8,9-Dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6] naphthyridin-6-one (ARC-111, topovale) is a new synthetic antitumor agent. In the current study, we show that ARC-111 is highly potent in scid mice carrying human tumor xenografts. ARC-111 was shown to be as active as camptothecin (CPT)-11 in the HCT-8 colon tumor model, and compared favorably with CPT-11 and topotecan in the SKNEP anaplastic Wilms’ tumor model. In tissue culture models, ARC-111 exhibited low nm cytotoxicity against a panel of cancer cells. ARC-111 cytotoxicity as well as ARC-111-induced apoptosis was reduced >100-fold in CPT-resistant topoisomerase I (TOP1)-deficient P388/CPT45 cells as compared with P388 cells. Similarly, ARC-111 cytotoxicity was greatly reduced in CPT-resistant CPT-K5 and U937/CR cells, which express CPT-resistant mutant TOP1, suggesting that the cytotoxic target of ARC-111 is TOP1. Indeed, ARC-111, like CPT, was shown to induce reversible TOP1 cleavage complexes in tumor cells as evidenced by specific reduction of the TOP1 immunoreactive band in a band depletion assay, as well as elevation of small ubiquitin modifier-TOP1 conjugate levels and activation of 26S proteasome-mediated degradation of TOP1. Unlike CPT, ARC-111 is not a substrate for the ATP-binding cassette transporter breast cancer resistance protein. In addition, ARC-111 cytotoxicity was not significantly reduced in the presence of human serum albumin. These results suggest that ARC-111 is a promising new TOP1-targeting antitumor drug with a different drug resistance profile than CPT.

CPT,4 the active component responsible for the anticancer activity in the extract of the plant Camptotheca acuminata(1), exhibits a broad spectrum antitumor activity against a panel of solid tumors in animal models (2, 3). Several CPT derivatives (e.g., irinotecan and TPT) have been developed recently into the clinic (4, 5). Extensive studies have firmly established that the nuclear protein DNA TOP1 is the molecular target for CPT (6). Mechanistic studies have demonstrated that CPT inhibits TOP1 by stabilizing a reversible covalent reaction intermediate, referred to as a cleavable or cleavage complex (7, 8, 9, 10).

CPT exhibits highly S phase-specific cytotoxicity and induces G2-M cell cycle arrest (11). A replication fork collision model has been proposed to explain the S phase cytotoxicity of CPT (11, 12). In this model, the collision between an advancing replication fork and the CPT-trapped TOP1 cleavable complex triggers replication fork arrest, breakage of the replication fork to generate a DNA double-strand break, and the formation of a covalent TOP1-DNA complex at the fork (12). In addition to S phase cytotoxicity, the same collision has also been suggested to be responsible for G2-M cell cycle arrest (13), and the generation of many DNA damage signals, such as nuclear factor κB activation, p53 up-regulation, replication protein A phosphorylation, Chk1 phosphorylation, and ATM/ATR activation (reviewed in Ref. 14). The S phase-specific cytotoxicity is presumably important for the antitumor activity of CPT (11, 12).

In addition to its S phase-specific cytotoxicity, the elevated TOP1 level in tumors has also been considered to be important for the antitumor activity of CPT (15, 16). More recently, CPT has been shown to induce transcription-dependent degradation of TOP1 (down-regulation) through a ubiquitin/26S proteasome pathway (17, 18, 19). CPT-induced TOP1 down-regulation, which is presumably a repair response to TOP1 cleavable complexes, occurs in normal nontransformed cells but not in most tumor cells (20). CPT-induced TOP1 down-regulation, which reduces TOP1-mediated DNA damage, results in recovery of RNA synthesis inhibition in normal cells (17) and, thus, reduced toxic side effects of CPT (17).

TOP1 cleavable complexes induced by CPT are rapidly (i.e., within minutes) SUMO-1 (also SUMO-2/3) conjugated by UBC9, the conjugating enzyme (E2) for SUMO (21). SUMO is a ubiquitin-like protein that forms conjugates with a limited number of proteins, most of which are nuclear proteins (21). The function of SUMO conjugation to TOP1 is unclear but has been suggested to represent a repair response to TOP1 cleavable complexes (21).

The elucidation of the mechanism of action of CPT has stimulated the development of other TOP1-directed anticancer drugs. This effort is particularly important, because CPTs are the only TOP1-directed anticancer drugs in the clinic. Despite their potency, CPTs are chemically unstable due to the labile β-hydroxy lactone ring, which is readily hydrolyzed to the inactive CPT carboxylate at neutral pH (22, 23). In addition, CPTs are substrates for the ABC transporter BCRP, known to be expressed in many human tumors (24, 25). Moreover, CPTs have been shown to bind to HSA in their carboxylate forms, which reduces their potency in humans as compared with mice (26, 27, 28). Our previous studies have demonstrated that a synthetic ARC-111 (see Fig. 3 A for structure) is as potent as CPT in stimulating TOP1-mediated DNA cleavage using purified hTOP1 and is more potent than irinotecan (CPT-11, a CPT derivative in clinical use) in nude mice carrying human breast MDA-MB-435 tumor xenografts (29, 30). In the current study, we show that ARC-111 rivals the CPTs in its potency in inducing tumor regression in nude mice bearing SKNEP anaplastic Wilms’ tumor xenografts. The molecular target of ARC-111 was shown to be TOP1 based on comparative studies on CPT and ARC-111 in a number of biochemical and cell-based assays. ARC-111 is as potent as CPT in inducing TOP1-mediated cytotoxicity and apoptosis in tissue culture models. Unlike CPT, ARC-111 is not a substrate for BCRP, a member of the ABC family of drug transporters. In addition, ARC-111 cytotoxicity is not significantly affected by HSA. Our results indicate that ARC-111 is a promising new TOP1-targeting anticancer drug with a different drug resistance profile than CPTs.

Chemicals and Antibodies.

CPT was kindly provided by Dr. Mansukh C. Wani (Research Triangle Institute, Research Triangle Park, NC). Etoposide (VP-16) was purchased from Sigma. ARC-111 is synthesized as described previously (30). Antibodies against BRCP were obtained from Dr. Jan H. M. Schellens (Netherlands Cancer Institute, Amsterdam, the Netherlands). Anti-TOP1 antibodies were obtained from sera of Scleroderma 70 patients.

Antitumor Activity against scid Mice Carrying Human Tumor Xenografts.

