Mo-CBP3-PepIII Demonstrates Selective Cytotoxicity in AGS Cells

The synthetic peptides used for the cytotoxicity analysis were Mo-CBP3-PepI, Mo-CBP3-PepIII, RcAlb-PepI, and RcAlb-PepII, evaluated against four gastric adenocarcinoma cell lines (AGP-01, ACP-02, ACP-03, and AGS) and one non-tumor gastric cell line (MNP-01) (Table 1 and supplementary Fig. 1). The IC₅₀ values were determined from the dose–response curve, corresponding to the concentrations required to reduce cell viability by 50% in the evaluated cell lines (Table 1 and supplementary Fig. 1).

Table 1 IC₅₀ values (μg/mL) and 95% confidence intervals (CI 95%) obtained after 72 h of treatment with the synthetic peptides in gastric tumor and non-tumor cell lines

The Mo-CBP3-PepIII showed the highest cytotoxicity and selectivity against the AGS cell line, with an IC₅₀ of 50.36 µg/mL. In the non-cancerous gastric cell line MNP-01, the Mo-CBP3-PepIII showed one of the highest IC₅₀ values (125.1 µg/mL), indicating selectivity toward AGS cells. Based on these values, the Selectivity Index (SI) was calculated at 2.48, indicating that this molecule is moderately selective. According to Abd El-Sattar et al. (2021), molecules with SI values greater than 2 are considered to display moderate selectivity, and values below 2 are considered low selective for tumor cells over healthy cells. Studies have shown that the SI values for doxorubicin and cisplatin are 0.05 and 1.8, respectively, demonstrating their low selectivity (Garbuz et al. 2025). Furthermore, the literature considers SI values below 2 as indicative of low selectivity (Abd El-Sattar et al., 2021).

This selectivity of Mo-CBP3-PepIII is even more relevant than that of conventional chemotherapeutics such as doxorubicin and cisplatin, which exhibit low selectivity and high toxicity, leading to several side effects and hindering treatment (El-Sayyad et al. 2009; Ibrahim et al. 2022). Due to these limitations, new molecules have been investigated to achieve greater selectivity and cytotoxicity against tumor cells while causing fewer adverse effects.

To allow comparison with the positive control, whose IC₅₀ values are in µM, the IC₅₀ values obtained for Mo-CBP3-PepIII were converted, yielding 72.7 µM for AGS and 180.6 µM for MNP-01. The literature also reports peptides that exhibit selective cytotoxicity toward tumor cells. For example, the combination of Enterocin A and Colicin E1 showed an IC₅₀ of 60.41 µg/mL and induced apoptosis in AGS cells (Lu et al., 2016; Fathizadeh et al. 2021). Although the study did not provide the molecular weight necessary for conversion to µM, comparison with the IC₅₀ of Mo-CBP3-PepIII (50.36 µg/mL) is still possible; values within this range are considered cytotoxic to tumor cells and sufficient to trigger severe cellular damage.

Another example is lactoferrin B, which displays an IC₅₀ of 64 µM and also induces apoptosis in AGS cells (Pan et al. 2013). The PepGAT exhibits an IC₅₀ of 125.4 µM after 72 h in colorectal cancer cells by interacting with the plasma membrane, forming pores, and inducing apoptosis (Mesquita et al., 2024). Likewise, the Ple-a peptide shows an IC₅₀ of 197 µM and demonstrates selectivity for tumor cells, while exhibiting low toxicity toward 3T3 fibroblasts (Hsu et al. 2022). Taken together, these data and literature evidence support the therapeutic potential of peptides as anticancer agents and highlight Mo-CBP3-PepIII for its selective cytotoxicity.

