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ORIGINAL ARTICLE

Cellular Topoisomerase I Inhibition and Antiproliferative Activity by MJ-III-65 (NSC 706744), an Indenoisoquinoline Topoisomerase I Poison

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (S.A., G.K., K.A., Y.P.); Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (M.J., M.C.); and Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York (S.C., F.D., Y.M.R.)

Received June 14, 2004; accepted November 5, 2004

Abstract

To overcome camptothecin's (CPT) lactone instability, reversibility of the drug-target interaction, and drug resistance, attempts to synthesize compounds that are CPT-like in their specificity and potency yet display a unique profile have been underway. In this pursuit, we have identified one of the idenoisoquinoline derivatives, MJ-III-65 (NSC 706744; 6-[3-(2-hydroxyethyl)amino-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline) with both similarities and differences from CPT. MJ-III-65 traps topoisomerase I (Top1) reversibly like CPT but with different DNA sequence preferences. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with MJ-III-65 at nanomolar concentrations. These MJ-III-65-induced protein-linked DNA breaks were resistant to reversal after an hour of drug removal, compared with CPT, which completely reversed. Studies in human cells in culture found MJ-III-65 to be cytotoxic. Furthermore, limited cross-resistance was observed in camptothecin-resistant cell lines. MJ-III-65 also exhibits antitumor activity in mouse tumor xenografts.

To discover, design, and develop novel Top1 inhibitors, a COMPARE analysis was carried out using CPT as a seed. As a result, we identified NSC 314622 (Fig. 1) as having a similar tumor cell growth inhibitory profile as CPT in the National Cancer Institute cytotoxicity screen. Further analysis showed NSC 314622 to be a Top1 inhibitor (Kohlhagen et al., 1998). Although not as potent as CPT, its chemical stability, unique Top1 cleavage sequence preference, and slower cleavage complex reversibility made NSC 314622 a good lead compound. By chemically modifying NSC 314622, we synthesized indenoisoquinoline derivatives to increase Top1 inhibition and cancer cell cytotoxicity (Strumberg et al., 1999; Cushman et al., 2000; Fox et al., 2003). As reported previously (Antony et al., 2003), one of the derivatives MJ-III-65 (NSC 706744), with an amino alcohol instead of a methyl at the N-6 position of the parent compound (Fig. 1), is a potent inhibitor of Top1. MJ-III-65 preferentially traps Top1 at sites with cytosine (C) immediately 5' from the Top1 cleavage site compared with a thymine (T) for CPT. Moreover, MJ-III-65 remains active against Top1 enzymes that are resistant to CPTs, homocamptothecins, and indolocarbazoles (Antony et al., 2003).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Structures of two indenoisoquinolines. NSC 314622 was discovered first. MJ-III-65 is the derivative studied in this report.

Having demonstrated MJ-III-65 to be a Top1 inhibitor comparable with CPT in its potency in vitro (Cushman et al., 2000; Antony et al., 2003), this study was carried out to ascertain MJ-III-65's in vivo potential. Here, we show that in human leukemic cells MJ-III-65 causes DNA-protein crosslinks and Top1 cleavage complexes that are markedly more resistant to reversal after drug removal than those produced by CPT. In addition, CPT-resistant cells remain sensitive to MJ-III-65 and MJ-III-65 exhibits antitumor activity in mouse tumor xenografts.

Materials and Methods

Drugs, Enzymes, and Chemicals. Camptothecin was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD). The synthesis of MJ-III-65 has been described previously (Cushman et al., 2000). Etoposide (VP-16) was purchased from Sigma-Aldrich (St. Louis, MO). Drug stock solutions were made in dimethyl sulfoxide at 100 mM for VP-16, 10 mM for CPT, and 5 mM for MJ-III-65. Aliquots were stored at –20°C, and further dilutions were made in dimethyl sulfoxide immediately before use. The final concentration of dimethyl sulfoxide in the reactions did not exceed 10% (v/v).

Recombinant human Top1 (Top1) was purified from TN5 insect cells (HighFive; Invitrogen, San Diego, CA) using a baculovirus construct for the N terminus-truncated human Top1 cDNA as described previously (Zhelkovsky and Moore, 1994). The C21 human Top1 monoclonal antibody was a generous gift from Yung-Chi Cheng (Yale University, New Haven, CT), and human Top2 antibodies were purchased from TopoGEN (Columbus, OH). DNA polymerase I (Klenow fragment), dNTP [where N is A (adenosine), C (cytosine), G (guanosine), or T (thymine)], agarose and polyacrylamide/bis were purchased from Invitrogen (Gaithersburg, MD) or New England Biolabs (Beverly, MA). DNA quick spin columns were purchased from Roche Diagnostics (Indianapolis, IN). [-³²P]deoxy-GTP was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA).

