Therapeutic Targeting of the Pentose Phosphate Pathway in Colorectal Cancer Using 6-Aminonicotinamide and 5-Fluorouracil

Abstract

Introduction: Despite the remarkable improvement in technology and healthcare applications, colorectal cancer (CRC) remains the third most prevalent cancer type worldwide with significant levels of mortality. 5-Fluorouracil (5-FU) is the standard treatment of this neoplasm. Unfortunately, treatment failure, tumor recurrence, and severe toxic side effects are significant calling for more efficient therapeutic strategies. Recent studies highlighted that colorectal neoplasia exhibits a significant upregulation of the pentose phosphate pathway (PPP). The oxidative phase of this metabolic route has both biosynthetic and antioxidant functions through supplying nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphates. NADPH supports tumor initiation and progression through supplying nucleotides for proliferation and scavenging reactive oxygen species (ROS) while ribose-5-phosphates satisfy the nucleotide synthetic demand of rapidly proliferating cancer cells. Glucose-6-phosphate dehydrogenase enzyme (G6PD) is the gatekeeper of the PPP and is associated with poor prognosis and drug resistance in cancer therapy. Therefore, we hypothesized that hijacking the PPP in CRC halts tumor progression and sensitizes treatment to conventional 5-FU therapy. We have previously demonstrated that 6-Aminonicotineamide (6-AN), a competitive inhibitor of G6PD, reduced CRC cell viability. 6-AN synergized with 5-FU in arresting the growth of CRC cells with different p53 and 5-FU resistance status (HCT116, HCT116 p53-/- , HCT116 5FUR), and inducing cell cycle arrest and cell death. Therefore, we aim to investigate the anti-tumor effects, cell death mechanism, and the mode of action of 6-AN and 5-FU alone or 6-AN/5-FU combination treatments using human CRC in vitro and in vivo models. Methods: Determine the mechanism of action of the combination treatment 6-AN/5-FU on cell cycle arrest and DNA damage by immunoblotting techniques, apoptosis using TUNEL, and on senescence via B-galactosidase staining on CRC cell lines of different p53 and 5-FU resistance status. ROS levels were evaluated by nitroblue tetrazolium (NBT) assay. Effect of the single and combination 6-AN/5-FU treatment was assessed on HCT116 5FUR cell cycle distribution using propidium iodide-based flow cytometric cell cycle analysis of DNA content. The G6PD activity was measured in the control and treated CRC cells. Mice were injected in the flank with HCT116 cells and divided into four groups: control, 6-AN, 5-FU, and combination of 6-AN and 5-FU. Tumor volumes and body weights were measured every other day. Results: 6-AN synergized with 5-FU in inducing oxidative stress in HCT116, HCT116 p53-/- , and HCT116 5FU resistant cells, as shown by the NBT assay. The treatment combination did not induce apoptosis in HCT116 cells as demonstrated by lack of 3 PARP cleavage and TUNEL positivity. Instead, it induced senescence when HCT116 cells were left at longer time points in culture. In contrast, the treatment combination induced a partial caspase-dependent apoptotic cell death in HCT116 p53-/- cells. Cell cycle analysis showed that 5-FU synergized with 6-AN in inducing cell death and cell cycle arrest in different phases in HCT116 5FUR cells. Furthermore, the 6-AN/5-FU combination reduced the activity of G6PD compared to single treatments in the tested CRC cells with different p53 and 5-FU resistance status. Preliminary findings showed that 6-AN alone or in combination with 5-FU reduced tumor growth in HCT116 xenografted animals. Conclusion: These results demonstrate that, regardless of p53 and 5-FU drug resistance status, interfering with G6PD activity further sensitizes CRC cells to 5-FU treatment and reduces chemotherapy resistance. This research may shed light on the clinical potential of combining 5-FU to agents that target the PPP, therefore impacting the future of cancer clinical management.