Camptothecin (CPT) analogues are powerful anticancer agents but are chemically unstable due to their α-hydroxylactone six-membered E-ring structure, which is essential for trapping topoisomerase I (Top1)-DNA cleavage complexes. To stabilize the E-ring, CPT keto analogues with a five-membered E-ring lacking the oxygen of the lactone ring (S38809 and S39625) have been synthesized. S39625 has been selected for advanced preclinical development based on its promising activity in tumor models. Here, we show that both keto analogues are active against purified Top1 and selective against Top1 in yeast and human cancer cells. The keto analogues show improved cytotoxicity toward colon, breast, and prostate cancer cells and leukemia cells compared with CPT. The drug-induced Top1-DNA cleavage complexes induced by the keto analogues show remarkable persistence both with purified Top1 and in cells following 1-h drug treatments. Moreover, we find that S39625 is not a substrate for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which sets S39625 apart from the clinically used CPT analogues topotecan or SN-38 (active metabolite of irinotecan). Finally, we show that nanomolar concentrations of S38809 or S39625 induce intense and persistent histone γ-H2AX. The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39625 a promising new anticancer drug candidate. Histone γ-H2AX could be used as a biomarker for the upcoming clinical trials of S39625. [Mol Cancer Ther 2007;6(12):3229–38]

The approval of two camptothecin (CPT) derivatives for use in anticancer therapy has underscored the validity of DNA topoisomerase I (Top1) as a therapeutic target. CPT could not be developed clinically because of its instability and toxicity, but modifications at selected sites in analogues have improved the pharmacologic and activity profile (1, 2). Topotecan has been approved for use in ovarian, cervical, and small cell lung cancer, whereas irinotecan has been approved for use in colorectal cancer. Most of the analogues developed to date have myelosuppression as a major toxicity due to activity of the compounds in dividing cells, and in some examples, there was more toxicity than anticancer activity. Still, the pursuit of analogues that may have a different spectrum of activity and toxicity is ongoing.

CPT is a pentacyclic alkaloid first isolated from the Chinese tree Camptotheca acuminata by Wall et al. (1). CPT and its clinical derivatives inhibit Top1 with exquisite selectivity (35). A single drug molecule is sufficient to trap Top1 as the enzyme relaxes DNA by forming a ternary complex with Top1-linked DNA break (6, 7). Those breaks are referred to as Top1-DNA cleavage (or cleavable) complexes (2, 3). Studies in yeast showed that Top1 is necessary and sufficient for the activity of CPT and therefore the sole cellular target of CPT (810). Three-dimensional structure analyses of a ternary complex formed between Top1, DNA, and CPT or topotecan by X-ray crystallography (6, 11) showed that one drug molecule interacts both with the DNA base pairs flanking the cleavage sites and three key amino acid residues of Top1 (Asn722, Arg364, and Asp533; ref. 12). Single mutation of any of those three amino acid residues is sufficient to confer high resistance to CPT and its analogues (13). The E-ring of CPT (Fig. 1A) is essential to the interaction of the drug with the Top1-DNA cleavage complex (6, 11, 12). Because CPT analogues are the only clinical drugs targeting Top1, and because of their clinical importance, the search for novel inhibitors of Top1 is ongoing (2).

Figure 1.

A, chemical structures of CPT, S38809, and S39625. The α-hydroxylactone E-ring of CPT is rapidly converted into the inactive carboxylate at physiologic pH (2, 14). Both S38809 and S39625 have a stable five-membered E-ring that cannot open. B, comparison of Top1-mediated DNA cleavage patterns induced by CPT, S38809, and S39625 using a 3′-end–labeled PvuII/HindIII fragment of the pBluescript SK(−) phagemid DNA at concentrations of 0.1, 1.0, 10, and 100 μmol/L. The numbers and arrows to the right indicate cleavage site positions (24, 30). C, sequence of the 3′-end–labeled 22-bp duplex oligonucleotide containing a single Top1 cleavage site (^). Asterisk, 3′-end label. The labeled oligonucleotide was reacted with Top1 in the presence of 0.01, 0.1, 1, 10, and 100 μmol/L of CPT, S38809, and S39625. Middle, arrows to the left, full-length (23 mer) and cleaved (13 mer) products; bottom, graph representing the percentage of drug-induced cleavage product from the gel above.

Figure 1.

A, chemical structures of CPT, S38809, and S39625. The α-hydroxylactone E-ring of CPT is rapidly converted into the inactive carboxylate at physiologic pH (2, 14). Both S38809 and S39625 have a stable five-membered E-ring that cannot open. B, comparison of Top1-mediated DNA cleavage patterns induced by CPT, S38809, and S39625 using a 3′-end–labeled PvuII/HindIII fragment of the pBluescript SK(−) phagemid DNA at concentrations of 0.1, 1.0, 10, and 100 μmol/L. The numbers and arrows to the right indicate cleavage site positions (24, 30). C, sequence of the 3′-end–labeled 22-bp duplex oligonucleotide containing a single Top1 cleavage site (^). Asterisk, 3′-end label. The labeled oligonucleotide was reacted with Top1 in the presence of 0.01, 0.1, 1, 10, and 100 μmol/L of CPT, S38809, and S39625. Middle, arrows to the left, full-length (23 mer) and cleaved (13 mer) products; bottom, graph representing the percentage of drug-induced cleavage product from the gel above.

