Molecular Cancer Therapeutics Targeting the PI3-Kinase Pathway in Cancer
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Molecular Cancer Therapeutics 6, 2092-2102, July 1, 2007. doi: 10.1158/1535-7163.MCT-07-0148
© 2007 American Association for Cancer Research
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Research Articles

Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein

Kazuhiro Katayama1, Sho Yoshioka1, Satomi Tsukahara2, Junko Mitsuhashi1 and Yoshikazu Sugimoto1,2

1 Department of Chemotherapy, Kyoritsu University of Pharmacy and 2 Division of Gene Therapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan

Requests for reprints: Yoshikazu Sugimoto, Department of Chemotherapy, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan. Phone: 81-3-5400-2670; Fax: 81-3-5400-2669. E-mail: sugimoto-ys{at}kyoritsu-ph.ac.jp

Abstract

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and 17-allylamino-17-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-15 and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH2-terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal growth factor and basic fibroblast growth factor enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal growth factor–stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance. [Mol Cancer Ther 2007;6(7):2092–2102]

Introduction

The acquisition of multidrug resistance to anticancer agents by tumor cells is characterized by a cross-resistance to structurally unrelated agents (1). Such multidrug-resistant cells express ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp)/ABCB1, breast cancer resistance protein (BCRP)/ABCG2, and multidrug resistance-related protein 1/ABCC1. These gene products pump out a variety of anticancer agents from the cell in energy-dependent manners. Human P-gp is a 170 to 180 kDa plasma membrane glycoprotein encoded by multidrug resistance gene 1 (MDR1), and contains two ATP-binding sites and two transmembrane domains. P-gp functions as an efflux pump for different anticancer agents such as anthracyclines, Vinca alkaloids, and taxanes (1–4). Thus, P-gp–expressing cells display low intracellular concentrations of these agents and are resistant to their cytotoxic effects. P-gp is widely expressed in human tissues, including the adrenal gland, colon, kidney, liver, angioendothelial cells, and hematopoietic precursor cells (5–7), and its expression has been shown to be significantly elevated in drug-resistant tumors of the colon, kidney, and liver (8). P-gp expression levels have also been reported to be augmented in some cancers following a recurrence after chemotherapy (8). Hence, the P-gp expression status can significantly affect the sensitivity of cancer cells to its substrate anticancer agents.

The mitogen-activated protein kinase (MAPK) pathways are activated by various kinds of stimuli, including those provided by growth factors, different types of stress, or inflammatory cytokines, and this can result in cell proliferation, differentiation, development, inflammation, or apoptosis (9). The principal components of the MAPK comprise three subfamilies: extracellular signal-regulated kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38MAPK. The activities of the MAPKs are regulated by two events, phosphorylation and dephosphorylation. The MAPK-associated pathways are composed of a growth factor–responsive pathway, including ERK1/2, and two stress-responsive pathways, including JNK/SAPK and p38MAPK. The former comprises Ras, Raf-1, MAPK/ERK kinase (MEK) 1/2, ERK1/2, and p90 ribosomal S6 kinase (RSK) 1/2/3/4. In cells stimulated by growth factors [e.g., epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)], the Ras proto-oncogene is activated via receptor protein tyrosine kinases such as EGF receptor (EGFR) and FGF receptor. Ras can associate with the plasma membrane by virtue of lipid modifications at its COOH terminus (10). This recruitment to the plasma membrane transduces Ras from an inactive form (GDP-Ras) to an active form (GTP-Ras), and is essential for activation by growth factors (10). GTP-Ras interacts with Raf-1, localizing the latter to the plasma membrane where it becomes activated by several kinases and phosphatases (9). Activated Raf-1 then induces the sequential activation of MEK1/2 and ERK1/2 in a phosphorylation-dependent manner. Activated ERK1/2 then translocate from the cytoplasm to the nucleus and activate a number of transcription factors associated with cell cycle progression, survival, development, and differentiation (9). ERK1/2 also transduce signals to RSKs and thereby positively regulate cell cycle progression by promoting the cyclic AMP response element binding protein–dependent transactivation of cyclin A and cyclin D1, and by inhibiting the nuclear translocation of the cyclin-dependent kinase inhibitor, p27Kip1 (11–13). Hence, the MEK-ERK-RSK pathway plays a central role during cell growth and survival.

In our previous studies, we have shown that estrogens down-regulate both P-gp and BCRP expression levels (14, 15). We have also further shown that the down-regulation of BCRP by 17ß-estradiol (E2) is dependent on the posttranscriptional inhibition of protein biosynthesis, but not on protein degradation (14). It has been shown by others that phosphatidylinositol 3-OH kinase (PI3K) inhibitors also down-regulate BCRP expression levels (16). Studies of small molecules or physiologic compounds that suppress ABC transporter protein expression are thus now ongoing. In our present study, we examine the effects of several signal transduction inhibitors upon the expression levels of P-gp, and find that U0126, PD98059, ERK small interfering RNA (siRNA), and RSK siRNA inhibit P-gp expression. Moreover, U0126 down-regulate P-gp expression by promoting its degradation, but does not affect its biosynthesis. Our present findings thus provide an increased understanding of P-gp biosynthesis and degradation and reveal potential new strategies for the circumvention of P-gp–mediated drug resistance.