Top1 Reactions. The 161-base pair fragment from pBluescript SK(–) phagemid DNA (Stratagene, La Jolla, CA) was cleaved with restriction endonucleases PvuII and HindIII (New England Biolabs) in supplied NE buffer 2 (50-µl reactions) for 1 h at 37°C. Reaction products were separated by electrophoresis in a 1% agarose gel made in 1x Tris/borate/EDTA buffer. The 161-base pair fragment was eluted from the gel slice using the QIAEX II kit (QIAGEN, Valencia, CA). The pSK fragment was singly 3' end-labeled by a fill-in reaction. In brief, linearized pSK (200 ng) was incubated with [-³²P]dGTP in 1x labeling buffer (0.5 mM each dATP, dCTP, dTTP in 50 mM Tris-HCl, pH 8.0, 100 mM MgCl₂, and 50 mM NaCl) in the presence of 0.5 units of the Klenow fragment of DNA polymerase I. Labeled DNA was purified using mini quick spin DNA columns (Roche Diagnostics).

For Top1 cleavage assays, labeled DNA (50 fmol/reaction) was incubated with 5 ng of recombinant Top1 with or without drug at 25°C in 10 µl of reaction buffer (10 mM Tris-Cl, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, and 15 µg/ml bovine serum albumin, final concentrations). For reversal experiments, the SDS (0.5%) stop was preceded by the addition of NaCl to a final concentration of 0.35 M followed by incubation for the indicated times at 25°C.

Samples were denatured by the addition of 3.3 volumes of Maxam Gilbert loading buffer (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue, pH 8.0). Aliquots were separated in 16% denaturing polyacrylamide gels (7 M urea) in 1x Tris/borate/EDTA (89 mM Tris-borate and 2 mM EDTA, pH 8.0) for 2 h at 40 V/cm at 50°C.

Imaging and quantitation were performed using a PhosphorImager (Amersham Biosciences Inc., Piscataway, NJ).

DNA-Protein Cross-Links and Single-Strand Breaks. Alkaline elution was performed to assess DNA damage by detecting DNA-protein and DNA-DNA cross-links as described previously (Kohn et al., 1981; Bertrand and Pommier, 1995; Pommier et al., 1995; Kohn, 1996). Before alkaline elution and drug treatments, human leukemic CEM cells were radiolabeled with 0.02 µCi/ml [³H]thymidine for one to two doubling times at 37°C and then chased in nonradioactive medium overnight. Cells were treated with appropriate concentrations of MJ-III-65 or CPT for 1 h. After drug treatments, cells were scraped in Hanks' balanced salt solution. For reversal experiments, the cells were cultured in drug-free medium for the appropriate time before scraping. After alkaline elution, filters were incubated at 65°C with 1 N HCl for 45 min, and then 0.04 M NaCl was added for an additional 45 min. Radioactivity in all fractions was measured with a liquid scintillation analyzer (PerkinElmer Life and Analytical Sciences).

DPC were analyzed under nondeproteinizing, DNA-denaturing conditions using protein-adsorbing filters (polyvinylchloride-acrylic copolymer filters, 0.8-µm pore size; Gelman Instrument Co., Ann Arbor, MI) and LS10 lysis solution (2 M NaCl, 0.2% Sarkosyl, and 0.04 M disodium EDTA, pH 10). All cell suspensions were irradiated with 30 Gy. The DNA was eluted from filters with tetrapropylammonium hydroxide-EDTA, pH 12.1, without SDS at a flow rate of 0.035 ml/min. Fractions were collected at 3-h intervals for 15 h. DPC frequencies were calculated according to the bound to one terminus model formula (Ross et al., 1979), which is represented as p_cD = (1/(1 – r) – 1/(1 – r_o)) p_bR, where p_cD is the frequency of drug-induced DNA-protein cross-links, p_bR is the frequency of X-ray-induced single-strand breaks (3000 when results are expressed in Rad-equivalents and 30 Gy is used before elution), and r and r_o are the fractions of the DNA eluting in the slow component in the presence and absence of drug, respectively.

