[CANCER RESEARCH 50, 1571-1575, March I. 1990]

Comparison of the Cytotoxic Effects of the High- and Low-Molecular-Weight

Anticancer Agents on Multidrug-resistant Chinese Hamster Ovary Cells inVitro1

Yoichi Miyamoto, Tatsuya Oda, and Hiroshi Maeda2

Department of Microbiology, Kumamoto University Medical School, Kumamoto 860, Japan

ABSTRACT

Neocarzinostatin (NCS), styrene-maleic acid copolymer-conjugatedneocarzinostatin (SMANCS), and ricin exhibited cytotoxicity against twodifferent types of Chinese hamster ovary cells, parental AUXB1 cellsand the multidrug-resistant (MI )K) subline CHRC5 cells at the nanomolar

range. These doses were much lower than those of the other anticancerdrugs tested (micromolar range), even after a short incubation. MDKCHRC5 cells were 20 to 900 times more resistant to Adriamycin, aclaci-

nomycin, vinblastine, and mitomycin C than were AUXB1 cells. However,the resistance of CHRC5 cells to NCS, SMANCS, or ricin was relatively

low: the 50% colony inhibitory concentration was only 5 to 10 timeshigher than that for parental AUXB1 cells. CHRC5 cells were notresistant to 5-fluorouracil and c/i-diamminedichloroplatinum(II), but theeffective doses of these agents to them were III1 -10" times higher, and

longer incubation times were required to produce the same cytotoxicityas NCS and SMANCS. Furthermore, cell-bound NCS, SMANCS, andricin were not released from AUXB1 or CHRC5 cells during a 120-minincubation, although Adriamycin was excreted very rapidly from CHRC5

cells after binding and internalization. These results strongly suggest thatNCS, SMANCS, and ricin, which are internalized into cells by endocy-tosis, were not excreted from the cells by active efflux and exhibited apronounced anticancer effect against MDR cells.

INTRODUCTION

One of the most serious problems in cancer chemotherapy isMDR3 of cancer cells. The active efflux of anticancer drugsmediated by the P-glycoprotein of MDR cells is now well knownto be a basic mechanism of MDR (1). One tactic to overcomeMDR is the inhibition of active efflux by suitable agents suchas calcium channel blockers (2-5), calmodulin inhibitors (4-8),synthetic isoprenoids (9), biscoclaurine alkaloids (10, 11), andlysomotropic agents (12), which directly or indirectly act on P-glycoprotein. However, there are problems with many of thetechniques involving the use of these agents, especially, maintenance of effective concentrations of such agents in tumortissue without side effects (1, 2).

Whether or not anticancer drugs can be excreted by P-glycoprotein may depend on the cell's uptake mechanism and

intracellular localization of the drug. It may be possible toovercome MDR by using anticancer drugs that are internalizedinto the target cells by endocytosis. Recently, we found that theproteinaceous anticancer drug NCS and its styrene-maleic acidcopolymer conjugate, SMANCS, were internalized into cells byendocytosis via a specific receptor (13-16). Endocytotic inter-

Received4/24/89;revised11/22/89;accepted12/4/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by a Cancer Research Grant from Japanese Mon-

busho.2To whom correspondence should be addressed.3The abbreviations used are: MDR, multidrug resistance: KRP. Krebs-Ringer

phosphate buffer; ID50, 50% of inhibitory dose; ID10o. 100% inhibitory minimaldose; NCS, neocarzinostatin; SMANCS, styrene-maleic acid copolymer-conjugated neocarzinostatin; ADM. Adriamycin; ACM, aclacinomycin; VBL. vinblastine; MMC, mitomycin C; 5-FU, 5-fluorouracil; CDDP, c/i-diamminedichloro-platinum(II); FITC. fluorescein isothiocyanate; F-NCS, FITC-labeled neocarzinostatin; F-SMANCS. FITC-labeled SMANCS; F-ricin, FITC-labeled ricin;GSH. glutathione.

nalization is also observed for ricin (17). Thus, we decided toinvestigate whether NCS, SMANCS, and ricin might not besubject to active efflux by P-glycoprotein. Furthermore, it isdifficult to maintain the effective concentration of most low-molecular-weight anticancer drugs in tumor tissues for a longperiod because of their rapid clearance, whereas we previouslyfound that macromolecular drugs, including SMANCS, accumulated in tumor tissue to a greater degree than low-molecular-weight anticancer drugs (18, 19). In addition, several pharmacological properties of SMANCS were found much improvedcompared with NCS in vitro and in vivo: increased stability,targeting efficiency to tumor, cell surface affinity, a rapid internalization into cells, and cell killing rate (19-22).

In the experiment described here, we evaluated whether NCSand SMANCS, which are internalized into tumor cells differently from low-molecular-weight agents, were effective antican-cer drugs against MDR cancer cells.

MATERIALS AND METHODS

Drugs and Chemicals. NCS (Mr 12,000) and SMANCS (M, 16,000)were obtained from Kayaku Antibiotics Research Ltd., Tokyo, andKuraray Ltd., Osaka, Japan, respectively. The activity of SMANCSresides in the NCS portion, which primarily affects DNA metabolism.The chemical entity of SMANCS is well established and the reproducible product is available (20). Ricin (M, 64,000) was from SeikagakuKogyo Ltd., Tokyo. ADM, MMC, and 5-FU were from Kyowa HakkoKogyo Ltd., Tokyo. ACM and CDDP were from Yamanouchi Pharmaceutical and Nippon Kayaku Ltd., Tokyo, respectively. Stanners'

modified mininal essential medium was purchased from Flow Laboratories, Mclean, VA. FITC-labeled NCS, FITC-labeled SMANCS, andFITC-labeled ricin were prepared by essentially the same method asdescribed (23). All other chemicals were from commercial sources. FreeFITC moiety was not liberated from F-NCS or F-SMANCS duringbinding experiments as confirmed previously (13-16).

