Reversal of P-glycoprotein–Mediated Multidrug Resistance ... · Reversal of...

Reversal of P-glycoprotein–Mediated Multidrug Resistance

in Cancer Cells by the c-Jun NH2-Terminal Kinase

Jun Zhou,1Min Liu,

1Ritu Aneja,

2,3Ramesh Chandra,

3

Hermann Lage,4and Harish C. Joshi

2,3

1Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China; 2Department of Cell Biology, EmoryUniversity School of Medicine, Atlanta, Georgia; 3BR Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India;and 4Institute of Pathology, Humboldt University Berlin, Charite Campus Mitte, Schumannstrasse, Berlin, Germany

Abstract

A significant impediment to the success of cancer chemother-apy is multidrug resistance (MDR). A typical form of MDR isattributable to the overexpression of membrane transportproteins, such as P-glycoprotein, resulting in an increaseddrug efflux. In this study, we show that adenovirus-mediatedenhancement of the c-Jun NH2-terminal kinase (JNK) reducesthe level of P-glycoprotein in a dose- and time-dependentmanner. Protein turnover assay shows that the decrease ofP-glycoprotein is independent of its protein stability. Instead,this occurs primarily at the mRNA level, as revealed by reversetranscription-PCR analysis. We find that P-glycoproteindown-regulation requires the catalytic activity of JNK and ismediated by the c-Jun transcription factor, as either pharma-cologic inhibition of JNK activity or dominant-negativesuppression of c-Jun remarkably abolishes the ability of JNKto down-regulate P-glycoprotein. In addition, electrophoreticmobility shift assay reveals that adenoviral JNK increases theactivator protein binding activity of themdr1 gene in the MDRcells. We further show that the decrease of P-glycoproteinlevel is associated with a significant increase in intracellulardrug accumulation and dramatically enhances the sensitivityof MDR cancer cells to chemotherapeutic agents. Our studyprovides the first direct evidence that enhancement of theJNK pathway down-regulates P-glycoprotein and reversesP-glycoprotein–mediated MDR in cancer cells. (Cancer Res2006; 66(1): 445-52)

Introduction

Multidrug resistance (MDR), by which cells resist manystructurally and functionally unrelated drugs, is a serious problemin chemotherapeutic management of cancer. The MDR phenotypeis most often due to overexpression of drug efflux pumps inthe plasma membrane of cancer cells. P-glycoprotein, a 170-kDatransmembrane glycoprotein encoded by the mdr1 gene, is thebest characterized drug efflux pump to date and is a member ofthe ATP-binding cassette transporter family (1–3). A wide rangeof anticancer drugs has been described to be substrates forP-glycoprotein (e.g., anthracyclines, Vinca alkaloids, and taxanes;ref. 3). Overexpression of P-glycoprotein has been shown to conferMDR in cultured cells and has also been implicated in the clinical

MDR (3, 4). In addition, P-glycoprotein overexpression correlateswith poor prognosis for a number of human cancers (3).It is believed that inhibition of P-glycoprotein function or

inhibition of its expression may reverse P-glycoprotein-mediatedMDR phenotype and improve the effectiveness of chemotherapy.Since the early 1980s, a broad spectrum of compounds has beenexamined for their capability to overcome P-glycoprotein-mediatedMDR. Notable examples include verapamil, phenothiazines,and cyclosporins. Unfortunately, despite their potent anti-P-glycoprotein activity in cultured cells, the clinical trials of thesecompounds have not yet been successful, in large part because oftheir confounded pharmacokinetic interaction with anticancerdrugs and their notorious side effects (3). Thus, it is of greatimportance to develop new compounds or strategies that arecapable of circumventing P-glycoprotein-mediated MDR withimproved clinical characteristics.c-Jun NH2-terminal kinase (JNK) is a member of the mitogen-

activated protein kinase family that binds the NH2-terminalactivation domain of the transcription factor c-Jun and phosphor-ylates c-Jun (5). The activity of JNK has been implicated in theregulation of embryonic morphogenesis, cell proliferation, tumortransformation, and apoptosis (5). We and others recently foundthat cancer cells overexpressing P-glycoprotein had minorendogenous JNK activity (Fig. 1; refs. 6, 7). In addition, c-Jun wasrecently reported to play a critical role in the down-regulation ofP-glycoprotein by salvicine, a synthesized diterpenoid quinine (8).These findings led us to hypothesize that elevation of the JNKpathway might be able to down-regulate P-glycoprotein and,if so, the JNK pathway might be exploited for overcomingP-glycoprotein-mediated MDR. This study was undertaken to testthese hypotheses directly.