Materials and Methods

Reagents
U0126 and PD98059 were purchased from Cell Signaling Technology. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) was purchased from Alomone Labs. Rapamycin, SP600125, EGF, bFGF, and rhodamine123 were purchased from Sigma. FTI-277, LY294002, and SB203580 were obtained from Calbiochem. Paclitaxel was obtained from Bristol-Myers Squibb.

Antibodies for Western blotting were purchased as follows: anti-MDR1+3 monoclonal antibody (C219; Zymed); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon); anti-p44/p42, anti–phospho-p44/p42 (Thr202/Tyr204), anti-Akt, and anti–phospho-Akt (Ser473) polyclonal antibodies and anti–phospho-EGFR (Tyr1068) monoclonal antibody (Cell Signaling Technology); anti-EGFR monoclonal antibody (Santa Cruz Biotechnology); and anti–poly(ADP-ribose) polymerase (PARP) p85 fragment polyclonal antibody (Promega).

Cells
The human cancer cell lines used in this study were obtained from the National Cancer Institute (Bethesda, MD). SW620-14 cells were isolated from human colorectal tumor SW620 cells by limiting dilution. The MDR1-transduced human breast cancer cell lines MCF-7/MDR and MDA-MB-231/MDR were established by transduction with the HaMDR retrovirus as described previously (15). MDA-MB-231 cells were transduced with the Ha3HisMDR retrovirus harboring 3'-His–tagged human MDR1 cDNA. The transduced cells were selected with 3 nmol/L vincristine for 10 days and the resulting mixed population was designated MDA-MB-231/3HisMDR. All cells were cultured in the growth medium consisting of 93% DMEM and 7% fetal bovine serum (FBS) at 37°C in 5% CO2.

Western Blotting and Immunoprecipitation
Cell membrane and cytoplasmic fractions were prepared from cells in lysis buffer [0.2% NP40, 10% glycerol, 137 mmol/L NaCl, 20 mmol/L Tris-Cl (pH 7.5), 1.5 mol/L MgCl2, 1 mmol/L EDTA (pH 8.0), 50 mmol/L NaF, 1 mmol/L Na3PO4, 12 mmol/L ß-glycerophosphate, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L aprotinin]. For immunoprecipitation of 3HisP-gp from MDA-MB-231/3HisMDR cells, 500 µg of protein were incubated with Ni-NTA agarose (Qiagen) for 2 h on ice with occasional tapping. The immunocomplexes precipitated with the Ni-NTA agarose were then washed five times with ice-cold wash buffer [1 mol/L Tris-Cl (pH 7.5), 1 mol/L NaCl, 1% Triton X-100]. Whole-cell lysates were prepared with SDS-containing lysis buffer [1% SDS, 10% glycerol, and 100 mmol/L Tris-Cl (pH 7.5)]. All cell lysates and immunoprecipitates were solubilized with sample buffer [2% SDS, 50 mmol/L Tris-HCl (pH 8.0), 0.2% bromophenol blue, 5% 2-mercaptoethanol] with boiling for 5 min at 100°C, separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies following by peroxidase-conjugated sheep anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences Corp.). Bands were visualized with the ECL (enhanced chemiluminescence) Plus detection kit (Amersham Biosciences Corp.).

siRNA Transfection
Nonsilencing control siRNA were purchased from Qiagen. ERK siRNA was obtained from Cell Signaling Technology and comprises a mixture of ERK1 and ERK2 siRNAs. RSK siRNA was purchased from Qiagen and was composed of RSK1, RSK2, and RSK3 siRNAs. Cells were transfected with these siRNAs using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's instructions.

Plasmid DNAs and Transfection
Human wild-type (WT) H-Ras, Raf-1, MEK1, MEK2, ERK1, ERK2, RSK1, RSK2, p85{alpha} regulatory subunit of PI3K, akt1, and phosphatase and tensin homologue deleted in chromosome 10 (PTEN) cDNAs were generated by PCR with a Human Liver BD Marathon-Ready cDNA (BD Biosciences Clontech) as the template. The PCR products were then digested and cloned into the pFLAG-CMV-2 vector (Sigma). Mutant PTEN cDNA was constructed by the substitution of Cys124 with Ser of PTEN (WT) using a QuickChange Site-Directed Mutagenesis kit (Stratagene). Cells were transfected with these plasmid DNAs using LipofectAMINE 2000 (Invitrogen).

Semiquantitative Reverse Transcription-PCR of MDR1
Cells were treated with either 10 µmol/L U0126 or 100 µg/L EGF, and total RNA was then extracted using an RNeasy kit (Qiagen). Reverse transcription-PCR (RT-PCR) was done using RNA LA PCR kit (Takara) as described previously (15). Real-time RT-PCR was done using SYBR Green PCR Master Mix and RT-PCR Reagents (Applied Biosystems) and an ABI PRISM 7700 Sequence Detection System (Applied Biosystems).

