Cytotoxic Effects of Gemcitabine-Containing Regimens Against Human Non-Small Cell Lung Cancer Cell Lines Which Express Different Levels of P185@Â€•1

Home , Gemcitabine

(CANCERRESEARCH56.794-801, FebruaryIS. 9961 Cytotoxic Effects of Gemcitabine-containing Regimens against Human Non-Small Cell Lung Cancer Cell Lines Which Express Different Levels of p185@â€•1

Chun-Ming Tsai,2 Kuo-Ting Chang, Jeou-Yuan Chen, Yuh-Min Chen, Mei-Hui Chen, and Reury-Perng Perng

Department of Medicine, School of Medicine IC.M. T., Y-M. C., R-P. P.] and Institute of Life Sciences If.)'. CI, National Yang-Ming University, Taipei, Taiwan 11217; Section of Thoracic Oncology [C.M. T., K.T. C.. Y.M. C.. M-H. C.J Chest Department fR-P. P.], Veterans General Hospital-Taipei, Taipei, Taiwan 11217; and Institute of Biomedical Sciences, Academia Sinica Taipei fJ-J. C.]. Taiwan 11529. Republic of China

ABSTRACT in small cell lung cancer tumors (10â€”12).Enhancedchemoresistance associated with the elevated levels of pl85n@@inHER-2/neu-trans A novelpyrimidineanalogue,gemcitabine,hasbeenfoundtoinhibit fected NSCLC cells suggested the possibility that a high level of DNA replicationandrepair.We speculatedthatgemcitabineincombina tion with DNA-damaging agentsmight be more active againsthigh- than p185neuconfers multiple-drug resistance to certain human cancers low-p18S'@â€•expressingnon-smallcell lung cancer(NSCLC) cellsbecause (13). thehigh-p185@â€•expressorswereproposedtopossesamoreeffectiveDNA Previously, we have demonstrated that caffeine enhances chemo repair ability. We therefore compared the combination effects of gemcit sensitivity to a greater degree in high- than in low-p l851'@'-expressing abinepluscisplatin,gemcitabineplusetoposide,andcisplatinplusetopo NSCLC cell lines (7). Caffeine is known to enhance cell killing by side in a panel of 12 NSCLC cell lines. We also investigatedthe correla overriding the cell cycle arrest triggered by DNA-damaging agents tionsbetweenthe level of plSS'@â€•andthe cytotoxicitiesofeachsingle (14, 15). It is presumed that cell cycle arrest induced by such agents agentand the three combinations.Wefound that as singleagentsthe would allow cells to repair DNA lesions (16, 17). Our findings (7), cytotoxicities of cisplatin and etoposide but not gemcitabine were signifi therefore, suggested that the chemoresistant high-pl 85@@'-expressing cantlycorrelatedwiththe levelof pl85â€•@'.Incontrasttothe tightcross resistance between cisplatin and etoposide, gemcitabine demonstrated cells might utilize DNA repair machinery more effectively, attenuat little cross-resistanceto either etoposideor cisplatin. Both gemcitabine ing the lethal effects of chemotherapeutic agents,and that this survival containing combinationsdemonstratedequivalentor more activecytotox advantage would be diminished by caffeine. icities compared to cisplatin plus etoposide, with gemcitabine plus cispla Gemcitabine (2'2'-difluorodeoxycytidine) is a novel pynmidine tin showinga greatersynergisticactivitywhichwas effect(dose) analogue with structural and metabolic similarities to cytarabine (ar dependent. The effect of cisplatin plus etoposide was not plSS@' related, abinofuranosylcytosine). Compared to cytarabine, gemcitabine is, whereas gemcitabine-containing regimens, especially gemcitabine plus however, markedly distinctive in its cellular pharmacology and bio cisplatin, had a greater cytotoxicity againstthe high- than the low-p185â€•@' logical action and exhibits a much wider spectrum of activity against expressors.Our findings indicate that gemcitabinein combination with cisplatin is active against NSCLC cellsin vitro. The gemcitabine-cisplatin munne solid tumors and human tumor xenografts ( 18, 19). Gemcit interactionismoreactivethantheetoposide-cisplatininteractionincells abine is a prodrug that requires the intracellular conversion by deoxy with high-p185'@expression. cytidine kinase to its active forms, gemcitabine diphosphate and gemcitabine tnphosphate. Gemcitabine tnphosphate competes di INTRODUCTION rectly with dCTP as a substrate for incorporation into DNA by DNA polymerases (20). The gemcitabine metabolic pathway exhibits an NSCLC3 tumors are notorious for their resistance to chemothera interesting and unique combination of self-potentiating mechanisms peutic agents. Most chemotherapeutic drugs, both alone and in com whereby gemcitabine diphosphate contributes to a reduction of cellu bination, are not particularly active against this type of tumor and are lar dCTP levels (2 1), facilitating both increased gemcitabine phos at best strictly palliative in the clinical setting. Although current phorylation by reducing the inhibition of deoxycytidine kinase and cisplatin-based combination chemotherapeutic regimens produce ob decreased elimination by inactivating dCMP deaminase (22). This jective tumor responses in up to 40% of patients with advanced results in the accumulation of the active metabolites within the cell, NSCLC, the routine use of chemotherapy in these patients remains and hence continuing inhibition of cellular DNA synthesis and repair. controversial ( 1, 2). To improve the likelihood of response, response The inherent ability of gemcitabine to inhibit DNA synthesis and duration, and survival, it is clear that more active drugs or drug repair led us to propose the following hypotheses: (a) the intrinsic combinations are required. chemoresistancefor gemcitabine of NSCLC cells might not be tightly Using large panels of lung cancer cell lines and determining their correlated with the level of pl8Sâ€•@';and(b) DNA-damaging agents chemosensitivity profiles for several commonly used cytotoxic drugs, when combined with gemcitabine might be more active against the we and others have demonstrated that in vitro activity of most, if not high-pl85â€•@'-expressingNSCLC cell lines, which are presumably all, of the drugs were highly correlated with each other (3â€”5).In able to repair damaged DNA more effectively as compared to the NSCLC, the spectrum of cross-resistance/sensitivity observed is low-pl 8sne@J@expressingcelllines. closely associated with the expression of the HER-2/neu gene (6â€”8). Currently, cisplatin in combination with etoposide is one of the The HER-2/neu gene encodes a transmembrane glycoprotein receptor most commonly used chemotherapeutic regimens in the treatment of (pl85nra@)with tyrosine kinase activity (9). Overexpression of the NSCLC. This combination has been shown to have synergistic or HER-2/neu gene is encountered in >30% of NSCLC tumors but not additive antitumor activity in NSCLC cells in a variety of experimen tal models (23â€”25).Thecombination of cisplatin and etopoide pro Received 8/28/95; accepted I 2/15/95. The costsof publicationof this article weredefrayedin part by the paymentof page duces an objective response in 30â€”35%of patients with advanced charges. This article must therefore be hereby marked ads'ertisement in accordance with NSCLC (1, 2). In the present study, we have evaluated and compared 18 U.S.C. Section 1734 solely to indicate this fact. the combination effects of gemcitabine plus cisplatin, gemcitabine I This work was supported by National Science Council Grant NSC-85-233l-B-075- 018 and by the Lung Cancer Foundation in memory of Dr. K. S. Lu. plus etoposide, and cisplatin plus etoposide at the cellular level using 2 To whom requests for reprints should be addressed. a panel of NSCLC cell lines. We have also investigated the correla 3 The abbreviations used are: NSCLC, non-small cell lung cancer; cisplatin, cis tions between the level of pl85@u and the cytotoxicities of each single diamminedichloroplatinum (II); HNU, human neu units; IC, inhibitory concentration; CI, combination index. agent and the three drug combinations. 794

