Novel Property Cytotoxicity and Mechanism of Food Preservative Brevilaterins against Human Gastric Cancer Cells

Brevilaterins, antimicrobial peptides produced by Brevibacillus laterosporus, are regarded as excellent food preservatives and are popular as antimicrobial applications. Recent research has uncovered their potent cytotoxic effects against diverse cancer cells, thereby underscoring the pressing need for more extensive and intensive investigations into this use. In this study, we explored their novel function in inducing cytotoxicity to cancer cells and systematically investigated the mechanism of action of Brevilaterin B/C (BB/BC) in vivo. Proliferation, membrane permeability, and apoptotic rate were evaluated using CCK-8 assay, LDH assay, and Annexin V-FITC/PI kits. ROS levels and mitochondrial membrane potential were detected using the fluorescent probe DCFH-DA and JC-1. Our results demonstrated that both BB and BC at concentrations of 4–6 µg/mL significantly inhibited the proliferation and migration of gastric cancer cells BGC-823. Treatment with 4 µg/mL of BB/BC rapidly increased LDH levels in the supernatant of BGC-823 cells, leading to further investigation of the mechanism of apoptosis. We found that the apoptotic rate of BGC-823 cells significantly increased upon treatment with BB/BC, demonstrating their potent induction of apoptosis. BB/BC-induced ROS production in BGC-823 cells impaired their growth and induced apoptosis, indicating a close association between apoptosis and ROS elevation. Additionally, JC-1 aggregates rapidly accumulated after treatment with 4 µg/mL of BB/BC, suggesting changes in mitochondrial membrane potential and early apoptosis. Taken together, our findings revealed that BB and BC exhibit significant anticancer effects against gastric cancer cells, highlighting the promising potential of Brevilaterins as anticancer agents.


Introduction
Cancer is a leading cause of human mortality, with gastric cancer ranking among the top five fatal cancers [1]. Despite recent advances in diagnosis and treatment, complete surgical resection remains the sole approach for early-stage gastric cancer management [2]. However, the five-year survival rate post-surgery remains low at approximately 20%, highlighting significant safety concerns [3]. Therefore, a multimodal approach combining radiotherapy, chemotherapy, novel targeted drugs, or a combination of these has emerged as a means of enhancing the efficacy of gastric cancer treatment. The search for a novel, safe, and effective gastric cancer therapeutic agent or adjuvant has gradually become a primary approach to adjuvant therapy. Studies have revealed that antimicrobial peptides (AMPs) constitute a crucial element of the innate immune defense system with selective inhibitory effects on specific tumor cells, offering renewed hope for the development of potent and innovative anticancer drugs [4,5].
AMPs, commonly used as food preservatives, and also known as Bacteriocins, are a diverse group of peptides secreted by bacteria with the primary function of inhibiting pathogenic microorganisms. These cationic peptides achieve their bactericidal effect by binding to the microbial cell membrane [6]. Recent research has demonstrated that AMPs W0: Original scratch width of cells without treatment with Brevilaterin. W1: Final scratch width of cells treated with Brevilaterin.

Effect of Brevilaterins on the Membrane Permeability
To investigate membrane permeability, the levels of LDH released from cells were measured. Logarithmic cells (1 × 10 5 cells/mL) were cultured for 24 h and subsequently treated with varying concentrations of Brevilaterins (0, 4, 6, 8 µg/mL) for an additional 24 h. Following treatment, the amount of LDH released into the supernatant of the treated cells was measured using an LDH Assay Kit.

Impact of Brevilaterins on the Ultrastructural Features of Cancer Cells
BGC-823 gastric cancer cells in the logarithmic growth phase were cultured at a density of 3 × 10 5 cells/mL in a 10 cm cell culture dish containing 10 mL of medium for 24 h to allow adhesion. Subsequently, varying concentrations of antibacterial peptides (0 and 6 µg/mL) were added, and after 24 h, the cells were washed twice with PBS and treated with trypsin for 2 min. Complete culture medium was added, and the cells were transferred to a centrifuge tube, washed with PBS, and fixed with 2.5% electron microscopy fixation solution for 24 h at 4 • C. Ultrathin sections of the cells were prepared by dehydrating them with ethanol and acetone, embedding them in epoxy resin, and staining them with uranyl acetate and lead citrate for contrast. Transmission electron microscopy was used to visualize the ultrastructure of the cells.

