Overexpression of the breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. To circumvent BCRP-mediated MDR, a common approach is the use of potent and specific inhibitors of BCRP transport such as fumitremorgin C, novobiocin, and GF120918. Here, we evaluated a new approach using RNA interference for the specific knockdown of BCRP. We designed and synthesized small interfering RNA (siRNA) using T7 RNA polymerase and showed that siRNAs markedly down-regulated both exogenous and endogenous expression of BCRP. As a functional consequence, knockdown of BCRP by siRNAs increased the sensitivity of human choriocarcinoma BeWo cells to mitoxantrone and topotecan by 10.5- and 8.2-fold, respectively. Using flow cytometry, we found that introduction of siRNAs also enhanced the intracellular accumulation of topotecan. We have previously identified an estrogen response element in the BCRP promoter and have shown that 17β-estradiol increased BCRP mRNA expression. Furthermore, in the present study, we found that expression of BCRP protein was inducible by 17β-estradiol and that this effect was ameliorated by the introduction of siRNAs. These studies indicate that siRNAs could modulate MDR in vitro and may present a new approach to overcome BCRP-mediated drug resistance.

Successful chemotherapy is often hampered by the development of multidrug resistance (MDR) in tumor cells. The bête noire of cancer therapeutics, MDR is a phenomenon whereby tumor cells acquire cross-resistance to a variety of structurally and functionally unrelated compounds. It is commonly known that some forms of MDR arise from the overexpression of ATP-binding cassette transporters such as P-glycoprotein (P-gp), MDR-associated protein 1, and/or breast cancer resistance protein (BCRP; ref. 1). Unlike P-gp and MDR-associated protein 1, BCRP (gene symbol ABCG2) is a half-transporter of 655 amino acids that consists of only one transmembrane domain with six transmembrane segments and one nucleotide-binding site (2–4, 9, 10). BCRP is believed to function as a homodimer or as a multimer (5–8). Some examples of BCRP substrates include topotecan, SN-38, mitoxantrone, and flavopiridol (2–4, 9, 10). Acting as an efflux pump in tumor cells, overexpression of BCRP leads to reduced intracellular drug concentration and decreased cytotoxicity. Clinically, it has been reported that a correlation exists between BCRP expression and patient outcome in some hematologic and solid tumors (11, 12).

One pharmacologic approach to circumvent BCRP-mediated drug resistance involves the use of potent inhibitors of BCRP transport such as fumitremorgin C and its analogues (13–15). Fumitremorgin C, described as the first BCRP-selective inhibitor, has been shown to reverse resistance to mitoxantrone, doxorubicin, and flavopiridol in cancer cell lines expressing BCRP (14, 15). Other inhibitors that have been described include, but are not limited to, novobiocin (16–18), GF120918 (19–22), estrogen agonists and anti-estrogens (23), and the tyrosine kinase inhibitors, CI1033 and imatinib mesylate (Gleevec; refs. 24, 25). A nonpharmacologic approach involving the use of a hammerhead ribozyme directed against BCRP to modulate the MDR phenotype has also been reported (26).

More recently, an alternative strategy employing the use of small interfering RNA (siRNA) targeted against the MDR1 gene is effective in reversing MDR (27–30). Both synthetic and vector-based siRNAs markedly down-regulated the expression of MDR1 gene and restored drug sensitivity in cancer cells (27–30). Based on an evolutionarily conserved mechanism termed RNA interference (RNAi), these dsRNA molecules can direct sequence-specific degradation of homologous mRNA to cause specific knockdown of genes (31, 32).

In the present study, we have designed and synthesized siRNAs for the specific knockdown of BCRP and reversal of BCRP-mediated MDR. We found that siRNAs generated using T7 RNA polymerase effectively down-regulated the expression of both exogenous and, importantly, endogenous BCRP and increased tumor cell drug sensitivity and accumulation. We have previously identified an estrogen response element in the BCRP gene promoter (33). Here, using estrogen receptor–positive human choriocarcinoma BeWo cells, we found that 17β-estradiol (E2) increased BCRP protein expression in a dose-dependent manner and that siRNAs were effective in ameliorating the stimulatory effect of E2.

Chemicals

E2 was purchased from Calbiochem (San Diego, CA) and topotecan was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). All other chemicals were purchased from Sigma (St. Louis, MO).

