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. 2025 Oct 13;36(11):2497–2506. doi: 10.1021/acs.bioconjchem.5c00459

Mitochondria-Targeting Antimicrobial Peptide (AMP) Regulating N6-Methyladenosine (m6A) Modification to Promote Colon Cancer Ferroptosis

Chenyu Li †,‡,§, Shuai Li †, Linlin Lv †, Yanwei Chen †, Shilei Yang †, Yan Lu ∥,*, Deshi Dong †,*
PMCID: PMC12637310  PMID: 41082909

Abstract

Colon cancer (COAD) is one of the common malignant tumors in the gastrointestinal tract; it is urgent to deeply study the mechanism of COAD and develop new therapeutic agents, which will provide new hope for improving the therapeutic efficacy and prolonging the survival of patients. Mitochondria are crucial organelles that play an important role in COAD, participating in cellular energy and material metabolism and playing a key role in the regulation of cell death, making mitochondria a potential target for COAD therapy. In this study, we designed an antimicrobial peptide (AMP) that can target tumor cells and act on mitochondria. The AMP is taken up by tumor cells and can achieve colocalization with mitochondria, reducing the mitochondrial membrane potential levels in tumor cells and inducing ferroptosis. The AMP affects N6-methyladenosine (m6A) methylation modification in cells and participates in the regulation of ferroptosis. During in vivo experiments on COAD, the AMP demonstrated a strong ability to inhibit tumor growth and good biosafety. Unlike peptide–drug conjugates that rely on toxin release, the synthetic AMP exerts direct targeted activity with improved biosafety and efficiency. The AMP effectively suppresses the development of COAD, providing a new reference method for the treatment of COAD.

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Introduction

Colon cancer (COAD) has become the most common malignant tumor in the gastrointestinal tract, accounting for 9.6% of all new cancer cases and causing about 900,000 deaths, with a mortality rate of 9.3% of all cancer deaths. , In the early stage of COAD, traditional surgical resection is still the treatment of choice, while for intermediate and advanced COAD, the combination of chemotherapy, targeted therapy and immunotherapy has become the mainstream treatment strategy. , However, relapse and drug resistance hamper the successful treatment of COAD, resulting in a relatively poor prognosis with a 5-year survival rate of approximately 60%. Therefore, it is crucial to delve into the molecular mechanisms associated with the onset and progression of COAD and explore new therapeutic targets. In recent years, a large number of studies have shown that mitochondria play an important role in the occurrence, development and treatment of COAD. − Mitochondria are vital organelles in the cell, extensively involved in the respiratory process and energy metabolism of the cell, which converts nutrients into adenosine triphosphate (ATP) required by the cell through oxidative phosphorylation, and is the main source of energy for the cell’s life activities. In addition to providing the energy needed by cells, they also play a key role in the regulation of cell death, which is especially important in the killing of tumor cells.

As the metabolic center of the cell, mitochondria, in addition to providing important precursors for biosynthesis, produce intermediate metabolites that can serve as substrates for epigenetic modifications and participate in processes such as methylation. , N6-methyladenosine (m6A) methylation is a common epigenetic modification of RNA, which affects the regulation of gene expression by modulating RNA stability, splicing, translational efficiency, translocation, and degradation. The metabolic state of mitochondria dynamically modulates m6A methylation modification, thereby regulating gene expression and influencing a range of biological functions. Following mitochondrial injury, changes in metabolite levels affect the maintenance of cellular homeostasis by regulating the epigenome and, consequently, the cellular homeostasis.

Regulating mitochondria to influence ferroptosis is an effective method for killing COAD cells. − Ferroptosis is a regulated cell death caused by iron-dependent lipid peroxidation at the cell membrane, and mitochondria play a key role in this process. , In mitochondria, iron overload generates large amounts of reactive oxygen species (ROS), and these ROS lead to lipid peroxidation in the cell membrane, which in turn disrupts membrane integrity and triggers ferroptosis. If the mitochondrial membrane is damaged it further inhibits oxidative phosphorylation, reduces ATP synthesis, and exacerbates ROS generation, creating a positive feedback loop that drives the ferroptosis process. , In addition, antioxidant enzymes in mitochondria, such as GPX4, usually play a protective role and reduce lipid peroxidation. The antioxidant defense system is unable to effectively scavenge ROS, which in turn exacerbates ferroptosis. Thus, damage to mitochondria is a driver of ferroptosis, providing a new target for the treatment of cancer.