Close modal

One of the major limitations of CPT and its clinical analogues is the instability of the α-hydroxylactone E-ring, which is rapidly converted into a carboxylate at physiologic pH (Fig. 1A; refs. 2, 14). The CPT carboxylate is inactive against Top1 (4). Moreover, it binds tightly to serum albumin (15), which limits the fraction of drug in the active lactone form. Early studies showed that it is impossible to stabilize the six-membered E-ring without losing the activity of CPT analogues. For instance, replacing the ring oxygen by nitrogen yields CPT lactam, which is inactive in spite of the overall conservation of the CPT structure (4, 14).

The first successful approach for stabilizing the CPT E-ring was to include an additional methylene in the E-ring, thereby generating seven-membered β-hydroxylactone E-ring analogues, which are referred to as homocamptothecins (16, 17). One homocamptothecin analogue, diflomotecan (BN80915), is in clinical development. The seven-membered β-hydroxylactone E-ring of homocamptothecins opens less readily than the six-membered α-hydroxylactone lactone E-ring of CPT and cannot reclose once it opens to the carboxylate. By using human cancer cell lines resistant to CPT due to point mutations of Top1, we previously showed that the Top1-DNA cleavage complexes formed in the presence of homocamptothecin are more stable than those induced by CPT (18). We also showed more limited cross-resistance to homocamptothecin and diflomotecan than to CPT analogues in cells overexpressing the ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporter (19). These studies showed that E-ring modifications were possible while retaining potent anti-Top1 activity and improving ABCG2-mediated drug resistance.

A second successful approach to stabilize the CPT E-ring was recently reported. It consists in removing the oxygen of the lactone E-ring, thereby generating five-membered CPT analogues (2, 20). We will refer to this class of drugs as CPT keto analogues (20). Here, we investigated two such analogues, S38809 and S39625 (Fig. 1A). S38809 contains a 10,11-methylenedioxy substitution, which is known to increase the activity of CPT analogues against Top1 (4). S38809 was previously shown to be active against purified Top1 (20, 21), but no data have yet been reported about its cellular activity. S39625 is a new analogue of S38809 with a cyclobutyl substitution at the 7-position. Because S39625 exhibits remarkable antitumor activity in animal models, it is currently in advanced preclinical development.4

4

J. Hickman, Institut de Recherches Servier, personal communication.

The present study describes the activity of those two drugs against purified Top1 and the remarkable stability of the Top1 cleavage complexes induced by S39625. Our study also shows the high potency of S39625 across a variety of cell lines and shows that Top1 is the cellular target of those two drugs. We also report that the keto analogues are not substrates for either the ABCB1 [P-glycoprotein (P-gp)/multidrug resistance-1 (MDR-1)] or the ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which differentiates them from topotecan and irinotecan (19, 22, 23).

Drugs, Enzymes, and Chemicals

CPT was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD). S38809 and S39625 were synthesized as described5

5

U.S. patent 6-509-345 G. Lavielle et al.

(20).

Stock solutions of CPT, S38809, and S39625 were made in DMSO at 10 mmol/L and aliquots were stored at −20°C, Further dilutions were made in DMSO immediately before use. The final concentration of DMSO in the reaction mixtures did not exceed 1% (v/v). Human Top1 was purified from Sf9 cells using a baculovirus construct (24).

Cell Lines and Cytotoxicity Assays

Human colon HCT116 and breast MCF-7 cancer cells were purchased from the American Type Culture Collection. The HCT116 Top1-small interfering RNA (siRNA; HCT116-siTop1) and MCF-7 Top1-siRNA (MCF-7-siTop1) cells are described in details separately (25). HCT116, MCF-7, DU145, and their CPT-resistant subclones were maintained in DMEM supplemented with 10% fetal bovine serum (25, 26). CEM and CEM/C2 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (27). Cytotoxicity of CPT, S38809, and S39625 in HCT116, MCF-7, DU145, and their CPT-resistant subclones was assessed by the sulforhodamine B (Sigma-Aldrich) assay, whereas in CEM and CEM/C2 cells cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich) colorimetric assay. Drug treatment was continuous for 3 days for both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sulforhodamine B assays. All experiments were done in quadruplicate and the results are expressed as mean ± SD. Percentage of growth was calculated relative to control (vehicle-treated cells) after 3 days of culture with control taken as 100%.

In vitro Top1-Mediated DNA Cleavage Reactions

A 161-bp 3′-end–labeled DNA fragment from pBluescript SK(−) phagemid DNA (Stratagene) and a 23-bp 3′-end scissile strand–labeled duplex oligonucleotide (5′-AAAAAGACTT^GGAAAAATTTTTA-3′) with a single Top1 cleavage site (^ in Fig. 1C) were generated as previously described (24, 2830). These DNA substrates were incubated with drugs in reaction buffer containing 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 15 μg/mL bovine serum albumin, and 0.2 mmol/L DTT at 25°C for 20 min in the presence of recombinant Top1. Reactions were stopped with SDS (0.5% final concentration). For reversal experiments, the addition of SDS was preceded by the addition of NaCl (final concentration of 0.35 mol/L at 25°C for the indicated times). The kinetics of reversal for each drug was calculated from the semilog plot of the percentage of cleavage product remaining after salt reversal. All quantification was carried out using a PhosphorImager and ImageQuant software.

Yeast Survival Experiments

The parental yeast strain selected in this study was Saccharomyces cerevisiae JN394 (31, 32). JN394-Top1Δ, which has a chromosomal deletion of Top1 gene (9), was used to determine the contribution of Top1 to drug cytotoxicity. Yeast cells were pregrown in YEPD medium (Quality Biological, Inc.) and serially diluted 10-fold. Aliquots (4 μL) of the diluted samples were spotted onto YPD agar plates (Quality Biological, Inc.) with or without drugs. The plates were incubated at room temperature for 3 days and photographed under white light.

