Anti-Proliferative Effects of Bioinspired Antimicrobial Peptides on MDA-MB-231 Triple- Negative Breast Cancer Cells

Anti-Proliferative Effects of Bioinspired Antimicrobial Peptides on MDA-MB-231 Triple- Negative Breast Cancer Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Anti-Proliferative Effects of Bioinspired Antimicrobial Peptides on MDA-MB-231 Triple- Negative Breast Cancer Cells Nouralhuda Alateyah, Sergio Crovella, Richa Gill, Maha Abdulla Al-Asmakh, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7426302/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Conventional strategies for treating breast cancer, including chemotherapy and radiotherapy, often face critical challenges such as severe adverse effects and the development of therapeutic resistance. These limitations have driven interest in exploring alternative and complementary approaches with improved safety profiles and targeted efficacy. Among these, antimicrobial peptides (AMPs), naturally occurring components of the innate immune system, are increasingly recognized for their selective cytotoxic activity against cancer cells and potential immunomodulatory effects. In particular, AMPs derived from extremophiles—organisms adapted to survive in extreme environmental conditions—present compelling therapeutic opportunities. These peptides tend to exhibit remarkable stability and enzymatic resistance, which enhances their functionality under physiological conditions. Compared to plant-derived defensins, which may suffer from poor bioavailability despite their cysteine-rich profiles, and bacterial or synthetic AMPs, which can present selectivity or toxicity issues, extremophile-inspired peptides offer a more robust and targeted anticancer profile. In this study, bioinspired short antimicrobial peptides (BSAMPs) were designed in silico using structural motifs from extremophilic organisms to enhance anticancer activity and tumor specificity. When tested on MDA-MB-231 triple-negative breast cancer (TNBC) cells, BSAMPs significantly reduced cell viability (by 70% at 400 µg/ml), inhibited cellular migration (by 80%), and decreased invasion (by 60%), while showing minimal toxicity (≤ 10%) to non-malignant fibroblasts and MCF10A epithelial cells. Mechanistically, BSAMPs promoted apoptosis via mitochondrial pathways, downregulating β-catenin and Bcl-xl while increasing Bax expression. While these results are currently limited to in vitro models, they highlight the therapeutic potential of BSAMPs and justify further validation in in vivo systems for breast cancer treatment. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Microbiology Breast cancer Antimicrobial peptides Bioinformatics Cell proliferation Migration Cell invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Breast cancer represents one of the most commonly diagnosed malignancies and continues to pose a global threat ( 1 ). According to GLOBOCAN 2020 data, it ranks as the fifth leading cause of cancer-related mortality globally, with an estimated 2.3 million new cases and 685,000 deaths reported in that year ( 2 ). In women specifically, it accounts for approximately 24.5% of all diagnosed cancers and is responsible for 15.5% of all cancer-related deaths ( 3 ). Multiple established risk factors are known to contribute to the development of breast cancer, including advanced age, obesity, tobacco consumption, physical inactivity, high-fat dietary intake, delayed childbirth, short breastfeeding duration, and positive family history ( 4 ). These trends emphasize the urgency of developing effective, precise therapies that are both effective and accessible to diverse populations. Conventional breast cancer treatment– including surgical intervention, chemotherapy, radiotherapy, and hormone-based or biological therapies ( 5 ) remain the cornerstone of clinical management, however, they present some limitations such as elevated costs, non-specific toxicity, and emergence of therapeutic resistance ( 6 ). These limitations have driven a growing interest in Complementary and Alternative Medicine (CAM), which explores the use of bioactive natural products and traditional medicinal practices as adjuncts or alternatives to enhance or replace conventional therapies. In this context, comprehensive phytochemical repositories, such as the Collection of Open Natural Products (COCONUT), the Traditional Chinese Medicine (TCM) Database@Taiwan, and the South African Natural Compounds Database (SANCDB), have become invaluable for identifying plant-derived compounds with potential anticancer activity ( 7 – 9 ). Antimicrobial peptides (AMPs) originating from extremophile organisms—particularly plants adapted to harsh environments such as high salinity, intense UV exposure, and prolonged drought—are gaining attention due to their inherent structural stability, resistance to proteolytic degradation, and selective bioactivity under stressful physiological conditions ( 10 – 11 ). Therapeutic peptides are defined as those generally consisting of fewer than 50 amino acid residues ( 12 ) and are emerging as promising agents for targeting and disrupting protein-protein interactions that are essential for cancer development ( 13 ). Owing to their high target specificity, strong binding affinity to cellular receptors, effective membrane permeability, and negligible immunogenicity, these peptides are being investigated as potential adjuncts or alternatives to conventional clinical cancer therapies ( 14 ). All this considered, we hypothesize that antimicrobial peptides (AMPs) derived from extremophilic plants will exhibit cytotoxicity against TNBC through the induction of mitochondrial apoptosis, thereby representing a novel approach to addressing the limitation of current breast cancer therapies. In this study, we evaluated the anticancer activity of two newly engineered antimicrobial peptides in MDA-MB-231, a TNBC cell line, using as controls the MCF10 A non-tumorigenic epithelial cell line and primary neonatal fibroblast cells. Specifically, we examined their impact on cellular proliferation, migration, and invasion, and investigated the underlying molecular pathways contributing to their anticancer mechanisms. Our work aims to support the advancement of peptide-based therapeutics and highlights the translational potential of extremophile-derived peptides as innovative candidates for next-generation anticancer agents. Materials and Methods 2.1. In Silico Peptide Design and Selection Two antimicrobial peptide sequences, designated as AF (sequence: AFSCGRCLGRRRRKCFCTRGAC), derived from defensin-like protein 1 of Ricinus communis , and GF (sequence: GFSGGKCKGFRRRRCFCTRLC), derived and modified from defensin-like protein 1 of Manihot esculenta , were selected. The selected plant species Ricinus communis and Manihot esculenta were chosen for their extremophilic traits, specifically due to their ability to survive and thrive in arid, nutrient-deficient environments. Prior evidence indicates that their defensin peptides exhibit notable bioactivity, including antimicrobial and immunomodulatory activity. The initial selection criteria for peptide candidates included a length of 20–25 amino acids, a net positive charge, and amphipathic structural characteristics. Sequence alignments were performed to pinpoint conserved motifs and regions suitable to targeted modifications. Coherent amino acid replacements were subsequently introduced to enhance bioactivity, structural integrity, and selective cytotoxicity towards cancer cells. Peptide candidates were screened for anticancer potential using established bioinformatics tools, including LMPred ( https://github.com/williamdee1/LMPred_AMP_Prediction ), AntiCP ( http://crdd.osdd.net/raghava/anticp/ ), CancerPPD ( http://crdd.osdd.net/raghava/cancerppd/ ), ACPred http://codes.bio/acpred/ ), and CPPsite 2.0 ( http://crdd.osdd.net/raghava/cppsite/ ). AF and GF peptides emerged as top-ranked candidates across these predictive models. Their molecular weights were determined computationally: AF = 2507.98 Da and GF = 2438.91 Da. In silico analysis using ToxiPred ( http://crdd.osdd.net/raghava/toxipred/ ) and the Antigenic Peptide Prediction Tool ( http://crdd.osdd.net/raghava/antigenic/ ) predicted both peptides to be non-toxic and non-immunogenic. Figure 1 shows the 3D structure of AF and GF peptides, obtained with the Pep Fold-3 ( https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/ ) web tool. 2.2. 3D Structural Prediction Three-dimensional conformers of AF and GF were predicted using ColabFold (AlphaFold2 implementation). Structural integrity and confidence were evaluated based on per-residue pLDDT and overall TM-scores. 2.3. Molecular Dynamics Simulations Molecular dynamics simulations were conducted using GROMACS 2019.4 with the GROMOS 53A6 force field. Peptides were solvated in SPC water within a cubic simulation box containing 0.15 M NaCl. After energy minimization, simulations were run at 300 K and 1 atm using a 2 fs timestep for 100 ns. Structural stability, RMSD, and secondary structure changes were analyzed post-simulation. Both structural prediction and molecular dynamics simulations have been performed according to the bioinformatics pipeline described in (Hashem S et.,al 2025) ( 34 ). 2.4. Peptide Synthesis and Stability Assessment Peptides AF and GF were synthesized by NovoPro Bioscience Inc. (Shanghai, China). Peptide identity and purity were validated via HPLC and mass spectrometry, confirming purities of > 95% (AF: 95.435%; GF: 95.740%). Blood stability and half-life were predicted using the PlifePred ( https://webs.iiitd.edu.in/raghava/plifepred/ ) tool, yielding estimates of 954.61 s for AF and 840.31 s for GF. 2.5. Cell culture MDA-MB-231 and MCF10 A cells were obtained from the American Type Culture Collection (ATCC, USA), and the primary neonatal fibroblast cells were kindly provided by Sidra Medicine-Qatar. Both MDA-MB-231 and primary neonatal fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and 1% L-Glutamine. All cell lines were cultured in the same optimum conditions in a humidified incubator adjusted to 37°C and 5% CO 2 . 