©2009 International Medical Press 1359-6535 (print) 2040-2058 (online) 1101

Antiviral Therapy 2009 14:1101–1111 (doi: 10.3851/IMP1468)

Background: The multidrug resistance proteins (MRPs) form a subfamily within the ATP binding cassette trans-porters that confer resistance to a variety of structurally unrelated compounds. MRP4 has been reported to trans-port antiretroviral drugs out of cells in an active process. Although the main therapeutic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are their ability to inhibit cyclooxygenase activity, in recent years, some phar-macological effects independent of this action have been described, such as inhibition of the activity of MRP4. Methods: Detection of MRP4 expression was carried out by Western blot analysis, immunofluorescence and flow cytometry in peripheral blood lymphocytes (PBLs). Cells were infected with HIV type-1NL4.3 isolate, and treated with anti-retroviral drugs plus different NSAIDs. Agp24 was measured by ELISA 3 days post-infection. Intracellular [3H] zidovudine (AZT) was quantified by a scintiller counter. Expression of different cell markers was assessed by flow cytometry.

Results: NSAIDs, as well as probenecid, were able to potentiate the antiretroviral effect of several nucleoside reverse transcriptase inhibitors (NRTIs). PBLs expressed MRP4 and treatment with ibuprofen did not affect this expression. However, MRP4 expression increased fol-lowing phytohaemaglutinin and AZT treatment. This decrease of Agp24 was correlated with an increase in the intracellular AZT concentration. This effect was unrelated to changes on expression of CD4, CXCR4, cell viability or activation. Interestingly, patients treated with highly active antiretroviral therapy, who had a detectable viral load, presented a higher expression of MRP4 than those with an undetectable viral load.Conclusions: NSAIDs can improve the antiretroviral activity of NRTIs, increasing their intracellular con-centration by blocking MRP4. This finding could have implications for success of antiviral therapy.

Treatment with highly active antiretroviral therapy (HAART) leads to emergence of drug-resistant virus [1]. Although therapy failure is primarily the result of viral mutations, some infected individuals present resistance symptoms without resistant virus. This fact is related to the possibility of existence of cellular mechanisms that contribute to HIV treatment failure [2]. For an effective therapy, it is necessary to reach an adequate intracellu-lar concentration of antiretroviral drugs, which is cont-rolled by influx and efflux processes [3], their balance being pivotal in overall therapeutic efficacy. Specifically,

the increased efflux of phosphorylated drugs has been proposed to explain decreased drug accumulation and resistance to retroviral inhibitors [4].

The ATP binding cassette (ABC) transporter super-family contains membrane proteins that translocate a wide variety of substrates across extra- and intracel-lular membranes, including metabolic products, lipids and sterols, and drugs. Although many are uncharac-terized, some of them have been shown to transport anionic substances against a concentration gradi-ent with the energy supplied by ATP hydrolysis [5].

Original article

Non-steroidal anti-inflammatory drugs increase the antiretroviral activity of nucleoside reverse transcriptase inhibitors in HIV type-1-infected T-lymphocytes: role of multidrug resistance protein 4Ma Isabel Clemente1, Susana Álvarez2, Ma Jesús Serramía1, Ombretta Turriziani3, Miguel Genebat4, Manuel Leal4, Manuel Fresno2 and Mª Ángeles Muñoz-Fernández1*

1Laboratory Inmuno-Biología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain2Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain3Department of Experimental Medicine, Virology Section ‘Sapienza’ University of Rome, Rome, Italy4Servicio de Enfermedades Infecciosas, Hospital Virgen del Rocío, Seville, Spain

*Corresponding author: e-mail: mmunoz.hgugm@madrid.salud.org

Introduction

©2009 International Medical Press1102

MI Clemente et al.

Multidrug resistance protein 4 (MRP4/ABCC4 [trivial name/Human Genome Organisation name]) is a mem-ber of the C subfamily of ABC transporters. The first functional properties of the MRP4/ABCC4 gene tran-script were described in 1999 in a human T-lymphoid cell line, in which overexpression of MRP4 was directly linked to impaired antiviral efficacy and enhanced efflux of antiviral nucleoside-based drugs [4]. Within the C subfamily, nine full MRPs have been identified so far, together with the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) and the sulfo-nylurea receptors (SUR1/ABCC8 and SUR2/ABCC9). Eight of them (MRP1–8) are localized to the plasma membrane of different cell types, in particular in tissues with a barrier function, such as intestine, liver, brain capillaries, placenta and kidney [6]. MRPs are integral plasma membrane proteins, which mediate the ATP-dependent export of organic anions from cells. In addi-tion to being important export pumps for physiological substances, MRP subfamily members are involved in the active efflux of organic anions of toxicological rel-evance [7,8]. Moreover, MRP transporters have been shown to confer resistance to cytotoxic and antiviral drugs (for a review, see [9]). The various MRPs show remarkable differences with respect to tissue distribu-tion, substrate specificity and physiological function [10]. MRP1, MRP4 and MRP5 have been reported to transport antiretroviral drugs commonly used to inhibit HIV replication out of cells in an active proc-ess [4,11–13]. Importantly, protease inhibitors (PIs) are transported mainly by Pgp and MRP1 [14–16], whereas MRP4 and MRP5 transport mainly purine-based antiviral drugs, such as 9-(2-phosphonomethox-yethyl) adenine and the phosphorylated derivatives of the nucleoside reverse transcriptase inhibitors (NRTIs), zidovudine monophosphate (AZT) and stavudine monophosphate [4,17]. It has also been reported that 9-(2- phosphonyl methoxyethyl) adenine (adefovir), is directly transported by both MRP4 and MRP5 [18,19]. Using membrane vesicles expressing MRP4, Imaoka et al. [20] demonstrated the ATP-dependent uptake of tenofovir (TFV), suggesting that this NRTI could be an MRP4 substrate.