Cell Lines and Culture

All cell lines were obtained from American Type Culture Collection (Manassas, VA). The human choriocarcinoma BeWo cells were maintained in Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 units/mL penicillin, and 100 units/mL streptomycin in a humidified atmosphere of 5% CO2 and 95% air. The human cervical carcinoma HeLa cells were maintained in DMEM containing 10% fetal bovine serum under similar conditions. All cell culture reagents were purchased from BioWhittaker (Walkersville, MD) unless otherwise stated.

siRNA Preparation

In vitro synthesis of siRNAs was done using T7 RNA polymerase in a previously described protocol (34). The siRNA sequence targeting BCRP lies within exon 7 and corresponds to nucleotides 904 to 923 relative to the transcription start site (Genbank accession no. NM_004827; sense: 5′-AAGATGATTGTTCGTCCCTGCTATAGTGAGTCGTATTA-3′ and antisense: 5′-AAGCAGGGACGAACAATCATCTATAGTGAGTCGTATTA-3′). Target sequences were scrambled and used as a negative control (sense: 5′-AAGCCTTGAGTAGTCGTCTTCTATAGTGAGTCGTATTA-3′ and antisense: 5′-AAGAAGACGACTACTCAAGGCTATAGTGAGTCGTATTA-3′). [The target sequences for the BCRP gene are italicized. All primers used contain the T7 promoter sequence (normal font) at their 3′ ends. The primer 5′-TAATACGACTCACTATAG-3′ was annealed to all templates to synthesize siRNA duplexes.] Desalted DNA oligonucleotides (Sigma-Genosys, The Woodlands, TX) encoding sense and antisense target sequences were used as templates. The concentration of the generated siRNAs was measured by absorbance at 260 nm and the double-stranded nature of the siRNAs was confirmed by agarose (2%, w/v) electrophoresis and ethidium bromide staining.

Transfection and E2 Treatment

To determine the effect of siRNA on the exogenous expression of BCRP, HeLa cells at a density of 5 × 105/well were simultaneously transfected with 0 to 10 μg siRNAs and 3 μg expression vector for wild-type, full-length BCRP (pcDNA3-BCRP; ref. 3) using LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. As a control, HeLa cells were also transfected with the same amount of empty pcDNA3 vector. Cells were harvested 24 hours after transfection. To determine the effect of siRNA on the endogenous expression of BCRP, BeWo cells at the same density were transfected with 0 to 10 μg siRNAs as described above. To examine BCRP expression under E2 stimulation, BeWo cells were cultured in phenol red–free Ham's F-12 medium (Biosource, Rockville, MD) supplemented with 10% charcoal/dextran-stripped fetal bovine serum (Gemini, Woodland, CA) for 3 days before subsequent siRNA transfection. Cells were then treated with 0 to 100 nmol/L E2 for 24 hours before being harvested.

Semiquantitative Reverse Transcription-PCR and Western Blot Analysis

Total RNA extraction and reverse transcription-PCR analysis of BCRP expression in BeWo and HeLa cells were done as described previously (33) using primer sets specific for the human BCRP gene (sense: 5′-TTCTCCATTCATCAGCCTCG-3′ and antisense: 5′-TGGTTGGTCGTCAGGAAGA-3′) and β-actin gene (sense: 5′-GAGAAGATGACCCAGATCATGT-3′ and antisense: 5′-TCGTCATACTCCTGCTTGCAG-3′). For Western blot analysis, cell lysates were prepared as described elsewhere (35), and 60 to 80 μg of protein per lane were loaded on a 9% gel. Following electrophoretic separation of proteins and the transfer to nitrocellulose, membranes were immunoblotted with monoclonal antibody BXP-21 (Chemicon, Temecula, CA) for human BCRP and monoclonal antibody AC-74 (Sigma) for human β-actin. A chemiluminescence detection kit from Amersham Biosciences (Piscataway, NJ) was used for detection.

Cytotoxicity Assays

BeWo cells were transfected with siRNAs as above before being seeded in 96-well plates at a density of 4,000 cells per well and were grown overnight. The cells were then treated with drugs at the concentrations indicated and incubated at 37°C for 3 to 4 days. Cytotoxicity was assessed by using the CellTiter 96 AQueous assay according to the manufacturer's instructions (Promega, Madison, WI).