2.6. Alamar-Blue Cell Proliferation Assay MDA-MB-231 breast cancer cells, primary neonatal fibroblast, and MCF10A cells were seeded in 96-well tissue culture plates with 10,000 cells in each well, in triplicates using DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (100 µl/well). Cells were incubated overnight prior to treatment with different concentrations of AF and GF peptides (100 µg/ml, 200 µg/ml, 300 µg/ml, and 400 µg/ml). The 48-hour treatment duration was chosen to allow sufficient time for the peptides to exert measurable cytotoxic effects on the cells, consistent with prior studies evaluating antimicrobial peptides where incubation times ranging from 24 to 72 hours are commonly used to assess anticancer activity (e.g., Smith et al., 2021; Lee et al., 2022). After 48 hours, cells were washed with sterile PBS and 10% Alamar-Blue reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells and incubated for 4–5 hours in the incubator according to the manufacturer’s protocol. Fluorescence measurements were taken at 560 nm and 590 nm using a TECAN infinite M200 plate reader after incubation with the dye. Relative cell proliferation was determined based on the fluorescence of treated cells relative to control cells. 2.7. Wound Healing Assay MDA-MB-231 cells were seeded in flat 6-well tissue culture plates and incubated overnight to allow for cell adherence and confluency before the addition of treatments. Upon reaching 50% confluency, as confirmed by microscopic observation, a linear scratch was introduced in each well using a sterile 200 µL pipette tip. The wells were subsequently washed with sterile phosphate-buffered saline (PBS) to eliminate cellular debris and undetached cells. Cells were then treated with AMPs at a concentration of 100 µg/mL, while untreated wells containing only culture medium served as controls. Images were captured at defined time points (0, 24, and 48 hrs) to monitor wound closure. Images were analyzed using ImageJ software, statistical analysis was performed, and the data were plotted as a graph. 2.8. Invasion Assay The cell invasion assay was performed using the Boyden Matrigel Chamber technique to assess the effect of AF and GF peptides on the invasion capacity of the MDA-MB-231 TNBC cell line. The experiment was executed in 24-well Biocoat Matrigel invasion chambers (8 µm pore size, Corning, USA) as per the manufacturer’s instructions. Untreated cells (medium only) served as the control, and peptide-treated MDA-MB-231 cells were seeded into the top chambers, meanwhile, the bottom chambers were filled with DMEM prepared with 10% fetal bovine serum (FBS) to act as a chemoattractant. The system was incubated at 37°C for 48 hours. Subsequently, the top chambers were washed with sterile PBS, and non-invasive cells were lightly removed employing a sterile cotton swab. Cells that had migrated to the base of the top membrane were fixed with methanol and formaldehyde for 10 minutes and stained with 0.5% crystal violet stain. After washing with PBS, invaded cells were imaged under an inverted microscope (OPTIKA Microscopes, Ponteranica, Italy) across four predefined fields. The percentage inhibition of invasion was determined relative to untreated control cells and quantified using ImageJ software. 2.9. Western Blot analysis To gain insight into the molecular pathways affected by treatment with AF and GF peptides, we examined key regulators involved in cell cycle progression and apoptosis, specifically β-catenin, Bcl-xl, and Bax. Briefly, MDA-MB-231 cells were seeded and treated with 100 ug/ul of AMP peptides for 48 hours. After 48 hours of treatment, protein lysates were extracted using RIPA buffer and quantified by Bradford assay. Equal amounts (30µg) of protein lysates were denatured at 95°C for 10 minutes and were resolved on 10% polyacrylamide gels and electroblotted onto PVDF membranes. The PVDF membranes were incubated overnight with the primary antibodies: antirabbit Bax (Cell Signaling Technology, CST #5023S, USA), anti Bcl-XL, and β-catenin (Cell Signaling Technology, CST #4970S). To confirm equal loading of protein samples, the membranes were re-probed with anti-rabbit β-actin (Cell Signaling Technology, CST #4970S, USA). Following overnight incubation, the membranes were washed twice in PBS and were then incubated for 2 hours in the corresponding secondary antibody. Immunoreactivity was detected by using ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer. Images were captured using the iBright machine (ThermoFisher Scientific, USA) and relative quantification of protein expressions from images acquired from Western blotting were analyzed using ImageJ software. The intensity of the bands relative to the β-actin bands was used to calculate a relative expression of proteins. 2.10. Statistical Analysis All data were analyzed using SPSS Statistics 23.0 software. Data were shown as an average of mean ± SEM (standard error of the mean). Each experiment was repeated two times (n = 2). Tukey’s post-hoc test was used to compare the difference between treated and untreated cells. and differences with p < 0.05 were considered as significant. Results The predicted three-dimensional structures of the AF and GF peptides revealed distinct conformational characteristics. As illustrated in Figure 1 , the AF peptide adopted a relatively compact and linear structure, whereas the GF peptide displayed a loop and a more flexible structure. Variations in the orientation of the key side chains between the two peptides suggest potential differences in their molecular interactions and biological activities. Both peptides exhibited defined secondary structural elements, including short α-helices and turns, with marked disparities in surface accessibility and possible interaction sites. Importantly, the enhanced structural flexibility and looped configuration of the GF peptide may facilitate dynamic engagement with diverse molecular targets, potentially explaining its pronounced anti-invasive activity observed in functional assays. Conversely, the AF peptide’s more rigid and linear structure may promote stable interactions with specific biomolecular partners, consistent with its strong anti-proliferative effects. 3.1 Establishment of the Optimal Dose of the AMPs: To determine the effect of AF and GF peptides on breast cancer cell line, MDAMB-231 cells were treated with different concentrations of both peptides (100 µg/ml, 200 µg/ml, 300 µg/ml, and 400 µg/ml) for 48 hours. Primary neonatal fibroblast cells and MCF10A epithelial cell lines were used as controls. 3.2 Effect of the AMPs on Cell Migration and Invasion: Subsequently, the impact of the antimicrobial peptides (AMPs) on breast cancer cell migration and invasion was evaluated using wound-healing and Boyden chamber assays, respectively. In the wound-healing assay, AMP-treated MDA-MB-231 cells demonstrated a pronounced decrease in migratory capacity compared to untreated controls. Quantitative measurements revealed that peptide exposure reduced wound closure by approximately 80% after 24 hours (p < 0.05, Fig. 4 ), indicating a significant disruption of cellular motility. Consistently, the Boyden chamber invasion assay showed that AMP treatment substantially impaired the ability of breast cancer cells to penetrate the Matrigel-coated membrane, with an observed ~ 60% reduction in invasive relative to control cells (p < 0.05, Fig. 5 ). These findings highlight the capacity of the AMPs to effectively suppress essential mechanisms underlying cancer metastasis. The engineered peptides inhibit key steps in breast cancer progression by impairing both migratory and invasive behaviors of tumor cells. 3.3 Molecular Mechanisms Mediating AMPs-Induced Apoptosis: To elucidate the molecular mechanisms underlying AMP-induced apoptosis, we examined the modulation of key apoptotic regulators by antimicrobial peptides (AMPs). Western blot analysis demonstrated a marked downregulation of the expression of β-catenin – a protein implicated in promoting cell proliferation and survival – in both AF and GF peptide-treated groups. Densitometric analysis indicated an approximate 3- to 3.75-fold reduction in β-catenin expression compared to untreated controls. Also, AMP treatment resulted in an upregulation of a pro-apoptotic gene, BAX, with expression increasing by ~ 1.25-fold with AF peptide and ~ 1.75-fold with GF peptide relative to control. In contrast, BCL-XL, an anti-apoptotic protein, was significantly suppressed, showing a ~ 3- to 3.75-fold decrease following AMP exposure. These combined effects resulted in an elevated BAX/BCL-XL ratio, particularly in response to GF peptide treatment, suggesting a shift toward a pro-apoptotic intracellular environment. Our findings indicate that AMPs promote apoptosis by modulating β-catenin, BAX, and BCL-XL expression, thereby highlighting a potential mechanistic basis for their anticancer activity. Discussion Due to the extremophile origin of the AF and GF peptides, they may possess enhanced stability under physiological conditions, potentially overcoming a major challenge associated with synthetic AMPs like melittin, which are prone to rapid degradation and systemic toxicity ( 35 ). This study presents evidence of the anticancer properties of these peptides engineered from defensin-like proteins of extremophile plants against the triple-negative breast cancer (TNBC) cell line MDA-MB-231. Both AF and GF peptides effectively suppressed cell proliferation, migration, and invasion while exhibiting selective cytotoxicity toward cancer cells, with minimal impact on normal fibroblasts and breast epithelial cells. Among the two peptides, GF exhibited a modestly superior anticancer profile, leading to a ~ 70% reduction in TNBC cell viability at 400 µg/mL, compared to ~ 65% by AF at the same dose. Importantly, GF also demonstrated a more favorable selectivity, causing negligible cytotoxic effects (< 10%) in normal fibroblasts and MCF10A cells, while AF induced slightly higher nonspecific toxicity (~ 12% cytotoxicity). These differences indicate GF’s potential advantage as a lead candidate for further in vivo exploration. The enhanced bioactivity of GF may be attributed to its structural dynamics; its flexible, looped architecture may promote more efficient interaction with cancer cell membranes, unlike the more linear and rigid