MATERIALS AND METHODS standard curve. Results were expressed as HNU4tg protein. The experiments were performedin triplicate. Each experimentwas performedin duplicate Cell Lines. Twelve NSCLC cell lineswere studied.Therewereeight wells. adenocarcinomas (NCI-H23, H322, H358, H522, H820, H1355, Hl435, and Study Design and Cytotoxicity Assays. We tested the cytotoxic effects of H1437); one adenosquamous carcinoma (Hl25); and three large cell carcino the single agentsgemeitabine(Eli Lilly Co., Indianapolis,IN), cisplatin mas (H460, Hl 155, and H1299). These cell lines expressed various levels of (FarmitaliaCarlo Erba,Milano, Italy), andetoposide(Bristol-MyersGMBH, pl85@ (6â€”8).Alllines wereestablishedfrompreviouslyuntreatedpatients Troisdorf,Germany)andof eachtwo-drugcombination.In vitro drugcombi and were grown in RPMI 1640medium(GIBCO) supplementedwith 10% nation testing was designed and performed using the 3-(4,5-dimethyl-2-thia fetal bovine serum and adapted in RPMI 1640 medium supplemented with 5% zolyl)-2,5-diphenyl-2H-tetrazolium bromde colorimethc assay as we de fetal bovine serum for >6 months before this study. All cell lines were in the scribedin detailpreviously(26, 27).The singledrugsweretestedoverseven logarithmicphaseof growth at the time of the drug sensitivityassays. concentrations(in0.5- or 1-logincrements)tocovertheentiredose-response @ Quantitative Measurement of the P185'@ Nearly confluent cells were harvestedby scraping,followed by centrifugationto form a cell curveswheneverpossible.Sixteensurvivalcurvesweregeneratedfromevery pellet.Cells wereincubatedinlysis buffer [10 mMTris-HCI (pH 7.6), 1.5mxi setof experiments,onefrom eachof thesingledrugsand14from the49 pairs EDTA, 10% glycerol, and 0.1% sodiumazide] and Douncehomogenized. of drugcombinations(27).ThedrugsweredissolvedinPBSto 1mM,andthen After homogenization the proteins were detergent extracted. After centrifuga all weresubsequentlydilutedin culturemedium.For gemcitabine-containing tion, supernatants were collected and protein concentrations determined. The regimens,gemcitabinewasadded20 mn beforethe additionof the second immunoassay for detection and quantitation of p1 85@e@*wasa sandwich assay drug. For cisplatin plus etoposide, etoposide was added 20 mm after the utilizing monoclonal antibodies NB-3 (coated onto mcrotiter wells) and TA-l addition of cisplatin. The percentage of control absorbance was considered to @ (biotin labeled) (human neu assay kits: Oncogene Science, Inc., Uniondale, be the surviving fraction of cells, and the IC@o- and IC70valueswere NY; Ref. 7). The biotin-labeled TA- I was detected using a streptavidin definedastheconcentrationsofdrugthatproduced30,50,and70%reduction horseradish peroxidase conjugate in PBS (pH 7.4), 1% BSA, and 0.1% chlo in control absorbance, respectively. The results were the means of three roacetamide.After the addition of O-phenylenediaminesubstrate,thecolor independentlyperformedassays.Eachassaywasperformedinfour wellsatthe change was measured at 490 nm using a microplate reader. A standard curve sametime. wasgeneratedbyusingstandardsolutions.Theconcentrationofp18Sâ€•@'intest Identification of the Combination Effects. We used the classical isobole samples was determined by interpolation of the sample absorbance from the method(26â€”28)todeterminethein vitro effectsofdrug combinations.TheCI