SSB were assessed by alkaline elution under deproteinizing, DNA denaturing conditions. In brief, after treatment, radiolabeled cells were harvested at 4°C, loaded onto polycarbonate filters (2-µm pore size; Poretics, Livermore, CA), and lysed with SDS buffer [0.1 M glycine, 0.025 M EDTA, 2% (w/v) SDS, and 0.5 mg/ml proteinase K, pH 10]. The lysis solution was washed from filters with 0.02 M EDTA, pH 10, and the DNA was eluted with tetrapropylammonium hydroxide-EDTA, pH 12.1, containing 0.1% SDS at a flow rate of 0.035 ml/min into five fractions at 3-h intervals.

Detection of Covalent Top1-DNA Complexes in CEM Cells. Top1-DNA adducts were isolated using the immunocomplex of enzyme (ICE) bioassay (Shaw et al., 1975; Subramanian et al., 1995; Pourquier et al., 2000). In brief, 10⁶ treated or untreated CEM cells were pelleted and immediately lysed in 1% sarkosyl. After homogenization with a Dounce homogenizer, cell lysates were gently layered on CsCl step gradients and centrifuged at 165,000g for 20 h at 20°C. Half-milliliter fractions were collected, diluted with an equal volume of 25 mM sodium phosphate buffer, pH 6.5, and applied to Immobilon-P membranes (Millipore Corporation, Billerica, MA) by using a slot-blot vacuum manifold as described previously (Pourquier et al., 2000). Top1-DNA complexes were detected using the C21 Top1 monoclonal antibody and Top2-DNA complexes using Top2 antibody using standard Western procedures.

Cell Lines and Cytotoxicity Assays. The human T-lymphoblastoid leukemia CEM cell line was purchased from American Type Culture Collection (Manassas, VA). The CEM/C2 cells were established as described previously (Fujimori et al., 1995, 1996). P388 and P388/CPT45 mouse leukemia cells were a kind gift from Michael R. Mattern and Randal K. Johnson (GlaxoSmithKline, King of Prussia, PA). P388/CPT45-resistant cells were obtained by exposing CPT-5 cells (Mattern et al., 1991, 1993) to stepwise increasing concentrations of CPT until they grew in the presence of 45 µM CPT. CEM and P388 cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) and 2 mM L-glutamine in a 5% CO₂ incubator at 37°C.

Cytotoxicity of MJ-III-65 in CEM, CEM/C2, P388, and P388/CPT45 cells was measured using the MTT (Sigma-Aldrich) colorimetric assay performed in 96-well plates. Cells were seeded (6000 cells for CEM and P388 and 30,000 cells for CEM/C2 and P388/CPT45) into each well with 180 µl of RPMI 1640 medium containing 10% fetal bovine serum. Twenty microliters of MJ-III-65 or CPT at each concentration was added to the wells, and incubations were continued for 3 days, after which 10 µl of MTT (5 mg/ml in phosphate-buffered saline) was added to each well. After an additional 4-h incubation, the resulting formazan was dissolved in 100 µl of 2-propanol containing 0.04 N HCl. Optical densities were read immediately at 570 nm using a Micro Plate Reader. Determinations for all experiments were made in duplicates, and the results were expressed as mean ± S.D. Percentage of growth was calculated relative to control (untreated cells) after 3 days of culture with control taken as 100.

Antitumor Activity and Toxicity of NSC 314622 and MJ-III-65 in Nude Mice Bearing Human A253 and FaDu Head and Neck Xenografts. Athymic nude mice (nu/nu, female, 20–25 g, 8–12 weeks old) from Harlan (Indianapolis, IN) were transplanted with A253 and FaDu human head and neck xenografts. In brief, 50 mg of non-necrotic tumor pieces was transplanted s.c. to nude mice. Both the A253 and FaDu tumors were transplanted in the same animals at different sides (right versus left). Treatment was initiated 6 to 8 days later when tumor weight reached 200 to 250 mg. Five mice per treatment group were included in all experiments. The animals were treated with either NSC 314622 (5 or 10 mg/kg/week) or MJ-III-65 (10, 25, or 50 mg/kg/week) administered i.v. push via tail vein once a week for four consecutive weeks. Both drugs were dissolved in dimethyl sulfoxide and diluted to 10% dimethyl sulfoxide solution. The two axes (millimeters) of tumor (L, longest axis; W, shortest axis) were measured with the aid of a Vernier caliper. Tumor weight (milligrams) was estimated as tumor weight = 1/2 (L x W²). Tumor measurements were taken daily for the first 10 days and at least three times a week the first 3 weeks of post-therapy and once a week thereafter. Estimates were made of the maximum tolerated dose, defined as the maximum dose that does not cause drug-related lethality in mice and maximum weight loss <20%; the antitumor activity, defined as the relative tumor volume of the treated animal over control; and the tumor doubling time, defined as the mean time for the tumor to reach twice the initial size.

