Abstract Breast cancer (BrCa), in particular, triple-negative breast cancer (TNBC), is a complex disease to treat and respond to conventional chemotherapy. Recently, thymidylate synthase (TYMS), has gained attention as important enzyme whose inhibition has been reported to be effective in the inhibition of the de novo production of 2 2-deoxythymidine-5-monophosphate (dTMP), involved in DNA synthesis in cancer cells. TYMS is a known target of 5-fluorouracil (5-FU), a widely used antimetabolite chemotherapeutic agent, effective against various tumors. Nevertheless, clinical application is commonly compromised by serious side effects, resistance to drugs, and upregulation of TYMS. Additionally, drug-induced activation of NF-κB contributes to the overexpression of TYMS, thereby promoting tumor cell proliferation and chemotherapeutic resistance. Therefore, identifying new anti-cancer agents that can be used alongside 5-FU to target cancer cells while sparing healthy cells is crucial. Recently, honokiol (HNK), a natural biphenolic compound from Magnolia officinalis, has shown promise as an anti-inflammatory, antioxidant, and anti-tumor agent. HNK is known to inhibit NF-κB activation in response to various stimuli. In this study, we investigated the synergistic effects of HNK and 5-FU in TNBC cells (MDA-MB-468 and MDA-MB-231), with a focus on the suppression of NF-κB-mediated TYMS expression. To determine the optimal IC50 values and synergy, TNBC cells were treated with HNK, 5-FU, or a combination of both at various time points and dosages, followed by a cell viability assay (MTT assay). Our results, which include cell cytotoxicity assays, apoptosis assays, cell cycle assays, RT-qPCR analysis, and Western blots, demonstrate that the combined treatment is most effective in inhibiting TYMS, reducing cell proliferation, and inducing apoptosis. The combination inhibits NF-κB p65 activation, thereby disrupting the transcriptional regulation of target genes associated with cell survival and communication with TYMS. This dual inhibition causes a cell cycle arrest at the G0/G1 phase, and upregulates the cell cycle inhibitors, including p21, which, in turn, downregulates the expression of the cyclin D1/CDK2 and CDK4 complexes. These findings suggest that NF-κB may stimulate TYMS transcription under inflammatory conditions. Additionally, the combinatorial treatment increased the number of apoptotic cells compared to the individual or control groups. These results suggest that the combination of HNK and 5-FU may alter DNA synthesis and repair, predisposing TNBC cells to apoptosis. This dual-targeting strategy, which involves direct inhibition of TYMS by 5-FU and suppression of its NF-κB-mediated upregulation by HNK, presents a promising approach to overcoming chemoresistance and improving therapeutic outcomes in patients with BrCa. Citation Format: Santosh K. Singh, Rajesh Singh. Enhancing the efficacy of 5-fluorouracil in targeting thymidylate synthase (TYMS) for breast cancer treatment using honokiol abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 588.
Singh et al. (Fri,) studied this question.