Effect of Brevilaterins on the Apoptosis of Cancer Cells
To assess apoptosis in BGC-823, we measured apoptotic rate, changes in ROS levels, and mitochondrial membrane potential using the Annexin V-FITC/PI Kit, DCFH-DA, and JC-1 fluorescent probes, respectively. The methods employed were previously described by Chen et al. [14]. Specifically, logarithmically growing cells (2 × 10 5 cells/mL) were cultured for 24 h, and then treated with various concentrations of Brevilaterins (0, 4, 6, 8 µg/mL) for an additional 24 h. To determine apoptotic rate, 100 µL of treated cells were mixed with 5 µL of Annexin V-FITC for 10 min, followed by the addition of 5 µL PI. The final mixture was diluted with 400 µL Binding buffer and quantitatively analyzed using a BD C6 flow cytometer. To measure ROS levels, treated cells were incubated with DCFH-DA for 20 min, washed twice with PBS (1×) buffer, harvested by trypsin digestion, and analyzed using a BD C6 flow cytometer. Finally, mitochondrial membrane potential was detected using the JC-1 fluorescent probe. Specifically, 500 µL of JC-1 and treated cells were mixed and incubated for 15 min. The cells were then harvested after trypsin digestion and analyzed using a BD C6 flow cytometer.

Statistical Analysis
Data were expressed as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) in SPSS version 10.0 for Windows (SPSS Inc., Chicago, IL, USA) to determine any significant differences. The significant differences (p < 0.05) were observed and further analyzed using the least significant difference multiple-range test.

Inhibition of BB/BC on the Proliferation of Gastric Cancer Cells
In this study, we aimed to investigate the impact of BB/BC, a spectrum anticancer peptide [14,15], on gastric cancer cells. Our results showed that BB/BC strongly inhibited cell proliferation in a dose-dependent manner ( Figure 1). Specifically, as the concentration increased from 2.0 µg/mL to 16.0 µg/mL, the viability of gastric cancer cells BGC-823 significantly decreased, whereas the corresponding gastric epithelial cells GES-1 were only slightly affected. Notably, 8.0 µg/mL of BB/BC strongly inhibited the viability of cancer cells BGC-823 while having minimal effect on its corresponding human normal gastric epithelial cells, GES-1, suggesting their potential for selective inhibition of human cells. Moreover, the inhibitory effects of BB/BC on BGC-823 were further elucidated through migration assays ( Figure 2). Compared to the control group, the antimicrobial peptides BB and BC exhibited a concentration-dependent enhancement in their inhibition effect on cell migration, leading to a decreased rate of cell scratch closure during the identical treatment duration. Specifically, treatment with 4.0 µg/mL and 6.0 µg/mL of BB for 48 h resulted in significantly decreased scratch-healing rates of BGC-823, at 58.91% and 51.53%, respectively, in contrast to the control group's rate of 94.57% ( Figure 2B). Similarly, treatment with BC also showed a similar trend with scratch-healing rates of 55.45% and 39.46%, respectively ( Figure 2C).
The final mixture was diluted with 400 µL Binding buffer and quantitatively analyzed using a BD C6 flow cytometer. To measure ROS levels, treated cells were incubated with DCFH-DA for 20 min, washed twice with PBS (1×) buffer, harvested by trypsin digestion, and analyzed using a BD C6 flow cytometer. Finally, mitochondrial membrane potential was detected using the JC-1 fluorescent probe. Specifically, 500 µL of JC-1 and treated cells were mixed and incubated for 15 min. The cells were then harvested after trypsin digestion and analyzed using a BD C6 flow cytometer.