CB17/Icr female scid mice were implanted with a single tumor fragment s.c. Tumor-bearing mice were randomized into groups of 5–10 animals before therapy. All of the mice were maintained under barrier conditions. All of the experiments were conducted using protocols and conditions approved by the Institutional Animal Care and Use Committee. Mice bearing s.c. SKNEP anaplastic Wilms’ tumor or HCT-8 colon adenocarcinoma xenografts received the agent when tumors were approximately 0.2–1 cm in diameter. The procedures have been reported previously (31). Briefly, two perpendicular diameters were determined at 7-day intervals using digital Vernier calipers interfaced with a Macintosh computer. Tumor volumes were calculated assuming tumors to be spherical using the formula [(π/6) × d3], where d is the mean diameter, and mice were followed for up to 12 weeks after starting treatment. For the HCT-8 experiments, CPT-11 and ARC-111 were compared at the maximum tolerated dose levels (approximately LD5). CPT-11 was injected i.v. every 3 days for five administrations. ARC-111 was administered three times per week for 2 consecutive weeks. For the SKNEP experiments, ARC-111 was administered as described. CPT-11 and TPT were administered i.v. 5 days per week for 2 consecutive weeks. Cycles of treatment were repeated every 21 days for a total of three cycles of treatment {designated [(d×5)2]3}. TPT and CPT-11 were formulated as described previously (1). ARC-111, as the monocitrate salt, was dissolved in 5% dextrose solution USP before administration.

Cell Lines and Cytotoxicity Assay.

The RPMI-8402 human lymphoblastoma cell line and its CPT-resistant variant CPT-K5 (32) were obtained from Toshiwo Andoh (Soka University, Tokyo, Japan). The P388 mouse leukemia cell line and its CPT-resistant TOP1-deficient variant P388/CPT45 were obtained from Michael R. Mattern and Randal K. Johnson (GlaxoSmithKline, King of Prussia, PA; Ref. 33). The U937 cell line and its CPT-resistant variant U937/CR were obtained from Dr. Eric H. Rubin (The Cancer Institute of New Jersey, New Brunswick, NJ; Ref. 34). The KB3–1 cell line and its multidrug-resistant variant KBV-1 were obtained from K. V. Chin (The Cancer Institute of New Jersey; Ref. 35). The KBH5.0 cell line was derived from KB3–1 by stepwise selection against Hoechst 33342. The CEM cell line and its VP-16-resistant variants CEM/V-1 and CEM/V-5 were obtained from William Beck (University of Illinois, Chicago, IL; Ref. 36). The colon cancer cell line HCT116 was obtained from American Type Culture Collection (Rockville, MD). The breast cancer cell line ZR-75–1 was obtained from K. V. Chin (The Cancer Institute of New Jersey). All of the cells were cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10% heat-inactivated FCS, penicillin (100 units/ml), streptomycin (100 μg/ml), and glutamine (2 mm) in a 5% CO2 incubator at 37°C. Cytotoxicity was measured by using MTT assay after continuous treatment with the drug for 4 days.

In Vitro Assay for TOP1 Cleavable Complexes.

The reaction mixture (20 μl each) containing 40 mm Tris-HCl (pH 7.5), 100 mm KCl, 0.5 mm DTT, 0.5 mm EDTA, 30 μg/ml BSA, 20 ng 3′-32P-end-labeled YEpG DNA, 10 ng of hTOP1, and drug was incubated at 23 for 30 min. The reactions were terminated by the addition of 5 μl of 5% SDS and 1 mg/ml of proteinase K with an additional 1-h incubation at 37°C. DNA samples were alkali-denatured by the addition of NaOH (final 0.1 N), sucrose (final 5%), and bromphenol blue (final 0.05 mg/ml), and analyzed by electrophoresis in 1% agarose gel in 0.5× Tris-phosphate-EDTA (TPE) electrophoresis buffer ≤45 mm Tris-phosphate and 1 mm EDTA (pH 8.0)]. After electrophoresis, gels were dried onto Whatman 3MM chromatographic paper and autoradiographed at −80 using Kodak XAR-5 film.

Comet Assay for TOP1 Cleavable Complexes.

The alkaline SCGE assay, or comet assay, has been used extensively to monitor the integrity of chromosomal DNA and the amount of topoisomerase cleavable complexes (37). Briefly, drug-treated RPMI-8402 cells (∼5 × 105) were pelleted and resuspended in 1 ml of ice-cold PBS buffer. Fifty μl of resuspended cells were mixed with 0.5 ml of prewarmed 0.7% low melting point agarose. This mixture (0.1 ml) was loaded onto a fully frosted slide that had been precoated with 0.7% agarose. A coverslip was then applied to the slide. The slides, after being kept for 10 min at 4°C, were submerged in prechilled lysis solution [1% N-lauryl sarcosine, 1% Triton X-100, 2.5 M NaCl, and 10 mm EDTA (pH 10.5)] for 1 h at 4°C. After soaking with electrophoresis buffer (0.3 N NaOH and 1 mm EDTA), the slides were subjected to electrophoresis for 10 min at 2 V/cm in the cold room. After electrophoresis, slides were stained with Sybr Gold (Molecular Probes) and nuclei were visualized under a microscope. The images of nuclei were captured by a CCD camera.

Band Depletion Assay for TOP1 Cleavable Complexes.

RPMI-8402 cells (106/sample) were treated with drug for 15 min at 37°C. Cells were pelleted and lysed immediately (without washing) using the alkaline lysis procedure as described previously (20). Briefly, cell pellets were lysed with 0.2 N NaOH containing 1 mm EDTA. Alkaline lysates were then neutralized with one-tenth volume of 2 N HCl and one-tenth volume of a solution containing 10% NP40, 1 M Tris (pH 7.4), 0.1 M MgCl2, 0.1 M CaCl2, 10 mm DTT, and 1 mm EGTA, and 100 μg/ml each of leupeptin, pepstatin, and aprotinin. To the neutralized lysates, half volume of 3× SDS-PAGE sample buffer was added. SDS lysates were analyzed by SDS-PAGE. Immunoblotting analysis of cell lysates was carried out using TOP1 antiserum from Scleroderma 70 patients as described previously using the enhanced chemiluminescence Western procedure (20).

Assay for TOP1 Down-Regulation.