Materials and Methods

Materials. All compounds were purchased from Sigma-Aldrich

(St. Louis, MO) and prepared in DMSO. Mouse monoclonal antibody

against P-glycoprotein (Calbiochem, La Jolla, CA), rabbit polyclonalantibody against h-actin (Sigma-Aldrich), and horseradish peroxidase–

conjugated anti-rabbit and anti-mouse antibodies (Sigma-Aldrich) were

obtained from the indicated sources.

Cell culture. The human gastric carcinoma cell line EPG85-257 andhuman pancreatic carcinoma cell line EPP85-181 and their MDR derivative

lines, EPG85-257RDB and EPP85-181RDB, were cultured in Leibowitz-15

medium supplemented with 10% fetal bovine serum and 2 mmol/LL-glutamine at 37jC in a humidified atmosphere with 5% CO2.

Cell proliferation assay. Cells were seeded in 96-well plates at a density

of 2 � 103 per well, and the sulforodamine B assay was then done as

described previously (9, 10). The percentage of cell survival as a function ofdrug concentration was then plotted to determine IC50, which stands for

the drug concentration needed to prevent cell proliferation by 50%.

Requests for reprints: Jun Zhou, Department of Genetics and Cell Biology, Collegeof Life Sciences, Nankai University, Tianjin 300071, China. Phone: 86-22-2350-8800;Fax: 86-22-2350-8800; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1779

www.aacrjournals.org 445 Cancer Res 2006; 66: (1). January 1, 2006

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Adenovirus preparation and infection. The replication-defectiverecombinant adenovirus was prepared using the Adeno-X expression

system (BD Biosciences, San Jose, CA). Briefly, the cDNA of JNK was first

cloned into the pShuttle2 vector. The JNK expression cassette was then

subcloned into the pAdenoX vector. To produce the adenovirus, the

recombinant pAdenoX-JNK plasmid was linearized by digestion with PacIand then transfected into low-passage HEK293 cells as described previously

(11). Adenovirus titer was determined with an adenovirus titer kit from BD

Biosciences. The multiplicity of infection (MOI) was defined as the ratio of

infectious units divided by the number of cells.Immunocomplex kinase assay. The activity of JNK was measured by

using the immunocomplex JNK kinase assay kit (Calbiochem). Briefly, cell

lysates were prepared in 20 mmol/L Tris (pH 7.4), 200 mmol/L NaCl, and

1% NP40 with the protease inhibitor cocktail (Roche Applied Science,Indianapolis, IN). The protein concentrations were determined by

bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL). To

immunoprecipitate JNK, cell lysate containing 100 Ag of total protein was

incubated with a JNK-specific antibody and protein A/G-agarose beads at4jC overnight. The JNK immunoprecipitates were washed thrice with the

cell lysis buffer and then used for the kinase assay, with purified c-Jun as a

substrate as described previously (12).Western blot analysis. Proteins were resolved by SDS-PAGE and

transferred onto polyvinylidene difluoride membranes (Millipore, Bedford,

MA). The membranes were blocked for 2 hours in TBS containing 0.2%

Tween 20 and 5% fat-free dry milk and then incubated first with primaryantibodies and then horseradish peroxidase–conjugated secondary anti-

bodies for 2 hours and 1 hour, respectively. Specific proteins were visualized

with enhanced chemiluminescence detection reagent according to the

manufacturer’s instructions (Pierce Biotechnology). The intensity of proteinbands was determined by densitometric analysis with a Lynx video

densitometer (Biological Vision).