Detection of Covalent Top1-DNA Complexes

Top1-DNA adducts were isolated using the in vivo immunocomplex of enzyme bioassay (3335). Briefly, 1 × 106 HCT116 cells treated with 100 nmol/L of each drug or untreated were pelleted and immediately lysed in buffer containing 1% sarkosyl, 8 mol/L guanidine hydrochloride, 30 mmol/L Tris, and 10 mmol/L EDTA (pH 7.5). After homogenization with a Dounce homogenizer, cell lysates were gently layered on cesium chloride step gradients and centrifuged at 165,000 × g for 20 h at 20°C. DNA fractions (0.5 mL) were collected and diluted with an equal volume of 25 mmol/L sodium phosphate buffer, including 8.8 mmol/L Na2HPO4 and 16.2 mmol/L NaH2PO4 (pH 6.5). Aliquots of each DNA fraction were applied to Immobilon-P membranes (Millipore) by using a slot-blot vacuum manifold as described previously (35). Top1-DNA complexes of each aliquot were detected using the C21 Top1 monoclonal antibody (a kind gift from Dr. Yung-Chi Cheng, Yale University, New Haven, CT) and standard Western procedures.

Alkaline Elution Assay for the Detection of DNA-Protein Cross-Links

Alkaline elution was done as previously described (36) to assess DNA damage by detecting DNA-protein cross-links (DPC). Before alkaline elution and drug treatments, human colon cancer HCT116 cells were radiolabeled with 0.2 μCi/mL of [14C]thymidine for one to two doubling times at 37°C and then chased in nonradioactive medium overnight. Cells were treated for 1 h with 0.1 or 1 μmol/L of S38809, S39625, or CPT. After drug treatment, cells were scraped in HBSS. For reversal experiments, the drug-treated cells were cultured in drug-free medium for the indicated times before scraping.

DPCs were analyzed under nondeproteinizing, DNA-denaturing conditions using protein-adsorbing filters (polyvinylchloride-acrylic copolymer filters, 0.8-μm pore size; Gelman Science) and LS10 lysis solution [2 mol/L NaCl, 0.2% sarkosyl, and 0.04 mol/L 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. After alkaline elution, filters were incubated at 65°C with 1 mol/L HCl for 45 min. Then, 0.04 mol/L NaCl was added for an additional 45 min of incubation. Radioactivity in each fraction was measured by liquid scintillation (Packard Instruments). DPC frequencies were calculated according to the bound to one terminus model formula (36), which is represented as

\[p_{\mathrm{cD}}=[1/(1{-}r){-}1/(1{-}r_{\mathrm{o}})]p_{\mathrm{bR}}\]

where pcD is the frequency of drug-induced DPCs, pbR is the frequency of X-ray–induced single-strand breaks (3,000 when results are expressed in rad equivalents and 30 Gy is used before elution), and r and ro are the fractions of the DNA eluting in the slow component in the presence and absence of drug, respectively.

Cytotoxic Assay in MDR Cells

Cytotoxicity assays to determine substrates of ABCG2 and P-gp/MDR-1/ABCB1 were done as previously described (37). Human embryonic kidney cells HEK293 were transfected with either empty pcDNA3 vector (Invitrogen) or pcDNA3 vector containing full-length ABCG2 or P-gp (or MDR-1) as described previously (38). Stable transfectants expressing wild-type ABCG2 (HEK293-R482-5 cells) and wild-type MDR-1/P-gp (HEK293-MDR-19 cells) were maintained in Eagle's MEM (American Type Culture Collection) supplemented with 10% fetal bovine serum with G418 (Invitrogen) at a concentration of 2 mg/mL. All cells were maintained in a 5% CO2 incubator at 37°C. Cells were plated in 96-well plates at a density of 3,000 per well and allowed to attach overnight. Drugs were added at the desired concentrations and the cells were allowed to incubate for 72 h. Subsequently, cells were fixed in 10% trichloroacetic acid. Plates were then stained with a sulforhodamine B solution [0.4% (w/v) sulforhodamine B in 1% acetic acid] and absorbances were read on a plate reader at an absorbency of 560 nm. Each concentration was tested in quadruplicate.

Measurement of Drug-Induced γ-H2AX

HCT116 cells were grown in culture medium in six-well plates. Cells were treated with or without 10 nmol/L of CPT, S38809, and S39625 for 1 h at 37°C. Cells were then washed and cultured in drug-free medium for 24 h. At 0, 4, or 24 h after washing, 1 × 105 cells were collected, washed twice with ice-cold PBS, and then fixed with 4% paraformaldehyde on glass slides (Superfrost Plus, Erie Scientific Co.). Fixed cells were then washed in PBS and permeabilized in 70% methanol. Slides were blocked for 1 h with PBS containing 8% bovine serum albumin and incubated with mouse monoclonal anti-γ-H2AX antibody (Upstate) for 2 h at a 500-fold dilution in the presence of 1% bovine serum albumin. After washing with PBS, the slides were incubated for 1 h with Alexa Fluor 488–conjugated goat anti-mouse IgG secondary antibody (Molecular Probes) at a 500-fold dilution with 1% bovine serum albumin. After washing with PBS, the slides were stained with propidium iodide and sealed with mounting medium (Vectashield, Vector Laboratories, Inc.). The slides were viewed using a PCM2000 laser scanning confocal microscope (Nikon Co.) with an ×40 objective (39). To quantify drug-induced γ-H2AX foci in cells, seven representative pictures from each time point, which included >500 cells, were used to measure average intensity of γ-H2AX staining. The average γ-H2AX signal was computed on each picture using the histogram mode of Adobe Photoshop version 7.0. The average intensity of γ-H2AX staining was then normalized to the number of cells on each picture.

