CANCERRESEARCH56,789-793,FebruaryII, 19961 Synergism between and I Inhibitors, NB-506 and SN-38, in Human Small Lung Cells'

Minoru Fukuda, Kazuto Nishio, Fumihiko Kanzawa, Hayato Ogasawara, Tomoyuki Ishida, Hitoshi Arioka, Krzysztof Bojanowski, Mikio Oka, and Nagahiro Sai.jo2

Medical Oncology, National (‘ancerCenter Hospital (M. F.. N. £1and Pharmacology Division, National C'ancer Center Research Institute (K. N., F. K., H. 0., T. 1., H. A., K. B., N. S.!, Tsukiji 5J.J Chuo-ku, Tokyo 104, and The Second Department ofinternal Medicine. Nagasaki University School ofMedicine, Nagasaki, (M. F., M. 0.], Japan

ABSTRACT among potent topoisomerase inhibitors and its cytotoxic mechanisms are not the same as those of CPT derivatives (6). Consequently, Topolsomerase I-targeting anticancer agents such as 7-ethyl-lO-[4-(l- NB-506 is considered an interesting anticancer agent, and single pipendyl)-1-pipendyljcarbonyloxy-eamptothecin (CPT-11) and 6-N- dosing schedule Phase I studies of NB-506 are now conducted in formylamlno-12,13-dihydro-1,11-dihydroxy-13-(fi-D-glucopyranosyl)-5H- indolo[2@-a]pyr@lo[3,4-c]carbazole-5,7(6H)-dione(NB-506)have been Japan. CDDP is a key agent for cancer , and some developedandshowstrong antitumor activity againstvariouscancers.We combination chemotherapy regimens have been prompted by a ther examinedthe interaction of thesedrugs and cisplatin (CDDP), and bio apeutic synergy between CDDP and (7—9)or CPT-l 1 chemicalmechanismsofsynergismbetweenthem.Interactionofdrugsin (10—12).CPT-ll is active against various human cancer patients; humansmallcelllungcancercells,SBC-3,wasanalyzedusingtheisobo however, preclinical and clinical studies suggest it behaves as a logram method. Combinations of CDDP with NB-506, CPT-11, and an prodrug in vivo, and the majority of antitumor activity may be attrib active metabolite of CPT-11, 7-ethyl-1O-hydroxy-CPT (SN-38), showed utable to its more active metabolite SN-38. In vitro, SN-38 is 250— synergistic effects. Formation of DNA interstrand cross-links (ICLs) on 1000-fold more potent than CVF-ll in the inhibition of topoisomerase thecellswasanalyzedusinganalkalineelutionassayandincreasedICLs I activity. wereobservedbysimultaneousexposuretoCDDP (1.5 gtM)and NB-506 Demonstration of supraadditive cell killing would imply an inter (10flM)comparedwiththatinresponsetoCDDPalone.DNArepairafter ICL formationInducedby3-hexposuretoCDDP(1.5gtM)wasreducedby action between two agents at a cellular level and have profound NB-506 (10 nM) exposure.On the other hand, a higher concentration of implications for biochemical study. In this study, we demonstrated CDDP (150 ELM)enhancedthetopolsomeraseIinhibitoryactivityof that the cytotoxic effects of CDDP combined with NB-506, CPT-11, NB-506 and SN-38determined by relaxation of supercoiledEscherichia and SN-38 were synergistic and elucidated the mechanisms respon coli DNA. These biological interactions might result in synergistic inter sible for the synergistic effects. actions between CDDP and NB-506 or SN-38. Topolsomerase I inhibitors and CDDP may be a key regimen for cancer chemotherapy and merit further examination. MATERIALS AND METHODS