Semiquantitative reverse transcription-PCR. Total cellular RNA wasprepared using the TRIzol reagent (Invitrogen, San Diego, CA) following the

manufacturer’s instructions. Reverse transcription-PCR (RT-PCR) analysis

of mdr1 mRNA expression was done as described previously (13). Equal

volumes of RT-PCR reactions were loaded for agarose gel electrophoresis,and the products were quantified by densitometry after ethidium bromide

staining.

Drug accumulation. Cells were collected by trypsinization and

resuspended in growth medium containing 20 Amol/L daunorubicin ordoxorubicin. After incubation at 37jC in 5% CO2 for 1 hour, cells were

spinned down and washed in PBS. Cells were resuspended in drug-free

growth medium and incubated for another 2 hours at 37jC in 5% CO2. Theaccumulation of drugs in the cells was then analyzed with fluorescence

microscopy, and the intracellular fluorescence was quantified with a

fluorescence plate reader (Millipore).

Results

Properties of human gastric and pancreatic carcinoma celllines that display MDR. The MDR cell lines EPG85-257RDB andEPP85-181RDB were derived from EPG85-257 and EPP85-181 celllines, respectively, by selection in increased concentrations ofdaunorubicin (14–16). These derivative cell lines were shown tohave MDR phenotype by their wide cross-resistance and defects inintracellular drug accumulation. In addition, their MDR phenotypeseemed mediated by increased expression of P-glycoprotein butnot by MRP-related proteins or BCRP (17, 18). In agreement withthe previous studies, cell proliferation assay revealed that the twoMDR cell lines exhibited 2,287- and 1,498-fold resistance todaunorubicin, respectively compared with their parental chemo-sensitive counterparts (Fig. 1A). Similarly, EPG85-257RDB andEPP85-181RDB cell lines were also 3,788- and 2,856-fold moreresistant to doxorubicin, respectively, compared with their parentalcounterparts (Fig. 1A). We examined the level of P-glycoprotein andthe level and activity of JNK in the MDR lines 7 days after the cellswere released to drug-free medium. The MDR cell lines showedrobust expression of P-glycoprotein, whereas in the parental celllines P-glycoprotein was not detectable (Fig. 1B). In addition, boththe activity and level of the JNK seemed lower in the MDR cell lines

Figure 1. Characterization of MDR cancer cell lines. A, IC50 values ofdaunorubicin and doxorubicin in human gastric (EPG85-257) and pancreatic(EPP85-181) carcinoma cell lines and their respective MDR cell linesEPG85-257RDB and EPP85-181RDB. IC50 values were measured by the cellproliferation assay as described in Materials and Methods. Note that the y axis isin logarithmic scale. B and C, comparison of the level of P-glycoprotein (Pgp)and the level and activity of JNK between the parental and MDR cells. Cells weremaintained in daunorubicin, and the levels of P-glycoprotein, h-actin,phosphorylated c-Jun (p-c-Jun), and JNK were examined 7 days after cells werereleased to drug-free medium. B, P-glycoprotein and h-actin levels wereexamined by Western blot analysis of the cell lysates. The levels ofphosphorylated c-Jun were determined by the immunocomplex kinaseassay as described in Materials and Methods, as a measure of JNK activity, andJNK protein levels were examined by Western blot analysis of the JNKimmunoprecipitates. C, experiments were done as in (B ), and the level ofphosphorylated c-Jun was measured by densitometry. D, examination of thelevel of P-glycoprotein and the level and activity of JNK in the MDR celllines 7 days after cells were released to drug-free medium (�) as in (B)or in the presence of continuous drug exposure (+). E, examination of thelevel of P-glycoprotein and the level and activity of JNK in SKOV3,SKOV3R24, and SKOV3R100 cells as in (B ). Columns, averages of threeindependent experiments; bars, SD.