Intracellular Drug Accumulation

Topotecan, a BCRP substrate, is commonly used to test for changes in drug accumulation mediated by BCRP (23, 25, 36, 37). Here, the effect of siRNA introduction on cellular accumulation of topotecan was determined by flow cytometry as described elsewhere (37). Briefly, BeWo cells (5 × 105) were transfected with 5 μg siRNAs as described above for 24 hours and incubated with 30 μmol/L topotecan for 15 minutes at 37°C. After washing twice with ice-cold PBS, cells were subjected to fluorescence analysis using a FACSCalibur (Becton Dickinson, San Jose, CA) with 488 nm excitation at the University of Illinois at Chicago Research Resources Center.

siRNA Knockdown of Exogenous BCRP mRNA and Protein Expression

To determine whether siRNA specific to the BCRP gene sequence could down-regulate gene expression, we first tested its effectiveness in cells exogenously expressing BCRP. Both the expression plasmid encoding the wild-type, full-length BCRP (pcDNA3-BCRP) and siRNAs targeting the coding sequence of BCRP (siBCRP) were simultaneously transfected into HeLa cells for 24 hours before being harvested for total RNA and protein. As shown in Fig 1A, levels of BCRP mRNA were decreased by at least 25% twenty-four hours after transfection. However, no change in mRNA levels was observed in HeLa cells that were simultaneously transfected with pCDNA3-BCRP and siRNAs containing target sequences that were scrambled (siBCRP-scram). On the protein level, a more dramatic decrease was observed with the introduction of siBCRP as shown in Fig. 1B. siBCRP-scram, on the other hand, did not result in any change in the levels of BCRP protein.

Figure 1.

siRNA knockdown of exogenous BCRP mRNA (A) and protein (B) expression in HeLa cells. HeLa cells were simultaneously transfected with 0–10 μg siRNAs and 3 μg expression vector for wild-type, full-length BCRP (pcDNA3-BCRP) using LipofectAMINE Plus according to the manufacturer's protocol. After 24 hours, cells were harvested for total RNA and protein as described in Materials and Methods. Total RNA (1.5 μg) was used for reverse transcription-PCR and 60 μg of protein were separated by 9% SDS-PAGE and then transferred onto nitrocellulose membrane. Membranes were immunoblotted with monoclonal antibody BXP-21 for human BCRP. β-Actin was used as a loading control. Representative of three independent experiments.

Figure 1.

siRNA knockdown of exogenous BCRP mRNA (A) and protein (B) expression in HeLa cells. HeLa cells were simultaneously transfected with 0–10 μg siRNAs and 3 μg expression vector for wild-type, full-length BCRP (pcDNA3-BCRP) using LipofectAMINE Plus according to the manufacturer's protocol. After 24 hours, cells were harvested for total RNA and protein as described in Materials and Methods. Total RNA (1.5 μg) was used for reverse transcription-PCR and 60 μg of protein were separated by 9% SDS-PAGE and then transferred onto nitrocellulose membrane. Membranes were immunoblotted with monoclonal antibody BXP-21 for human BCRP. β-Actin was used as a loading control. Representative of three independent experiments.

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siRNA Knockdown of Endogenous BCRP mRNA and Protein Expression

BeWo cells were the first human trophoblastic endocrine cell type to be maintained in continuous culture and were initiated from a malignant gestational choriocarcinoma of the placenta (38). They were also shown to express high endogenous levels of BCRP, comparable with that found in topotecan-resistant human ovarian Igrov/T8 cells (39), and thus were used to examine the effect of siRNA on the endogenous expression of BCRP. Similar knockdown of BCRP expression in both mRNA and protein levels was observed. As shown in Fig. 2A, levels of BCRP mRNA were decreased by at least 50% in BeWo cells transfected with siBCRP. A similar decrease in protein levels is shown in Fig. 2B. Control cells that were either mock transfected or transfected with siBCRP-scram did not show any change in BCRP expression.

Figure 2.

siRNA knockdown of endogenous BCRP mRNA (A) and protein (B) expression in BeWo cells. Human choriocarcinoma BeWo cells were transfected with 0–10 μg siRNAs and harvested for total RNA and protein after 24 hours as described in Materials and Methods. Total RNA (1.5 μg) was used for reverse transcription-PCR and 60 μg of protein were separated by 9% SDS-PAGE and then transferred onto nitrocellulose membrane. Membranes were immunoblotted with monoclonal antibody BXP-21 for human BCRP. β-Actin was used as a loading control. Representative of three independent experiments.