conformation observed in AF peptide (Fig. 1 ). Notably, our experimental results confirmed the in-silico findings based on tools like AntiCP, ACPred, and CancerPPD, showing that both peptides show strong anticancer potential, but GF showed slightly better performance in decreasing migration and invasion, likely due to better internalization and interaction with intracellular targets. Mechanistically, both AF and GF influenced critical apoptotic pathway components, particularly by suppressing the expression of β-catenin and Bcl-xl while upregulating levels of the pro-apoptotic protein Bax, suggesting activation of mitochondrial-mediated apoptosis ( 15 ) ( 16 ) ( 17 ). Quantitative densitometry revealed a 3- to 3.75-fold reduction in β-catenin and Bcl-xl, along with a Bax upregulation of approximately ~ 1.25-fold with AF and ~ 1.75-fold for GF. These changes resulted in an increased BAX/BCL-XL ratio, a sign of apoptotic commitment. This mechanistic profile is consistent with prior findings that describe antimicrobial peptides as possessing dual functionality: not only antimicrobial defense but also selective cytotoxicity against cancer cells ( 18 ). Well-characterized AMPs such as LL-37, magainins, and defensins preferentially target cancer cells by exploiting specific membrane features, including the abnormal externalization of negatively charged phosphatidylserines ( 19 )( 20 ). The potent anticancer effects observed for AF and GF likely stem from their net positive charge and amphipathic structures, which facilitate specific binding to and disruption of cancer cell membranes ( 21 )( 22 ). Corroborating our findings, Răileanu and Bacalum ( 23 ) reported that plant-derived antimicrobial peptides exhibit broad-spectrum cytotoxicity against colorectal cancer cells, primarily through mechanisms involving membrane disruption and mitochondrial impairment, highlighting the therapeutic potential of thermophile-sourced peptides in cancer treatment ( 23 ). The observed suppression of β-catenin in our study is particularly significant, as dysregulation of the Wnt/β-catenin pathway is a well-established driver of TNBC progression and therapy resistance ( 24 ). Our data are consistent with the work of Leite et al. (2018), who demonstrated that AMP-mediated β-catenin inhibition contributed to cell cycle arrest and induction of apoptosis in cancer cells ( 25 ). Furthermore, the substantial reduction in cell migration (80%) and invasion (60%) observed in this study supports previous findings showing that AMPs can inhibit epithelial-to-mesenchymal transition (EMT) and suppress metastatic behavior, hallmarks of highly invasive and aggressive TNBC ( 26 )( 27 ). Notably, AF and GF exhibited minimal cytotoxic effects on non-cancerous cells thereby demonstrating a favorable therapeutic window consistent with other AMP candidates of plant and animal origin ( 28 ). Unlike melittin, which, despite its potent anticancer activity, is limited by hemolytic and systemic toxicities, both AF and GF were predicted and experimentally validated to be non-toxic and non-immunogenic in silico , reinforcing their potential as safe and selective anticancer agents ( 29 )( 30 ). This study constitutes an early attempt to exploit in-silico engineered plant defensins as therapeutic agents against a TNBC cell line. Nonetheless, this study has several important limitations. The small sample size (n = 2) reduces statistical robustness and may affect the reproducibility of the observed effects. Furthermore, the lack of in vivo validation precludes a comprehensive assessment of pharmacokinetics, systemic toxicity, and immune system interactions. To address these gaps, future research should prioritize in vivo validation using TNBC xenograft models to confirm therapeutic efficacy and safety within a physiological context. Additionally, the application of omics-based approaches, such as RNA sequencing and proteomic profiling, would enable a more detailed characterization of AMP-mediated signaling pathways and potential mechanisms of resistance. Moreover, investigating combination therapies that integrate AF and GF peptides with immune checkpoint inhibitors or standard chemotherapeutic agents offers a promising strategy to overcome therapeutic resistance in TNBC and improve clinical outcomes. These synergistic approaches may exploit both the direct anticancer activity of AMPs and their capacity to modulate the tumor immune microenvironment, potentially enhancing overall treatment efficacy. Conclusion Our study highlights the anticancer potential of two antimicrobial peptides, AF and GF, engineered from extremophile plant defensins. Both peptides showed inhibition of proliferation, migration, and invasion in TNBC MDA-MB-231 cells, with minimal toxicity to normal breast epithelial cells and fibroblasts. Mechanistically, they induced mitochondrial apoptosis by downregulating β-catenin and Bcl-xl and upregulating Bax, consistent with AMP-mediated disruption of oncogenic pathways and cancer cell membranes. Compared to traditional chemotherapies, AF and GF offer targeted, less toxic alternatives, with potential application for TNBC. Their plant origin, small size, and predicted non-immunogenicity enhance translational promise. Future work should prioritize in vivo testing, omics analyses, and combination therapies to confirm efficacy and optimize clinical potential. Overall, AF and GF represent promising, rationally designed candidates for treating aggressive breast cancer. Declarations Author Contributions: Nouralhuda Alateyah methodology, validation, formal analysis, writing—original draft preparation; Sergio Crovella, conceptualization, software, writing—review and editing; Richa Gill methodology, data curation, writing—original draft preparation; Maha Abdulla Al-Asmakh supervision, investigation, writing—review and editing; Haissam Abou Saleh conceptualization, investigation, resources, project administration, supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Qatar National Research Foundation (GSRA11-L-1-0530-24081). Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, or in the decision to publish the results. Data availability: All raw data generated or analyzed during this study, including original gel images, are provided in the supplementary information files. References -Wilkinson, L. & Gathani, T. Understanding Breast Cancer as a Global Health Concern. Br. J. Radiol. 95 (1130). https://doi.org/10.1259/bjr.20211033 (2021). -Sung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J. Clin. 71 (3), 209–249 (2021). -Sung, H. et al. Lei, S., Zheng, R., Zhang, S., Wang, S., Chen, R., Sun, K., … Wei, W. (2021). Global patterns of breast cancer incidence and mortality: A population-based cancer registry data analysis from 2000 to 2020. Cancer Communications . -Fakhri, N. et al. Risk factors for breast cancer in women: an update review. Med. Oncol. 39 (12). https://doi.org/10.1007/s12032-022-01804-x (2022). -Maughan, K. L., Lutterbie, M. A. & Ham, P. S. Treatment of Breast Cancer. Am. Family Phys. 81 (11), 1339–1346 (2010). https://www.aafp.org/pubs/afp/issues/2010/0601/p1339.html -Marqus, S., Pirogova, E. & Piva, T. J. Evaluation of the use of therapeutic peptides for cancer treatment. J. Biomed. Sci. 24 (1), 1–15 (2017). -Cassileth, B. R. & Deng, G. Complementary and Alternative Therapies for Cancer. Oncologist 9 (1), 80–89. https://doi.org/10.1634/theoncologist.9-1-80 (2004). -Scalbert, A. et al. Databases on Food Phytochemicals and Their Health-Promoting Effects. J. Agric. Food Chem. 59 (9), 4331–4348. https://doi.org/10.1021/jf200591d (2011). -Mani, S. et al. The Updated Review on Plant Peptides and Their Applications in Human Health. Int. J. Pept. Res. Ther. 28 (5). https://doi.org/10.1007/s10989-022-10437-7 (2022). -Kochhar, N. et al. Perspectives on the microorganism of extreme environments and their applications. Curr. Res. Microb. Sci. 3 , 100134. https://doi.org/10.1016/j.crmicr.2022.100134 (2022). -Duarte-Mata, D. I. & Salinas-Carmona, M. C. Antimicrobial peptides´ immune modulation role in intracellular bacterial infection. Front. Immunol. 14 https://doi.org/10.3389/fimmu.2023.1119574 (2023). -Nugrahadi, P. P., Hinrichs, J., Frijlink, H. W., Schöneich, C. & Avanti, C. Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review. Pharmaceutics 15 (3), 935–935. https://doi.org/10.3390/pharmaceutics15030935 (2023). -Damodaran, S. & Parkin, K. L. Amino acids, peptides, and proteins. In Fennema’s food chemistry (235–356). CRC. (2017). -Shemetov, A. A., Nabiev, I. & Sukhanova, A. Molecular interaction of proteins and peptides with nanoparticles. ACS nano . 6 (6), 4585–4602 (2012). -Hoskin, D. W. & Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. Biochim. Biophys. Acta . 1778 (2), 357–375 (2008). -Saha, S., Ghosh, M. & Dutta, S. K. Role of metabolic modulator Bet-CA in altering mitochondrial hyperpolarization to suppress cancer-associated angiogenesis and metastasis. Sci. Rep. 6 (1). https://doi.org/10.1038/srep23552 (2016). -Prakash, E. & Gupta, D. K. In vitro study of extracts of Ricinus communis Linn on human cancer cell lines. J. Med. Sci. Pub Health . 2 (1), 1520 (2014). -Parchebafi, A. et al. The dual interaction of antimicrobial peptides on bacteria and cancer cells; mechanism of action and therapeutic strategies of nanostructures. Microb. Cell. Fact. 21 (1). https://doi.org/10.1186/s12934-022-01848-8 (2022). -Zare-Zardini, H. et al. From defense to offense: antimicrobial peptides as promising therapeutics for cancer. Front. Oncol. 