Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide.

Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of ß5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional ß5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the ß5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis.

Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential antirheumatic agent. Its reported side effects, however, make it unappealing for long-term administration, and resistance may also develop. To overcome this, second-generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs, including carfilzomib, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide (ONX0912), and (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanamide (ONX0914), might escape two established BTZ-resistance mechanisms: 1) mutations in the proteasome ß5 subunit (PSMB5) targeted by these PIs, and 2) drug efflux mediated by ATP-binding cassette transporters. THP1 myeloid sublines with acquired resistance to BTZ (54- to 235-fold) caused by mutations in the PSMB5 gene displayed marked cross-resistance but less pronounced cross-resistance to carfilzomib (9- to 32-fold), ONX0912 (39- to 62-fold), and ONX0914 (27- to 97-fold). As for ATP-binding cassette transporter-mediated efflux, lymphoid CEM/VLB cells with P-glycoprotein (Pgp)/multidrug resistance 1 overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX0912 (23-fold), and ONX0914 (162-fold), whereas less resistance to BTZ (4.5-fold) was observed. Consistently, ß5 subunit-associated chymotrypsin-like proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo analysis of peripheral blood mononuclear cells from therapy-naive patients with rheumatoid arthritis revealed that, although basal Pgp levels were low, P-glycoprotein expression compromised the inhibitory effect of carfilzomib and ONX0914. However, the use of P121 (reversin 121), a Pgp transport inhibitor, restored parental cell inhibitory levels in both CEM/VLB cells and peripheral blood mononuclear cells. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.

T lymphocytes impair P-glycoprotein function during neuroinflammation.

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp; ABCB1) is highly expressed at the blood-brain barrier (BBB). P-gp actively secretes and keeps the central nervous system (CNS) safe from body-born metabolites, but also from drugs and food components, emphasising the importance of its optimal function to maintain brain homeostasis. Here we demonstrate that vascular P-gp expression and function are strongly decreased during neuroinflammation. In vivo, the expression and function of brain endothelial P-gp in experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), were significantly impaired. Strikingly, vascular P-gp expression was decreased in both MS and EAE lesions and its disappearance coincided with the presence of perivascular infiltrates consisting of lymphocytes. Our data strongly suggest that activated CD4(+) T cells induce impaired function of brain endothelial P-gp. Notably, lymphocyte interaction through endothelial intracellular adhesion molecule -1 (ICAM-1) resulted in activation of a nuclear factor kappa B (NF-kappaB) signaling pathway, which resulted in endothelial P-gp malfunction. Our study provides first evidence that CD4(+) T cells are able to affect endogenous molecular protection mechanisms of brain endothelium. Loss of vascular P-gp function during neuroinflammation may disturb brain homeostasis and thereby aggravate disease progression via exposure of vulnerable CNS cells to detrimental compounds.

A role for multidrug resistance protein 4 (MRP4; ABCC4) in human dendritic cell migration.

The capacity of dendritic cells (DCs) to migrate from peripheral organs to lymph nodes (LNs) is important in the initiation of a T cell-mediated immune response. The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; ABCB1) and the multidrug resistance protein 1 (MRP1; ABCC1) have been shown to play a role in both human and murine DC migration. Here we show that a more recently discovered family member, MRP4 (ABCC4), is expressed on both epidermal and dermal human skin DCs and contributes to the migratory capacity of DCs. Pharmacological inhibition of MRP4 activity or down-regulation through RNAi in DCs resulted in reduced migration of DCs from human skin explants and of in vitro generated Langerhans cells. The responsible MRP4 substrate remains to be identified as exogenous addition of MRP4's known substrates prostaglandin E(2), leukotriene B(4) and D(4), or cyclic nucleotides (all previously implicated in DC migration) could not restore migration. This notwithstanding, our data show that MRP4 is an important protein, significantly contributing to human DC migration toward the draining lymph nodes, and therefore relevant for the initiation of an immune response and a possible target for immunotherapy.

Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein.

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45- to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome beta5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to beta5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) restoration of bortezomib sensitivity in bortezomib-resistant cells by siRNA-mediated silencing of PSMB5 gene expression. Collectively, these findings establish a novel mechanism of bortezomib resistance associated with the selective overexpression of a mutant PSMB5 protein.

