The Effects of Low-Dose Methotrexate on Metabolism in Primary Human PBMCs
Citation:O'FARRELL, LAURA, The Effects of Low-Dose Methotrexate on Metabolism in Primary Human PBMCs, Trinity College Dublin.School of Biochemistry & Immunology.BIOCHEMISTRY, 2017
Laura O'Farrell Final thesis 2017.pdf (PhD Thesis) 6.955Mb
Low-dose Methotrexate (MTX) is a powerful anti-arthritic drug used in the treatment of a variety of inflammatory diseases. It is an economical drug and is easily administered, but incomplete knowledge into its mechanism of action limits its therapeutic capability. As an anti-folate drug, MTX has been shown to have a variety of effects including effects on the one-carbon metabolism pathway, including purine and thymidine nucleotide synthesis. MTX has also been shown to reverse the aberrant glycosylation patterns in Rheumatoid arthritis (RA) patients. It is not known exactly which effect of MTX has therapeutic potential nor is it known which aspect of this anti-folate is responsible. Elucidating new effects of MTX and whether purine and thymidine inhibition is the mechanism by which these effects occur, would provide new insight into the workings of this drug and expand its therapeutic potential. Using a model of activated T cells, the effects of MTX on a variety of biochemical processes were investigated and compared to unactivated PBMCs to see if MTX could preferentially affect activated PBMCs, such as the over-active T cells in RA. Flow cytometric analysis revealed that MTX displayed activation-dependent effects on cell viability, proliferation, transferrin receptor expression and blastogenesis, but dissipated the mitochondrial inner-membrane potential (∆Ѱm) in both activated and unactivated PBMCs. The MTX-mediated effects were either partially or completely reversed upon purine and thymidine rescue, which indicated that insults to this biosynthesis pathway were the mechanism in which these effects occurred. No direct effect of low-dose MTX on cellular glycosylation has ever been shown. In this study, flow cytometry was also employed to quantify ManNAz incorporation on the-cell-surface, using click chemistry and was used to show how changes to surface sialylation can occur during activation which can thus be used to evaluate the effects of drug treatment that limit cellular activation. MTX was found to significantly affect cell-surface sialylation, which was reversed by purine and thymidine rescue. In the absence of extensive metabolic profiling upon low-dose MTX treatment, HPLC was used to quantify any changes to intracellular nucleotides and sugar-nucleotides in both unactivated and anti-CD3-activated PBMCs. MTX inhibited CDP and NAD/NADH nucleotides in activated PBMCs but no changes were found in the sugar-nucleotides detected. NAD/NADH did not increase during activation, which suggested that MTX could exert effects which were activation-independent. Purine and thymidine inhibition was also shown to be the mechanism in which MTX reduced the levels of NAD/NADH nucleotides. Combined, the findings shown here suggest that the MTX-mediated inhibition of purine and thymidine synthesis has far-reaching consequences, encompassing effects on cellular activation and on cell-surface sialylation. A state of folate-deficiency was found under these MTX-treated conditions, illustrating the powerful effect of this anti-folate, even at low-doses. This finding has implications for any future effects shown by MTX whereby a consequence of folate-deficiency would have to be disproven. Therapeutically, these results highlight a new link between the anti-proliferative effect of MTX and the altered glycosylation patterns found in RA patients treated with MTX. Although the therapeutic value of these insults by MTX was not measured, the global effects of purine and thymidine inhibition strengthen the anti-proliferative effect as a potent mechanism of action.
Author: O'FARRELL, LAURA
Publisher:Trinity College Dublin. School of Biochemistry & Immunology. Discipline of Biochemistry
Type of material:Thesis
Availability:Full text available