TOP1 down-regulation was monitored as described previously (20). Briefly, RPMI-8402 cells were treated with drug for 3 and 6 h at 37°C. Drug-treated cells were then incubated in drug-free fresh medium for another 30 min at 37°C before lysis using the alkaline lysis procedure as described above for band depletion assay. Neutralized cell lysates were incubated with Staphylococcus aureus nuclease S7 (60 units/reaction) for 20 min on ice to release any residual TOP1 from TOP1-DNA covalent complexes. SDS-PAGE and immunoblotting with anti-hTOP1 antibodies were performed as described above.

Assay for TOP1-SUMO Conjugates.

RPMI-8402 cells (106/sample) were treated with drug for 15 min at 37°C. Cells were pelleted and lysed immediately (without washing) using the alkaline lysis procedure. Neutralized lysates were then treated with S. aureus nuclease S7 and analyzed by immunoblotting using anti-hTOP1 antibodies as described above.

FACS Analysis for Caspase Activation.

Caspase activity was measured using the CaspACE FITC-VAD-FMK in situ marker (Promega). Briefly, 106 cells were treated with drug for indicated time periods. Drug-treated cells were treated with 25 μm FITC-VAD-FMK at 37°C for 30 min. Cells were then resuspended in 1× PBS buffer and analyzed for FITC fluorescence at excitation of 488 nm and emission of 525–550 nm by FACS.

ARC-111 Suppresses Tumor Growth in Mouse Xenograft Models.

ARC-111 was compared with CPT-11 in two tumor models, HCT-8 colon tumors and SKNEP (a model of the diffuse anaplastic Wilms’ tumor). In the HCT-8 model, CPT-11 at the highest dose level of 50 mg/kg (Fig. 1,B; administered every 3 days) caused slight inhibition of tumor growth (4.3 ± 0.5 weeks). This CPT-11 dose (50 mg/kg) was considered as the maximum tolerated dose (1 of 6 deaths) under our treatment conditions. CPT-11 administered at 33 mg/kg (Fig. 1,C) or 22 mg/kg (Fig. 1,D) did not significantly inhibit growth of HCT-8 tumors. ARC-111 (2 mg/kg; Fig. 1,F) inhibited growth of HCT-8 tumors (P = 0.024). However, the dose of ARC-111 at either 1 mg/kg (Fig. 1,G) or 0.5 mg/kg (Fig. 1 H) exhibited no significant antitumor activity. There was no statistical difference between the antitumor activity of CPT-11 (50 mg/kg) and ARC-111 (2 mg/kg; P = 0.55). Our results suggest that ARC-111 is more potent than CPT-11 and is as efficacious as CPT-11 in the HC T-8 model.

The SKNEP anaplastic Wilms’ tumor xenograft was chosen for comparison of ARC-111 with CPT-11 and TPT (Table 1). TPT was administered at the maximum tolerated dose (2 mg/kg) on the [(dx5)2]3 schedule (Fig. 2,C), whereas CPT-11 was administered at 1.25 mg/kg (Fig. 2,B), yielding systemic exposure to SN-38 equivalent to 20 mg/m2 in patients treated on this schedule (38). At these doses, TPT had a relatively minor effect on the growth of this tumor line (Fig. 2,C), whereas CPT-11 did induce some initial regressions (Fig. 2,B). However, tumors progressed during subsequent cycles of treatment (Fig. 2, B and C). At 2 mg/kg (Fig. 2,E), ARC-111 brought about complete regression without regrowth of advanced SKNEP tumors, and was significantly more active than either CPT-11 or TPT (P < 0.001). Like CPT-11 and TPT, ARC-111 at a lower dose (1 mg/kg; Fig. 2 F) induced some initial tumor regressions, but tumors progressed during subsequent courses of treatment.

ARC-111 Traps TOP1 Cleavable Complexes in Vitro.

We compared the ability of ARC-111 and CPT to inhibit TOP1 in vitro using purified recombinant hTOP1. As shown in Fig. 3 B, CPT and ARC-111 have similar TOP1-targeting activity, as revealed by neutral agarose gel electrophoresis of alkali-denatured 32P-labeled linearized plasmid DNA. The cleavage pattern induced by ARC-111 was not identical to that by CPT (see bands marked by ∗), which indicates that the ARC-111 consensus sequence is not identical to that of CPT.

TOP1-mediated DNA breaks induced by ARC-111 appear to be reversible, because a brief heat treatment (65°C for 7 min; marked HR) in a second incubation completely reversed DNA cleavage (Fig. 3 B, right), suggesting that ARC-111, like CPT, traps reversible TOP1 cleavable complexes.

We have also examined the inhibitory effect of ARC-111 on hTOP2α. ARC-111 exhibited minimal activity in stimulating TOP2α-mediated DNA cleavage (at least 1000-fold less potent than the prototypical TOP2-targeting agent VM-26; data not shown), suggesting that ARC-111 is a TOP1- but not TOP2-specific targeting agent.

ARC-111 Induces TOP1 Cleavable Complexes in Tumor Cells.

A number of assays were used to test whether ARC-111, like CPT, induces reversible TOP1 cleavable complexes in tumor cells.

Comet Assay.

Alkaline SCGE, or comet assay, was used to monitor TOP1-cleavable complexes in human lymphoblastoma RPMI-8402 cells. As shown in Fig. 4, A and C, within 1 h of treatment, ARC-111 (1 μm) and CPT (1 μm) both induced extensive strand breaks on chromosomal DNA in RPMI-8402 cells (∼90% of cells showed the comet image, which corresponds to damaged chromosomal DNA; P < 0.001). The majority of these strand breaks (>60%) were shown to be reversible on a second incubation at 55°C for 10 min (Fig. 4 B). These results suggest that ARC-111 induces topoisomerase-cleavable complexes in cells. However, the comet assay cannot distinguish between TOP1- and TOP2-cleavable complexes.

Band Depletion Assay.

The band depletion assay has been used to demonstrate the formation of TOP1 cleavable complexes (20). In this assay, free TOP1 is detected as an immunoreactive band at Mr 100,000. TOP1-cleavable complexes, which are trapped as TOP1-DNA covalent complexes by alkaline lysis, are detected as a smear migrating slower than Mr 100,000 TOP1 band. As shown in Fig. 5 A, both ARC-111 (100 and 300 nm) and CPT (100 and 300 nm) reduced the band intensity of the Mr 100,000 TOP1 band with about equal efficiency. This reduction of TOP1 band intensity was abolished when drug-treated RPMI-8402 cells were subjected to a second incubation at 65°C for 7 min (labeled HR), suggesting that ARC-111, like CPT, induces reversible TOP1-cleavable complexes.

TOP1 Down-Regulation.