Peptide drugs are widely used in the treatment of cancer and other diseases due to their high specificity, low toxicity and good biocompatibility. , However, they also face the challenges of stability, targeting and bioavailability. KLA peptide is a small molecule peptide that consists of the amino acid sequence KLAKLAKKLAKLAK. It is a mitochondria-toxic peptide with antitumor effects that induces intracellular mitochondrial dysfunction, thereby triggering cell death. , However, KLA peptides have some drawbacks, such as poor cell membrane penetration and low selectivity for tumor cells, which prevent them from being widely used in clinical practice. Generally, KLA is modified to achieve better therapeutic effects, and it has been demonstrated that the combination of KLA peptide with the cell membrane penetrating peptide TAT and the anticancer peptide HPRP-A1 enhances the cell membrane penetration ability of peptide drugs to achieve better anticancer effects. , Epithelial growth factor receptor (EGFR) is located on the surface of cell membrane and is an important receptor for cell proliferation and signaling. Therefore, EGFR receptor has become a key target in tumor therapy. , GE11 peptide (YHWYGYTPQNVI) is a binding peptide for EGFR receptor, which can bind specifically to tumor cells with high EGFR expression. In tumor therapy, GE11 peptide, by binding to EGFR, can mediate improved targeting of drugs and ensure that drugs act more effectively on tumor cells, thus enhancing therapeutic capacity and reducing toxic side effects on normal cells.

In this study, we designed an antimicrobial peptide (AMP) sequence, YHWYGYTPQNVI-GSGS-KLAKLAKKLAKLAK, which consists of KLA peptide and GE11 peptide together through a linker (GSGS). First, the GE11 peptide binds to the EGFR receptor on the surface of the tumor cell, ensuring that it specifically targets the tumor cell, thus effectively differentiating normal cells from tumor cells. Then, after entering the tumor cell through the cell membrane, the KLA peptide acts intracellularly to further damage the mitochondria, disrupting their function and ultimately inducing tumor cell death. This design, by combining targeting and cytotoxicity, improves therapeutic efficacy while reducing the killing of normal cells. As a synthetic Peptide drug, AMP contains no exogenous toxins. Compared to peptide–drug conjugate (PDC) drugs that require complex mechanisms to release their toxic effects, AMP offers higher biosafety and more direct, efficient targeted biological activity. We designed and synthesized AMP peptides to further explore the ferroptosis effects and molecular mechanisms triggered by its action on mitochondria while investigating its antitumor ability in COAD. This study will reveal the therapeutic potential of AMP peptide in COAD and provide new ideas for precision therapy of COAD, contributing to the development of targeted therapy and individualized treatment strategies.

Results

Bioinformatics Validation of the Value of Granules as a Therapeutic Target for COAD and AMP Synthesis

Differential gene screening of 119 mitochondria-related genes collected from the literature summary with the expression data of COAD in the TCGA database yielded a total of 50 up-regulated and 20 down-regulated genes mitochondria-related genes, and these 50 up-regulated genes were enumerated in the form of a heatmap (Figure S1). Functional enrichment analysis of the up-regulated differential genes showed that KEGG pathway enrichment analysis indicated that the differential genes mainly targeted the aminoacyl-tRNA biosynthesis pathway; bioprocess analysis indicated that the differential genes were mainly enriched in mitochondrial gene expression, RNA metabolism and modification; cellular component enrichment analysis indicated that the differential genes were mainly enriched in the inner mitochondrial membrane; and molecular function enrichment analysis indicated that the differential genes were significantly associated with the action on the RNA and tRNA catalytic activity (Figure S2). Given that bioprocess analyses indicate that differential genes mainly affect RNA metabolism and modification, with m6A methylation modification being the most common modification in RNA. To analyze whether mitochondria affect m6A modification and to verify the potential association between COAD and m6A methylation modification, we selected m6A-related genes (METTL3, IGF2BP1, IGF2BP2, IGF2BP3, YTHDF1, and YTHDF2) to perform bioprocess analysis. The results showed (Figure S3) that the expression levels of all these genes were higher in COAD tissues than in normal tissues. Taking the above bioinformatics analysis data together, it can be concluded that mitochondria play an important role in the process of COAD development and progression, and have a profound impact on the progression of COAD. Therefore, mitochondria can be regarded as an important target for the treatment of COAD.

