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Volume 18
Issue 8
10.3390/ph18081203
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

PEPAD: A Promising Therapeutic Approach for the Treatment of Murine Melanoma (B16F10-Nex2)

by
Camila de Oliveira Gutierrez
1,
Rafael Araujo Pereira
1,
Claudiane Vilharroel Almeida
1,
Luís Henrique de Oliveira Almeida
1,
Caio Fernando Ramalho de Oliveira
1,
Ana Cristina Jacobowski
1,
Patrícia Maria Guedes Paiva
2,
Durvanei Augusto Maria
3,
Rodrigo Juliano Oliveira
4,
Thais de Andrade Farias Rodrigues
4,
Tamaeh Monteiro-Alfredo
1,
Ana Paula de Araújo Boleti
1 and
Maria Ligia Rodrigues Macedo
1,*
1
Laboratory of Protein Purification and Their Biological Functions, Faculty of Pharmaceutical Sciences, Food and Nutrition, Federal University of Mato Grosso do Sul (UFMS), Campo Grande 79070-900, MS, Brazil
2
Department of Biochemistry, Federal University of Pernambuco, Recife 50670-420, PE, Brazil
3
Laboratory of Molecular Biology, Butantan Institute, São Paulo 03178-200, SP, Brazil
4
Center for Stem Cell Studies, Cell Therapy and Genetic Toxicology (CeTroGen), Faculty of Medicine, Federal University of Mato Grosso do Sul (UFMS), Campo Grande 79070-900, MS, Brazil
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2025, 18(8), 1203; https://doi.org/10.3390/ph18081203 (registering DOI)
Submission received: 14 July 2025 / Revised: 6 August 2025 / Accepted: 8 August 2025 / Published: 14 August 2025
(This article belongs to the Special Issue Peptide Synthesis and Drug Development: Exploring Progress and Potential)
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Abstract

Background/Objectives: Cancer is one of the leading causes of death worldwide, and skin cancer is especially prevalent and lethal in Brazil. Despite advancements in treatment, there is still a need for new anticancer agents that are effective, selective, and less toxic. This study aimed to evaluate the cytotoxic and therapeutic potential of the peptide PEPAD. Methods: The cytotoxicity of PEPAD was assessed by MTT assay in murine melanoma (B16F10-Nex2), human melanoma (SK-MEL-28), breast (MCF-7), and cervical (HeLa) cancer cell lines. Selectivity was evaluated in healthy cells (RAW 264.7 and FN1). Morphological changes were analyzed by microscopy. Cell migration was assessed using scratch assays. Apoptotic features were evaluated using MitoTracker Deep Red, NucBlue, CaspACETM labeling, and flow cytometry. Immunogenic cell death was investigated by calreticulin and HMGB1 release. Molecular dynamics simulations explored peptide structure and interaction with lipid membranes. Results: PEPAD showed IC50 values of 7.4 µM and 18 µM in B16F10-Nex2 and SK-MEL-28 cells, respectively, and >60 µM in MCF-7 and HeLa cells. Low toxicity was observed in healthy cells (IC50 > 56 µM), indicating high selectivity. Apoptotic morphology and reduced cell migration were observed. Flow cytometry and fluorescence probes confirmed apoptosis and mitochondrial swelling. Calreticulin and HMGB1 release indicated immunogenic cell death. Simulations showed that PEPAD maintains a stable α-helical conformation and interacts with membranes. Conclusions: These findings highlight PEPAD’s selective cytotoxicity and its potential as an anticancer agent with apoptotic and immunogenic properties, making it a promising candidate for therapeutic development.
Keywords: peptide anticancer; cell migration; selectivity; immunogenic cell death; Adenanthera pavonina peptide anticancer; cell migration; selectivity; immunogenic cell death; Adenanthera pavonina
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MDPI and ACS Style

Gutierrez, C.d.O.; Pereira, R.A.; Almeida, C.V.; Almeida, L.H.d.O.; de Oliveira, C.F.R.; Jacobowski, A.C.; Paiva, P.M.G.; Maria, D.A.; Oliveira, R.J.; de Andrade Farias Rodrigues, T.; et al. PEPAD: A Promising Therapeutic Approach for the Treatment of Murine Melanoma (B16F10-Nex2). Pharmaceuticals 2025, 18, 1203. https://doi.org/10.3390/ph18081203

AMA Style

Gutierrez CdO, Pereira RA, Almeida CV, Almeida LHdO, de Oliveira CFR, Jacobowski AC, Paiva PMG, Maria DA, Oliveira RJ, de Andrade Farias Rodrigues T, et al. PEPAD: A Promising Therapeutic Approach for the Treatment of Murine Melanoma (B16F10-Nex2). Pharmaceuticals. 2025; 18(8):1203. https://doi.org/10.3390/ph18081203

Chicago/Turabian Style

Gutierrez, Camila de Oliveira, Rafael Araujo Pereira, Claudiane Vilharroel Almeida, Luís Henrique de Oliveira Almeida, Caio Fernando Ramalho de Oliveira, Ana Cristina Jacobowski, Patrícia Maria Guedes Paiva, Durvanei Augusto Maria, Rodrigo Juliano Oliveira, Thais de Andrade Farias Rodrigues, and et al. 2025. "PEPAD: A Promising Therapeutic Approach for the Treatment of Murine Melanoma (B16F10-Nex2)" Pharmaceuticals 18, no. 8: 1203. https://doi.org/10.3390/ph18081203

APA Style

Gutierrez, C. d. O., Pereira, R. A., Almeida, C. V., Almeida, L. H. d. O., de Oliveira, C. F. R., Jacobowski, A. C., Paiva, P. M. G., Maria, D. A., Oliveira, R. J., de Andrade Farias Rodrigues, T., Monteiro-Alfredo, T., Boleti, A. P. d. A., & Macedo, M. L. R. (2025). PEPAD: A Promising Therapeutic Approach for the Treatment of Murine Melanoma (B16F10-Nex2). Pharmaceuticals, 18(8), 1203. https://doi.org/10.3390/ph18081203

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