14. https://doi.org/10.3389/fonc.2024.1463088 (2024). -Arafa, N. M., Moawad, M. & El-Shabrawi, H. E. Comparison of the organic and inorganic solvents effect on phenolic compounds extraction and the activity against breast carcinoma cell lines from callus cultures of Manihot esculenta. International Journal of PharmTech Research , 2016,9(12): 380–396. (2016). -Manrique-Moreno, M., Santa-González, G. A. & Gallego, V. Bioactive cationic peptides as potential agents for breast cancer treatment. Biosci. Rep. 41 (12). https://doi.org/10.1042/bsr20211218c (2021). -Lei, J. et al. The antimicrobial peptides and their potential clinical applications. Am. J. Transl Res. 11 , 3919–3931 (2019). -Răileanu, M. & Bacalum, M. Cancer Wars: Revenge of the AMPs (Antimicrobial Peptides), a New Strategy against Colorectal Cancer. Toxins 15 (7), 459 (2023). -Zhan, T., Rindtorff, N. & Boutros, M. Wnt signaling in cancer. EMBO Rep. 18 (4), 698–711 (2017). -Leite, M. L., da Cunha, N. B. & Costa, F. F. Antimicrobial peptides, nanotechnology, and natural metabolites as novel approaches for cancer treatment. Pharmacol. Ther. 183 , 160–176 (2018). -Scocchi, M., Mardirossian, M., Runti, G. & Benincasa, M. Non-Membrane permeabilizing modes of action of antimicrobial peptides on bacteria. Curr. Top. Med. Chem. 16 , 76–88 (2016). [Google Scholar]. -Xiong, N., Wu, H. & Yu, Z. Advancements and challenges in triple-negative breast cancer: a comprehensive review of therapeutic and diagnostic strategies. Front. Oncol. 14 https://doi.org/10.3389/fonc.2024.1405491 (2024). -Guryanova, S. V. & Ovchinnikova, T. V. Immunomodulatory and allergenic properties of AMPs. Int. J. Mol. Sci. 23 (5), 2499 (2022). -Mihaylova-Garnizova, R., Davidova, S., Hodzhev, Y. & Satchanska, G. Antimicrobial Peptides Derived from Bacteria: Classification, Sources, and Mechanism of Action against Multidrug-Resistant Bacteria. Int. J. Mol. Sci. 25 (19), 10788. https://doi.org/10.3390/ijms251910788 (2024). -Marqus, S., Pirogova, E. & Piva, T. J. Evaluation of therapeutic peptides for cancer. J. Biomed. Sci. 24 (1), 1–15 (2017). -Sorokina, M. & Steinbeck, C. COCONUT: The COlleCtion of Open Natural prodUcTs database. J. Cheminform. , 12 (20). (2020). https://coconut.naturalproducts.net -TCM Database@Taiwan. (Accessed. May (2025). http://tcm.cmu.edu.tw/ -South African Natural Compounds Database (SANCDB). (Accessed. May (2025). http://sancdb.rubi.ru.ac.za/ -South African Natural Compounds Database (SANCDB). (Accessed. El-Serag, H., Kanwal, F., Ning, J., Powell, H., Khaderi, S., Singal, A. G., Asrani,S., Marrero, J. A., Amos, C. I., Thrift, A. P., Luster, M., Alsarraj, A., Olivares,L., Skapura, D., Deng, J., Salem, E., Najjar, O., Yu, X., Duong, H., Scheurer, M.E., … Kaochar, S. (2024). Serum biomarker signature is predictive of the risk of hepatocellular cancer in patients with cirrhosis. Gut , gutjnl-2024-332034. Advance online publication. https://doi.org/10.1136/gutjnl-2024-332034. -Li, Y. Mechanism of melittin for anti-tumor effects: Research status and future perspectives. J. Clin. Technol. Theory . 2 (1), 33–37. https://doi.org/10.54254/3049-5458/2025.21076 (2025). Additional Declarations No competing interests reported. Supplementary Files Originalblots.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7426302","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":519301541,"identity":"18078222-77b3-46d2-8f29-39e6fbcfdec3","order_by":0,"name":"Nouralhuda Alateyah","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Nouralhuda","middleName":"","lastName":"Alateyah","suffix":""},{"id":519301542,"identity":"fb707597-ace9-4e10-8fc8-3f528d9235dc","order_by":1,"name":"Sergio Crovella","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Crovella","suffix":""},{"id":519301543,"identity":"f0e41f95-2c1f-48b4-b44d-99dd143b8615","order_by":2,"name":"Richa Gill","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Richa","middleName":"","lastName":"Gill","suffix":""},{"id":519301545,"identity":"7f4936da-6544-430d-b887-4870159cbba9","order_by":3,"name":"Maha Abdulla Al-Asmakh","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Maha","middleName":"Abdulla","lastName":"Al-Asmakh","suffix":""},{"id":519301546,"identity":"d601c6bb-7757-4c6c-84a4-cf7c46fa02ca","order_by":4,"name":"Ajaz A. 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13:12:33","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89256,"visible":true,"origin":"","legend":"","description":"","filename":"f4e8a4630c764f86bbd4784e4d3421751structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/3485fdc295096adf1432244a.xml"},{"id":92264363,"identity":"ce06a326-424f-4452-90cc-834f8c91b000","added_by":"auto","created_at":"2025-09-26 13:12:33","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102012,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/33c569b6dc9a82fe6471077c.html"},{"id":92264336,"identity":"dce97cf4-3524-4b8f-9d5c-1fd34ac67206","added_by":"auto","created_at":"2025-09-26 13:12:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":293184,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted 3D structures of AF and GF peptides. The figure illustrates the modeled conformation of AF and GF peptides generated through structural prediction analysis. Blue regions represent nitrogen atoms, red for oxygen, and black for carbon atoms, highlighting the spatial orientation of amino acid side chains and potential interaction sites.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/6dcff72ab3029ece4043730b.png"},{"id":92264339,"identity":"c65b79cf-ff7e-43a1-b215-bb1e99b3291a","added_by":"auto","created_at":"2025-09-26 13:12:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53017,"visible":true,"origin":"","legend":"\u003cp\u003eshowing the effect of AMPs MDA-MB-231 breast cancer cells and normal Neonatal fibroblast cells which reduced the cell viability of MDA-MB-231 BC cells compared to normal neonatal fibroblast cells. Notably, concentration of 100 μg /ml showed a substantial decrease in cell viability of MDA-MB-231 cells by ~50% Interestingly, less reduction rate in cell viability was noted in the normal neonatal cells after 48 hours of exposure compared to cancer cells.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/44debdee6d649c9ca8ebaea6.png"},{"id":92264718,"identity":"6409c529-1337-4691-8e43-150020fdb157","added_by":"auto","created_at":"2025-09-26 13:20:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18816,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 100 µg/ml of both AF and GF peptides on the proliferation of MDA-MB-231 compared with MCF10-A normal breast cells. The presented data shows a decrease in MDA-MB-231 proliferation compared with normal breast epithelial cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/0e5b42725e7a8fa4d5182dbf.png"},{"id":92265743,"identity":"f711e0bc-0a76-4e3b-9ea0-5fbe56df59fd","added_by":"auto","created_at":"2025-09-26 13:28:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":554329,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AMPs on MDA-MB-231 cell migration using scratch wound-healing assay. (A) AMPs inhibited MDA-MB-231 cell migration. (B) Semi-quantitative analysis of the relative wound closure (%) showing an increase in the wound closure by the AMPs, indicating an inhibition of MDA-231 cell migration.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/0501cd5cac1a6d0da0605338.png"},{"id":92264721,"identity":"b26782cb-2442-4f4d-bbe2-99f52958c1e3","added_by":"auto","created_at":"2025-09-26 13:20:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":319189,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AF and GF peptides on cell invasion of MDA-MB-231 breast cancer cells using Boyden chamber assay. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBoth AF and GF peptides significantly decreased cell invasion ability of MDA-MB-231 by ~60% in comparison to control cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/341fa987fc2ee598235d0814.png"},{"id":109296322,"identity":"f7ca0220-d340-44dc-9cfb-a49c34bc250a","added_by":"auto","created_at":"2026-05-15 08:46:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1386849,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/a5632328-02f4-45d0-9c32-4caf56d12879.pdf"},{"id":92264719,"identity":"9a736b89-5deb-46de-907b-7c907ccbf5d1","added_by":"auto","created_at":"2025-09-26 13:20:32","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1819374,"visible":true,"origin":"","legend":"","description":"","filename":"Originalblots.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7426302/v1/ad9e36167bfc14fd62ce612e.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-Proliferative Effects of Bioinspired Antimicrobial Peptides on MDA-MB-231 Triple- Negative Breast Cancer Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer represents one of the most commonly diagnosed malignancies and continues to pose a global threat (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). According to GLOBOCAN 2020 data, it ranks as the fifth leading cause of cancer-related mortality globally, with an estimated 2.3\u0026nbsp;million new cases and 685,000 deaths reported in that year (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In women specifically, it accounts for approximately 24.5% of all diagnosed cancers and is responsible for 15.5% of all cancer-related deaths (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Multiple established risk factors are known to contribute to the development of breast cancer, including advanced age, obesity, tobacco consumption, physical inactivity, high-fat dietary intake, delayed childbirth, short breastfeeding duration, and positive family history (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These trends emphasize the urgency of developing effective, precise therapies that are both effective and accessible to diverse populations.