Statistical Analysis
Data were expressed as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) in SPSS version 10.0 for Windows (SPSS Inc., Chicago, Illinois, USA) to determine any significant differences. The significant differences (p < 0.05) were observed and further analyzed using the least significant difference multiple-range test.

Inhibition of BB/BC on the Proliferation of Gastric Cancer Cells
In this study, we aimed to investigate the impact of BB/BC, a spectrum anticancer peptide [14,15], on gastric cancer cells. Our results showed that BB/BC strongly inhibited cell proliferation in a dose-dependent manner ( Figure 1). Specifically, as the concentration increased from 2.0 µg/mL to 16.0 µg/mL, the viability of gastric cancer cells BGC-823 significantly decreased, whereas the corresponding gastric epithelial cells GES-1 were only slightly affected. Notably, 8.0 µg/mL of BB/BC strongly inhibited the viability of cancer cells BGC-823 while having minimal effect on its corresponding human normal gastric epithelial cells, GES-1, suggesting their potential for selective inhibition of human cells. Moreover, the inhibitory effects of BB/BC on BGC-823 were further elucidated through migration assays ( Figure 2). Compared to the control group, the antimicrobial peptides BB and BC exhibited a concentration-dependent enhancement in their inhibition effect on cell migration, leading to a decreased rate of cell scratch closure during the identical treatment duration. Specifically, treatment with 4.0 µg/mL and 6.0 µg/mL of BB for 48 h resulted in significantly decreased scratch-healing rates of BGC-823, at 58.91% and 51.53%, respectively, in contrast to the control group's rate of 94.57% ( Figure 2B). Similarly, treatment with BC also showed a similar trend with scratch-healing rates of 55.45% and 39.46%, respectively ( Figure 2C).  In recent years, the potential of AMPs as a source of effective cancer treatment has garnered significant attention. Originally identified as a component of the human innate defense system, AMPs have been extensively investigated for their therapeutic applications against various diseases, including cancer. Numerous studies have demonstrated that AMPs exhibit potent anticancer properties that surpass those of currently available therapies, such as the AMP named Bogorol B-JX that is secreted by the strain of Br. laterosporus, which efficiently inhibits proliferation of the human histiocytic lymphoma cell line U-937 [11,[16][17][18]. Our previous research has also identified Brevilaterins, produced by Br. laterosporus S62-9, as a promising new class of molecules with potent anticancer activity. Indeed, Brevilaterins could display variable inhibition towards 27 cancer cells in a dose-dependent manner [14,15]. In this study, we specifically focused on the inhibitory effects of Brevilaterins on gastric cancer cells and described their properties as potential anticancer agents. This marks the first report that Brevilaterins demonstrate activity against gastric cancer cells, thereby further underscoring their potential as a novel class of AMPs for cancer therapy. In recent years, the potential of AMPs as a source of effective cancer treatm garnered significant attention. Originally identified as a component of the human defense system, AMPs have been extensively investigated for their therapeutic a tions against various diseases, including cancer. Numerous studies have demon that AMPs exhibit potent anticancer properties that surpass those of currently a therapies, such as the AMP named Bogorol B-JX that is secreted by the strain of B osporus, which efficiently inhibits proliferation of the human histiocytic lympho line U-937 [11,[16][17][18]. Our previous research has also identified Brevilaterins, pr by Br. laterosporus S62-9, as a promising new class of molecules with potent an activity. Indeed, Brevilaterins could display variable inhibition towards 27 cancer a dose-dependent manner [14,15]. In this study, we specifically focused on the in effects of Brevilaterins on gastric cancer cells and described their properties as p

Effect on the Changes in Gastric Cancer Cell Morphology of BB/BC
In comparison to the control group that was not treated with antimicrobial peptides (AMPs), both BB and BC demonstrated a significant increase in LDH levels in BGC-823 cells ( Figure 3). The LDH release rate in BGC-823 cells increased from 113.41% to 153.78% as the concentration of BB increased from 4 µg/mL to 8 µg/mL. Similarly, the LDH release rate in BGC-823 cells increased from 103.68% to 163.8% at the same concentrations of BC. These results indicate that BC is more effective than BB in damaging the cell membrane of gastric cancer cells BGC-823, which may be due to their differences in their amino acid sequence and molecular structure. In instances of cell apoptosis or necrosis, various cellular mechanisms are disrupted, leading to changes in the structural and functional integrity of the cell membrane. As a consequence, the cell membrane may become compromised, resulting in the release of intracellular contents, such as enzymes and other cytoplasmic proteins, into the extracellular environment. This phenomenon is often associated with an increase in the activity of certain cellular enzymes, such as LDH, which is released into the culture medium and serves as a typical marker of cell membrane damage and the extent of cell death [19,20]. Herein, the LDH release from BGC-823 cells confirms that both BB and BC induce apoptosis, leading to cell death. This precise feedback was also observed in human promyelocytic leukemia HL-60 cells after treatment with the AMP cecropin A [21].