Fluorescence-Activated Cell Sorting Analysis
The expression levels of P-gp on the cell surface after treatment with 10 µmol/L U0126 for 72 h were detected by fluorescence-activated cell sorting analysis using a human-specific monoclonal antibody, MRK16, raised against P-gp. Cells were incubated with or without a biotinylated F(ab')2 fragment of MRK16 (100 µg/mL). The cells were then washed and incubated with R-phycoerythrin–conjugated streptavidin (400 µg/mL; Becton Dickinson and Company). Fluorescence staining levels were detected using FACSCalibur (Becton Dickinson and Company).

The cellular accumulation of rhodamine123, a substrate of P-gp, was determined by flow cytometry. Cells were treated with 10 µmol/L U0126 for 72 h, and the medium was changed every 24 h. After trypsinization, the cells (1 x 105) were washed with ice-cold PBS, resuspended in 1 mL of DMEM supplemented with 300 nmol/L rhodamine123, and incubated for 20 min at 37°C. The cells were then washed twice with ice-cold PBS, and the intracellular accumulation of rhodamine123 was measured using FACSCalibur.

Metabolic Labeling of P-gp in MDA-MB-231/3HisMDR Cells
To examine the biosynthesis profile of P-gp, MDA-MB-231/3HisMDR cells (1 x 106 cells/25 cm2 flask) were incubated in methionine- and cysteine-free DMEM (Invitrogen) supplemented with 7% dialyzed charcoal/dextran-treated FBS (HyClone; labeling medium) for 1.5 h just before the beginning of the experiments. The cells were then incubated in the labeling medium containing 300 µCi/mL of [35S]methionine/cysteine for either 0.5 or 1 h. For U0126-treated cells, 10 µmol/L U0126 was added at 4 h before the start of the experiment. Cells were then harvested and lysed with lysis buffer. 3HisP-gp was immunoprecipitated with Ni-NTA agarose and solubilized with 2x sample buffer as described above. The labeled protein was then subjected to SDS-PAGE and autoradiographed. The band intensities of the labeled P-gp were quantified using the NIH-Image densitometric program. Each column represents the mean ± SD from three independent experiments.

To examine the degradation of P-gp, MDA-MB-231/3HisMDR cells (1 x 106 cells/25 cm2 flask) were incubated in the labeling medium for 1.5 h just before starting the experiment and then incubated in the labeling medium containing 300 µCi/mL of [35S]methionine/cysteine for 1 h. The labeling medium was then replaced with the growth medium, and the cells were chased for 2 to 12 h. For U0126-treated cells, 10 µmol/L U0126 was added to the medium throughout the experiment. The band intensities of the labeled P-gp were again quantified using NIH Image and calculated as a percentage of the control (labeled sample with no chase; 0 h).

Results

The Inhibitors of MEK-ERK-RSK Pathway Suppress P-gp Expression
The human colorectal tumor cell lines HCT-15 and SW620-14, which both express endogenous P-gp, were treated with either 10 µmol/L FTI-277 (an inhibitor of farnesyltransferase that activates Ras), 10 µmol/L U0126 (a MEK1/2 inhibitor), 100 nmol/L 17-AAG (an inhibitor of heat shock protein 90 that stabilizes both Raf-1 and PDK1), 50 µmol/L LY294002 (a PI3K inhibitor), or 100 nmol/L rapamycin [an inhibitor of mammalian target of rapamycin (mTOR)] for 12 h. The P-gp expression levels were then determined by Western blot analysis. As shown in Fig. 1A , the cells treated with FTI-277, U0126, and 17-AAG showed 5- to 20-fold lower levels of P-gp compared with untreated cells. However, neither LY294002 nor rapamycin treatments affected P-gp expression. To exclude the possibility that the down-regulation of P-gp in these analyses was due to an alteration in the status and solubility of the protein in 0.2% NP40, we also analyzed its expression using whole cell lysates. In this experiment, U0126 and 17-AAG again suppressed P-gp expression in HCT-15 and SW620-14 cells (Fig. 1B). We next examined whether either SB203580 (a p38MAPK inhibitor) or SP600125 (a JNK inhibitor) affected P-gp expression levels in HCT-15 and SW620-14 cells. However, upon treatment with 10 µmol/L SB203580 or 20 µmol/L SP600125 for 12 h, the P-gp expression levels were found to be unchanged in these cells (Fig. 1C).