\u003c/p\u003e\u003cp\u003eConventional breast cancer treatment\u0026ndash; including surgical intervention, chemotherapy, radiotherapy, and hormone-based or biological therapies (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) remain the cornerstone of clinical management, however, they present some limitations such as elevated costs, non-specific toxicity, and emergence of therapeutic resistance (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These limitations have driven a growing interest in Complementary and Alternative Medicine (CAM), which explores the use of bioactive natural products and traditional medicinal practices as adjuncts or alternatives to enhance or replace conventional therapies. In this context, comprehensive phytochemical repositories, such as the Collection of Open Natural Products (COCONUT), the Traditional Chinese Medicine (TCM) Database@Taiwan, and the South African Natural Compounds Database (SANCDB), have become invaluable for identifying plant-derived compounds with potential anticancer activity (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAntimicrobial peptides (AMPs) originating from extremophile organisms\u0026mdash;particularly plants adapted to harsh environments such as high salinity, intense UV exposure, and prolonged drought\u0026mdash;are gaining attention due to their inherent structural stability, resistance to proteolytic degradation, and selective bioactivity under stressful physiological conditions (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherapeutic peptides are defined as those generally consisting of fewer than 50 amino acid residues (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and are emerging as promising agents for targeting and disrupting protein-protein interactions that are essential for cancer development (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Owing to their high target specificity, strong binding affinity to cellular receptors, effective membrane permeability, and negligible immunogenicity, these peptides are being investigated as potential adjuncts or alternatives to conventional clinical cancer therapies (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll this considered, we hypothesize that antimicrobial peptides (AMPs) derived from extremophilic plants will exhibit cytotoxicity against TNBC through the induction of mitochondrial apoptosis, thereby representing a novel approach to addressing the limitation of current breast cancer therapies. In this study, we evaluated the anticancer activity of two newly engineered antimicrobial peptides in MDA-MB-231, a TNBC cell line, using as controls the MCF10 A non-tumorigenic epithelial cell line and primary neonatal fibroblast cells. Specifically, we examined their impact on cellular proliferation, migration, and invasion, and investigated the underlying molecular pathways contributing to their anticancer mechanisms. Our work aims to support the advancement of peptide-based therapeutics and highlights the translational potential of extremophile-derived peptides as innovative candidates for next-generation anticancer agents.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. In Silico Peptide Design and Selection\u003c/h2\u003e\u003cp\u003eTwo antimicrobial peptide sequences, designated as AF (sequence: AFSCGRCLGRRRRKCFCTRGAC), derived from defensin-like protein 1 of \u003cem\u003eRicinus communis\u003c/em\u003e, and GF (sequence: GFSGGKCKGFRRRRCFCTRLC), derived and modified from defensin-like protein 1 of \u003cem\u003eManihot esculenta\u003c/em\u003e, were selected. The selected plant species \u003cem\u003eRicinus communis\u003c/em\u003e and \u003cem\u003eManihot esculenta\u003c/em\u003e were chosen for their extremophilic traits, specifically due to their ability to survive and thrive in arid, nutrient-deficient environments. Prior evidence indicates that their defensin peptides exhibit notable bioactivity, including antimicrobial and immunomodulatory activity. The initial selection criteria for peptide candidates included a length of 20\u0026ndash;25 amino acids, a net positive charge, and amphipathic structural characteristics. Sequence alignments were performed to pinpoint conserved motifs and regions suitable to targeted modifications. Coherent amino acid replacements were subsequently introduced to enhance bioactivity, structural integrity, and selective cytotoxicity towards cancer cells.\u003c/p\u003e\u003cp\u003ePeptide candidates were screened for anticancer potential using established bioinformatics tools, including LMPred (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/williamdee1/LMPred_AMP_Prediction\u003c/span\u003e\u003cspan address=\"https://github.com/williamdee1/LMPred_AMP_Prediction\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), AntiCP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/anticp/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/anticp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), CancerPPD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/cancerppd/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/cancerppd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ACPred \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://codes.bio/acpred/\u003c/span\u003e\u003cspan address=\"http://codes.bio/acpred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and CPPsite 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/cppsite/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/cppsite/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). AF and GF peptides emerged as top-ranked candidates across these predictive models. Their molecular weights were determined computationally: AF\u0026thinsp;=\u0026thinsp;2507.98 Da and GF\u0026thinsp;=\u0026thinsp;2438.91 Da. In silico analysis using ToxiPred (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/toxipred/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/toxipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the Antigenic Peptide Prediction Tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/antigenic/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/antigenic/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predicted both peptides to be non-toxic and non-immunogenic. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the 3D structure of AF and GF peptides, obtained with the Pep Fold-3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/\u003c/span\u003e\u003cspan address=\"https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) web tool.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. 3D Structural Prediction\u003c/h2\u003e\u003cp\u003eThree-dimensional conformers of AF and GF were predicted using ColabFold (AlphaFold2 implementation). Structural integrity and confidence were evaluated based on per-residue pLDDT and overall TM-scores.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Molecular Dynamics Simulations\u003c/h2\u003e\u003cp\u003eMolecular dynamics simulations were conducted using GROMACS 2019.4 with the GROMOS 53A6 force field. Peptides were solvated in SPC water within a cubic simulation box containing 0.15 M NaCl. After energy minimization, simulations were run at 300 K and 1 atm using a 2 fs timestep for 100 ns. Structural stability, RMSD, and secondary structure changes were analyzed post-simulation. Both structural prediction and molecular dynamics simulations have been performed according to the bioinformatics pipeline described in (Hashem S et.,al 2025) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Peptide Synthesis and Stability Assessment\u003c/h2\u003e\u003cp\u003ePeptides AF and GF were synthesized by NovoPro Bioscience Inc. (Shanghai, China). Peptide identity and purity were validated via HPLC and mass spectrometry, confirming purities of \u0026gt;\u0026thinsp;95% (AF: 95.435%; GF: 95.740%). Blood stability and half-life were predicted using the PlifePred (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webs.iiitd.edu.in/raghava/plifepred/\u003c/span\u003e\u003cspan address=\"https://webs.iiitd.edu.in/raghava/plifepred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) tool, yielding estimates of 954.61 s for AF and 840.31 s for GF.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cell culture\u003c/h2\u003e\u003cp\u003eMDA-MB-231 and MCF10 A cells were obtained from the American Type Culture Collection (ATCC, USA), and the primary neonatal fibroblast cells were kindly provided by Sidra Medicine-Qatar. Both MDA-MB-231 and primary neonatal fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and 1% L-Glutamine. All cell lines were cultured in the same optimum conditions in a humidified incubator adjusted to 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Alamar-Blue Cell Proliferation Assay\u003c/h2\u003e\u003cp\u003eMDA-MB-231 breast cancer cells, primary neonatal fibroblast, and MCF10A cells were seeded in 96-well tissue culture plates with 10,000 cells in each well, in triplicates using DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (100 \u0026micro;l/well). Cells were incubated overnight prior to treatment with different concentrations of AF and GF peptides (100 \u0026micro;g/ml, 200 \u0026micro;g/ml, 300 \u0026micro;g/ml, and 400 \u0026micro;g/ml). The 48-hour treatment duration was chosen to allow sufficient time for the peptides to exert measurable cytotoxic effects on the cells, consistent with prior studies evaluating antimicrobial peptides where incubation times ranging from 24 to 72 hours are commonly used to assess anticancer activity (e.g., Smith et al., 2021; Lee et al., 2022). After 48 hours, cells were washed with sterile PBS and 10% Alamar-Blue reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells and incubated for 4\u0026ndash;5 hours in the incubator according to the manufacturer\u0026rsquo;s protocol. Fluorescence measurements were taken at 560 nm and 590 nm using a TECAN infinite M200 plate reader after incubation with the dye. Relative cell proliferation was determined based on the fluorescence of treated cells relative to control cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Wound Healing Assay\u003c/h2\u003e\u003cp\u003eMDA-MB-231 cells were seeded in flat 6-well tissue culture plates and incubated overnight to allow for cell adherence and confluency before the addition of treatments. Upon reaching 50% confluency, as confirmed by microscopic observation, a linear scratch was introduced in each well using a sterile 200 \u0026micro;L pipette tip. The wells were subsequently washed with sterile phosphate-buffered saline (PBS) to eliminate cellular debris and undetached cells. Cells were then treated with AMPs at a concentration of 100 \u0026micro;g/mL, while untreated wells containing only culture medium served as controls. Images were captured at defined time points (0, 24, and 48 hrs) to monitor wound closure. Images were analyzed using ImageJ software, statistical analysis was performed, and the data were plotted as a graph.