In comparison to the control group that was not treated with antimicrobial peptides (AMPs), both BB and BC demonstrated a significant increase in LDH levels in BGC-823 cells (Figure 3). The LDH release rate in BGC-823 cells increased from 113.41% to 153.78% as the concentration of BB increased from 4 µg/mL to 8 µg/mL. Similarly, the LDH release rate in BGC-823 cells increased from 103.68% to 163.8% at the same concentrations of BC. These results indicate that BC is more effective than BB in damaging the cell membrane of gastric cancer cells BGC-823, which may be due to their differences in their amino acid sequence and molecular structure. In instances of cell apoptosis or necrosis, various cellular mechanisms are disrupted, leading to changes in the structural and functional integrity of the cell membrane. As a consequence, the cell membrane may become compromised, resulting in the release of intracellular contents, such as enzymes and other cytoplasmic proteins, into the extracellular environment. This phenomenon is often associated with an increase in the activity of certain cellular enzymes, such as LDH, which is released into the culture medium and serves as a typical marker of cell membrane damage and the extent of cell death [19,20]. Herein, the LDH release from BGC-823 cells confirms that both BB and BC induce apoptosis, leading to cell death. This precise feedback was also observed in human promyelocytic leukemia HL-60 cells after treatment with the AMP cecropin A [21]. Morphological alterations are the cornerstone for identifying apoptosis and represent the most straightforward approach, featuring chromatin condensation, fragmented DNA, swollen mitochondria, or the emergence of apoptotic bodies. The cellular ultrastructure can provide a more detailed observation of the fine changes in cell structure following AMP treatment, thus corroborating the influence of AMPs on cell morphology. Observation of the effects of BB and BC on the ultrastructure of BGC-823 cells was conducted using transmission electron microscopy (TEM) (Figure 4). BGC-823 cells without treatment displayed abundant and intact organelles, evenly distributed chromosomes in the nucleus, and clear nucleoli. The overall cell morphology was intact, with a rich population of microvilli on the cell surface. However, after treatment with 6 µg/mL of BB, the number of Morphological alterations are the cornerstone for identifying apoptosis and represent the most straightforward approach, featuring chromatin condensation, fragmented DNA, swollen mitochondria, or the emergence of apoptotic bodies. The cellular ultrastructure can provide a more detailed observation of the fine changes in cell structure following AMP treatment, thus corroborating the influence of AMPs on cell morphology. Observation of the effects of BB and BC on the ultrastructure of BGC-823 cells was conducted using transmission electron microscopy (TEM) (Figure 4). BGC-823 cells without treatment displayed abundant and intact organelles, evenly distributed chromosomes in the nucleus, and clear nucleoli. The overall cell morphology was intact, with a rich population of microvilli on the cell surface. However, after treatment with 6 µg/mL of BB, the number of microvilli on the cell surface decreased, and numerous autophagic vacuoles appeared inside the cells. The nuclear membrane displayed a "wrinkled" appearance, with heterochromatin in the nucleus increasing and forming chunky edges near the nuclear membrane. Some organelles exhibited a loss of their internal structure and assumed a "linear" configuration, displaying typical features of apoptosis. Treatment with 6 µg/mL of BC resulted in evident swelling of both the cell volume and nucleus of BGC-823 cells, with many vacuoles appearing. Internal structures such as mitochondrial cristae disappeared. These findings suggest that 6 µg/mL of BB and BC can modulate the ultrastructure of BGC-823 cells.
