Antitumor Activities of a New Indolocarbazole Substance, NB-506, and Establishment of NB-506-Resistant Cell Lines, SBC-3/NB'

[CANCER RESEARCH 55, 2806-2813, July 1, 19951 Antitumor Activities of a New Indolocarbazole Substance, NB-506, and Establishment of NB-506-resistant Cell Lines, SBC-3/NB'

Fumthiko Kanzawa, Kazuto Nishio, Naohiro Kubota, and Nagahiro Saijo2

Pharmacology Division. National Cancer Center Research Institute. 1-1, Tsukiji 5 Chome, Chuo-ku, Tokyo 104. Japan

ABSTRACT somerase II in proliferating cells and very low levels in quiescent cells appear to explain the selective sensitivities of proliferative tumor cells The novel anticancer glucosyl derivative of indolo-carbazole (NB-506), to the cytotoxic effects of topoisomenase LI-targeting drugs (3). In an inhibitor of DNA topoisomerase I, exhibited strong in vitrocytotoxicity contrast, the intracellular levels of topoisomerase I have been reported against various human cancer cell lines. In order to elucidate its cytotoxic to be largely unaffected by cultured cell growth conditions. However, mechanisms, we established nine NB-506-resistant sublines with different resistance ratios from human small cell lung cancer cells (SBC-3/P) by the findings of Giovanella et a!. (7) that topoisomenase I levels were stepwise and brief exposure (24 h) to NB-506. Among them, SBC-3INB#9 elevated in advanced stage human colon cancer tissues compared with was 454 times more resistant to NB-506 than the parent cell line. The those in normal colon tissues lend support to the possibility that this SBC-3/NB#9cellsshowedcross-resistanceonlyto topoisomeraseIinhib enzyme also is an important target for antitumor drugs. In agreement itors, such as 11,7-ethyl-10-[4-(1.piperidino)-1-piperidlno] carbonyloxy with these results, recent clinical trials of camptothecin derivatives camptothecinand 7-ethyl-lO-hydroxy-camptothecin,andnot to other an have shown that these drugs are potentially promising new antitumor ticancer drugs, such as vincristine, vinblastine, Adriamycin, etoposide, agents (8â€”10). and teniposide. These results indicate that the difference on the effect of Therefore, the identification of new drugs that induce the formation topoisomerase I was considered to be related to a resistance mechanism. of cleavable topoisomerase I complexes is now viewed as a promising The topoisomerase I activities of nuclear extracts eluted from SBC-3/ approach to fmding clinically effective antitumor agents. In an attempt NB#9 cells was only one-tenth of the parent cell activity. A Western blotting study indicated that this lower activity was due to a lower amount to discover new antitumor agents that target topoisomerase I, Kojiri et of DNA topoisomerase I. Furthermore, we found correlations between a!. (1 1) screened Actinomycetes culture supennatants and isolated a topoisomeraseI activityand sensitivityto NB-506in subilneswith differ novel indolocarbazole antibiotic, BE-13793C. Although BE13793C ent degrees of resistance. Accumulation of 3H-labeled NB-506 by SBC-3/ showed topoisomenase-inhibiting activity and good activity against NB#9 cells was only one-fifth of that by the parent cells, whereas intra Ehrlich ascites tumor cells, its low solubility in water hampered cellular accumulation of 3H-labeled camptothecin by both cell lines did further evaluation. Therefore, they synthesized a glucosyl derivative not differ. The reduction of accumulation was specific to NB-506, and this of BE-13793C, NB-506 (the chemical structure of which is shown in result may explain why the resistance ratio for NB-506 was higher than Fig. 1), which was more water soluble. NB-506 was shown to have those for 11,7-ethyl-1O-[4-(1-piperidlno)-1-piperidino] carbonyloxycamp strong effects in inhibiting the growth of various experimental tumors, tothecin and 7-ethyl-lO-hydroxy-camptothecin. including solid tumors, not only in vitro but also in vivo (12). Its cytotoxicity was demonstrated to lead by the induction of topoisomerase INTRODUCTION I-mediated DNA cleavage (13). In this study, we evaluated the activity of NB-506 against human The DNA topoisomerases are nuclear enzymes that catalyze the tumor cells in vitro. Furthermore, we tried to establish NB-506- concerted breaking and rejoining of DNA strands, thereby controlling resistant cell lines and elucidate the likely mechanisms of their drug the topological states of DNA. Topoisomerase I catalyzes the passage resistance, because development of drug resistance is likely be a major of DNA strands through transient single-strand breaks, whereas to limiting factor in determining the clinical success of NB-506. poisomerase II catalyzes the passage of DNA double strands through transient double-strand breaks. These topoisomerases are known to be involved in many DNA metabolic processes, including replication, MATERIALS AND METHODS recombination, transcription, and chromosome segregation at mitosis (1). In addition to their normal cellular functions, both topoisomerases Materials. The indolocarbazole derivative, NB-506, and its tnitium-labeled compound were obtained from Banyu Tsukuba Research Institute (Tsukuba, I and II have generated extensive clinical interest as targets for cancer Japan). CVF-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy camp chemotherapy. tothecin and SN-38 (7-ethyl-1-hydroxy-CPT) were obtained from Yakult Co., Eukaryotic topoisomerase II is the target for intercalative antitumor Ltd. (Tokyo, Japan). Cisplatin, etoposide, and teniposide were gifts from agents, such as 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and Bristol Myers Japan (Tokyo, Japan), and 5-fluorouracil was supplied by Mitsui Adriamycin, as well as nonintercalative agents like etoposide and Pharmaceuticals, Inc. (Tokyo, Japan). Adriamycin, vincristine, vinblastine, teniposide (2, 3). Eukaryotic topoisomerase I is the target for the meiphalan, and bleomycin were purchased from Kyowa Hakko Kogyo Co., antitumor plant alkaloid camptothecin (2â€”4)and its synthetic deny Ltd. (Tokyo, Japan), Shionogi Pharmaceutical Co. (Osaka, Japan), Kyorin atives, such as CPT-1 i@ (5) and topotecans (6). High levels of topoi Pharmaceutical Co., Ltd. (Tokyo, Japan), Sigma Chemical Co. (St. Louis, MO), and Nihon Kayaku Co., Ltd. (Tokyo, Japan), respectively. Plasmid pBR322 DNA was purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan), and Received 12/30/94; accepted 5/1/95. The costs of publication of this article were defrayed in part by the payment of page tnitium-labeled 12-3H(N)-camptothecin was purchased from Moravek charges. This article must therefore be hereby marked advertisement in accordance with Biochemicals, Inc. (Brea, CA). 18 U.S.C. Section 1734 solely to indicate this fact. Cell CUltUreand Isolation of NB-506-resistant Cell Lines. The cell line I This work was supported by Grants-in-Aid for cancer research from the Compre used as the parent for obtaining NB-506-resistant sublines was SBC-3fP, which hensive Ten-Year Strategy for Cancer Control, from the Ministry of Health and Welfare, Japan. is sensitive to NB-506 and CPT-1 1 and was derived from a human small cell 2 To whom requests for reprints should be addressed. lung carcinoma (kindly provided by Professor Kimura, Okayama University, 3 The abbreviations used are: CVF-1 1; 1 1,7-ethyl-10-[4-(1-piperidino)-1-piperidinoj School of Medicine, Okayama, Japan). The cells were propagated in RPMI @ carbonyloxycamptothecin; CPT, camptothecin; SN-38; 7-ethyl-lO-hydroxy-camptoth 1640 supplemented with 10% FCS, 100 g/ml streptomycin, and 100 units ecin; IC5@,theconcentrations required to inhibit cell proliferation by 50% compared with control cultures; PKC, protein kinase C; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe penicillin/ml in an incubator under a humidified atmosphere of 5% CO2andair, nyltetrazolium bromide. as described previously (14). 2806