Cisplatin Gemcitabine Gemcitabine vs. Etoposide vs. Etoposide vs. Cisplatin â€” :3 R = 0.790 A = 0.231 R=0.545 @ 2 P = 0.009 P=0.444 2 P=0.070

1'S' c.l@ 0)1 @1 @ .2 .. ,.. .2 0 .1 @ @oo @0 .@ @- @o .@â€¢@â€˜ 0. .@ ..,.. . ..@ 0 @3 ...... @ Ui Ui . @e) -2 @ i .3 :@ -1 0 1 2 -5 -3 -1 1 3 Cisplatin log (pM) Gomcftabino log (pM) Gemcitabine log (pM) Fig. I . Correlations of the cytotoxicities of cis platin, etoposide, and gemcitabine in 12 NSCLC â€” cell lines at the 30, 50, and 70% effect levels.In 4? R=0.902 R = 0.687 R=0.552 vitro drug testing was performed using the 3-(4,5- 4? P=0.003 P=0.025 P = 0.067 dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium @ bromide colorimetric assay.The IC@0,IC50,and 0) 0) . C.? .21 .21 S IC70 valueswere defined as the concentrationsof 0 drug that produced 30, 50, and 70% reduction in 4? 0 @0 . .,@ Cl) 0 . ....â€¢ control absorbance, respectively. The results were ...... @ .., the meansof three independentlyperformedas a @ a â€¢@1 0 ... . .â€¢â€¢â€¢â€˜â€¢â€¢â€¢.. says.Eachassaywasperformedinfour wellsatthe -1 c@i-i @ sametime.TheRandP valuescalculatedusingthe Ui Spearman rank correlation test are labeled. @@@ In -2 .5 :3 -1 0 1 2 5@:3@:1@ @3 Cisplatin log (pM) Gemcitabine log (pM) Gemcitabine log (pM)

â€” 3 @ R=0.853 A = 0.615 R=0.203 @@ 2 P0.005@ P=0.0413 -@, 2 P=0.501 2 . 0) . 0 1 0)1 â€¢SS...... â€¢' 0 .f.... .2 â€¢...... *@â€¢â€¢ @i .,.@ @0 0 0 .@ . @ ...i ...... S 0. .. . @o â€¢ 0 @ 2 .Ã³ â€”1 @ Ui_i ..â€˜ Ui C-) N - ______:3@.'1'r@3 .53@1 @ â€”1 a -5 3 Cisplatin log (pM) Gemcitabine log (pM) Gemcitabine log (pM:

795

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. GEMCrrABINE-CONTAINING REGIMENS AGAINST NSCLC CELLS wasusedto expressthecytotoxiceffectsof drugcombinations.Fordetecting wasusedto testfor differencesin the IC valuesof drugsandtheCI valuesof true in vitro synergy between drug combinations, there is still controversy over drug combinations of the six cell lines with low and the other six cell lines with whichmethodisbest(29).Theclassicalisobolemethodwithapplicationofthe high p185ncuexpression.All tests were two sided, and significance was CI is oneof themostcommonlyusedmethods.Byusingthis methodto testa assumedif P < 0.05. variety of drug combinations, our previous findings were in accordance with the findingsobtainedfrom otherexperimentalmodelsystemsaswell aswith RESULTS the clinical observations (7, 26, 27). The CI was defined as the sum of the relative dosesof eachdrug which yields an isoeffectcell kill when added Little Cross-resistance between Gemcitabine and Either Cia together.Forexample,for thecombinationof drug A + drugB, CI@= (dose platin or Etoposide. In this panel of cell lines, we found that the of drugA/ICr of drugA) + (doseof drugB/ICXof drugB). Here,thedoseof inhibitory concentrations of cisplatin versus etoposide were closely drug A and dose of drug B when added together yielded an isoeffect (x%) cell correlated through the effect levels studied (all P values 0.009, kill. The CI at the 30, 50, and 70% effect levels were determined and Fig. I, left set ofpanels). Gemcitabine toxicity correlated with eto designatedasCI@,Cl50,andCl70.Within thedesignedassayrange,a setof poside toxicity (P = 0.025 and 0.0413 for IC50and IC70,respectively, CI valuesor data points was generatedbecausetherewere multiple drug concentrations that achieved the same isoeffect. In the present study, the mean Fig. 1, middle center and middle bottom panels) but correlated weakly valuesof thesurvivalfractionsof thethreereplicatetestswereusedtogenerate if at all with cisplatin toxicity (all P values 0.067, Fig. I , right set thesetof CI valuesandconstructtheisobolefor aparticularcell line anddrug ofpanels). As compared to the tight cross-resistancebetween cisplatin combination.The meanCI value for this set was reportedas the summary and etoposide, gemcitabine showed little cross-resistance to either measure.Upperandlower boundsof I.05and0.95wereselectedasbeingof cisplatin or etoposide. interest (based on a preset â€œnullâ€•intervalof 0.95â€”1.05), so that mean CI values Correlation of the Level of plSS@â€• with the Cytotoxic Effects of >1.05 or <0.95 are interpreted as being suggestive of antagonism and syn- Cisplatin and Etoposide, but not with the Effect of Gemcitabine. ergism, respectively. The 12 cell lines utilized were divided into two equal groups accord Data Analysis. Sign testswere performedon eachset of CI valuesto ing to the level of p185@seuexpressed,high and low expressors (Table formally evaluatewhetherantagonismorsynergismwasevidentfor a partic- 1) The values of pl85neu, IC30, IC50, and IC70 of gemcitabine, ularcell line,drugcombination,andeffectlevels.In addition,Wilcoxonsigned ranktestswerecomputedtoevaluatewhethersignificantdifferencesinthecell cisplatin, and etoposide of individual cell lines as well as their mean line meansoccurredascomparedtoa null hypothesizedCIof 1andto analyze values of the total and the two subgroups are shown in Table 1. In the thedifferencesofdruginteractionsofthetestedregimens.TheSpearmanrank entire panel of I 2 cell lines, the cytotoxicities of cisplatin and etopo correlation was used to analyzethe correlationsbetweenpl85â€•@'protein side but not of gemcitabine showed a statistically significant correla production and the IC values of the drug. The Mann-Whitney U rank sum test tion with the level of pl85neu at all of the effect levels studied (Fig. 2).

linesCell Table 1 Cliemosensitivitiesfor single agents of NSCLC cell

(pM)IC@0'IC50aIC70a (@sM) Cisplatin (aiM)Etoposide

linePl85@U(HNU/pg)Gemcitabine IC@()IC70IC301C5()IC70Low-pl85â€•@'IC30 linesHl expression, 6 cell 1550.62 1.079 1.843 (0.007)H232.95 (0.05)0.404 (0.001)0.672 (0.078)1.151 (0.018) (0.057) (0.206)2.894 (0.059)0.337 (0.007)0.558 (0.095)0.951 0.325 0.683 19 (0.023)H12993.57 (0.12)0.076 (0.002)0.185 (0.045)0.305 (0.003) (0.010) (0.033)2.170 (0.277)0.1 (0.003)0.224 (0.027)0.535 1.472 2.872 (0.959)H46O4.81 (0.29)0.0910 (0.020)0.193 (0.041)0.597 (0.018) (0.003) (0.151)6.151 (0.168)0.351 (0.008)0.718 (0.047)3.992 1 0.208 0.448 (0.035)H3581 (1.08)0.01 (0.013)0.003 (0.003)0.005 (0.003) (0.021) (0.021)0.866 (0.034)0.029 (0.007)0.062 (0.008)0.152 1.62 0.513 1.528 (0.974)H12512.65 (1.65)0.0003(0.0001)0.0008 (0.0002)0.0029 (0.001) (0. 160) (0. 192)5.935 ( I .141)0.091 (0.035)0.228 (0.024)2.009 1.060 3.298 (1.010)Mean6.04 (0.74)0.0135 (0.003)0.035 (0.008)0.904 (0.061) (0.105) (0.312)5.809 (0.829)0.434 (0.083)1.377 (0.211)5.505 0.776 1.779 (2.01)0.099 (0.063)0.181 (0.104)0.494 (0.87)High@pl85@c@@(0.194) (0.204) (0.467)3.971 (0.932)0.227 (0.068)0.528 (0.196)2.191 linesH82026.34 expression, 6 cell 0.751 2.382 (2.677)H143729.16(1.76)0.029 (0.004)1.141 (0.672)9.868 (0.386) (0.137) (0.597)4.677 (0.858)0.876 (0.047)3.951 (0.973)10.456 1.709 3.991 (0.116)H32235.75 (5.04)0.047 (0.003)0.097 (0.018)0.248 (0.001) (0.090) (0.307)9.856 (0.922)0.633 (0.064)2.541 (0.995)9.013 0.649 2.192 (2.195)H135542.06(3.85)0.002 (0.0002)0.0043 (0.001)0.0151 (0.018) (0.151) (0.256)10.841(1.602)0.446 (0.048)1.546 (0.631)14.695 1 2.888 6.799 (1.807)Hl43553.70 (2.31)0.453 (0.010)0.958 (0.150)2.61 (0.718) (0.335) (1.572)10.523 (2.131)0.727 (0.079)8.866 (1.099)19.902 7.583 19.734 (5.592)H52273.94 (3.28)0.097 (0.002)0.399 (0.078)1.519 (0.229) (0.089) (4.477)39.871 (3.767)4.316 (0.868)13.522 (3.962)60.045 2.279 4.658 (3.317)Mean43.50 (4.00)0.007 (0.003)12.214 (5.789)610.31 (264.7) (0.093) (0.820)6.538 (0.570)2.197 (0.085)17.287 (1.526)32.606 2.643 6.626 (7.29)0.106 (0.071)2.469 (1.958)104.1 (101.3)(7.92)Total,(1.049) (2.71)13.718 (5.325)1.532 (0.613)7.952 (2.616)24.45 linesMean24.77 12cell 1.71 4.20 (5.07)P, (6.70)0.103 (0.045)1.325 (0.996)52.30 (50.74) (0.58) (1.50)8.84 (0.35)0.88 (0.35)4.24 (1.68)13.32 low vs. highâ€•0.0040.8730.1090.109 0.055 0.0250.0160.0040.0040.004