Induction of Top1-Mediated DNA Cleavage Complexes by S38809 and S39625 In vitro

Initial examination of Top1-mediated DNA cleavage by S38809 and S39625 in vitro was done using a PvuII/HindIII fragment of the pBluescript SK(−) phagemid DNA, which enabled the determination of the cleavage site distribution (Fig. 1B; ref. 24). Both S38809 and S39625 showed a cleavage pattern similar to that of CPT, which suggests that the three drugs have comparable binding modes within the Top1-DNA cleavage complex. To investigate the relative potency of Top1-mediated DNA cleavage induced by these drugs, we used a 22-bp double-stranded oligonucleotide that contains a single CPT-induced Top1-DNA cleavage site. Cleavage intensity can be measured by the generation of a 13-mer cleavage product labeled at the 3′-end with [32P]cordycepin (Fig. 1C, top; refs. 28, 29). Figure 1C (middle) illustrates that all three drugs generate the 13-mer cleavage product in a dose-dependent manner in the presence of recombinant Top1. The rank order of potency in stabilizing the Top1 cleavage complex in vitro was CPT > S38809 > S39625 (Fig. 1C, bottom). These experiments show that the CPT keto analogues are potent Top1 inhibitors with similar base sequence selectivity as CPT.

S38809 and S39625 Target Top1 in Yeast and Human Cells

To determine whether Top1 is the cellular target of S38809 and S39625, we used a yeast genetic system that allows manipulation of Top1 activity (810). The cytotoxic effects of CPT, S38809, and S39625 were seen on the wild-type yeast JN394 (Fig. 2A). However, high concentrations of the keto analogues were necessary to observe cytotoxicity, probably because those drugs failed to penetrate yeast. As expected, JN394-Top1Δ yeast cells were completely resistant to CPT (8, 9). Under conditions that corresponded to the highest drug concentrations that could be used while keeping the keto analogues in solution, JN394-Top1Δ yeast cells were completely resistant to both keto analogues, indicating that the target of both S38809 and S39625 is Top1.

Figure 2.

Top1 is the primary cytotoxic target for both S38809 and S39625 in cells. A, the sensitivity of wild-type (WT; JN394) and Top1-deleted yeast strains (JN394-Top1Δ) to CPT, S38809, and S39625 was examined using drug-containing plates. Aliquots of 10-fold diluted samples were spotted onto YPD agar plates with or without drug. B, HCT116 cells were treated with 0.1 μmol/L of CPT, S38809, and S39625 for 1 h at 37°C. Cells were lysed and submitted to the immunocomplex of enzyme assay (see Materials and Methods). The DNA fractions were pooled and serial dilutions were blotted. The Top1-DNA covalent complexes were detected using Top1 C21 monoclonal antibody.

Figure 2.

Top1 is the primary cytotoxic target for both S38809 and S39625 in cells. A, the sensitivity of wild-type (WT; JN394) and Top1-deleted yeast strains (JN394-Top1Δ) to CPT, S38809, and S39625 was examined using drug-containing plates. Aliquots of 10-fold diluted samples were spotted onto YPD agar plates with or without drug. B, HCT116 cells were treated with 0.1 μmol/L of CPT, S38809, and S39625 for 1 h at 37°C. Cells were lysed and submitted to the immunocomplex of enzyme assay (see Materials and Methods). The DNA fractions were pooled and serial dilutions were blotted. The Top1-DNA covalent complexes were detected using Top1 C21 monoclonal antibody.

Close modal

To determine whether Top1 was targeted by S38809 and S39625 in human cells, we used the immunocomplex of enzyme assay (33, 34). Cells were treated for 1 h with 0.1 μmol/L of CPT, S38809, or S39625 and then processed in the immunocomplex of enzyme bioassay. Immunoblotting of the DNA-containing fractions isolated from cesium chloride gradients revealed the presence of Top1 in these DNA fractions for the CPT-, S38809-, and S39625-treated cells (Fig. 2B). These experiments indicate that S38809 and S39625 produce Top1-DNA cleavage complexes in human cells. Moreover, they suggested that S39625 was more efficient at producing Top1 cleavage complexes than S38809, which was slightly more effective than CPT. Quantitation of the Top1 cleavage complexes by alkaline elution confirmed the rank order potency of the three compounds (see below and Fig. 3).

Figure 3.

DPCs and Top1 cleavage complexes induced by CPT, S38809, and S39625. A, amount of DPCs induced by CPT, S38809, and S39625 at 0.1 and 1 μmol/L following a 1-h drug exposure. DPC frequencies are expressed in DPC rad equivalent (36). B, persistence of Top1 cleavage complexes (DPCs) induced by CPT, S38809, and S39625 in HCT116 cells after drug removal. Cells were prelabeled with [14C]thymidine and treated with either 1 μmol/L S38809, S39625, or CPT for 1 h at 37°C. After drug treatment, cells were cultured in drug-free medium and assayed for DPCs at the indicated time points. Points, DPCs induced by S38809, S39625, and CPT as an average of two independent experiments; bars, SD. The half-time of reversal (t1/2) for S39625, S38809, and CPT was calculated as 20, 6, and <4 min, respectively. C, reversibility of Top1-mediated DNA cleavage induced by CPT, S38809, and S39625. Samples were reacted with Top1 for 20 min in the presence of 10 μmol/L CPT, 50 μmol/L S38809, or 100 μmol/L S39625. DNA cleavage was reversed by adding 0.35 mol/L NaCl (final concentration) and monitored over time. D, semilog plot of the percentage of Top1-mediated cleavage product remaining after reversal for CPT, S38809, and S39625. The amount of drug-induced cleavage product before NaCl reversal is taken as 100% for each drug individually.