Figure 2.

siRNA knockdown of endogenous BCRP mRNA (A) and protein (B) expression in BeWo cells. Human choriocarcinoma BeWo cells were transfected with 0–10 μg siRNAs and harvested for total RNA and protein after 24 hours as described in Materials and Methods. Total RNA (1.5 μg) was used for reverse transcription-PCR and 60 μg of protein were separated by 9% SDS-PAGE and then transferred onto nitrocellulose membrane. Membranes were immunoblotted with monoclonal antibody BXP-21 for human BCRP. β-Actin was used as a loading control. Representative of three independent experiments.

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Increase in Drug Sensitivity in siRNA-Transfected Cells

To test whether siRNA-mediated knockdown of BCRP resulted in increased drug sensitivity, we compared IC50 values of mock- and siRNA-transfected BeWo cells obtained from cytotoxicity assays. As shown in Table 1 and Fig. 3A-C, the sensitivity of siBCRP-transfected BeWo cells to two BCRP substrates (i.e., mitoxantrone and topotecan) increased when compared with mock- and siBCRP-scram-transfected cells. More specifically, siRNAs caused a 10.5- and 8.2-fold sensitization to mitoxantrone and topotecan, respectively. On the other hand, the sensitivity to etoposide, a non-BCRP substrate, was not affected by siRNA knockdown of BCRP.

Table 1.

Effect of siRNA on IC50 values of drugs in BeWo cells

siRNAMitoxantrone IC50Topotecan IC50Etoposide IC50
Mock 0.294 ± 0.066 0.164 ± 0.055 2.07 ± 0.095 
siBCRP 0.028 ± 0.075 (10.5) 0.020 ± 0.077 (8.2) 1.77 ± 0.079 (1.2) 
siBCRP-scram 0.241 ± 0.077 (1.2) 0.158 ± 0.058 (1.0) 1.87 ± 0.113 (1.1) 
siRNAMitoxantrone IC50Topotecan IC50Etoposide IC50
Mock 0.294 ± 0.066 0.164 ± 0.055 2.07 ± 0.095 
siBCRP 0.028 ± 0.075 (10.5) 0.020 ± 0.077 (8.2) 1.77 ± 0.079 (1.2) 
siBCRP-scram 0.241 ± 0.077 (1.2) 0.158 ± 0.058 (1.0) 1.87 ± 0.113 (1.1) 

NOTE: In vitro cytotoxicity assays (CellTiter 96 AQueous assay) were done as described in the manufacturer's protocol. IC50 is the concentration (μmol/L) that produced 50% inhibition of cell growth. Results represent the mean ± SE of three experiments done in triplicate. Numbers in parentheses represent fold sensitization of drug resistance.

Increase in Drug Accumulation in siRNA-Transfected Cells

With the introduction of siRNAs into BeWo cells, intracellular drug accumulation was enhanced (Fig. 3D). Using flow cytometry, we showed that intracellular accumulation of topotecan was increased in siBCRP-transfected BeWo cells when compared with those that were mock transfected. As expected, cells that were transfected with siBCRP-scram did not result in any change in intracellular accumulation of topotecan.

Figure 3.

Effect of siRNA on drug sensitivity and accumulation in BeWo cells. BeWo cells were transfected with siRNAs before being seeded in 96-well plates at a density of 4,000 cells per well and grown overnight. Cells were then treated with (A) mitoxantrone, (B) topotecan, or (C) etoposide at the concentrations indicated and incubated at 37°C for 3–4 days. Cytotoxicity was assessed by using the CellTiter 96 AQueous assay according to the manufacturer's instructions. Representative of three independent experiments done in triplicate. D, mock- or siRNA-transfected BeWo cells were incubated with 30 μmol/L topotecan for 15 minutes at 37°C. Cellular content of topotecan was measured by fluorescence-activated cell sorting. Bold lines, shaded area, siBCRP transfected; bold lines, unshaded area, mock transfected; dotted lines, siBCRP-scram transfected. See Materials and Methods for details.

Figure 3.