Figure 1
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  		Figure 1. Screening of the effects of various kinase inhibitors upon P-gp expression levels. A, Western blot analysis of endogenous P-gp expression levels in both cell membrane and cytoplasmic fractions of cells treated with inhibitors against the MEK-ERK-RSK or PI3K-Akt-mTOR pathway. HCT-15 or SW620-14 cells were treated with either medium alone (Cont.), 10 µmol/L FTI-277, 10 µmol/L U0126, 100 nmol/L 17-AAG, 20 µmol/L LY294002, or 10 µmol/L rapamycin for 12 h. Cellular membrane and cytoplasmic fractions were prepared with lysis buffer containing 0.2% NP40 and subjected to Western blotting with the indicated antibodies. B, Western blot analysis of P-gp expression levels in whole-cell lysates of U0126- or 17-AAG–treated cells. HCT-15 or SW620-14 cells were treated with either medium alone, 10 µmol/L U0126, or 100 nmol/L 17-AAG. After 12 h, whole-cell lysates were obtained using lysis buffer containing 1% SDS and subjected to Western blotting with the indicated antibodies. C, Western blot analysis of P-gp expression levels in cells treated with p38MAPK or JNK inhibitor. HCT-15 or SW620-14 cells were treated with either medium alone, 10 µmol/L SB203580, or 20 µmol/L SP600125 for 12 h. Cells were harvested and divided into two groups. One was lysed with lysis buffer containing 1% SDS to analyze the expression levels of total c-Jun and phosphorylated c-Jun, and another was lysed with lysis buffer containing 0.2% NP40 to analyze the other indicated proteins. The lysates were then subjected to Western blotting with the indicated antibodies.

 
MEK Inhibitors Down-regulate P-gp Expression in a Time-Dependent Manner
We next examined the suppressive effects of U0126 in exogenous P-gp–expressing cells. We also examined the time dependency of the down-regulation of P-gp by U0126. HCT-15, SW620-14, MCF-7/MDR, and MDA-MB-231/MDR cells were treated with 10 µmol/L U0126 for 2 to 16 h, and P-gp expression levels were determined by Western blotting. U0126 was found to down-regulate exogenous P-gp expression also in these MDR1-transduced cell lines (Fig. 2A ). The time dependency of this suppression varied among the cell lines, as it was evident within 4 h in SW620-14 cells, but required 8 h in HCT-15 and MCF-7/MDR cells and 12 h in MDA-MB-231/MDR cells. As a control experiment, we examined the time dependency of P-gp expression in the absence of the inhibitor. The same cells were cultured for a further 2 to 16 h after changing the medium to the flesh growth medium without inhibitors, and the P-gp levels were found to be unchanged at each time point for each cell line (Fig. 2B).


Figure 2
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  		Figure 2. Down-regulation of P-gp by treatment with MEK inhibitors or by RNA interference of MEK and ERK. A, Western blot analysis of P-gp expression levels after a time course of U0126 treatment. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were treated with medium alone or with 10 µmol/L U0126 for 2 to 16 h. Cellular membrane and cytoplasmic fractions were prepared using lysis buffer containing 0.2% NP40 and subjected to Western blotting with the indicated antibodies. B, Western blot analysis of P-gp expression levels after a time course of treatment with medium alone. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were treated with medium alone for 2 to 16 h and processed as described in A. C, RT-PCR analysis of MDR1 mRNA levels over a time course of U0126 treatment. After treatment with medium alone or with 10 µmol/L U0126 for 2 to 16 h in HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells, total RNAs were extracted from the cells. The mRNA levels of the MDR1 and GAPDH genes were analyzed by RT-PCR as described in Materials and Methods. D, P-gp expression levels in PD98059-treated HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells. The cells were treated with medium alone or with 50 µmol/L PD98059 for 8 or 12 h and processed as described in A. E, the effects of ERK and RSK siRNAs on P-gp expression levels. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were transfected with siRNAs in the following combinations: 50 µmol/L negative control siRNA (Cont.); 25 or 50 µmol/L (each 12.5 or 25 µmol/L) of an ERK1+ERK2 siRNA combination; or 25 or 50 µmol/L (each 8.3 or 16.7 µmol/L) of an RSK1/RSK2/RSK3 siRNA mixture. After transfection for 48 h, the cells were harvested and lysed with lysis buffer containing 0.2% NP40. Cell lysates were then subjected to Western blotting with the indicated antibodies.

 
We next examined MDR1 mRNA expression levels by RT-PCR. Each of the HCT-15, SW620-14, MCF-7/MDR, and MDA-MB-231/MDR cells were treated with U0126 under the same conditions used for the experiments in Fig. 2A. Total RNAs were isolated from these cells and RT-PCR was done using MDR1 or GAPDH-specific oligonucleotides. As shown in Fig. 2C, the MDR1 mRNA levels were unchanged after U0126 treatment at all time points in each of the cell lines. Similar results were obtained using real-time PCR analysis (Supplementary Fig. S1A).3 P-gp expression levels were then examined in these cells after treatment with another MEK inhibitor, PD98059, at a concentration of 50 µmol/L for 8 or 12 h. Similar to the findings shown in Fig. 2A, all of the cell lines tested in this experiment expressed lower levels of P-gp after PD98059 treatment, compared with untreated cells (Fig. 2D).