The cell nucleus, being a critical organelle in cells, plays a pivotal role in cell growth and metabolism. Upon the occurrence of cell apoptosis, notable changes in cell morphology ensue, including a reduction in cell volume, the aggregation of chromatin, and the formation of apoptotic bodies. To investigate whether the inhibitory effect of AMPs on BGC-823 cells is caused by cell apoptosis, Hoechst 33258 fluorescence--a blue fluorescent dye that can pass through the cell membrane, and commonly used for detecting cell apoptosis--staining was used to observe the morphological changes of the cell nucleus, as depicted in Figure 5. In the control group, chromatin is uniformly distributed, and the cell nuclei exhibits uniform light blue staining. However, exposure of BGC-823 cells to different concentrations of BB and BC results in nuclear condensation and fragmentation of chromatin, which are hallmarks of apoptosis. Moreover, as the concentration of BB and BC increases, there is a decrease in the overall number of cell nuclei, accompanied by a relative increase in the number of cells with densely stained chromatin. Notably, exposure to 6 µg/mL and 8 µg/mL of BC leads to a significant reduction in the overall number of cells compared to the control group, with most cell nuclei becoming deformed, fragmented, and clustering together. These changes in cell nuclei morphology clearly demonstrate that both BB and BC can induce apoptosis in BGC-823 cells, with BC being more potent in inducing apoptosis compared to BB. microvilli on the cell surface decreased, and numerous autophagic vacuoles appeared inside the cells. The nuclear membrane displayed a "wrinkled" appearance, with heterochromatin in the nucleus increasing and forming chunky edges near the nuclear membrane. Some organelles exhibited a loss of their internal structure and assumed a "linear" configuration, displaying typical features of apoptosis. Treatment with 6 µg/mL of BC resulted in evident swelling of both the cell volume and nucleus of BGC-823 cells, with many vacuoles appearing. Internal structures such as mitochondrial cristae disappeared. These findings suggest that 6 µg/mL of BB and BC can modulate the ultrastructure of BGC-823 cells. The cell nucleus, being a critical organelle in cells, plays a pivotal role in cell growth and metabolism. Upon the occurrence of cell apoptosis, notable changes in cell morphology ensue, including a reduction in cell volume, the aggregation of chromatin, and the formation of apoptotic bodies. To investigate whether the inhibitory effect of AMPs on BGC-823 cells is caused by cell apoptosis, Hoechst 33258 fluorescence--a blue fluorescent dye that can pass through the cell membrane, and commonly used for detecting cell apoptosis--staining was used to observe the morphological changes of the cell nucleus, as depicted in Figure 5. In the control group, chromatin is uniformly distributed, and the cell nuclei exhibits uniform light blue staining. However, exposure of BGC-823 cells to different concentrations of BB and BC results in nuclear condensation and fragmentation of chromatin, which are hallmarks of apoptosis. Moreover, as the concentration of BB and BC increases, there is a decrease in the overall number of cell nuclei, accompanied by a relative increase in the number of cells with densely stained chromatin. Notably, exposure to 6 µg/mL and 8 µg/mL of BC leads to a significant reduction in the overall number of cells compared to the control group, with most cell nuclei becoming deformed, fragmented, and clustering together. These changes in cell nuclei morphology clearly demonstrate that both BB and BC can induce apoptosis in BGC-823 cells, with BC being more potent in inducing apoptosis compared to BB.

