in molecular targeted drug research. These advancements are crucial for enhancing clinical outcomes and prognosis in cancer patients.

Astaxanthin is a carotenoid widely present in marine organisms and microorganisms. It has been extensively utilized in nutritional supplements, cosmetics, and animal feed, while also exhibiting antioxidant and antitumor properties. Due to its unique molecular characteristics, astaxanthin holds broad research potential across multiple fields (3-5). Furthermore, astaxanthin exhibits therapeutic benefits against various human and animal diseases, such as anti-diabetic, anti-inflammatory, hepatoprotective, and cardiovascular protective effects (6,7). Experimental studies have demonstrated its antitumor activity (8), with significant preventive and therapeutic effects on dextran sulfate sodium (DSS)-induced colitis and CRC (9,10). These effects are potentially mediated through antioxidant and anti-inflammatory mechanisms, as well as modulation of multiple signaling pathways. Notably, astaxanthin serves as an ideal multi-target agent. Its tissue distribution, absorption, and toxicity profile have been thoroughly investigated in experimental animal models, confirming excellent safety with no adverse events reported in any clinical studies to date (11). Therefore, astaxanthin was selected as the intervention agent in this study based on three primary considerations: first, as a natural carotenoid, astaxanthin has demonstrated excellent safety and tolerability in multiple clinical studies; second, existing literature indicates that astaxanthin can induce tumor cell death by regulating oxidative stress pathways, yet its interaction with m6A methylation modifications remains unreported. Finally, our preliminary experimental data confirm that astaxanthin exhibits significant antitumor activity in CRC cell lines, with this effect demonstrating clear dose-dependent characteristics.

N6-methyladenosine (m6A) methylation, the most abundant post-transcriptional modification in eukaryotic messenger RNA (mRNA), plays pivotal roles in regulating mRNA splicing, translation, stability, and decay (12,13). The methyltransferase-like (METTL) protein family, characterized by their S-adenosylmethionine (SAM)-binding domains, comprises over 30 methyltransferase proteins. Among these, methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14) are the most extensively studied. Notably, methyltransferase 16 (METTL16) has also been reported to deposit m6A modifications on transcripts of target genes (e.g., MALAT1, XIST, and MAT2A). Emerging evidence demonstrates METTL16’s involvement in diverse physiological and pathological processes, including DNA damage repair, stem cell differentiation, circadian rhythm regulation, and embryonic development (14,15). Recent studies highlight METTL16’s critical functions in the initiation and progression of various cancers, positioning it as a promising novel biomarker and potential therapeutic target (16-18). However, although METTL16’s role in m6A modification has been documented, its function in regulating ferroptosis in CRC remains unclear. Existing studies primarily focus on METTL16’s involvement in neuronal differentiation and pancreatic cancer, leaving its role at the intersection of tumor metabolism and cell death largely unexplored.

Ferroptosis is a recently identified form of iron-dependent regulated cell death characterized by increased phospholipid peroxidation, polyunsaturated fatty acid accumulation, iron ion overload, elevated malondialdehyde (MDA) levels, and glutathione peroxidase 4 (GPX4) inactivation, collectively leading to lethal lipid peroxidation (19-21). Distinct from conventional apoptosis and autophagy, this cell death modality is regulated by multiple intracellular signaling pathways. Notably, aberrant lipid metabolism, reactive oxygen species (ROS) overproduction, and iron accumulation constitute hallmark metabolic features differentiating tumor cells from normal cells (22,23). Emerging research demonstrates that ferroptosis exerts tumor-suppressive effects through diverse mechanisms. Several tumor suppressors and oncogenic signaling pathways have been shown to either promote or inhibit ferroptosis. Consequently, as a novel inducible cell death pathway, ferroptosis plays a positive role in overcoming tumor drug resistance and enhancing cancer therapy, highlighting its significant potential for anticancer treatment development.

Although significant progress has been made in the diagnosis and treatment of CRC in recent years, the 5-year survival rates for CRC patients and those with metastatic disease remain only 48% and 10%, respectively (24). While conventional chemotherapeutic agents demonstrate efficacy during initial treatment cycles, the inevitable development of drug resistance after repeated administrations poses a major clinical challenge (25,26). Therefore, the discovery and identification of new biomarkers, early disease diagnosis, and the development of novel treatment strategies are particularly important in CRC research. Emerging evidence from both international studies and our preliminary research suggests that METTL16 represents a promising therapeutic target for CRC. Meanwhile, the natural compound astaxanthin offers new perspectives for anti-CRC drug development. Compelling data indicate that ferroptosis mediates tumor suppression through multiple mechanisms. Therefore, systematic investigation of the functional interplay between astaxanthin, METTL16 and ferroptosis—with particular focus on elucidating their molecular mechanisms in colorectal carcinogenesis—may reveal novel therapeutic targets and inform innovative treatment paradigms for clinical management of CRC. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2002/rc).

