Hydroxychavicol in combination with 5- fluorouracil modulates purine metabolism in colorectal cancer cells by reducing hypoxanthine, xanthine oxidoreductase, and reactive oxygen species levels.

The development of resistance to 5-fluorouracil (5-FU) led to the need for combination therapies in colorectal cancer patients. In recent years, natural products have gained a lot of attention as adjuvants for chemotherapies due to their multi-targeting properties. Hydroxychavicol (HC), a bioactive compound found in Piper betle leaves, was reported to be a potent anti-apoptotic agent. The mechanism by which HC induces cell death is not fully understood, particularly in relation to purine metabolism. Hence, this study aims to elucidate the effect of 5-FU, HC, and HC in combination with 5-FU (HC+5-FU) in modulating the purine metabolism pathway of colorectal cancer (CRC) cell lines, HT-29 (Duke’s B) and DLD-1 (Duke’s C). The inhibitory concentration (IC50) of HC, 5-FU, and HC+5-FU was determined by measuring the CRC cell viability via MTT assay. The most effective doses were then chosen in the subsequent experiments. The effect of HC+5-FU on the purine metabolism pathway was determined by measuring the levels of hypoxanthine (HPX), xanthine oxidoreductase (XOR) via Amplex xanthine/xanthine oxidoreductase assay, reactive oxygen species (ROS) via DCFDA assay in HT-29 and DLD-1 cells and elucidating the mitochondrial activity by analysing its mitochondrial membrane potential via JC-1 assay. Furthermore, to validate the findings on functional analysis, the expression of genes, equilibrative nucleoside transporter 1 and 2 (ENT1 and ENT2), caspase 3 (CASP3), and B-cell lymphoma 2 (BCL2) were measured via qPCR. ENT1 and ENT2 transport hypoxanthine, whereas CASP3 and BCL2 regulate apoptotic cell death. The results showed that the treatments affect the cell viability of HT-29 and DLD-1 treated for 24 and 48h in a time-dose-dependent manner. The inhibition concentration of 50% (IC50) of HC in HT-29 was 695.3 µM (24h) and 201.3 µM (48h), while for 5-FU, 244.8 µM (24h), and 123.1 µM (48h) respectively. In DLD-1, the IC50 of HC were 718.3 µM (24h) and 225.1 µM (48h), while for 5-FU, 98.3 µM (24h), 72.7 µM (48h). Following this, sub-effective doses of HC and 5-FU (55 µM) were combined to determine the IC50 of combination treatment. The IC50 of HC + 55 µM 5-FU treatment for HT-29 was 180.9 µM (24h) and 132.8 µM (48h). In DLD-1, the IC50 of HC + 55 µM 5-FU was 152.6 µM (24h) and 72.69 µM (48h). All concentration values were predicted by GraphPad Prism. HC+5-FU showed synergistic interaction in HT-29 treated for 24h, with a combination interaction (CI) of 0.49. However, treatment for 48h showed additive interaction in HT-29 (1.11). In both time points, treatment of HC+5-FU in DLD-1 showed additive interaction (0.77 and 1.08). The HPX, XOR, and ROS levels in HT-29 and DLD-1 treated with HC+5-FU (24 and 48h) were decreased compared to untreated cells. The combination treatment induced high mitochondrial depolarization. Gene expression analysis in HT-29 showed that ENT1 was downregulated in 24h but upregulated in 48h HC+5-FU treatment compared to untreated and single doses (HC / 5-FU) treatment. Interestingly, these observations are contradictory in DLD-1. On the other hand, ENT2 was downregulated in both HT-29 and DLD-1. Expression of CASP3 was induced in both HT-29 and DLD-1 treated with HC+5-FU for 24 and 48h compared to untreated and single doses. BCL2, the anti-apoptotic gene, was downregulated in HT-29 and DLD-1 when treated with HC+5-FU. In conclusion, the induction of cell death in HT-29 and DLD-1 treated with HC+5-FU may be via the modulation of purine metabolism and the apoptotic pathway, in which the decrease of HPX and XOR may cause an imbalance of ROS and lead to the disruption of mitochondrial membrane potential. These observations were further supported by the downregulation of ENT2, which plays a role in HPX transport, the downregulation of the anti- apoptotic gene BCL2, and the upregulation of CASP3, a pro-apoptotic gene. Further investigation is needed on the potential of targeting the purine metabolism pathway as a novel therapeutic approach for colorectal cancer by elucidating its role in cancer formation and response to treatment