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Vol. 1, 1327-1334, December 2002     Molecular Cancer Therapeutics
© 2002 American Association for Cancer Research

Overcoming Multidrug Drug Resistance in P-Glycoprotein/MDR1-overexpressing Cell Lines by Ecteinascidin 743

Atsuko Kanzaki, Yuji Takebayashi, Xiao-Qin Ren, Hitoshi Miyashita, Shiro Mori, Shin-ichi Akiyama and Yves Pommier1

Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan [A.K., Y.T., H.M.]; Graduate School of Dentistry, Department of Oral and Maxillofacial Surgery, Tohoku University, Aobaku, Sendai 980-8575, Japan [X-Q.R., S-i.A.]; Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan [S.M.]; and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [Y.P.]


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ecteinascidin 743 (Et-743) is a novel anticancer agent forming covalent guanine adducts at specific sites in the DNA minor groove. Et-743 has a unique mechanism of action because it kills cancer cells by poisoning transcription-coupled nucleotide excision repair. Recent studies suggested a complex relationship between P-glycoprotein (P-gp)/MDR1 and Et-743. On one hand, Et-743 was reported to down-regulate the MDR1 promoter in vitro. On the other hand, P-gp overexpression was hypothesized to contribute to Et-743 resistance in an ovarian cell line. The present study was performed to further investigate the relationship between P-gp/MDR1 and the activity of Et-743. First, we found no P-gp/MDR1 overexpression (mRNA and protein levels) in two independently generated Et-743-resistant human colon carcinoma cell lines (HCT116/ER5 and SW480/ER0.5). Secondly, we found no cross-resistance to Et-743 in two well-characterized P-gp/MDR1-overexpressing cell lines (KB-8-5 and KB-C-2). Third, Et-743 pretreatment enhanced the cytotoxicity and the cellular accumulation of doxorubicin and vincristine in P-gp/MDR1-overexpressing KB-8-5/KB-C-2 cell lines. Fourth, we observed P-gp/MDR1 down-regulation by Et-743 in KB-C-2 cells. These results indicate that Et-743 does not select for the emergence of a P-gp phenotype in all cell lines made resistant to Et-743 and that P-gp overexpression is not sufficient to confer resistance to Et-743. Furthermore, Et-743 is an effective agent in P-gp-overexpressing cells. Et-743 can potentiate the activity of other chemotherapeutic agents by down-regulating P-gp/MDR1, suggesting that the combination of Et-743 and chemotherapeutic agents that are substrates for P-gp/MDR1 may be valuable in the clinic.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Et-7432(Fig. 1) is a marine alkaloid isolated from the Caribbean sea squirt Ecteinascidia turbinata. Although the extraordinary antiproliferative activity of extracts from this tunicate was discovered in the late 1960s, the purification and chemical structure of active compounds were not completed until 1990 (Refs.1 and 2, for review, see Refs. 3 and 4). Preclinical studies revealed that Et-743 is remarkably active in diverse solid tumors including ovarian cancer, breast cancer, non-small lung carcinomas, melanoma, sarcomas, and renal cancers (5–7). Data from the National Cancer Institute Drug Discovery Screen using the 60 human tumor cell line panel demonstrate a unique activity profile of Et-743 when Et-743 is compared with standard agents presently used in cancer chemotherapy (8). Martinez and Corey (9) also synthesized derivatives, the phthalascidins, that might be important for clinical development. Recently, Phase II/III clinical trials demonstrated that Et-743 exhibits potent activity in human solid tumors, particularly in soft tissue sarcomas, a disease for which there is currently no effective chemotherapy (10–13).



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  		Fig. 1. Chemical structure of Et-743.

 
Et-743 binding to DNA was discovered using biochemical, X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular modeling studies. Et-743 binds in the DNA minor groove by a pattern of hydrogen bond acceptors and donors (14, 15). Et-743 contains a carbinolamine center, and elimination of the adjacent hydroxyl group results in a Schiff base vulnerable to nucleophilic attack, leading to alkylation of the exocyclic 2-amino group of guanine (Fig. 1; Refs. 14–16). The filling of the minor groove by this bulky drug, the formation of a sharp DNA bend toward the major groove by the Et-743 adduct (17), and the unique sequence specificity and reversibility of the Et-743-DNA adducts when the two DNA strands become separated (16, 18) set Et-743 apart from the DNA alkylating agents presently used in cancer chemotherapy.