By contrast, non-steroidal anti-inflammatory drugs (NSAIDs) are specific inhibitors of the enzymatic activ-ity of cyclooxygenase (COX), the rate-limiting enzyme in the synthesis of prostaglandins. Two isoforms have been identified: Cox type-1 (COX-1), which is consti-tutively expressed, and Cox type-2 (COX-2), which is induced by different stimuli [21]. The selectivity for COX-1 and COX-2 differs among different NSAIDs, ranging from COX unselective compounds (for exam-ple, ibuprofen) to COX-2 selective drugs (for example, NS398) [22,23]. However, it has been suggested that NSAIDs might have pharmacological effects that are

independent of the inhibition of COX activity [24]. Among those activities, they might inhibit the transport activity of MRP4, the active efflux transporter for the release of prostaglandins from the cells that produce them [25].

We investigated whether MRP4 inhibition by NSAIDs could alter antiviral activity of several NRTIs, such as AZT, lamivudine (3TC), abacavir and TFV. We found an increase in antiviral efficacy after treatment with ibu-profen or indomethacin. Moreover, we show that cell activation induces MRP4 expression in T-lymphocytes and that multitreated HIV type-1 (HIV-1) patients with detectable viral load (VL) present higher levels of MRP4 than responders, suggesting a new mechanism of resistance of NRTIs.

Methods

PatientsSamples from patients were kindly provided by the HIV BioBank integrated in the Spanish AIDS Research Network. All patients participating in the study gave their informed consent and protocols were approved by institutional ethical committees. All patients at the time of study had HAART, seven of them had detectable plasma VL (>50 copies/ml) and the remaining three had undetectable VL.

Cell culturesHuman peripheral blood lymphocytes (PBLs) were isolated by Ficoll-Hypaque centrifugation. The PBLs were grown in RPMI 1640 (Biochrom KG Seromed, Berlin, Germany) supplemented with 10% heat- inactivated fetal calf serum at 37°C and activated for 3 days with 2 µg/ml phytohaemagglutinin (PHA) and 60 µg/ml interleukin-2 (Murex Diagnostics Corp., Norcross, GA, USA).

Cell linesThe human T-lymphoid cell line CEMs and its MRP4 overexpressing variant, CEM3TC, were cultured as described previously [26,27].

ReagentsMRP inhibitor probenecid (PBCD), ibuprofen, indomethacin, and 3′-azido-3′-deoxythymidine-methyl-3H [3H]AZT were purchased from Sigma (St Louis, MO, USA). The NRTIs used in this study were AZT (3-azido-3-deoxythymidine; Sigma) 3TC (2,3-dideoxy-3-thiacytidinel; Moravek Biochemicals, Brea, CA, USA), abacavir (1592U89; Moravek Biochemi-cals) and TFV (Gilead Sciences, Inc., Foster City, CA, USA). The selective inhibitor of COX-2, NS398, was from Cayman Chemical Company (Ann Arbor, MI, USA). Ibuprofen and indomethacin were dissolved in

NSAIDs increase NTRI activity through MRP4

Antiviral Therapy 14.8 1103

distilled water, NS398 in dimethyl sulfoxide and PBCD just before use in NaOH 1M, pH 7.4. Monoclonal anti-body rat anti-human anti-MRP4 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-human CD4- phycoerythrin, anti-CD4-PC5, anti-CD25-ECD and anti-CD69-PC5 antibodies were from Beckman– Coulter (Marseille, France). Anti-CXCR4 monoclonal antibody was from R&D Systems (Abingdon, Oxon, UK). As secondary antibodies, we used fluorescein iso-thiocyanate (FITC)- conjugated AffinityPure donkey anti-rat Immunoglobulin G (IgG; Jackson ImmunoRe-search, West Grove, PA, USA), horseradish peroxidase (HRP)-conjugated anti-rat (Chemicon International, Temecula, CA, USA) and HRP-conjugated anti-mouse (Amersham Biosciences, Chalfont, St Giles, UK).

HIV-1 infection of PBLsInfection experiments were performed in triplicate using PHA-activated PBLs as target cells. Those cells were infected with a T-lymphocytotropic strain, HIV-1NL4.3 (X4), at 0.5 multiplicity of infection (MOI) for 120 min. After this time, cells were washed with phosphate-buffered saline (PBS) and fresh medium was added to each well. When indicated, PBLs were pretreated overnight with ibuprofen, indomethacin or PBCD, and after the infection incubated with ibupro-fen, indomethacin or PBCD, as MRP4 inhibitors and AZT, 3TC, abacavir or TFV. The cells were washed and resuspended in fresh medium. Cell supernatants were harvested 3 days post-infection to monitor p24 viral core production using an antigen capture (Innotest HIV-1 Antigen; Innogenetic, Ghent, Belgium).

HIV-1 infection of CEM and CEM3TC cellsCEM or CEM3TC cells were infected with a T- lymphocytotropic strain, HIV-1NL4.3 (X4), at 0.5 MOI for 2 h. Cells were then washed with PBS and fresh medium was added to each well. When indi-cated, CEM and CEM3TC cells were pretreated with ibuprofen overnight and, after the infection, were incubated with ibuprofen plus AZT. Cell superna-tants were harvested 3 days post-infection to monitor p24 viral core production using an antigen capture (Innotest HIV-1 Antigen).

Western blot analysisMRP4 expression was studied in PHA-activated PBLs treated or not with ibuprofen for 3 days. After this time, cells were washed twice with PBS and lysed using Nonidet- P40 buffer (NP-40), adequate buffer for membrane protein extraction. Protein contents were measured using a bicinchoninic acid protein assay Kit (Pierce, Rockford, IL, USA) according to the manu-facturer’s instructions. For western blotting, 25 µg of protein from each sample was subjected to SDS-PAGE

on a 7.5% gel. Proteins were then transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) by humidified transference blotting. The membrane was blocked overnight at 4°C using Roti-block (Carl Roth, Karlsruhe, Germany) and for 1 h at room temperature before incubation with the primary antibodies: a mouse anti-human MRP4 (Santa Cruz Biotechnology), or a mouse anti-human α-tubulin (Sigma). Membranes were washed in Tris-buffered saline with Tween and incubated with secondary anti-bodies as appropriate. Visualization of protein bands was performed using enhanced chemiluminescencerea-gents (Amersham Biosciences). All western blot experi-ments were carried out at least 3×.