a IC@, IC50, and IC70 designated for the inhibitory concentrations of drugs required to inhibit cell growth by 30, 50, and7O%, respectively. Numbers in parentheses, SE. b Statistical evaluation using the Mann-Whitney U rank sum test for the differences between the low- and the high-pl85'@â€• groups. All statistical tests were two-sided, and statistical significance was assumed if P < 0.05. 796

GEMCITABINE-CONTAININGREGIMENSAGAINST NSCLC CELLS 30%EffectLevel50%EffectLevel70%EffectLevel (A) Cisplatin

0 0 R=0.615 R = 0.769 R = 0.783 @ P=0.0413 P=0.011 P=0.0094 2 2 ...... 2 z . I . 4 â€¢dIL.@@@â€¢ S... .,I. 1 Q.O@ 1 â€¢.O 1 .0

Lf) Co 1@ 0 0.....@ 0 0 . 0 ...... 0

-1 0 1 2 -1 0 1 ? â€”1 0 1 2 log 1C30 (jiM) log IC5o (jiM) log IC7o (jiM) (B)Etoposide Fig. 2. Correlations between the level of pl85@' and chemosensitivities for cisplatin. etoposide, and gemcitabine R=0.832 = 0.881 R=0.923 of the I2 testedcell lines at the 30, 50, and 70% effect P=0.006 P=0004 P = 0.002 .,...1%. levels. The immunoassay for detection and quantitation of 2 2R 2 z 0...@,..... pl85@@e@@wasa sandwich assay utilizing monoclonal anti I bodies NB-3 and TA-I . Results expressed as HNU/pg pro tein were the mean values of three separate assays (each I Q..O @ assaywas performed in duplicate wells). (â€¢). C .o...e 0 [email protected] o..0...... 0 expressors: (0), low-pl85@ expressors. The R and P val to ues calculated using the Spearman rank correlation test are CO 0 0..'@@ .. 0 0 .. 0 labeled. 0

â€”1 @@ -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 2 log 1C30 (jiM) log 1C50 (jiM) log IC7o (jiM) (C) Gemcitabine 3 R = -0i4 R = 0.336 R=0.441 C) P=0.64 P=0.266 P=0.144 :D 2 @ z 2 â€¢ 2 S I ...1...,...... ,S @ 1 0 I 1

to og CO 0 0 0 0 g) â€”1 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 log 1C30 (jiM) log IC5o (jiM) log IC7o (jiM)

The high-pl 85neugroup was more chemoresistant when treated with combinations demonstrated synergistic interactions, but the mean CI cisplatin and etoposide but not with gemcitabine as compared to the values of the gemcitabine-containing regimes were smaller than those low@pl85r@@@group(Table I). The differences of the mean IC values of cisplatin plus etoposide (Fig. 3A), indicating a greater degree of of cisplatin and etoposide but not of gemcitabine between the two cell synergy. At the 70% effect level, gemcitabine plus cisplatin was line groups were statistically significant for all three effect levels statistically more active than cisplatin plus etoposide (P 0.015, Fig. studied, except that of cisplatin at the 30% effect level where the 3A). We also found that the synergistic combination effects of gem difference was marginally significant (P = 0.055; Table I). citabine plus cisplatin was effect dependent (Fig. 3A; r â€”0.998, More Active Cytotoxicitiesof Gemcitabine-containingRegi P = 0.037 by simple correlation). mens, Especially Gemcitabine Plus Cisplatin. Inthe entirepanelof Greater Enhancementofthe CytotoxicitiesofDNA-damaging cell lines, gemcitabine-containing regimens showed an additive effect, Agents by Gemcitabine in the High-p185@-expressing Cell Lines. and cisplatin plus etoposide demonstrated a synergistic effect at the In the low-pl85'@â€•group,the threecombinationregimens,in general, 30% effect level (Table 2); however, the difference between the mean demonstrated additive or marginally synergistic effects at the 30 and CI valuesof any two combinationswasminimal and not statistically 50% effect levels and synergistic effects at the 70% effect level (Table significant (Fig. 3A ). At higher (50 and 70%) effect levels, all of the 2 and Fig. 3B), but the differences between the mean CI values of 797