Results

MJ-III-65 Induces Top1-Mediated DNA Cleavage Complexes with a Different Pattern from CPT. Induction of DNA cleavage in the presence of Top1 was tested in the PvuII/HindIII fragment of pBluescript SK(–) phagemid DNA (pSK) (Fig. 2A). The DNA cleavages produced by the indenoisoqinolines were different from the pattern observed with CPT. The lined wedge to the right of the Fig. 2A marks a CPT cleavage site that is not observed with the indenoisoquinolines. In contrast, the two open wedges show indenoisoquinoline DNA cleavage sites that are not seen with CPT. The solid wedge indicates common cleavage sites that are observed with both CPT and the indenoisoquinolines. The bands corresponding to the indenoisoquinoline-stabilized DNA cleavage sites varied in intensity between the indenoisoquinolines. Previous studies have shown the lead indenoisoquinoline NSC 314622 to be a Top1 inhibitor, although not as potent as CPT. However, MJ-III-65 (Fig. 2A) exhibits Top1 inhibition comparable with CPT with differential DNA cleavage preferences for sites 44 and 62 compared with sites 37, 97, and 119 for CPT.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Reversible trapping of Top1 by MJ-III-65. A, DNA corresponds to the 3' end-labeled PvuII/HindIII fragment of pBluescript SK(–) phagemid DNA (pSK). DNA fragments were reacted with Top1 in the absence of drug (Top1) or presence of the indicated concentrations (micromolar) of CPT, NSC 314622, or MJ-III-65. Reactions were at 30°C for 20 min and stopped by adding 0.5% SDS. DNA fragments were separated in 16% denaturing polyacrylamide gels. B, 3' end-labeled pSK DNA used was the same as in Fig. 2A. Reactions were performed with Top1 in the absence of drug (Top1) or in the presence of 1 µM CPT (CPT) or 1 µM MJ-III-65 (MJ-III-65). After incubation at 30°C for 20 min, reactions were reversed by adding NaCl to a final concentration of 0.35 M for the indicated times (minutes) before addition of 0.5% SDS and proteinase K digestion. Time 0 refers to the samples taken immediately before NaCl addition. DNA fragments were then separated in a 16% denaturing polyacrylamide gel. Arrows indicate cleavage sites that are unique to CPT (striped arrowhead) or unique to indenoisoquinolines (open arrowhead) or common to both (closed arrowhead). Numbers to the side of the gels indicate the migration position of DNA fragments cleaved at this position in the DNA fragment analyzed.

Reversibility of Top1-DNA Cleavage Complexes Induced by MJ-III-65. To compare the stability of the Top1-DNA cleavage complexes induced by CPT and MJ-III-65, salt-reversal experiments were carried out (Tanizawa et al., 1995). In pSK DNA (Fig. 2B) salt-reversal of Top1-DNA cleavage complexes was slower for MJ-III-65 (sites 44 and 62) than for CPT (sites 70 and 119). Site 92, which is targeted by both drugs, showed similar reversal. This reversibility of Top1-DNA cleavage complexes is consistent with the reversible trapping of Top1 cleavage complexes by MJ-III-65 (Antony et al., 2003).

MJ-III-65 Induces Top1-DNA Complexes in Cells. The ICE bioassay can detect topoisomerase-DNA covalent complexes in tissue culture cells or in vivo samples (Shaw et al., 1975; Subramanian et al., 1995). We used this assay to evaluate whether Top1-DNA complexes were detectable in MJ-III-65-treated cells. Exponentially growing CEM cells were treated with 1, 10, or 100 µM MJ-III-65; 1 µM CPT; or 100 µM VP-16 for 1 h and processed in the ICE bioassay. Fractionation of the CsCl gradient showed DNA band in fractions 7 to 10, and immunoblotting revealed the presence of Top1 signals in these DNA fractions for the MJ-III-65- and CPT-treated cells but not in the untreated or VP-16-treated cells (Fig. 3). Immunoblotting against Top2 was positive in the DNA fractions of the VP-16-treated cells but not in the CPT- or MJ-III-65-treated cells. These data indicate that MJ-III-65 produces Top1- but not Top2-DNA cleavage complexes in cells and demonstrate that Top1 is a cellular target for MJ-III-65.