Recently, the cellular consequences of the Et-743-DNA adducts have been further investigated. Whereas DNA alkylation by Et-743 can trap topoisomerase I cleavage complexes (19, 20), this effect does not appear critical for the drug’s antiproliferative activity because topoisomerase I cleavage complexes are only detectable at micromolar concentrations (i.e., well above the pharmacological concentrations, which are in the nanomolar range) and because topo I-deficient cells remain sensitive to Et-743 (8, 21). Interference of the Et-743 adducts with other DNA-binding factors, such as transcription-coupled protein complexes, seems more relevant for the antiproliferative activity of the drug at pharmacological concentrations. Et-743 is a potent inhibitor of the NF-Y, sp1, and SXR transcription factors (2224), and Et-743 specifically blocks the transcription of certain genes including the P-gp/MDR1 promoter (23, 24). Recently, the identification of NER deficiency in two Et-743-resistant cell lines (HCT116/ER5 and SW480/ER0.5) and the resistance of NER-deficient cell to Et-743 suggested a unique mechanism of action for Et-743 related to the poisoning of transcription-coupled NER (25). Independently, Erba et al. (21) found that NER-deficient cells are resistant to Et-743. We have also demonstrated that Et-743 induces the formation of transcription-coupled repair-dependent DNA single-strand breaks and proposed that the Et-743 adducts trap the transition-coupled NER complex after incision of the transcribed DNA possibly by XPG and the XPF NER endonucleases (25).

P-gp/MDR1 is expressed in a variety of human solid tumors and hematological malignancies (26, 27). The aim of the present study was to elucidate the relationship(s) between P-gp/MDR1 and the mechanisms of resistance to Et-743. A prior study reported that P-gp was overexpressed in the human ovarian carcinoma Igrov-1 cell line selected for resistance to Et-743 in vitro and that this resistance was attenuated by the P-gp-reversing drug agent PSC, a cyclosporine analogue (28). In the present study, we investigated the activity of Et-743 in two well-characterized P-gp/MDR1-overexpressing cell lines, KB-8-5 and KB-C-2, and we found that neither cell line was cross-resistant to Et-743. Furthermore, we found that subtoxic concentrations of Et-743 reversed resistance to DOX and VCR in these cells. The results suggest the usefulness of Et-743 alone or in combination with other agents in P-gp-overexpressing tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals.
[14C]DOX (54 mCi/mmol) and [14H]VCR (2200 Ci/mmol) were obtained from Amersham International Ltd. (Buckinghamshire, United Kingdom). Et-743 was provided by the National Cancer Institute. Cepharanthine was a gift from Kakenshoyaku (Tokyo, Japan). Other chemicals were obtained from Sigma (St. Louis, MO).

Cells and Cell Culture.
The human epidermoid KB carcinoma cell line KB-3-1 (29) is the parental drug-sensitive cell line. The P-gp-overexpressing cell lines, KB-8-5 and KB-C-2, which have increased levels of MDR1 mRNA, were originally selected from KB-3-1 cells and cultured in medium containing 10 ng/ml and 2 µg/ml colchicine, respectively (29). HCT116/ER5 cells were obtained by continuous exposure of human colon carcinoma HCT116 cells to stepwise increasing concentrations of Et-743 for a period of 1 year, until the cells grew in the presence of 1.5 nM Et-743. After three rounds of subcloning, one clone, referred to as HCT116/ER5, was selected for additional studies. HCT116/ER5 cells are 23-fold resistant to Et-743 compared with the parental HCT116 cells in MTT assays (IC50 = 3.66 ± 0.43 versus 0.16 ± 0.05 nM). SW480-0.5n cells were generated from human colon carcinoma SW480 cells after 12 months of exposure to stepwise increasing concentrations of Et-743. The SW480-0.5n cells grow in the presence of 0.5 nM Et-743 and are approximately 10-fold resistant compared with the parental SW480 cells in MTT assays (IC50 = 6.78 ± 0.35 versus 0.68 ± 0.03 nM). Et-743 resistance is stable in both cell lines because further passage of both cell lines in drug-free medium for 1 year had no effect on resistance to Et-743.