CPT has been shown to induce transcription-dependent degradation of TOP1 (TOP1 down-regulation) through a ubiquitin/26S proteasome pathway (17). TOP1 down- regulation is indicative of TOP1 cleavable complex formation in cells (20). As shown in Fig. 5 B, ARC-111, like CPT, induced TOP1 down-regulation in RPMI-8402 cells as evidenced by the reduction of the Mr 100,000 TOP1 immunoreactive band. In this assay, the reduction of the Mr 100,000 TOP1 immunoreactive band was the result of TOP1 degradation rather than TOP1-DNA covalent complex formation, because TOP1-DNA covalent complexes were dissociated (reversed) by a second incubation in a drug-free medium. In addition, lysates were treated with S. aureus nuclease S7 to ensure the release of any residual TOP1-DNA covalent complexes.

Formation of TOP1-SUMO Conjugates.

CPT is also known to induce the formation of TOP1-SUMO conjugates within minutes of treatment. SUMO conjugation occurs primarily on covalent TOP1-DNA complexes and reflects the formation of TOP1-cleavable complexes in cells. However, it has been shown that not all of the TOP1-targeting agents induce TOP1-SUMO conjugates (39). The different cleavage pattern induced by ARC-111 compared with CPT prompted us to examine whether ARC-111 also induces the formation of TOP1-SUMO conjugates. As shown in Fig. 5 C, ARC-111, like CPT, induced a ladder of bands migrating slower than Mr 100,000 TOP1 within 15 min of drug treatment in RPMI-8402 cells. These bands have been identified previously as TOP1-SUMO conjugates (21).

ARC-111 Induces TOP1-Mediated Cytotoxicity and Apoptosis.

As shown in Table 2, ACR-111 rivals CPT in its cytotoxicity against many tumor cells. The cytotoxic target of ARC-111 was investigated using the mouse leukemia P388 cell line and its CPT-resistant TOP1-deficient variant P388/CPT45 cell line. The absence of Mr 100,000 TOP1 in P388/CPT45 was confirmed by immunoblotting with anti-hTOP1 antibodies, which cross-reacted with mouse TOP1 in P388 cells (Fig. 6,A). The effect of ARC-111 on apoptosis in P388 and P388/CPT45 cells was investigated by monitoring caspase activation using the FITC-conjugated caspase inhibitor FITC-VAD-FMK followed by FACS analysis. As shown in Fig. 6,B, left, ARC-111 induced dose-dependent increase of the P388 cell population with activated caspase (see the shifted peak with increasing ARC-111 concentrations). At the highest concentration of ARC-111 (1 μm), ∼65 + 1.7% of P388 cells exhibited activated caspase (P = 0.01). As positive controls, both CPT (2 μm) and VP-16 (a TOP2-targeting agent; 20 μm) were shown to induce caspase activation in the majority of the P388 cell population (71.1 + 4.8 and 81.1 + 5.6%, respectively; P < 0.05; Fig. 6,B, left). ARC-111- and CPT-induced caspase activation was almost entirely absent in P388/CPT45 cells (7.0 + 2.4% and 4.8 + 0.6%, respectively; P > 0.05), whereas VP-16-induced caspase activation was only slightly affected (52.4 + 3.6%; P < 0.05) in P388/CPT45 cells (Fig. 6 B, right). These results demonstrate that ARC-111, like CPT, induces TOP1-mediated caspase activation, suggesting that ARC-111, like CPT, induces TOP1-mediated apoptosis.

We have also measured ARC-111 cytotoxicity using the MTT assay. As shown in Fig. 7,B, ARC-111 had an IC50 of 1 nm against P388 cells and an IC50 of 300 nm against TOP1-deficient P388/CPT45 cells (a 300-fold difference in cytotoxicity). As a positive control, CPT was shown to have an IC50 of 5 nm against P388 and >10 μm against P388/CPT45 (Fig. 7,A). As a negative control, VP-16 was shown to have about the same IC50 (11 nm) against either P388 or P388/CPT45 cells (Fig. 7 C). These results suggest that TOP1 is the cytotoxic target of ARC-111.

Additional support for this conclusion came from studies of two other CPT-resistant cell lines, CPT-K5 and U937/CR, both of which are known to express CPT-resistant mutant TOP1 (32, 34). As shown in Table 2, both CPT-K5 and U937/CR cells were found to be cross-resistant to ARC-111 when compared with their respective wild-type cells, RPMI-8402 and U937 cells (P < 0.05). By contrast, these CPT-resistant cells showed no cross-resistance to VP-16. As negative controls, ARC-111 cytotoxicity was also measured in CEM, CEM/V1, and CEM/V5 cells. CEM/V1 and CEM/V5 cells were selected for resistance to VP-16 and had been shown to express mutant TOP2 (36). Neither CEM/V1 nor CEM/V5 exhibited any cross-resistance to CPT or ARC-111. These results are consistent with the notion that both CPT and ARC-111 act by targeting TOP1.

ARC-111 Cytotoxicity Is Not Significantly Affected by HSA.

CPT carboxylate has been shown to bind HSA but not mouse serum albumin (26, 27, 28). As shown in Fig. 8,A, the presence of 1% HSA reduced CPT cytotoxicity >100-fold (4 nmversus 500 nm IC50) in RPMI-8402 cells as measured by MTT assay. By contrast, ARC-111 cytotoxicity was minimally affected by 1% HSA (∼2-fold reduction) in RPMI-8402 cells (Fig. 8 B). Similar results were obtained in RPMI-8402 cells with 3% HSA (data not shown).

ARC-111 Cytotoxicity Is Not Affected by the Activity of BCRP and MDR1.

CPT is a substrate for the ABC transporter protein BCRP (24, 25). The effect of BCRP on ARC-111 cytotoxicity was tested using KBH5.0 cells. KBH5.0 cells were derived from KB3–1 cells by stepwise selection for resistance to Hoechst 33342. As shown in Fig. 5,C, KBH5.0 cells greatly overexpress BCRP as evidenced by Western blotting analysis. KBH5.0 cells were ∼14-fold more resistant to TPT (P < 0.001; Table 3). However, KBH5.0 cells were as sensitive as KB3–1 cells to ARC-111, suggesting that ARC-111, unlike TPT, is not a substrate for BCRP.