We further examined the effects upon P-gp expression when either ERK or RSK was specifically suppressed by siRNA. Each of our four test cell lines was transfected with either a nonsilencing control, ERK siRNA, or RSK siRNA, and the P-gp expression levels were then determined by Western blotting after 48 h. P-gp was found to be down-regulated by the knockdown of both the ERK and RSK proteins in a dose-dependent manner (Fig. 2E). The suppression of RSK in particular had a significant negative effect upon P-gp expression (Fig. 2E). Fluorescence-activated cell sorting analysis also revealed that cells treated with U0126 for 72 h expressed lower amounts of cell surface P-gp compared with untreated cells (Fig. 3 ). From these data, we conclude that P-gp expression is suppressed by a blockade of the MEK-ERK-RSK pathway.


Figure 3
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  		Figure 3. The down-regulation of cell surface P-gp expression by U0126. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were treated with medium alone or with 10 µmol/L U0126 for 72 h, with medium replacement every 24 h. The cells were then harvested with trypsin, washed with PBS, and incubated with (closed areas) or without (open areas) a biotinylated F(ab')2 fragment of the MRK16 antibody. The cells were then washed with PBS and incubated with R-phycoerythrin–conjugated streptavidin. Fluorescence staining was then evaluated using FACSCalibur.

 
EGF or bFGF Stimulation Enhances P-gp Expression but Does Not Affect MDR1 Transcription
Given that the inhibition of the MEK-ERK-RSK pathway suppressed P-gp expression, we next investigated whether the activation of this pathway would in fact enhance P-gp. HCT-15, SW620-14, MCF-7/MDR, and MDA-MB-231/MDR cells were cultured in serum-free DMEM for 6 h and incubated in the growth medium supplemented with 100 µg/L EGF for a further 2 to 16 h. Western blot analysis revealed that EGF activated EGFR (increased levels of phosphorylated EGFR are detectable), and that upon induction of the MEK-ERK-RSK pathway (increased phosphorylation of ERK) there was an observable increase in the P-gp expression levels in all cells (Fig. 4A ). To examine the effects of serum starvation itself on P-gp expression, the cells were cultured in serum-free DMEM for 6 h and then incubated in the growth medium only for a further 2 to 16 h. Phosphorylated ERK was found to have slightly increased in each of the four cell lines by the addition of FBS to the serum-starved culture, and this occurred simultaneously with increased P-gp expression levels (Fig. 4B). These data indicate that FBS itself activates the MEK-ERK-RSK pathway and increases P-gp expression. Moreover, the strong enhancement of P-gp expression by the stimulation of EGF and FBS persisted for a longer duration compared with the stimulation with FBS alone (Fig. 4A and B). However, although the P-gp levels were enhanced by EGF stimulation, the corresponding MDR1 mRNA levels were unchanged, as indicated by our RT-PCR analysis (Fig. 4C; Supplementary Fig. S1B).3 We next did bFGF treatments at 10 µg/L for 8 or 12 h using the same procedures described for Fig. 4A and a bFGF-dependent enhancement of P-gp expression was also observed in each cell line (Fig. 4D).


Figure 4
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  		Figure 4. The up-regulation of P-gp by EGF or bFGF in a time-dependent manner. A, Western blot analysis of P-gp expression levels after the EGF-dependent activation of the MEK-ERK-RSK pathway. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were cultured in medium without serum for 6 h (Serum starved; +). The medium was then replaced with the growth medium supplemented with 100 µg/L EGF, and the cells were incubated for a further 2 to 16 h. Negative control cells were not serum starved and were untreated (Serum starved; – and EGF; –). Cellular membrane and cytoplasmic fractions were lysed with lysis buffer containing 0.2% NP40 and subjected to Western blotting with the indicated antibodies. B, Western blot analysis of P-gp expression levels after FBS-dependent activation of the MEK-ERK-RSK pathway. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were cultured in medium without serum for 6 h (Serum starved; +). The medium was then replaced with the flesh growth medium, and the cells were incubated for a further 2 to 16 h. Negative control cells and cell processing were as described in A. C, RT-PCR analysis of MDR1 mRNA levels after EGF treatment. After treatment with EGF as in A, total RNAs were extracted from the cells and the mRNA levels of the MDR1 and GAPDH genes were analyzed by RT-PCR. D, Western blot analysis of P-gp expression levels after bFGF treatment. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were cultured in medium without serum (Serum starved; +) and after incubation for 6 h, the medium was replaced with the growth medium supplemented with 10 µg/L bFGF, and the cells were cultured for a further 8 or 12 h. Negative control cells and cell processing were as described in A.

 
We next determined the effects upon P-gp expression levels by Western blotting when HCT-15 cells were transiently transfected with cDNAs corresponding to genes of either the MEK-ERK-RSK or PI3K-Akt signaling pathways. The activation of the MEK-ERK-RSK pathway by transfection with WT cDNAs for H-Ras, Raf-1, MEK1, MEK2, ERK1, ERK2, RSK1, or RSK2 was found to enhance the P-gp expression levels, whereas the activation of PI3K-Akt signaling pathway by transfection with p85{alpha} (WT) regulatory subunit of PI3K, akt1 (WT), PTEN (C124S), or PTEN (WT) DNA did not affect P-gp expression levels (Supplementary Fig. S2).3 These results further indicate that P-gp expression is positively regulated by the MEK-ERK-RSK pathway.