drugs in the agar throughout the growth period in vitro. The exponentially growing cells (9 x 10'@)weresuspended in 2.7 ml RPMI 1640 containing the required drug, to which 0.3 ml hot agar solution (3%) was added. The resulting o@ mixture was plated as the top layer on an underlayer of RPMI 1640 supple mented with 0.5% agar in a 35-mm plastic dish, and the plates were incubated under a humidified atmosphere of 5% carbon dioxide and 95% air at 37Â°Cfor 9 days, after which the number of colonies on the triplicate control and drug-treated plates were counted by a computerized image analyzer (Colony Analyzer CA-7A; Oriental Instrument Ltd., Tokyo, Japan). (c) The MTF assay was performed as follows. Aliquots (100 pA) of an exponentially growing cell suspension (1 X 10@cells/mi) were seeded in 96-well microtiter plates, and 100-pi aliquots of the required drug solution OH (various concentrations) were added. After exposure to the drugs for 96 h at 37Â°C,20 @LlMTF solution (5 mg/ml in PBS) was added to each well. The plates were incubated at 37Â°Cfor an additional 4 h and centrifuged at 800 X g for 5 mm. The medium was aspirated from each well as completely as possible. DMSO (200 pi) was added to each well to dissolve the formazan,and the Fig. 1. Chemical structure of NB-506. absorbances of the solution in each well were measured at 562 and 630 nm using Delta-soft ELISA analysis for a Macintosh computer interfaced to a Bio-Tek Microplate Reader (EL-340; Bio Metallics, Princeton, NJ.). Wells Each NB-506-resistant cell line was selected from a subculture that had containing only RPMI-FBS and M'Vf were used as controls. Each experiment acquired resistance to NB-506 as a result of stepwise and brief exposure to was performed using six replicate wells for each drug concentration, and three NB-506. cultured SBC-3 cells were exposed to NB-506 at an initial concen independent experiments were earned out (16). tration of 2.5 ,.&g/ml for 24 h and then were washed and cultured in drug-free Accumulation Study. To evaluate drug accumulation, exponentially grow medium for 6 days. This treatment was repeated when the treated cells grew at ing SBC-3/P and SBC-3/NB#9 cells were harvested. Cells (2 X 10@)in0.98 the same proliferation rate as untreated cells, and the resulting cells were ml culture medium were preincubated at 37Â°Cina water bath for 45 mm and exposed to a concentration of the drug about three times higher than the then exposed to 75 p@ [3H]NB-506or camptothecin at 37Â°Cina humidified preceding one. Each such procedure was termed an episode of drug treatment, incubator with 5% CÂ°2.Aftervarious incubation times, the cells were collected and after several successive episodes, nine NB-506-resistant sublines were by low-speed centrifugation, washed once with cold PBS, and recentrifuged. established using the limiting dilution technique (Fig. 2). The resulting cell pellet was dissolved in 0.5 ml formic acid and 3 ml Clearsol Drug Sensitivity Test. The sensitivity and resistance of each cell line to 1 solution was added (Nacalai Tesque, Kyoto, Japan)to each tube, and the NB-506 and other drugs were estimated using three different methods. (a) We radioactivity was measured with a liquid scintillation counter (LS6000TA; evaluatedthecytotoxiceffect of continuousdrugexposureon cell proliferation Beckman, Irvine, CA; Ref. 17). using the regrowth assay described previously (15). Duplicate 10-mi culture Preparation of Nuclear Extracts and DNA TopoisomeraseI Activity flasks,initiallycontaining2.5 X 10@cells/mimediumand the requireddrugs Assay. Crude nuclear extracts were prepared, as described by Deffie et a!. at various concentrations, were incubated for 7 days at 37Â°Cundera humid (18). The cells were collected by centrifugation and washed twice with ice-cold ified atmosphere of5% carbon dioxide and 95% air, after which the cells were nuclear buffer (NB; pH 6.5, 2 mMK2HPO45 mMMgC12150 mMNaCl, 1 mM counted either with a TOA Microcell counter CC-108 (TOA Medical Elec EGTA,and0.1 mMDli'), recentrifuged,resuspendedin 1 ml cold NB, and9 tronica Co., Kobe, Japan) or under a microscope, and the cell proliferation ratio ml cold NB containing 0.35% Triton X-100 and 1 mMphenylmethylsulfonyl of treated to control cultures was calculated. The antiproliferative activities fluoride were added. The cell suspension was kept on ice for 10 mm and were expressed as IC@s. washed with Triton X-100-free cold NB; then the nuclear protein was eluted (b) Forthesoftagarcolonyinhibitionassay(15),thecellswereexposedto for 1 h at 4Â°Cwithcold NB containing 0.35 MNaCl. A nuclear protein solution