The mitochondria-targeting peptide AMP is a multifunctional peptide chain consisting of two functional modules, which each have unique biological roles and work synergistically to enhance their effectiveness in targeting tumor therapy. (1) KLA peptide: a known mitochondrial toxicity peptide that can directly enter the cell and disrupt the mitochondrial function through interaction with mitochondria thereby killing the cell. (2) GE11 peptide: it can target the EGFR receptor highly expressed on the surface of the tumor, and play the role of targeted treatment of tumors. During the synthesis of AMP peptide, we also synthesized KLA peptide and purchased GE11 peptide to be used as a comparison experiment. Subsequently, it was purified by HPLC and identified using MS to confirm its molecular weight. The results showed (Figure A–D) that the molecular weight of the KLA peptide was 1524.1 Da and the molecular weight of the AMP peptide was 3332.88 Da. These data indicated that we successfully synthesized KLA peptide and AMP peptide with high purity.

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AMP synthesis and cellular uptake. (A) HPLC analysis of KLA; (B) HPLC analysis of AMP; (C) MS analysis of KLA; (D) MS analysis of AMP; (E) AMP uptake and mitochondrial colocalization experiments.

To assess the uptake of AMP peptides in vitro and the binding of AMP to mitochondria upon entry into cells, we performed cellular uptake and mitochondrial colocalization experiments. Mito-Tracker Green is a green fluorescent probe specifically designed for mitochondrial staining of living cells. Its staining process does not depend on the mitochondrial membrane potential and therefore can specifically label mitochondria. After treating the cells with AMP labeled by Cy3 fluorescence (Cy3-AMP) for 6 h, HCT116 cells were stained using Mito-Tracker Green working solution with nuclear staining working solution. The results showed (Figure E) that Cy3-AMP was successfully taken up in COAD cells and clearly overlapped with mitochondria. We observed that the red fluorescence of the AMP peptide showed good colocalization with the green fluorescence of the mitochondria, indicating that the AMP peptide was able to effectively enter the HCT116 cells and target the mitochondria. Line scan fluorescence intensity profiles indicated partial overlap between red and green signals, suggesting a moderate degree of colocalization. Thus, the targeting property of AMP peptide in tumor cells was proved to be able to exert its therapeutic effect through mitochondrial localization in cells.

Antitumor Effects of AMP at the Cellular Level

To evaluate the safety of AMP peptide and its ability to kill tumor cells, we performed CCK8 experiments to compare the toxic effects of KLA peptide, GE11 peptide and AMP peptide on HCT116 COAD cells. Normal cell line 293T cells were also treated to further verify the safety and selective killing effect of AMP peptide. In our experiments, we treated HCT116 cells with KLA, GE11 and AMP peptides respectively for 24 h, and used AMP peptides for the same treatment time on 293T cells. The results showed (Figure A,B) that KLA and GE11 peptides had an effect on the viability of COAD cells only at high concentrations for 24 h after treatment. In contrast (Figure C), AMP peptide significantly inhibited the growth of HCT116 cells at a concentration of 50 μM, showing an obvious killing effect. The cell viability gradually decreased with the increase of AMP concentration. For the normal cell line 293T (Figure D), the AMP peptide hardly affected its cell viability, indicating that the toxicity of AMP to normal cells was extremely low, showing a good safety profile. Additionally, we selected two COAD cell lines, SW480 and HT29. Results showed that at an AMP concentration of 50 μM, both cell lines exhibited strong cytotoxic effects (Figure E,F). In summary, AMP peptide showed a semilethal effect on HCT116 COAD cells at a concentration of 50 μM and possessed antitumor activity superior to that of KLA and GE11 peptides. Therefore, we decided to use a concentration of 50 μM for AMP peptide in subsequent experiments to further explore its ability in tumor therapy.

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Effect of AMP on COAD cells. (A) Relative cell viability of HCT116 cells treated with different concentrations of KLA; (B) Relative cell viability of HCT116 cells treated with different concentrations of GE11; (C) Relative cell viability of HCT116 cells treated with different concentrations of AMP; (D) Relative cell viability of 293T cells treated with different concentrations of AMP; (E) Relative cell viability of SW480 cells treated with different concentrations of AMP; (F) Relative cell viability of HT29 cells treated with different concentrations of AMP; (G) EdU cell proliferation assay; (H) Cell Death Assay; (I) Cell cloning assay.

