A multifunctional 5-fluorouracil prodrug: synthesis, biological evaluation, and antitumor mechanism investigation

As FDA-approved chemotherapeutic agents, paclitaxel (PTX) and 5-fluorouracil (5-FU) are widely used in the clinic but suffer from dose-limiting toxicities. To overcome these limitations, we designed and synthesized two novel 5-FU prodrugs conjugated with the microtubule inhibitor PTX. The hybrids exhibited potent cytotoxicity against cancer cell lines while demonstrating favorable selectivity over normal human cells, outperforming the individual agents or their combinations. As a representative, FP2 potently induced DNA damage through downregulation of thymidylate synthase (TS), arrested the cell cycle at S-phase, and suppressed metastasis of HCT-116 cells. Moreover, FP2 triggered mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential, increased ROS generation, downregulation of Bcl-2, and upregulation of Bax. FP2 also triggered oxidative stress, elevating MDA and NO levels, depleting GSH and SOD, downregulating Nrf2, and upregulating HO-1. Additionally, FP2 activated the PERK/ATF4/CHOP signaling pathway, leading to endoplasmic reticulum (ER) stress. Furthermore, FP2 induced autophagic cell death, as evidenced by the formation of autophagosomes, an increased LC3 and decreased p62 expression. Collectively, these findings established FP2 as a novel compound with a promising in vitro anticancer profile that promotes HCT-116 cell death through an integrated mechanism involving DNA damage, mitochondrial dysfunction, ER stress, oxidative stress, and autophagy.