Method: Large-scale expression profiling by cDNA Stanford microarrays, containing 43000 elements, was performed on peripheral blood from 35 RA patients and 15 healthy individuals (van der Pouw Kraan, et al., Ann Rheum Dis 2007). The RA patient group included 25 patients treated with methotrexate (MTX+ group) and 10 patients untreated for methotrexate (MTX- group). As a control, a group of healthy, age and sex-matched, individuals (n=15) were arrayed. The array data was filtered and normalized in the Stanford microarray database. Subanalysis on this dataset was preformed for a set of genes involved in the methotrexate/folate pathway (van der Heijden, et al., Nat Clin Pract Rheumatol. 2007), in particular those involved in the cellular uptake (FR’s) and efflux (MRP1-5, BCRP and P-gP) of MTX, as well as the metabolism and intracellular targeting of MTX (FPGS, GGH, DHFR, TYMS, and GART). Statistical analysis was performed using Student’s t test or Mann-Whitney U test, p-values of ≤0.05 were considered to be statistically significant.
Result: Several folate/MTX-related genes were markedly and significantly altered between the three study groups. Interestingly, the metabolic enzymes FPGS and GGH were significantly up-regulated in the MTX- RA group compared to the healthy control group (HC group), whereas GART expression was markedly down-regulated. Following MTX treatment, these alterations in expression levels were normalized to those observed in the HC group. Furthermore, the MTX-efflux transporters multidrug resistance protein-2 (MRP2) and MRP3 showed an increased expression in the MTX+ group compared to the MTX- group and the HC group, suggesting that cellular extrusion may contribute to a diminished MTX response in the MTX+ group.
Conclusion: Collectively, these results indicate that, under inflammatory conditions basal folate metabolism is altered in blood cells of RA patients vs HC. Treatment with MTX restores expression of these genes to the levels within the range of the HC group. Finally, our results provide the first indication that multidrug resistance protein efflux transporters could contribute to an attenuated MTX response in MTX-based RA treatment.
Acknowledgements: Prof. Dr. Y.G. Assaraf (Technion, Haifa, Israel) was a recipient of a visiting professor fellowship, provided by the Nationaal Reumafonds, to the VU University Medical Center Amsterdam. This study is partly supported by the Center for Translational and Molecular Medicine and the Dutch Arthritis Foundation.
Disclosure: M. Blits, None; G. Jansen, None; Y. G. Assaraf, None; C. L. Verweij, None.