Table 2 Mean values of C!' of two-drug combinations

Gemcitabine + cisplatin Gemcitabine + etoposide Cisplatin + etoposide

b b Cell line 30 50 Cl70b CI@ Cl50 Cl70 Cl30 Cl50 Cl70 Low@pl85@@euexpression,6 cell lines HI 155 1.035 0.995 0.902 0.987 0.927 0.857 I .035 1.003 1.033 (0.02) (0.018) (0.019) (0.026) (0.011) (0.017) (0.03 1) (0.022) (0.014) H23 1.022 0.988 0.781 0.949 0.923 0.827 0.984 0.968 0.777 (0.033) (0.012) (0.057) (0.034) (0.015) (0.042) (0.024) (0.012) (0.047) H1299 0.825 0.813 0.743 0.867 0.855 0.625 0.968 0.977 0.933 (0.028) (0.037) (0.035) (0.031) (0.034) (0.035) (0.017) (0.015) (0.01) H460 1.100 I .005 0.969 1.151 1.081 1.054 0.889 0.887 0.867 (0.056) (0.037) (0.035) (0.071) (0.041) (0.024) (0.033) (0.019) (0.021) H358 0.980 I.036 0.961 0.945 0.953 0.911 0.856 0.916 0.969 (0.035) (0.041) (0.029) (0.021) (0.012) (0.037) (0.019) (0.018) (0.032) H125 0.922 0.935 0.889 0.930 0.912 0.881 0.897 0.903 0.882 (0.046) (0.036) (0.048) (0.026) (0.027) (0.024) (0.044) (0.037) (0.03) MeanC 0.981 0.962 0.874 0.972 0.942 0.859 0.938 0.942 0.910 (0.039) (0.033) (0.038) (0.039) (0.031) (0.057) (0.028) (0.019) (0.036) pC 0.92 0.293 0.028 0.35 0.173 0.046 0. 12 0.046 0.075 Groupresultsâ€• A A S A A S A S A High@pl85@@euexpression,6 cell lines H820 0.774 0.491 0.496 0.892 0.788 0.721 0.908 0.887 0.799 (0.048) (0.097) (0.103) (0.040) (0.076) (0.084) (0.040) (0.019) (0.035) H1437 0.923 0.919 0.788 0.978 0.799 0.843 I .044 0.929 0.980 (0.017) (0.015) (0.037) (0.009) (0.047) (0.038) (0.035) (0.041) (0.017) H322 1.052 0.973 0.886 1.131 0.946 0.910 0.908 0.780 1.015 (0.041) (0.02) (0.063) (0.044) (0.063) (0.05) (0.02 1) (0.032) (0.024) H1355 0.830 0.812 0.627 0.978 0.802 0.822 0.847 0.867 0.793 (0.045) (0.031) (0.058) (0.014) (0.028) (0.036) (0.067) (0.017) (0.042) H1435 0.871 0.815 0.636 0.666 0.721 0.852 0.893 0.901 0.723 (0.037) (0.044) (0.049) (0.047) (0.066) (0.053) (0.027) (0.055) (0.09) H522 1.034 0.618 0.549 0.616 0.575 0.778 0.646 0.968 0.901 (0.069) (0.122) (0.112) (0.089) (0.122) (0.084) (0.067) (0.018) (0.028) Mean' 0.914 0.771 0.664 0.877 0.772 0.821 0.874 0.889 0.869 (0.045) (0.075) (0.060) (0.081) (0.050) (0.027) (0.053) (0.026) (0.047) PC 0.12 0.028 0.028 0.17 0.028 0.028 0.046 0.028 0.046 Groupresults' A S S A S S S S S Total, 12 cell lines Mean' 0.947 0.867 0.769 0.924 0.857 0.840 0.906 0.915 0.889 (0.030) (0.048) (0.046) (0.045) (0.038) (0.030) (0.030) (0.017) (0.029) PC 0.182 0.011 0.002 0.10 0.008 0.003 0.012 0.003 0.008 Groupresultsâ€• A S S A S S S S S P. low vs. highe 0.423 0.025 0.025 0.521 0.025 0.262 0.521 0.108 0.631 @ a text. b CI@, CI@, and Cl70 were designated for the group mean values of CI at the 30, 50, and 70% effect levels, respectively. Mean CI values > 1.05 or < 0.95 are interpreted as being suggestive of antagonism and synergism, respectively. Numbers in parentheses,SE. C Wilcoxon signed rank tests were computed to evaluate whether significant differences in the group means occurred as compared to a null hypothesized CI of 1. d Group results (analyzed using the Wilcoxon signed rank test). A, additive; Ant, antagonistic; 5, synergistic. e Statistical evaluations using the Mann-Whitney U rank sum test for the differences between the low- and the high@pl85@@cu groups. All statistical tests were two-sided, and significance was assumed if P < 0.05.

