Quercetin and 5-Fluorouracil Synergistically Suppress ZEB1 and Restore Epithelial Integrity in Triple-Negative Breast...

Triple-negative breast cancer (TNBC) remains a therapeutic challenge due to its metastatic features and resistance to conventional therapies. This study investigated the synergistic potential of quercetin (QC), a natural flavonoid, and 5-fluorouracil (5-FU) in targeting epithelial-mesenchymal transition (EMT) to inhibit proliferation and migration of the MDA-MB-231 TNBC cell line.

Cytotoxicity and selectivity were assessed using the MTT assay in MDA-MB-231 cells and normal MRC-5 fibroblasts. Synergy was quantified using combination index (CI) values. Scratch assay was assessed for migration inhibition, while quantitative RT-PCR analyzed EMT-related gene expression (Vimentin, ZEB1, N-cadherin, and E-cadherin). Western blotting confirmed protein expression of ZEB1 and E-cadherin following treatment with QC (50 μM), 5-FU (25 nM), or their combination.

QC and 5-FU demonstrated concentration- and time-dependent cytotoxicity in MDA-MB-231 cells, with QC being highly selective for cancer cells. Toxicity in normal MRC-5 fibroblasts occurred only at 800 µM (25% viability). The combination of 50 µM of QC and 25 nM of 5-FU exhibited the strongest synergy (CI = 0.08), reducing viability by 75% and migration by 87.8%. QC alone downregulated ZEB1, Vimentin, and N-cadherin, while 5-FU further reduced these markers. Combined treatment enhanced suppression (ZEB1: 0.46-fold; Vimentin: 0.47-fold; N-cadherin: 0.68-fold) and significantly upregulated E-cadherin (3.75-fold), indicating EMT reversal. Western blotting confirmed decreased ZEB1 and increased E-cadherin by over four-fold with QC + 5-FU, indicating a shift to an epithelial phenotype.

Combining QC and 5-FU effectively inhibited TNBC proliferation and migration while minimizing toxicity to normal cells. This dual-action strategy offers a promising low-toxicity therapeutic approach for aggressive TNBC.