Transport inhibition assaysTo evaluate the inhibitory effects of NSAIDs on AZT transport mediated by MRP4, the transport assays were performed using 1 µCi of [3H]AZT, in the absence or presence of NSAIDs (10 µM) or PBCD (200 µM). After the incubation, the medium was removed, and cells were washed twice with PBS. In the final wash, cells were lysed in ice-cold methanol (60%) and Tween (10%) overnight, and radioactivity was measured in a Microbeta Luminiscence Counter (Walex Trilux, Perk-inElmer, Waltham, MA, USA).

Flow cytometryThe effect of different stimuli on expression levels of cell surface receptors was determined by staining with monoclonal antibodies as indicated. Fluores-cence staining was measured and analysed with the FAC Sort (Epics XL-MCL, Beckman–Coulter, Brea, CA, USA).

Immunofluorescence assayTotal lymphocytes were left to adhere to slides treated with poli-l-lysine (Sigma) during 2 h at 37°C, fixed for 20 min in 4% paraformaldehyde, and then stained with specific primary antibodies (anti-MRP4 and anti-CD4) for 1 h. Following washing with PBS, the monolayers were incubated in the dark for 30 min with FITC-conjugated goat anti-rat. The nuclei of all cells were stained for 5 min with 4′,6-diamidine-2-phenylindole (Calbiochem, San Diego, CA, USA). Fol-lowing a final PBS wash, the slides were mounted with DAKO® Ultramount, Aqueous Permanent Mounting Medium (DAKO North America Inc., Carpinteria, CA, USA), and observed with a Nikon Microphot fluorescence microscope.

Statistical analysisAt least three independent experiments were used for data analysis. Differences were analysed using non- parametric tests (Mann–Whitney U).

MI Clemente et al.

©2009 International Medical Press1104

Results

NSAIDs increase the antiretroviral activity of NRTIs in PBL culturesIt has been proposed that some NSAIDs could increase the effectiveness of anti-HIV drugs, blocking the action of MRP4 [28]. To test this hypothesis, we first evalu-ated whether ibuprofen could improve antiviral effect of different NRTIs, such as AZT, abacavir, 3TC and TFV on HIV-1 replication. Therefore, we determined the effects of ibuprofen on AZT efficacy in a dose– response manner (Figure 1A), with a maximal dose of 50 µM. Although we found the maximal effects at the higher dose used, the dose of 10 µM was used in fur-ther experiments for studying the regulation of HIV-1 replication for considering an intermediate dose. After this, PHA-activated PBLs were pretreated with NRTI alone (abacavir, 3TC or TFV) or in combination with ibuprofen, and Agp24 liberation was measured 3 days post- infection. The same results were obtained at 6 days post-infection (data not shown). As expected, NRTIs alone strongly decreased HIV-1 replication, their effect being significantly potentiated by ibuprofen (Figure 1B, 1C and 1D). Moreover, no effect on viral replication was observed in presence of ibuprofen alone (Figure 1E). Similar results were found when we studied the effect of indomethacin, another reported MRP4 inhibi-tor [25], on HIV-1 replication after AZT treatment (Figure 1F).

To further analyse the influence of MRP4 inhibition on diminished HIV-1 replication in response to NSAIDs, we used PBCD, an agent that blocks the activity of the several MRP transporters including MRP4 [29,30]. As expected, AZT treatment reduced HIV-1 replication over 60%, and AZT plus PBCD potentiated this inhibi-tion (Figure 1G).

Ibuprofen increases the antiretroviral activity of AZT in MRP4 overexpressing T-cellsTo confirm the implication of the MRP4 pump in the increase of the antiretroviral effect described above, we performed some experiments using CEM parental and CEM3TC cells. As shown in Figure 2, ibuprofen (50 µM) was able to reduce Agp24 levels in CEM3TC cells without altering HIV-1 replication in parental ones. As expected, we needed a higher dose of ibupro-fen to obtain the same effect observed in PBL because CEM3TC cells express much more MRP4.

Expression of MRP4 in lymphocytesThe above results suggested that MRP4 is blocked by NSAIDs, promoting the antiviral activity of NRTIs. It has been clearly shown that MRP4 is involved in the resist-ance to antiretroviral drugs [17], but MRP4 expression in PBLs is not well determined. To investigate this, we first

examined MRP4 expression in primary lymphocytes. At the protein level, we detected by immunoblot MRP4 in total cell extracts (Figure 3A). To evaluate whether ibu-profen could affect surface MRP4 levels, we determined MRP4 expression in PHA-activated PBLs stimulated with ibuprofen. We did not find any change in MRP4 expression after NSAID treatment. Immunofluorescence analysis of PHA-activated cells showed that the protein is predominantly located on the plasma membrane and, interestingly, we observed MRP4 expression in most of the CD4- positive cells (Figure 3B). Moreover, flow cytometry analysis in PBLs from different healthy donors revealed an expression of approximately 6–8%, and, interestingly, PHA treatment led to a substantial increase on MRP4 expression (approximately 25–60%) depending on the healthy donor (P<0.05; Figure 3C).

It has been previously described that therapy might contribute to the modulation of the messenger RNA (mRNA) expression of different MRPs [31], so we investigated whether treatment with AZT could modify cell surface MRP4 expression on short term cultures. We found that following incubation with AZT, MRP4 levels were increased by approximately 10% in total lymphocytes in vitro (Figure 3D).

