Abstract
BackgroundRheumatoid arthritis fibroblast-like synovial cells (RA-FLS) show resistance to methotrexate (MTX) treatment. To better understand the mechanisms of this resistance, RA-FLS and osteoarthritis fibroblast-like synovial cells (OA-FLS) were isolated and exposed to MTX. We analyzed the autophagy induced by MTX in vitro and its relationship to apoptosis.MethodsCell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was detected by flow cytometry and Western blot analysis. Autophagy was determined by transmission electron microscopy as well as Western blot analysis. The expression levels of Beclin-1, LC3, Akt, p-Akt, mammalian target of rapamycin (mTOR), p-mTOR, high mobility group box chromosomal protein 1 (HMGB1), and an 85 kDa caspase cleaved fragment of poly(ADP-ribose) polymerase were measured by Western blotting.ResultsMTX-induced apoptosis was increased in OA-FLS compared with RA-FLS. However, MTX stimulated the autophagy response in RA-FLS by inducing autophagosome formation, but not in OA-FLS. In RA-FLS, transfection with Beclin-1 small interfering RNA inhibited autophagy and increased susceptibility to MTX, which induces cell death. MTX upregulated autophagy through its ability to enhance the expression of HMGB1 and Beclin-1 rather than through the Akt/mTOR pathway.ConclusionsAutophagy induction contributes to resistance to MTX treatment in fibroblasts from patients with rheumatoid arthritis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0892-y) contains supplementary material, which is available to authorized users.
Highlights
Rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) show resistance to methotrexate (MTX) treatment
MTX inhibited cell viability and induced apoptosis rheumatoid arthritis (RA)-FLS and osteoarthritis fibroblast-like synovial cells (OA-FLS) were treated with MTX at concentrations ranging from 0.01 μM to 10 μM for 48 h
MTX induced autophagosome formation Both RA-FLS and OA-FLS were treated with MTX at concentrations ranging from 0.01 μM to 10 μM for 48 h and at times ranging from 12 h to 96 h at a concentration of 0.1 μM
Summary
Rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) show resistance to methotrexate (MTX) treatment. To better understand the mechanisms of this resistance, RA-FLS and osteoarthritis fibroblast-like synovial cells (OA-FLS) were isolated and exposed to MTX. We analyzed the autophagy induced by MTX in vitro and its relationship to apoptosis. Increasing evidence indicates that autophagy protects various tumor cells from apoptosis induced by chemotherapy drugs, both in vivo and in vitro [12,13,14]. Compared with osteoarthritis (OA), both enhanced autophagy in RA synovial tissues and increased induction of autophagy in RA-FLS were recently described [16, 17]. Autophagy exerted protective effects in these studies. This raises a question about the role of autophagy in the process of MTX treatment of RA-FLS
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