Cellular Resistance to the Antitumor DNA Topoisomerase II Inhibitor S16020-2: Importance of the N-[2(Dimethylamino)ethyl]carbamoyl Side Chain

The new olivacine derivative S16020-2 (NSC-659687) is a DNA topoisomerase II inhibitor endowed with a remarkable antitumor activity against various experimental tumors. In vitro physicochemical properties of this compound, in particular its interaction with DNA and DNA topoisomerase II, were very similar to those of ellipticine derivatives, except for a strictly ATP-dependent mechanism of cleavable complex induction. From the Chinese hamster lung fibroblast cell line DC-3F, a subline resistant to S16020-2, named DC-3F/S16, was selected by adding stepwise increasing concentrations of the drug to the cell growth medium. Whereas DC-3F/9-OH-E cells, a DC-3F subline resistant to 9-hydroxy-ellipticine, are cross-resistant to S16020-2, DC-3F/S16 cells are only very weakly cross-resistant to ellipticine derivatives, indicating that, despite their structural similarity, these compounds may differ in their mechanisms of action. Uptake and efflux rates of S16020-2 were identical in the resistant and the sensitive cells. Topoisomerase IIa was expressed at the same level in both sensitive and resistant cells, whereas expression of the b-enzyme was approximately 50% lower in the resistant cells. Sequencing of both aand b-isoform cDNAs revealed a point mutation that converts Arg to a Gly in the a cDNA, whereas the b cDNA was not modified. This amino acid substitution in a highly conserved sequence of the enzyme appears to be responsible for the resistance to S16020-2. Comparative analysis of the properties of the ellipticine and S16020-2-resistant cells suggests that S16020-2, which is a DNA intercalator, might also interact with this enzyme amino acid sequence through its side chain. DNA topoisomerase II inhibitors used in human cancer chemotherapy constitute a group of structurally unrelated compounds that share a common property, the capacity to induce a significant increase of the number of covalent enzyme-DNA complexes (cleavable complexes) present on the cell genome at a given time, thus triggering a cell response eventually leading to cell death by apoptosis (Liu, 1989; Froelich-Ammon and Osheroff, 1995). However, probably as a consequence of their structural diversity, DNA topoisomerase II inhibitors have been shown to be active in vitro through different mechanisms. It is generally admitted that these compounds are involved in the formation of a drugenzyme-DNA ternary complex. Some drugs enter this complex by interacting predominantly with the protein (etoposide) or the DNA (ellipticine), but it is likely that most DNAintercalating drugs bind to both the DNA, through intercalation of the chromophore at the enzyme-DNA interface, and the enzyme, through side chains, which are usually essential to their pharmacological activity. Some drugs [49(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), etoposide] can inhibit the religation of the cleaved DNA, whereas others (ellipticine, genistein, quinolones) are presumed to accelerate the forward rate of complex formation (Froelich-Ammon and Osheroff, 1995). The relatively simple structure of ellipticine has prompted chemists to design various structural modifications to synthesize compounds endowed with increased activity and specificity (Le Pecq et al., 1974; Auclair, 1987a). More than 70 molecules were thus obtained, but only one, 2-N-methyl9-hydroxy-ellipticinium (NMHE), was finally used in human clinics with a moderate efficacy (Paoletti et al., 1980; Rouessé et al., 1993). Olivacine, a natural ellipticine isomer, differing by the shift of the methyl group from the 11 to the 1 position, This work was supported in part by grants from Association pour la Recherche sur le Cancer (Villejuif, France), Ligue Nationale Française contre le Cancer (Comité de l’Essonne), and Fondation de France (Paris). J-M.S. is supported by Institut National de la Santé et de la Recherche Médicale, and S. L was awarded fellowships from Association pour la Recherche sur le Cancer, Ligue Nationale Française contre le Cancer, and Société Française du Cancer. ABBREVIATIONS: m-AMSA, 49-(9-acridinylamino)methanesulfon-m-anisidide; 9-OH-E, 9-hydroxy-ellipticine; NMHE, 2-N-methyl-9-hydroxyellipticinium; MDR, multidrug resistance; SSB, single-strand break. 