View larger version (20K): [in this window] [in a new window]   Fig. 3. MJ-III-65 induces Top1 cleavage complexes in CEM cells. CEM cells were treated with 1, 10, and 100 µM MJ-III-65; 1 µM CPT; or 100 µM VP-16 for 1 h at 37°C. Equal numbers of cells were lysed in 1% sarkosyl and submitted to the ICE assay (see Materials and Methods). DNA-containing fractions were collected from the bottom of the gradients. Fractions (7–10) were blotted, and Top1-DNA and Top2-DNA covalent complexes were detected using Top1 C21 monoclonal and Top2 antibody. Treatment with 1 µM CPT and 100 µM VP-16 was used as positive control for Top1 and Top2 cleavage complexes, respectively.

DPC Induced by MJ-III-65 in Cells Persists after Drug Removal. To quantitate the Top1-DNA complexes in drug-treated cells, alkaline elutions were carried out to detect DPC (Covey et al., 1989; Pommier et al., 1994a,b; Kohn and Pommier, 2000; Kohn et al., 2000). Figure 4 shows that MJ-III-65 produced DPC in a concentration-dependent manner. DPC were detectable after 1-h exposure to concentrations as low as 30 nM. The induction of DPC increased almost linearly as a function of MJ-III-65 concentration up to 1 µM. At higher concentrations, a saturation of DPC was observed. Because Top1 cleavages are characterized by an equivalent frequency of SSB and DPC (Covey et al., 1989; Kohn, 1996; Kohn and Pommier, 2000), we also measured MJ-III-65-induced SSB by alkaline elution. Table 1 shows a near equivalence between SSB and DPC, which is consistent with Top1 inhibition by MJ-III-65.

View larger version (14K): [in this window] [in a new window]   Fig. 4. DNA-protein cross-links induced by MJ-III-65 in human leukemic CEM cells. Cells were prelabeled with [3H]thymidine and were treated with 0.03, 0.1, 0.3, 1, 10, or 100 µM MJ-III-65 for 1 h at 37°C. DPC were assayed by alkaline elution. Results from three different experiments (, , ) are plotted.

We then studied the reversal of the DPC induced by MJ-III-65. MJ-III-65 was compared with CPT. Figure 5 shows that MJ-III-65-induced DPC were not reversible within 1 h after drug removal. By contrast, CPT-induced DNA crosslinks reversed completely within 30 min of drug removal (Covey et al., 1989; Tanizawa et al., 1994). Thus, MJ-III-65-induced DNA cross-links persist after drug removal.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Persistence of DPC induced by MJ-III-65 in human leukemic CEM cells after drug removal. Cells were prelabeled with [3H]thymidine and were treated with either 1 µM MJ-III-65 or 1 µM CPT for 1 h at 37°C. DPC were assayed immediately before drug removal (T0) and after drug treatment followed by culturing in drug-free medium for 30 min (T30) or 60 min (T60), respectively. DPC induced in MJ-III-65 and CPT treated cells () are plotted as an average of two independent experiments ± S.D. Elution of untreated cells () is also shown.