Mo-CBP3-PepIII Induces Membrane Permeability and Pore Formation on AGS Cells

To investigate the impact of Mo-CBP3-PepIII on the cell membrane, the PI uptake assay, which indicates increased membrane permeability, and FITC–Dextran, which indicates pore formation, were employed (Fig. 1). The PI assay showed increased fluorescence intensity in cells treated with Mo-CBP3-PepIII compared to the DMSO control, indicating altered membrane permeability (Fig. 1A). The FITC–Dextran assay also showed increased fluorescence in Mo-CBP3-PepIII-treated cells. FITC–Dextran is a high–molecular–weight marker (6 kDa) that indicates the formation of membrane pores and the passage of molecules at least 6 kDa (Fig. 1B). These results demonstrate that the peptide compromises the highly selective membrane barrier, allowing the passage of molecules that would usually be impermeable.

Fig. 1The alternative text for this image may have been generated using AI.

Mechanism of action analysis of Mo-CBP3-PepIII using PI and FITC–Dextran. A Increased uptake of propidium iodide (PI) after treatment with Mo-CBP3-PepIII indicates membrane damage. B internalization of FITC–dextran (6 kDa) in treated cells suggests increased membrane permeability. Fluorescence images: data are presented as mean ± standard deviation. Significant differences: *P < 0.05; ***P < 0.001

A previous study by our group demonstrated that peptides derived from the Mo-CBP3 protein, including Mo-CBP3-PepIII, exhibit cytotoxicity by targeting the cell membrane. The investigation was performed on the membranes of pathogenic microorganisms such as Staphylococcus aureus and Candida parapsilosis, where the peptides showed selectivity, as they did not affect normal human cells and also induced membrane pore formation. A bioinformatic analysis revealed that the Mo-CBP3-PepIII peptide is cell-penetrating (Oliveira et al. 2019).

Given this mechanism of action, already reported in microorganisms, interest arose in investigating these peptides for anticancer activity due to their selective toxicity. In this context, the PepGAT and PepKAA peptides were evaluated using the same markers in colorectal cancer cell lines, revealing similar mechanisms of membrane permeabilization, pore formation, and apoptosis induction (Mesquita et al., 2024). Tumor cell resistance represents a significant challenge; therefore, it has been observed that the physical damage caused by peptides may overcome these resistance mechanisms, since pore formation in the membrane is their initial action and does not depend on intracellular pathways (Jahanafrooz and Mokhtarzadeh 2022; Mesquita et al., 2024).

The ppM1 peptide formed irreversible pores of 4–8 kDa in several tumor cells, leading to the release of intracellular lactate dehydrogenase (LDH) and to immunogenic cell death (LI et al. 2022a, b, c). Another pore-forming peptide, melittin, initially increases membrane permeability and induces pore formation, leading to ion efflux, membrane disorder, increased oxidative stress, and induction of ferroptosis (Yu et al. 2023). Altogether, these findings suggest that Mo-CBP3-PepIII has strong potential as a therapeutic peptide for gastric cancer.

Mo-CBP3-PepIII Induces Oxidative Stress in AGS Cells

The evaluation of reactive oxygen species (ROS) production was performed using the fluorescent probe DCFH-DA. An increase in fluorescence was observed, indicating the accumulation of hydrogen peroxide (H₂O₂), a hallmark of oxidative stress (LAM et al. 2020). Fluorescence microscopy images showed higher green fluorescent intensity in cells treated with Mo-CBP3-PepIII (Fig. 2).

Fig. 2The alternative text for this image may have been generated using AI.

Evaluation of reactive oxygen species (ROS) production in AGS cells treated with Mo-CBP3-PepIII. Quantification of fluorescence intensity showed a significant increase in ROS production in the treated group compared to the DMSO control. Representative fluorescence microscopy images demonstrate higher green fluorescence emission in treated cells, indicating increased oxidative stress. Scale bar: 100 µm. Data are presented as mean ± standard deviation

The accumulation of ROS may be associated with its cytotoxic potential, as high levels lead to lipid, protein, and DNA damage, favoring apoptosis and cellular necrosis (Trachootham et al. 2009). These data complement the PI and FITC-dextran results, showing that Mo-CBP3-PepIII, in addition to destabilizing the membrane and forming pores, also induces ROS accumulation, thereby disrupting intracellular redox homeostasis (Lam et al. 2020). This mechanism is already well known for conventional chemotherapeutic agents, such as irinotecan, which, through ROS accumulation, induces autophagy and apoptosis in gastric cells (Zhu et al. 2020). The same occurs with 5-fluorouracil (5-FU), which reduces histone methyltransferase G9a, thereby increasing ROS production and triggering apoptosis via the ROS/JNK pathway (Lou et al. 2019).

