Shahrekord University of Medical Sciences Journal of Herbmed Pharmacology 2345-5004 15 4 2026 10 01 Methanolic extract of Karo-derived Trigonella foenum-graecum induces apoptosis and cell-cycle arrest in breast cancer cells through modulation of PI3K/AKT/mTOR signaling 445 457 10.34172/jhp.53833 EN Cicik Suriani https://orcid.org/0009-0008-2816-9410 Fauziyah Harahap https://orcid.org/0000-0003-0860-5736 Syahmi Edi https://orcid.org/0009-0001-8551-9746 Ahmad Shafwan S Pulungan https://orcid.org/0000-0002-4628-3515 Armansyah Maulana Harahap https://orcid.org/0000-0001-5221-7648 Journal Article 10.34172/jhp.53833 2026 04 15 2026 08 10 Introduction: Breast cancer therapy often faces challenges with drug resistance and severe side effects, highlighting the urgent need for novel therapeutic agents. Trigonella foenum-graecum (TFG) contains steroid sapogenins with potential as anti-cancer agents. This study assessed the methanolic extract of TFG (METFG) against eight breast cancer cell lines. The novelty lies in uncovering p53-independent mechanisms and demonstrating its capacity to overcome initial therapeutic resistance by simultaneously blocking PI3K/AKT/mTOR survival pathways. Methods: TFG seeds were extracted using methanol, and the chemical components were analyzed via GC-MS. Cytotoxicity across eight breast cancer cell lines was evaluated using MTT assays across 24 and 48 hours. Furthermore, apoptosis, cell cycle progression, and the phosphorylation of DNA damage and PI3K/AKT/mTOR marker proteins were analyzed using flow cytometry and Western blot. Results: METFG demonstrated significant time-dependent cytotoxicity, reducing the IC₅₀ in MCF7 -/-p53 cells from 172.5±2.4 µg/mL at 24 hours to 134.3±7.2 µg/mL at 48 hours. The extract successfully induced apoptosis and triggered a cell-cycle phase shift, increasing the percentage of G2/M-phase cells to 29% in MDA-MB cell lines. Additionally, METFG significantly reduced phosphorylation of CHK1, CHK2, AKT, and mTOR, suggesting a potential inhibition of the DNA damage response and cell survival signaling. Conclusion: The endemic Karo METFG exhibits significant cytotoxic activity by inducing apoptosis, promoting cell cycle arrest, and suppressing DDR and PI3K/AKT/mTOR pathways. Future investigations must assess its selective toxicity against normal mammary epithelial cells to establish its safety.