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Invasion Assay\u003c/h2\u003e\u003cp\u003eThe cell invasion assay was performed using the Boyden Matrigel Chamber technique to assess the effect of AF and GF peptides on the invasion capacity of the MDA-MB-231 TNBC cell line. The experiment was executed in 24-well Biocoat Matrigel invasion chambers (8 \u0026micro;m pore size, Corning, USA) as per the manufacturer\u0026rsquo;s instructions. Untreated cells (medium only) served as the control, and peptide-treated MDA-MB-231 cells were seeded into the top chambers, meanwhile, the bottom chambers were filled with DMEM prepared with 10% fetal bovine serum (FBS) to act as a chemoattractant. The system was incubated at 37\u0026deg;C for 48 hours. Subsequently, the top chambers were washed with sterile PBS, and non-invasive cells were lightly removed employing a sterile cotton swab. Cells that had migrated to the base of the top membrane were fixed with methanol and formaldehyde for 10 minutes and stained with 0.5% crystal violet stain. After washing with PBS, invaded cells were imaged under an inverted microscope (OPTIKA Microscopes, Ponteranica, Italy) across four predefined fields. The percentage inhibition of invasion was determined relative to untreated control cells and quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Western Blot analysis\u003c/h2\u003e\u003cp\u003eTo gain insight into the molecular pathways affected by treatment with AF and GF peptides, we examined key regulators involved in cell cycle progression and apoptosis, specifically β-catenin, Bcl-xl, and Bax. Briefly, MDA-MB-231 cells were seeded and treated with 100 ug/ul of AMP peptides for 48 hours. After 48 hours of treatment, protein lysates were extracted using RIPA buffer and quantified by Bradford assay. Equal amounts (30\u0026micro;g) of protein lysates were denatured at 95\u0026deg;C for 10 minutes and were resolved on 10% polyacrylamide gels and electroblotted onto PVDF membranes. The PVDF membranes were incubated overnight with the primary antibodies: antirabbit Bax (Cell Signaling Technology, CST #5023S, USA), anti Bcl-XL, and β-catenin (Cell Signaling Technology, CST #4970S). To confirm equal loading of protein samples, the membranes were re-probed with anti-rabbit β-actin (Cell Signaling Technology, CST #4970S, USA). Following overnight incubation, the membranes were washed twice in PBS and were then incubated for 2 hours in the corresponding secondary antibody.\u003c/p\u003e\u003cp\u003eImmunoreactivity was detected by using ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer. Images were captured using the iBright machine (ThermoFisher Scientific, USA) and relative quantification of protein expressions from images acquired from Western blotting were analyzed using ImageJ software. The intensity of the bands relative to the β-actin bands was used to calculate a relative expression of proteins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using SPSS Statistics 23.0 software. Data were shown as an average of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). Each experiment was repeated two times (n\u0026thinsp;=\u0026thinsp;2). Tukey\u0026rsquo;s post-hoc test was used to compare the difference between treated and untreated cells. and differences with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered as significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe predicted three-dimensional structures of the AF and GF peptides revealed distinct conformational characteristics. As illustrated in Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the AF peptide adopted a relatively compact and linear structure, whereas the GF peptide displayed a loop and a more flexible structure. Variations in the orientation of the key side chains between the two peptides suggest potential differences in their molecular interactions and biological activities. Both peptides exhibited defined secondary structural elements, including short α-helices and turns, with marked disparities in surface accessibility and possible interaction sites. Importantly, the enhanced structural flexibility and looped configuration of the GF peptide may facilitate dynamic engagement with diverse molecular targets, potentially explaining its pronounced anti-invasive activity observed in functional assays. Conversely, the AF peptide\u0026rsquo;s more rigid and linear structure may promote stable interactions with specific biomolecular partners, consistent with its strong anti-proliferative effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Establishment of the Optimal Dose of the AMPs:\u003c/h2\u003e\u003cp\u003eTo determine the effect of AF and GF peptides on breast cancer cell line, MDAMB-231 cells were treated with different concentrations of both peptides (100 \u0026micro;g/ml, 200 \u0026micro;g/ml, 300 \u0026micro;g/ml, and 400 \u0026micro;g/ml) for 48 hours. Primary neonatal fibroblast cells and MCF10A epithelial cell lines were used as controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effect of the AMPs on Cell Migration and Invasion:\u003c/h2\u003e\u003cp\u003eSubsequently, the impact of the antimicrobial peptides (AMPs) on breast cancer cell migration and invasion was evaluated using wound-healing and Boyden chamber assays, respectively. In the wound-healing assay, AMP-treated MDA-MB-231 cells demonstrated a pronounced decrease in migratory capacity compared to untreated controls. Quantitative measurements revealed that peptide exposure reduced wound closure by approximately 80% after 24 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating a significant disruption of cellular motility. Consistently, the Boyden chamber invasion assay showed that AMP treatment substantially impaired the ability of breast cancer cells to penetrate the Matrigel-coated membrane, with an observed\u0026thinsp;~\u0026thinsp;60% reduction in invasive relative to control cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings highlight the capacity of the AMPs to effectively suppress essential mechanisms underlying cancer metastasis. The engineered peptides inhibit key steps in breast cancer progression by impairing both migratory and invasive behaviors of tumor cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Molecular Mechanisms Mediating AMPs-Induced Apoptosis:\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular mechanisms underlying AMP-induced apoptosis, we examined the modulation of key apoptotic regulators by antimicrobial peptides (AMPs). Western blot analysis demonstrated a marked downregulation of the expression of β-catenin \u0026ndash; a protein implicated in promoting cell proliferation and survival \u0026ndash; in both AF and GF peptide-treated groups. Densitometric analysis indicated an approximate 3- to 3.75-fold reduction in β-catenin expression compared to untreated controls. Also, AMP treatment resulted in an upregulation of a pro-apoptotic gene, BAX, with expression increasing by ~\u0026thinsp;1.25-fold with AF peptide and ~\u0026thinsp;1.75-fold with GF peptide relative to control. In contrast, BCL-XL, an anti-apoptotic protein, was significantly suppressed, showing a\u0026thinsp;~\u0026thinsp;3- to 3.75-fold decrease following AMP exposure. These combined effects resulted in an elevated BAX/BCL-XL ratio, particularly in response to GF peptide treatment, suggesting a shift toward a pro-apoptotic intracellular environment. Our findings indicate that AMPs promote apoptosis by modulating β-catenin, BAX, and BCL-XL expression, thereby highlighting a potential mechanistic basis for their anticancer activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the extremophile origin of the AF and GF peptides, they may possess enhanced stability under physiological conditions, potentially overcoming a major challenge associated with synthetic AMPs like melittin, which are prone to rapid degradation and systemic toxicity (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). This study presents evidence of the anticancer properties of these peptides engineered from defensin-like proteins of extremophile plants against the triple-negative breast cancer (TNBC) cell line MDA-MB-231. Both AF and GF peptides effectively suppressed cell proliferation, migration, and invasion while exhibiting selective cytotoxicity toward cancer cells, with minimal impact on normal fibroblasts and breast epithelial cells.\u003c/p\u003e\u003cp\u003eAmong the two peptides, GF exhibited a modestly superior anticancer profile, leading to a\u0026thinsp;~\u0026thinsp;70% reduction in TNBC cell viability at 400 \u0026micro;g/mL, compared to ~\u0026thinsp;65% by AF at the same dose. Importantly, GF also demonstrated a more favorable selectivity, causing negligible cytotoxic effects (\u0026lt;\u0026thinsp;10%) in normal fibroblasts and MCF10A cells, while AF induced slightly higher nonspecific toxicity (~\u0026thinsp;12% cytotoxicity). These differences indicate GF\u0026rsquo;s potential advantage as a lead candidate for further in vivo exploration. The enhanced bioactivity of GF may be attributed to its structural dynamics; its flexible, looped architecture may promote more efficient interaction with cancer cell membranes, unlike the more linear and rigid conformation observed in AF peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNotably, our experimental results confirmed the \u003cem\u003ein-silico\u003c/em\u003e findings based on tools like AntiCP, ACPred, and CancerPPD, showing that both peptides show strong anticancer potential, but GF showed slightly better performance in decreasing migration and invasion, likely due to better internalization and interaction with intracellular targets.