NSAID effects on PBL culturesTo further discard that NSAIDs could alter the expression of the two main cellular receptors involved in HIV-1 infection, we analysed the expression of CD4 and CXCR4 in PHA-activated PBLs treated or not with ibuprofen. Neither significant alteration on the CD4 nor the CXCR4 surface expression was observed in stimulated cells PBLs (Table 1). Moreover, similar results were obtained with the other NRTIs used in this study (data not shown).

It has been described that COX-2-specific inhibitors decrease T-cell activation [32], which in turn modu-lates HIV-1 replication; thus, we wanted to study whether ibuprofen could inhibit lymphocyte activation and subsequently HIV-1 replication in our cultures. Therefore, PBLs were pretreated with NS398, a spe-cific COX-2 inhibitor, before infection and cultured for 3 days in combination with AZT. In contrast with previous results show above, NS398 did not potenti-ate AZT activity, but rather prevented it (Figure 4). To discard that ibuprofen treatment could change cellu-lar activation, we performed flow cytometry analysis of expression of early activation molecules as CD69 and CD25. Similar expression of both markers was observed in PBLs treated with ibuprofen over con-trol cells (Table 2). Our data confirm that the effect of NSAIDs on HIV-1 replication were not the result of reduced cellular activation.

Furthermore, we discarded that the effects shown above could be ascribed to an increase in cell toxicity,

NSAIDs increase NTRI activity through MRP4

Antiviral Therapy 14.8 1105

a

a

b

b

19,000

25,000

20,000

15,000

10,000

5,000

7,000

6,000

5,000

4,000

3,000

2,000

1,000

0

0HIV-1NL4.3 +

-

-

-

+

+ +

+ + + + +

+-++ -

+ + +

Ibuprofen 10 µM

+- -Ibuprofen 10 µM

+ +-HIV-1NL4.3 HIV-1NL4.3

+- + + +AZT 0.5 µM ++--AZT 0.5 µM

+-+-PBCD 200 µM

+ + + +HIV-1NL4.3

3TC 250 nM 250 nM 1 µM 1 µM 5 µM 5 µM

25,000

20,000

15,000

10,000

5,000

NS

0

17,000

15,000

13,0003,5003,0002,5002,0001,5001,000

5000

HIV-1NL4.3

Ibuprofen 10 µM

6,000

5,000

4,000

3,000

2,000

HIV-1NL4.3

Ibuprofen 10µM

TFV -

-

- + + +

+- -

1 µM 1 µM-

1,000

0

+HIV-1NL4.3 + + + + +

- -

+

Ibuprofen 1 µM 10 µM 50 µM- -

AZT 0.5 µM +++- +

Agp

24, p

g/m

l

Agp

24, p

g/m

lA

gp24

, pg/

ml

Agp

24, p

g/m

l

Agp

24, p

g/m

lA

gp24

, pg/

ml

Agp

24, p

g/m

l

A B

C D

E F G

- -Indomethacin 1 µM 10 µM 50 µM

129,000260,000

210,000

160,0004,5004,0003,5003,0002,5002,0001,5001,000

5000

127,000128,000

126,000125,000

9,0008,0007,0006,0005,0004,0003,0002,0001,000

0

-

+ +

+ -

+

+

+

Abacavir - 15 µM2 µM 2 µM 10 µM 10 µM 15 µM

+

+

a

a

a

a

a

a

Figure 1. NSAIDs potentiate antiretroviral activity of NRTIs in primary lymphocytes

Phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBLs) were preincubated overnight with ibuprofen 10 µM before infection with HIV type-1 (HIV-1 NL4.3 isolate at a multiplicity of infection (MOI) of 0.5. After 2 h, virus was extensively washed and the cultures were left in the presence of ibuprofen (10 µM) alone or in combination with (A) zidovudine (AZT), (B) abacavir or (C) lamivudine (3TC) at different doses and (D) tenofovir (TFV; 1 µM). (E) Effect of ibuprofen 10 µM on HIV-1 replication in infected PBLs. PHA-activated PBLs were preincubated overnight with indomethacin 10 µM before infection with HIV-1NL4.3 isolate at an MOI of 0.5. After 2 h, virus was extensively washed and the cultures were left for 3 days. (F) Viral production at 3 days in PHA-activated PBLs treated with different doses of indomethacin plus AZT (0.5 µM). (G) Effect of probenecid (PBCD) on HIV-1 infection. Cells were preincubated with PBCD (200 µM) before being infected with HIV-1NL4.3 (0.5 MOI) for 2 h, and treated with AZT (0.5 µM). Data shown are the mean ±sem of three independent experiments. We compared the results of each point incubated with antiretroviral alone and antiretroviral plus ibuprofen 10 µM. aP<0.05. bP=0.02. NRTI, nucleoside reverse transcriptase inhibitor; NS, non-significant; NSAIDs, non-steroidal anti-inflammatory drugs.

MI Clemente et al.

©2009 International Medical Press1106

1,600

1,400

1,200

1,000

800

600

400

200

0

HIV-1NL4.3 - + + + +

+ ++

- - - 10 µM 50 µM

--

Ibuprofen

AZT 0.5 µM

Agp

24, p

g/m

l

A B

1,800

1,400

1,200

1,000

800

600

400

200

0

1,600

HIV-1NL4.3 -

-

-

-

-

-

+ +

+ +

+ +

+

50 µM10 µMIbuprofen

AZT 0.5 µMA

gp24

, pg/

ml

a

Figure 2. Effect of ibuprofen on AZT antiviral activity in CEM and CEM3TC cells

(A) CEM parental cells and (B) CEM3TC cells were preincubated overnight with ibuprofen (10 µM or 50 µM) before infection with HIV type-1 (HIV-1)NL4.3 isolate (multiplicity of infection of 0.5). After 2 h, virus was extensively washed and the cultures were left in the presence of ibuprofen alone or in combination with zidovudine (AZT) 0.5 µM. Agp24 levels were measured after 3 days post-infection. aP<0.05.