1.1

1.0 Fig. 3. The mean values of the CI (see text) of gemcitab @ inc plus cisplatin (G + C), gemcitabine plus etoposide @ (G + V), and cisplatin plus etoposide (C + V) at the 30, 50, Ã˜@9 and 70% effect levels are plotted to demonstrate the differ encesof drug interactions of the tested regimens which were o statistically analyzed using the Wilcoxon signed rank test. Shaded area, additivity based on null interval of 0.95â€”1.05. .@ 0.8 A, the entire panel of cell lines; B, the low-pl85â€•@'expres sors; and C, the high@pl85@@c@[email protected]+ C versus o C+V,P=0.0l5;**,G+CversusG+VandG+C 0 07 versus C + V. P = 0.028. In the entire panel of cell lines, gemcitabine-containing regimens (particularly, gemcitabine o @ plus cisplatin)demonstratedgreatersynergisticeffectsthan the combinationof cisplatin plus etoposideat middle and 0.6 higher effect levels, presumably primarily related to their moreactiveeffectsagainstthehigh-pl85'@â€•-expressingcells. 0.5

30 50 70 30 50 70 30 50 70 % Inhibition 798

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. GEMCITABINE-CONTAINING REGIMENS AGAINST NSCLC CELLS Low-pi85neu expressingcell Highp185neU expressingcell lineNCI-H460 lineNCI-H1355

(A) Gemcitabine + Cisplatin

* mCI3o = 1.100Â±0.056 (7) mCI3o = 0.830Â±0.045(7) @ 1 mCI5o = 1.005Â±0.037(7) 1.0. mCI5o = 0.812Â± 0.031 (8) mCI7o= 0.969Â±0.035(9) C mCI7O= 0.627Â± 0.058 (9) *** 0 0 @@@ 05 k,@1,,,,, C C Cu Cu 0@ Cl) Cl) V

0 0 o@o -.. 1.0 0.0 0.5 1.0 Gemcitabine (IC unit) Gemcitabine(ICunit)

(B) Gemcitabine + Etonoside

mCI3o = 1.151 Â±0.071 (6) mCI3O=0.978Â±0.014(6) mCI5o = 1.081 Â±0.041 (7) 1.0. mCI5O=0.802Â±0028(8) C mCI7o = 1.054 Â±0024 (9) C mCI7o = 0.822Â± 0.036 (1O)** 0 0 0.5.

Cl) U) 0 0 0@ 0 0 w w 0.0 0.5 1.0 0.0 0.5 1.0 Gemcitabine (IC unit) Gemcitabine(ICunit)

10 mCI3o= mCI3o = 0.847Â±0067(6) 0.889Â±0.033(6) 1.0 @ 4-' â€˜ mCI5o = 0.867Â± 0.017(8) C . â€˜ mCI5o= 0.887Â±0.019(7)* C @ 0.867Â±0.021(8)** 0.042 (9) *** 0 0

C) C) 0.5 V V Cl) Cl) 0 0 0 0 4-' w w AA 0.0 0.5 1.0 Cisplatin(ICunit) Cisplatin(ICunit)

@ Fig. 4. The isobolograms of gemcitabine plus cisplatin (A), gemcitabine plus etoposide (B), and cisplatin plus etoposide (C) agalnst the NCI-H460 (left panel) and the high-pl85â€•-expressing NCI-HI355 (right panel) cell lines at 30, 50, and 70% effect levels are shown. The values of the mean CI (see text) for a particular cell line, drugcombination,andeffect levelsarealsolabeled.mCI,@,mCI50,andmCI70weredesignatedforthemeanCI valuesat the30,50, and70%effect levels,respectively.Numbersin parentheses,number of data points. Data points at 30, 50, and 70% effect levels (signified as x, 0, and S. respectively] were generated from the mean survival fractions of the three replicate experiments and were fitted to construct the isoboles (by 3 order of polynomial, with all R values >0.964). Data points above the dashed diagonal line of the additive effect in the isobole suggest antagonism and those below the diagonal suggest synergism. Sign tests were applied on each set of data points to formally evaluate whether synergism or @ antagonism was evident for a particular cell line, drug combination, and effect levels. @,P< 0.05; @,P< 0.01; P < 0.005. Although the effects of gemcitabine-containing regimens were additive against NCI-H460 and statistically synergistic against NCI-Hl355, the effects of cisplatin plus etoposide were synergistic against both cell lines. In general, thecytotoxiceffectsof all combinationsagainstthesetwo cell linestendto bemoreactiveat highereffect levels(isobolesshift leftwardtowardtheaxisanddecreaseinthemeanCI valueswith the increaseof the combinationeffects),with gemcitabinepluscisplatinagainstNCI-H1355showingthe greatestactivity.