I ParentSBC-3/Pcells

2.5 @igJmI,3courses

5 @ig/ml,3courses

10jsg/mI,2courses Fig. 2 Genealogy of the NB-506-resistant sublines, SBC-3/NB. Nine NB-506-resistant SBC-3 sublines were established after several drugtreatmentepisodes,asdescribedinâ€œMaterialsandMethods.â€• Thedrugtreatmentwascarriedoutas follows.Cellswereexposed 10pig/mI,3courses 25 jsg/ml,2 courses to the indicatedconcentrationsfor24 h, washed,and culturedin drug-free medium for 6 days; and the procedure was repeated with the next concentration. Resistant sublines were cloned using the cloning 50 jsg/m!, 2 courses limiting dilution technique. I#i@234 100 @tg/ml,1courses

250 jsg/ml, 7 courses 100 @sg/ml,7courses cloning I#56cloning I#789

2807

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1995 American Association for Cancer Research. ESTABUSHMENTOF CELLS RESISTANTTO NB-506 was obtained by centrifugation at 18,000 X g for 10 min, and mtsprotein topoisomerase I, because the CPT-1 1 resistance mechanism in these concentration was determined using the method of Bradford (19) with bovine cells has been reported to be attributable to reduced topoisomerase I plasma -y-globulinas the standard. activity (14). The DNA topoisomerase I activity was determined by measuring the relax Establishment of NB-506-resistant SBC-3 Cells. Several cell ation of supercoiled Escherichia coli DNA (jBR322), essentially as described lines resistant to NB-506 were established using the technique de by Liu and Miller (20). The reaction mixtures for measuring the total topoi scribed previously for selecting of the PC-7tCPT sublines (14). Cul somerase I activities in both cell lines comprised 100 m@tKCI,10 m@iMgCl2, 1 mM DTF, 0.1 mM EDTA, 10% glycerol, 50 mM Tris-HCI (pH 7.4), 1.7 @g tured SBC-3/P cells were exposed to NB-506 at an initial concentra pBR322 DNA, and crude nuclear extracts (0.001â€”2.0p@gprotein). tion of 2.5 ,.tg/ml for 24 h, washed, and cultured in drug-free medium The reaction mixtures for measuring the inhibition of DNA relaxation by until cell proliferation resumed. Then, the resulting cells were exposed NB-506 contained the specified concentrations of drug and amounts of nuclear to about three times the previous drug concentration. This procedure, extracts (0.01 @gforSBC-31Pand 0.05 g.@gforSBC-3INB#9) in addition to termed a drug treatment episode, was repeated when the cells showed the above components. The reaction mixtures were incubated at 37Â°Cfor20 the same proliferation ratio as untreated cultures. After the treatment mm, and the reactions were terminated by adding 5 @ildyesolution (2.5% SDS episode with 10 @.tWmlNB-506, the cultured cells were cloned using and 0.01% bromophenol blue in water). These samples were applied to 0.7% a dilution technique, which resulted in the establishment of four agarose gel and electrophoresed for 3 h with a running buffer of Tris-acetate EDTA, after which the gel was stained with 2 @Methidium bromide and NB-506-resistant SBC-3 sublines (designated SBC-3INB#1, #2, #3, photographed under transillumination with 300 nm UV light. and #4). Furthermore, SBC-3INB#5 and #6 sublines were derived DNA Topoisomerase I Content. We used Westernblottinganalysiswith from a subculture that acquired resistance to NB-506 by passaging topoisomerase I antiserum (20) to determine the cellular topoisomerase I through three more successive episodes with 25, 50, and 100 ,i@g/ml contents. In brief, stored nuclear extracts (â€”80Â°C)were analyzed on 12.5% NB-506; and SBC-3INB#7, #8, and #9 sublines were established polyacrylamide slab gels containing SDS (21). Equivalent amounts of nuclear from a subculture that was passaged through an additional episode protein from each type of cell were applied to the gel and electrophoresed; then with 250 ,Lg/ml NB-506 (shown in Fig. 2). the proteins on the gels were transferred electrophoretically to nitrocellulose At concentrations of 0.01, 0.025, 0.05, and 0.25 @giml,NB-506 membranes (22), which were placed in 50 ,@MTris/400 @MNaC1(pH 7.5) inhibited proliferation of the parent SBC-3 cells, determined using the buffer containing 0.05% Tween 20 and rinsed. All the rinses lasted 45 mm with three changes of buffer, and all the incubations lasted 1 h. The membranes regrowth assay, by 1, 25, 55, and 87%, respectively. The concentra were rinsed and incubated with peroxidase-conjugated goat anti-rabbit IgG tion-response curves of the nine NB-506-resistant sublines against (Bio-Rad Laboratories, Richmond, VA) in the above buffer containing 3% NB-506 are shown in Fig. 4, and the resistance ratios obtained from