Figure 3.

DPCs and Top1 cleavage complexes induced by CPT, S38809, and S39625. A, amount of DPCs induced by CPT, S38809, and S39625 at 0.1 and 1 μmol/L following a 1-h drug exposure. DPC frequencies are expressed in DPC rad equivalent (36). B, persistence of Top1 cleavage complexes (DPCs) induced by CPT, S38809, and S39625 in HCT116 cells after drug removal. Cells were prelabeled with [14C]thymidine and treated with either 1 μmol/L S38809, S39625, or CPT for 1 h at 37°C. After drug treatment, cells were cultured in drug-free medium and assayed for DPCs at the indicated time points. Points, DPCs induced by S38809, S39625, and CPT as an average of two independent experiments; bars, SD. The half-time of reversal (t1/2) for S39625, S38809, and CPT was calculated as 20, 6, and <4 min, respectively. C, reversibility of Top1-mediated DNA cleavage induced by CPT, S38809, and S39625. Samples were reacted with Top1 for 20 min in the presence of 10 μmol/L CPT, 50 μmol/L S38809, or 100 μmol/L S39625. DNA cleavage was reversed by adding 0.35 mol/L NaCl (final concentration) and monitored over time. D, semilog plot of the percentage of Top1-mediated cleavage product remaining after reversal for CPT, S38809, and S39625. The amount of drug-induced cleavage product before NaCl reversal is taken as 100% for each drug individually.

Close modal

The Activity of S38809 and S39625 Is Top1 Dependent in Human Cancer Cell Lines, and S39625 Is Markedly More Potent than CPT in All Cell Lines Examined

For the purpose of determining the relationship between cellular sensitivity to S38809 and S39625 and Top1, we examined four pairs of isogenic cell lines with genetic alterations of Top1 that are known to confer resistance to CPT (Table 1). Those cell line pairs belong to different tissue types: human colon cancer (HCT116), breast cancer (MCF-7), prostate cancer (DU145), and leukemia (CEM). The HCT116 and MCF-7 cells (HCT116-siTop1 and MCF-7-siTop1) correspond to cell lines with stable down-regulation of Top1 (∼5-fold) by siRNA against TOP1 (25). DU145/RC1 cells have a point mutation R364H (26) and the CEM/C2 cells have a point mutation N722S (see Materials and Methods; ref. 27).

Table 1.

Cytotoxicity of CPT keto analogues (S38809 and S39625) and CPT in human cancer cell lines

Cell lineDrugIC50 (nmol/L)
RR
Control cellsResistant cells
HCT116 CPT 60.6 ± 22.0 169.5 ± 80.0 2.8 
 S38809 28.9 ± 11.1 92.0 ± 23.6 3.2 
 S39625 5.8 ± 3.0 15.7 ± 7.4 2.7 
MCF-7 CPT 29.6 ± 9.6 180.3 ± 126.5 6.1 
 S38809 17.0 ± 10.2 111.7 ± 72.4 6.6 
 S39625 3.2 ± 1.9 17.8 ± 12.8 5.6 
DU145 CPT 21.5 ± 15.2 15,700 ± 1,300 730 
 S38809 13.6 ± 11.1 1,600 ± 100 120 
 S39625 0.5 ± 0.1 96.0 ± 53.3 190 
CEM CPT 4.9 ± 2.5 13,600 ± 6,300 2,800 
 S38809 11.6 ± 9.6 >10,000 >860 
 S39625 1.3 ± 1.1 720 ± 118 560 
Cell lineDrugIC50 (nmol/L)
RR
Control cellsResistant cells
HCT116 CPT 60.6 ± 22.0 169.5 ± 80.0 2.8 
 S38809 28.9 ± 11.1 92.0 ± 23.6 3.2 
 S39625 5.8 ± 3.0 15.7 ± 7.4 2.7 
MCF-7 CPT 29.6 ± 9.6 180.3 ± 126.5 6.1 
 S38809 17.0 ± 10.2 111.7 ± 72.4 6.6 
 S39625 3.2 ± 1.9 17.8 ± 12.8 5.6 
DU145 CPT 21.5 ± 15.2 15,700 ± 1,300 730 
 S38809 13.6 ± 11.1 1,600 ± 100 120 
 S39625 0.5 ± 0.1 96.0 ± 53.3 190 
CEM CPT 4.9 ± 2.5 13,600 ± 6,300 2,800 
 S38809 11.6 ± 9.6 >10,000 >860 
 S39625 1.3 ± 1.1 720 ± 118 560 

NOTE: Cytotoxic effect of S38809 and S39625 to human cancer cells is shown. IC50 values are in nanomolars. RR: relative resistance values obtained by dividing the IC50 values of each resistant cell line by parental cell line of control. In HCT116, MCF-7, or DU145 cells, sulforhodamine B assay was done after 72 h of continuous drug exposure. In CEM cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done after 72 h of continuous drug exposure. Values obtained were from four separate experiments as mean ± SD.