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Cancer Res 2006; 66: (1). January 1, 2006 446 www.aacrjournals.org



than in the parental lines (Fig. 1B). Densitometric quantificationshowed that the activity of JNK in EPG85-257RDB cells was only38.1% of that in EPG85-257 cells, and the activity in EPP85-181RDBcells was only 35.6% of that in EPG85-181 cells (Fig. 1C). The levelof P-glycoprotein and the level and activity of JNK in the MDR lineswere also examined in the presence of continuous drug exposureand were found similar to those obtained in the absence ofcontinuous drug exposure (Fig. 1D). Additionally, we found thatanother two P-glycoprotein-overexpressing MDR cell lines,SKOV3R24 and SKOV3R100, which possess 24- and 100-foldresistance respectively, also had lower JNK level and activity thanthe parental line, SKOV3 (Fig. 1E).Elevated JNK decreases P-glycoprotein levels in MDR cells in

a dose- and time-dependent manner. To test a possible role forJNK in regulating P-glycoprotein, we examined the P-glycoproteinlevel in EPG85-257RDB and EPP85-181RDB cells treated withdifferent MOI adenoviral JNK. Strikingly, adenoviral JNK decreasedthe P-glycoprotein level in both cell lines in a dose-dependentmanner (Fig. 2A and B). For example, in EPG85-257RDB cells, theP-glycoprotein level was reduced by 61.3% ( from 10.1 � 104 to3.91 � 104, arbitrary unit) upon treatment with 10 MOI adenoviralJNK for 24 hours (Fig. 2B). A similar effect of adenoviral JNKwas seen in EPP85-181RDB cells. In contrast, the adenoviralh-galactosidase control did not have obvious effect on theP-glycoprotein level even at a dose as high as 50 MOI (Fig. 2B).We also did a time course analysis for the down-regulatory effectof 10 MOI adenoviral JNK on P-glycoprotein. As shown in Fig. 2Cand D , the reduction of P-glycoprotein in EPG85-257RDB andEPP85-181RDB cells was clearly seen as early as 12 hours of

adenoviral JNK treatment. The P-glycoprotein level decreasedfurther upon longer treatment and reached a plateau after24 hours. To test whether JNK could down-regulate the expressionof endogenous P-glycoprotein, we examined the effect of adeno-viral JNK in HCT15 cells. As shown in Fig. 2E , the expression ofendogenous P-glycoprotein in HCT15 cells was also reduced byJNK in a dose-dependent manner.Down-regulation of P-glycoprotein by JNK is independent of

P-glycoprotein protein stability. Like several other membraneproteins (19, 20), the level of P-glycoprotein seems regulated byprotein stability (21, 22). To investigate whether adenoviralJNK down-regulated P-glycoprotein through this mechanism, weexamined P-glycoprotein stability in EPG85-257RDB and EPP85-181RDB cells. Protein turnover assay revealed a half-life of 18.3hours for P-glycoprotein in EPG85-257RDB cells, which was notsignificantly altered by adenoviral JNK (Fig. 3A). Similarly, inEPP85-181RDB cells, the half-life of P-glycoprotein was 19.1 hours,and it was only slightly reduced by adenoviral JNK (Fig. 3B). TheP-glycoprotein half-life values obtained in these cells were veryclose to that reported previously (14-17 hours; ref. 23). These resultsthus suggested that protein stability might play a minor role, if any,in the down-regulation of P-glycoprotein by JNK.JNK down-regulates P-glycoprotein at the mRNA level. To

examine whether P-glycoprotein was down-regulated by JNK at themRNA level, we analyzed mdr1 mRNA expression by semiquanti-tative RT-PCR. As shown in Fig. 4A and B , 5 and 10 MOI adenoviralJNK decreased mdr1 mRNA expression to similar degrees in bothEPG85-257RDB and EPP85-181RDB cells, and 50 MOI adenoviralJNK had a stronger effect, whereas there was no obvious effect for