Methods

Cell culture and transfection

The CRC cell lines HCT-116 and RKO (Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in a humidified incubator at 37 ℃ with 5% CO₂. HCT-116 cells were maintained in RPMI-1640 medium (Gibco Thermo Fisher Scientific, Inc., Waltham, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. RKO cells were cultured in DMEM medium (Gibco Thermo Fisher Scientific, Inc.) containing 10% FBS and 1% penicillin/streptomycin (Beyotime Biotechnology, Shanghai, China). For plasmid transfection, Lipofectamine 8000 (Beyotime Biotechnology) was used according to the manufacturer’s protocol. For small interfering RNA (siRNA) transfection, EZ TransRNA transfection reagent (Life-iLab, Inc., Shanghai, China) was employed following the recommended guidelines.

Cell Counting Kit-8 (CCK8) assay

Cell viability was analyzed using the CCK8 assay. The two CRC cell lines were respectively seeded into 96-well plates (LABSELECT, Lanjieke Technology Co.,Ltd, Beijing, China). On the second day, different concentrations of astaxanthin (HY-B2163, MCE, Shanghai, China) were added. On the third day, the prepared cell CCK8 reagent was added, and after 1 hour of incubation, the absorbance at 450 nm was measured. The same procedure was repeated on the fourth day: the prepared CCK8 reagent (Biosharp, Anhui, China) was added, followed by 1 hour incubation and measurement of absorbance at 450 nm.

Wound healing assay

Cell migration ability was detected using the wound healing assay. The two cell lines were seeded into 6-well plates (LABSELECT, Lanjieke Technology Co.,Ltd). After reaching 90% confluence, a 200 µL pipette tip was used to make scratches on the cell monolayer surface, ensuring consistent pressure to maintain uniform scratch width. Microscopic images were then taken to record the wound area at 0 hour. Astaxanthin was then added at half maximal inhibitory concentration (IC₅₀) and IC₇₅ concentrations. After 24 hours, images were taken again. The wound closure area was measured using ImageJ software (designed by National Institutes of Health) to calculate the percentage of wound closure.

Transwell assay

The Transwell assay was used to examine cell migration and invasion capabilities. After cell counting, the cells were seeded into the upper chamber (Corning, New York, USA) containing serum-free medium. The lower chamber of the control group was filled with complete medium, while the experimental groups received complete medium containing astaxanthin at IC₅₀ and IC₇₅ concentrations, respectively. After 48 hours of incubation, the cells were fixed with paraformaldehyde (Biosharp) and stained with crystal violet (Beyotime Biotechnology). Following air-drying, images were captured using an inverted microscope (Olympus, Tokyo, Japan).

Western blotting

Cells were seeded in 6-well plates and treated accordingly. After 48 hours, proteins were extracted and quantified using the BCA method (Beyotime Biotechnology). The proteins were separated by SDS-PAGE gel electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (TransGen Biotech, Beijing, China). The membranes were blocked, followed by overnight incubation with primary antibodies at 4 ℃. After washing three times with Tris-Buffered Saline with Tween 20 (TBST) buffer (Biosharp), the membranes were incubated with secondary antibodies for 2 hours. Finally, the protein bands were visualized by exposure. Antibodies used: anti-beta Actin Rabbit (Servicebio, Wuhan, China; #GB11001-100; 1:2,000); anti-GPX4 (Affinity, Jiangsu, China; #DF6701; 1:2,000); anti-XCT (Affinity; #DF12509; 1:2,000); anti-METTL16 (Proteintech, Wuhan, China; #19924-1-AP; 1:1,000).

ROS measurement

ROS were measured using flow cytometry. Cells were seeded in 6-well plates and treated accordingly. After 48 hours, 0.5 µL of DCFH probe (Beyotime Biotechnology) was added following the manufacturer’s instructions, followed by incubation at 37 ℃ for 30 minutes. The cells were then trypsinized, collected into flow cytometry tubes (LABSELECT, Lanjieke Technology Co.,Ltd), and centrifuged at 1,500 rpm for 5 minutes. After supernatant removal, the cells were washed three times with 1 mL phosphate-buffered saline (PBS) and finally resuspended in 300 µL PBS for flow cytometric analysis.

Fe2+ measurement

The iron colorimetric assay kit (E-BC-K881-M, Elabscience, Wuhan, China) was used to determine total intracellular ferrous iron content. Prior to use, all kit reagents were equilibrated to room temperature. Collected cells were placed in 1.5 mL centrifuge tubes, mixed with Reagent 1, and lysed on ice for 10 minutes, followed by centrifugation at 15,000 g for 10 minutes. The supernatant was collected for subsequent analysis. According to the manufacturer’s protocol, corresponding reagents were added to standard wells, test wells, and control wells, respectively. After thorough mixing and incubation at 37 ℃ for 10 minutes, absorbance at 593 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The ferrous iron concentration was calculated based on the standard curve.

MDA measurement

The MDA assay was performed using the MDA test kit (A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Prior to the experiment, Reagent 1, Reagent 2, and Reagent 3 were prepared according to the manufacturer’s instructions. Cells were collected into 1.5 mL centrifuge tubes, and the reagents were added to blank tubes, standard tubes, and test tubes as specified in the protocol. Small holes were punctured on the tube caps using a needle, followed by thorough mixing using a vortex mixer. The samples were then incubated in a 95 ℃ water bath for 40 minutes. After cooling under running water, the samples were centrifuged at 4,000 rpm for 10 minutes. Subsequently, 200 µL of supernatant was transferred to a 96-well plate, and the absorbance at 532 nm was measured. MDA concentration was calculated according to the standard formula.