chloro-4-naphthol and H2O2in the above buffer containing 11% methanol. The their concentration-response curves are summarized in Table 2. This reaction was stopped by rinsing with water. table showed a tendency that the large number of treatment and the high concentration of NB-506 produced a high resistance. The SBC RESULTS 3INB#9 cells were the most resistant of these cell lines to NB-506; its IC50was 21.3 @.tg/ml,whichwas 454-fold that against SBC-3/P cells. Cytotoxicity of NB-506 against Human Tumors and Their CPT Therefore, SBC-3INB#9 was selected for further study. The IC50 of resistant Cell Lines in Vitro NB-506 against subline SBC-3tNB#9f cells, which were cultured in The cytotoxicity of NB-506 was examined on various human drug-free medium for 10 weeks, was 21.0 Â±0.52 p@g/ml.Therefore, tumors, namely human small cell (SBC-3 and H69) and non-small the acquired resistance to NB-506 was stable after prolonged growth cell (PC-7, PC-9, and PC-14) lung cancer and human leukemia in nonselective medium. (K562 and RPM18402) cell lines, and the results are shown in The colony formation assay (human tumor clonogenic cell assay) Table 1. The IC50s of NB-506 against RPMI84O2, H69, and SBC-3 yielded an IC50 for NB-506 against SBC-31P cells of 0.045 p.gtml were 0.15, 0.14, and 0.046 @Wml,respectively, which were sig (Fig. 5A), whereas that against SBC-3/NB#9 cells was 38 p@g/ml, nificantly lower than those of CPT-11 (14). The IC50 was lowest more than 800-fold that against the parent cells. The resistance ratio, against the SBC-3 small cell lung cancer cell line, which, therefore, determined using the MU assay, was 4000-fold that of the parent was used as the parent cell line for establishing NB-506-resistant cells (300 @gimlfor SBC-3INB#9 versus 0.06 @g/mlfor SBC-31P; cells. Fig. Sb). Next, we examined whether RPM18402/K5 and PC-7tCPT cells, Characterization of NB-506-resistant Sublime SBC-3/NB#9 which are resistant to CPT-11, were cross-resistant to NB-506 (Fig. Cells. The doubling times of SBC-3/P and SBC-3/NB#9 cells were 3). Both these cells showed cross-resistance to NB-506, which mdi cates that the cytotoxic mechanism of NB-506 is associated with 15.2 and 16.4 h, respectively, their respective plating efficiencies were 1.3 and 1.2%, and the parent and resistant cell diameters were 1.13 Â±0.089 and 1.13 Â±0.091 @m,respectively. None of these differences were significant. These fmdings were important for the linesCellTable 1 1n vitro cytotoxicity of NB-506 against various human tumor cell study of mechanisms of resistance to NB-506, because the cytotoxic pg/mI)NB-506(IC50 Â±SD; activity of NB-506 was cell cycle specific. 1SBC-3(SmalllinesCytotoxicity CPT-1 Sensitivities of SBC-3INB#9 Sublime Cells to Other Anticancer 0.02H69(Small cell lung cancer)0.046 Â±0.0083 0.11 Â± Drugs. Table 3 shows the cytotoxic effects (IC50; @giml)of various anticancer drugs against SBC-31P and its resistant subline, SBC-3/ 0.11PC-7(Non-small Â±0.057 0.7 Â± cell lung cancer)0.14 NB#9. The IC50 of CPT-11 against SBC-3fP was 0.11 @g/ml,one 0.0021PC-9(Non-small cell lung cancer)0.11 Â±0.0069 0.037 Â± eighth that against SBC-3/NB#9. This subline also showed cross resistance of about 22-fold to SN-38, which is an active metabolite of 0.061PC-14(Non-small cell lung cancer)6.9 Â±1.56 1.27 Â± CPT-11. It was noticeable that SBC-3tNB#9 cells showed cross 2.6Â±0.99K562(Leukemia)0.83cell lung cancer)1.8 Â±0.14 resistance only to topoisomerase I inhibitors and none towards cis

0.6Â±0.11RPM18402(Leukemia)0.15 Â±0.15 platin, etoposide, teniposide, vinblastine, or vincristine. These results indicate that the resistance mechanism of these cells is unlikely to be Â±0.04 0.76 Â±0.048 that associated with typical multidrug resistance. 2808

A) NB-506 SN-38 100

0 60

.@ 40 0 C,

Fig 3. CytOtOxicityof NB-5O6, CPT-11, and Concentration (@tg/mi) SN-38againstRPM18402andPC-7andtheircor responding @fl-11-resistantcells The antitumor activities of the drugs were measured using the â€”0---@ RPM18402 . K5 regrowth assay, as described in â€œMaterialsand Methods.â€•Exponentiallygrowing cells (3 X 1O@'/ test) were incubated in medium containing the re quired drug at the indicated concentrations at 37Â°C for7 days afterwhich,thecellswerecounted,and cell growth ratios were calculated. A, concentra tins-response curves of NB-506 and SN-38 against RPM18402 and K5. B, concentration-response CPT-11 curves of NB.506, CFf-11, and SN-38 against B) NB-506 SN-38 PC-7/P and PC-7/CPT. 100 100 100

80 80

0 60 60

.@ 40 40

0 C, 20

0@ .001.01 .1 I 10 .0001 001 .01 .1 Concentration (hg/mi)