INTRODUCTION and Chemicals. CVF-l 1 and SN-38 were provided by Daiichi Co. Ltd. (Tokyo, Japan),NB-506wasprovidedby the BanyuTsukubaResearch The DNA are enzymes that can alter the topology Institute (Tsukuba, Japan), CDDP was purchased from Nippon Kayaku Co. of DNA by transiently breaking one or two strands of DNA, passing Ltd. (Tokyo, Japan), and etoposidewere obtainedfrom Bristol a single- or double-stranded DNA through the break, and finally MyersSquibbCo.Ltd. (Tokyo,Japan),vindesinewasobtainedfromShionogi resealing the breaks. These enzymes are involved in a number of Co. Ltd. (Osaka,Japan),and plasmid DNA pBR322 was purchasedfrom crucial cellular processes, including replication, transcription, and ToyoboCo. Ltd. (Osaka,Japan). Cell Line and CUltUre. The human small cell lung cancer cell line SBC.3, recombination, and they are now viewed as important therapeutic originally established at the Okayama University School of Medicine, was targets for cancer chemotherapy. In particular, after demonstrating donatedby the JapaneseCancerResearchResourcesCellBank.The SBC-3 that CPT-l l@, a semisynthetic derivative of CPT, showed strong cellsweregrownasattachedculturesin RPM! 1640medium(GIBCO,Grand antitumor activity against , lymphoma (1), small cell lung Island,NY) supplementedwith10%v/v heat-inactivatedfetalbovineserum (2), non-small-cell lung (3), colorectal (4), and ovarian and cervical (SigmaChemicalCo. SL Louis, MO), penicillin (100 units/mI),andstrepto (5) . It is therefore probable that topoisomerase I inhibitors are mycin (100 @.&g/ml)ina humidified atmosphere of 5% CO2 in air at 37°C.The promising anticancer agents. NB-506 is a novel cells wereharvestedroutinelyby trypsinizationanddiluted with the medium anticancer agent. The primary target of this agent is considered to be to the appropriateconcentrations.Thecell sizesandnumbersweremeasured topoisomerase I. The indolocarbazole of NB-506 is unique by a CoulterChannalyzerC-256system(CoulterElectronics,Hialeah,FL). Growth Inhibition Assay. We used the tetrazoliumdye (MU) assay described previously (13) to evaluate the growth inhibitory effects of the Received 8/14/95; accepted 12/6/95. The costsof publicationof this article weredefrayedin part by the paymentof page cytotoxic agents. In brief, l60-@d aliquots of an exponentially growing cell charges. This article must therefore be hereby marked advertisement in accordance with suspension(6.3X l0@cells/mI)wereseededinto96-wellmicrotiterplates,and 18 U.S.C. Section 1734 solely to indicate this fact. thecellswereincubatedfor 12h, afterwhich 20-@alaliquotsof solutions @ This work was supported in part by Grants-in-Aid for Cancer Researchand from the of variousconcentrationswereadded.Followingexposuretothedrugsfor 72 SecondTermComprehensive10-YearStrategyforCancerControl,theMinistry of Health andWelfare,theMinistryofEducationScienceofJapan,atrustfund,theAdultDisease h, 20 p3 MTF solution(5 mg/mi in PBS)wereaddedto eachwell, andthe MemorialFoundation,andthe Bristol-MyersSquibbFoundation. plateswereincubatedat37°Cforanother4h.After centrifugationoftheplates 2 To whom requests for reprints should be addressed. Phone: 81-3-3542-251 1; Fax: at 1200X g for 8 mm,themediumwasaspiratedfromeachwell ascompletely 81-3-3542-I 886. aspossible,200pi DMSO wereaddedto eachwell to dissolvetheformazan, 3 The abbreviations used are: CPT-l 1, 7-ethyl-l0-[4-(l-piperidyl)-l-piperidyl]car andtheabsorbancewasmeasuredat562and630nmusingaDeltaSoftELISA bonyloxy-; CPT, camptothecin; NB-506, 6-N-formylamino-l2,l3-dihydro 1,11-dihydroxy-I3-(j3-o-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole analysis program for a Macintosh computer interfaced with a Bio-Tek Micro 5,7(6H)-dione; CDDP, cis-diamminedichloroplatimum(ll); SN-38, 7-ethyl-lO-hydroxy plate Reader(EL-340; BioMetallics, Princeton,NJ). Wells containingonly camptothecin; IC50, drug concentration that inhibited cell growth by 50%; MT1', 3-(4,5- RPMI 1640-fetal bovine serum and MU were used as controls. Each exper dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ICL, interstrand cross-link; Cl, imentwasperformedusingsix replicatewellsfor eachdrugconcentrationand cross-link index; NB, nuclear buffer, comprising 2 inst KH2PO4@5msi MgCl2, 150 inst NaCI, I mM ethyleneglycol bis(@3-aminoethylether)-N,N,N',N'-tetraacetic acid, and 1 inst carried out independently three times. The IC@ was defined as the concentra dithiothreitol (adjusted to pH 6.5). tion that reduced the absorbance in each test by 50%. The absorbance was 789