U0126 Promotes P-gp Degradation but Does Not Inhibit Its Biosynthesis
Based on our observation that U0126 suppresses P-gp expression levels without affecting its gene transcription (Fig. 2A and C), we further examined the biosynthesis and degradation of P-gp using pulse-chase experiments in which MDA-MB-231/3HisMDR cells were treated with (+) or without (–) 10 µmol/L U0126. During the pulse labeling procedure for 0.5 or 1 h, the labeled P-gp levels were observed to gradually increase in both the untreated and U0126-treated cells (Fig. 5A and B ), and were found to be almost equivalent in both cases. To subsequently examine the effects of U0126 on P-gp degradation, 35S metabolic labeling was done for 1 h using MDA-MB-231/3HisMDR cells in the absence (–) or presence (+) of 10 µmol/L U0126. The cells were then chased for 2 to 12 h in the growth medium without (–) or with (+) 10 µmol/L U0126. The labeled P-gp expression levels were found to be largely unchanged in the untreated cells, but a significant reduction in P-gp was observed in the U0126-treated cells at the 8 and 12 h time points of the chase period (Fig. 5C and D). Moreover, the quantities of labeled P-gp at 12 h were ~50% of the 0 h levels (no chase; Fig. 5C and D). These results suggest that U0126 promotes P-gp degradation but does not affect its biosynthesis.


Figure 5
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  		Figure 5. U0126 promotes the degradation of P-gp but does not suppress its biosynthesis. A and B, biosynthesis of P-gp. MDA-MB-231/3HisMDR cells were cultured in methionine- and cysteine-free medium for 1.5 h and then metabolically labeled with 300 µCi/mL of 35S for 0.5 or 1 h. For the U0126 treatment, the cells were treated with 10 µmol/L U0126 from 4 h before beginning the experiments to the end of the 35S-labeling period. 35S-labeled P-gp was immunoprecipitated from 500 µg of cell lysates, subjected to SDS-PAGE, and autoradiographed. Band intensities were measured using an NIH Image densitometer (B). Columns, means; bars, SD; calculated from three independent experiments. C and D, degradation of P-gp. MDA-MB-231/3HisMDR cells were cultured in methionine- and cysteine-free medium for 1.5 h, metabolically labeled with 300 µCi/mL of 35S for 1 h, and chased for a further 2 to 12 h. For U0126 treatment, the cells were treated with 10 µmol/L U0126 from the time of 35S labeling to the end of the chase period. 35S-labeled P-gp was immunoprecipitated from 500 µg of cell lysates, subjected to SDS-PAGE, and autoradiographed. The cells without 35S labeling were also prepared, and unlabeled P-gp was analyzed in the same manner as the 35S-labeled P-gp (Chase; –). Band intensities were again measured using an NIH Image densitometer (D).

 
The U0126-Mediated Down-regulation of P-gp Enhances the Sensitivity of Cells to Paclitaxel and Rhodamine123 Uptake
To examine whether the U0126-mediated down-regulation of P-gp has any effect on anticancer agent–mediated apoptosis, we treated HCT-15, SW620-14, MCF-7/MDR, and MDA-MB-231/MDR cells with (+) or without (–) 10 µmol/L U0126 for 72 h to down-regulate P-gp expression on cell surface. We then treated the cells with (+) or without (–) 10 µmol/L U0126 combined with different doses of paclitaxel for an additional 24 h. In this experiment, the IC50 values (the dosage at which a 50% inhibition of cell growth occurs) for paclitaxel had been previously determined for each cell line, and the cells were treated with both this dose and a 3-fold higher concentration of the drug. As shown in Fig. 6A , paclitaxel increased the levels of cleaved PARP, which is a substrate of caspase-3, as reported previously (17). When the cells were pretreated with U0126, the extent of this PARP cleavage by paclitaxel was enhanced in each cell lines, particularly at the 3-fold IC50 concentrations (Fig. 6A). These results indicate that paclitaxel-mediated apoptosis signaling via the activation of caspase-3 is enhanced by the U0126-mediated suppression of P-gp.