Sensitivity in the parent (control) cell lines indicated that S39625 and S38809 were about 10- or 2-fold more potent than CPT, respectively. Thus, the CPT keto analogues exhibit potent activity with IC50 values in the low nanomolar range in the different cell lines examined (Table 1). Table 1 also shows that all the CPT-resistant cell lines exhibited cross-resistance to S38809 and S39625. The resistance ratio (RR) for S38809 and S39625 was comparable with the RR for CPT in the two cell lines with Top1 down-regulation by siRNA (∼3 for the HCT116-siRNA cells; ∼6 for the MCF-7-siRNA cells), which is consistent with Top1 being the only target of S38809 and S39625 in those cells. Moreover, IC50 values in the two CPT-resistant cell lines with Top1 point mutations showed high cross-resistance (over a 100-fold) to both S38809 and S39625. Together, the results shown in Fig. 2 and Table 1 indicate that Top1 is the cellular target of the CPT keto analogues.

Induction of High Levels of Top1-DNA Cleavage Complexes by S39625 in Human Cells

We next used alkaline elution to quantitate Top1 cleavage complexes as DPCs (36, 40). Figure 3A shows that S38809 and S39625 efficiently produced DPC in a concentration-dependent manner. S39625 seemed remarkably more potent than CPT. It produced approximately the same levels of DPC at 0.1 μmol/L that CPT produced at 10-fold higher concentration (1 μmol/L; Fig. 3A). The potency of S38809 was intermediate between the potency of S39625 and that of CPT (Fig. 3A). These results are consistent with the immunocomplex of enzyme bioassay results (see Fig. 2B). Thus, the CPT keto analogues are potent Top1 inhibitors in cells.

Persistence of Top1 Cleavage Complexes Induced by S39625 after Drug Removal

To evaluate the stability of Top1 cleavage complexes induced by S38809 and S39625 in cells, we quantitated DPC at several time points following drug removal. Figure 3B shows that the reversibility of S39625-induced DPCs following drug-treated cells into fresh medium was much slower (t1/2 = 20 min) than that of CPT (t1/2 ≤ 4 min; refs. 40, 41). The Top1 cleavage complexes induced by S39625 remained detectable for at least 24 h, which is much longer than those induced by CPT, which were within limit of detection after 20 min (Fig. 3B) following drug removal (40, 41). The reversal of S38809-induced DPC was intermediate between S39625 and CPT. These results show that the keto analogue S39625 produces high levels of Top1 cleavage complexes (Fig. 3A) and that these cleavage complexes persist for hours following drug removal (Fig. 3B).

Next, we determined whether the slow reversal of the cellular Top1 cleavage complexes induced by S39625 was related to a direct effect of the drug on Top1. It has been previously shown that increasing the salt concentration can initiate the reversal of drug-stabilized Top1 cleavage complexes (5, 24, 42). Therefore, to further compare CPT with S38809 and S39625, the stability of Top1 cleavage complexes was assessed using a salt reversal assay. In this assay, different concentrations of each drug were used to produce an initial cleavage product of similar intensity. As shown in Fig. 3C, the reversal of Top1-DNA cleavage was much slower for S39625 than for S38809 and CPT. The apparent half-time of reversal for CPT, S38809, and S39625 was 2, 3, and 15 min, respectively (Fig. 3D). Thus, S39625 is markedly more efficient than CPT in stabilizing Top1 cleavage complexes.

Lack of Resistance of ATP-Binding Cassette Transporter-Transfected Cells to S39625

Previous studies have shown that the ATP-binding cassette (ABC) transporter ABCG2 mediates resistance to the clinical CPT analogues topotecan and SN-38 (the active metabolite of irinotecan; refs. 19, 23, 43). More recently, by using ABC transporter-transfected HEK293 cell lines, we showed that homocamptothecins were substrates for the ABCG2 transporter, although less than SN-38 (19). In the present study, we compared the involvement of the drug efflux ABC transporter family members ABCG2 and MDR-1/P-gp/ABCB1 for S38809 and S39625. For comparison, we used topotecan and SN-38, as CPT is not a preferred substrate for those transporters (19, 23).

Table 2 shows that cells overexpressing ABCB1 (MDR-1/P-gp) did not exhibit appreciable CPT resistance, whereas cell lines overexpressing the ABC half-transporter ABCG2 were found to be resistant to CPT, although not to a great extent (1.8-fold; P = 0.04). The clinical CPT analogues SN-38 and topotecan showed relatively high resistance in ABCG2-transfected HEK cells (SN-38, 46-fold; topotecan, 8.6-fold). In contrast, S38809 and S39625 showed lower relative resistance values in the ABCG2 transporter-infected cells (S38809, 5.8-fold; S39625, 3.7-fold). MDR-1–transfected cells only showed limited resistance to S38809 (∼3-fold; P = 0.26) but showed no resistance to S39625. We conclude that both CPT keto analogues are weaker substrates for the ABCG2 drug efflux transporter than the clinical CPT analogues (topotecan and SN-38).

Table 2.