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1996 American Association for Cancer Research. SYNERGISM OF CDDP AND TOPOI5OMERA5E I INHIBITORS NB-506 AND SN-38 calculatedas(meanabsorbanceofsix wells containingdrug —absorbanceof v/v glycerol, and 50 M Iris-HCI (pH 7.4), 0.7 @.tgpBR322,and crude nuclear six control wells)/(mean absorbance of six drug-free wells —absorbance of six extract. The reactionmixtures used for measuringthe inhibition of DNA control wells) X 100. relaxationby topoisomeraseIinhibitors comprisedthe specifiedamountof Analysisof CombinationEffects.On thebasisofthegrowthinhibition nuclearextract(1.0p@g/mlprotein)anddrugsolution,or theequivalentvolume curve for each single drug, we analyzed the effects of drug combinations using of water,in addition to the abovecomponents.Thereactionmixtureswere the isobologrammethodof Steeland Peckham(14).Three isoeffectcurves incubated at 37°Cfor 10 mm, and the reactions were terminated by adding 45 (modesI, hA, andIIB) weredrawnasdescribedpreviously(15),andthetotal @ldyesolutioncomprising2.5%w/v SDS,0.01%w/v bromphenolblue,and area enclosed by these three lines represents an “envelopeofadditivity.― 50% v/v glycerol. The mixtures were applied to 0.7% w/v agarose gel and Actual IC50values,which were the concentrationsof the two drugs to electrophoresedfor4 h with a runningbuffer of Iris-acetateEDTA. The gel produce50%growth inhibition, wereobtainedfrom growthinhibition curves was stainedwith 2 @LMethidiumbromideand photographedundertransillu afterexposureof variousconcentrationsofthedrugs.Whentheexperimental minationwith 300 nm UV light. IC50concentrationofadrugcombinationplottedliesin theareaontheleft side of the envelope,thetwo-drugcombinationis consideredtoshowa supraad RESULTS ditive (synergistic) interaction. When the experimental data point plotted lies within the envelope, this combination is considered additive, when it is in the Analysis of Cytotoxic Effects of Two-Drug Combinations. To areato theright of theenvelope,butwithin thesquareproducedby0—1(IC50 determine whether topoisomerase I inhibitors and CDDP combina unit), the combination is considered subadditive, and when the point falls tions had synergistic cytotoxic effects, we incubated the cells with outside the square, the two drugs are considered to be protective to each other. various concentrations of two drugs simultaneously for 72 h. The IC50 Alkaline Elution Assay. Exponentially growing SBC-3 cells were radio labeledby incubationwith Lmethyl-'4C]thym'dine(0.02MCi/mi) for 24 h, values of CDDP, NB-506, CPT- 11, and SN-38 against SBC-3 cells washedwith nonradioactivemedium,and incubatedin fresh mediumfor at using the MU assay were 410 ± 100, 18.5 ± 1.0, 250 ±60 and least 2 h before drug treatment and alkaline elution analysis. 0.34 ±0.11 nM, respectively. On the basis of each single-drug growth For the determinationof drug-inducedICL formation by CDDP-treated inhibitory curve, we drew three isoeffect curves (modes I, IIA, and IIB SBC-3cells, the alkalineelution techniquewasperformedasdescribedpre lines), the area enclosed with these curves called envelope of addi viously (16). To introduce a frequency of y-ray-induced DNA single-strand tivity, and plotted the IC50 values obtained from growth inhibitory breaksbeforeanalysis,approximately3 X 10@drug-treatedcellswereirradi curves. The isobolograms of CDDP combined with NB-506 (Fig. 1A), ated with a total doseof 4 Gy by t'°Co‘y-irradiationata doserate of 1.0 SN-38 (Fig. 1B), and CPT-l 1 demonstrated that these combinations Gy/mrn. Before and after irradiation, the cells were kept on ice to prevent DNA had synergistic cytotoxic effects, whereas the effect of CDDP com repair.For the assay,they werediluted with cold PBSanddepositedgently ontoa polycarbonatefilter.Thecellswerelysedon thefilter for 1h with 5 ml bined with etoposide, , and paclitaxel were additive (data not lysis solution comprising 2% w/v SDS, 25 mM disodium EDTA, 50 mM Iris, shown). 50msiglycine,and0.5mg/mIprotemnaseK(pH9.6),whichwasthenallowed Effects of Topoisomerase I Inhibitors on CDDP-induced ICL to flow through the filter by gravity, and then the filter was rinsed three times Formation. We examined the ICL formation induced by CDDP with with 3 ml 20 mM disodium EDTA (pH 9.6) to remove most of the cell , NB-506andSN-38usingthealkalineelution assayatpH 12.1,which membranes,andRNA. The remainingDNA (over97% of that appliedto the enables the relative lengths of damaged DNA strands to be evaluated filter) waselutedwith tetrapropylammonium-tetrahydroxy-EDIA(pH12.1)at by determining the rate of elution of DNA from the filter (16). These a constantflow rate of 0.025—0.030ml/min.Ten eluatefractionswerecol independent experiments were replicated three times. ‘y-Irradiation lecteddirectly into scintillationvials on a fractioncollectorat 1.5-hintervals induced DNA single-strand breaks and reduced the amount of DNA for 15 h. Eachfraction was mixed with 5 volumesof Clear-solI (Nacarai retained on the filter in a dose-dependent manner, whereas the treat lesque, Inc., Kyoto, Japan)containing0.5%v/v aceticacid,andthe radioac ment with CDDP before irradiation increased it in a concentration tivity was counted using a LS38O1 liquid scintillation counter. Analysisof AlkalineElution Data. ForquantitationofDNA, ICLs, the dependent manner, suggesting that CDDP induced ICL formation, as amountsof [‘4CIDNAfromsamplecellsretainedonthefilter werecalculated it induced no single-strand breaks (data not shown). The Cls of SBC-3 as a percentage of the amount retained after elution for 15 h; these values were cells exposed to CDDP with or without NB-506 and SN-38 for 0, 3, called“relativeretention.―Thefrequencyof CDDP-inducedICLswascalcu 8, and24 h are shownin Fig. 2. The Cls of the cellstreatedwith lated using the following formula (17): CDDPandNB-506for8 and24h werehigherthantheClswhenthe cells were treated with CDDP alone (P < 0.05). The combination of Cl= [(I —Rradiation)/(I_RCDDP)l@@'2_1 CDDP and SN-38 did not resultin a statisticallysignificantincrease in comparison to the control values. in which Cl is the CI of CDDP-treated cells, Rradiation is the relative retention Effects of Topoisomerase I Inhibitors on DNA Repair after of 4-Gy-irradiatedcells,andRCDDPis thatof CDDP-treatedcellsafter4-Gy CDDP-induced ICL Formation. The effects of NB-506 (10 nr@i)on irradiation. All of the values are expressed as means ±SDs and were analyzed DNA repair after ICL formation in SBC-3 cells induced by 3-h using the two-tailed t test. Differences at P values of <0.05 were considered to be significant. exposure to 1.5 LM CDDP were examined using the alkaline elution Preparation of Nuclear Extracts. Crudenuclearextractswerepreparedas technique (Fig. 3). The cells were incubated with no drug, 10 ni@i describedpreviouslyby Deffie et al. (18). In brief, cells were collectedby NB-506 after 3-h exposure to 1.5 p.MCDDP, and the CIs at 0, 2, 4, 6, centrifugation, washed twice with cold NB, resuspended in 1 ml cold NB, and 8, 24, and48 h afterwashingtheCDDP out werecalculated.The 9 ml cold NB containing0.35%v/v Triton X-l00, and 1 mMphenylmethyl maximum Cl value was observed after 6 h in controls after the sulfonyl fluoride was added. The cell suspension was kept on ice for 10 mm, washout, whereas in NB-506-treated cells, it occurred 2 h earlier (4 h washed with Triton X-lOO-free cold NB, the nuclear protein was eluted for 1 after washout). After 24 and 48 h, the Cls of the NB-506-treated cells h at 4°Cwithcold NB containing0.35 M NaCI, and a solution of nuclear were higher than those of the drug-free cells (P < 0.05). protein.The supernatantwasobtainedby centrifugationat 18,000X g for 10 Effects of CDDP on the Inhibitory Effects of NB-506 and SN-38 mm.The proteinconcentrationwasdeterminedusingthemethodof Bradford on TopoisomeraseI. The inhibitoryeffectsof NB-506 andSN-38 (19). with and without CDDP on the catalytic activity of topoisomerase I DNA TopoisomeraseIActivity. TheactivityofDNA topoisomeraseIwas determinedbymeasuringtherelaxationof supercoiledEscherichiaco/i DNA are shown in Fig. 4. Over 90% of the plasmid DNA was in the (pBR322),essentiallyasdescribedbyLiu andMiller (20).Formeasurementof superhelical form (Fig. 4A, Lanes 1 and 11; Fig 4B, Lane 1). Nuclear the total topoisomeraseIactivity in SBC-3cells, the reactionmixturescoin extract (1.8—2 @Wml)fromuntreated SBC-3 cells relaxed the super prised100mMKCI, 10mMMgCl2, 1mMdithiothreitol,0.1 mistEDTA, 10% coiled DNA (Fig 4A, Lanes 2 and 12; Fig. 4B, Lane 2), whereas 790