Sensitivity of CPT-Resistant Cell Lines to MJ-III-65. Because MJ-III-65 had been previously shown to be effective in trapping CPT-resistant and mutant Top1 enzymes in DNA cleavage assays (Antony et al., 2003), we evaluated the sensitivity of human leukemia CEM/C2 cells (having the mutation N722S in Top1 and silencing of the normal Top1 allele) (Fujimori et al., 1996) to both MJ-III-65 and CPT. We used an additional cell line P388/CPT45. This cell line is cultured with 45 µM CPT and is highly resistant to CPT (Urasaki et al., 2000, 2001). Western blot analysis using monoclonal antibody against Top1 does not detect Top1 in P388/CPT45. The antiproliferative activity of MJ-III-65 and CPT was evaluated by MTT assays. Limited cross-resistance was seen in the CEM/C2 cell line (Fig. 6). The Top1-deficient cells (P388/CPT45 only) showed considerable resistance to MJ-III-65 at low drug concentrations (<1 µM) but not at higher doses, whereas resistance to CPT was >2000-fold (Fig. 7). These results demonstrate that MJ-III-65-mediated cell cytotoxicity is Top1-dependent at low concentrations and that MJ-III-65 can overcome CPT resistance resulting from Top1 mutations.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Sensitivity of CPT-resistant Top1-mutant CEM/C2 cells to MJ-III-65. Growth inhibition in CEM () and CEM/C2 () cells was measured by MTT assay after treatment with MJ-III-65 (A) or CPT (B) for 1 h. MJ-III-65 and CPT concentrations used were as follows: 0.01, 0.1, 1, and 10 µM. Percentage of growth of two independent experiments is represented as the mean ± S.D.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Resistance of Top1-deficient P388/CPT45 cells to MJ-III-65. Growth inhibition in P388 () and P388/CPT45 () cells was measured by MTT assay after treatment with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM MJ-III-65 (A) or CPT (B) for 3 days. Percentage of growth of two independent experiments is represented as the mean ± S.D.

Antitumor Activity and Toxicity of MJ-III-65 and NSC 314622 in Nude Mice Bearing A253 and FaDu Human Tumor Xenografts. The data in Table 2 and Figs. 8 and 9 are a summary of the antitumor activity and toxicity of MJ-III-65 (10–50 mg/kg) and NSC 314622 (5–10 mg/kg) administered i.v. push once a week for 4 weeks in nude mice bearing human head and neck xenografts of A253 and FaDu. Although these agents have limited solubility, both agents were moderately active against human A253 and FaDu tumor xenografts without significant toxicity. NSC 314622 shows about 65% tumor growth inhibition and delayed tumor growth (increased tumor doubling time). MJ-III-65 was slightly more active with similar response rate against both A253 and FaDu tumor xenografts. Defining accurately the potential therapeutic activity of these compounds is perhaps limited by the limited solubility of these compounds relative to other Top1 poisons such as irinotecan.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Antitumor activity (A) and toxicity (B) of NSC 314622 in nude mice bearing human A253 and FaDu head and neck tumor xenografts. Control (untreated) (); NSC 314622, 5 mg/kg (); and NSC 314622, 10 mg/kg ().

View larger version (23K): [in this window] [in a new window]   Fig. 9. Antitumor activity (A) and toxicity (B) of MJ-III-65 in nude mice bearing human A253 and FaDu head and neck tumor xenografts. Control (untreated) (); MJ-III-65, 10 mg/kg (); MJ-III-65, 25 mg/kg (); and MJ-111-65, 50 mg/kg ().

Discussion

Work on design and synthesis of potent Top1 inhibitors that overcome the limitations of CPT and its derivatives has been ongoing. Non-CPT Top1 inhibitors such as the indolocarbazoles NB-506 and J-107088 are in clinical trials (Meng et al., 2003). More recently, indenoisoquinolines and minor groove binders (benzimidazoles) have been reported to be promising Top1 inhibitors (Strumberg et al., 1999; Cushman et al., 2000; Rangarajan et al., 2000; Jayaraman et al., 2002; Fox et al., 2003; Nagarajan et al., 2003). Preliminary screening of approximately 100 derivatives of our parent indenoisoquinoline compound NSC 314622 has identified MJ-III-65 (NSC 706744) to be a potent inhibitor of purified Top1 in biochemical assay (Cushman et al., 2000; Antony et al., 2003). This study was carried out to further characterize and determine the molecular mechanism by which MJ-III-65 exhibits its cytotoxicity in cellular systems.

Our results indicate that both the parent compound NSC 314622 and MJ-III-65 trap Top1 at similar sites, with MJ-III-65 being more potent and producing Top1 cleavage complexes even at the lowest concentration of 30 nM (Fig. 2A). MJ-III-65's potency, which increases and saturates with dose, is comparable with that of CPT, although differing in the sequence/cleavage sites preferred.

We find that MJ-III-65 also inhibits Top1 in human leukemic cells because Top1-DNA complexes were detected by the ICE bioassay and by alkaline elution for DPC and SSB (Table 1; Figs. 3 and 4). Therefore, MJ-III-65 traps Top1 both in vitro and in cells.