Similarly, as observed for Mo-CBP3-PepIII, many peptides reported in the literature act by accumulating ROS, thereby inducing cell death, as seen with chemotherapeutic agents. One example is PepGAT and PepKAA, which induced accumulated ROS production, promoting cell death in colorectal cancer cell lines (Mesquita et al., 2024). The same mechanism is observed in other peptides, such as the synthetic peptide Moricin, which induced apoptosis by accumulating ROS in the breast cancer cell line MDA-MB-231. This increased ROS production may lead to higher p53 protein expression, a tumor suppressor gene that triggers cell death (Ahmad et al. 2023). These data demonstrate the imbalance caused by Mo-CBP3-PepIII in the AGS cell line and suggest that this mechanism may induce cell death; however, further in-depth investigation is required to confirm this.

Morphological Alterations Induced by Mo-CBP3-PepIII in Gastric Cancer Cells

Scanning electron microscopy (SEM) was used to evaluate the morphological changes induced by the treatment in AGS cells. Images at different magnifications were obtained, allowing visualization of the general pattern of cell clustering and details of the cell surface (Fig. 3). In the control cells (Fig. 3A), a group of spherical, compact, and highly organized cells is observed. In cells treated with Mo-CBP3-PepIII, morphological alterations were observed, including increased cell surface roughness (Fig. 3B and D), depressions in the cellular structure (Fig. 3C), and irregular contours, indicating compromised membrane integrity and pore formation.

Fig. 3The alternative text for this image may have been generated using AI.

Scanning electron microscopy (SEM) images of the analyzed samples. A Untreated cells at 3,000 × magnification, showing cell clustering with spherical morphology, smooth surface, and compact organization. B Treated cells at 8,000 × magnification, showing a rough surface and well-defined contours, suggesting the onset of morphological alterations. C Treated cell visualized at 10,000 × , with evident morphological changes, presence of associated smaller structures, and irregular contours. D Treated cells at 10,000 × magnification, showing surface roughness and possible impairment of structural integrity

These results reinforce the literature, which shows that several bioactive peptides can induce cell death through membrane damage (Oliveira et al. 2019; Branco et al. 2022; Mesquita et al., 2024). A study analyzed, by scanning electron microscopy, two peptides, PepGAT and PepKAA, in colorectal cancer and observed changes in cell shape, surface roughness, cellular deformation, pore formation, and membrane damage, similar to those observed in the microscopy analysis of Mo-CBP3-PepIII (Mesquita et al., 2024).

The same type of analysis was performed with the peptide melittin in AGS, COLO205, and HCT-15 cell lines, where pore formation in the membrane, granularity, and irregular membrane shape were observed, indicating loss of membrane integrity (Soliman et al. 2019). Another peptide that exhibited the same mechanism was magainin II in bladder cancer, in which pores and membrane rupture were visualized by SEM (Lehmann et al., 2006). The SEM data obtained for the Mo-CBP3-PepIII peptide reinforce its cytotoxicity, as evidenced by these morphological characteristics, and indicate strong potential for evaluation as an anti–gastric cancer peptide.