Animal experiment

Five-week-old female nude mice (GemPharmatech Co., Ltd., Nanjing, China) were used for subcutaneous xenograft experiments. HCT-116 cells with stable expression of either METTL16 or control vectors were subcutaneously injected into the mice. After one week of tumor formation, the body weight of the mice was measured every three days, and tumor size was monitored. Meanwhile, the mice were administered astaxanthin solution (25 mg/mL) daily via oral gavage according to their body weight. The general condition of the mice was closely observed throughout the experiment. After 15 days, the mice were euthanized, and the tumor tissues were excised for size measurement and weighing. Experiments were performed under a project license (No. 129 [2022]) granted by the Animal Ethics Committee of Bengbu Medical University, in compliance with Bengbu Medical University guidelines for the care and use of animals.

Untargeted proteomics

Quantitative proteomic analysis was performed using data-independent acquisition (DIA) mode. Significantly differentially expressed proteins were screened based on the following criteria: fold change >1.5 (up-regulated) or <0.67 (down-regulated) with P value <0.05. Based on the acquired data, systematic bioinformatics analysis (protein functional annotation) was conducted for all identified proteins. Functional enrichment and protein-protein interaction network analysis were performed for all differentially expressed proteins. Additionally, volcano plot analysis, cluster heatmap analysis, and pathway enrichment analysis [Gene Ontology (GO), InterPro (IPR), and Kyoto Encyclopedia of Genes and Genomes (KEGG)] were carried out for the differentially expressed proteins.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 8.0; designed by GraphPad Software). Results were expressed as mean ± standard error of the mean (SEM). Statistical significance was set at P<0.05.

Results

METTL16 is identified as an oncogenic driver in CRC cells

Bioinformatic analysis of METTL16 expression in normal versus cancerous tissues revealed that METTL16 expression was significantly higher in CRC tissues compared to normal tissues (Figure 1A). Similarly, in paired samples, METTL16 expression was markedly elevated in CRC tissues relative to adjacent normal tissues (Figure 1B). Furthermore, higher expression levels of METTL16 protein are observed in colon cancer (Figure 1C). These findings underscore the clinical significance of METTL16 in CRC and highlight its potential as a therapeutic target for CRC.

Figure 1 METTL16 was identified as an oncogenic driver in colorectal cancer cells. (A) Expression of METTL16 in normal tissues and colorectal cancer tissues from the TCGA database. (B) Expression of METTL16 in paired samples from normal tissues and colorectal cancer tissues. (C) Protein expression levels of METTL16 in colorectal cancer. Compared with control: ***, P<0.001. COAD, colon adenocarcinoma; METTL16, methyltransferase 16; TCGA, The Cancer Genome Atlas; TPM, transcript per million.

Astaxanthin inhibits the growth and metastasis of CRC cells

To elucidate the effects of astaxanthin on CRC cells, we treated CRC cell lines with varying concentrations of astaxanthin. The results demonstrated that cell viability decreased in a dose-dependent manner with increasing astaxanthin concentrations (Figure 2A,2B), indicating that astaxanthin can inhibit CRC cell activity. Subsequent experiments were conducted using IC50 and IC75 concentrations. The wound healing assay revealed that astaxanthin treatment reduced the migratory capacity of colon cancer cells compared to the control group (Figure 2C,2D). Similarly, the Transwell assay showed that astaxanthin treatment decreased both migratory and invasive abilities of the cells relative to controls (Figure 2E,2F). Collectively, these results demonstrate that astaxanthin inhibits CRC growth and metastasis.

Figure 2 Astaxanthin inhibits colorectal cancer growth and metastasis in vitro. (A,B) CCK8 assay for changes in activity of colorectal cancer cells after addition of different concentrations of astaxanthin. (C,D) Wound healing assay to detect changes in wound healing ability of colorectal cancer cell lines after addition of different concentrations of astaxanthin (×100). (E,F) Transwell assay to detect changes in migration and invasion ability of colorectal cancer cell lines after addition of different concentrations of astaxanthin (stained with crystal violet, ×200). Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK8, Cell Counting Kit-8; OD, optical density.

To determine astaxanthin’s effects on tumor growth in vivo and its regulatory role on METTL16, we established subcutaneous xenograft tumors in 4–5-week-old female nude mice using METTL16-overexpressing HCT-116 cells. After tumor formation, the mice received daily astaxanthin treatment via oral gavage (Figure 3A). Finally, we found that after METTL16 overexpression, both the weight and volume of tumors increased. After astaxanthin treatment, the weight and volume of tumors significantly decreased (Figure 3B,3C). Simultaneously, Western blot experiments demonstrated that astaxanthin downregulated METTL16 expression (Figure 3D). All results indicate that astaxanthin plays an inhibitory role in the occurrence and development of CRC, and downregulates METTL16 expression.

