Hydroxychavicol in Combination with 5-Fluorouracil Induced Apoptosis by Inhibiting Purine Metabolism in HT-29 and DLD-1 Cell Lines.

Objective: To elucidate the effect of hydroxychavicol (HC) in combination with 5-fluorouracil (5-FU) on purine metabolism and apoptosis in colorectal cancer cell lines HT-29 and DLD-1.

Methods: The viability of HT-29 and DLD-1 cells when treated with HC, (0-1,000 µmol/L) 5-FU (0-100 µmol/L) alone, and HC+5-FU for 24 and 48 h was determined. Hypoxanthine (HPX) and xanthine oxidoreductase (XOR) were evaluated, as well as reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP). The expression levels of genes including nucleoside transporters equilibrative nucleotide transport 1 and 2 (ENT1 and ENT2), the proapoptotic gene Caspase-3 (CASP3), and the anti-apoptotic gene BCL2 were analysed by quantitative polymerase chain reaction.

Results: Both HPX and XOR levels in cells treated with HC+5-FU were significantly decreased (P<0.05) after 24 and 48 h compared to control cells. ROS levels in HT-29 and DLD-1 treated with HC+5-FU for 24 and 48 h were 26.2% and 21.4%, and 9.1% and 20.5%, respectively, significantly lower than control cells. MMP assays indicated mitochondrial depolarisation. In HT-29 cells, ENT1 and BCL2 were downregulated at 24 h, and CASP3 was upregulated at 48 h. In DLD-1 cells, ENT1 and ENT2 were downregulated, while CASP3 showed a transient decrease at 24 h.

Conclusions: The combination of HC + 5-FU demonstrated synergistic effects in HT-29 and DLD-1 cells, disrupting oxidative balance and purine metabolism, as reflected in reduced hypoxanthine levels, XOR activity, and ROS production. This treatment also induced mitochondrial membrane depolarisation and altered apoptosis-related gene expression, supporting its role in apoptosis induction.