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0.12-

0.08- I I 0.04- 0@

-0.04 I U 0 0.2 0.4 0.6 0.8 40 50 re@ lime @i) NB-506 IC@unft Fig. 3. Repair of DNA after ICL formation induced by CDDP with and without NB-506. The SBC-3 cells were incubated with and without 10 nst NB-506 after 3-h B 1 exposureto1.5@LMCDDP,thealkalineelutionassaywasperformedat0,2,4,6,8,24, and48h afterwashingouttheCDDP,andtheClswerecalculated.0,withoutdrug;•, with 10nstNB-506.Bars,SD. @,P< 0.05 versuscontrolcells not treatedwith drug. 0.8

NB-506 and SN-38 (both 50 p.M) inhibited this relaxation (Fig. 4A, Lanes 17, Fig 4B, Lane 7). One hundred fifty @u@iCDDPincreased the inhibition of topoisomerase I by NB-506 and SN-38 Fig 4A, Lanes 6, 20.4 10, and 16; Fig. 4B, Lane 6. C) DISCUSSION 0.2- The combinationof CDDP and a topoisomeraseI inhibitor seems to be a very interesting strategy for cancer chemotherapy. 0 High clinical response rates for non-small cell lung cancer has 0 0.2 0.4 0.6 0.8 been reported with CDDP and CPT-11 (10). In the present study, SN-38 IC5tjJflft we elucidated the molecular mechanism responsible for this syn Fig. 1. Effects of combinations of CDDP and topoisomerase I inhibitors NB-506 (A) ergistic effect. We used the isobologram method, which enables andSN-38(B) on SBC-3cells.On thebasisof theconcentration-effectcurvedata,the the combination effects of drugs with nonlinear dose-response effects of two-drug combinations were analyzed at the point corresponding to the IC50 curves to be evaluated (14, 15, 21), because the concentration value. The drug concentrations relative to the IC50 were plotted on a linear scale. response curves of anticancer agents do not always follow first order kinetics and the additivity range cannot be shown by a simple additive method. Some studies have demonstrated synergistic ef fects of CDDP with CPT-11 or SN-38, which is an active metab olite of CPT-1l in serum (22, 23). Itoh et a!. (12) observed synergistic effects of SN-38 combined with CDDP on five of six human lung cancer cell lines, and Kano et al. (24) reported syn ergistic effects of CPT-1 1 and SN-38 combined with CDDP on acute lymphoblastic leukemia cells. However, CPT-1 1 did not always enhance the antitumor effect of CDDP significantly in mice @0 in vivo (25, 26). In the present study, we demonstrated synergistic C