We have also tested the effect of the ABC transporter protein MDR1 on ARC-111 cytotoxicity. KBV-1 cells were derived from KB3–1 cells by selection for resistance to colchicines and have been shown to overexpress MDR1 (40). As shown in Table 3, KBV-1 cells were 12-fold more resistant to TPT as compared with KB3–1 cells (P < 0.001). By contrast, KBV-1 cells were as sensitive to ARC-111 as KB3–1 cells, suggesting that ARC-111 is not a substrate for MDR1.

We have shown that the synthetic compound ARC-111 (topotvale) is more potent than CPT-11 and is as efficacious as CPT-11 in the HCT colon tumor xenograft model. We have also shown that the antitumor activity of ARC-111 compares favorably to that of TPT and CPT-11 in scid mice carrying SKNEP anaplastic Wilms’ tumors. These results are consistent with the previous result obtained in nude mice carrying human breast cancer MDA-MB-435 xenografts (29, 30).

To identify the molecular target of ARC-111, we have carried out a number of biochemical and cell-based studies. Using purified hTOP1, we have shown that ARC-111, like CPT, induces TOP1-mediated DNA breaks. These breaks were shown to be reversible on a brief heat treatment (65°C for 7 min), suggesting that ARC-111 traps reversible TOP1 cleavable complexes. We have noticed that the cleavage pattern induced by ARC-111 is not identical to that by CPT, suggesting that the cleavage specificity of these two drugs is not the same. The molecular basis for the difference in cleavage specificity is currently under investigation.

The ability of ARC-111 to trap reversible TOP1-cleavable complexes in tumor cell has been evaluated by two different methods, the alkaline SCGE and the band depletion method. Alkaline SCGE was used to monitor strand breaks on chromosomal DNA in ARC-111-treated RPMI-8402 cells. Both ARC-111 and CPT were shown to induce DNA strand breaks. These strand breaks were shown to be heat reversible, suggesting that ARC-111, like CPT, traps reversible topoisomerase cleavable complexes in tumor cells. However, this technique does not distinguish between TOP1- and TOP2-cleavable complexes. The most convincing evidence that ARC-111 traps TOP1 cleavable complexes came from the band depletion assay. The reduction of the immunoreactive TOP1 band is indicative of either degradation of TOP1 or formation of TOP1-DNA covalent complexes. Because the immunoreactive TOP1 band was completely recovered on a brief heat treatment, it is likely that the reduction of the immunoreactive TOP1 band was due to the formation of TOP1-cleavable complexes rather than TOP1 degradation.

CPT-induced TOP1 cleavable complexes have been shown to undergo two different covalent modifications to form ubiquitin-TOP1 and SUMO-TOP1 conjugates (18, 21). The difference in the cleavage pattern between ARC-111 and CPT could suggest a difference in the nature of their respective cleavable complexes. In fact, it has been reported that not all of the TOP1-cleavable complexes can be modified by SUMO (39). Consequently, we have investigated whether TOP1-cleavable complexes induced by ARC-111 can be similarly modified by ubiquitin and SUMO. The formation of ubiquitin-TOP1 conjugates is difficult to demonstrate directly. However, ubiquitin-TOP1 conjugates can be readily degraded by 26S proteasome. Consequently, degradation of TOP1 could be used as an indicator for the formation of ubiquitin-TOP1 conjugates. Indeed, both ARC-111 and CPT induced TOP1 degradation in RPMI-8402 cells, suggesting the formation of ubiquitin-TOP1 conjugates in these drug-treated cells. This result is somewhat surprising, because TOP1 degradation through the ubiquitin/26S proteasome pathway is normally defective in most solid tumor cells (20).

We have shown in this study that ARC-111, like CPT, can rapidly (within 15 min) induce the formation of TOP1-SUMO conjugates. These results provide additional support for the notion that ARC-111 induces TOP1-DNA cleavable complexes in tumor cells. In addition, these results suggest that the immediate cellular responses to TOP1-cleavable complexes induced by ARC-111 and CPT appear quite similar.

In this study, we have provided evidence that the major cytotoxic target of ARC-111 is TOP1 using several assays. Using the MTT assay, we have shown that TOP1-deficient P388/CPT45 cells were >100-fold more resistant to ARC-111 than P388 cells. It might be arguable that resistance of P388/CPT45 cells to ARC-111 is due to mutations in either the DNA repair or cell death pathways in addition to TOP1 deficiency. However, this possibility seems unlikely because P388/CPT45 cells are not significantly more resistant to VP-16 than P388 cells, suggesting that there is no significant defect in the DNA repair or cell death pathways in P388/CPT45 cells. The simplest explanation is that TOP1 is the major cytotoxic target of both ARC-111 and CPT, and the absence of TOP1 in P388/CPT45 cells confers resistance to both of these TOP1-directed drugs. This conclusion is also supported by results from our studies using the apoptosis assay, because P388/CPT45 cells were more resistant to ARC-111-induced apoptosis than P388 cells. Studies using other CPT-resistant cell lines (i.e., CPT-K5 and U937/CR) have provided similar results and supported this conclusion.

CPTs are known to be substrates for BCRP (24, 25). The two clinically prescribed CPTs, TPT and irinotecan, are also weak substrates for MDR1 (24, 25). In this study, we have shown that ARC-111 is a substrate for neither BCRP nor MDR1 based on cytotoxicity measurement in cells overexpressing BCRP or MDR1. This could have important implications in the future development of this drug.

Another interesting finding is that ARC-111 cytotoxicity is not affected by 1% HSA, whereas CPT cytotoxicity is >100-fold reduced. Previous studies have shown that the inactive CPT carboxylate binds human but not mouse serum albumin (26, 28). Binding of CPT carboxylate to HSA drives lactone hydrolysis, reducing effective serum concentration of active CPT (26). It has been suggested that the impressive antitumor activity of CPT in mice, which cannot be completely duplicated in humans, may be partly due to this effect (41). The lack of a substantial HSA effect on ARC-111 cytotoxicity may indicate a lack of binding of ARC-111 to HSA.

ARC-111 belongs to a new class of compounds, which can be readily synthesized (29, 30). Many ARC-111 analogues have been synthesized and shown to be potent TOP1-targeting agents (30).5 The ease of synthesis of ARC-111 coupled with its chemical stability (29, 30) is likely to facilitate its development in the future. The different drug resistance profile and the lack of HSA binding make ARC-111 a promising clinical candidate. Additional studies are under way to characterize its pharmacological and toxicological properties.

Grant support: NIH grants CA39662 (L. F. L.), CA077433 (L. F. L.), and CA23099 (P. J. H.).

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.