Figure 3 Astaxanthin inhibits colorectal cell growth and metastasis in vivo. (A) Tumor size was observed by the naked eye in the EV group, the METTL16, and the METTL16 combined with astaxanthin group. (B) Scatterplot of tumor weight. (C) Scatterplot of tumor volume. (D) Western blot analysis of tumor tissue METTL16 as well as iron death-related proteins. Compared with control: ***, P<0.001. asta, astaxanthin; EV, empty vector; GPX4, glutathione peroxidase 4; METTL16, methyltransferase 16; XCT, cystine-glutamate transporter.

Astaxanthin induces ferroptosis in CRC cells

To investigate the role of astaxanthin in ferroptosis of CRC, we treated both HCT-116 and RKO cell lines with astaxanthin at IC50 and IC75 concentrations. Western blot analysis revealed that astaxanthin treatment downregulated the expression of ferroptosis-related inhibitory proteins GPX4 and cystine-glutamate transporter (XCT) (Figure 4A,4B). Compared with the control group, astaxanthin treatment significantly increased ROS levels in both cell lines (Figure 4C,4D). Additionally, we measured intracellular MDA and ferrous ion levels. The results demonstrated that astaxanthin treatment elevated both MDA and ferrous ion levels in HCT-116 and RKO cells compared to controls (Figure 4E,4F). These findings collectively indicate that astaxanthin induces ferroptosis in CRC cells, while simultaneously downregulating METTL16 expression.

Figure 4 Astaxanthin induces ferroptosis in colorectal cancer cells. (A,B) Western blot to detect the expression levels of METTL16 and iron death-related proteins after addition of different concentrations of astaxanthin in HCT-116 and RKO cell lines. (C,D) Lipid ROS levels after treatment of HCT-116, RKO with different concentrations of astaxanthin. (E) Ferrous ion content in HCT-116 and RKO after treatment with different concentrations of astaxanthin. (F) MDA content in HCT-116 and RKO after treatment with different concentrations of astaxanthin. Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001. EV, empty vector; GPX4, glutathione peroxidase 4; MDA, malondialdehyde; METTL16, methyltransferase 16; ROS, reactive oxygen species; XCT, cystine-glutamate transporter.

Astaxanthin promotes ferroptosis in CRC by regulating METTL16

To further investigate the relationship between METTL16, astaxanthin, and ferroptosis, we first knocked down and overexpressed METTL16 in both HCT-116 and RKO cell lines. Western blot analysis revealed that METTL16 knockdown reduced the expression levels of ferroptosis-related inhibitory proteins. While METTL16 overexpression increased the expression levels of ferroptosis-related inhibitory proteins (Figure 5A,5B). We then measured ROS levels following METTL16 modulation. The results showed that METTL16 knockdown increased ROS levels, while METTL16 overexpression decreased them (Figure 5C-5F). Additionally, we assessed MDA and ferrous ion levels in both cell lines. Compared to controls, METTL16 knockdown elevated MDA and ferrous ion levels, whereas METTL16 overexpression decreased these markers (Figure 5G-5J). Collectively, these data demonstrate that METTL16 inhibits ferroptosis in CRC cells. Overall, our experimental data demonstrate that METTL16 can inhibit ferroptosis in colon cancer cells.

Figure 5 METTL16 inhibits ferroptosis in colorectal cancer cells. (A) Western blot detection of changes in iron death-related proteins XCT and GPX4 in HCT-116 and RKO after knockdown of METTL16. (B) Changes in the levels of iron death-related proteins XCT and GPX4 in HCT-116 and RKO after overexpression of METTL16. (C,D) Lipid ROS levels in HCT-116 and RKO after knockdown of METTL16. (E,F) Lipid ROS levels in HCT-116 and RKO after overexpression of METTL16. (G) Ferrous ion content in HCT-116 and RKO after knockdown of METTL16. (H) Ferrous ion content in HCT-116 and RKO after overexpression of METTL16. (I) MDA content in HCT-116 and RKO after knockdown of METTL16. (J) MDA content in HCT-116 and RKO after overexpression of METTL16. Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001. EV, empty vector; GPX4, glutathione peroxidase 4; MDA, malondialdehyde; METTL16, methyltransferase 16; NC, negative control; ROS, reactive oxygen species; XCT, cystine-glutamate transporter.

As previously shown by Western blot results, astaxanthin can downregulate METTL16 expression levels. To further investigate the relationship between these factors and ferroptosis, we treated the two cell lines with the following conditions: METTL16 knockdown, astaxanthin treatment, and combined METTL16 knockdown with astaxanthin treatment. Subsequently, we detected the expression levels of ferroptosis-related proteins XCT and GPX4 by Western blot. The results showed that both METTL16 knockdown and astaxanthin treatment reduced XCT and GPX4 levels, and more notably, the combined METTL16 knockdown with astaxanthin treatment group showed a more pronounced decrease (Figure 6A,6B). Consistent with previous experiments, we also measured ROS levels. The results demonstrated that the METTL16 knockdown plus astaxanthin treatment group showed a more significant increase in ROS (Figure 6C,6D). Furthermore, we examined changes in ferrous ion and MDA levels. The results consistently showed that the METTL16 knockdown plus astaxanthin treatment group had more markedly elevated MDA and ferrous ion levels (Figure 6E,6F).