\u003c/p\u003e\u003cp\u003eMechanistically, both AF and GF influenced critical apoptotic pathway components, particularly by suppressing the expression of β-catenin and Bcl-xl while upregulating levels of the pro-apoptotic protein Bax, suggesting activation of mitochondrial-mediated apoptosis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Quantitative densitometry revealed a 3- to 3.75-fold reduction in β-catenin and Bcl-xl, along with a Bax upregulation of approximately\u0026thinsp;~\u0026thinsp;1.25-fold with AF and ~\u0026thinsp;1.75-fold for GF. These changes resulted in an increased BAX/BCL-XL ratio, a sign of apoptotic commitment. This mechanistic profile is consistent with prior findings that describe antimicrobial peptides as possessing dual functionality: not only antimicrobial defense but also selective cytotoxicity against cancer cells (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Well-characterized AMPs such as LL-37, magainins, and defensins preferentially target cancer cells by exploiting specific membrane features, including the abnormal externalization of negatively charged phosphatidylserines (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The potent anticancer effects observed for AF and GF likely stem from their net positive charge and amphipathic structures, which facilitate specific binding to and disruption of cancer cell membranes (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCorroborating our findings, Răileanu and Bacalum (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) reported that plant-derived antimicrobial peptides exhibit broad-spectrum cytotoxicity against colorectal cancer cells, primarily through mechanisms involving membrane disruption and mitochondrial impairment, highlighting the therapeutic potential of thermophile-sourced peptides in cancer treatment (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The observed suppression of β-catenin in our study is particularly significant, as dysregulation of the Wnt/β-catenin pathway is a well-established driver of TNBC progression and therapy resistance (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Our data are consistent with the work of Leite et al. (2018), who demonstrated that AMP-mediated β-catenin inhibition contributed to cell cycle arrest and induction of apoptosis in cancer cells (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, the substantial reduction in cell migration (80%) and invasion (60%) observed in this study supports previous findings showing that AMPs can inhibit epithelial-to-mesenchymal transition (EMT) and suppress metastatic behavior, hallmarks of highly invasive and aggressive TNBC (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Notably, AF and GF exhibited minimal cytotoxic effects on non-cancerous cells thereby demonstrating a favorable therapeutic window consistent with other AMP candidates of plant and animal origin (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Unlike melittin, which, despite its potent anticancer activity, is limited by hemolytic and systemic toxicities, both AF and GF were predicted and experimentally validated to be non-toxic and non-immunogenic \u003cem\u003ein silico\u003c/em\u003e, reinforcing their potential as safe and selective anticancer agents (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study constitutes an early attempt to exploit \u003cem\u003ein-silico\u003c/em\u003e engineered plant defensins as therapeutic agents against a TNBC cell line. Nonetheless, this study has several important limitations. The small sample size (n\u0026thinsp;=\u0026thinsp;2) reduces statistical robustness and may affect the reproducibility of the observed effects. Furthermore, the lack of \u003cem\u003ein vivo\u003c/em\u003e validation precludes a comprehensive assessment of pharmacokinetics, systemic toxicity, and immune system interactions. To address these gaps, future research should prioritize \u003cem\u003ein vivo\u003c/em\u003e validation using TNBC xenograft models to confirm therapeutic efficacy and safety within a physiological context. Additionally, the application of omics-based approaches, such as RNA sequencing and proteomic profiling, would enable a more detailed characterization of AMP-mediated signaling pathways and potential mechanisms of resistance.\u003c/p\u003e\u003cp\u003eMoreover, investigating combination therapies that integrate AF and GF peptides with immune checkpoint inhibitors or standard chemotherapeutic agents offers a promising strategy to overcome therapeutic resistance in TNBC and improve clinical outcomes. These synergistic approaches may exploit both the direct anticancer activity of AMPs and their capacity to modulate the tumor immune microenvironment, potentially enhancing overall treatment efficacy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights the anticancer potential of two antimicrobial peptides, AF and GF, engineered from extremophile plant defensins. Both peptides showed inhibition of proliferation, migration, and invasion in TNBC MDA-MB-231 cells, with minimal toxicity to normal breast epithelial cells and fibroblasts. Mechanistically, they induced mitochondrial apoptosis by downregulating β-catenin and Bcl-xl and upregulating Bax, consistent with AMP-mediated disruption of oncogenic pathways and cancer cell membranes. Compared to traditional chemotherapies, AF and GF offer targeted, less toxic alternatives, with potential application for TNBC. Their plant origin, small size, and predicted non-immunogenicity enhance translational promise. Future work should prioritize in vivo testing, omics analyses, and combination therapies to confirm efficacy and optimize clinical potential. Overall, AF and GF represent promising, rationally designed candidates for treating aggressive breast cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eNouralhuda Alateyah methodology, validation, formal analysis, writing—original draft preparation; \u0026nbsp;Sergio Crovella, conceptualization, software, writing—review and editing; \u0026nbsp;Richa Gill methodology, data curation, writing—original draft preparation; Maha Abdulla Al-Asmakh supervision, investigation, writing—review and editing; \u0026nbsp;Haissam Abou Saleh conceptualization, investigation, resources, project administration, supervision, writing—review and editing.\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by the Qatar National Research Foundation (GSRA11-L-1-0530-24081).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eAll raw data generated or analyzed during this study, including original gel images, are provided in the supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e-Wilkinson, L. \u0026amp; Gathani, T. Understanding Breast Cancer as a Global Health Concern. \u003cem\u003eBr. J. Radiol.\u003c/em\u003e \u003cb\u003e95\u003c/b\u003e (1130). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1259/bjr.20211033\u003c/span\u003e\u003cspan address=\"10.1259/bjr.20211033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Sung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCancer J. Clin.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e (3), 209\u0026ndash;249 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Sung, H. et al. Lei, S., Zheng, R., Zhang, S., Wang, S., Chen, R., Sun, K., \u0026hellip; Wei, W. (2021). Global patterns of breast cancer incidence and mortality: A population-based cancer registry data analysis from 2000 to 2020. \u003cem\u003eCancer Communications\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Fakhri, N. et al. Risk factors for breast cancer in women: an update review. \u003cem\u003eMed. Oncol.\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e (12). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12032-022-01804-x\u003c/span\u003e\u003cspan address=\"10.1007/s12032-022-01804-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Maughan, K. L., Lutterbie, M. A. \u0026amp; Ham, P. S. Treatment of Breast Cancer. \u003cem\u003eAm. Family Phys.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e (11), 1339\u0026ndash;1346 (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.aafp.org/pubs/afp/issues/2010/0601/p1339.html\u003c/span\u003e\u003cspan address=\"https://www.aafp.org/pubs/afp/issues/2010/0601/p1339.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Marqus, S., Pirogova, E. \u0026amp; Piva, T. J. Evaluation of the use of therapeutic peptides for cancer treatment. \u003cem\u003eJ. Biomed. Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (1), 1\u0026ndash;15 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Cassileth, B. R. \u0026amp; Deng, G. Complementary and Alternative Therapies for Cancer. \u003cem\u003eOncologist\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (1), 80\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1634/theoncologist.9-1-80\u003c/span\u003e\u003cspan address=\"10.1634/theoncologist.9-1-80\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Scalbert, A. et al. Databases on Food Phytochemicals and Their Health-Promoting Effects. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e (9), 4331\u0026ndash;4348. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf200591d\u003c/span\u003e\u003cspan address=\"10.1021/jf200591d\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Mani, S. et al. The Updated Review on Plant Peptides and Their Applications in Human Health. \u003cem\u003eInt. J. Pept. Res. Ther.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10989-022-10437-7\u003c/span\u003e\u003cspan address=\"10.1007/s10989-022-10437-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Kochhar, N. et al. Perspectives on the microorganism of extreme environments and their applications. \u003cem\u003eCurr. Res. Microb. Sci.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 100134. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.crmicr.2022.100134\u003c/span\u003e\u003cspan address=\"10.1016/j.crmicr.2022.100134\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Duarte-Mata, D. I. \u0026amp; Salinas-Carmona, M. C. Antimicrobial peptides\u0026acute; immune modulation role in intracellular bacterial infection. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2023.1119574\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2023.1119574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Nugrahadi, P. P., Hinrichs, J., Frijlink, H. W., Sch\u0026ouml;neich, C. \u0026amp; Avanti, C. Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (3), 935\u0026ndash;935. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pharmaceutics15030935\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics15030935\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Damodaran, S. \u0026amp; Parkin, K. L. Amino acids, peptides, and proteins. In Fennema\u0026rsquo;s food chemistry (235\u0026ndash;356). CRC. (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Shemetov, A. A., Nabiev, I. \u0026amp; Sukhanova, A. Molecular interaction of proteins and peptides with nanoparticles. \u003cem\u003eACS nano\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e (6), 4585\u0026ndash;4602 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Hoskin, D. W. \u0026amp; Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e. \u003cb\u003e1778\u003c/b\u003e (2), 357\u0026ndash;375 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Saha, S., Ghosh, M. \u0026amp; Dutta, S. K. Role of metabolic modulator Bet-CA in altering mitochondrial hyperpolarization to suppress cancer-associated angiogenesis and metastasis. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep23552\u003c/span\u003e\u003cspan address=\"10.1038/srep23552\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Prakash, E. \u0026amp; Gupta, D. K. \u003cem\u003eIn vitro\u003c/em\u003e study of extracts of Ricinus communis Linn on human cancer cell lines. \u003cem\u003eJ. Med. Sci. Pub Health\u003c/em\u003e. \u003cb\u003e2\u003c/b\u003e (1), 1520 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Parchebafi, A. et al. The dual interaction of antimicrobial peptides on bacteria and cancer cells; mechanism of action and therapeutic strategies of nanostructures. \u003cem\u003eMicrob. Cell. Fact.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12934-022-01848-8\u003c/span\u003e\u003cspan address=\"10.1186/s12934-022-01848-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Zare-Zardini, H. et al. From defense to offense: antimicrobial peptides as promising therapeutics for cancer. \u003cem\u003eFront. Oncol.\u003c/em\u003e 14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2024.1463088\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2024.1463088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Arafa, N. M., Moawad, M. \u0026amp; El-Shabrawi, H. E. Comparison of the organic and inorganic solvents effect on phenolic compounds extraction and the activity against breast carcinoma cell lines from callus cultures of Manihot esculenta. \u003cem\u003eInternational Journal of PharmTech Research\u003c/em\u003e, 2016,9(12): 380\u0026ndash;396. (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Manrique-Moreno, M., Santa-Gonz\u0026aacute;lez, G. A. \u0026amp; Gallego, V. Bioactive cationic peptides as potential agents for breast cancer treatment. \u003cem\u003eBiosci. Rep.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e (12). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1042/bsr20211218c\u003c/span\u003e\u003cspan address=\"10.1042/bsr20211218c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Lei, J. et al. The antimicrobial peptides and their potential clinical applications. \u003cem\u003eAm. J. Transl Res.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 3919\u0026ndash;3931 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Răileanu, M. \u0026amp; Bacalum, M. Cancer Wars: Revenge of the AMPs (Antimicrobial Peptides), a New Strategy against Colorectal Cancer. \u003cem\u003eToxins\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (7), 459 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Zhan, T., Rindtorff, N. \u0026amp; Boutros, M. Wnt signaling in cancer. \u003cem\u003eEMBO Rep.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (4), 698\u0026ndash;711 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Leite, M. L., da Cunha, N. B. \u0026amp; Costa, F. F. Antimicrobial peptides, nanotechnology, and natural metabolites as novel approaches for cancer treatment. \u003cem\u003ePharmacol. Ther.\u003c/em\u003e \u003cb\u003e183\u003c/b\u003e, 160\u0026ndash;176 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Scocchi, M., Mardirossian, M., Runti, G. \u0026amp; Benincasa, M. Non-Membrane permeabilizing modes of action of antimicrobial peptides on bacteria. \u003cem\u003eCurr. Top. Med. Chem.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 76\u0026ndash;88 (2016). [Google Scholar].\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Xiong, N., Wu, H. \u0026amp; Yu, Z. Advancements and challenges in triple-negative breast cancer: a comprehensive review of therapeutic and diagnostic strategies. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2024.1405491\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2024.1405491\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Guryanova, S. V. \u0026amp; Ovchinnikova, T. V. Immunomodulatory and allergenic properties of AMPs. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (5), 2499 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Mihaylova-Garnizova, R., Davidova, S., Hodzhev, Y. \u0026amp; Satchanska, G. Antimicrobial Peptides Derived from Bacteria: Classification, Sources, and Mechanism of Action against Multidrug-Resistant Bacteria. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (19), 10788. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms251910788\u003c/span\u003e\u003cspan address=\"10.3390/ijms251910788\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Marqus, S., Pirogova, E. \u0026amp; Piva, T. J. Evaluation of therapeutic peptides for cancer. \u003cem\u003eJ. Biomed. Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (1), 1\u0026ndash;15 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Sorokina, M. \u0026amp; Steinbeck, C. COCONUT: The COlleCtion of Open Natural prodUcTs database. \u003cem\u003eJ. Cheminform.\u003c/em\u003e, \u003cb\u003e12\u003c/b\u003e(20). (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://coconut.naturalproducts.net\u003c/span\u003e\u003cspan address=\"https://coconut.naturalproducts.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-TCM Database@Taiwan. (Accessed. May (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tcm.cmu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://tcm.cmu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-South African Natural Compounds Database (SANCDB). (Accessed. May (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sancdb.rubi.ru.ac.za/\u003c/span\u003e\u003cspan address=\"http://sancdb.rubi.ru.ac.za/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-South African Natural Compounds Database (SANCDB). (Accessed. El-Serag, H., Kanwal, F., Ning, J., Powell, H., Khaderi, S., Singal, A. G., Asrani,S., Marrero, J. A., Amos, C. I., Thrift, A. P., Luster, M., Alsarraj, A., Olivares,L., Skapura, D., Deng, J., Salem, E., Najjar, O., Yu, X., Duong, H., Scheurer, M.E., \u0026hellip; Kaochar, S. (2024). Serum biomarker signature is predictive of the risk of hepatocellular cancer in patients with cirrhosis. \u003cem\u003eGut\u003c/em\u003e, gutjnl-2024-332034. Advance online publication. https://doi.org/10.1136/gutjnl-2024-332034.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e-Li, Y. Mechanism of melittin for anti-tumor effects: Research status and future perspectives. \u003cem\u003eJ. Clin. Technol. Theory\u003c/em\u003e. \u003cb\u003e2\u003c/b\u003e (1), 33\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.54254/3049-5458/2025.21076\u003c/span\u003e\u003cspan address=\"10.54254/3049-5458/2025.21076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Antimicrobial peptides, Bioinformatics, Cell proliferation, Migration, Cell invasion","lastPublishedDoi":"10.21203/rs.3.rs-7426302/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7426302/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eConventional strategies for treating breast cancer, including chemotherapy and radiotherapy, often face critical challenges such as severe adverse effects and the development of therapeutic resistance. These limitations have driven interest in exploring alternative and complementary approaches with improved safety profiles and targeted efficacy. Among these, antimicrobial peptides (AMPs), naturally occurring components of the innate immune system, are increasingly recognized for their selective cytotoxic activity against cancer cells and potential immunomodulatory effects.\u003c/p\u003e\u003cp\u003eIn particular, AMPs derived from extremophiles\u0026mdash;organisms adapted to survive in extreme environmental conditions\u0026mdash;present compelling therapeutic opportunities. These peptides tend to exhibit remarkable stability and enzymatic resistance, which enhances their functionality under physiological conditions. Compared to plant-derived defensins, which may suffer from poor bioavailability despite their cysteine-rich profiles, and bacterial or synthetic AMPs, which can present selectivity or toxicity issues, extremophile-inspired peptides offer a more robust and targeted anticancer profile.\u003c/p\u003e\u003cp\u003eIn this study, bioinspired short antimicrobial peptides (BSAMPs) were designed in silico using structural motifs from extremophilic organisms to enhance anticancer activity and tumor specificity. When tested on MDA-MB-231 triple-negative breast cancer (TNBC) cells, BSAMPs significantly reduced cell viability (by 70% at 400 \u0026micro;g/ml), inhibited cellular migration (by 80%), and decreased invasion (by 60%), while showing minimal toxicity (\u0026le;\u0026thinsp;10%) to non-malignant fibroblasts and MCF10A epithelial cells. Mechanistically, BSAMPs promoted apoptosis via mitochondrial pathways, downregulating β-catenin and Bcl-xl while increasing Bax expression. While these results are currently limited to in vitro models, they highlight the therapeutic potential of BSAMPs and justify further validation in in vivo systems for breast cancer treatment.\u003c/p\u003e","manuscriptTitle":"Anti-Proliferative Effects of Bioinspired Antimicrobial Peptides on MDA-MB-231 Triple- Negative Breast Cancer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 13:12:28","doi":"10.21203/rs.3.rs-7426302/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"efc50885-dc28-4a98-a879-a6f133eb3179","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-15T01:07:11+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55182500,"name":"Biological sciences/Biochemistry"},{"id":55182501,"name":"Biological sciences/Biotechnology"},{"id":55182502,"name":"Biological sciences/Cancer"},{"id":55182503,"name":"Biological sciences/Drug discovery"},{"id":55182504,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-15T01:24:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 13:12:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7426302","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7426302","identity":"rs-7426302","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}