PHA-activated PBLs

Ibuprofen

10 µM

MRP4

Tubulin

MRP4-FITC

A B

C D

CD4-PE DAPI

MRP4 expression, %

PBLs

PHA-activatedPBLs

0 10 20 30 40 50

a

100 101 102 103

75.3%

397

PHA-activated PBLsAZT 2 µM

100 101 102 103

65%G G

379

PHA-activated PBLs

Number of cells

Flou

resc

ence

inte

nsity

Flou

resc

ence

inte

nsity

Number of cells

Figure 3. Expression of MRP4 in primary lymphocytes

(A) Western blot analysis of multidrug resistance protein (MRP)4 expression in phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBLs) incubated or not with ibuprofen (10 µM). (B) Study of MRP4 expression by immunofluorescence. PHA-activated PBLs were stained with an anti-MRP4 or anti-CD4 monoclonal antibodies. Nuclei are staining with 4′,6-diamidine-2-phenylindole (DAPI). Original magnification ×100. (C) Flow cytometry analysis of MRP4 expression in non-activated and PHA-activated PBLs of healthy donors. (D) Flow cytometry analysis of MRP4 expression after zidovudine (AZT) treatment for 3 days. The results are representative of three independent experiments with different PBLs donors. aP<0.05. FITC, fluorescein isothiocyanate.

NSAIDs increase NTRI activity through MRP4

Antiviral Therapy 14.8 1107

as no reduced cell viability was shown in any instance at any dose used in this study (data not shown).

Taken together, our data indicate that the inhibi-tion of HIV-1 infection was not to the result of down- regulation of cell surface receptors, changes on cell acti-vation or cellular toxicity.

Effect of NSAIDs on efflux transporter activityIt has been previously described that ibuprofen or indomethacin can inhibit MRP4 transporter, thus increasing effective intracellular concentrations of NRTIs [25]. To investigate this hypothesis, we studied whether the action of NSAIDs on HIV-1 infection could be associated with an effect on MRP4 increasing capa-bility of the cells to retain AZT, thereby improving its antiviral activity. PHA-activated PBLs were pretreated with ibuprofen, indomethacin or PBCD for 16 h, loaded with [3H]AZT and levels of radioactivity intracellular were determined at different time set points. Interest-ingly, the NSAIDs tested, as well as the PBCD used as control, produced a marked increase in the accumula-tion of [3H]AZT to pretreated cells (Figure 5A and 5B). Therefore, the effect of NSAIDs described in this study is associated, at least in part, with an increase capability of cells to retain antiretroviral drugs.

MRP4 expression is increased in HAART-treated patients who failed to control HIV-1 replicationNext, we addressed whether MRP4 expression was modified in HIV-1-positive patients by long-term treat-ment with HAART. Therefore, we performed flow cytometry analysis on total leukocytes from whole blood samples from 3 healthy controls and 10 multi-treated HIV-1 patients (3 of them with undetectable VL and 7 with detectable VL). A representative profile from each group of patients is shown in Figure 6A. No differ-ences were observed between HIV-1-positive individu-als with undetectable plasma VL and healthy donors, but MRP4 expression was significantly higher in HIV-1 patients who not control viral replication. In Figure 6B we show the mean of MRP4 expression ±se from all patients tested. Our data suggest that patients with no control of viral replication suffer a continued exposi-tion to antiretroviral therapy, which could increase MRP4 expression contributing to treatment failure.

Discussion

Significant advances in the treatment of HIV-1 infection have been achieved with the success of HAART. Unfortunately, the emergence of drug resist-ance frequently occurs because of rapid mutation of viral genome. In addition, host cellular factors might also be involved in the resistance to antiretroviral drugs [2,33]. Two main host cellular mechanisms

contribute to resistance: altered metabolism of nucleo-side analogues caused by impaired nucleoside phos-phorylation and increased efflux of the compounds by membrane transport mechanisms [4,18].

35,000

30,000

25,000

20,000

4,0003,5003,0002,5002,0001,5001,000

5000

HIV-1NL4.3

NS398 5 µM

AZT 0.5 µM

Agp

24, p

g/m

l

+

-

- - +

+ - +

+

+ + +

Figure 4. NSAIDs do not alter cell activation in PBLs

Phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBLs) were pretreated with NS398 (5 µM) for 2 h before infection with HIV type-1 (HIV-1)NL4.3. After this, cells were subsequently incubated with NS398 alone or plus zidovudine (AZT; 0.5 µM). Agp24 was quantified in the supernatants of cultures 3 days post-infection. The results are representative of three independent experiments with different PBL donors. NSAIDs, non-steroidal anti-inflammatory drugs.

Table 1. NSAIDS do not alter cell receptors as CD4/CXCR4 in PBLs

NSAIDS, non-steroidal anti-inflammatory drugs; PBLs, peripheral blood lymphocytes.

Cell receptorTreatment CD4, % CXCR4, %

Control 66 68Ibuprofen 10 µM 65 66Zidovudine 0.5 µM 66 70Ibuprofen 10 µM and zidovudine 0.5 µM 67 72

Table 2. NSAIDS do not alter activation markers as CD69 and CD25 in PBLs

NSAIDS, non-steroidal anti-inflammatory drugs; PBLs, peripheral blood lymphocytes.

Activation markerTreatment CD89, % CD25, %

Control 12 63Ibuprofen 10 µM 13 58Zidovudine 0.5 µM 14 58Ibuprofen 10 µM and zidovudine 0.5 µM 14 53

MI Clemente et al.

©2009 International Medical Press1108

MRP4, also known as ABC transporter family class C4 (ABCC4), is a member of a large family of trans-membrane proteins involved in active transport of physiological substrates out of cells, such us glutath-ione, glucuronate or sulfate conjugates [34]. MRP4 has also been described as an active transporter of cyclic nucleotides [35–37] and prostaglandins [25]. More-over, MRP4 might mediate the efflux of antiviral [4,20] and anticancer [35] purine nucleotide analogues, and is involved in storage and release of ADP in platelet-dense granules [38].

