Vet World   Vol.18   August-2025  Article - 8 

Annonacin induces apoptosis and modulates programmed death-ligand 1 and interferon-gamma expression in triple-negative breast cancer: Integrated in silico and in vitro analysis

Background and Aim: Triple-negative breast cancer (TNBC) presents therapeutic challenges due to its aggressive nature and lack of targeted treatments. Programmed death-ligand 1 (PD-L1) and interferon-gamma (IFN-γ) are key immune modulators in tumor immune evasion. Annonacin, a natural acetogenin from Annona species, has shown promising anticancer properties, though its immunomodulatory mechanisms remain underexplored. This study aimed to investigate the dual apoptotic and immunomodulatory effects of annonacin on PD-L1 and IFN-γ expression using combined molecular docking and in vitro assays in TNBC (4T1) cells.

Materials and Methods: Molecular docking simulations were conducted to assess annonacin’s interaction with PD-L1 (Protein Data Bank [PDB] ID: 6PV9) and IFN-γ (PDB ID: 1FG9). In vitro experiments using 4T1 cells involved 3-(4,-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays for cytotoxicity, Annexin V-fluorescein isothiocyanate staining for apoptosis, and flow cytometry to analyze PD-L1 and IFN-γ expression following treatment with annonacin (1.5–25 μg/mL).

Results: Docking scores indicated moderate binding affinities of annonacin to IFN-γ (–5.2 kcal/mol) and PD-L1 (–5.0 kcal/mol), involving both hydrogen bonds and hydrophobic interactions. Annonacin exhibited a selective cytotoxic effect on 4T1 cells with a half-maximal inhibitory concentration of 15 μg/mL and a selectivity index of 2.6. Apoptosis was induced in a concentration-dependent manner, with late apoptotic populations peaking at 25 μg/mL. PD-L1 and IFN-γ expression peaked at 6.25 μg/mL, followed by a decline at higher doses, suggesting a dose-dependent immunomodulatory shift from immune activation to suppression.

Conclusion: Annonacin modulates immune checkpoint (PD-L1) and cytokine (IFN-γ) expression while promoting apoptosis in TNBC cells. These results highlight its potential as a dual-function anticancer agent, warranting further preclinical evaluation for use as a monotherapy or in combination with immunotherapies.

Keywords: annonacin, apoptosis, cytotoxicity, immune modulation, in silico, interferon-gamma, programmed death-ligand 1, triple-negative breast cancer.

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How to cite this article: Yunani R, Sudjarwo SA, Effendi MH, Kurnijasanti R, and Hidayah N (2025) Annonacin induces apoptosis and modulates programmed death-ligand 1 and interferon-gamma expression in triple-negative breast cancer: Integrated in silico and in vitro analysis, Veterinary World, 18(8): 2241-2251.

Received: 20-04-2025   Accepted: 14-07-2025   Published online: 09-08-2025

Corresponding author: Sri Agus Sudjarwo    E-mail: ags158@yahoo.com

DOI: 10.14202/vetworld.2025.2241-2251

Copyright: Yunani, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.