0026-895X/00/040709-10$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 4 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 13080/854113 Mol Pharmacol 58:709–718, 2000 Printed in U.S.A. 709 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from was not nearly as extensively studied, although it displayed a significant antitumor activity on various experimental tumors (Pierré et al., 1997). Recently, a new series of 6H-pyrido[4,3b]carbazole derivatives, characterized by a basic N-dialkylaminoalkyl carboxamido side chain grafted onto an olivacine chromophore, was synthesized (Jasztold-Howorko et al., 1994). Some of these compounds displayed a remarkable activity against various experimental tumors. The most active of these compounds, S16020-2 (NSC-659687) (Fig. 1), has demonstrated a broad range of antitumor activity against a panel of murine and human tumor xenografts, being particularly active against the Lewis lung carcinoma and the human non-small cell lung carcinoma NCI-H460, two highly aggressive and chemoresistant models. In these models, NMHE was inactive and S16020-2 was at least as active as adriamycin taken as a reference in preclinical studies. Recently, two orthotopic models were used to confirm the potential activity of S16020-2 against experimental lung cancer. Murine Lewis lung carcinoma and human A549 tumor cells were grafted by the i.v route, the latter into SCID mice, to obtain lung metastases, which progressively invade the lungs resulting in the death of the animals. S16020-2 totally inhibited the growth of lung metastases and cured 89% of Lewis lung carcinoma-bearing mice and increased by 50% the survival of A549-bearing mice (Guilbaud et al., 1997). In addition, the drug retained its activity on several sublines expressing the multidrug resistance (MDR) phenotype (Guilbaud et al., 1996; Pierré et al., 1998). All these observations made S16020-2 a very promising antitumor agent. Because of its favorable pharmacokinetic characteristics and acceptable toxicity in different species (Pierré et al., 1997), S16020-2 is presently in clinical trials. The molecular basis for the remarkable antitumor activity of S16020-2 is currently unknown. In vitro, S16020-2 displays strong similarities with NMHE (Le Mée et al., 1998). S16020-2 intercalates between adjacent DNA base pairs with an affinity comparable to that of NMHE. This new olivacine derivative is a specific topoisomerase II inhibitor that stimulates the cleavable complex formation in a concentration range close to that of NMHE and has no detectable effect on the DNA religation rate. However, S16020-2 differs markedly from any other topoisomerase II poison by its absolute ATP requirement to stimulate the enzyme-mediated DNA cleavage. In contrast with the results obtained in vitro, S16020-2 was at least 30-fold more cytotoxic than NMHE on the DC-3F cell line, and in these cells, the formation of the cleavable complexes was detected at concentrations 500-fold lower than with NMHE. We now report the selection and properties of S16020-2resistant cells derived from the Chinese hamster fibrosarcoma cell line DC-3F. Comparison of these cells with DC-3F/9-OH-E cells, a DC-3F variant resistant to 9-OH-E, led us to propose a new model for the mechanism of action of S16020-2. Materials and Methods Cell Lines and Culture Conditions. The parental Chinese hamster lung fibroblast cell line DC-3F, the S16020-2-resistant subline DC-3F/S16, the 9-OH-E-resistant subline DC-3F/9-OH-E (Salles et al., 1982), clone 24 (Khélifa et al., 1999), and clone 11 cells (Dereuddre et al., 1997) were maintained as monolayer cultures in Eagle’s minimal medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 50 mg/ml streptomycin (Dereuddre et al., 1997). The cells were incubated at 37°C in a humidified incubator with 5% CO2 in air. The resistant