Results
AC potentiated the antitumor efficacy of 5-FU in vivo and did not exacerbate 5-FU-associated hepatic or renal biochemical toxicity under the tested conditions. In vitro, AC-containing serum extracts inhibited CRC cell viability, induced reversible G2/M-phase arrest, and enhanced 5-FU sensitivity in HCT116, CT26, and HCT15 cells. AC treatment caused limited early apoptosis, whereas delayed apoptosis was observed after prolonged exposure, suggesting that cell-cycle arrest represents the primary early response. RNA-seq and integrative analyses showed that AC suppressed pathways associated with DNA replication, mismatch repair, ribosome biogenesis, and cell-cycle progression. AC downregulated key regulators involved in cell-cycle control, DNA repair, and stress-survival signaling, including PLK1, CDK1, CCNB1, CDC25A, c-JUN, MSH2, BRCA1, and p-AKT. Combination treatment further increased γ-H2AX accumulation and reduced cell-cycle regulatory proteins. Functional validation indicated that c-JUN overexpression partially attenuated AC-mediated 5-FU sensitization, whereas pharmacological AKT inhibition partially mimicked the sensitizing effect of AC. Among the serum-exposed constituents, Demethoxycurcumin and Kumatakenin were identified as bioavailable active compounds that partially recapitulated the effects of AC by inducing G2/M-phase arrest and enhancing the antiproliferative activity of 5-FU. Molecular docking, molecular dynamics simulation, and CETSA supported their target engagement with cell-cycle regulators, particularly CCNB1 and CDK1.