Relative resistance to CPTs S38809 and S39625 in cell lines overexpressing ABCG2 and MDR-1/P-gp transporter proteins

DrugHEK293 control cells
HEK293-ABCG2–transfected cells
HEK293-MDR-1–transfected cells
Transporter implicated
IC50 (nmol/L)IC50 (nmol/L)RR1IC50 (nmol/L)RR2
CPT 19.6 ± 8.3 34.3 ± 8.3* 1.8 13.8 ± 8.5 0.7 ABCG2 
S38809 7.0 ± 2.6 40.6 ± 11.5* 5.8 19.1 ± 16.1 2.7 ABCG2 
S39625 1.3 ± 1.0 4.8 ± 1.2* 3.7 1.3 ± 0.7 1.0 (ABCG2) 
SN-38 5.3 ± 1.2 244.8 ± 67.7 46.0 7.3 ± 0.1* 1.4 ABCG2 and MDR-1 
Topotecan 35.8 ± 7.5 308.8 ± 10.2 8.6 51.3 ± 5.5* 1.4 ABCG2 and MDR-1 
DrugHEK293 control cells
HEK293-ABCG2–transfected cells
HEK293-MDR-1–transfected cells
Transporter implicated
IC50 (nmol/L)IC50 (nmol/L)RR1IC50 (nmol/L)RR2
CPT 19.6 ± 8.3 34.3 ± 8.3* 1.8 13.8 ± 8.5 0.7 ABCG2 
S38809 7.0 ± 2.6 40.6 ± 11.5* 5.8 19.1 ± 16.1 2.7 ABCG2 
S39625 1.3 ± 1.0 4.8 ± 1.2* 3.7 1.3 ± 0.7 1.0 (ABCG2) 
SN-38 5.3 ± 1.2 244.8 ± 67.7 46.0 7.3 ± 0.1* 1.4 ABCG2 and MDR-1 
Topotecan 35.8 ± 7.5 308.8 ± 10.2 8.6 51.3 ± 5.5* 1.4 ABCG2 and MDR-1 

NOTE: IC50 represents the concentration of drug that is required for 50% growth inhibition. RR1: relative resistance values obtained by dividing the IC50 values of the ABCG2-overexpressing cell line (HEK293-482R-5) by the IC50 of the respective parental cell line. RR2: relative resistance values obtained by dividing the IC50 values of the MDR-1/P-gp/ABCB1–overexpressing cell line (HEK293-MDR-19) by the IC50 value of the respective parental cell line. Values obtained were from four separate experiments as mean ± SD. Each P value was analyzed by unpaired t test analysis.

*

P < 0.05.

P < 0.01.

Formation of Histone γ-H2AX Foci in Cells Treated with S38809 and S39625

As histone H2AX phosphorylation (at its COOH terminus on Ser139; γ-H2AX) appears within minutes after ionizing radiation (44, 45) and is induced by CPT (39), γ-H2AX focus production is considered to be a sensitive and selective marker for the existence of DNA damage in cells. Moreover, γ-H2AX could serve as a biomarker for clinical trials (2). To investigate whether S38809 and S39625 induced γ-H2AX, cells were treated with 10 nmol/L drug concentrations for 1 h, and γ-H2AX signal was followed for several hours after drug removal. Figure 4 shows that both S38809 and S39625 were potent inducers of γ-H2AX. The γ-H2AX was already strong 1 h after drug exposure (time 0 in Fig. 4). The γ-H2AX signals produced by 10 nmol/L S39625 were almost the same intensities as those produced by 100 nmol/L CPT (data not shown). Furthermore, γ-H2AX persisted for up to 24 h after drug removal. Together, these results show potent and durable induction of histone γ-H2AX by the CPT keto analogues. They also show that S39625 produced the most intense and durable γ-H2AX signal.

Figure 4.

Production of histone γ-H2AX foci in HCT116 cells treated with 10 nmol/L of CPT, S38809, or S39625. A, representative pictures of drug-induced γ-H2AX foci (green) in HCT116 cells treated for 1 h in the presence or absence of drug. Following this treatment, the drug was removed and the cells were incubated with drug-free medium for 0, 4, or 24 h. Green, cells were stained with mouse anti-γ-H2AX antibody and goat anti-mouse antibody conjugated with Alexa Fluor 488. Red, nuclei were stained with propidium iodide. B, graphical representation of the percentage of γ-H2AX–positive cells at 0, 4, and 24 h after drug removal. γ-H2AX foci-positive cells were determined from independent images, which included >500 cells for each time point. C, graphical representation of the average intensity of γ-H2AX staining at 0, 4, and 24 h after drug removal. The average intensity of γ-H2AX was measured by histogram mode for green signal on Adobe Photoshop normalized to the number of cells.

Figure 4.

Production of histone γ-H2AX foci in HCT116 cells treated with 10 nmol/L of CPT, S38809, or S39625. A, representative pictures of drug-induced γ-H2AX foci (green) in HCT116 cells treated for 1 h in the presence or absence of drug. Following this treatment, the drug was removed and the cells were incubated with drug-free medium for 0, 4, or 24 h. Green, cells were stained with mouse anti-γ-H2AX antibody and goat anti-mouse antibody conjugated with Alexa Fluor 488. Red, nuclei were stained with propidium iodide. B, graphical representation of the percentage of γ-H2AX–positive cells at 0, 4, and 24 h after drug removal. γ-H2AX foci-positive cells were determined from independent images, which included >500 cells for each time point. C, graphical representation of the average intensity of γ-H2AX staining at 0, 4, and 24 h after drug removal. The average intensity of γ-H2AX was measured by histogram mode for green signal on Adobe Photoshop normalized to the number of cells.

Close modal

The present study shows that the novel nonlactone CPT keto analogues S38809 and S39625 are potent and selective Top1 inhibitors that induce stable and persistent Top1-DNA cleavage complexes. They are antiproliferative at nanomolar concentrations and overall more active than CPT in the various cancer cell lines examined in the present study. The CPT keto analogues are not substrates for the drug efflux transporters and induce the formation of histone γ-H2AX, which may serve as a pharmacodynamic biomarker. S39625 is currently in advanced preclinical development.