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theseregimens were not statistically significant throughout the studied view that high levels of pl85'@â€•maypromote DNA repair. Never effect levels (Fig. 3B). In contrast, in the @gh-pl85@@â€•group(Fig. theless, thus far the pathway mediating the relationship between 3C), while all combinationsshowed synergisticinteractionsacrossthe pl85'@' and DNA repair remains unclear. Whether the more active effect levels studied, the mean CI values of gemcitabine-containing interactions between gemcitabine and either cisplatin or etoposide in regimens were smaller than those of cisplatin plus etoposide at the 50 high- than in low- p185neuexpressors is due to more inhibition on and 70% effect levels. Gemcitabine plus cisplatin was statistically DNA repair must be verified in additional mechanistic studies. more active than the other two regimens at the 70% effect level (both Through the past decade,the increasedknowledge of tumor biology P values = 0.028). Moreover, we found that cisplatin plus etoposide of lung cancer has enabled us not only to develop new therapeutic demonstrated no differential effects between the high- and the low modalities for clinical intervention (e.g., gene therapy), but also to p185@lC@@groups(Table 2). In contrast, the combination effects of maximize the therapeutic benefit of currently available combination gemcitabine plus cisplatin at the 50 and 70% effect levels and that of chemotherapy. Overexpression of HER-2/neu has been detected in a gemcitabine plus etoposide at the 50% effect level were statistically subgroup of NSCLCs (10â€”12)andis associated with advanced dis more active in the high- than in the low-pl85'@'@group (all P val easestage (11), shortened survival (12), and multidrug resistance (6). ues = 0.025; Table 2). These findings demonstrated that gemcitabine Gemcitabine alone or in combination with other agents is currently was able to enhance the cytotoxicities of cisplatin and etoposide to a being evaluated in Phase I and Phase II clinical trials in a variety of greater degree in the high- than in the low-pl85'@â€•-expressingcell human cancers, including NSCLC (33â€”40).Severalpreliminary din lines. Fig. 4 demonstrates the representative examples of the drug ical studies have shown that the combination of gemcitabine and combination isoboles of one low- and one high-pl85'@'-expressing cisplatin may induce a considerable response rate (in the range of cell line, NCI-H460 and NCI-H1355, respectively. 36â€”58%)with modest side effects in the treatment of advanced NSCLC (36â€”40).Our results in the present study indicate that gem citabine-containing regimens (especially gemcitabine plus cisplatin) DISCUSSION are highly cytotoxic against NSCLC cell lines in vitro. The gemcit Despite the fact that dozens of chemotherapeutic agents have been abine-cisplatin interaction is more active than the etoposide-cisplatin introduced over the past four decades, only a few of them, such as interaction in cells with high-pl85'@â€•expression.This work clearly ifosfamide, mitomycin C, vinblastine, and vindesine, have consis provides a rationale to test the gemcitabine-cisplatin combination in tently producedsingle-agent response rates greaterthan 15% in pa PhaseII and PhaseIII clinical trials in a subgroup of NSCLC patients tients with NSCLC (1, 2). Nevertheless, several newly developed with tumors expressing high levels of pl8sneu and which therefore drugs, including navelbine, Taxol, and taxotere, the camptothecins may be more chemoresistant to commonly used combination therapy. CPT-1 1 and topotecan, and gemcitabine, have also been able to produce single-agent responserates above 20% (2, 30). Among these ACKNOWLEDGMENTS new drugs, gemcitabine attracted our attention becauseof its ability to inhibit DNA replication and repair. It is shown that many DNA We thankDr. BarnettS. Kramerfor reviewingthemanuscriptandEli Lilly replicative processestargeted by gemcitabine in growing cells are also Co. for kindly providinggemcitabine. required for the repair of DNA damage in growth-arrested cells (18â€”21).Therefore,itis plausiblethatDNArepairsiteselicitedby REFERENCES DNA-damaging agentscould be the target sites for gemcitabine. Cells that are more actively undergoing DNA repair may be more suscep 1. Minna, J. D., Pass, H., Glatstein, E., and Ihde, D. C. Cancer of the lung. In: V. T. Devita,Jr., S. Hellman,andS. A. Rosenberg(eds.),Cancer:PrinciplesandPractice tible to the blocking effect of gemcitabine. Gemcitabine, therefore, of Oncology, pp. 673â€”722.Philadelphia:J. B. Lippincott Co., 1993. would be a good candidate to combine with DNA-damaging agents 2. Shepherd, F. A. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1996 American Association for Cancer Research. Cytotoxic Effects of Gemcitabine-containing Regimens against Human Non-Small Cell Lung Cancer Cell Lines Which Express Different Levels of p185neu

Chun-Ming Tsai, Kuo-Ting Chang, Jeou-Yuan Chen, et al.

Cancer Res 1996;56:794-801.

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