Escherichia coli DH5α-loaded fenugreek extract: an effective anticancer combination against human breast cancer cell lines.

Breast cancer is the most frequently diagnosed cancer in women and remains a leading cause of cancer-related mortality worldwide. Recently, bacteria-based delivery systems have emerged as promising strategies for targeted cancer therapy due to their biocompatibility, low systemic toxicity, and ability to overcome drug resistance. In this study, bacterial ghosts (BGs) derived from Escherichia coli DH5-α were successfully prepared using a modified sponge-like reduced protocol, achieving approximately 92% reduction in optical density and releasing 246.3 ± 5.46 µg/mL DNA and 2.5 ± 0.054 mg/mL proteins, confirming efficient cytoplasmic evacuation. Heat-killed E. coli (HKEc) was prepared via thermal inactivation. Ethanolic extract of fenugreek seeds (Trigonella foenum-graecum) was obtained with a yield of 14% and characterized by GC-MS, identifying 26 bioactive compounds, including linoleic acid ethyl ester (19.39%) and β-sitosterol (14.14%). LC-MS/MS analysis further confirmed the presence of key metabolites such as trigonelline, diosgenin, orientin, and vitexin. The extract was successfully loaded into BGs and HKEc with loading capacities of 14 µg/mg and 11.6 µg/mg, and entrapment efficiencies of 21% and 13.4%, respectively. In vitro release studies demonstrated sustained release behavior from BGs, with only 14% released after 4 h at pH 7.4 compared to 90% for free extract, indicating effective controlled delivery. Cytotoxicity analysis using the WST-1 assay revealed enhanced anticancer activity of FEE-loaded BGs, with IC₅₀ values of 1.2 mg/mL in MDA-MB-231 and 0.87 mg/mL in ZR-75-1 cells, compared to 2.2 and 1.74 mg/mL for free extract, respectively. Mechanistically, treatment resulted in significant downregulation of STAT3, mTOR, and β-catenin, alongside upregulation of STAT5, and a twofold increase in caspase-3 activity, confirming apoptosis induction. Overall, these findings highlight E. coli-derived bacterial ghosts as an effective and low-cost delivery platform for plant-derived anticancer compounds, offering enhanced therapeutic efficacy and controlled release for breast cancer treatment. KEY POINTS: • Escherichia coli ghosts are safe, versatile carriers with high drug-loading capacity. • E. coli ghosts enhance natural extract delivery, improving anticancer efficacy and targeting. • Fenugreek extract-loaded E. coli ghosts modulate key cancer pathways.