We describe here for the first time that the anti-HIV-1 activity of NRTIs is potentiated by some NSAIDs, such as ibuprofen and indomethacin, at pharmacological con-centrations (1–50 µM). Although it has been previously

described that various NSAIDs inhibit both MRP2 and MRP4 [39], no data exist about the relation among NSAIDs, MRP4 and HIV-1 infection. To our knowl-edge, it is the first time that this relation is described. Here, we show that NSAIDs tested improve NRTI activity, likely reflecting MRP4 inhibition, as PBCD mimics the NSAID-induced effects. Thus, it is possible that blocking MRP4 function by using NSAIDs leads to an increase in the efficacy of antiretroviral treatment.

PBLs were found to express MRP4 as assessed by Western blot analyses, indirect immunoflourescence, and flow cytometry and, more importantly, most of the CD4+ T-cells also coexpress MRP4. Sassi et al. [40] have described that there is a MRP4 up- regulation in prolif-erating primary cultures of human coronary artery that

5,0004,5004,0003,5003,0002,5002,0001,5001,000

5000

cpm

cpm

Control [3H]AZT

Indomethacin10 µM

[3H]AZTControl

8,000

7,000

6,000

5,000

4,000

3,000

2,000

1,000

0[3H]AZT

12,000

10,000

8,000

6,000

4,000

2,000

0

0 h 1 h 3 h 5 h

cpm

A

B C

Control

Ibuprofen 10 µM and [3H]AZT

24 h cpm 24 h cpm

[3H]AZT

PBCD 200 µM

[3H]AZT

Figure 5. Effect of NSAIDs on efflux transporter activity

(A) Phytohemagglutinin (PHA)-activated peripheral blood lymphocytes (PBLs) were incubated overnight with ibuprofen (10 µM) and then, [3H]zidovudine (AZT; 1 µCi) was added to the culture. Intracellular levels of [3H]AZT were determined at the indicated times. (B) Intracellular levels of [3H]AZT were determined at 24 h in PHA-activated PBLs treated with indomethacin (10 µM) for 16 h. (C) PHA-activated PBLs were incubated with probenecid (PBCD; 200 µM), overnight and [3H]AZT was added to the culture. Intracellular levels of [3H]AZT were determined at the indicated time points. Data are representative of at least three independent experiments. cpm, count per million.

NSAIDs increase NTRI activity through MRP4

Antiviral Therapy 14.8 1109

correlate with the expression of cyclin D1, a marker of aortic smooth muscle cell proliferation. In this work, we described that the MRP4 levels were increased by 25–60% in PHA-activated PBLs over unstimulated cells. It seems clear that in these cells, MRP4 is expressed at a low level and is up-regulated in response to prolifera-tive stimuli as PHA.

Several ABC membrane transporters exhibit an increase of the expression upon drug exposure. It has been described that overexpression of MRP4 was directly linked to impaired antiviral efficacy and enhanced efflux of antiviral nucleoside-based drugs [4] and, in patients with neuroblastoma, increased MRP4 expression was shown to be of prognostic value [41]. In peripheral blood mononuclear cells isolated from healthy volunteers, various PIs were shown to increase the cell surface expression of Pgp after 72 h of incuba-tion [42]. However, at the moment, there is no conclu-sive evidence for a causal relationship between clini-cally relevant drug resistance and MRP4 transport activity. The factors regulating MRP4 expression are not yet known, but in hepatocytes, MRP4 was shown to be induced by oxidative stress [43], and the COX-2 inhibitor celecoxib induced mRNA and protein expres-sion in lung cancer cells [44]. Interestingly, we found that short-term treatment of PBLs with AZT leads to a significant increase in the number of MRP4-positive

cells. These results fit with previous data showing that in vitro culturing of PBLs with nucleoside analogues increases MRP4 expression [26]. Moreover, in macro-phages, AZT has been shown to increase the mRNA levels of several multidrug transporters, including MRP4, thereby decreasing AZT pharmacological activity [45].

It is possible that the results described here are caused by an effect of ibuprofen on long terminal repeat acti-vation, as it has been described that this NSAID inhib-its nuclear factor-κB activation, the transcription factor required for LTR-dependent transcription. However, much higher doses (approximately 1–3 mM concentra-tions) are necessary than those required for the effects observed here [24,46], making it an unlikely mecha-nism of inhibition.

By contrast, it has been described that blocking COX-2 activity prevents T-cell activation [32], which is clearly an important factor for HIV-1 replication. However, we discard this fact because NS398, a COX-2- specific inhibitor, did not potentiate NRTI anti-viral effect in contrast to ibuprofen or indomethacin. Moreover, none of the NSAIDs tested had any effect on T-cell activation as detected by CD69 and CD25 expression. Therefore, it is unlikely that changes in cell activation are responsible for the effects observed on HIV-1 replication. More importantly, we found

MRP4 expression, %5 10 15 200

HIV-1-positive VL

HIV-1-positive uVL

HIV-1-negative

Number of cells100 101 102 103

HIV-1-negativeA

B

6.8%

1.40

Number of cells100 101 102 103

HIV-1-positive uVL

102

7.8%

Number of cells100 101 102 103

HIV-1-positive VL

79

17%

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Figure 6. MRP4 expression increases in HIV-1-treated patients who failed to control viraemia

(A) Multidrug resistance protein (MRP)4 differential expression in response to antiretroviral treatment. Flow cytometry analysis of whole blood samples of three healthy donors, three patients treated heavily with highly active antiretroviral therapy with undetectable viral load (uVL) and seven with detectable viral load (VL), was performed. (B) Mean ±se of MRP4 expression of all patients. FITC, fluorescein isothiocyanate. HIV-1, HIV type-1.

MI Clemente et al.