Notes: Dr. Li is currently in the Department of Microbiology, School of Medicine, National Taiwan University, Taipei, Republic of China.

Requests for reprints: Leroy F. Liu, Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-4592; E-mail: lliu@umdnj.edu

4

The abbreviations used are: CPT, camptothecin; TOP1, topoisomerase I; SUMO, small ubiquitin modifier; ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; HSA, human serum albumin; ARC-111, 5H-dibenzo[c,h][1,6]-naphthyridin-6-one; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SCGE, single cell gel electrophoresis; FACS, fluorescent activated cell sorting; hTOP1, human topoisomerase I; TPT, topotecan.

5

Unpublished observations.

Fig. 1.

ARC-111 induces regression of HCT-8 human colon carcinoma xenografts. CB17/Icr female scid mice bearing s.c. HCT-8 xenografts were treated i.v. with CPT-11 every 3 days for five administrations, or with ARC-111 three times weekly for 2 consecutive weeks. Cycles of treatment were repeated every 21 days. A, E: vehicle controls; B: CPT-11, 50 mg/kg; C: CPT-11, 33 mg/kg; D: CPT-11, 22 mg/kg; F: ARC-111, 2 mg/kg; G: ARC-111, 1 mg/kg; H: ARC-111. 0.5 mg/kg. Each curve represents the growth of an individual tumor.

Fig. 1.

ARC-111 induces regression of HCT-8 human colon carcinoma xenografts. CB17/Icr female scid mice bearing s.c. HCT-8 xenografts were treated i.v. with CPT-11 every 3 days for five administrations, or with ARC-111 three times weekly for 2 consecutive weeks. Cycles of treatment were repeated every 21 days. A, E: vehicle controls; B: CPT-11, 50 mg/kg; C: CPT-11, 33 mg/kg; D: CPT-11, 22 mg/kg; F: ARC-111, 2 mg/kg; G: ARC-111, 1 mg/kg; H: ARC-111. 0.5 mg/kg. Each curve represents the growth of an individual tumor.

Close modal
Fig. 2.

ARC-111 induces tumor regression in scid mice bearing human tumor xenografts. CB17/Icr female scid mice bearing s.c. SKNEP anaplastic Wilms tumor xenografts were treated i.v. with ARC-111 and other drugs using the [(dx5)2]3 schedule of administration. A and D, vehicle controls; B, CPT-11, 1.25 mg/kg; C, TPT, 2 mg/kg; E, ARC-111, 2 mg/kg; F, ARC-111, 1 mg/kg. Each curve represents the growth of an individual tumor.

Fig. 2.

ARC-111 induces tumor regression in scid mice bearing human tumor xenografts. CB17/Icr female scid mice bearing s.c. SKNEP anaplastic Wilms tumor xenografts were treated i.v. with ARC-111 and other drugs using the [(dx5)2]3 schedule of administration. A and D, vehicle controls; B, CPT-11, 1.25 mg/kg; C, TPT, 2 mg/kg; E, ARC-111, 2 mg/kg; F, ARC-111, 1 mg/kg. Each curve represents the growth of an individual tumor.

Close modal
Fig. 3.

ARC-111 induces reversible hTOP1 cleavable complexes in vitro. A, the chemical structure of ARC-111 and CPTs. B, ARC-111 induces hTOP1-mediated DNA cleavage. Assay for TOP1-cleavable complexes using purified hTOP1 was performed as described in “Materials and Methods.” After incubation at 23°C for 30 min, reactions were terminated with SDS/proteinase K. Three concentrations of CPT and ARC-111, 0.01, 0.1, and 1.0 μm, were used in this experiment. The triangle above the lanes indicates increasing concentrations of drugs with the wider end representing the highest drug concentration (1 μm). The last two lanes show the results of the heat reversal experiment for ARC-111 (1 μm). In this reversal experiment, the incubated reactions (23οC for 30 min) were immediately shifted to 65°C for 7 min before termination with SDS/proteinase K. HR indicates the heat reversal treatment.

Fig. 3.

ARC-111 induces reversible hTOP1 cleavable complexes in vitro. A, the chemical structure of ARC-111 and CPTs. B, ARC-111 induces hTOP1-mediated DNA cleavage. Assay for TOP1-cleavable complexes using purified hTOP1 was performed as described in “Materials and Methods.” After incubation at 23°C for 30 min, reactions were terminated with SDS/proteinase K. Three concentrations of CPT and ARC-111, 0.01, 0.1, and 1.0 μm, were used in this experiment. The triangle above the lanes indicates increasing concentrations of drugs with the wider end representing the highest drug concentration (1 μm). The last two lanes show the results of the heat reversal experiment for ARC-111 (1 μm). In this reversal experiment, the incubated reactions (23οC for 30 min) were immediately shifted to 65°C for 7 min before termination with SDS/proteinase K. HR indicates the heat reversal treatment.

Close modal
Fig. 4.

ARC-111 induces reversible chromosomal DNA strand breaks as revealed by comet assay. RPMI-8402 cells treated with CPT (1 μm) or ARC-111 (1 μm) for 1 h. After treatments, comet assay was performed to analyze the chromosomal DNA integrity in RPMI-8402 cells. A, ARC-111 induces strand breaks on chromosomal DNA. Representative comet images are shown (see C for quantitation). B, ARC-111-induced strand breaks on chromosomal DNA are reversible. Reversal of DNA strand breaks was performed by shifting the treated cells to 55°C for 10 min before termination of the reaction. Representative images after heat reversal are shown (see C for quantitation). C, quantitation of the results shown in A and B. Bars, ±SD of n = 4 samples. Statistical relationships between groups were determined by Student’s t test. ∗, statistically significant comparisons of drug-treated cells to control (P < 0.001). ∗∗, statistically significant comparisons of heat reversal-treated cells to nontreated cells (P < 0.001).

Fig. 4.

ARC-111 induces reversible chromosomal DNA strand breaks as revealed by comet assay. RPMI-8402 cells treated with CPT (1 μm) or ARC-111 (1 μm) for 1 h. After treatments, comet assay was performed to analyze the chromosomal DNA integrity in RPMI-8402 cells. A, ARC-111 induces strand breaks on chromosomal DNA. Representative comet images are shown (see C for quantitation). B, ARC-111-induced strand breaks on chromosomal DNA are reversible. Reversal of DNA strand breaks was performed by shifting the treated cells to 55°C for 10 min before termination of the reaction. Representative images after heat reversal are shown (see C for quantitation). C, quantitation of the results shown in A and B. Bars, ±SD of n = 4 samples. Statistical relationships between groups were determined by Student’s t test. ∗, statistically significant comparisons of drug-treated cells to control (P < 0.001). ∗∗, statistically significant comparisons of heat reversal-treated cells to nontreated cells (P < 0.001).

