Methotrexate Combined with 4-Hydroperoxycyclophosphamide Downregulates Multidrug-Resistance P-Glycoprotein Expression Induced by Methotrexate in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via the JAK2STAT3 Pathway

الملخص EN

Objective.

Rheumatoid arthritis (RA) multidrug resistance is associated with P-glycoprotein (P-gp) overexpression.

We investigated the effects of methotrexate (MTX) alone and combined with 4-hydroperoxycyclophosphamide (4-HC) on P-gp expression in fibroblast-like synoviocytes (FLSs) from patients with RA and examined the signaling pathway involved.

Methods.

RA-FLSs were treated with MTX, MTX + 4-HC, AG490 + MTX, or AG490 + MTX + 4-HC for 72 h.

Proliferation inhibition rates were determined by MTT assay; P-gp expression was measured by flow cytometry and real-time polymerase chain reaction (RT-PCR); JAK2 and STAT3 were measured by RT-PCR and cell-based ELISA to assess STAT3 signaling.

Results.

MTX alone significantly induced P-gp expression and mRNA production in RA-FLSs.

P-gp expression and mRNA levels were lower in the MTX + 4-HC group than in the MTX-alone group.

In contrast to MTX, MTX + 4-HC reduced the STAT3 phosphorylation and downregulated JAK2 and STAT3 mRNA production.

Inhibition of constitutively active STAT3 accompanied by 4-HC suppressed P-gp levels in RA-FLSs.

The MTT assays revealed no significant differences in proliferation inhibition rates among groups.

Conclusions.

The increased anti-P-gp effect of MTX + 4-HC versus MTX alone in RA-FLSs was mediated via inhibition of the JAK2/STAT3 pathway and may have helped reverse MDR in refractory RA patients with high-P-gp levels.