Further Study on the Apoptotic Mechanism of BB/BC towards Gastric Cancer Cells
Building on previous research findings, we hypothesized that BB/BC induces apoptosis in gastric cancer cells. In this study, we aimed to investigate the mechanism behind this process by examining the apoptotic rate, ROS levels, and mitochondrial membrane potential as assessment indicators.
As depicted in Figure 6A, treatment with BB/BC resulted in a gradual increase in apoptotic rates of BGC-823 cells. At concentrations of 4 µg/mL, 6 µg/mL, and 8 µg/mL of BB, approximately 18.68%, 22.25%, and 36.69% of cancer cells underwent apoptosis, respectively ( Figure 6B). Similarly, treatment with BC at the same concentrations led to even higher apoptotic rates of BGC-823 cells, reaching 28.31%, 31.46%, and 51.61%, respectively, indicating that BC exhibited stronger inhibitory effects ( Figure 6B). These findings suggest that BB and BC may induce apoptosis in BGC-823 cells, highlighting their potential as effective anticancer agents. Apoptosis is a crucial physiological process that regulates the balance between cell division and cell death to maintain cellular homeostasis and prevent uncontrolled proliferation of cancer cells [22]. During the early stages of cellular apoptosis, a critical event is the translocation of phosphatidylserine from the inner to the outer leaflet of the cell membrane. This phenomenon can be detected using Annexin V-FITC staining, which specifically binds to phosphatidylserine and produces green fluorescence in apoptotic cells. In contrast, necrotic or late-stage apoptotic cells can be identified using PI staining, which penetrates cells with compromised plasma membrane integrity and emits red fluorescence upon binding to nucleic acids. The relative distribution of green and red fluorescence provides essential parameters for quantifying the level of apoptosis in cells, allowing for a direct measurement of cell death. This approach has been widely employed in various studies and is considered a robust tool for monitoring apoptosis in vitro and in vivo. As evidenced by our previous research, BB/BC could induce apoptosis in epidermal carcinoma cells A431, with a remarkable apoptotic rate exceeding 70% [14,15]. Moreover, AMPs from various origins, including ranatuerin-2PLx and temporin-1CEa, were found to elevate the apoptotic rate during the induction of cell apoptosis in PC-3 M and Bcap-37 cell lines [23,24].

Further Study on the Apoptotic Mechanism of BB/BC towards Gastric Cancer Cells
Building on previous research findings, we hypothesized that BB/BC induces apoptosis in gastric cancer cells. In this study, we aimed to investigate the mechanism behind this process by examining the apoptotic rate, ROS levels, and mitochondrial membrane potential as assessment indicators.
As depicted in Figure 6A, treatment with BB/BC resulted in a gradual increase in apoptotic rates of BGC-823 cells. At concentrations of 4 µg/mL, 6 µg/mL, and 8 µg/mL of BB, approximately 18.68%, 22.25%, and 36.69% of cancer cells underwent apoptosis, respectively ( Figure 6B). Similarly, treatment with BC at the same concentrations led to even higher apoptotic rates of BGC-823 cells, reaching 28.31%, 31.46%, and 51.61%, respectively, indicating that BC exhibited stronger inhibitory effects ( Figure 6C). These findings suggest that BB and BC may induce apoptosis in BGC-823 cells, highlighting their potential as effective anticancer agents. Apoptosis is a crucial physiological process that regulates the balance between cell division and cell death to maintain cellular homeostasis and prevent uncontrolled proliferation of cancer cells [22]. During the early stages of cellular apoptosis, a critical event is the translocation of phosphatidylserine