Enhancing colorectal cancer treatment with a triple therapy approach involving 5-fluorouracil, thymoquinone, Polygonum minus.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality globally, with chemoresistance to 5-fluorouracil (5-FU) and dose-limiting toxicities posing major therapeutic challenges. This study investigates the synergistic potential of 5-FU combined with bioactive compounds from Polygonum minus (PM) extract and thymoquinone (TQ) in free and calcium carbonate (CaCO)-nanoparticle-encapsulated forms (5-FU-CaCO₃, TQ-CaCO) against SW480 and SW620 CRC cell lines. Cytotoxicity (MTT assay), combination index (CI), and apoptosis/necrosis (Annexin V/PI flow cytometry) were evaluated at 24 -72 h. Both free-drug (FTP: 5-FU+TQ+PM) and CaCO-encapsulated (FT-NP/PM: 5-FU-CaCO+TQ-CaCO+PM) combinations exhibited dose- and time-dependent cytotoxicity in SW480 and SW620 cells. In SW620 cells, the encapsulated FT-NP/PM showed superior cytotoxicity compared to the free combination, with IC values decreasing from 11.75 µg/mL (24 h) to 6.70 µg/mL (72 h), whereas FTP declined from 17.75 µg/mL to 6.54 µg/mL. In SW480 cells, both formulations demonstrated comparable effects, maintaining cytotoxicity over time (FTP: 15.38-7.68 µg/mL; FT-NP/PM: 16.98-8.81 µg/mL). Combination index analysis confirmed strong synergism at lower concentrations (CI < 0.4) in both cell lines, with FT-NP/PM showing slightly greater synergy, particularly in SW620. Higher concentrations tended toward additive or antagonistic effects. Apoptosis and necrosis analyses further supported these results, where FT-NP/PM induced higher necrosis and late apoptosis compared to FTP, especially after 72 h in SW620 cells. In SW480, apoptosis remained the predominant mode of cell death, with nanoencapsulation sustaining the response over time. These findings underscore the therapeutic advantage of combining natural compounds with conventional chemotherapy. Overall, the synergistic interaction between 5-FU, TQ, and PM, enhanced by CaCO-mediated delivery, improved cytotoxic and apoptotic effects, especially in the chemoresistant SW620 cells, suggesting the potential of FT-NP/PM as an optimized combination strategy for colorectal cancer treatment.