Proteomic Analysis Reveals Changes in the Proteomic Profile of AGS Cells

To better understand the effect of Mo-CBP3-PepIII on AGS cells, a proteomic analysis was performed to assess changes in their protein profile. In the Venn diagram (Fig. 4A), alterations in the protein profile are already evident, separated into two groups: the control group (CN) with 1970 detected proteins and the Mo-CBP3-PepIII-treated group with 1532 proteins. At the intersection of the two groups, 1189 shared proteins were identified. Among the proteins exclusively detected in the CN group, 781 proteins were identified that were no longer detected after treatment with Mo-CBP3-PepIII, demonstrating that the treatment reduced several proteins below the instrument’s detection limit, which may be associated with cell growth.

Fig. 4The alternative text for this image may have been generated using AI.

Proteomic profile analysis and GO pathways associated with differentially expressed proteins. A Venn diagram showing the shared and exclusive proteins between the control and treated groups. B Volcano plot represents the proteins with differential expression between the analyzed groups. C Functional enrichment (GO) analysis of upregulated proteins between the control and treated groups, distributed into the categories: biological process (BP), cellular component (CC), and molecular function (MF). D Functional enrichment (GO) analysis of downregulated proteins between the control and treated groups, distributed into the categories: BP, CC, and MF. E A protein–protein interaction network was constructed using the STRING database with the differentially expressed proteins between the analyzed groups

The proteins detected exclusively in the treated group numbered 343, indicating that their activation occurred after exposure to the peptide. This result suggests that the Mo-CBP3-PepIII peptide induces cellular reprogramming in the AGS cell line, possibly altering several internal processes in cancer cells. The volcano plot was constructed from log₂ fold change (log₂ FC) values obtained from PatternLab analyses. The colors represent differential protein regulation between the groups (Fig. 4B). Proteins highlighted in red are upregulated, indicating increased protein accumulation after treatment with Mo-CBP3-PepIII. Proteins indicated in blue were downregulated, suggesting reduced protein accumulation after treatment with Mo-CBP3-PepIII. Proteins shown in gray did not differ significantly between the groups (Fig. 4B).

In the functional enrichment (GO) analysis of upregulated proteins, the biological processes (BP) in the control and Mo-CBP3-PepIII-treated groups were related to chromatin structure and DNA organization. In the cellular component (CC), they were associated with nuclear structures and cell–extracellular matrix interactions, and in the molecular functions (MF), they were linked to DNA interaction and structural regulation of chromatin (Fig. 4C).

In the GO analysis of downregulated proteins, biological processes (BP) were associated with maintaining homeostasis and controlling cellular stress. In the cellular component (CC), associations with cell adhesion and the cytoskeleton were observed, and the molecular functions (MF) were related to protein quality control, protein folding, and regulation of translation (Fig. 4D). Finally, in Fig. 4E, the STRING analysis shows the protein–protein interaction network, which revealed differentially expressed genes after treatment forming connected modules, indicating their involvement in important cellular pathways.

Impact of Mo-CBP3-PepIII on AGS proteome

The proteins identified in both groups, considered shared proteins, were subjected to fold change analysis to assess their abundance. According to the criteria established in a reference study (Branco et al. 2022), proteins with a fold change ≥ 1.5 (p < 0.05) were identified as increased in abundance after exposure to Mo-CBP3-PepIII and are represented in red in Fig. 4B. Proteins with a fold change ≤ 0.5 (p < 0.05) showed reduced abundance and are indicated in blue. Proteins shown in gray correspond to fold change values between 0.5 and 1.5 (p < 0.05) and were considered as having no significant change in their accumulation (Fig. 4B).

In light of these results, the identified proteins were grouped by biological function to facilitate understanding of this discussion. The defined categories included proteins involved in energy metabolism, proliferation, the cytoskeleton, and stress response. Among the proteins associated with energy metabolism, pyruvate kinase (PKM), enolase 1 (ENO1), and thioredoxin (TXN) showed a significant reduction in abundance, with fold changes of − 5.41, − 16.93, and − 5.39, respectively, and their downregulation directly affects cell growth and adaptation to the hostile tumor microenvironment.

