Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells

Abstract

Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance.

Figure 1. HegG2 MTT assay of 5-FU.

Figure 2. HegG2 FANN results for 60 cell samples before and after 5-FU using concentrations of 27 ribonucleotide and deoxyribonucleotide Pools sizes as input variables.

Figure 3. Self-organizing map (SOM) analysis revealing an effect of -5FU on the RN and dRN pool sizes.

Figure 4. Feature maps of adenosine and deoxyadenosine pool sizes as input attributes.

(A) ATP; (B)ADP; (C)AMP; (E) dATP; (F) dADP; (F) dAMP.

Figure 5. Ratio of ATP/ADP (A) ATP/dATP (B) before and after incubation of HepG2 cells with 50 μM of 5-FU.

Each data point is an average of two independent experiments (done in triplicate) and is reported as mean ± standard deviation.

Figure 6. Feature maps of uridine and deoxyuridine pool sizes as input attributes.

(A) UTP; (B) UDP; (C) UMP; (E) dUMP.

Figure 7. Western blot analysis of the effect of 5-FU on expression of dUTPase.

Figure 8. Feature maps of thymidine pool sizes as input attributes.

(A) dTTP; (B) dTDP; (C) dTMP.

Figure 9. Percent change in thymidine pool after incubation of HepG2 cells with 50 μM of 5-FU.

Each data point is an average of two independent experiments (done in triplicate) and is reported as mean ± standard deviation.

Figure 10. Feature maps of cytidine and deoxycytidine pool sizes as input attributes.

(A) CTP; (B) CDP; (C) CMP; (E) dCTP; (F) dCDP; (F) dCMP.

Figure 11. 5-FU induced cell cycle arrest at G2/M phase in HepG2.

(A) Control group; (B) 5-FU group; (C) The percentages of HepG2 cells at different phases.

Figure 12. Ratio of CTP/dCTP before and after incubation of HepG2 cells with 50 μM of 5-FU.

Each data point is an average of two independent experiments (done in triplicate) and is reported as mean ± standard deviation.

Figure 13. Feature maps of guanosine and deoxyguanosine pool sizes as input attributes.

(A) GTP; (B) GDP; (C) GMP; (E) dGTP; (F) dGDP; (F) dGMP.

Figure 14. Percent change in guanosine pool after incubation of HepG2 cells with 50 μM of 5-FU.

Each data point is an average of two independent experiments (done in triplicate) and is reported as mean ± standard deviation.