.@ cytotoxic effects of CDDP combined not only with CPT-l1 and C SN-38, but also with NB-506, on SBC-3 cells. Therefore, we would guess that CDDP and topoisomerase I inhibitor combina g tions have synergistic effects. The DNA topoisomeraseare now considered to be important cancer chemotherapeutic targets. Although a number of antitumor agents interact with topoisomerase H and form drug-induced cleavable com plexes, camptothecin and its derivatives are known only as topoi somerase I-targeting anticancer agents. A new indolocarbazole anti 0 5 10 15 20 25 tumor agent, NB-506, at 0.01 ,.LM,enhanced the DNA cleavage exposure @me(h) catalyzed by HeLa S3 topoisomerase I, but it did not inhibit that by Fig. 2. ICLs in SBC-3 cells treated with CDDP with and without NB-506 and SN-38 topoisomerase II even at 300 @LM(6),and some CPT and CPT-11- using the alkaline elution technique at pH 12.1. 0, cells treated with 1.5 @iMCDDP;@, cells treated with 1.5 p@MCDDPand 10 flM NB-5O6; •,cellstreated with 1.5 @.tMCDDP resistant cell lines show cross-resistance to NB-506 (27). Therefore, and0.05 nMSN-38.*, P < 0.05 versuscontrolcells treatedwith CDDP.Bars, SD. topoisomerase I is considered the major target of NB-506, and the 791

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A j I- + - 15 150 C-)1.5 15 150 CDDP(IiM) @ a a 5 5 10 10 10 10 NB-506(@tM)

II-@ Ir I—

I 2 3 4 5 0 7 S 9 10 Fig. 4. A, inhibitory effects of NB-506 with and without CDDP on catalytic DNA relaxation induced by topoisomerase I extracted from SBC-3 cells.Supercoiledplasmid(pBR322) DNA wastreatedwith no 0 agentand no enzyme(Lanes1 and 11), no agent(Lanes2 and 12), 0. 0 NB-5O6 (Lanes 3, 7. 13, and 17); and NB-506 and CDDP (Lanes 4-6, I- @ 8—10.14—16,and 18—20)in the presence of 2 @.tg/mlnuclear extract l@ (•) 1.5 15 150 (-) 1.5 15 150 CDDP(ILM) from SBC-3cells incubatedat 37°Cfor12rein, after which electro @@ 20 20 20 20 50 50 50 50 NB-506(@M) phoresis was performed on 0.7% w/v agarose gel for 12 h. I, Jr. and I!, supercoiled, relaxed, and nicked DNA, respectively. Concentra tionsof NB-506:Lanes3—6,5psi; Lanes7—1010ELM;Lanes13—16, 20 psi; Lanes 17—20,50psi. Concentrations ofCDDP: Lanes 4, 8, 14, and 18, 1.5 p.si; Lanes 5, 9. 15. and 19, 15 psi; Lanes 6, 10, 16, and Ir 20, 150 LM.B. inhibitory effects of SN-38 with and without CDDP on catalytic DNA relaxation induced by topoisomerase I extracted from I—, SBC-3 cells. Supercoiledplasmid(pBR322) DNA wastreatedwith no agent and no enzyme (Lane 1); no agent (Lane 2); SN-38 (Lanes 3 and 11 12 13 14 15 16 17 18 19 20 7); and SN-38 and CDDP (Lanes 4—6and 8—10) in the presence of 1.8 @Wmlnuclearextract from SBC-3 cells incubated at 37'C for 12 mm. Concentrations of SN-38: Lanes 3—6,5 pat; Lanes 7—10,50 psi. Concentrations of CDDP: Lanes 4 and 8, 1.5 @.sMLanes5 and 9, 15 lAM;lJJnes 6 and JO, 150 LM. B a.0

@@ (.,) 1.5 15 150 (-) 1.5 15 150 CDDP(@LM) a o 5 5 5 5 50 50 50 50 SN-38 (iiM) II—,, .@ I, •$4@ Ir {