Cells with elevated PKM levels exhibit a higher proliferative rate, a characteristic of neoplastic and embryonic cells. According to Dai et al. (2022), the overexpression of this protein is associated with increased malignancy and the Warburg effect, both of which are essential for gastric cancer survival. On the other hand, PKM inhibition reduces tumor growth and decreases the Warburg effect (Lv et al. 2021).

The oncoprotein ENO1 exhibits multifunctional characteristics. When expressed on the cell surface, it contributes to tumor invasion and metastasis; when intracellular, it promotes glucose metabolism, sustaining cell proliferation, contributing to chemoresistance, and reducing the migration of T lymphocytes, B lymphocytes, and natural killer cells into the tumor microenvironment. In addition, ENO1 activates pathways associated with cell survival and angiogenesis via PI3K/AKT and HIF-1α signaling (Li et al. 2024). The inhibition of ENO1 has been shown to reduce glucose consumption and increase sensitivity to the chemotherapeutic agent cisplatin (Yang et al. 2020a, b). In light of this, the peptide Mo-CBP3-PepIII reduced this protein by 16-fold, suggesting interference with metabolic pathways important for gastric cancer progression.

Complementarily, the TXN protein was also reduced after treatment with Mo-CBP3-PepIII. Its overexpression has been associated with unfavorable clinical outcomes and increased tumor aggressiveness in patients with gastric cancer (Shang et al. 2019). Studies performed in the AGS cell line have shown that downregulation of TXN inhibits proliferation, migration, and invasion, and decreases activation of the AKT pathway, which plays a fundamental role in tumor growth (Sun et al. 2025).

This pattern of downregulation was also observed in proteins involved in cell proliferation and survival, such as intracellular chloride channel 1 (CLIC1), eukaryotic translation initiation factor 4A1 (EIF4A1), and heterogeneous nuclear ribonucleoprotein C (HNRNPC), whose fold changes were − 20.05, − 15.59, and − 22.10, respectively. These proteins reinforce the potential of Mo-CBP3-PepIII to modulate central targets in tumor biology, since they exhibited the greatest reductions in abundance.

The CLIC1 protein promotes the progression of gastric cancer and is associated with cell migration, invasion, and survival. Its reduction after treatment with Mo-CBP3-PepIII was approximately 20-fold, indicating a strong capacity to restrict gastric tumor aggressiveness. This result is supported by the literature, as inhibition of CLIC1 has been shown to regulate integrins and signaling pathways, including PI3K/AKT and MAPK/ERK (Li et al. 2018). High expression of CLIC1 induces resistance to cisplatin and vincristine in AGS cells, whereas its downregulation can restore this sensitivity (Nong et al. 2024).

Another protein that showed downregulation was EIF4A1, with approximately a 15-fold decrease; it plays an important role in protein translation. In gastric cancer, its overexpression is associated with lymph node metastasis, unfavorable prognosis, and poor tumor differentiation (Gao et al. 2020). Studies show that EIF4A1 stimulates migration, invasion, and epithelial–mesenchymal transition (EMT) through the modulation of E-cadherin and N-cadherin (Gao et al. 2020). Additionally, EIF4A1 has been shown to promote increased VEGFA expression and NF-κB activation, contributing to remodeling of the tumor microenvironment and favoring angiogenesis (Zhu et al. 2025). In this context, the suppression of EIF4A1 has been associated with reduced tumor progression (Wei et al. 2019).

HNRNPC stood out for presenting the greatest reduction in abundance, with a fold change of − 22.10. When highly upregulated, this protein increases aerobic glycolysis, promotes lactate accumulation, and induces resistance to ferroptosis and to the chemotherapeutic agent oxaliplatin (Huang et al., 2015; Yang et al. 2025). In light of this evidence from the literature, it is evident that the reduction caused by Mo-CBP3-PepIII affects mechanisms crucial to cancer progression, particularly chemotherapy resistance, which remains a major challenge in the treatment of gastric cancer.

