Objective:

2

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:

2

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 pro-apoptotic gene Caspase-3 (CASP3)

and the anti-apoptotic gene BCL2 were analysed by quantitative polymerase chain reaction.

Results:

2

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:

2

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.