1 2 3 4 5 6 7 8 9 10

finding in the present study that CDDP increased the inhibition of adducts, and interstrand and intrastrand DNA cross-links (34). In the topoisomerase I catalytic activity by NB-506 (Fig. 4A) appears to be present study, we demonstrated that treatment with NB-506 for 8 and the mechanism responsible for the cytotoxic synergy of the CDDP and 24 h increased the ICL formation induced by CDDP, whereas SN-38 NB-506 combination. Furthermore, unlike CPT, NB-506 intercalated treatment did not (Fig. 2). There are three possible reasons for the with DNA. In view of its unique structure, cytotoxic mechanism, and enhancing effect of NB-506 on ICL formation (a) increased CDDP: synergistic effect with CDDP, NB-506 is considered an interesting nucleotide ratio around the DNA; (b) increased affinity of DNA for antitumor drug. CPT inhibits DNA topoisomerase I through the for CDDP; and (c) decreasedrepair after CDDP adduct formation. Repair mation of stable topoisomerase I-DNA cleavable complexes (28—30), of CDDP adducts is mediated primarily by the nucleotide excision and it induces accumulation of topoisomerase I-DNA cleavable com repair pathway (35), and some drugs have been reported to interfere plexes (31). Since the inhibition of topoisomerase I-mediated DNA with excision repair. Arabinofuranosylcytosine, hydroxyurea, and relaxation by CPT analogues correlates with their antitumor activities 5- inhibit the repair of CDDP-induced ICLs (36, 37). (32), the present data suggest that the CDDP-enhanced inhibition of Furthermore, enhanced cross-link repair has been correlated with topoisomerase I catalytic activity by SN-38 (Fig. 4B) is the mecha CDDP resistance (38, 39). These data suggest an inverse relationship nism responsible for the cytotoxic synergy of the CDDP and SN-38 between sensitivity to CDDP and repair of CDDP adducts. Although combination. The mechanism of CDDP modulation against inhibition the mechanism responsible remains unclear, the present observations of the topoisomerase I catalytic activity is still unclear. X-ray diffrac shown in Fig. 3 indicate that NB-506 may interfere with DNA repair tion of the cross-linked dinucleotide cis-Pt(N1-13)2(d(pGpG)) reveales protein, which removes CDDP-induced DNA adducts, thereby in that the two guanines are completely destacked,the deoxynbose sugar creasing cross-linking of DNA with CDDP and enhancing its antitu of the 5'-deoxyguanosine is in a C3'-endo pucker (33). Thus, the mor activity. intrastrand CDDP cross-link produces a severe local distortion in the The combinations of CDDP with NB-506 and SN-38 showed DNA double helix, leading to unwinding and kinking, and it may therapeutic synergy against SBC-3, a human small cell lung cancer modulate the stabilization of topoisomerase I-drug-DNA cleavable cell line. The biochemical mechanisms responsible for these interac complexes. tions were suggested to be: (a) NB-506 modulated the repair of The cytotoxicity of CDDP is believed to be due to the formation of CDDP-induced DNA adducts and (b) CDDP enhanced the topoi DNA adducts, which include DNA protein cross-links, DNA mono someraseI inhibitory effects of NB-506 and SN-38. 792