Figure 6
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  		Figure 6. The physiologic effects of U0126 upon the function of P-gp as an efflux pump. A, Western blot analysis of cleaved PARP after paclitaxel treatment combined with U0126.HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were treated with medium alone (U0126; –) or with 10 µmol/L U0126 (U0126; +) for 72 h, with medium replacement every 24 h. The cells were then further treated with or without 10 µmol/L U0126 and/or paclitaxel as the indicated combinations for 24 h. Paclitaxel was used at both 1x and 3x its predetermined IC50 concentration for each cell line. The cells were harvested with lysis buffer containing 0.2% NP40 and the cell lysates were subjected to Western blotting with the indicated antibodies. B, fluorescence-activated cell sorting analysis of rhodamine123 uptake in U0126-treated cells. HCT-15, SW620-14, MCF-7/MDR, or MDA-MB-231/MDR cells were treated with medium alone [U0126 (–)] or with 10 µmol/L U0126 [U0126 (+)] for 72 h, with medium replacement every 24 h. The cells were then trypsinized and adjusted to a concentration of 5 x 105/mL with medium. The cells were incubated with 300 nmol/L rhodamine123 for 20 min at 37°C and washed twice with ice-cold PBS. The intracellular accumulation of rhodamine123 was detected using FACSCalibur.

 
Finally, we examined the effects of U0126 on rhodamine123 uptake in P-gp–expressing cells. As shown in Fig. 6B, cells treated with U0126 accumulated higher levels of rhodamine123 compared with untreated cells. Hence, the U0126-mediated down-regulation of P-gp leads to an increase in the intracellular concentration of P-gp substrates.

Discussion

Many previous studies have evaluated P-gp inhibitors to effectively reverse P-gp–mediated drug resistance. In the 1980s, verapamil was initially identified as a P-gp inhibitor (18, 19), as it increases the intracellular concentration of various anticancer agents in multidrug-resistant cells by binding P-gp and inhibiting drug efflux. Subsequently, many P-gp inhibitors such as valspoder (PSC-833), dofequidar fumarate (MS-209), tariquidar (XR9576), and thiose-micarbazone derivative (NSC73306), have been developed (20–23). Clinical trials using such P-gp inhibitors have shown in vivo increases in the intracellular concentrations of coadministered anticancer agents in P-gp–positive tumor cells (24). However, phase III trials of these agents have not been successful, and no significant survival benefit as a result of P-gp inhibition has yet been achieved (25, 26). Further clinical studies using new P-gp inhibitors and new combination-treatment regimens have been devised, and some are currently ongoing.

The regulatory mechanisms underlying the expression of ABC transporters, including P-gp and BCRP, have not yet been well clarified. We have previously shown that physiologic levels of estrogens suppress P-gp and BCRP in estrogen receptor {alpha}–expressing breast cancer cells via posttranscriptional processes, and that this occurs without any affects upon transcription (14, 15). Other groups have also shown that the stability of P-gp is regulated by the ubiquitin-proteasome system (27, 28). Furthermore, Akt signaling has been shown to modulate a side population cell phenotype by regulating the expression of Bcrp1 in mouse (29). The PI3K inhibitor, LY294002, and a dominant-negative form of Akt have also been reported to down-regulate BCRP expression levels (16). Thus, the association between the expression of ABC transporter proteins and either cell growth signaling or the ubiquitin-proteasome system has recently generated some interest. In our present study, we have attempted to further clarify the regulatory mechanisms underlying ABC transporter protein expression, focusing on P-gp, to identify inhibitors that specifically target these expression mechanism(s).

We initially found that inhibitors of the MEK-ERK-RSK pathway suppressed P-gp expression (Fig. 1). In particular, the MEK inhibitor U0126 was found to potently down-regulate endogenous P-gp expression (Fig. 1), and both the U0126- and PD98059-mediated down-regulation of P-gp could be observed in both endogenous and exogenous P-gp–expressing cells (Fig. 2A and D). Moreover, these phenomena were found not to be the result of transcriptional regulation (Fig. 2C; Supplementary Fig. S1A).3 The suppression of P-gp by MEK inhibitors was also far more rapid compared with estrogens (Fig. 2A and D, compared with ref. 15). ERK and RSK knockdown by siRNAs also down-regulated P-gp expression (Fig. 2E). Conversely, the activation of the MEK-ERK-RSK pathway by the overexpression of WT proteins for H-Ras, Raf-1, MEK1, MEK2, ERK1, ERK2, RSK1, or RSK2 enhanced P-gp expression in HCT-15 cells, whereas the activation of the PI3K-Akt signaling pathway by overexpressing the phosphatase inactive form of PTEN (C124S), p85{alpha} (WT) regulatory subunit of PI3K, or Akt (WT) did not affect P-gp in these cells (Supplementary Fig. S2).3 These results suggest that the stability of P-gp is regulated by the MEK-ERK-RSK pathway, and that the kinase activities of RSK are necessary for this stabilization. Although BCRP has been shown to be regulated by the PI3K-Akt signaling pathway (16), it is likely that P-gp is regulated by different mechanisms. We additionally examined P-gp expression in cells treated with EGF and bFGF, which are activators of the MEK-ERK-RSK pathway via EGFR and FGF receptor, respectively. As expected, the stimulation of either EGF or bFGF enhanced P-gp expression levels without affecting its transcription (Fig. 4A–D; Supplementary Fig. S1B).3 Moreover, the presence of FBS in the growth medium slightly enhanced the P-gp expression levels with the activation of the MEK-ERK-RSK pathway (Fig. 4B). These data thus support our earlier findings that P-gp expression is positively regulated by the MEK-ERK-RSK pathway.

