Methanolic extract of Karo-derived Trigonella foenum-graecum induces apoptosis and cell-cycle arrest in breast cancer cells through modulation of PI۳K/AKT/mTOR signaling

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 p۵۳-independent mechanisms and demonstrating its capacity to overcome initial therapeutic resistance by simultaneously blocking PI۳K/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 ۲۴ and ۴۸ hours. Furthermore, apoptosis, cell cycle progression, and the phosphorylation of DNA damage and PI۳K/AKT/mTOR marker proteins were analyzed using flow cytometry and Western blot. Results: METFG demonstrated significant time-dependent cytotoxicity, reducing the IC₅₀ in MCF۷ -/-p۵۳ cells from ۱۷۲.۵±۲.۴ µg/mL at ۲۴ hours to ۱۳۴.۳±۷.۲ µg/mL at ۴۸ hours. The extract successfully induced apoptosis and triggered a cell-cycle phase shift, increasing the percentage of G۲/M-phase cells to ۲۹% in MDA-MB cell lines. Additionally, METFG significantly reduced phosphorylation of CHK۱, CHK۲, 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 PI۳K/AKT/mTOR pathways. Future investigations must assess its selective toxicity against normal mammary epithelial cells to establish its safety.