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REFERENCES quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248—254,1976. I . Ohno, R., Okada, K., Masaoka, T., Kuramoto, A., Arima, T., Yoshida, Y., Ariyoshi, 20. Liu, L. F., and Miller, K. G. Eukaryotic DNA topoisomerase; two forms of type I H., Ichimaru, M., Sasaki, Y., Oguro, M., Ito, Y., Morishima, Y., Yokomaku, S., and DNA topoisomerasefromHeLacell nuclei.Proc.NatI. Acad.Sci. USA, 78: 3487— Ota,K. An earlyphaseII studyof CPT-l 1:a newderivativeof camptothecinforthe 3491,1981. treatmentofleukemiaandlymphoma.J.Clin.Oncol.,8:1907—1912,1990. 21. Chou, T-C., and Talalay, P. Quantitative analysis of dose-effects relationships: the 2. Ma.suda,N., Fukuoka, M., Kusunoki, Y., Matsui, K., Takifuji, N., Kudoh, S., Negoro, combined effect of multiple drugs on enzyme inhibitors. Adv. Enzyme Regul., 22: S., Nishioka, M., Nakagawa,K., and Takada,M. CPT-l 1: a new derivative of 27—55,1985. camptothecin for the treatment of refractory or relapsed small-cell lung cancer. J. 22. Kaneda, N., Nagata, H., Furuta, T., and Yokokura, T. Metabolism and pharmacoki Clin. Oncol., 10: 1225—1229,1992. netics of the camptothecin analogue CPT-l I in the mouse. Cancer Res., 50: 1715— 3. FukuokaM.,Niitani,H.,Suzuki,A.,Motomiya,M.,Hasegawa,K.,Nishiwaki,Y., 1720, 1990. Kuriyama, T., Ariyoshi. Y., Negoro, S., Masuda, N., Nakajima, S., and Taguchi, T. 23. Rothenberg, M. L., Kuhn, J. G., Bums, H. A., Ill, Nelson, J., Eckardt, J. R., A phase II study of CPT-l I , a new derivative of camptothecin for previously Tristan-Morales,M.,Hilsenbeck,S.G., Weiss,0. R., Smith,L. S.,Rodriguez,G.I., untreated non-small-cell lung cancer. J. Clin. Oncol., 10: 16—20,1992. Rock, M. K., and Von Hoff, D. D. Phase I and pharmacokinetic trial of weekly 4. Shimada, Y., Yoshino, M., Wakui, A., Nakao, I., Futatsuki, K., Sakata, Y., Kambe, CPT-1l. J. Clin. Oncol., 11: 2194—2204,1993. M., Taguchi.T., and Ogawa,N. PhaseII study of CPT-l 1, a new camptothecin 24. Kano, Y., Suzuki, S., Akutsu, M., Suds, K. Inoue, Y., Yoshida, M., Sakamoto, S., and derivative, in metastatic colorectal cancer. J. Clin. Oncol., ii: 909—913,1993. Miura, Y. Effects of CPT-l I in combination with other anti-cancer agents in culture. 5. Takeuchi, S., Takamizawa, H., Takeda, Y., Okawa, T., Tamaya, T., Noda, K., Sugawa, T., Sekiba, K., Yakushiji, M., and Taguchi, T. Clinical study of CPT-l 1, Int. J. Cancer,50: 604—610,1992. camptothecin derivative, on gynecological malignancy (Abstract 617). Proc. Am. 25. Kudoh, S., Takada, M., Masuda, N., Nakagawa, K.. Itoh, K., Kusunoki, Y., Negoro, Soc. Clin. Oncol., 10: 189, 1991. S., Matsui, K., Takifuji, N., Morino, H., and Fukuoka,M. Enhancedantitumor 6. Yoshinari, T., Matsumoto, M., Arakawa, H., Okada, H., Noguchi, K., Suda, H., efficacy of a combination of CPT-l 1, a new derivative of camptothecin, and cisplatin Okura, A., and Nishimura, K. Novel antitumor indolocarbazole compound 6-N- against human lung tumor xenografts. Jpn. J. Cancer Res., 84: 203—207,1993. formylamino- 12,13-dihydro- I ,I I -dihydroxy- 13-(/3-D-glucopyranosyl)-5H-in 26. Kim, R., Hirabayashi, N., Nishiyama, M., Jinushi, K., Toge, T., and Okada, K. dolol2,3-ajpyrrolol3,4-clcarbazole-5,7(6H)-dione (NB-506): induction of topoi Experimental studies on biochemical modulation targeting topoisomerase I and II in somerase I-mediated DNA cleavage and mechanisms of cell line-selective human tumor xenografts in nude mice. mt. J. Cancer, 50: 760—766,1992. cytotoxicity. Cancer Res., 55: 1310—1315,1995. 27. Kanzawa, F., Nishio, K., Kubota, N., and Saijo, N. Antitumor activities of a new 7. Evans, W. K., Shepherd, F. A., Feld, R., Osoba, D., and Deboer, G. VP-16 and indolocarbazolesubstance,NB-506-resistantcellline, SBC-3/NB.CancerRca.,55: cisplatin as first-line therapy for small-cell lung cancer. J. Clin. Oncol., 3: 1471—1477, 2806—2813,1995. 1985. 28. Hsiang, Y-H., Hertzberg, R., Hecht, S., and Liu, L. F. Camptothecin induces protein 8. Splinter. T., Kok, T., Kho, T., Lameris, H., Kate, F., Dalesio, 0., Dolman, B., linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem., 260: Palmen, F.. Bouvy, J., Burghouts, J., Simonis, F., Harper, P.. Rankin, E., Reijs 14873—14878,1985. woud, I., and Hoogenhuijze, J. A multicenter phase II trial of cisplatin and oral 29. Hsiang, Y-H., and Liu, L F. Identification of mammalian DNA topoisomerase I as an etoposide (VP-16) in inoperable non-small-cell lung cancer. Semin. Oncol., 13 intracellular target of the anticancer drug camptothecin. Cancer Res., 48: 1722—1726, (Suppl. 3): 97—103.1986. 1988. 