Figure 6 Astaxanthin can promote ferroptosis in colorectal cancer by modulating METTL16. (A,B) Western blot to detect changes in METTL16 as well as iron death-associated proteins XCT and GPX4 after knockdown of METTL16, addition of astaxanthin, and both treatments. (C,D) Changes in lipid ROS levels in HCT-116 and RKO after knockdown of METTL16, addition of astaxanthin, and both treatments. (E) Ferrous ions content in HCT-116 and RKO after knockdown of METTL16, addition of astaxanthin, and both treatments. (F) MDA levels in HCT-116 and RKO after knockdown of METTL16, addition of astaxanthin, and both treatments simultaneously. Compared with control: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. asta, astaxanthin; GPX4, glutathione peroxidase 4; MDA, malondialdehyde; METTL16, methyltransferase 16; NC, negative control; ROS, reactive oxygen species; XCT, cystine-glutamate transporter.

Proteomics reveals the molecular mechanism by which METTL16 and astaxanthin regulate ferroptosis in CRC cells

Under various factors in the tumor microenvironment, cancer cells activate immune evasion mechanisms and undergo metastasis. Our preliminary experimental results demonstrated that astaxanthin could inhibit the growth and migration capabilities of CRC cells both in vivo and in vitro. To further explore the mechanisms by which METTL16 and astaxanthin regulate ferroptosis. We compared protein expression changes between the “METTL16 overexpression group vs. control group” and the “METTL16 overexpression + astaxanthin group vs. METTL16 overexpression group”. We distinguished the effects of METTL16 acting alone versus those following astaxanthin intervention. This design facilitates the identification of downstream molecules directly regulated by METTL16 and further screens for key proteins associated with ferroptosis. Based on proteomics results, we have identified key proteins across multiple pathways that may be involved in regulating ferroptosis (Table S1). In the principal component analysis (PCA) plot, the three groups (normal control group, METTL16 group, and METTL16 + Ast group) were clearly separated into three distinct clusters (component 1: 19.68%; component 2: 15.4%) (Figure 7A). Venn diagram analysis revealed 11 common proteins among the empty vector (EV) group, METTL16 group, and METTL16 plus astaxanthin treatment group (Figure 7B). GO analysis showed that: in biological processes (BPs), the differentially expressed proteins were mainly enriched in DNA metabolic processes, lipid transport, amino acid metabolism, and lipoprotein metabolic processes; In cellular components (CCs), they were primarily enriched in the extracellular region, NF, nucleosomes, and chromatin; In molecular functions (MFs), they were enriched in metal ion binding, ion binding, iron ion binding, and DNA binding (Figure 7C). We compared protein changes between the METTL16 plus astaxanthin group and the control group. Proteomics analysis identified 643 differentially expressed proteins, including 282 upregulated proteins and 361 downregulated proteins (fold change ≥1.5, P<0.05) (Figure 7D). Furthermore, proteins affected by METTL16 plus astaxanthin treatment were primarily involved in the NF-kappaB signaling pathway and DNA replication (Figure 7E).

Figure 7 Proteomics reveals the molecular mechanism by which METTL16 and astaxanthin regulate ferroptosis in colorectal cancer cells. (A) PCA analysis of total samples. (B) Differential protein Wayne plots. (C) GO enrichment histogram. (D) Volcano plot of differential proteins in METTL16 co-added astaxanthin group vs. EV group. (E) KEGG-enriched bubble plot of differential proteins in METTL16 co-added astaxanthin group vs. EV group. asta, astaxanthin; BP, biological process; CC, cellular component; EV, empty vector; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; METTL16, methyltransferase 16; MF, molecular function; PC, principal component; PCA, principal component analysis.

Ferroptosis-related pathways

Through proteomics analysis, we found that transferrin receptor 1 (TFRC) was downregulated after METTL16 overexpression. This suggests that METTL16 may affect the mRNA stability or translation efficiency of this protein by regulating m6A modification, thereby inhibiting iron uptake, reducing intracellular free iron levels, and suppressing ferroptosis. Simultaneously, we observed that compared to the METTL16 overexpression group, the expression of TFRC was upregulated when METTL16 overexpression was combined with astaxanthin treatment. This led to increased intracellular free iron levels and promoted ferroptosis, indicating that astaxanthin may regulate METTL16 through TFRC to induce ferroptosis. Additionally, we found that solute carrier family 3 member 2 (SLC3A2) was upregulated after METTL16 overexpression. SLC3A2 can form a heterodimer with solute carrier family 3 member 11 (SLC7A11), and SLC7A11 is known to inhibit ferroptosis. Therefore, we speculate that METTL16 may suppress ferroptosis by regulating SLC3A2 expression. Furthermore, compared to the METTL16 overexpression group, SLC3A2 expression was downregulated when METTL16 overexpression was combined with astaxanthin treatment. This demonstrates that astaxanthin promotes ferroptosis occurrence.