â€”a-â€”. PC-71P â€”p PC-7/CPT

Intracellular Accumulation of NB-506. The time course of NB Relaxed forms were observed in the presence of only 0.005 @g 506 uptake by SBC-31P cells is shown in Fig. 6A. When incubated SBC-3/P nuclear extract, whereas the SBC-3INB#9 cell nuclear with 10 @&g/mlE3HINB-506(0.68 @tCi),uptakeby SBC-3INB#9 cells extract did not catalyze supercoiled DNA relaxation, even when 0.02 was relatively slow, and steady-state was reached after 30 mm, ,i.g was used, which indicates that the topoisomerase I activity of whereas SBC-3/P cells had not reached steady-state after 2 h. The rate SBC-3/NB#9 cells was about one-tenth that of SBC-3/P cells. The constant determined from the initial uptake rate was 3.59 Â±0.10 topoisomerase I activities of the other NB-506-resistant sublines also min@1(mean Â±SE) for the SBC-31P cells and 0.89 Â±0.15 min@ for were lower; the amount of topoisomerase I protein required to relax the SBC-3/NB#9 cells, which reflects the finding that the initial all of the supercoiled DNA completely was 0.05 @gforthe SBC-3/#6 uptake by the NB-506-resistant cells (SBC-3INB#9) was only 24.8% and #9 sublines; 0.02 p.g for SBC-3/NB#3, #4, #7, and #8; and of that by the SBC-31P cells. The accumulation rate constants of 0.01 g.i.gforSBC-3INB#1 and #5. We concluded that the resistance [3H]CPT for SBC-31P and SBC-3/NB#9 cells were 53.3 Â±10.1 and of these sublines to NB-506 is due to topoisomerase I reduced activity. 58.7 Â±5.8 min', respectively (shown in Fig. 6B), and the cellular To determine whether the reduced DNA topoisomerase I activities uptake of camptothecin by these two cell lines did not differ were due to reductions in the cellular contents, the DNA topoisomer significantly. The decreased drug uptake by SBC-3INB#9 ase I contents of the nuclear extracts were measured using an immu compared with its parent cells was found to be specific for noblotting assay. From the results of this study in Fig. 8, it appeared NB-506. that the NB-506-resistant sublines contained a reduced amount of ACtiVity and Content of Topoisomerase I and the Effects of topoisomerase I protein in comparison to the parent cells. Further CPT-11 on Them. The total cellular activities of DNA topoisomer more, the degree of its reduction was correlated to the activity of am I of SBC-31Pand its NB-506-resistantsublines in the crude topoisomerase I, indicating that the reduced activity was caused by nuclear extracts eluted with 0.35 M NaCI were measured. The relax reduction of amount of DNA topoisomerase I. ation of pBR322 DNA incubated with different amounts of SBC-3/P The effects of NB-506 on the catalytic activities of DNA topoi and SBC-3/NB#9 cell nuclear protein extracts is shown in Fig. 7. In somerase I extracted from SBC-3/P and SBC-3/NB#9 cells were this experiment, the relaxation of supercoiled DNA catalyzed by determined using 10 and 50 ng, respectively, of their nuclear extacts topoisomerase I was monitored using gel electrophoresis. to adjust for their different DNA relaxation potencies in the absence 2809

Growth ratio (%) NB-506 on topoisomerase I-catalyzed DNA relaxation was stronger than that of CPT-11.

DISCUSSION SBC-3/P clone#1 The new semisynthetic antitumor agent NB506 is derived from a â€”0- clone #2 novel indolocarbazole antibiotic produced by an Actinomycete (12). â€”0- clone #5 Recently, several indolocarbazole compounds were reported to show activity against experimental tumors in vivo. Two such compounds, 7-hydroxystauro sporine (UCN-01; Refs. 23 and 24) and 4'-benzoyl staurosporine (CGP 41 251; Ref. 25) appeared to exert antitumor

.001 .01 .1 1 10 100 1000 activity via inhibition of protein kinases, including PKC, whereas rebeccamycin (26) and AT2433 (27) exerted their antitumor activity via interaction with DNA but demonstrated little inhibition of PKC (28). In a cell-free system, PKC activity was virtually unaffected by NB-506, which was postulated to act via inhibition of topoisomerase .. ..w.. SBC-3/P 1-mediated DNA cleavage (29). .â€”eâ€”â€”clone#4 One of the aims of this study was to evaluate the efficacy of â€”â€¢0â€” clone #5 NB-506 by comparing it with another topoisomerase I inhibitor, â€”0@â€”clone #6 CPT-11, currently undergoing Phases I and II clinical evaluation in adult and pediatric patients with malignant disease (8â€”10).Another aim was to establish an NB-506-resistant tumor cell line and elucidate its resistance mechanism, because the development of drug resistance

....w.. SBC-3/P â€”0-- SBC-3/P clone #7 . SBC-3/NB#9 â€”0- clone#8 A) â€”0- clone#9 100 ---.--. clone#9f 80 at 0 60 C

Â£ 40 .001 .01 .1 1 10 100 1000 0 20 Conc.ntratlon of NB.506(@tg/ml) C,

Fig. 4. Concentration-response curves of NB-5O6 against the SBC-3 parent cell line 0 (SBC-31P)and its NB-5O6-resistantsublines. The cytotoxicity of NB-506 was measured .001 .01 .1 1 10 100 1000 10000 using the procedure described in the legend to Fig. 3. The SBC-3/NB#91 subline cells Concentration of NB-506 (@tg/mi) were cultured in drug-free medium for 10 weeks.