©2009 International Medical Press1110

neither an altered expression of CD4 or CXCR4, nor changes in cellular viability in the presence of ibupro-fen or indomethacin.

To test the specific effect of ibuprofen in MRP4, we used the MRP4 overexpressing T-cell CEM3TC. As Schuetz et al. [4] described, we have found that HIV-1 replication is less effectively inhibited by NRTIs in cell lines overexpressing MRP4. Our data showed that AZT inhibition in CEM3TC cells was approximately 60%, whereas AZT inhibition in PBLs and in CEM parental cells was approximately 90%. The AZT inhibition was increased when we added ibuprofen 50 µM and the inhibition increased by >40% com-pared with AZT alone. As we expected, we needed a higher dose of ibuprofen to obtain the same effect observed in PBL, as CEM3TC cells express much more MRP4. In CEM parental cells we did not obtain an increase in the antiretroviral effect. These results indicated that MRP4, as we expected, awarded resist-ance to AZT and that ibuprofen was inhibiting MRP4 and not other MRP members.

Data on the role of MRP4 on HIV-1 infection are also scarce. Therefore, it has been reported that HIV-1 infection can increase the transcription of MRP1, MRP4 and MRP5 in human monocyte-derived macrophages [47]. Moreover, ectopic MRP4 over expression impairs the antiviral efficacy of 3TC and AZT [27]. In line with this, it has been described that MRP4 restrict permea-tion of antiretroviral nucleosides in microglia [19].

Turriziani et al. [31] demonstrated that HIV-1- positive individuals failing treatment, presented higher levels of MRP mRNA expression than healthy donors. However, this study was performed only on samples from patients failing antiretroviral treatment. Here, we examined MRP4 expression in both patients with and without control of VL. Although it is clear that we need to perform the study on a higher number of patients, our data suggest that multitreated patients with detectable VL present higher expression of MRP4 than patients with undetectable VL. On the basis of our data and others [31], it is possible that HIV-1 infection and antiretroviral therapy are responsible for MRP expression.

Taken together, our data imply that the use of MRP4 inhibitors, including some well known and safe NSAIDs, in conjunction with current HIV-1 therapies, could be advantageous to patients by increasing intrac-ellular drug concentrations and preventing host cellular mechanisms that contribute to drug resistance. Thus, the importance of MRP4 in antiretroviral therapy is becoming evident. Further studies with higher num-bers of patients are needed to confirm our results and to determine which antiretroviral(s) might cause altera-tion on MRP4 expression in HIV-1-patients, in turn reducing HAART efficacy.

Acknowledgements

We thank Dr Laura Díaz for helpful analysis of flow cytometry results. This work was supported by grants from Fondos de Investigación Sanitaria (FIS PI061479), Red Temática de Investigación Coopera-tiva Sanitaria ISCIII (RETIC RD06/0006/0035), Fun-dación para la Investigación y Prevención del SIDA en España, FIPSE (36514/05, 36536/05) and Fun-dación Caja Navarra to MAMF. From RED RICET (RD06/0021), Programa Nacional de Salud of Spain (SAF2005-02220), Laboratorios del Dr ESTEVE, the 6th EU Framework Programme European Com-mission (integrated project EICOSANOX, LSH-CT-2004-005033 and MAIN network of excellence) and the Fundación Ramón Areces to MF. From Fondo de Investigación Sanitaria (PI040883) and Comunidad Autónoma de Madrid (S-SAL-0159/2006) to both MAMF and MF. MIC is the holder of a fellowship from FIS (FI0501093), and SA is supported by a fel-lowship of FIS (CD06/00321).

Disclosure statement

The authors declare no competing interests.

References1. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance

genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet 1999; 353:2195–2199.

2. Groschel B, Cinatl J, Cinatl J, Jr. Viral and cellular factors for resistance against antiretroviral agents. Intervirology 1997; 40:400–407.

3. Hoggard PG, Owen A. The mechanisms that control intracellular penetration of the HIV protease inhibitors. J Antimicrob Chemother 2003; 51:493–496.

4. Schuetz JD, Connelly MC, Sun D, et al. MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nat Med 1999; 5:1048–1051.

5. Borst P, Elferink RO. Mammalian ABC transporters in health and disease. Annu Rev Biochem 2002; 71:537–592.

6. Russel FG, Koenderink JB, Masereeuw R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharmacol Sci 2008; 29:200–207.

7. Leslie EM, Deeley RG, Cole SP. Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters. Toxicology 2001; 167:3–23.

8. Konig J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1999; 1461:377–394.

9. Haimeur A, Conseil G, Deeley RG, Cole SP. The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation. Curr Drug Metab 2004; 5:21–53.

10. Dallas S, Miller DS, Bendayan R. Multidrug resistance-associated proteins: expression and function in the central nervous system. Pharmacol Rev 2006; 58:140–161.

11. Borst P, de Wolf C, van de Wetering K. Multidrug resistance-associated proteins 3, 4, and 5. Pflugers Arch 2007; 453:661–673.

NSAIDs increase NTRI activity through MRP4

Antiviral Therapy 14.8 1111

12. Guo Y, Kotova E, Chen ZS, et al. MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic nucleotide efflux pump and a resistance factor for fluoropyrimidines 2′,3′-dideoxycytidine and 9′-(2′-phosphonylmethoxyethyl)adenine. J Biol Chem 2003; 278:29509–29514.

13. Kruh GD, Belinsky MG. The MRP family of drug efflux pumps. Oncogene 2003; 22:7537–7552.

14. Srinivas RV, Middlemas D, Flynn P, Fridland A. Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters. Antimicrob Agents Chemother 1998; 42:3157–3162.

15. Jones K, Bray PG, Khoo SH, et al. P-Glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance? AIDS 2001; 15:1353–1358.

16. Janneh O, Owen A, Chandler B, et al. Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP. AIDS 2005; 19:2097–2102.

17. Reid G, Wielinga P, Zelcer N, et al. Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol 2003; 63:1094–1103.

18. Wijnholds J, Mol CA, van Deemter L, et al. Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs. Proc Natl Acad Sci U S A 2000; 97:7476–7481.