Cell migration-related pathways

Proteomics data analysis revealed that Ras homolog family member B (RHOB) was upregulated following METTL16 overexpression. RHOB regulates actin contraction and stress fiber formation, thereby influencing migration velocity. We therefore hypothesize that METTL16 may promote CRC cell migration by modulating the mRNA stability of epithelial-mesenchymal transition (EMT) transcription factors (such as Snail and Twist1) through m6A modification. Additionally, transforming growth factor beta receptor 3 (TGFBR3) showed upregulated expression in the METTL16 overexpression group compared to the control group. Since transforming growth factor beta (TGF-β) can induce EMT and migration while promoting tumor invasion, we speculate that METTL16 may enhance the migratory and invasive capacities of CRC cells by regulating the mRNA stability of EMT transcription factors through m6A modification.

Mitochondrial energy metabolism-related pathways

The occurrence of ferroptosis is closely associated with alterations in energy metabolism. Through proteomics data analysis, we observed that the mitochondrial citrate transporter (SLC25A1) was downregulated following METTL16 overexpression but upregulated when METTL16 overexpression was combined with astaxanthin treatment. SLC25A1, located in the mitochondrial inner membrane, is responsible for transporting citrate from the mitochondrial matrix to the cytoplasm—a process crucial for energy metabolism, the tricarboxylic acid (TCA) cycle, and fatty acid synthesis. Furthermore, we found that mitochondrial adenosine triphosphate (ATP) synthase subunit F (ATP5MF) was downregulated after METTL16 overexpression and similarly upregulated when combined with astaxanthin treatment. ATP5MF serves as a core component of mitochondrial ATP synthase and catalyzes ATP generation. These results potentially reveal the mechanism by which astaxanthin combined with METTL16 overexpression may induce ferroptosis through regulation of mitochondrial lipid ROS production.

Oxidative stress-related pathways

Through analysis of the proteomics data, we found that compared to the METTL16 group, cytochrome P450 2S1 (CYP2S1) expression was upregulated after combined treatment with astaxanthin. CYP2S1 is a member of the cytochrome P450 superfamily and primarily functions in drug metabolism and tumor microenvironment regulation. Therefore, astaxanthin may promote ferroptosis in CRC cells by regulating CYP2S1 to enhance cellular oxidative stress levels. Additionally, we observed that compared to the control group, cytochrome P450 4F11 (CYP4F11) expression was downregulated following METTL16 overexpression. More notably, when METTL16 overexpression was combined with astaxanthin treatment, CYP4F11 expression showed upregulation compared to the METTL16-only group. CYP4F11, another cytochrome P450 superfamily member, is primarily involved in biological metabolic processes, including fatty acid metabolism, drug metabolism, and vitamin metabolism. Thus, we speculate that astaxanthin may regulate METTL16 through CYP4F11 to promote ferroptosis in CRC.

Fatty acid and amino acid metabolism-related protein pathways

Analysis of proteomics data revealed that, compared to the METTL16 group, stearoyl-CoA desaturase (SCD) expression was downregulated following combined treatment with astaxanthin. Previous studies have shown that SCD inhibition increases the membrane polyunsaturated fatty acid (PUFA)/saturated fatty acid (SFA) ratio and promotes ferroptosis. Therefore, we speculate that astaxanthin may promote ferroptosis by regulating SCD expression to further modulate METTL16 activity. Regarding amino acid metabolism, solute carrier family 7 member 1 (SLC7A1) expression was upregulated after METTL16 overexpression compared to the control group. SLC7A1 functions as a cystine/glutamate antiporter that regulates glutathione (GSH) synthesis. Inhibition of SLC7A1 leads to GSH depletion, a key trigger for ferroptosis. Thus, we hypothesize that METTL16 may inhibit ferroptosis in CRC by upregulating SLC7A1. Additionally, glutamate-cysteine ligase catalytic subunit (GCLC) was upregulated following METTL16 overexpression. GCLC serves as the rate-limiting enzyme in GSH synthesis. Increased GCLC expression directly suppresses GSH synthesis and promotes ferroptosis. We postulate that METTL16 may regulate ferroptosis in CRC cells by modulating GCLC mRNA stability through m6A modification.

METTL16 suppresses ferroptosis in CRC cells by downregulating 5'-aminolevulinate synthase 1 (ALAS1)

Proteomic analysis identified 11 overlapping proteins among the EV group, METTL16 group, and METTL16-plus-astaxanthin treatment group. Notably, ALAS1, the rate-limiting enzyme of heme biosynthesis whose expression correlates closely with intracellular iron levels, emerged as a ferroptosis-associated protein. Bioinformatics analysis revealed that ALAS1 is downregulated in CRC and exhibits tumor-suppressive effects (Figure 8A,8B), contrasting with METTL16’s oncogenic role. To further investigate the function of ALAS1 and its regulatory relationship with METTL16, we established cell models in CRC cells that overexpressed ALAS1 alone and co-expressed ALAS1 with METTL16. CCK-8 assay results showed that ALAS1 overexpression significantly reduced cell viability, while co-expression of METTL16 effectively reversed the ALAS1-induced decline in cell viability (Figure 8C,8D). Transwell assays further demonstrated that ALAS1 overexpression markedly suppressed the migration and invasion capabilities of CRC cells, whereas co-expression of METTL16 significantly attenuated the ALAS1-mediated inhibition of migration and invasion (Figure 8E,8F). Collectively, these findings indicate that METTL16 functionally antagonizes the suppression of malignant phenotypes in CRC cells mediated by ALAS1. Western blot analysis demonstrated that METTL16 downregulates ALAS1 expression, while astaxanthin treatment attenuates this effect (Figure 8G,8H). Furthermore, co-immunoprecipitation (co-IP) assays confirmed a specific physical interaction between METTL16 and ALAS1 in both HCT-116 and RKO cell lines (Figure 8I,8J). To further validate whether METTL16 inhibits ferroptosis by regulating ALAS1, we detected the expression levels of the key ferroptosis proteins GPX4 and XCT via Western blot. Results showed that ALAS1 overexpression significantly reduced GPX4 and XCT protein expression, while co-expression with METTL16 effectively reversed ALAS1’s inhibitory effect on both proteins (Figure 8K,8L). These findings further indicate that METTL16 maintains GPX4 and XCT expression levels by regulating ALAS1, thereby suppressing ferroptosis.