At the present time, the CPT derivatives topotecan and irinotecan are the only Top1 inhibitors available for cancer therapy (2). CPT derivatives penetrate cells readily and target Top1 selectively within minutes of exposure (40). The exquisite selectivity of CPT for Top1 and its unique mechanism of action led us to propose that CPT represents a paradigm for “interfacial inhibitors” (i.e., natural products that bind at the interface of macromolecular complexes and reversibly trap catalytic or/and structural intermediates; refs. 46, 47). In spite of these remarkable features, CPT and its clinical derivatives have three critical limitations. First, the α-hydroxylactone E-ring of CPTs is rapidly converted within minutes into its inactive carboxylate form in blood (see Fig. 1A; refs. 4, 15). Second, because CPTs bind the Top1-DNA complexes noncovalently, they dissociate and diffuse from the Top1 cleavage complexes rapidly (3, 5, 4042), resulting in the need for prolonged infusions to maintain persistent cleavage complexes that in turn generate DNA damage and death-initiating signals (2). Third, irinotecan and topotecan are substrates for the drug efflux ABC transporters ABCG2 (mitoxantrone resistance/breast cancer resistance protein; refs. 19, 23) and, to a lesser extent, ABCB1 (MDR-1; ref. 22), which may confer intrinsic or acquired resistance to those CPT derivatives.

The keto analogues seem to overcome the main limitations of the CPT derivatives. First, removal of the oxygen from the CPT lactone E-ring stabilizes the E-ring (20). Second, the Top1 cleavage complexes induced by the keto derivative S39625 are markedly more persistent than those induced by CPT both in cells and at the molecular Top1 level (see Fig. 3). It is likely that the cyclobutyl substitution at the 7-position of S39625 (see Fig. 1A) contributes markedly to the retention of S39625 into the Top1-DNA complexes as S38809 does not exhibit these favorable characteristics (see Fig. 3). Crystal structure and molecular modeling analyses of S39625 in the Top1 cleavage complex6

6

Unpublished crystal structure results from Dr. Lance Stewart and coworkers at DeCode Biostructures, Brainbridge Island, WA, and our own molecular docking results.

show that the cyclobutyl substitution occupies an open space in the DNA major groove. Thus, by filling this cavity, the cyclobutyl group may slow the drug dissociation from the Top1 cleavage complex. Finally, we found that S39625 is not a substrate for either of the drug efflux transporter implicated in CPT resistance, ABCG2 (mitoxantrone resistance/breast cancer resistance protein; refs. 19, 23) and ABCB1 (P-gp/MDR-1; see Table 2; ref. 22). Therefore, our results suggest that S39625 may hold the promising possibility to overcome the limitations of CPTs.

We also found that S39625 exerts antiproliferative activity at low nanomolar concentrations in all the human cancer and transformed cell lines examined (Tables 1 and 2). Those inhibitory concentrations are lower than those required for CPT and topotecan to exert similar effects. Additional experiments showed similarly high potency for S39625 by clonogenic assays in HCT116 cells (data not shown), which shows the efficacy of the keto analogues at killing tumor cells. To further show that the cytotoxicity of S39625 does translate into in vivo antitumor activity, HCT116 cells were grafted to nude mice and treated by S39625, administered either i.v. (once a week for 3 weeks) or p.o. (once a day, 5 days a week for 3 weeks). S39625, at its optimal dose of 12.5 mg/kg i.v., completely inhibited tumor growth. This effect lasted for at least 35 days after the last injection. The weight losses were lower than 15%. Administered at 6.25 mg/kg p.o., a similar antitumoral effect was observed, without any weight loss. S39625 was also active in five other xenografts, inducing long-term tumor-free animals in prostate carcinoma and breast carcinoma models. Overall, S39625 was more active than topotecan and was as active as irinotecan and Taxol (data not shown). Thus, the keto derivative S39625 represents a promising candidate Top1 inhibitor for development (2).

As previously published, histone γ-H2AX is formed within an hour in cells treated with CPT. This response has been attributed to the conversion of Top1 cleavage complexes into replication-mediated DNA double-strand breaks (39, 48). The present data show that the nanomolar concentrations of the keto analogues also produced intense γ-H2AX signals within 1 h (see Fig. 4). Moreover, the γ-H2AX signals persisted and even increased following drug removal and were the most intense for S39625, which is also the most effective antiproliferative derivative. The increase in signal is likely due to secondary fragmentation related to apoptosis (49). Thus, γ-H2AX could be considered as a potential pharmacodynamic biomarker for monitoring the activity of S39625 in the upcoming clinical trials.

The studies presented here show that the CPT keto analogues, and particularly S39625, are more stable than CPT and are more potent in cytotoxicity and γ-H2AX assays. Further, the cleavage complexes induced are significantly more stable. These differences offer the prospect of significant differences from the already developed agents. This increased potency could be accompanied by increased toxicity, and that will be answered in time. Hopefully, however, the increased potency and stability will allow a greater therapeutic window. This question will have to be resolved in clinical trials.

Grant support: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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.

We thank Dr. Neil Osheroff for providing us with the yeast cells, Dr. Yung-Chi Cheng for generous gift of the C21 monoclonal Top1 antibodies, Robert W. Robey (Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, NIH) for doing assistance with the ABCB1 and ABCG2 cell lines, and Dr. John Hickman (Centre de Recherches Servier) for helpful discussions and continuous support of this project.

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