Photoaffinity Labeling with [3H]Azidopine and SDS-PAGE.
Membrane vesicles (100 µg of protein) were incubated with 1 µM [3H]azidopine for 15 min at room temperature in the presence of the indicated concentrations of Et-743 or verapamil. After continuous irradiation at 366 nm for 30 min at 25°C, samples were solubilized in a SDS buffer as described previously (30). Electrophoresis was performed in 7.5% polyacrylamide gels without heating the samples. Gels were fixed, stained with 0.25% Coomassie Blue in 50% trichloroacetic acid, and subjected to fluorography using ENLIGHTNING (DuPont-New England Nuclear, Wilmington, DE). The dried gels were exposed to Kodak XAR film (Kodak, Rochester, NY) at 80°C.

Immunoblotting.
Membrane vesicles were prepared from each cell line. Cells were washed once and scraped into PBS containing 1% aprotinin. Cells were then washed at 4°C in PBS, collected by centrifugation (4,000 x g for 10 min), suspended in buffer A [10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, and 0.2 mM CaCl2], and equilibrated at 4°C for 30 min. EDTA at a final concentration of 1 mM was added to the suspension of lysed cells, which was then diluted 1:4 with buffer B [10 mM Tris-HCl (pH 7.4) and 0.25 M sucrose] and centrifuged at 1,000 x g for 10 min at 4°C to remove nuclei and cellular debris. The supernatant was layered onto a 35% sucrose cushion [10 mM Tris-HCl (pH 7.4), 35% sucrose, and 1 mM EDTA] and centrifuged for 30 min at 16,000 x g at 4°C. The interface was collected, diluted 1:5 in buffer B, and then centrifuged for 45 min at 100,000 x g at 4°C. The vesicle pellet was resuspended in buffer B by repeated passages through a 25-gauge needle. Protein concentration in the vesicles was determined by the method of Bradford, and vesicles were stored at -80°C. To prepare the total cell lysates, cells were washed with PBS and scraped into PBS containing 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 1% aprotinin (Sigma), and 1 mM p-amidinophenyl methanesulfonyl fluoride hydrochloride (APMSF; Wako, Osaka, Japan). The lysates were passed through a 21-gauge needle to shear the DNA, incubated for 30 min on ice, and centrifuged at 15,000 x g for 20 min at 4°C. Protein concentrations were determined by the method of Bradford. Membrane vesicles (20 µg of protein) for the analysis of P-gp and total cell lysates (20 µg of protein) for the analysis of lung related protein (LRP) were prepared from cells, and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The transferred proteins were reacted with a monoclonal antibody against P-gp (C-219; Ref. 31) and incubated for 1 h at room temperature for with horseradish peroxidase-conjugated goat antirabbit IgG. Antibody binding was visualized with the enhanced chemiluminescence Western blotting detection system (Amersham, Little Chalfont, United Kingdom).

Conventional RT-PCR.
Conventional RT-PCR for the detection of MDR1 gene was performed as described previously (32). Total RNA was prepared by the Trizol (Life Technologies, Inc., Gaithersburg, MD). cDNA was synthesized from 3 µg of total RNA using random hexadeoxynucleotide primers (Life Technologies, Inc.) in a 20-µl solution containing reverse transcriptase (Life Technologies, Inc.). cDNA was diluted 4-fold in water and stored at -20°C until use. PCR was performed with cDNA derived from 30 ng of RNA in a total volume of 25 µl containing cDNA, dGTP, dATP, dTTP, and dCTP at a concentration of 200 µM, 4 µM of each primer and 0.25 unit of ExTaq polymerase (Takara Shuzo, Otsu, Shiga, Japan). The PCR conditions consisted of 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, followed by 72°C for 10 min. The PCR primer sequences of MDR1 and GAPDH (used as internal control) were as follows: MDR1, sense primer 5`-CCCATCATTGCAATAGCAGG-3` and antisense primer 5`-GTTCAAACTTCTGCTCCTGA-3` corresponding to 167 bp (residues 2733–2752); GAPDH, sense primer 5`-CCCCTGGCCAAGGTCATCCATGACAACTTT-3` and antisense primer 5`-GGCCATGAGGTCCACCACCCTGTTGCTGTA-3` corresponding to 513 bp (residues 515–1027). To evaluate the amplified PCR products semiquantitatively, the optimal conditions for the detection of MDR1 and GADPH genes were determined using cDNA derived from placenta. At 40 cycles of PCR in each molecule, the relative yield of PCR products was similar, indicating that the number of cycles used corresponded to a plateau. At 25 cycles or less, each gene expression could not be clearly distinguished (data not shown). Thus, we used 35 PCR cycles for the detection of each gene. The amplified cDNA fragments were electrophoresed on 2% agarose gels.