Effect of siRNA on drug sensitivity and accumulation in BeWo cells. BeWo cells were transfected with siRNAs before being seeded in 96-well plates at a density of 4,000 cells per well and grown overnight. Cells were then treated with (A) mitoxantrone, (B) topotecan, or (C) etoposide at the concentrations indicated and incubated at 37°C for 3–4 days. Cytotoxicity was assessed by using the CellTiter 96 AQueous assay according to the manufacturer's instructions. Representative of three independent experiments done in triplicate. D, mock- or siRNA-transfected BeWo cells were incubated with 30 μmol/L topotecan for 15 minutes at 37°C. Cellular content of topotecan was measured by fluorescence-activated cell sorting. Bold lines, shaded area, siBCRP transfected; bold lines, unshaded area, mock transfected; dotted lines, siBCRP-scram transfected. See Materials and Methods for details.

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Circumvention of BCRP Expression under E2 Stimulation by siRNA

We have previously identified an estrogen response element in the BCRP gene promoter and have shown that E2 enhanced expression of BCRP mRNA in estrogen receptor–positive cells (33). To determine whether BCRP protein expression is similarly inducible by E2, we first placed estrogen receptor–positive BeWo cells in phenol red–free medium supplemented with charcoal/dextran-stripped fetal bovine serum (Gemini) for 3 days before subsequent siRNAs transfection and E2 treatment. It is important to note that, with the removal of estrogens found in phenol red and fetal bovine serum, the levels of BCRP protein decreased dramatically. This is shown in Fig. 4A whereby the amount of BCRP protein in cell lysate obtained from untreated BeWo cells was much lower than that in the same amount of lysate obtained from cells cultured in phenol red–containing medium (Fig. 2B). With the addition of 1 to 100 nmol/L E2, we observed a dose-dependent increase in the levels of BCRP protein from 1.0- to 7.8-fold in BeWo cells (Fig. 4A). In contrast, when BeWo cells were transfected with siBCRP and then subjected to E2 treatment, only a marginal 1.5-fold increase was observed at maximal E2 concentration of 100 nmol/L (Fig. 4B).

Figure 4.

Effect of siRNA on estrogen stimulation of BCRP in BeWo cells. BeWo cells were placed in phenol red–free medium for 3 days (A) before treatment with 0–100 nmol/L E2 for 24 hours or (B) before transfection with siBCRP. siBCRP-transfected cells were cultured for an additional 24 hours before E2 treatment. Protein (60–80 μg) was separated by 9% SDS-PAGE, and separated proteins were then transferred onto nitrocellulose membrane. Membranes were immunoblotted with monoclonal antibody BXP-21 for human BCRP. β-Actin was used as a loading control. Fold change, change in band density of BCRP normalized with that of β-actin. See Materials and Methods for details.

Figure 4.

Effect of siRNA on estrogen stimulation of BCRP in BeWo cells. BeWo cells were placed in phenol red–free medium for 3 days (A) before treatment with 0–100 nmol/L E2 for 24 hours or (B) before transfection with siBCRP. siBCRP-transfected cells were cultured for an additional 24 hours before E2 treatment. Protein (60–80 μg) was separated by 9% SDS-PAGE, and separated proteins were then transferred onto nitrocellulose membrane. Membranes were immunoblotted with monoclonal antibody BXP-21 for human BCRP. β-Actin was used as a loading control. Fold change, change in band density of BCRP normalized with that of β-actin. See Materials and Methods for details.

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To date, clinical success using functional modulators to reverse MDR has been modest due in part to toxicity problems and pharmacokinetic interactions (40). Whereas the search for effective small molecule inhibitors continues, molecular strategies via the use of hammerhead ribozymes and antisense oligonucleotides have also been proposed. Although experimental results indicate that these strategies are effective in vitro, their clinical utility is limited due to difficulties in delivery, stability, and potency. With our increasing understanding of the mechanism of RNAi, a third approach of using siRNAs in combating MDR should not be left unexplored. Here, we have designed and synthesized siRNAs against human BCRP and showed that siRNAs could specifically and effectively down-regulate BCRP expression in tumor cells. We found that siRNAs transfected for an optimal time of 24 hours were effective in down-regulating BCRP expression not only in HeLa cells containing enforced expression of wild-type BCRP but also in human choriocarcinoma BeWo cells displaying high endogenous expression of BCRP (39). It is interesting to note that, when the BCRP cDNA and siRNAs were simultaneously transfected into HeLa cells, a 25% decrease in mRNA levels resulted in a dramatic decrease in protein levels. This may be because the HeLa cells were overwhelmed with plasmid DNA during transient transfection such that BCRP mRNA was transcribed in great excess over the translational machinery in positively transfected cells. The relatively small proportion of mRNA that was knocked down by the siRNAs might be representative of the population of transcripts that were actually translated in the cells; hence, we observed a dramatic decrease in protein levels. On the other hand, we do not rule out the possibility that Western blotting is not sensitive enough to detect subtle changes in the protein levels. Most important, siRNAs effectively down-regulated endogenous BCRP in BeWo cells with decreases in mRNA and protein levels that were in good agreement with changes in the MDR phenotype. siRNAs sensitized BeWo cells to BCRP substrates such as mitoxantrone and topotecan and enhanced the intracellular accumulation of topotecan.