TableNB-506ClonesCytotoxicity 2 Drugsensitivity of various clones of SBC-3 cellsresistant to B) MTT assay ratioSBC-3/#l0.78 (IC50 Â±SD; @Wml)Resistance 100 0.1617SBC-3/#21.12 Â± 0.2824SBC-3/#30.7 Â± 80 0.3815SBC-3/#40.91 Â± at 0.2519SBC-3/#51.33 Â± 0 0.0628SBC-3/#616.33 Â± 60 2.89347SBC-3/#74.97 Â± 0.15106SBC-3/#812.33 Â± Â£ 40 2.08262SBC-3/#921.33 Â± 0 2.08454SBC-3/#9 Â± 20 3.33406SBC-31P0.047Â±0.0021f19.09 Â± C,

Concentration of NB-506 (@tg/mi) of the inhibitor. The DNA relaxation catalyzed by topoisomerase I Fig. 5. Concentration-response curves of NB-5O6 on the growth of the SBC-31P and isolated from SBC-3fP cells was inhibited by NB-506 in a concen SBC-3INB#9, measured using the colony-forming and MiT assays. A, colony forming tration-dependent manner and was inhibited completely at 5 p@g/ml assay: exponentially growing cells (3 X 1O@/dish)were seeded in soft agar medium containing NB-506 at the indicated concentrations and cultured for 9 days, after which the (Fig. 9). It also inhibited the topoisomerase I activities of the resistant colonies were counted with a computerized image analyzer. B, MTI' assay: exponentially cell nuclear extracts at the same concentrations. In contrast, 25 p.g/ml growing cells (1 X 1O@/well)wereseeded in 96-well microtiter plates and exposed to CPT-1 1 had no effect on the activities of topoisomerase I extracted NB-506. After a 4-day culture period, MiT was added to each well, the plates were incubated for a further 4 h, and then centrifuged; then the medium was removed, DM50 from both cell lines; this concentration was approximately 220 times was added to each well to dissolve the pigment (formazan) crystals, and the absorbances greater than its IC50 (Table 3). Therefore, the inhibitory effect of of the solutions were measured at 562 and 630 nm using a Bio-Tek Microplate Reader. 2810

Table 3 Cytotoxicityof various antitumor drugs against SBC-3/P and doxorubicin (14 mg/kg), mitomycin C (8 mg/kg), and cisplatin (15 SBC-3/NB#9 cells mg/kg). This indicates that NB-506 is a drug with low toxicity and is (IC@ Â±SD;pg/mi)Resistance a superior anticancer agent to CPT-1 1 against experimental animal SBC-3/NB#9NB..506Drugsâ€•CytotoxicityratioSBC-3/P tumors. Â±0.0083 21.3 Â±1.53â€• In this study using human cancer cell lines, NB-506 showed anti cP'r-ii 0.11Â±0.02 0.84Â±0.14â€• 7.60 tumor activity similar to or better than CPT-11. In particular, the 22.00CDDP0.094SN-380.047 0.00016 Â±0.00012 0.0023 Â±[email protected] cytotoxicity of NB-506 against human small cell lung cancer cell 0.0120.69VP-160.031 Â±0.016 0.065 Â± 0.0291.34VM-260.0037 Â±0.011 0.042 Â± lines, such as SBC-3 and H69, was 5-fold that of CPT-11. Further 0.00350.94ADM0.0071 Â±0.0013 0.0035 Â± more, we expected NB-506 to show cytotoxicity against CPT-11- 0.0053.24VBL0.00053 Â±0.0013 0.023 Â± resistant cells, because its structure and ability to intercalate double 0.0000450.64VCR0.00059Â±0.00015 0.00034 Â± 0.000070.80L-PAM0.17Â±0.085Â±0.00006 0.00047 Â± stranded DNA differ from those of CPT-11. Regrettably, the 0.16Â±0.0570.915-PU0.14 CPT-11-resistant PC-7 and RPM18402 (K5) cell lines showed cross 0.0141.02BLM0.12Â±0.021Â±0.006 0.14 Â± 0.14Â±0.0571.14-ti.c-diamminedich1omnIatinnm(II1@ resistance to NB-506 (Fig. 3). However, cells with the typical mdrl phenotype, such as the Adriamycin-resistant K562 and etoposide @@ a â€”@_.--_----- _-@1@@ @;;, VP-16, etoposide; ADM, Adriamycin; VBL, vinbiastine; VCR, vincristine; I-PAM, L-phenylalaninemustard; 5-Hi, 5-fluorou resistant H69, were not cross-resistant to NB-506 (data not shown). racil; BUd, bleomycin. These results indicate that NB-506 is superior to CPT-1 1 against bp<0001 human tumor cells as well as experimental animal tumor cells and pin our expectation of considerable cytotoxic effects on drug-resistant A) NB-506 tumor cells. A major limitation to cancer chemotherapy is the development of â€”â€¢0â€”SBC.3/P

â€”..--- SBC.3/NB#9 0 I

@ 0.2 SBC-31P @@p@r-1F!p1p,r,p-. j ir z @ 0.1 SBC-3/NB#1 J S â€”

0.0 SBC-3/NB#2 __@;..i3 Ir 0 30 60 â€”â€” Time (mm) @ SBC-3/NB#3 !,â€˜ Jir B) NB-506 C) Camptothecin

@@@@@@ 4' ,., 1.@@ri @: - SBC-3/NB#4 :i ir .E 3 C E E 0 @2 E SBC-3/NB#5 J ir C

a S S a 0. @ . ] ir SBC-3/NB#6 @__@; -C--i 0 S BC-31P SBC-3/NB#9 SBC-3/P SBC-3/NB#9

Fig. 6. Uptake of NB-506 and camptothecin by SBC-3/P and SBC-3/NB#9 cells. SBC-3/NB#7 3 ir @ Exponentially sowing cells (2 x 107/test) were preincubated at 37Â°Cfor45 min and â€” .4â€” i incubated with [3HJNB-506or camptothecin at 37Â°Cforthe indicated time; after which, the cells werecollected,washedwithPBS,anddissolvedin formicacid.Thenthe @ radioactivity was measured with a liquid scintillation counter. A, time course of NB-5O6 SBC-3/NB#8 J ir incorporation into SBC-3/P (0) and SBC-3/NB#9 (â€¢)cells.Accumulation of NB-506 @1@m (B) and camptothecin (C) by SBC-3/P and SBC-3/NB#9 cells.

