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.