In order to verify the effect of AMP on the growth ability of tumor cells, we conducted a series of experiments. The first one was the cell proliferation EdU experiment, in which the cells were divided into the control group, KLA group, GE11 group, and AMP group for comparison. The results showed (Figure E) that the AMP group significantly reduced the EdU positivity rate of HCT116 cells compared with other groups, indicating that AMP has a strong inhibitory effect on the proliferation of tumor cells. It can significantly reduce the proliferation ability of tumor cells, and then inhibit tumor growth. To further assess the role of AMP on the clone formation ability of tumor cells, we performed plate cloning experiments. The results showed (Figure F) that there was no significant difference between the KLA group and the GE11 group compared with the control group, which did not significantly affect the clone formation ability of HCT116 cells, which was significantly inhibited in the AMP-treated group. Finally, the cell live–dead staining experiment, this experiment used Calcein-AM and PI staining reagents to stain live and dead cells, respectively. Calcein can pass through the cell membrane of living cells under the action of Calcein-AM to produce strong green fluorescence, while PI cannot penetrate the cell membrane and is excluded from living cells, but can pass through the damaged cell membrane and then stain the cell nucleus. Therefore, it is possible to use both of them for staining and identification of live and dead cells. The results showed (Figure G) that there was no significant difference between the KLA and GE11 groups compared with the control group, while in the AMP group, the amount of live cells was significantly reduced and the amount of dead cells increased. In summary, AMP has a certain killing ability on HCT116 cells, which can inhibit the proliferation and cloning of COAD cells and promote cell death.

AMP Peptide Induces Cellular Ferroptosis

To further verify whether AMP disrupts mitochondria. We performed a mitochondrial membrane potential assay by means of a fluorescent probe for JC-1. JC-1 exists in normal cells mainly as a multimeric form in the mitochondrial matrix, generating red fluorescence and reflecting a high mitochondrial membrane potential. If the mitochondrial membrane potential is lowered by damage to the mitochondria, JC-1 is converted to the monomeric form and produces green fluorescence in the matrix. This change can be used to assess the alteration of mitochondrial membrane potential. The results showed (Figure A) that the red fluorescence was significantly weakened and the green fluorescence was significantly enhanced in the AMP group compared with the control group and the other groups, suggesting that AMP can target and damage the mitochondria of tumor cells.

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AMP induces ferroptosis in COAD cells. (A) Detection of mitochondrial membrane potential; (B) ROS content assay in HCT116 cells; (C) Kit to detect GSH content level in HCT116 cells; (D) Kit to detect MDA content level in HCT116 cells; (E) RT-qPCR to detect the expression level of ferroptosis-related genes; (F) Western Blot to detect the expression level of ferroptosis-related proteins.

Next, in order to verify whether mitochondrial damage induces ferroptosis in cells, we measured a series of ferroptosis-related indicators. First, ROS reactive oxygen species content was assayed, and the results showed (Figure B) that the intracellular reactive oxygen species content was significantly elevated in the AMP group compared with the control group.

JC-1 staining was employed to evaluate the changes in mitochondrial membrane potential by flow cytometry. Compared with the control group, both AMP and the positive control drug rotenone significantly reduced the red fluorescence intensity (Figure S4), indicating a loss of mitochondrial membrane potential, further suggesting its ability to disrupt mitochondrial function. Flow cytometric analysis using a ROS-sensitive fluorescent probe DCFH-DA demonstrated that both AMP and the positive control drug rotenone markedly increased green fluorescence intensity compared with the control group (Figure S4), indicating enhanced intracellular reactive oxygen species production. These findings suggest that AMP promotes ROS accumulation within cells, thereby contributing to oxidative stress.

Next, GSH and MDA kits were used to detect GSH and MDA levels in the cells of each group, respectively. The results showed (Figure C,D) that GSH levels were reduced in the KLA, GE11 and AMP groups compared to the control group and the most significant decrease was observed in the AMP group. Meanwhile, MDA content was also significantly increased in the AMP group compared to the control group. The above results demonstrated that the use of AMP caused significant oxidative damage to cancer cells.

Subsequently, seven genes related to ferroptosis (CISD1, ACSL4, PTGS2, FTH1, TP53, GPX4, and SLC7A11) were selected for RT-qPCR experiments. The results are shown in the figure (Figure E), in which ACSL4, PTGS2 and TP53 genes showed a significant upward trend, and CISD1, FTH1, GPX4 and SLC7A11 genes showed a significant downward trend. The expression of all these genes was consistent with the hallmark of ferroptosis. For further verification, four proteins related to ferroptosis were selected for Western Blot (Figure F). The above experimental results indicated that AMP treatment disrupted the mitochondria of HCT116 cells and caused oxidative damage, which in turn induced the cells to undergo ferroptosis.