Figure 2. Adenoviral JNK decreases P-glycoprotein (Pgp )protein level in a dose- and time-dependent manner in theMDR cancer cells. A, Western blot analysis of P-glycoproteinand h-actin levels in cells untreated or treated for 24 hours with5, 10, or 50 MOI adenoviral JNK, or 50 MOI adenoviralh-galactosidase (b-gal ) as a control. The level and activity ofJNK were also examined to confirm that adenoviral JNK wasexpressed and functional. B, experiments were doneas in (A), and the intensity of P-glycoprotein was quantified bydensitometric analysis of the Western blot bands. C, Westernblot analysis of P-glycoprotein and h-actin levels in cellstreated with 10 MOI adenoviral JNK for 0, 12, 24, 36, or48 hours. D, experiments were done as in (C ), and theintensity of P-glycoprotein was quantified by densitometry.E, adenoviral JNK decreases endogenous P-glycoproteinexpression in HCT15 cells. P-glycoprotein and h-actinexpression was examined by Western blot analysis in cellsuntreated or treated with 5, 10, or 50 MOI adenoviralJNK for 24 hours.

Reversal of MDR by JNK




adenoviral h-galactosidase treatment. The dose-effect pattern ofadenoviral JNK on mdr1 mRNA was slightly different from that onP-glycoprotein protein, for which 10 and 50 MOI adenoviral JNKhad similar effects (Fig. 2A and B). We also found that adenoviralJNK inhibited mdr1 mRNA expression in a time-dependentmanner; the mdr1 mRNA kept decreasing over 48 hours oftreatment (Fig. 4C and D). This time-effect pattern of adenoviral

JNK was also slightly different from that on P-glycoprotein protein,for which P-glycoprotein protein stopped decreasing after 24 hoursof adenoviral treatment (Fig. 2C and D). Nevertheless, these dataindicated that adenoviral JNK down-regulated P-glycoprotein at themRNA level.JNK activity and c-Jun are required for the down-regulatory

effect of JNK on P-glycoprotein. We then examined whether the

Figure 3. Down-regulation ofP-glycoprotein (Pgp ) by JNK isindependent of P-glycoprotein proteinstability. A, decrease of P-glycoproteinover time in EPG85-257RDB cells.Top, Western blot analysis ofP-glycoprotein levels. Cells were treatedwith 10 MOI adenoviral JNK orh-galactosidase (control ) for 12 hours andwere then treated with the proteinsynthesis inhibitor cycloheximide(CHX , 20 Ag/mL) for 0, 12, 24, 36,or 48 hours. Bottom, quantification ofremaining P-glycoprotein at different timepoints. % Initial P-glycoprotein levels(0 hour of cycloheximide treatment).B, decrease of P-glycoprotein over time inEPP85-181RDB cells. Experiments andquantifications were done as in (A ).

Figure 4. Adenoviral JNK decreasesP-glycoprotein at the mRNA level.A, RT-PCR analysis of mdr1 and GAPDHmRNA expression levels in cellsuntreated or treated with 5, 10, or 50 MOIadenoviral JNK, or 50 MOI adenoviralh-galactosidase (b-gal ) for 24 hours.B, experiments were done as in (A),and the intensity of the mdr1 RT-PCRproduct was quantified by densitometry.C, RT-PCR analysis of mdr1 and GAPDHmRNA expression levels in cells treatedwith 10 MOI adenoviral JNK for 0, 12, 24,36, or 48 hours. D, experiments weredone as in (C), and the intensity ofmdr1 RT-PCR product was quantifiedby densitometry.

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down-regulation of P-glycoprotein by JNK required JNK activity. Wetreated cells with adenoviral JNK in the presence of SP600125, asmall molecule inhibitor of JNK (24). As shown in Fig. 5A , SP600125not only inhibited the basic activity of endogenous JNK but alsoinhibited the JNK activity induced by exogenous JNK expression.Furthermore, SP600125 remarkably abolished the down-regulatoryeffect of JNK on P-glycoprotein (Fig. 5A ). Following thisobservation, we investigated whether the transcription factorc-Jun, the substrate for JNK, played a role for the effect of JNK(Fig. 5B). We treated cells with adenoviral JNK in the presence ofdominant-negative c-Jun and then examined the P-glycoproteinlevel. We found that dominant-negative c-Jun was able to preventpartially JNK-induced down-regulation of P-glycoprotein (Fig. 5B).This result thus indicated a role for c-Jun in mediating the effect ofJNK on P-glycoprotein.The promoter of the mdr1 gene was reported previously to