Here, we showed that the sensitivity to etoposide in BeWo cells remained unchanged with the introduction of siRNAs. Various levels of cross-resistance to etoposide have been observed in several drug-selected cell lines overexpressing BCRP/Bcrp1 (14, 21, 41, 42), although there is no conclusive evidence that this phenomenon could be attributed solely to BCRP/Bcrp1 activity, because the cells in those studies were heavily selected for drug resistance. The contribution of other mechanisms of resistance, such as changes in topoisomerase II and overexpression of other drug transporters, may confound those results and should therefore be taken into account. BeWo cells, however, express high levels of endogenous BCRP and, more importantly, were not selected for resistance with drugs. Instead of evaluating substrate specificities and transport properties of a specific transporter by using cell lines with a gain-of-function, the present study shows that RNAi is also useful in performing this task by the specific knockdown of individual transporter genes (i.e., loss-of-function).

We reported previously the presence of an estrogen response element in the BCRP promoter and showed that E2 was able to activate BCRP transcription in estrogen receptor–positive breast and ovarian cancer cells (33). BCRP is highly expressed in the placenta (43) where steroid hormones such as estrogens are produced and our earlier findings suggested that estrogens may induce the endogenous expression of BCRP in the placenta. Here, using estrogen receptor–positive BeWo cells derived from the placenta, we found that E2 was also able to induce BCRP expression at the protein level. This is shown by a noticeable decrease in BCRP protein levels in BeWo cells on removal of estrogens in the culture system by using phenol red–free medium containing charcoal-stripped serum. Following treatment with 1 to 100 nmol/L E2, the levels of BCRP protein levels in BeWo cells then increased dramatically to 7.8-fold at the maximal E2 concentration. However, with the introduction of siRNAs, we found that the effect was ameliorated with only 1.5-fold induction of BCRP expression at 100 nmol/L E2. These results further confirm that siRNAs are effective in modulating the functional phenotype mediated by BCRP.

Given the exploitation of a naturally occurring machinery that is both efficient and specific, RNAi seems to be more potent than the other types of RNA strategies (44) and may have an impact as a therapeutic tool. As with antisense and gene therapy approaches, the major challenge in translating RNAi into useful therapeutic strategy is the delivery of siRNAs. However, considerable progress has recently been made in this field and several genes have been successfully down-regulated by RNAi in in vivo models due to improved delivery systems (45–47). Given sufficient research into delivery methods, siRNA may indeed become a viable treatment modality.

To our knowledge, this is the first report describing evidence of silencing the human BCRP gene by siRNAs in cancer cells. Several studies employing the same strategy against the MDR1 gene have been published recently (27–30). Although selection for drug resistance in cell lines usually results in predominance of a single ATP-binding cassette transporter, such a situation is not observed in clinical samples. For example, in some cases of adult leukemias, combined overexpression of P-gp and MDR-associated protein 1 have been implicated in the MDR phenotype (48), and in others, even when P-gp-positive blasts were eliminated, a non-P-gp mechanism of resistance emerged in relapsed patients (49). It is also suggested that BCRP may have a role in recurrent tumors, especially in hematologic malignancies in which patients have been concurrently treated with chemotherapy and with inhibitors of P-gp (50). Taken together, it may be feasible to use siRNAs therapeutically in combination against the repertoire of ATP-binding cassette transporters to combat MDR.