9. Durand, R. E., and Goldie, J. H. Interaction of etoposide and cisplatin in an in vitro 30. Hertzberg, R. P., Caranfa, M. J., and Hecht, S. M. On the mechanism of topoisomer tumor model. Cancer Treat. Rep.. 72: 673—679,1987. ase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex. 10. Masuda, N., Fukuoka, M., Takada, M., Kusunoki, Y., Negoro. S., Matsui, K., Kudoh, Biochemistry, 28: 4629—4638,1989. S., Takifuji, N., Nakagawa,K., and Kishimoto, S. CPT-l I in combinationwith 31. Hsiang, Y-H., Liu, L. F., Wall, M. E., Wami, M. C., Nicholas, A. W., Manikumar, G., displatin for advanced non-small-cell lung cancer. J. Clin. Oncol., 10: 1775—1780, Kirschenbaum, S., Silber, R., and Potmesil, M. DNA topoisomerase I-mediated DNA 1992. cleavage and cytotoxicity of camptothecin analogues. Cancer Res., 49: 4385—4389, 11. Fujiwara, Y., Yamakido, M., Fukuoka, M., Kudoh, S., Furuse, K., Ikegami, H., and 1989. Ariyoshi. Y. Phase II study of (CPT-l I) and cisplatin (CDDP) in patients 32. Jaxel, C., Kohn, K. W., Wani, M. C., Wall, M. E., and Pommier, Y. Structure-activity with small-cell lung cancer. Proc. Am. Soc. Clin. Oncol., 13: 335, 1994. study of the actions of camptothecin derivatives on mammalian topoisomerase I: 12. Itoh, K., Takada, M., Kudo, S., Masuda, N., Nakagawa, K., Matsui, K., Takifuji, N., evidence for a specific receptor site and a relaxation to antitumor activity. Cancer Kusunoki, Y., Fukuoka, M., and Kishimoto S. Synergistic effects of CPT-l 1 and Res.,49: 1465—1469,1989. cisplatin or etoposide on human lung cancer cell lines demonstrated by computer 33. Bellon, S. F., Coleman, J. H., and Lippard, S. J. DNA unwinding produced by analysis (Abstract). Lung Cancer, 7 (Suppl.): 124, 1991. site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplati 13. Horiuchi, N., Nakagawa. K., Sasaki, Y., Minato, K., Fujiwara, Y., Nezu, K., Ohe, Y., num(II). Biochemistry,30: 8026—8035,1991. and Saijo, N. In vitro antitumor activity of derivative (RM-49) and new 34. Fichtinger-Schepman, A. M. J., van der Veer, J. L., den Hartog, J. H. J., Lohman, anticancer antibiotics (FK973) against lung cancer cell lines determined by tetrazo P. H. M., and Reedijk, J. Adductsof the antitumordrug cis-diamminedichloroplati hum dye (MiT) assay. Cancer Chemother. Pharmacol.. 22: 246—250,1988. num(II) with DNA: formation, identification, and quantitation. Biochemistry, 24: 14. Steel, G. G., and Peckham, M. J. Exploitable mechanisms in combined radiotherapy 707—713,1985. chemotherapy: the concept of additivity. Int. J. Radiat. Oncol. BioI. Phys., 5: 85—91, 1979. 35. Gilbert, C. Cellular responsesto cisplatin. J. Biol. Chem., 269: 787—790,1994. 15. Chun-Ming. T.. Gazdar. A. F.. Venzon, D. J., Steinberg, S. M., Dedrick, R. L., 36. Swinnen, L. J., Ellis, N. K., and Erichson, L. C. Inhibition of cis-diammine-l,l Mulshine, J. L., and Kramer, B. S. Lack of in vitro synergy between etoposide and cyclobutane dicarboxylatoplatinum(ll)-induced DNA interstrand cross-link removal cis-diamminedichloroplatinum(II). Cancer Res., 49: 2390—2397,1989. and potentiation of cis-diammine-l,l-cyclobutane dicarboxy-Iatoplatinum(II) cyto 16. Bungo, M., Fujiwara, Y., Kasahara, K., Nakagawa, K., Ohe, Y., Sasaki, Y., Irino, S., toxicity by hydroxyurea and l-/3-D-arabinofuranosylcytosine. Cancer Res., 51: and Saijo, N. Decreased accumulation as a mechanism of resistance to cis-diam 1984—1989,1991. minedichloroplatinum(II) in human non-small-cell lung cancer cell line: relation to 37. Esaki, T., Nakano, S., Tatsumoto, T., Kuroki-Migita, M., Mitsugi, K., Nakamura. M., DNA damage and repair. Cancer Res.. 50: 2549—2553,1990. and Niho, Y. Inhibition by 5-fluorouracil of cis-diamminedichloroplatinum(II)-in 17. Zwelling, L. A., Anderson, T., and Kohn, K. W. DNA-protein and DNA interstrand duced DNA interstrand cross-link removal in a HST-l human squamouscarcinoma cross-linking by cis- and trans-platinum(II)diamminedichloride in L12l0 mouse cell line. CancerRes.,52: 6501—6506,1992. leukemia cells and relation to cytotoxicity. Cancer Res., 39: 365—369,1979. 38. Eastman, A., and Schulte, N. Enhanced DNA repair as a mechanism of resistance to 18. Deffie, A. M., Batra, J. K., and Goldenberg, G. J. Direct correlation between DNA cis-diamminedichloroplatinum(ll). Biochemistry, 27: 4730—4734,1988. topoisomerase II activity and cytotoxicity in Adriamycin-sensitive and-resistant P388 39. Parker, R. J., Eastman, A., Bostick, B. F., and Reed, E. Acquired cisplatin resistance leukemia cell lines. Cancer Res., 49: 58—62,1989. in human ovarian cancer cells is associated with enhanced repair of cisplatin-DNA 19. Bradford,M. M. A rapid and sensitivemethodfor the quantitationof microgram lesionsandreduceddrugaccumulation.J.Clin. Invest.,87: 772—777,1991.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1996 American Association for Cancer Research. Synergism between Cisplatin and Topoisomerase I Inhibitors, NB-506 and SN-38, in Human Small Cell Lung Cancer Cells

Minoru Fukuda, Kazuto Nishio, Fumihiko Kanzawa, et al.

Cancer Res 1996;56:789-793.

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