A series of experiments were further conducted to verify ferroptosis in HCT116 cells. First, we assessed the level of lipid peroxidation in HCT116 cells using the BODIPY 581/591 C11 fluorescent probe (Figure S5A). This probe can reflect lipid peroxidation levels in living cells through changes in its fluorescence spectrumits reduced form (581/591 nm) emits primarily red fluorescence, while its oxidized form (488/510 nm) emits primarily green fluorescence. The ratio of their intensities allows quantification of intracellular lipid peroxidation levels. The results showed that compared with the control group, the AMP-treated group exhibited the weakest red fluorescence and the strongest green fluorescence, suggesting diminished cellular antioxidant capacity and a significant increase in lipid peroxidation levels.

To verify the ferroptosis-specific nature of the observed cell death, we further performed a ferroptosis rescue experiment using the ferroptosis-specific inhibitor Ferrostatin-1 (Figure S5B). Detection with the Calcein-AM/PI viability staining assay showed that compared with the AMP-treated group, the inhibitor-treated group had a significantly reduced percentage of PI-positive dead cells, with a marked attenuation of cell death signals. These results effectively confirm the mechanism of ferroptosis in tumor cells.

To further characterize the ferroptosis-related cellular damage phenotype, we subsequently evaluated membrane integrity (Figure S5C). PI fluorescent staining revealed that the AMP-treated group displayed the strongest red fluorescence compared with the solvent control group. Combined with the aforementioned lipid peroxidation results, these findings suggest that AMP treatment causes disruption of the cell membrane phospholipid bilayer and membrane rupture, and this form of cell death may be associated with ferroptosis.

AMP Regulates m6A Methylation Modification in COAD Cells

In order to gain a preliminary understanding of the molecular mechanism by which the mitochondria-targeting peptide AMP exerts antitumor effects, we performed transcriptome sequencing of COAD cells treated with AMP. The results showed (Figure A) that a total of 162 genes were up-regulated and 149 genes were down-regulated after AMP treatment. Subsequently, the above differential genes were subjected to functional enrichment analysis, in which the results of GO enrichment analysis showed (Figure B) that AMP was significantly associated with biological processes such as systemic growth and cellular development process, etc. The results of KEGG enrichment analysis showed (Figure C) that AMP affected the metabolic pathway, MAPK, and other important tumor-related signaling pathways. This suggests that AMP may affect cellular metabolic processes and exert antitumor effects by damaging mitochondria.

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Effect of AMP on m6A methylation modifications. (A) Volcano plot of differentially expressed genes in the AMP group compared with the control group; (B) GO bioprocess enrichment analysis of the different genes; (C) KEGG pathway functional enrichment analysis of the different genes; (D) Dot Blot Experiment; (E) Expression of m6A methylation modification-related proteins.

In order to confirm whether AMP affects m6A modification in HCT116 cells, we divided HCT116 cells into NC and AMP groups and performed Dot Blot experiments, which showed (Figure D) that the m6A expression level in the AMP group decreased. From this, it can be preliminarily proved that AMP inhibits m6A methylation modification occurring in COAD cells. Western Blot experiments were performed on m6A methylation-related proteins, and the results were as shown (Figure E), only METTL3 and IGF2BP3 proteins showed a significant decreasing trend, however, IGF2BP2, YTHDF1, YTHDF2 and YTHDF3 proteins did not show any significant trend compared with the control group. Here we concluded that AMP was inhibiting HCT116 cells from undergoing m6A methylation modification by affecting METTL3 and IGF2BP3 proteins, and therefore METTL3 and IGF2BP3 proteins were also selected for the subsequent experiments.

AMP Promotes Ferroptosis in Relation to the Regulation of m6A Methylation

To verify the correlation between AMP promotes ferroptosis and m6A methylation, we subsequently selected two key genes for ferroptosis, FTH1 and GPX4, for MeRIP experiments. The direct binding interactions of m6A proteins with FTH1 mRNA and GPX4 mRNA were detected by MeRIP-qPCR, and the results showed that both FTH1 and GPX4 underwent m6A modification in HCT116 cells, and AMP inhibited the level of m6A modification in both genes compared with the control group (Figure A,B).