In conclusion, siRNAs effectively down-regulated BCRP expression in tumor cells and modulated their functional phenotype. These findings may present a new approach in combating BCRP-mediated drug resistance.

Grant support: National Cancer Institute grants CA30103 and CA40570(W.T. Beck), VA Merit Review grant (D.D. Ross), and University of Illinois at Chicago.

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 toindicate this fact.

We thank Drs. Takeo Nakanishi for the wild-type pcDNA3-BCRP construct, Yin-Yuan Mo for helpful discussion, and Karen Hagen for assistance in the flow cytometry experiments and Martina Vaskova for outstanding administrative support.

1
Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters.
Nat Rev Cancer
2002
;
2
:
48
–58.
2
Allikmets R, Schriml LM, Hutchinson A, Romano-Spica V, Dean M. A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance.
Cancer Res
1998
;
58
:
5337
–9.
3
Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells.
Proc Natl Acad Sci U S A
1998
;
95
:
15665
–70.
4
Miyake K, Mickley L, Litman T, et al. Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes.
Cancer Res
1999
;
59
:
8
–13.
5
Honjo Y, Hrycyna CA, Yan QW, et al. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells.
Cancer Res
2001
;
61
:
6635
–9.
6
Kage K, Tsukahara S, Sugiyama T, et al. Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization.
Int J Cancer
2002
;
97
:
626
–30.
7
Ozvegy C, Litman T, Szakacs G, et al. Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells.
Biochem Biophys Res Commun
2001
;
285
:
111
–7.
8
Xu J, Liu Y, Yang Y, Bates S, Zhang JT. Characterization of oligomeric human half-ABC transporter ATP-binding cassette G2.
J Biol Chem
2004
;
279
:
19781
–9.
9
Maliepaard M, van Gastelen MA, de Jong LA, et al. Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line.
Cancer Res
1999
;
59
:
4559
–63.
10
Kawabata S, Oka M, Shiozawa K, et al. Breast cancer resistance protein directly confers SN-38 resistance of lung cancer cells.
Biochem Biophys Res Commun
2001
;
280
:
1216
–23.
11
Yoh K, Ishii G, Yokose T, et al. Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer.
Clin Cancer Res
2004
;
10
:
1691
–7.
12
Nakanishi T, Karp JE, Tan M, et al. Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients.
Clin Cancer Res
2003
;
9
:
3320
–8.
13
Allen JD, van Loevezijn A, Lakhai JM, et al. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C.
Mol Cancer Ther
2002
;
1
:
417
–25.
14
Rabindran SK, Ross DD, Doyle LA, Yang W, Greenberger LM. Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein.
Cancer Res
2000
;
60
:
47
–50.
15
Robey RW, Medina-Perez WY, Nishiyama K, et al. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells.
Clin Cancer Res
2001
;
7
:
145
–52.
16
Hausner P, Venzon DJ, Grogan L, Kirsch IR. The “comparative growth assay”: examining the interplay of anti-cancer agents with cells carrying single gene alterations.
Neoplasia
1999
;
1
:
356
–67.
17
Shiozawa K, Oka M, Soda H, et al. Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic.
Int J Cancer
2004
;
108
:
146
–51.
18
Yang CH, Chen YC, Kuo ML. Novobiocin sensitizes BCRP/MXR/ABCP overexpressing topotecan-resistant human breast carcinoma cells to topotecan and mitoxantrone.
Anticancer Res
2003
;
23
:
2519
–23.
19
de Bruin M, Miyake K, Litman T, Robey R, Bates SE. Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR.
Cancer Lett
1999
;
146
:
117
–26.
20
Ishii M, Iwahana M, Mitsui I, et al. Growth inhibitory effect of a new camptothecin analog, DX-8951f, on various drug-resistant sublines including BCRP-mediated camptothecin derivative-resistant variants derived from the human lung cancer cell line PC-6.
Anticancer Drugs
2000
;
11
:
353
–62.
21
Allen JD, Brinkhuis RF, Wijnholds J, Schinkel AH. The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin.
Cancer Res
1999
;
59
:
4237
–41.
22
Maliepaard M, van Gastelen MA, Tohgo A, et al. Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF120918.
Clin Cancer Res
2001
;
7
:
935