Although CPT derivatives are also potent Top1 inhibitors in cells, they are limited by the rapid reversibility of the cleavage complexes upon drug removal, imposing prolonged drug treatments. On the other hand, MJ-III-65-induced Top1-cleavage complexes in cells persisted after drug removal, indicating stability of the MJ-III-65-induced Top1-DNA complexes (Fig. 5). This is also supported by the in vitro data (Antony et al., 2003), where Top1-mediated DNA cleavage complexes trapped by MJ-III-65 are more stable (4-fold) than those induced by CPT. Moreover, MJ-III-65 enhanced the DNA cleavage rate of Top1 (2-fold) more than CPT. Hence, we propose that tight binding of MJ-111-65 to the Top1-DNA cleavage complexes could account for the prolonged stability of the MJ-III-65-induced DNA-Top1 complexes in living cells. Like the homocamptothecins BN80915 and BN80927 (Philippart et al., 2000; Demarquay et al., 2004), MJ-III-65 stabilizes Top1-DNA cleavage complexes to a greater extent after drug removal compared with CPT but differs from BN80927 that can also inhibit topoisomerase II-mediated DNA relaxation (Demarquay et al., 2004).

Another frequently encountered therapeutic limitation of CPT derivatives is drug resistance. CPT-resistant (Fig. 6B) human leukemic cells CEM/C2 with a point mutation in Top1 (Fujimori et al., 1995, 1996) were sensitive to MJ-III-65 even at 0.1 µM drug concentration (Fig. 6A). The murine P388/CPT45 cells that do not have any detectable Top1 were also sensitive to MJ-III-65, although at higher doses (>1 µM; Fig. 7A). This implies that MJ-III-65 has additional targets beside Top1 at high concentrations. As shown previously (Cushman et al., 2000), MJ-III-65 intercalates at high concentrations. The ability to bind DNA could account for the additional targets that mediate MJ-III-65 cytotoxicity.

Both the parent NSC 314622 and the derivative MJ-III-65 are active against human head and neck tumor xenografts of A253 and FaDu with about 64 to 72% tumor growth inhibition (Table 2; Figs. 8 and 9) compared with untreated control in spite of their limited solubility. In xenografts bearing FaDu and A253 tumors, the response to the maximum tolerated dose of irinotecan (CPT-11, 100 mg/kg/week x 4) is 30 and 0% complete tumor regression, respectively. In contrast, these tumors were resistant to Topotecan. MJ-III-65 is slightly more potent in terms of tumor inhibition compared with NSC 314622, which may be caused, at least in part, by its higher solubility and higher dose (10 mg/kg for NSC 314622 versus 50 mg/kg for MJ-III-65). Because the toxicity (animal body weight loss) is very minimal in the highest tested dose with both agents increasing the bioavailability by improving the solubility or changing the route of administration may increase the efficacy of the drugs.

Based on the stability of MJ-III-65-induced Top1-DNA cleavage complexes and its ability to remain active even in CPT-resistant cell lines, MJ-III-65 is a promising non-CPT Top1 inhibitor whose therapeutic potential should be further explored.

Footnotes

This work was made possible with Research Grant U01-CA89566 and training grant ST32-CA09634 from the National Institutes of Health.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.003889.

ABBREVIATIONS: Top1, topoisomerase I; CPT, camptothecin; NSC 314622, 5,6-dihydro-5,11-diketo-2,3-dimethoxy-6-methyl-8,9-methylenedioxy-11H-indeno (1,2-c)isoquinoline; MJ-III-65, 6-[3-(2-hydroxyethyl)amino-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline; DPC, DNA-protein cross-link(s); SSB, single-strand break(s); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; ICE, immuno complex of enzyme; NB-506,6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(-D-glucopyranosyl)-5H-indolol[2,3-]pyrrolo-[3,4-c]carbazole-5,7-(6H)-dione; J-107088,6-N-(1-hydroxymethyla-2-hydroxy)ethylamino-12,13-dihydro-13-(-D-glucopyranosyl)-5H-indolo[2,3-a]-pyrrolo[3,4-c]-carbazole-5,7(6H)-dione.

Address correspondence to: Dr. Yves Pommier, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, 37 Convent Dr., Bldg 37, Rm 5068, National Institutes of Health, Bethesda, MD 20892-4255. E-mail: pommier{at}nih.gov

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