Figure 8 METTL16 inhibits ferroptosis in colorectal cancer cells by downregulating ALAS1. (A) Expression of ALAS1 in normal tissue and colorectal cancer tissue in the TCGA database. (B) Expression of ALAS1 in colorectal cancer based on individual cancer staging in the TCGA database. (C,D) CCK8 assay detected changes in the viability of colorectal cancer cells HCT-116 and RKO following co-overexpression of ALAS1 and METTL16. (E,F) The Transwell assay assessed changes in migration and invasion capabilities of colorectal cancer cells HCT-116 and RKO following co-overexpression of ALAS1 and METTL16 (stained with crystal violet, ×200). (G,H) Western blot analysis of changes in ALAS1 expression levels in HCT-116 and RKO cells upon overexpression of METTL16, as well as upon overexpression of METTL16 combined with astaxanthin addition. (I,J) Western blot analysis of changes in ALAS1 expression levels in HCT-116 and RKO cells following transfection with specific plasmids. (K,L) Western blot analysis was performed to detect changes in the expression of ferroptosis-related proteins GPX4 and XCT in colorectal cancer cells HCT-116 and RKO following co-overexpression of ALAS1 and METTL16. Compared with control: **, P<0.01; ***, P<0.001. ALAS1, 5'-aminolevulinate synthase 1; CCK8, Cell Counting Kit-8; COAD, colon adenocarcinoma; GPX4, glutathione peroxidase 4; METTL16, methyltransferase 16; TCGA, The Cancer Genome Atlas; XCT, cystine-glutamate transporter.

Discussion

CRC ranks as the fourth most commonly diagnosed cancer and the third leading cause of cancer-related deaths worldwide (1). In recent years, the incidence and mortality rates of CRC in China have shown an increasing trend. This phenomenon may result from the combined effects of multiple factors, including economic development, lifestyle changes, and dietary structure alterations (27,28). Surgery, chemotherapy, and radiotherapy have remained the standard treatments for CRC. However, these therapeutic approaches present significant drawbacks, often causing numerous side effects. Moreover, many patients still succumb to the disease due to its high recurrence rate, making it imperative to explore natural compounds and novel targets to provide new directions for CRC treatment. Previous studies have demonstrated that METTL16 plays crucial biological roles in CRC carcinogenesis and progression. Additionally, astaxanthin has been reported to possess multifaceted anti-tumor properties.

Numerous studies have demonstrated that astaxanthin exhibits anti-cancer or disease-alleviating effects in various cancers and diseases. For instance, astaxanthin demonstrates protective effects in rats with 5-fluorouracil (5-FU)-induced liver injury, mitigating hepatotoxicity and drug resistance during treatment (29). Astaxanthin-loaded nanoparticles exhibit remarkable stability and can inhibit the carcinogenesis and progression of gastric cancer (30). When breast cancer cells were treated with different concentrations of astaxanthin, elevated levels of caspase 3, Bax, and superoxide dismutase (SOD) enzymes were observed alongside reduced levels of Bcl-2 and MDA enzymes. Both in vitro and in vivo experiments confirmed that astaxanthin could serve as a potential therapeutic approach for breast cancer (31). Furthermore, astaxanthin can induce apoptosis in osteosarcoma cells, thereby restricting their development (32). In addition, research indicates that the optimal oral dosage of astaxanthin may be 8–12 mg/day in micellar formulation, taken with meals (33). Additionally, astaxanthin can enhance the cytotoxic effects of 5-FU by downregulating thymidine synthase (TYMS) through inhibition of the NF-κB/p38-MAPK pathway. Combination therapy increases tumor volume suppression rates while reducing intestinal epithelial damage (34). This suggests its potential for application in the treatment of CRC. These collective findings illustrate astaxanthin’s diverse therapeutic effects across multiple cancer types, providing valuable insights for our research on CRC.

Ferroptosis has garnered increasing attention in recent years. As an iron-dependent form of cell death characterized by lipid peroxidation accumulation and redox imbalance (35), ferroptosis holds potential significance for cancer treatment. In CRC, METTL16 functions as an oncogene while astaxanthin acts as a tumor growth inhibitor. We hypothesized an intimate relationship between these two factors and ferroptosis.