Quantitative Real-Time PCR.
For quantification, cDNA was used as template in a TaqMan real-time PCR assay (Perkin-Elmer Applied Biosystems), according to the manufacturer’s protocol. The housekeeping GADPH gene served as the reference and was amplified parallel to the MDR1 gene for every sample in separate vessels. The oligonucleotide primers for the detection of the MDR1 and GADPH genes were the same as those described above. The constituents of each PCR (15 µl) were 1 µl of cDNA (or distilled H2O), 0.5 µl of primer 15 pmol/liter each, 6.0 µl of distilled H2O, and 7.5 µl of TaqMan Universal PCR Mastermix (Perkin-Elmer Applied Biosystems).

Product amplification was done up to 50 PCR cycles, after uracil removal (2 min at 50°C) and polymerase activation (15 min at 95°C). Each three-step PCR cycle comprised denaturing (30 s at 95°C), annealing (30 s at 49°C) and extending (1 min at 74°C). A water control was added in every experiment. All assays were run in triplicate.

Cell Survival by the MTT Assay.
Chemosensitivity in vitro was measured by means of the MTT colorimetric assay performed in 96-well plate. To determine the effect of Et-743, cells in 180 µl of culture medium with or without reversing agents were added into each well. After a 24- to 48-h incubation (37°C, 5% CO2), 20 µl of various concentrations of DOX or VCR were added, and the plates were incubated for 4 days. Fifty µl of MTT (1 mg/ml in PBS) were added to each well. Plates were incubated for 4 h, and the resulting formazan was dissolved in 100 µl of DMSO after aspiration of the culture medium. The plates were shaken mechanically for 5 min and read immediately at 570 nm using a model 550 Micro Plate Reader (Bio-Rad, Richmond, CA).

Drug Accumulation.
Accumulation of DOX and VCR in cells was measured using [3H]VCR and [14C]DOX. Confluent cell monolayers in 24-well plates were incubated with the indicated concentrations of 0.1 µM [3H]VCR (54 mCi/mmol) or 1 µM [14C]DOX (2200 mCi/mmol) in MEM with or without 2-day preincubation with reversing agents (3 µM cepharanthine for 1 h or 0.1 nM Et-743 for 48 h) at 37°C after they had been incubated in MEM with or without the reversing agents for 30 min at 37°C. After three washes with ice-cold PBS, the cells were resuspended in 10 mM phosphate buffer (pH 7.4) containing 1% Triton X-100 and 0.2% SDS, and the incorporated radioactivity was determined.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Et-743 Resistance Is Not Associated with Alterations in P-gp/MDR1.
P-gp/MDR1 is a transporter implicated in multidrug resistance (33), and VCR and Taxol are substrates for P-gp (34). Table 1 shows that the two Et-743-resistant human colon carcinoma cell lines, HCT116/ER5 and SW480/ER0.5 (25), are not cross-resistant to the tubulin inhibitors Taxol and VCR, suggesting that P-gp/MDR1 is not involved in the resistance to Et-743. Fig. 2, A and B, demonstrates no detectable difference in P-gp and comparable MDR1 mRNA expression between HCT116 and HCT116/ER5 cells and between SW480 and SW480/ER0.5 cells. These data indicate that resistance to Et-743 is not related to overexpression of P-gp/MDR1 in either HCT116/ER5 or SW480/ER0.5 cells. Next, we examined whether P-gp overexpression could confer Et-743 resistance in two well-characterized P-gp-overexpressing cell lines, KB-8-5 and KB-C-2, in comparison with their parental cell line, KB 3-1 (29, 35). As expected, both cell lines were resistant to VCR (Fig. 2C). However, we observed no difference in sensitivity to Et-743 between the P-gp-overexpressing cells and the parental KB 3-1 cells (Fig. 2D).