SBC-3/NB#9 .â€”[email protected]@-:@lJ:â€”i@--' ir is likely be a major limiting factor in determining the clinical success ofNB-506. LaneNo. 1 2 3 4 5 6 7 8 9101112 In transplantable rodent tumor models, such as P388, L1210, colon @@@@@@@@@ 26, andM5076, NB-506 was reportedto showa broadspectrumand Nuclearextract(j&g) Â° markedly high degree of antitumor activity (12). The life span of mice a dOe bearing P388 murine leukemia cells given 100 mg/kg/day NB-506 Fig. 7. Topoisomerase I activities of NB-506-sensitive and -resistant SBC-3 cells. The was prolonged by over 326%, and cures (60-day survivors) were enzyme activity was determined by measuring the relaxation of supercoiled DNA (pBR322) during incubation with nuclear extract at 37Â°Cfor 10 mm, using electrophoresis observed in 4 of 5 (80%). Such high antitumor activity was not on 0.7% agarose gel for 4 h. I and Jr. supercoiled and relaxed DNA, respectively. Lane attained with any dose of CPT-11 (30, 31). Furthermore, the LD50 of 1, no nuclear extract (control); Lane 2, 2.0 @g;Lane3, 1.0 @sg;Lane 4, 0.5 @g;Lane5. NB-506, administered i.p., in mice was 500 mg/kg, which was much 0.2 gig; Lane 6, 0.1 @sg;Lane 7, 0.05 @.tg;Lane8, 0.02 @g;Lane9, 0.01 ,.@g;Lane 10, 0.005 ,Lg; Lane II. 0.002 gig; and Lane 12, 0.001 @tgnuclearprotein from SBC-3/P cells and higher than those of CPT-1 1 (177 mg/kg), etoposide (65 mgfkg), its NB-506 resistant sublines were added. 2811

100 resistance to chemotherapeutic drugs, which may be due to decreased drug uptake (17), efficient drug removal (mediated by P-glycopro 80 tein), drug detoxification, e.g., by increased levels of cellular gluta 0 thione (16), enhanced DNA repair, gene amplification, and subse 0. 0 quent overexpression of the target molecule or drug target specificity I- 60 0 changes. The resistance to topoisomerase inhibitors has been found to correlate with decreased topoisomerase activity (32). Topoisomerase 0 inhibitors are cytotoxic by virtue of producing DNA lesions as a result E 40 of inhibiting topoisomerases during the course of DNA replication, and therefore, drug sensitivity is directly proportional to enzyme 20 0 activity. We established a topoisomerase I inhibitor, CPT-11-resistant rir'ir'iri cell line (PC-7/CPT) from a human non-small cell lung cancer cell 0 line, PC-71P. The CPT-1 1-resistant cells exhibited about 10-fold re P 1 23456789 sistance to CPT-11; their topoisomerase I activity, determined using Subi me the DNA relaxation assay, was lower, and the topoisomerase I from the resistant cells was 5 times more resistant to the inhibitory effect of Fig. 8. Contents of topoisomerase I of SBC-3/P and its NB-506-resistant sublines. Topoisomerase I content was measured by Western blot assay described in â€œMaterialsand CPT-1 1 compared with the parent cells (14). The topoisomerase I Methods.â€•Theresulting autoradiographs were quantified by scanning densitometry. The activity and level changes were demonstrated, using single-strand results were confirmed by normalization to @3-actinsignals. Contents of topoisomerase I were presented as the relative amount of parent cells. conformation polymorphismlPCR analysis, to result from topoisomer ase I gene mutation (33). Thus, qualitative changes in topoisomerase I can lead to resistance to camptothecin and its derivatives (34, 35).

A) NB-506

LaneNo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

@ Nuclear extract (p.tg) o SBC-3/NB #9 (O.O5@tg 0 SBC-3/P (O.O1@ig)

In *0 In U) @@@@ Concentration of NB-506 (j.tg/ml) In - 0 â€˜Â°@a@- _

B) CPT-11

@ Ir [ @1P@@!!!

LaneNo. 1 2 3 4 5 6 7 8 9 10111213141516

@ Nuclear extract (@tg) 0 1 SBC3/NB#9 (O.O5jig) 0 SBC-3/P([email protected]

@ 0 10 0 tfl 0 0 0 tg@ 0 11) @@@ Concentration of CPT-1 1 (j.tg/ml) Â° . @tn @@ji-. csj

Fig. 9. Inhibitory effects of NB-5O6 and CPT-1 1 on DNA relaxation catalyzed by topoisomerase I from SBC-31P and SBC-3/NB#9 cells. The experiments are the same as those described in the legend to Fig. 7. 1 and Jr. supercoiled and relaxed DNA, respectively. Lanes I and 9. no nuclear extract (negative controls). Lanes 2-8 and 10â€”6were loaded in the presence of 0.05 and 0.01 @gSBC-3/NB#9and SBC-3/P nuclear extracts, respectively. Lanes 2 and JO. no drug (positive controls); Lanes 3 and 11, 4 and 12, 5 and 13. 6 and 14. 7 and 15, and 8 and 16 contained 25, 10, 5, 2.5, 1, and 0.5 @WmlNB-5O6and CPT-11, respectively. A and B. the effects of NB-5O6and CPT-11 on the topoisomerase I activities of SBC-3/P and SBC-3JNB#9 cells, respectively. 2812