Another small group consisted of proteins linked to cytoskeletal remodeling, such as beta-actin (ACTB), gamma-actin 1 (ACTG1), and alpha-actin 1 (ACTA1), all with a fold change of − 4.88. When ACTB is inhibited, reduced migration and invasion were observed through the NF-κB and Wnt/β-catenin pathways (Gu et al. 2021; Saha et al. 2025). ACTG1 is strongly associated with EMT and proliferation (Gu et al., 2020; Wang et al. 2024), while ACTA1, little explored in gastric cancer, shows relevant evidence in other tumor types (Suresh et al., 2021).

Finally, another group of reduced proteins was those related to stress response and cell signaling, such as HSP90B1 and HSPA1B, with fold changes of − 7.03 and − 9.31, respectively. These proteins act as chaperones and are involved in tumor cell adaptation to the microenvironment, contributing to immune evasion and resistance to chemotherapeutic agents (Huang et al. 2024; Teng et al., 2018). Thus, treatment with Mo-CBP3-PepIII decreased the levels of these chaperones, potentially increasing tumor cell vulnerability and making them more susceptible to ROS-mediated mechanisms, as observed in this study (Fig. 5).

Fig. 5The alternative text for this image may have been generated using AI.

Functional analysis and network of proteins exclusive to the negative control group. A Functional enrichment analysis (GO) of proteins exclusive to the control: biological process (BP), cellular component (CC), and cellular function (MF). B The protein–protein interaction network was generated using the STRING platform to generate the interaction of proteins exclusive to the control

The HSP90B1 protein participates in protein folding and stabilization, and is important for maintaining tumor homeostasis. When overexpressed, it favors cellular metabolism, cell cycle control, and activation of immune checkpoint genes, promoting immune escape and tumor progression. Its inhibition suppresses tumor proliferation and PD-L1 expression (Huang et al. 2024). HSPA1B, an isoform of the HSP70 family, is mainly associated with resistance to chemotherapeutic agents, is more highly expressed in tumor cells, and is associated with poorer survival and with modulation of immune cells that regulate the tumor microenvironment (He and Wang 2019). The reduction observed with Mo-CBP3-PepIII suggests that the peptide may interfere with chemoresistance mechanisms, as reported by Teng et al. (2018), who found that HSP70 inhibition decreased the resistance of gastric tumor cells to apatinib.

Conversely, an interesting result was observed: proteins with tumor-suppressor functions showed greater abundance after treatment with Mo-CBP3-PepIII. This result suggests modulation of Mo-CBP3-PepIII in the expression of tumor suppressor proteins, although its impact on the studied model warrants further investigation. Among them, the mitochondrial ATP-Mg/phosphate transporter 1 (SLC25A24) showed an 18-fold increase in abundance. Initially described as pro-tumor, recent studies in colorectal cancer suggest that it may also exert a suppressive effect, reducing proliferation and metastasis through the cGMP/PKG1 pathway, thereby promoting mitochondrial apoptosis (Gao et al. 2025). Although no evidence of its role in gastric cancer was found, the results of this study, combined with data from the literature, suggest that SLC25A24 may act similarly to what has been seen in colorectal cancer, reinforcing the need for further investigation.

Another protein found was heterogeneous nuclear ribonucleoprotein K (HNRNPK), which increased by 2.36. HNRNPK suppresses tumor proliferation, colony formation in vitro, and in vivo growth when overexpressed via the p53/p21/CCND1 axis. It showed a negative association with angiogenesis, TGF-β, and activation of the Hedgehog pathway, which are important pathways for proliferation (Huang et al. 2017). These results point to hypotheses about possible suppressive protein effects induced by the peptide.

Mo-CBP3-PepIII Suppresses Control-Exclusive Proteins Associated With Tumor Progression

As seen in the Venn diagram (Fig. 4A), 1970 proteins were identified in untreated AGS cells, while 781 proteins were detected in the exclusive cells. This s