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  		Table 1 Cytotoxicity of taxol and VCR in Et-743-resistant cell lines

IC50 (mean ± S.D.) values were determined from three independent experiments using MTT assay after 4-day drug exposure.

 


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  		Fig. 2. Et-743 resistance is not correlated with overexpression of P-gp/MDR1. A, immunoblot of P-gp in Et-743-resistant HCT116/ER5 and SW480/ER0.5 cells. The number of cells/sample is indicated above the lanes. The KB-8-5 cells were used as a positive control. B, MDR1 expression measured by RT-PCR in HCT116/ER5 and SW480/ER0.5 cells. GAPDH was used as an internal control. C, lack of cross-resistance to Et-743 in the P-gp-overexpressing KB-8-5 and KB-C-2 cells. D, resistance to VCR in KB-8-5 and KB-C-2 cells.

 
Et-743 Partially Reverses the Cytotoxicity of DOX and VCR in P-gp-overexpressing Cells.
The ability of Et-743 to reverse drug resistance in cells overexpressing P-gp was examined and compared with that of cepharanthine, a well-known reversing agent for P-gp-mediated multidrug resistance (36). The cytotoxic effects of DOX and VCR were examined by MTT assays in the absence or presence of 3 µM cepharanthine or 0.1 nM Et-743. These concentrations were chosen because they were the highest noncytotoxic concentrations in KB-3-1, KB-8-5, and KB-C-2 cells (data not shown). Et-743 or cepharanthine was added to cell cultures either 2 days before DOX or VCR (protocol A, Table 2) or at the same time (protocol B, Table 2). Table 2 shows that cepharanthine reversed the resistance of KB-8-5 and KB-C-2 cells to DOX and VCR. Et-743 also increased the sensitivity of KB-8-5 and KB-C-2 cells to DOX and VCR, but the effect of Et-743 was less than that for cepharanthine (see Table 2). Moreover, in contrast to cepharanthine, Et-743 was more effective when cells were pretreated with Et-743 for 2 days before being exposed to DOX or VCR (see Table 2, compare protocols 1 and 2).


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  		Table 2 Effects of Et-743 on DOX and VCR cytotoxicity as a reversing agent for P-gp

 
Et-743 Increases the Cellular Accumulation of DOX and VCR and Reduces P-gp Levels.
To investigate how Et-743 reduced the resistance of KB-8-5 and KB-C-2 cells to DOX and VCR, we examined the cellular accumulation of VCR and DOX in KB-3-1, KB-8-5, and KB-C-2 (Fig. 3). The accumulation of VCR in KB-C-2 cells was 17.7% of that in KB-3-1 cells, and cepharanthine enhanced the accumulation of VCR in KB-C-2 cells to a level 3.1-fold higher than that seen in KB-C-2 cells not treated with cepharanthine. Et-743 at 0.1 nM also enhanced the accumulation of VCR in KB-C-2 cells to a level 1.9-fold higher than that in KB-C-2 cells not treated with Et-743. Similar effects of Et-743 were observed in KB-8-5 cells (Fig. 3). The effect of Et-743 on the accumulation of DOX was also similar to that of VCR in both KB-C-2 and KB-8-5 cells.



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  		Fig. 3. Effect of Et-743 on photoaffinity labeling of P-gp in membrane vesicles from KB-C-2 cells. Membrane vesicles (100 µg of protein) were incubated with 1 µM [3H]azidopine (47 Ci/mmol) for 15 min at room temperature in the presence of the indicated concentrations of Et-743 or verapamil and then processed as described under "Materials and Methods."

