4-Pyridone-3-carboxamide-1-β-D-ribonucleoside Reduces Cyclophosphamide Effects and Induces Endothelial Inflammation in Murine Breast Cancer Model

Abstract

4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) is a nicotinamide derivative, considered a new oncometabolite. 4PYR formation induced a cytotoxic effect on the endothelium. Elevated blood 4PYR concentration was observed in patients with cancer. Still, little is known about the metabolic and functional effects of 4PYR in this pathology. The study aimed to investigate whether this toxic accumulation of 4PYR may affect the activity of anticancer therapy with cyclophosphamide in the orthotropic model of breast cancer. Female Balb/c mice were injected with 4T1 breast cancer cells and assigned into three groups: treated with PBS (Control), cyclophosphamide-treated (+CP), 4PYR-treated (+4PYR), and mice treated with both 4PYR and CP(+4PYR+CP) for 28 days. Afterward, blood and serum samples, liver, muscle, spleen, heart, lungs, aortas, and tumor tissue were collected for analysis of concentrations of nucleotides, nicotinamide metabolites, and 4PYR with its metabolites, as well as the liver level of cytochrome P450 enzymes. 4PYR treatment caused elevation of blood 4PYR, its monophosphate and a nicotinamide adenine dinucleotide (NAD+) analog-4PYRAD. Blood 4PYRAD concentration in the +4PYR+CP was reduced in comparison to +4PYR. Tumor growth and final tumor mass were significantly decreased in +CP and did not differ in +4PYR in comparison to Control. However, we observed a substantial increase in these parameters in +4PYR+CP as compared to +CP. The extracellular adenosine deamination rate was measured to assess vascular inflammation, and it was higher in +4PYR than the Control. Treatment with 4PYR and CP caused the highest vascular ATP hydrolysis and adenosine deamination rate. 4PYR administration caused significant elevation of CYP2C9 and reduction in CYP3A4 liver concentrations in both +4PYR and +4PYR+CP as compared to Control and +CP. In additional experiments, we compared healthy mice without cancer, treated with 4PYR (4PYR w/o cancer) and PBS (Control w/o cancer), where 4PYR treatment caused an increase in the serum proinflammatory cytokine expression as compared to Control w/o cancer. 4PYR accumulation in the blood interferes with cyclophosphamide anticancer activity and induces a pro-inflammatory shift of endothelial extracellular enzymes, probably by affecting its metabolism by cytochrome P450 enzymes. This observation may have crucial implications for the activity of various anticancer drugs metabolized by cytochrome P450.

Keywords: breast cancer; cyclophosphamide; nicotinamide metabolism.

Figure 1

Cyclophposphamide metabolic pathway. Hepatic cytochrome P450 enzymes activate CP (prodrug) to 4-hydroxycyclophosphamide, which is in an equilibrium state with aldophosphamide. Both metabolites diffuse into cells, where aldophosphamide is converted to phosphoramide mustard and acrolein.

Figure 2

(a) Blood 4PYR and 4PYR metabolite concentration of mammary 4T1 carcinoma mice (Control) treated with cyclophosphamide (+CP), 4PYR (+4PYR) and 4PYR with cyclophosphamide (+4PYR+CP); (b) 4PYMP and (c) 4PYRAD concentration in the tissues of 4PYR (+4PYR) and 4PYR with cyclophosphamide (+4PYR+CP) receiving mice. Mean ± SEM, n = 10; two-way ANOVA with post hoc Tukey test and Student t-test: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

(a) The kinetics of murine mammary 4T1 carcinoma tumor growth and (b) tumor weight of mammary 4T1 carcinoma mice (Control) treated with cyclophosphamide (+CP), 4PYR (+4PYR) and 4PYR with cyclophosphamide (+4PYR+CP). Mean ± SEM, n = 10; two-way ANOVA with post hoc Tukey test and Student t-test: *** p < 0.001; ** p < 0.01; * p < 0.05 vs. control; ### p < 0.001; ## p < 0.01; vs. CP and 4PYR+CP and $$$ p < 0.001; vs. 4PYR and CP.

Figure 4

(a) ATP, (b) AMP hydrolysis and (c) adenosine deamination on the aorta of mammary 4T1 carcinoma mice (Control) treated with cyclophosphamide (+CP), 4PYR (+4PYR) and 4PYR with cyclophosphamide (+4PYR+CP). Mean ± SEM, n = 10; two-way ANOVA with post hoc Tukey test and Student t test: *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 5

Changes in nicotinamide metabolites in the serum of mammary 4T1 carcinoma mice (Control) treated with cyclophosphamide (+CP), 4PYR (+4PYR) and 4PYR with cyclophosphamide (+4PYR+CP): the concentration of (a) nicotinamide (NA); (b) N-methylnicotinamide (MetNA); (c) nicotinamide riboside (NR); (d) N-methyl-2-pyridone-5-carboxamide (Met2PY); (e) N-methyl-4-pyridone-3-carboxamide (Met4PY) and (f) 4PYR. Mean ± SEM, n = 10; two-way ANOVA with post hoc Tukey test and Student t-test: *** p < 0.001; ** p < 0.01; * p < 0.05. Mean ± SEM, n = 10; *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 6

Liver concentration of cytochrome P450 enzymes involved in cyclophosphamide metabolism: (a) CYP2A6, (b) CYP2C9 and (c) CYP3A4 of mammary 4T1 carcinoma mice (Control) treated with cyclophosphamide (+CP), 4PYR (+4PYR) and 4PYR with cyclophosphamide (+4PYR+CP). Mean ± SEM, n = 5; two-way ANOVA with post hoc Tukey test and Student t-test: * p < 0.05.

Figure 7

Additional measurements of the serum expression of cytokines associated with inflammation or cancer progression: CCL19, CCL3, SDF-1α, CX3CL1, CCL5, FAS-ligand, IL-1β and IL-1α in healthy mice without cancer treated with 4PYR (4PYR w/o cancer) or PBS (Control w/o cancer). Mean ± SEM, n = 5; Student’s t-test: * p < 0.05.