19. Dallas S, Schlichter L, Bendayan R. Multidrug resistance protein (MRP) 4- and MRP 5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine by microglia. J Pharmacol Exp Ther 2004; 309:1221–1229.

20. Imaoka T, Kusuhara H, Adachi M, et al. Functional involvement of multidrug resistance-associated protein 4 (MRP4/ABCC4) in the renal elimination of the antiviral drugs adefovir and tenofovir. Mol Pharmacol 2007; 71:619–627.

21. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 1998; 38:97–120.

22. Han S, Inoue H, Flowers LC, Sidell N. Control of COX-2 gene expression through peroxisome proliferator-activated receptor gamma in human cervical cancer cells. Clin Cancer Res 2003; 9:4627–4635.

23. Stichtenoth DO, Wagner B, Frolich JC. Effects of meloxicam and indomethacin on cyclooxygenase pathways in healthy volunteers. J Investig Med 1997; 45:44–49.

24. Tegeder I, Pfeilschifter J, Geisslinger G. Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J 2001; 15:2057–2072.

25. Reid G, Wielinga P, Zelcer N, et al. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc Natl Acad Sci U S A 2003; 100:9244–9249.

26. Turriziani O, Pagnotti P, Pierangeli A, et al. The effects of prolonged treatment with zidovudine, lamivudine, and abacavir on a T-lymphoblastoid cell line. AIDS Res Hum Retroviruses 2006; 22:960–967.

27. Turriziani O, Schuetz JD, Focher F, et al. Impaired 2′,3′-dideoxy-3′-thiacytidine accumulation in T-lymphoblastoid cells as a mechanism of acquired resistance independent of multidrug resistant protein 4 with a possible role for ATP-binding cassette C11. Biochem J 2002; 368:325–332.

28. Warner TD, Mitchell JA. Nonsteroidal antiinflammatory drugs inhibiting prostanoid efflux: as easy as ABC? Proc Natl Acad Sci U S A 2003; 100:9108–9110.

29. Courtois A, Payen L, Lagadic D, Guillouzo A, Fardel O. Evidence for a multidrug resistance-associated protein 1 (MRP1)-related transport system in cultured rat liver biliary epithelial cells. Life Sci 1999; 64:763–774.

30. Payen L, Courtois A, Vernhet L, Guillouzo A, Fardel O. The multidrug resistance-associated protein (MRP) is over-expressed and functional in rat hepatoma cells. Int J Cancer 1999; 81:479–485.

31. Turriziani O, Gianotti N, Falasca F, et al. Expression levels of MDR1, MRP1, MRP4, and MRP5 in peripheral blood mononuclear cells from HIV infected patients failing antiretroviral therapy. J Med Virol 2008; 80:766–771.

32. Iniguez MA, Punzon C, Fresno M. Induction of cyclooxygenase-2 on activated T lymphocytes: regulation of T cell activation by cyclooxygenase-2 inhibitors. J Immunol 1999; 163:111–119.

33. Swanstrom R, Erona J. Human immunodeficiency virus type-1 protease inhibitors: therapeutic successes and failures, suppression and resistance. Pharmacol Ther 2000; 86:145–170.

34. Deeley RG, Westlake C, Cole SP. Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol Rev 2006; 86:849–899.

35. Chen ZS, Lee K, Kruh GD. Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J Biol Chem 2001; 276:33747–33754.

36. van Aubel RA, Smeets PH, Peters JG, Bindels RJ, Russel FG. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol 2002; 13:595–603.

37. Wielinga PR, van der Heijden I, Reid G, et al. Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells. J Biol Chem 2003; 278:17664–17671.

38. Jedlitschky G, Tirschmann K, Lubenow LE, et al. The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. Blood 2004; 104:3603–3610.

39. El-Sheikh AA, van den Heuvel JJ, Koenderink JB, Russel FG. Interaction of nonsteroidal anti-inflammatory drugs with multidrug resistance protein (MRP) 2/ABCC2- and MRP4/ABCC4-mediated methotrexate transport. J Pharmacol Exp Ther 2007; 320:229–235.

40. Sassi Y, Lipskaia L, Vandecasteele G, et al. Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation. J Clin Invest 2008; 118:2747–2757.

41. Norris MD, Smith J, Tanabe K, et al. Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro. Mol Cancer Ther 2005; 4:547–553.

42. Chandler B, Almond L, Ford J, et al. The effects of protease inhibitors and nonnucleoside reverse transcriptase inhibitors on p-glycoprotein expression in peripheral blood mononuclear cells in vitro. J Acquir Immune Defic Syndr 2003; 33:551–556.

43. Maher JM, Dieter MZ, Aleksunes LM, et al. Oxidative and electrophilic stress induces multidrug resistance-associated protein transporters via the nuclear factor-E2-related factor-2 transcriptional pathway. Hepatology 2007; 46:1597–1610.

44. Gradilone A, Pulcinelli FM, Lotti LV, et al. Celecoxib induces MRP-4 in lung cancer cells: therapeutic implications. J Clin Oncol 2007; 25:4318–4320, author reply 4320.

45. Jorajuria S, Dereuddre-Bosquet N, Becher F, et al. ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages. Antivir Ther 2004; 9:519–528.

46. Takada Y, Bhardwaj A, Potdar P, Aggarwal BB. Nonsteroidal anti-inflammatory agents differ in their ability to suppress NF-kappaB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 2004; 23:9247–9258.

47. Jorajuria S, Dereuddre-Bosquet N, Naissant-Storck K, Dormont D, Clayette P. Differential expression levels of MRP1, MRP4, and MRP5 in response to human immunodeficiency virus infection in human macrophages. Antimicrob Agents Chemother 2004; 48:1889–1891.

Accepted for publication 15 July 2009