Close modal
Fig. 5.

ARC-111 induces TOP1-cleavable complexes in tumor cells. A, ARC-111 induces reversible TOP1-cleavable complexes as revealed by a band depletion assay. RPMI-8402 cells were treated with either CPT (100 and 300 nm) or ARC-111 (100 and 300 nm) for 15 min. Cell lysates were immunoblotted with anti-hTOP1 antibodies. The reduction of TOP1 immunoreactive band (band depletion) is indicative of the formation of TOP1-DNA covalent complexes. A brief heating (65°C for 7 min; marked +HR on top of the two rightmost lanes) of drug-treated cells was performed before alkaline lysis to demonstrate the (heat) reversibility of TOP1-DNA covalent complexes. B, ARC-111 induces TOP1 down-regulation. RPMI-8402 cells were treated with CPT (10 μm) or ARC-111 (10 μm) for 3 and 6 h. TOP1 down-regulation was monitored in ARC-111-treated cells by immunoblotting with anti-hTOP1 antibodies as described in the “Materials and Methods.” C, ARC-111 induces rapid sumoylation of TOP1. RPMI-8402 cells were treated with 0, 1, 5, and 10 μm ARC-111 or CPT for 15 min. TOP1-SUMO conjugates were assayed as described in “Materials and Methods.”

Fig. 5.

ARC-111 induces TOP1-cleavable complexes in tumor cells. A, ARC-111 induces reversible TOP1-cleavable complexes as revealed by a band depletion assay. RPMI-8402 cells were treated with either CPT (100 and 300 nm) or ARC-111 (100 and 300 nm) for 15 min. Cell lysates were immunoblotted with anti-hTOP1 antibodies. The reduction of TOP1 immunoreactive band (band depletion) is indicative of the formation of TOP1-DNA covalent complexes. A brief heating (65°C for 7 min; marked +HR on top of the two rightmost lanes) of drug-treated cells was performed before alkaline lysis to demonstrate the (heat) reversibility of TOP1-DNA covalent complexes. B, ARC-111 induces TOP1 down-regulation. RPMI-8402 cells were treated with CPT (10 μm) or ARC-111 (10 μm) for 3 and 6 h. TOP1 down-regulation was monitored in ARC-111-treated cells by immunoblotting with anti-hTOP1 antibodies as described in the “Materials and Methods.” C, ARC-111 induces rapid sumoylation of TOP1. RPMI-8402 cells were treated with 0, 1, 5, and 10 μm ARC-111 or CPT for 15 min. TOP1-SUMO conjugates were assayed as described in “Materials and Methods.”

Close modal
Fig. 6.

ARC-111 induces TOP1-dependent apoptosis. A, the TOP1 level is greatly reduced in the CPT-resistant P338/CPT45 cell line as compared with its parental P388 cell line. Equal numbers of P388 and P388/CPT45 cells (106 each) were analyzed for TOP1 level by immunoblotting with anti-hTOP1 antibodies. B, ARC-111-induced apoptosis is reduced in TOP1-deficient P388/CPT45 cells. P388 and p388/CPT45 cells were treated with drug for 14 h. Apoptosis was then monitored by FACS analysis of activated caspase using the FITC-conjugated caspase inhibitor FITC-VAD-FMK. The concentrations used for various drugs were: ARC-111 (0.01, 0.1, and 1 μm), CPT (2 μm), and VP-16 (20 μm). The triangle to the right of ARC-111 lanesindicates increasing concentrations with the wider end being the highest concentration (1 μm). C, Hoechst 33342-resistant KBH5.0 cells overexpress BCRP. KBH5.0 cells were derived from KB3–1 cells by stepwise selection for resistance to Hoechst 33342. Lysates of KBH5.0 cells were immunoblotted with anti-BCRP antibodies.

Fig. 6.

ARC-111 induces TOP1-dependent apoptosis. A, the TOP1 level is greatly reduced in the CPT-resistant P338/CPT45 cell line as compared with its parental P388 cell line. Equal numbers of P388 and P388/CPT45 cells (106 each) were analyzed for TOP1 level by immunoblotting with anti-hTOP1 antibodies. B, ARC-111-induced apoptosis is reduced in TOP1-deficient P388/CPT45 cells. P388 and p388/CPT45 cells were treated with drug for 14 h. Apoptosis was then monitored by FACS analysis of activated caspase using the FITC-conjugated caspase inhibitor FITC-VAD-FMK. The concentrations used for various drugs were: ARC-111 (0.01, 0.1, and 1 μm), CPT (2 μm), and VP-16 (20 μm). The triangle to the right of ARC-111 lanesindicates increasing concentrations with the wider end being the highest concentration (1 μm). C, Hoechst 33342-resistant KBH5.0 cells overexpress BCRP. KBH5.0 cells were derived from KB3–1 cells by stepwise selection for resistance to Hoechst 33342. Lysates of KBH5.0 cells were immunoblotted with anti-BCRP antibodies.

Close modal
Fig. 7.

ARC-111 selectively kills P388 but not TOP1-deficient P388/CPT45 cells. MTT assay was used to examine the cytotoxic effect of CPT (A), ARC-111 (B), and VP-16 (C). Each dot represents the mean of three independent experiments, and the range is 95% confident interval.

Fig. 7.

ARC-111 selectively kills P388 but not TOP1-deficient P388/CPT45 cells. MTT assay was used to examine the cytotoxic effect of CPT (A), ARC-111 (B), and VP-16 (C). Each dot represents the mean of three independent experiments, and the range is 95% confident interval.

Close modal
Fig. 8.

CPT but not ARC-111 cytotoxicity is reduced in the presence of HSA. The cytotoxicities of CPT and ARC-111 in RPMI-8402 cells were determined by a 4-day MTT assay in the presence or absence of 1% HSA. Each dot represents the mean of three independent experiments, and the range is 95% confident interval.

Fig. 8.

CPT but not ARC-111 cytotoxicity is reduced in the presence of HSA. The cytotoxicities of CPT and ARC-111 in RPMI-8402 cells were determined by a 4-day MTT assay in the presence or absence of 1% HSA. Each dot represents the mean of three independent experiments, and the range is 95% confident interval.