In the present study, we have shown that U0126 treatment down-regulated P-gp expression for 12 h, but that SP600125 (a JNK inhibitor) and SB203580 (a p38MAPK inhibitor) did not alter these expression levels in HCT-15 and SW620-14 cells (Fig. 1). The components of MAPK comprise three subfamilies, ERK, JNK, and p38MAPK. In previous studies, the JNK or p38MAPK pathways have been reported to alter P-gp expression levels. In addition, adenoviral transduction of JNK has been shown to down-regulate P-gp expression, whereas SP600125 treatment for 24 h did not affect its expression in human gastric and pancreatic cancer cells (30). In another report, SP600125 treatment for 24 h was shown to enhance P-gp expression in human prostate cancer DU145 spheroids (31). SB203580 has been shown to decrease P-gp expression levels in DU145 spheroids and vincristine-resistant murine leukemia L1210/VCR cells (31, 32). U0126 treatment was also reported to up-regulate P-gp expression after 24 h in DU145 spheroids (31). The discrepancies between the data from these previous reports and our present experiments may be due to differences in the cell lines and treatment protocols used. We observed that the down-regulation of phosphorylated ERK by U0126 was slightly recovered at 24 h in each of the cell lines tested in this study (data not shown), suggesting that U0126 may be degraded. Therefore, we replenished the U0126-containing medium every 24 h in the experiments shown in Figs. 3 and 6.

Although U0126 suppressed both endogenous and exogenous P-gp, the MDR1 mRNA levels were unaffected by treatment with this agent (Fig. 2C; Supplementary Fig. S1A).3 These data strongly indicate the existence of U0126-mediated posttranscriptional P-gp regulation mechanism(s), most likely to be translation and degradation processes. To further elucidate this, we established MDA-MB-231/3HisMDR cells and found that the rate of P-gp biosynthesis in U0126-treated cells was virtually equivalent to the untreated cells (Fig. 5A and B). In contrast, however, the degradation rate of P-gp in U0126-treated cells was higher than in untreated cells (Fig. 5C and D). We subsequently did a pulse-chase experiment to confirm these observations and found that the P-gp expression levels in untreated cells were constant up to the 12 h time point, but that the levels in the U0126-treated cells had decreased by ~50% during the same 8 to 12 h chase period (Fig. 5C and D). These results indicate that U0126 suppresses P-gp expression by promoting its degradation.

To analyze the physiologic responses to the U0126-mediated down-regulation of P-gp, we examined the possible effects of this response upon the enhancement of apoptosis by paclitaxel, and also upon the accumulation of rhodamine123, both of which are substrates of P-gp (33). Because PARP is cleaved by active caspase-3, an apoptosis inducer (17, 34), we used this as the index of the paclitaxel-mediated enhancement of apoptosis signaling. P-gp expression on the cell surface decreased when the cells were treated with U0126 for 72 h (Fig. 3). The cells in this experiment were therefore pretreated with U0126 for 72 h and then cotreated with U0126 and paclitaxel for a further 24 h. The combination of U0126 and paclitaxel was found to enhance the levels of cleaved PARP, compared with paclitaxel exposure alone (Fig. 6A). In addition, the intracellular rhodamine123 levels were also found to accumulate after U0126 treatment for 72 h (Fig. 6B). These results indicate that the U0126-mediated down-regulation of P-gp can reverse the P-gp–mediated resistance to anticancer agents.

Although we show that inhibition of the MEK-ERK-RSK pathway suppresses P-gp expression, and that this involves the kinase activities of RSKs (Fig. 2E; Supplementary Fig. S2A),3 it remains unclear how MEK inhibitors promote P-gp degradation or whether the RSKs directly regulate P-gp expression. If these molecules indirectly regulate P-gp degradation, the question of which factors are associated with this mechanism remains to be elucidated and will require further molecular analyses.

Many P-gp inhibitors that have been developed are competitors of anticancer agents that are also P-gp substrates. Because RSKs have been shown to positively regulate P-gp expression in our present study, we speculate that MEK, ERK, and RSK inhibitors, and also RNA interferences may have potential as novel therapeutic agents for the reversal of P-gp–mediated anticancer drug resistance. During cellular hyperplasia, the MAPK pathway is often activated and provides a variety of growth signals, promotes cell cycle progression, and suppresses apoptosis (9). Inhibitors of the MEK-ERK-RSK pathway would thus be expected to have significant benefits as chemotherapeutics against P-gp–mediated drug-resistant cancer cells.

In conclusion, we show that a blockade of the MEK-ERK-RSK pathway suppresses cell surface P-gp expression by promoting its degradation. Our data therefore provide new insights into the regulation of P-gp expression and suggest potential new strategies for the reversal of P-gp–mediated anticancer drug resistance.

Footnotes

Grant support: The Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labor and Welfare, Japan.

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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Back

Received 3/ 5/07; accepted 5/30/07.

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