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Correlation between m6A methylation modifications and ferroptosis. (A) MeRIP-qPCR detection of direct binding interaction of m6A protein with FTH1 mRNA; (B) MeRIP-qPCR detection of direct binding interaction of m6A protein with GPX4 mRNA; (C, D) RIP-qPCR detection of direct binding interactions of METTL3 and IGF2BP3 proteins with FTH1 mRNA; (E, F) RIP-qPCR detection of direct binding interactions of IGF2BP3 proteins with GPX4 mRNA.

To further confirm that AMP regulates FTH1 and GPX4 genes by affecting METTL3, we performed RIP experiments. The direct binding interactions of METTL3 protein with FTH1 mRNA and GPX4 mRNA were detected by RIP-qPCR. The results showed (Figure C,D) that both FTH1 mRNA and GPX4 mRNA bound to METTL3 protein in COAD cells compared to IgG, and both mRNAs bound to this protein were decreased in the AMP group compared to the control group. Also to confirm whether IGF2BP3 is a recognition protein for m6A methylation of FTH1 and GPX4 genes, we also detected the direct binding interactions of IGF2BP3 protein with FTH1 mRNA and GPX4 mRNA by RIP-qPCR. The results showed (Figure E,F) that both FTH1 mRNA and GPX4 mRNA bound to IGF2BP3 protein in COAD cells, and both mRNAs bound to IGF2BP3 protein were significantly lower in the AMP group compared to the control group. IGF2BP3 acts as a reading protein for m6A, which recognizes m6A modifications on mRNAs to promote the target mRNAs’ stability as a way to regulate their expression. AMP reduces the level of m6A modifications in ferroptosis -related genes, which may lead to down-regulation of the expression of related genes. Therefore, we believe that there is a certain association between ferroptosis and m6A methylation modification in COAD, and AMP can be involved in the regulation of ferroptosis in colon tumor cells by inhibiting the occurrence of m6A methylation modification in COAD cells and affecting the expression of intracellular ferroptosis -related genes.

In Vivo Antitumor Effects of AMP

To evaluate the antitumor effect of AMP in vivo, we randomly divided 9 hormonal mice into 3 groups and injected saline, KLA (2.4 mg/kg), and AMP (5.2 mg/kg) via tail vein, in which KLA and AMP were equimolar concentrations. The results showed (Figure A–D) that after four consecutive treatments, tumor growth was inhibited in both the KLA-treated group and the AMP-treated group compared to the control group, with more significant inhibition of tumor growth in the AMP-treated group. During the treatment period, we observed and recorded the body weight changes of the mice in each group, and the data showed (Figure E) that the body weight of the mice in each group did not change significantly during this period, indicating that there was no significant toxicity of both KLA and AMP.

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In vivo antitumor effects of AMP. (A) Holotype mice in physiological saline, KLA, and AMP treatment groups; (B) images of mouse tumors; (C) differences in tumor mass between groups; (D) tumor growth curves; (E) changes in body weight of mice in each group; (F) HE staining of tumors, hearts, livers, spleens, lungs, and kidneys of holotype mice.

In order to further investigate the effect of the drug on tumor tissues and toxicity to major organs, HE staining of tumors, heart, liver, spleen, lungs, and kidneys of three groups of mice was performed for histopathological observation. The results showed (Figure F) that the tumor tissues were necrotic after treatment with KLA and AMP with the stronger effect of AMP. Meanwhile, no significant damage was observed in other organs in each group. Additionally, the results of ex vivo imaging showed (Figure S6) that 24 h after drug injection, KLA still did not accumulate at the tumor site, while AMP still accumulated. The results demonstrated that AMP had more obvious tumor targeting ability in vivo. The above results proved that AMP has high biosafety in vivo, almost no damage to normal tissues and organs, and has strong antitumor ability.

Discussion

There has been some progress in the treatment of COAD, but there are still problems such as treatment resistance, limitations of precision therapy, and lack of new drugs. The heterogeneity of COAD and the complexity of gene mutations make precision therapy challenging, and existing targeted drugs fail to cover all patients. , In addition, despite the efficacy of existing drugs, they have greater side effects and drug resistance, and there is an urgent need to develop new targeted drugs. Therefore, in-depth research on the mechanisms of COAD and the development of new therapeutic targets and drugs are key to solving these bottlenecks. In this study we designed and synthesized AMP peptides and verified the uptake ability and mitochondrial colocalization of AMP in colon tumor cells using HCT116 cells, and AMP showed good antitumor effects in vitro. In addition, we performed xenograft experiments on nude mice to investigate the in vivo targeting ability and antitumor activity of AMP. The results show that AMP can effectively target tumors and exert good therapeutic effects in vivo, and AMP also has a good biosafety, which can effectively avoid the side effects of drugs.