Close modal
Table 1

The antitumor activity of ARC-111, TPT, and CPT-11 against SKNEP anaplastic Wilms’ tumor xenografts

TreatmentAverage week tumor achieved 4× initial volume ± SDPaNumber PRsbNumber CRsNumber MCRs
Control 2.0 ± 0.0  
CPT-11 (1.25 mg/kg) 4.7 ± 1.6 0.005 
Topotecan (2 mg/kg) 6.0 ± 0.0 0.01 
Control 2.1 ± 1.1  
ARC-111 (2 mg/kg) >12 0.000 10 10 
ARC-111 (1 mg/kg) 4.8 ± 2.0 0.017 
TreatmentAverage week tumor achieved 4× initial volume ± SDPaNumber PRsbNumber CRsNumber MCRs
Control 2.0 ± 0.0  
CPT-11 (1.25 mg/kg) 4.7 ± 1.6 0.005 
Topotecan (2 mg/kg) 6.0 ± 0.0 0.01 
Control 2.1 ± 1.1  
ARC-111 (2 mg/kg) >12 0.000 10 10 
ARC-111 (1 mg/kg) 4.8 ± 2.0 0.017 
a

Level of significance relative to controls.

b

PR, partial response (≥50% volume regression); CR, complete response; MCR, maintained CR at week 12.

Table 2

ARC-111 cytotoxicity against various drug-resistant cells

Cell LinesCPTEtoposideARC-111
RPMI-8402 0.004 ± 0.002 (n = 10)a 0.88 ± 0.19 (n = 7) 0.003 ± 0.001 (n = 6) 
CPT-K5 60.0 ± 8.2 (n = 4) 1.5 ± 0.6 (n = 4) 1.43 ± 0.72 (n = 4) 
U937 0.005 ± 0.002 (n = 6) 0.13 ± 0.12 (n = 7) 0.004 ± 0.0009 (n = 4) 
U937/CR 0.47 ± 0.21 (n = 6) 0.056 ± 0.04 (n = 6) 0.13 ± 0.04 (n = 3) 
CEM 0.003 ± 0.002 (n = 8) 0.22 ± 0.13 (n = 8) 0.002 ± 0.0003 (n = 3) 
CEM/V-1 0.005 ± 0.003 (n = 6) 9.7 ± 3.5 (n = 4) 0.002 ± 0.7× 10 (n = 2) 
CEM/V-5 0.005 ± 0.002 (n = 6) 32 ± 2.1 (n = 3) 0.003 ± 6.0× 10 (n = 2) 
HeLa 0.004 ± 0.001 (n = 5) 0.42 ± 0.11 (n = 2) 0.004 ± 0.0006 (n = 4) 
ZR-75-1 0.003 ± 0.002 (n = 5) 0.57 ± 0.19 (n = 2) 0.005 ± 0.003 (n = 4) 
HCT116 0.005 ± 0.003 (n = 4) 1.8 ± 1.2 (n = 4) 0.006 ± 0.004 (n = 5) 
Cell LinesCPTEtoposideARC-111
RPMI-8402 0.004 ± 0.002 (n = 10)a 0.88 ± 0.19 (n = 7) 0.003 ± 0.001 (n = 6) 
CPT-K5 60.0 ± 8.2 (n = 4) 1.5 ± 0.6 (n = 4) 1.43 ± 0.72 (n = 4) 
U937 0.005 ± 0.002 (n = 6) 0.13 ± 0.12 (n = 7) 0.004 ± 0.0009 (n = 4) 
U937/CR 0.47 ± 0.21 (n = 6) 0.056 ± 0.04 (n = 6) 0.13 ± 0.04 (n = 3) 
CEM 0.003 ± 0.002 (n = 8) 0.22 ± 0.13 (n = 8) 0.002 ± 0.0003 (n = 3) 
CEM/V-1 0.005 ± 0.003 (n = 6) 9.7 ± 3.5 (n = 4) 0.002 ± 0.7× 10 (n = 2) 
CEM/V-5 0.005 ± 0.002 (n = 6) 32 ± 2.1 (n = 3) 0.003 ± 6.0× 10 (n = 2) 
HeLa 0.004 ± 0.001 (n = 5) 0.42 ± 0.11 (n = 2) 0.004 ± 0.0006 (n = 4) 
ZR-75-1 0.003 ± 0.002 (n = 5) 0.57 ± 0.19 (n = 2) 0.005 ± 0.003 (n = 4) 
HCT116 0.005 ± 0.003 (n = 4) 1.8 ± 1.2 (n = 4) 0.006 ± 0.004 (n = 5) 
a

IC50m) was measured using MTT assay.

Table 3

ARC-111 cytotoxicity against tumor cells overexpressing ABC transporters

Cell linesTopotecanARC-111EtoposideTaxol
KB3-1 0.038 ± 0.021 (n = 9)a 0.005 ± 0.001 (n = 9) 0.30 ± 0.06 (n = 7) 0.0013 ± 0 (n = 2) 
KBV-1 0.47 ± 0.12 (n = 6) 0.005 ± 0.001 (n = 7) 7.8 ± 5.0 (n = 5) >1 (n = 1) 
KBH5.0 0.55 ± 0.31 (n = 9) 0.005 ± 0.0009 (n = 6) 0.35 ± 0.18 (n = 7) 0.0003 ± 0 (n = 1) 
Cell linesTopotecanARC-111EtoposideTaxol
KB3-1 0.038 ± 0.021 (n = 9)a 0.005 ± 0.001 (n = 9) 0.30 ± 0.06 (n = 7) 0.0013 ± 0 (n = 2) 
KBV-1 0.47 ± 0.12 (n = 6) 0.005 ± 0.001 (n = 7) 7.8 ± 5.0 (n = 5) >1 (n = 1) 
KBH5.0 0.55 ± 0.31 (n = 9) 0.005 ± 0.0009 (n = 6) 0.35 ± 0.18 (n = 7) 0.0003 ± 0 (n = 1) 
a

IC50m) was determined using MTT assay. KBV-1 cells are known to overexpress MDR1 while KBH5.0 cells are shown to overexpress BCRP (this study).

We thank Drs. Rajeev Rajendra (The Cancer Institute of New Jersey) for technical help and William Beck, Toshiwo Andoh, Khew-Voon Chin, and Eric H. Rubin for providing us with various cell lines. Statistical analysis of xenograft data were performed by Catherine Billups, Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, TN.

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