Effect of age on functional P-glycoprotein in the blood-brain barrier measured by use of (R)-[(11)C]verapamil and positron emission tomography.

INTRODUCTION: P-glycoprotein (P-gp) is an efflux transporter responsible for the transport of various drugs across the blood-brain barrier (BBB). Loss of P-gp function with age may be one factor in the development and progression of neurodegenerative diseases. The aim of this study was to assess the effect of aging on BBB P-gp function. Furthermore, the relationship between BBB P-gp activity and peripheral P-gp activity in CD3-positive leukocytes was investigated. Finally, plasma pharmacokinetics of carbon 11-labeled (R)-verapamil was evaluated. METHODS: (R)-[(11)C]verapamil and positron emission tomography were used to assess gray matter P-gp function. Because (R)-[(11)C]verapamil is a substrate for P-gp, the volume of distribution of (R)-[(11)C]verapamil in the brain inversely reflects P-gp function in the BBB. RESULTS: Mean volume of distribution values for 5 young healthy volunteers (age range, 21-27 years) and 5 elderly healthy volunteers (age range, 59-68 years) were 0.62+/-0.10 and 0.73+/-0.07, respectively (P=.03). The activity index of P-gp activity in CD3-positive leukocytes was 2.88+/-0.77 in young volunteers and 1.76+/-0.58 in elderly volunteers (P=.02). CONCLUSION: This study showed decreased P-gp activity during aging. Consequently, the brain may be exposed to higher drug and toxin levels in elderly subjects.

ABC drug transporters and immunity: novel therapeutic targets in autoimmunity and cancer.

ABC transporters were identified originally for their contribution to clinical MDR as a result of their capacity to extrude various unrelated cytotoxic drugs. More recent reports have shown that ABC transporters can play important roles in the development, differentiation, and maturation of immune cells and are involved in migration of immune effector cells to sites of inflammation. Many of the currently identified, endogenous ABC transporter substrates have immunostimulating effects. Increasing the expression of ABC transporters on immune cells and thereby enhancing immune cell development or functionality may be beneficial to immunotherapy in the field of oncology. On the contrary, in the treatment of autoimmune diseases, blockade of these transporters may prove beneficial, as it could dampen disease activity by compromising immune effector cell functions. This review will focus on the expression, regulation, and substrate specificity of ABC transporters in relation to functional activities of immune effector cells and discusses implications for the treatment of cancer on the one hand and autoimmune diseases on the other.

Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients.

OBJECTIVE: To determine the expression of folate receptor beta (FRbeta) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FRbeta than methotrexate (MTX). METHODS: Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FRbeta protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FRbeta messenger RNA (mRNA) levels were determined by reverse transcription-polymerase chain reaction analysis. Binding affinities of FRbeta for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FRbeta-transfected cells. Efficacy of FRbeta-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FRbeta-transfected cells. RESULTS: Immunohistochemical staining of RA synovial tissue showed high expression of FRbeta on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FRbeta mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FRbeta had a high binding affinity (20-77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FRbeta-transfected cells. CONCLUSION: Abundant FRbeta expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity-binding folate antagonists, of which BCG 945 may be a prototypical representative.

Involvement of breast cancer resistance protein expression on rheumatoid arthritis synovial tissue macrophages in resistance to methotrexate and leflunomide.