In this study, we developed, established and characterized nine Kawamura, K., Okura, A., Suds, H., and Okanishi, M. A new antitumor substance, NB-506-resistant sublines (SBC-3/NB#1â€”9) from a human small cell BE-13793C, produced by a Streptomycete. J. Antibiot., 44: 723â€”728,1991. 12. Arakawa, H., Iguchi, T., Morita, M., Yoshinari, T., Kojiri, K., Suds, H., Okura, A., lung cancer cell line (SBC-31P). Their resistance ratios ranged from and Nishimura, S. Novel indolocarbazole compound 6-N- formylamino-12,13..dihy 15- to 454-fold. Of these, SBC-3/NB#9 was selected for comparative dro-1,11-dihydroxy-13-(@-o-glucopyranosyl)- 5H-indolo[2,3-aJ pyrrolo-[3,4-c@car bazole-5,7(6H)-dione (NB-506): its potent antitumor activities in mice. Cancer Res., studies because of its high degree of resistance and its growth char 55: 1316â€”1320,1995. acteristics, namely the doubling time, cloning efficiency, and cell size, 13. Yoshinari, T., Matsumoto, M., Arakawa, H., Okada, H., Noguchi, IC, Suda, H., did not differ from those of the parental cells. The total DNA topoi Okura, A., and Nishimura, S. Novel antitumor indolocarbazole compound 6-N- formylamino-1213-dihydro-1,1 1-dihydroxy-13-(@-D- glucopyranosyl)-5H-indolo[2,3-a] somerase I activity of SBC-3/NB#9 cells was shown to be only pyrrolo-(3,4-cJcarbazoie-5,7(6H)-dione(NB-5O6):induction of topoisomerase I-mull one-tenth that of the parent cells, and this reduced activity was ated DNA cleavageand mechanismsof cell lire-selectivecytotoxicity.Cancer Res., 55: 1310â€”1315,1995. attributable to a lower DNA topoisomerase I content. These results 14. Kanzawa, F., Sugimoto, Y., Minato, K., Kasahara, K., Bungo, M., Nakagawa, K., concur with those for the CPT-1 1 resistance mechanisms. Further Fujiwara, Y., Liu, L. F., and Saijo, N. Establishment of a camptothecin analogue more, we found a tendency that the high frequency of treatment and (CPT-11)-resistantcell line of human non-small-cell lung cancer: characterization and mechanism of resistance. Cancer Res., 50: 5919â€”5924,1990. the high drug concentration in the selection of the NB-506-resistant 15. Kanzawa, F., Maeda, M., Sasaki, T., Hoshi, A., and Kuretani, K. Murine lymphoma cells produced the low activity and low content of topoisomerase I, L5178Y cells resistant to purine antagonists: differences in cross-resistance to thio and consequently, high resistance (Table 2; Figs. 7 and 8). However, guanine-platinum(II) and selenoguanine platinum(II). J. Natl. Cancer Inst., 68: 287â€”291,1982. the affinities of the topoisomerase I enzymes isolated from the resist 16. Fujiwara, Y., Sugimoto, Y., Kasahara, K., Bungo, M., Yamakido, M., Tew, K. D., and ant and parental cells for NB-506 did not differ, which agreed with the Saijo, N. Determinants of drug response in a cisplatin- resistant human lung cancer single-strand conformation polymorphismfPCR results that showed cell line. Jpn. J. Cancer Res., 81: 527â€”535,1990. 17. Bungo, M., Fujiwara, Y., Kasahara, K., Nakagawa, K., Ohe, Y., Sasaki, Y., Irino, S., no topoisomerase I gene changes had occurred (data not shown). and Saijo, N. Decreased accumulation as a mechanism of resistance to cis-diam Changes in the cellular uptake/efflux of drugs are known to be minedichloroplatinum(lI) in human non-small cell lung cancer cell lines: relation to DNAdamageandrepair.CancerRes.,50: 2549â€”2553,1990. common drug resistance mechanisms. In this experiment, we ob 18. Deffie, A. M., Batra, J. K., and Goldenberg, G. 3. Direct correlation between DNA served that less NB-506 was accumulated by the resistant than the topoisomerase II activity and cytotoxicity in Adriamycin-sensitive and resistant P388 parent cells, although the intracellular camptothecin accumulation by leukemia cell lines. Cancer Res., 49: 58â€”62,1989. 19. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram SBC-3/P and SBC-3/NB#9 did not differ. The SBC-3INB#9 was quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., distinguishable from multidrug-resistant cells, because the multidrug 72: 248â€”254,1976. resistant K562/ADM subline, which is known to overexpress P 20. Liu, L. F., and Miller, K. G. Eukaryotic DNA topoisomerases from HeLa cell nuclei. Proc. NatI. Acad. Sci. USA, 78: 3487â€”3491,1981. glycoprotein (gpl7O), did not show cross-resistance to it. Therefore, 21. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the low intracellular drug accumulation by SBC-3INB#9 compared bacteriophage Y4. Nature (Land.), 227: 680â€”685,1970. with its parent cells appears to be attributable to reduced drug uptake, 22. Towbin, H., Staehelm, T., and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. not a p-glycoprotein-mediated change in effiux. The SBC-3INB#9 Natl. Acad. Sci. USA, 76: 4350-4354, 1979. cells also were distinguishable from CPT-11-resistant cells (PC-7/ 23. Akinaga, S., Gomi, K., Morimoto, M., Tamaoki, 1., and Okabe, M. Antitumor activity of UCN-O1 , a selective inhibitor of protein kinase C, in murine and human CF1'), the resistance mechanisms of which are considered to be a low tumor models. Cancer Res., 51: 4888â€”4892, 1991. affinity of topoisomerase I for CPT-11, as well as reduced enzyme 24. 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Fumihiko Kanzawa, Kazuto Nishio, Naohiro Kubota, et al.

Cancer Res 1995;55:2806-2813.

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