from the inner to the outer leaflet of the cell membrane. This phenomenon can be detected using Annexin V-FITC staining, which specifically binds to phosphatidylserine A fluorescent probe DCFH-DA was utilized to quantify the levels of ROS, and the outcomes were presented in Figure 7. The detected levels of ROS in BGC-823 gastric cancer cells increased significantly in a dose-dependent manner following treatment with BB/BC. Specifically, the DCFH positive ratio of BGC-823 cells showed an increase, peaking at 14.28%, 54.35%, and 72.77% after 24 h of exposure to 4, 6, and 8 µg/mL of BB, respectively. Likewise, a dose-dependent increase in the DCFH positive ratio was also observed in the same cancer cells treated with BC, reaching 28.5%, 61.57%, and 89.38% after 24 h exposure to 4, 6, and 8 µg/mL of BC, respectively. Mitochondria serve as the primary energy source of cells and play a critical role in cellular growth, proliferation, and differentiation. In the field of cancer treatment, several anti-tumor drugs activate mitochondrial pathways to exert their anti-cancer effects, which frequently entail the production of ROS. During apoptosis, there is a significant accumulation of ROS, which can affect the metabolic activities of cells and cause characteristic morphological changes, as established in previous research [25][26][27]. Therefore, the evaluation of drug-induced cell damage can be effectively assessed, and treatment adjustments can be made in real-time by detecting ROS levels during the use of drugs to combat cancer cells [28]. Studies using AMPs to induce apoptosis in different cancer cells have consistently demonstrated this typical biological feature, as seen in BB/BC inducing apoptosis and causing an increase in ROS levels in epidermal carcinoma cells A431 [14,15]. Similarly, AMP such as Bogorol B-JX from Br. Laterosporus can cause ROS accumulation in U-937 cells [11], another pentapeptide can induce apoptosis in HL-60 cells and increase their intracellular ROS levels [29], and the AMP CM4 has the same effect on breast cancer cells MX-1, MCF-7, and MDA-MB-231 [30,31]. A fluorescent probe DCFH-DA was utilized to quantify the levels of ROS, and the outcomes were presented in Figure 7. The detected levels of ROS in BGC-823 gastric cancer cells increased significantly in a dose-dependent manner following treatment with BB/BC. Specifically, the DCFH positive ratio of BGC-823 cells showed an increase, peaking at 14.28%, 54.35%, and 72.77% after 24 h of exposure to 4, 6, and 8 µg/mL of BB, respectively. Likewise, a dose-dependent increase in the DCFH positive ratio was also observed in the same cancer cells treated with BC, reaching 28.5%, 61.57%, and 89.38% after 24 h exposure to 4, 6, and 8 µg/mL of BC, respectively. Mitochondria serve as the primary energy source of cells and play a critical role in cellular growth, proliferation, and differentiation. In the field of cancer treatment, several anti-tumor drugs activate mitochondrial pathways to exert their anti-cancer effects, which frequently entail the production of ROS. During apoptosis, there is a significant accumulation of ROS, which can affect the metabolic activities of cells and cause characteristic morphological chang- To evaluate the mitochondrial membrane potential of BGC-823 cells, fluorescence probe JC-1, which exists in monomeric and aggregated forms, was used ( Figure 8). Consistent with its effect on ROS levels, treatment with a concentration of 4 µg/mL of BB/BC effectively prevented the formation of JC-1 aggregates, indicating the induction of earlystage apoptosis in BGC-823 cells. However, with higher concentrations of BB/BC, the formation of JC-1 aggregates increased, accompanied by increased apoptotic rates and ROS levels.
