Sulfasalazine sensitises human monocytic/macrophage cells for glucocorticoids by upregulation of glucocorticoid receptor alpha and glucocorticoid induced apoptosis.

BACKGROUND: Glucocorticoids (GCs) are commonly used in the treatment of (chronic) inflammatory diseases and cancer, but inherent or acquired resistance to these drugs limits their optimal efficacy. The availability of drugs that could modulate GC resistance is therefore of potential clinical interest. OBJECTIVE: To explore the molecular basis of GC sensitisation of GC resistant monocytic/macrophage cells after chronic exposure to sulfasalazine. METHODS: Human monocytic/macrophage THP1 and U937 cells represent a cell line model system characterised by inherent resistance to the GCs dexamethasone and prednisolone. Both cell lines were chronically exposed in vitro to 0.3-0.6 mM sulfasalazine (SSZ) for approximately 3 months, after which they were characterised for GC sensitivity, expression levels of GC receptor and components of the nuclear factor kappa B (NFkappaB) signalling pathway, and their ability to undergo GC induced apoptosis. RESULTS: Chronic exposure to SSZ markedly sensitised both U937 and THP1 cells to dexamethasone (781-fold and 1389-fold, respectively) and prednisolone (562-fold and 1220-fold, respectively). Restoration of GC sensitivity in cells exposed to SSZ was provoked via GC induced apoptosis, coinciding with inhibition of NFkappaB activation. Moreover, western blot analysis revealed a markedly increased expression of glucocorticoid receptor alpha (GRalpha) in cells exposed to SSZ. Since GRalpha mRNA levels were only marginally increased, these results suggest that an altered post-transcriptional mechanism was operable which conferred a stable GRalpha protein on SSZ exposed cells. CONCLUSION: These results suggest that chronic targeting of the NFkappaB signalling pathway by SSZ may be exploited as a novel strategy to stabilise GRalpha expression and thereby sensitise primary resistant cells to GCs.

Acquired resistance to chloroquine in human CEM T cells is mediated by multidrug resistance-associated protein 1 and provokes high levels of cross-resistance to glucocorticoids.

OBJECTIVE: To explore the onset and molecular mechanism of resistance to the antimalarial disease-modifying antirheumatic drug (DMARD) chloroquine (CQ) in human CEM T cells. METHODS: Human CEM cells were used as an in vitro model system to study the development of CQ resistance by growing cells in stepwise increasing concentrations of CQ. RESULTS: Over a period of 6 months, CEM cell lines developed 4-5-fold resistance to CQ. CQ resistance was associated with the specific overexpression of multidrug resistance-associated protein 1 (MRP-1), an ATP-driven drug efflux pump. This was illustrated by 1) overexpression of MRP-1 by Western blotting and 2) the complete reversal of CQ resistance by the MRP-1 transport inhibitors MK571 and probenecid. Importantly, CQ-resistant CEM cells retained full sensitivity to other DMARDs, including methotrexate, leflunomide, cyclosporin A, and sulfasalazine, but exhibited a high level of cross-resistance (>1,000-fold) to the glucocorticoid dexamethasone. The mechanistic basis for the latter was associated with aberrant signaling via the cAMP-protein kinase A pathway, since the cAMP-inducing agent forskolin reversed dexamethasone resistance. Finally, CQ-resistant CEM cells displayed a markedly reduced capacity to release proinflammatory cytokines (tumor necrosis factor alpha) and chemokines (interleukin-8). CONCLUSION: Induction of overexpression of the multidrug resistance efflux transporter MRP-1 can emerge after long-term exposure to CQ and results in CQ resistance and collateral resistance to dexamethasone. These findings warrant further detailed investigations into the possible role of MRP-1 and other members of the superfamily of drug efflux pumps in diminishing the efficacy of DMARDs in rheumatoid arthritis treatment.

The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14lambda and the PP2C phosphatase KAPP.

Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda. In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14lambda.