In our study, we demonstrated that METTL16 inhibits ferroptosis in CRC cells. Proteomic sequencing analysis suggested this effect might involve multiple pathways. For instance, in iron metabolism-related pathways, SLC3A2 showed upregulated expression following METTL16 overexpression. SLC3A2 forms a heterodimer with SLC7A11, which is known to suppress ferroptosis (36,37). We therefore speculate that METTL16 may inhibit ferroptosis by regulating SLC3A2 expression. Regarding mitochondrial energy metabolism, the SLC25A1 was downregulated after METTL16 overexpression. Located in the mitochondrial inner membrane, SLC25A1 mediates citrate transport from the mitochondrial matrix to the cytoplasm—a process crucial for energy metabolism (38,39). Our previous Western blot results confirmed that astaxanthin downregulates METTL16 expression, prompting further investigation into their relationship with ferroptosis. The findings revealed that astaxanthin not only induces ferroptosis in CRC cells but also modulates METTL16-mediated ferroptosis.

Astaxanthin is conventionally known to exert cytoprotective effects in cells. However, recent studies have revealed that under certain conditions, particularly within specific tumor microenvironments, astaxanthin may potentiate ferroptosis. Based on our proteomic sequencing data, we propose that this effect may be mediated through the following mechanisms. First, regarding iron metabolism, astaxanthin upregulates the expression of TFRC, consequently increasing intracellular labile iron levels and thereby inducing ferroptosis. Second, in mitochondrial energy metabolism: astaxanthin enhances the expression of ATP5MF. As an essential component of mitochondrial ATP synthase, ATP5MF catalyzes ATP production (40,41). Furthermore, in oxidative stress regulation, astaxanthin elevates the expression of CYP4F11, which is principally involved in fatty acid metabolism and drug metabolism (42). We postulate that astaxanthin may modulate ferroptosis through regulation of CYP4F11 expression. Finally, in fatty acid metabolism, our comparative analysis with the METTL16 group demonstrated that co-treatment with astaxanthin downregulates SCD expression. Established evidence indicates that SCD inhibition increases the membrane PUFA/SFA ratio and promotes ferroptosis. Therefore, we hypothesize that astaxanthin may facilitate ferroptosis by regulating SCD expression to subsequently influence METTL16 activity. Furthermore, although both in vitro experiments and animal model results in this study suggest that astaxanthin has the potential to downregulate METTL16 expression, the exploration of its mechanism remains primarily based on preclinical models due to the limited availability of paired clinical tumor tissue samples and constraints imposed by the study timeline. The universality and clinical relevance of this regulatory relationship in human CRC require systematic validation through rigorously designed prospective clinical trials. It should also be noted that the specific effects of astaxanthin may vary depending on cell type, tumor microenvironment, and combination with other therapeutic approaches. This ferroptosis-upregulating effect holds potential application value in cancer treatment, but further experimental validation and clinical research are required to precisely define its mechanism of action and optimize therapeutic strategies.

ALAS1 serves not only as the rate-limiting enzyme in heme biosynthesis but also plays a significant role in microRNA regulation (43). Existing studies have demonstrated that ALAS1 can inhibit drug-induced ferroptosis in normal cardiomyocytes; however, it should be noted that this effect may vary depending on cell type, drug characteristics, and tumor microenvironment conditions. Our research reveals that METTL16 likely suppresses ferroptosis in CRC cells by downregulating ALAS1, suggesting that ALAS1 may exert a pro-ferroptotic function in tumor contexts. Ferroptosis induction has emerged as a novel strategy to overcome chemotherapy resistance in CRC. Multiple preclinical studies have demonstrated that targeting GPX4 or System Xc⁻ effectively eliminates drug-resistant tumor cells. The METTL16-ALAS1 regulatory axis identified in this study provides a novel combination target for ferroptosis-directed therapy. Currently, these conclusions are primarily derived from in vitro CRC cell line models and have not been systematically validated in patient-derived organoids or clinical samples with different molecular subtypes or mutational backgrounds, potentially underestimating the impact of tumor heterogeneity on the ALAS1-METTL16 axis functionality. Further investigation is required to elucidate the precise mechanisms and functions involved.

In conclusion, our investigation of the METTL16-astaxanthin-ferroptosis axis reveals that astaxanthin may induce CRC ferroptosis through METTL16 regulation via multiple mechanisms. These findings provide novel research directions and therapeutic perspectives for clinical CRC treatment.

Conclusions

Our research reveals that METTL16 exerts a pro-cancer role in CRC and could suppress ferroptosis by downregulating ALAS1 expression. Additionally, we found that in vitro cell experiments, astaxanthin inhibits the viability, migration and invasion capabilities of CRC cells, and also suppresses tumor growth in mouse models. Furthermore, astaxanthin promotes ferroptosis by downregulating METTL16 expression. In summary, the discovered astaxanthin-METTL16-ALAS1 axis regulating ferroptosis may provide novel therapeutic directions for CRC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2002/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2002/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2002/prf

Funding: This work was supported by the National Natural Science Foundation of China (No. 32200047) and Anhui Provincial Department of Education Outstanding Young Teachers Development Program in Universities (No. YQZD2024028).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-2002/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. 129 [2022]) granted by the Animal Ethics Committee of Bengbu Medical University, in compliance with Bengbu Medical University guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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