 
We studied the effect of Et-743 on the photolabeling of P-gp in membrane vesicles from KB-C-2 cells to observe whether Et-743 directly inhibited the function of P-gp. As expected, verapamil at 100 µM inhibited the labeling of P-gp. However, at the same concentrations, Et-743 did not inhibit P-gp photolabeling (Fig. 4).



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  		Fig. 4. Effects of Et-743 on the accumulation of VCR or DOX in KB-3-1, KB-C-2, and KB-8-5 cells. Drug intracellular levels were measured without (dark gray bars) or with pretreatment for 48 h with 3 µM cepharanthine (black bars) or 0.1 nM Et-743 (light gray bars). Bars, SEs for triplicate determinations. *, P < 0.01; **, P < 0.05.

 
We next examined the expression levels of MDR1/P-gp after treatment with Et-743. As shown in Fig. 5, A and B, MDR1 gene expression was progressively decreased until 24 h after Et-743 treatment, and the decreased MDR1 expression level persisted until at least 72 h. Decreased P-gp protein expression level was also observed after 72 h of treatment with 0.1 nM Et-743 (Fig. 5B). These findings are consistent with the hypothesis that Et-743 enhances the accumulation of VCR/DOX by down-regulating MDR1/P-gp expression.



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  		Fig. 5. Down-regulation of MDR1/P-gp by Et-743 in KB-C-2 cells. A, mRNA levels of the MDR1 gene were observed by RT-PCR in KB-C-2 cells after Et-743 treatment. B, immunoblot analysis of P-gp after Et-743 treatment. C, mRNA levels of the MDR1 gene were observed by real-time-PCR in KB-C-2 cells after Et-743 treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to determine the relationship between P-gp/MDR1 and the cytotoxicity of the novel anticancer drug Et-743, presently in Phase II/III clinical trials. Analysis of two cell lines resistant to Et-743 revealed no detectable expression of P-gp. Additional studies using KB-8-5 and KB-C-2 cells overexpressing P-gp/MDR1 demonstrated that a nontoxic concentration of Et-743 partially reversed the resistance to both VCR and DOX while down-regulating MDR1/P-gp. This activity of Et-743 is unique among anticancer drugs in clinical trials.

MDR1/P-gp and Resistance to Et-743.
A recent study described an Et-743-resistant cell line (Igrov-1/25ET), which had been selected from ovarian carcinoma Igvor-1 cells (28). The overexpression of MDR1/P-gp was observed in this cell line by flow cytometric analysis (28). However, no direct evidence was provided that Et-743 was a substrate for P-gp. Therefore, we examined the expression of P-gp in two independently derived Et-743-resistant cell lines, HCT116/ER5 and SW480/ER0.5. P-gp was not detected in the two Et-743-resistant cell lines, and neither of these two cell lines were resistant to Taxol and VCR (Table 1). We also found that the two well-characterized P-gp-overexpressing cell lines, KB-8-5 and KB-C-2, were not cross-resistant to Et-743. These results indicate that P-gp/MDR1 is not directly involved in Et-743 resistance. The cause of the discrepancy between our findings and those by Erba et al. (28) is unknown. Actually, the induction of P-gp by Et-743 and the restoration of the sensitivity of Igrov/25ET to Et-743 by the cyclosporine analogue PSC833, a potent MDR1 reversing agent, were observed in Igrov/25ET cells (28). The resistance to Et-743 was not reversed while maintaining Igrov/25ET cells in drug-free medium for 24 months (28). These findings might be explained as follows. Some molecule(s) other than P-gp/MDR1 might be involved in the resistance to Et-743 in Igrov-1/25ET cells, and the cyclosporine analogue PSC833 might have interacted with this molecule(s) to reverse the Et-743 resistance. Alternatively, MDR1 might have been mutated in the Igrov-1/25ET cells, and the substrate specificity of the mutated P-gp may be different from that of wild-type P-gp. In any case, taken together, these observations suggest that Et-743 may not be a simple substrate for P-gp.