–41.
23
Sugimoto Y, Tsukahara S, Imai Y, Ueda K, Tsuruo T. Reversal of breast cancer resistance protein-mediated drug resistance by estrogen antagonists and agonists.
Mol Cancer Ther
2003
;
2
:
105
–12.
24
Erlichman C, Boerner SA, Hallgren CG, et al. The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux.
Cancer Res
2001
;
61
:
739
–48.
25
Houghton PJ, Germain GS, Harwood FC, et al. Imatinib mesylate is a potent inhibitor of the ABCG2 (BCRP) transporter and reverses resistance to topotecan and SN-38 in vitro.
Cancer Res
2004
;
64
:
2333
–7.
26
Kowalski P, Stein U, Scheffer GL, Lage H. Modulation of the atypical multidrug-resistant phenotype by a hammerhead ribozyme directed against the ABC transporter BCRP/MXR/ABCG2.
Cancer Gene Ther
2002
;
9
:
579
–86.
27
Duan Z, Brakora KA, Seiden MV. Inhibition of ABCB1 (MDR1) and ABCB4 (MDR3) expression by small interfering RNA and reversal of paclitaxel resistance in human ovarian cancer cells.
Mol Cancer Ther
2004
;
3
:
833
–8.
28
Yague E, Higgins CF, Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1.
Gene Ther
2004
;
11
:
1170
–4.
29
Nieth C, Priebsch A, Stege A, Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi).
FEBS Lett
2003
;
545
:
144
–50.
30
Wu H, Hait WN, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells.
Cancer Res
2003
;
63
:
1515
–9.
31
Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA. Targeted mRNA degradation by double-stranded RNA in vitro.
Genes Dev
1999
;
13
:
3191
–7.
32
Hannon GJ. RNA interference.
Nature
2002
;
418
:
244
–51.
33
Ee PL, Kamalakaran S, Tonetti D, He X, Ross DD, Beck WT. Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene.
Cancer Res
2004
;
64
:
1247
–51.
34
Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.
Nucleic Acids Res
2002
;
30
:
e46
.
35
Ifergan I, Shafran A, Jansen G, Hooijberg JH, Scheffer GL, Assaraf YG. Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis.
J Biol Chem
2004
;
279
:
25527
–34.
36
Imai Y, Tsukahara S, Asada S, Sugimoto Y. Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance.
Cancer Res
2004
;
64
:
4346
–52.
37
Kawabata S, Oka M, Soda H, et al. Expression and functional analyses of breast cancer resistance protein in lung cancer.
Clin Cancer Res
2003
;
9
:
3052
–7.
38
Pattillo RA, Gey GO. The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro.
Cancer Res
1968
;
28
:
1231
–6.
39
Bailey-Dell KJ, Hassel B, Doyle LA, Ross DD. Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene.
Biochim Biophys Acta
2001
;
1520
:
234
–41.
40
Fojo T, Bates S. Strategies for reversing drug resistance.
Oncogene
2003
;
22
:
7512
–23.
41
Hazlehurst LA, Foley NE, Gleason-Guzman MC, et al. Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line.
Cancer Res
1999
;
59
:
1021
–8.
42
Volk EL, Rohde K, Rhee M, et al. Methotrexate cross-resistance in a mitoxantrone-selected multidrug-resistant MCF7 breast cancer cell line is attributable to enhanced energy-dependent drug efflux.
Cancer Res
2000
;
60
:
3514
–21.
43
Maliepaard M, Scheffer GL, Faneyte IF, et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.
Cancer Res
2001
;
61
:
3458
–64.
44
Scherer LJ, Rossi JJ. Approaches for the sequence-specific knockdown of mRNA.
Nat Biotechnol
2003
;
21
:
1457
–65.
45
Caplen NJ. RNAi as a gene therapy approach.
Expert Opin Biol Ther
2003
;
3
:
575
–86.
46
McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice.
Nature
2002
;
418
:
38
–9.
47
Herweijer H, Wolff JA. Progress and prospects: naked DNA gene transfer and therapy.
Gene Ther
2003
;
10
:
453
–8.
48
Schuurhuis GJ, Broxterman HJ, Ossenkoppele GJ, et al. Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-β-d-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia.
Clin Cancer Res
1995
;
1
:
81
–93.
49
Leith CP, Kopecky KJ, Chen IM, et al. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein, MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study.
Blood
1999
;
94
:
1086
–99.
50
Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2).
Oncogene
2003
;
22
:
7340
–58.