contain a negative binding site of the heterodimeric transcriptionfactor activator protein (AP-1; notably the c-Jun/c-Fos dimer; ref.25). To test whether JNK down-regulates P-glycoprotein throughenhanced AP-1 binding to the mdr1 promoter, we did electropho-retic mobility shift assay. Our result revealed that adenoviral JNKindeed increased the AP-1 binding activity of the mdr1 gene in theMDR cells (Fig. 5C).JNK-induced down-regulation of P-glycoprotein enhances

the intracellular drug accumulation in the MDR cells. Wethen asked whether the decrease of P-glycoprotein by JNK inthe MDR cells could enhance the intracellular accumulation ofanticancer drugs. The natural fluorescence of daunorubicin anddoxorubicin allowed us to examine their accumulation in cellswith fluorescence microscopy. As shown in Fig. 6A , adenoviralJNK resulted in a 3.23-fold increase of daunorubicin in EPG85-257RDB cells and a 4.52-fold increase in EPP85-181RDB cells.Similarly, adenoviral JNK also dramatically enhanced the accumu-

lation of doxorubicin in EPG85-257RDB and EPP85-181RDB cells(Fig. 6B).Adenoviral JNK increases the sensitivity of MDR cells to

anticancer drugs. The effects of adenoviral JNK on the P-glycoprotein level and on the accumulation of daunorubicinand doxorubicin suggested that it might be able to sensitize theMDR cells to these drugs. We thus measured the IC50 values ofdaunorubicin and doxorubicin in EPG85-257RDB and EPP85-181RDB cells treated with adenoviral JNK (Fig. 7). We found thatadenoviral JNK decreased the IC50 values of daunorubicin in theMDR cells by >10-fold in these MDR cells, from 7.32 to 0.563 Amol/Lin EPG85-257RDB cells and from 11.4 to 0.887 Amol/L in EPP85-181RDB cells (Fig. 7A). Similarly, adenoviral JNK also greatlyincreased the sensitivity of the MDR cells to doxorubicin by >10-fold, from 52.6 to 3.26 Amol/L in EPG85-257RDB cells and from 34.7to 2.25 Amol/L in EPP85-181RDB cells (Fig. 7B). Because JNK activitywas shown to mediate apoptosis induced by a wide variety of drugs(12, 26, 27), one could argue that adenoviral JNK might simplysensitize MDR cancer cells to anticancer drugs by lowering thethreshold for apoptosis independently of its down-regulatory effecton P-glycoprotein. However, this is very unlikely because adenoviralJNK did not have obvious proapoptotic effect in non-MDR cells,including EPG85-257, EPP85-181, and SKOV3 (Fig. 7C).

Discussion

Chemotherapy is the most effective treatment for patientswho suffer from metastatic cancers. The effectiveness of chemo-therapy, however, is seriously limited by MDR. Overexpression ofP-glycoprotein, an integral membrane protein, represents one ofthe major mechanisms that contribute to the MDR phenotype.P-glycoprotein functions as a drug efflux pump that activelytransports drugs from the inside to the outside of cells and causes

Figure 5. JNK activity and c-Jun are required for P-glycoprotein (Pgp) downregulation. A, P-glycoprotein levels in cells treated for 24 hours with 10 MOI adenoviralJNK in the absence or presence of JNK inhibitor SP600125 (SP , 20 Amol/L). P-glycoprotein levels were examined by Western blot analysis and quantified bydensitometry. The levels of phosphorylated c-Jun (p-c-Jun) were determined by the immunocomplex kinase assay as a measure of JNK activity, and JNK proteinlevels were examined by Western blot analysis of the JNK immunoprecipitates. B, P-glycoprotein levels in cells treated for 24 hours with 10 MOI adenoviralJNK in the absence or presence of dominant-negative c-Jun (dn-c-Jun ). P-glycoprotein levels were examined by Western blot analysis and quantified bydensitometry. The levels of dominant-negative c-Jun and JNK were also examined by Western blot analysis to confirm the expression. C, electrophoretic mobilityshift assay showing that adenoviral JNK increases the AP-1 binding activity of the mdr1 gene in the MDR cells.