causing an increase in ROS levels in epidermal carcinoma cells A431 [14,15]. Similarly, AMP such as Bogorol B-JX from Br. Laterosporus can cause ROS accumulation in U-937 cells [11], another pentapeptide can induce apoptosis in HL-60 cells and increase their intracellular ROS levels [29], and the AMP CM4 has the same effect on breast cancer cells MX-1, MCF-7, and MDA-MB-231 [30,31]. To evaluate the mitochondrial membrane potential of BGC-823 cells, fluorescence probe JC-1, which exists in monomeric and aggregated forms, was used ( Figure 8). Consistent with its effect on ROS levels, treatment with a concentration of 4 µg/mL of BB/BC effectively prevented the formation of JC-1 aggregates, indicating the induction of early-stage apoptosis in BGC-823 cells. However, with higher concentrations of BB/BC, the formation of JC-1 aggregates increased, accompanied by increased apoptotic rates and ROS levels. Apoptosis is a complex cellular death process regulated by multiple factors that involves the participation of reactive oxygen species (ROS). ROS can function as an external inducer or an intermediate product of other stimuli that trigger apoptosis in cells. While ROS can be produced through various pathways, the mitochondria are the primary intracellular source of ROS generation, occurring within the mitochondrial electron transport chain [11]. Accumulation of intracellular ROS can disrupt the oxidative respiratory chain, alter the mitochondrial membrane potential, and ultimately result in mitochondrial dysfunction and apoptosis [25]. During apoptosis, the dissipation of mitochondrial membrane potential is a characteristic early signal of apoptosis. This process can be detected by the change in JC-1 fluorescence from red to green, which allows for the formation of JC-1 aggregates in the mitochondrial matrix and the quantification of mitochondrial membrane potential. The addition of BB/BC has been shown to effectively decrease the mitochondrial membrane potential in BGC-823 cells, indicating their ability to induce early apoptosis in these cancer cells. These findings are consistent with those observed in epidermal carcinoma cells A431 [14,15] and other AMPs such as Pardaxin and AMP B11, which have been shown to induce early apoptosis in cancer cells HT-1080 and Hela, respectively [31,32]. With increasing concentrations and duration of BB/BC treatment, an upward trend in mitochondrial membrane potential was observed in BGC-823 cells, which may be attributed to the fact that BB/BC triggers apoptosis in these cells from early to late stages at increasing concentrations [11]. Apoptosis is a complex cellular death process regulated by multiple factors that involves the participation of reactive oxygen species (ROS). ROS can function as an external inducer or an intermediate product of other stimuli that trigger apoptosis in cells. While ROS can be produced through various pathways, the mitochondria are the primary intracellular source of ROS generation, occurring within the mitochondrial electron transport chain [11]. Accumulation of intracellular ROS can disrupt the oxidative respiratory chain, alter the mitochondrial membrane potential, and ultimately result in mitochondrial dysfunction and apoptosis [25]. During apoptosis, the dissipation of mitochondrial membrane potential is a characteristic early signal of apoptosis. This process can be detected by the change in JC-1 fluorescence from red to green, which allows for the formation of JC-1 aggregates in the mitochondrial matrix and the quantification of mitochondrial membrane potential. The addition of BB/BC has been shown to effectively decrease the mitochondrial membrane potential in BGC-823 cells, indicating their ability to induce early apoptosis in these cancer cells. These findings are consistent with those observed in epidermal carcinoma cells A431 [14,15] and other AMPs such as Pardaxin and AMP B11, which have been shown to induce early apoptosis in cancer cells HT-1080

Conclusions
In conclusion, this study investigated the efficacy of two newly discovered antimicrobial peptides (AMPs), BB and BC, against BGC-823 gastric cancer cells. The findings revealed that treatment with 4-6 µg/mL of BB/BC effectively hindered cell migration, altered the ultrastructure and nuclear morphology of cancer cells, and induced the release of a significant amount of LDH, evidencing their potential to induce apoptosis in BGC-823 cells. Further analysis unveiled that BB and BC both triggered a noteworthy accumulation of reactive oxygen species (ROS) in cancer cells and disturbed mitochondrial function. At lower concentrations, BB/BC induced early apoptosis in BGC-823 cells, whereas at higher concentrations, they fostered late-stage apoptosis. Building on the aforementioned findings, we have ascertained that BB and BC exert pronounced inhibitory effects on BGC-823 gastric cancer cells. This breakthrough discovery is poised to significantly advance the development and exploitation of Brevilaterins as a promising anticancer drug component, thereby earmarking it as a pivotal avenue for future research. Moving forward, our investigations into Brevilaterins will encompass diverse test hosts, including an in depth exploration of its stability within the host during utilization, as well as an evaluation of its toxicity potency against gastric cancer cells during in vivo application.