cells DC-3F/S16 and DC-3F/9OH-E were permanently grown in the presence of 100 nM S16020-2 and 0.6 mg/ml 9-OH-E, respectively. Before each experiment, the cells were grown for two or three passages in the absence of drug. Drugs and Chemicals. S16020-2 and S30761, the ellipticine analog of S16020-2 with the side chain grafted at position 1, were provided by Institut de Recherches Servier (Suresnes, France); NMHE was purchased from Pasteur-Mérieux (Lyon, France), doxorubicine (Adriblastine) from Farmitalia (Rueil Malmaison, France), vincristine (Oncovin) from Lilly France S.A. (St. Cloud, France), and actinomycine D (Lyovac Cosmegen) from MSD (Whitehouse Station, NJ). These drugs were dissolved in H2O. 9-OH-E was kindly provided by Dr. E. Lescot (Institut Gustave Roussy, Villejuif, France) and dissolved in HCl 10 M. Etoposide and m-AMSA, obtained from Sigma Aldrich Chimie (Fallavier, France), were dissolved in dimethylsulfoxide. Stock solutions at 0.01 M were prepared for each drug. [H]S16020-2 (39 Ci/mmol) was synthesized by the CEA (Saclay, France). Population-Doubling Time. DC-3F cells (10) and DC-3F/S16 cells (5 3 10) were plated in 60-mm-diameter Petri dishes and incubated at 37°C. The cells were counted in duplicate every 12 h for 4 days. The data were analyzed by plotting the logarithm of the cell number as a function of time, and the population generation time was calculated from the exponential part of the growth curve. Tumorigenicity. For determination of tumorigenicity, 1 or 2 3 10 cells were injected s.c. into the left posterior flank of 6to 8-week-old female nu/nu mice. The mice were examined biweekly for development of tumors. Cells were scored as tumorigenic if a palpable nodule appeared and grew progressively at the injection site within 10 weeks. Otherwise the mice were kept for at least 3 months after injection before being scored as nontumorigenic. Assay of Drug Sensitivity. All of the experiments were carried out on exponentially growing cells. Two methods were used for determination of drug sensitivity: 1) The 72-h assay. The sensitivity of the DC-3F and DC-3F/S16 cells to the different drugs was determined by cell counting after 72-h drug exposure (Salles et al., 1982). Wells (16 mm) of a 24-well Falcon dish, containing 1 ml of minimal essential medium with graded drug concentrations, were inoculated with 1 ml of cell suspension containing either 10 parental cells or 2 3 10 resistant cells. After 72 (62) h of drug exposure, the number Fig. 1. Structures of olivacine and ellipticine derivatives. 710 Le Mée et al. at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from of cells per well was determined with a model ZM Coulter Counter (Coultronic France, Margency, France). 2) The colony formation assay. This assay determines the cloning efficiency of the cell survival fraction after 3-h drug exposure. Parental cells or resistant cells (2 to 10 or 4 to 10, respectively) were grown into 60-mm-diameter Petri dishes (Falcon, Becton-Dickinson, Lincoln Park, NJ) for 18 h at 37°C before drug treatment. After 3 h of exposure to the drugs, the cells were trypsinized, and appropriate dilutions were made to seed 250 or 500 treated cells into 60-mm-diameter Petri dishes. Colonies were stained and counted 6 to 8 days later. Drug Uptake and Efflux Studies. Cells were grown in 35-mm wells containing 4 ml of growth medium for 24 h to a cell density of 5 3 10 to 1 3 10 cells/well. The growth medium was then removed and replaced with 1 ml of fresh medium containing H-labeled S16020-2 at a concentration of 50 nM (specific activity adjusted to 2 Ci/mmol). At various times thereafter, the cells were washed three times with 1 ml of PBS at 4°C and lysed overnight with 1 ml of 2% Triton X-100 at room temperature. The cells lysates were withdrawn, and each well was washed with 1 ml of PBS. Each lysate and wash was collected in a scintillation vial and mixed with 10 ml of Ultima Gold (Packard Instruments Co., Meriden, CT) for liquid scintillation counting. Cell counting was carried out on duplicate samples grown in the same conditions with a model ZM Coulter