a defect in the intracellular accumulation of drugs necessaryfor cancer cell killing. Therefore, inhibition of P-glycoproteintransporter function or inhibition of its expression may reverse theMDR phenotype through enhancing intracellular accumulation ofanticancer drugs. In the past two decades, there has been aworldwide effort investigating chemical agents for their ability toovercome MDR through interacting with P-glycoprotein andinhibiting its transporter function. These MDR modulators includecalcium channel blockers, calmodulin inhibitors, and other classesof compounds. However, none of these compounds has beensuccessful thus far in clinical trials, primarily due to their toxicitiesat the doses necessary for suppression of P-glycoprotein functionand their effects on the pharmacokinetics of anticancer drugs (3).On the other hand, compounds or strategies capable of decreasingP-glycoprotein expression might be useful in modulating the MDRphenotype and improving chemotherapy. This notion is supportedby the data in the present study showing that adenovirus-mediatedenhancement of the JNK pathway circumvented P-glycoprotein-mediated MDR in cancer cells through down-regulatingP-glycoprotein.

The JNK pathway is known to play a critical role in diverse cellularprocesses. JNK is activated when cells are exposed to proinflamma-tory cytokines or environmental stress (e.g., UVand g-radiation, heatshock, osmotic shock, redox stress, etc.), treated with variousanticancer drugs, or undergo growth factor withdrawal. Interest-ingly, we found that MDR gastric and pancreatic carcinoma cell lineswith P-glycoprotein overexpression, EPG85-257RDB and EPP85-181RDB, respectively, had lower JNK level and activity than theirparental counterparts. In addition, SKOV3R24 and SKOV3R100,another two P-glycoprotein-overexpressing MDR cell lines, also

Figure 6. JNK-induced downregulation of P-glycoprotein enhances theintracellular drug accumulation in the MDR cells. A, accumulation ofdaunorubicin in cells treated for 24 hours with 10 MOI adenoviral JNKor h-galactosidase (control ). B, accumulation of doxorubicin in cells untreated ortreated for 24 hours with 10 MOI adenoviral JNK. Drug accumulation wasmeasured by fluorescence microscopy as described in Materials and Methods.JNK expression was examined by Western blot analysis.

Figure 7. Adenoviral JNK increases the sensitivity of the MDR cells todaunorubicin and doxorubicin. A, IC50 values of daunorubicin in cells treated for24 hours with 10 MOI adenoviral JNK or h-galactosidase (control ). B, IC50

values of doxorubicin in cells untreated or treated for 24 hours with 10 MOIadenoviral JNK. JNK expression was examined by Western blot analysis.C, IC50 values of daunorubicin in EPG85-257, EPP85-181 and SKOV3 cellstreated for 24 hours with 10 MOI adenoviral JNK or h-galactosidase (control ).

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displayed lower JNK level and activity than the parental line. It wasreported previously that MDR mouse mammary carcinoma cell lineFM3A/M also had lower basal and drug-stimulated JNK activitiesthan the parental cell line (6). In addition, reactive oxygen specieswere shown to down-regulate P-glycoprotein expression accompa-nied by an increase in JNK activity in multicellular prostate tumorspheroids (7). In another study, salvicine was found to decreaseP-glycoprotein expression and increase c-Jun expression; moreover,elevated c-Jun level seemed to be a prerequisite for P-glycoproteindown-regulation by salvicine (8). These studies together suggested apotential negative correlation of JNK activity with cellular levels ofdrug resistance and that the JNK pathway might be able to down-regulate P-glycoprotein. In this study, we provided the first directevidence showing that the JNK pathway could indeed reduce theexpression of P-glycoprotein in MDR cancer cells.How might P-glycoprotein expression be down-regulated by