Recent studies have shown that the abnormal function of mitochondria is closely related to tumorigenesis and progression, and therefore, specific targeting of cancer cell mitochondria has become one of the important directions in cancer therapy. − KLA peptide is a peptide molecule with the ability to target mitochondria and disrupt their function, but its ability to target tumor cells is weak and it is unable to distinguish between normal and tumor cells. Since the EGFR receptor is overexpressed in many tumor cells, the GE11 peptide was found to specifically target the EGFR receptor. Therefore, we designed a novel peptide AMP designed to simultaneously target tumor cells and act on mitochondria. The experimental results showed that AMP was able to effectively enter tumor cells, exert mitochondrial toxicity, induce cells to undergo ferroptosis, and reduce the level of m6A methylation modification, thus significantly inhibiting cancer progression. Further mechanistic studies showed that there is a correlation between ferroptosis and m6A methylation modification. This finding provides new targets and strategies for tumor therapy. ferroptosis has attracted much attention as a new mode of cell death discovered in recent years, and is considered to be a factor that inhibits tumor cell growth. The main manifestations of ferroptosis are elevated intracellular ROS and MDA content, decreased GSH content, and decreased mitochondrial membrane potential. − In this study, HCT116 cells were selected as the in vitro research object, and through a series of experiments, it was found that the GSH content of AMP-treated HCT116 cells was reduced, the MDA and ROS content were both significantly increased, and the mitochondrial membrane potential was also significantly reduced. The expression levels of four ferroptosis-related proteins, mainly GPX4 and FTH1, and seven ferroptosis-related genes were verified at the molecular level, confirming that AMP peptides can damage mitochondria and induce ferroptosis in cells. In addition, it has been suggested that mitochondrial damage has epigenetic effects, especially on the regulation of m6A methylation modification. , Therefore, we verified that the m6A methylation modification level of cells was significantly reduced after AMP treatment by Dot Blot experiments. Subsequently, the experiments further analyzed the expression of METTL3 and IGF2BP3 proteins and their involvement in the regulation of ferroptosis in AMP-treated cells, and the results showed that these two proteins regulate the expression of ferroptosis genes through their involvement in the modification of m6A and thus (Figure ). Mitochondrial injury may generate metabolic products that regulate the expression of METTL3, whereas the expression of YTHDF family proteins appears not to be directly influenced by these metabolites. This observation underscores the intricate interplay between cellular metabolism and epigenetic regulation, suggesting that the precise mechanisms underlying this crosstalk remain to be elucidated in future studies. Whether AMP affects ferroptosis by damaging mitochondria and triggering an increase in ROS, or by regulating ferroptosis-related genes through m6A methylation modification, or by a combination of the two, needs to be further investigated. On the other hand, the structure of AMP needs to be optimized and modified for further research and clinical translation of peptide drugs. Improving the half-life and enhancing the antitumor activity of peptide drugs are the issues that need to be addressed in the future.

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Schematic representation of AMP action on COAD cells.

In conclusion, we designed and synthesized AMP, a peptide drug that targets tumor cells and acts on mitochondria. This drug induces ferroptosis in cells by damaging mitochondria and reduces m6A methylation modification, thereby effectively inhibiting COAD progression. This study proposed a potential link between ferroptosis and m6A methylation modification, which provides a new strategy and research idea for COAD treatment. In the future, we will further optimize the drug properties of AMP to improve its application in clinical treatment and explore its potential application in other types of tumor therapy.

Supplementary Material

bc5c00459_si_001.pdf (800.3KB, pdf)

Acknowledgments

Thanks to Figdraw for providing some elements for schematic diagrams.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00459.

  • Materials and methods and Figures S1–S6 (PDF)

#.

C.L. and S.L. contributed equally to this paper and are the co-first-authors. All listed authors have contributed significantly, directly, and intellectually to the work and have approved it for publication.

The studies involving animal subjects were reviewed and approved by the Ethical Committee of Dalian Medical University.

The authors declare no competing financial interest.

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