OBJECTIVE: To determine whether multidrug-resistance efflux transporters are expressed on immune effector cells in synovial tissue from patients with rheumatoid arthritis (RA) and compromise the efficacy of methotrexate (MTX) and leflunomide (LEF). METHODS: Synovial tissue biopsy samples obtained from RA patients before treatment and 4 months after starting treatment with MTX (n = 17) or LEF (n = 13) were examined by immunohistochemical staining and digital image analysis for the expression of the drug efflux transporters P-glycoprotein, multidrug resistance-associated protein 1 (MRP-1) through MRP-5, MRP-8, MRP-9, and breast cancer resistance protein (BCRP), and the relationship to clinical efficacy of MTX and LEF was assessed. RESULTS: BCRP expression was observed in all RA synovial biopsy samples, both pretreatment and posttreatment, but not in control noninflammatory synovial tissue samples from orthopedic patients. BCRP expression was found both in the intimal lining layer and on macrophages and endothelial cells in the synovial sublining. Total numbers of macrophages in RA patients decreased upon treatment; in biopsy samples with persistently high macrophage counts, 2-fold higher BCRP expression was observed. Furthermore, median BCRP expression was significantly increased (3-fold) in nonresponders to disease-modifying antirheumatic drugs (DMARDs) compared with responders to DMARDs (P = 0.048). Low expression of MRP-1 was found on synovial macrophages, along with moderate expression in T cell areas of synovial biopsy specimens from one-third of the RA patients. CONCLUSION: These findings show that the drug resistance-related proteins BCRP and MRP-1 are expressed on inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP-1, and LEF is a high-affinity substrate for BCRP, these transporters may contribute to reduced therapeutic efficacy of these DMARDs.

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.

Sulfasalazine is a potent inhibitor of the reduced folate carrier: implications for combination therapies with methotrexate in rheumatoid arthritis.

OBJECTIVE: To investigate whether interactions of sulfasalazine (SSZ) with reduced folate carrier (RFC), the dominant cell membrane transporter for natural folates and methotrexate (MTX), may limit the efficacy of combination therapy with MTX and SSZ in patients with rheumatoid arthritis. METHODS: Human RFC-(over)expressing CEM cells of T cell origin were used to analyze the effect of SSZ on the RFC-mediated cellular uptake of radiolabeled MTX and the natural folate leucovorin. Moreover, both cells with and those without acquired resistance to SSZ were used to assess the antiproliferative effects of MTX in combination with SSZ. RESULTS: Transport kinetic analyses revealed that SSZ was a potent noncompetitive inhibitor of RFC-mediated cellular uptake of MTX and leucovorin, with mean +/- SD K(i) (50% inhibitory concentration) values of 36 +/- 6 microM and 74 +/- 7 microM, respectively. Consistent with the inhibitory interaction of SSZ with RFC, a marked loss of MTX efficacy was observed when MTX was coadministered with SSZ: up to 3.5-fold for CEM cells in the presence of 0.25 mM of SSZ, and >400-fold for SSZ-resistant cells in the presence of 2.5 mM of SSZ. Importantly, along with diminished efficacy of MTX, evidence for cellular folate depletion was obtained by the demonstration of an SSZ dose-dependent decrease in leucovorin accumulation. CONCLUSION: At clinically relevant plasma concentrations, interactions of SSZ with RFC provide a biochemical rationale for 2 important clinical observations: 1) the onset of (sub)clinical folate deficiency during SSZ treatment, and 2) the lack of additivity/synergism of the combination of SSZ and MTX when these disease-modifying antirheumatic drugs are administered simultaneously. Thus, when considering use of these drugs in combination therapies, the present results provide a rationale both for the use of folate supplementation and for spacing administration of these drugs over time.

Sulfasalazine down-regulates the expression of the angiogenic factors platelet-derived endothelial cell growth factor/thymidine phosphorylase and interleukin-8 in human monocytic-macrophage THP1 and U937 cells.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) and interleukin-8 (IL-8) are angiogenic factors produced by tumor infiltrating macrophages. Here, we show that prolonged exposure of human monocytic/macrophage THP1 and U937 cells to sulfasalazine, an anti-inflammatory drug and inhibitor of nuclear factor-kappaB (NF-kappaB), resulted in down-regulation of PD-ECGF/TP and IL-8 (mRNA, protein and activity) along with elimination of their induction by tumor necrosis factor-alpha and interferon-gamma. Concomitantly, sulfasalazine-exposed cells were markedly resistant to 5'-deoxyfluorouridine, the last intermediate of capecitabine requiring activation by PD-ECGF/TP. This is the first report suggesting that disruption of NF-kappaB-dependent signaling pathways can provoke a marked and sustained down-regulation of macrophage-related angiogenic factors. However, this may also negatively affect capecitabine efficacy.