elevated JNK? Our data revealed that this might occur primarilyat the mdr1 mRNA level and was independent of P-glycoproteinprotein stability. Our data also showed that pharmacologicinhibition of JNK activity or dominant-negative inhibition ofc-Jun significantly prevented the down-regulatory effect of JNKon P-glycoprotein. These results thus indicated a crucial require-ment for JNK activity and c-Jun in mediating JNK-induced down-regulation of P-glycoprotein. It was reported previously that thepromoter of the mdr1 gene possesses a negative binding site of AP-1 (c-Jun/c-Fos, etc.; ref. 25). We showed in this study that adenoviralJNK increased the AP-1 binding activity of the mdr1 gene in theMDR cells. It is therefore very likely that enhanced JNK activitypromoted the phosphorylation of c-Jun, which in turn stimulatedc-Jun/c-Fos binding to the AP-1 element of the mdr1 gene, therebyleading to the repression of mdr1 mRNA expression and ultimatelythe repression of P-glycoprotein protein expression. It should benoted, however, that under hypoxia, JNK activity seemed topositively correlate with P-glycoprotein/mdr1 expression (28–30).Therefore, more remains to be learned to generate a clear pictureabout the molecular basis underlying the regulation of P-glycoprotein/mdr1 expression by JNK. In addition, many othermechanisms have been presented previously for the regulation ofP-glycoprotein/mdr1. For example, cross-coupled nuclear factor-nB/p65 and c-Fos transcription factors have been reported tonegatively regulate the promoter activity of mdr1 (31). On the otherhand, the heat-shock transcription factor HSF1, the Y-box bindingprotein YB1, and the Sp1 transcription factor have been shown to

positively regulate P-glycoprotein/mdr1 expression (32–34). Inaddition, the transcription factor NF-Y has been shown to mediatethe regulation of mdr1 by histone acetyltransferase and deacetylase(35). These studies, together with our finding that JNK/c-Junnegatively regulates P-glycoprotein/mdr1, reflect the complexnature of P-glycoprotein/mdr1 regulation.The data in this study showed that adenoviral JNK significantly

sensitized P-glycoprotein-overexpressing MDR cancer cells tochemotherapeutic agents. This might be primarily attributed tothe observed increase in intracellular drug accumulation resultingfrom P-glycoprotein down-regulation by JNK, a feature similarto that displayed by inhibition of P-glycoprotein transporterfunction via chemical agents. However, alternative mechanismsmight exist mediating the JNK-induced sensitization of MDRcells. For instance, P-glycoprotein was reported to have anantiapoptotic function in addition to its drug efflux activity(36, 37). It is possible that the down-regulation of P-glycoproteinby JNK might potentiate MDR cells to chemotherapy-inducedcell death through antagonizing the antiapoptotic activity ofP-glycoprotein.In summary, we have shown that JNK activity negatively cor-

relates with P-glycoprotein level in MDR gastric and pancreaticcancer cells, and adenovirus-mediated enhancement of JNKdown-regulates P-glycoprotein in a dose- and time-dependentmanner, increases drug accumulation, and sensitizes MDR cancercells to chemotherapeutic agents. In addition, we have shown thatthe down-regulation of P-glycoprotein occurs at the messengerlevel and requires the activity of JNK and the c-Jun transcriptionfactor. These findings support the notion that decreasingP-glycoprotein expression may be a useful approach for reversingMDR in addition to the conventional approach that employs theinhibition of P-glycoprotein function. In vivo studies arewarranted to examine whether the JNK pathway has a clinicalpotential in modulating the MDR phenotype during cancerchemotherapy.

Acknowledgments

Received 5/25/2005; revised 10/10/2005; accepted 10/21/2005.Grant support: NIH (H.C. Joshi) and Nankai University startup fund (J. Zhou).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank members of the Joshi and Zhou laboratories and Dr. Ceshi Chen forhelpful discussions and Dr. Paraskevi Giannakakou for encouragement.

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Cancer Research




2006;66:445-452. Cancer Res Jun Zhou, Min Liu, Ritu Aneja, et al.

-Terminal Kinase2Cancer Cells by the c-Jun NHMediated Multidrug Resistance in−Reversal of P-glycoprotein

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