Overcoming of VCR/DOX Resistance.
Et-743 was recently reported to down-regulate the MDR1 promoter by interfering with the NF-Y, Sp1, and SXR transcription factors (2224). We examined the effect of a noncytotoxic concentration of Et-743 on MDR1/P-gp-overexpressing cells. Pretreatment with Et-743 was required to partially reverse the resistance to doxorubicin and VCR (Table 2). Pretreatment with Et-743 increased the accumulation of these two drugs, probably by down-regulating expression of P-gp (Figs. 4 and 5). In addition, overcoming of VCR/DOX resistance was not due to the direct inhibition of P-gp activity, as determined by photoaffinity labeling experiments (Fig. 3). Tsuruo et al. (37) first demonstrated that verapamil reverses P-gp-mediated MDR. Since then, many agents such as 8-Cl-cAMP (38), cepharanthine (36), and bryostatin 1 (39) have been reported to reverse P-gp-mediated MDR. Verapamil is an inhibitor of the slow channel of Ca2+ transport across the membrane, and it controls the transcriptional level of MDR1 (37). Recently, Kaura and Dey (40) reported that P-gp-like protein was overexpressed in arsenite-resistant Leishmania and that verapamil regulated drug sensitivity by down-regulating the expression of the P-gp-like protein. 8-Cl-cAMP, a differentiating agent, is a site-selective analogue that down-regulates MDR1 expression by regulation of protein kinase A in MCF-7TH cells (38). Bryostatin, a macrocyclic lactone isolated from the marine invertebrate Bugula neritina, exhibits significant antitumor activity in preclinical studies. Bryostatin down-regulates protein kinase C activity by ubiquitination of protein kinase C and subsequent targeting of the protein to the proteasome (41). Interestingly, this antitumor agent also down-regulates MDR1 and potentiates VCR cytotoxicity (39) as well as Et-743. Et-743 and bryostatin are different from other anticancer agents and P-gp-reversing agents because they both down-regulate MDR1/P-gp and exhibit antitumor activity by themselves.

Jin et al. (23) reported that NF-Y is a central mediator for activation of human MDR1 by several inducers and that NF-Y functions by recruiting the histone acetyltransferase PCAF to the MDR1 promoter (23). They also demonstrated that Et-743 blocks activation of the MDR1 promoter by interfering with the NF-Y/PCAF complex. These observations suggest that Et-743 is the prototype for a distinct class of transcription-targeted chemotherapeutic agents. In addition, the promoter of MDR1 may be one of the preferential targets of Et-743 because it contains two distinct guanine-rich regions (-121 to -88 and -73 to -51) that are potential targets for Et-743 (16) and are important for MDR1 promoter activity by Sp1 (42). Further, Synold et al. (24) demonstrated that Et-743 suppresses MDR1 transcription by acting as an inhibitor of SXR. Our present study revealed that preincubation with a subtoxic concentration (0.1 nM, 48 h) of Et-743 produced synergism for the cytotoxicity of DOX and VCR in cells overexpressing MDR1/P-gp and that this effect was mediated through the down-regulation of MDR1/P-gp expression. After Et-743 binding to DNA, single-strand breaks and DNA damages take place. The DNA lesions induced by Et-743 are very persistent in the cells (8). Therefore, if they take place at MDR1 promoter region, MDR1/P-gp expression would be persistently decreased by the interference of transcriptional factors such as Sp1, NF-Y, or SXR by Et743 in cells.

Conclusions.
The present study suggests that Et743 is an effective cytotoxic agent in P-gp/MDR1-overexpressing cells and that the combination of Et743 with chemotherapeutic agents that are substrates for MDR1/P-gp may be valuable in the clinic because P-gp/MDR1 is expressed in a variety of human malignancies.


   Footnotes
 
1 To whom requests for reprints should be addressed, at Building 37, Room 5068, NIH, Bethesda, MD 20892-4255. Phone: (301) 496-5944; Fax: (301) 402-0752; E-mail: pommier{at}nih.gov Back

2 The abbreviations used are: Et-743, ecteinascidin 743; P-gp, P-glycoprotein; NER, nucleotide excision repair; DOX, doxorubicin; VCR, vincristine; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; MTT, 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide; RT-PCR, reverse transcription-PCR. Back

Received 11/16/01; revised 9/17/02; accepted 10/16/02.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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