Cell Lines. An auxotrophic mutant of Chinese hamster ovary cells,AUXB1, and its MDR subline containing a high content of P-glycoprotein, CHRC5, were generous gifts from Dr. Victor Ling, Ontario

Cancer Institute, Toronto, Ontario, Canada (24, 25). The cells werecultured in Stanners' modified minimal essential medium containing10% heat-inactivated fetal calf serum (growth medium) at 37°Cas

usual.Cytotoxicity Studies. Cytotoxicity was measured by inhibition of

colony formation (15, 16). The cells were plated in 96-well flat-bottomplates (Falcon no. 3072, 6.4-mm diameter) at a density of 2 x IO4cells/

well and were cultured overnight in the growth medium in the bottomof the wells. After removal of the medium, cells were exposed to variousconcentrations of drugs in the growth medium for 1 or 24 h at 37°C.

Then, drug-containing medium was removed by aspiration, the cells ineach well were trypsinized and diluted with growth medium, and about500 cells were seeded into 35-mm diameter dishes (Falcon no. 3001).After incubation for 5-6 days, the numbers of colonies formed werestained and counted.

To study the time dependency of drug-induced cytotoxicity, 500 cellswere placed in 35-mm diameter dishes and were cultured overnight ingrowth medium. Then, the cells were exposed to a fixed concentrationof the drug that would completely inhibit colony formation in 24 h(IDioo). followed by incubation with the drug at 37°Cfor periods ranging

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CYTOTOXIC EFFECTS OF MULTIDRUG-RESISTANT CELLS IN VITRO

from 0 to 240 min. At the end of each incubation time, the supernatantwas removed and the cells were rinsed once with 0.01 M phosphate-buffered 0.15 M saline, pH 7.4, followed by incubation in fresh, drug-free growth medium at 37°Cfor 5-6 days. Drug-induced inhibition of

colony formation was determined as described above.Binding Studies (15, 16). AUXB1 or CHRC5 cells were plated at a

density of 1 x 10*cells/well in 24-well plates (Falcon; 16-mm diameter,no. 3047) and were cultured for 2 days at 37°C.The medium was

replaced with 500 ^1 of the growth medium, which contained a constantamount of ADM, F-NCS, F-SMANCS, or F-ricin at a pH of 7.4(adjusted with 20 mivi jV-(2-hydroxyethyl)-l-piperazineethanesulfonicacid) (see Fig. 3). The cells were then incubated at 37°Cfor different

times. At the end of the incubation period, the supernatant was discarded and the cells were washed three times with cold KRP, pH 7.4,to remove unbound drug. For quantification of cell-associated FITCconjugates, cells were lysed in 1 ml of 20 miviTris-HCl buffer, pH 8.5,containing 0.5% sodium dodecyl sulfate, and the fluorescence intensityof the lysate was measured with the fluorescence spectrophotometerwith an excitation at 490 nm and an emission at 520 nm (15, 16).Aliquots of the lysates were also used to quantify the protein contentby the usual method (26), with bovine serum albumin as the standard.In ADM-binding studies, rinsed cells were lysed in 250 ¿ilof 2% TritonX-100, after which 750 p\ of 4 N HC1 in 67% ethanol was added toextract ADM. The fluorescence intensity of the supernatant was thenmeasured with the excitation at 470 nm and the emission at 585 nm.

Efflux Studies. AUXB1 cells and CHRC5 cells were exposed to aconstant amount of ADM, F-NCS, F-SMANCS, or F-ricin for l h at37°Cin the same way as described above (see Fig. 4). Cells were rinsedthree times with cold KRP and were further incubated at 37°Cin drug-

free growth medium. At various times, the medium was discarded, cellswere washed with cold KRP three times, and cell-associated drugs andcellular protein were quantified after lysing the cells as described above.

RESULTS

Cytotoxicity of Drugs to AUXB1 and CHRC5 Cells. Cytotox-

icity of ADM to the parental cell line AUXB1 and to theresistant cell line CHRC5 after a 1-h drug exposure is shown inFig. 1. The ID50 of ADM for CHRC5 cells was 720-fold greater

than that for AUXB1 cells. The ID50values of both cell lines incolony-forming assays after 1- and 24-h incubations with ADMand the other anticancer drugs are summarized in Table 1.

NCS, SMANCS, and ricin effectively inhibited the colonyformation of the two cell lines at very low concentrationscompared with the other drugs (Table 1). The ID50of NCS orSMANCS for AUXB1 cells was 3-4 HMand that of ricin was1.5 nM for the 1-h treatment; ID5o values for the other drugs

100

98765432Anticancer drug ( -log M )

Fig. 1. Cytotoxicity of ADM, NCS, and SMANCS to AUXBI and CH"CScells. AUXBI cells (O, V, A) and CH"C5 cells (•,T, A) were exposed toappropriate concentrations of ADM (O, •¿�),NCS (V, T), and SMANCS (A, A)for I h at 37*C. and survival was measured by the technique of colony formation.

were l ¿IMto 25 IHM.ID50 values for 1-h exposure of CHRC5

cells to NCS or SMANCS and to ricin were about 30 nM and5.5 nM, respectively, whereas those of the other drugs were 12MMto 15 HIM,that is, 400 to 500,000 times higher.

CHRC5 cells were 20 to 900 times more resistant to ADM,

ACM, MMC, and VBL than were the AUXBI cells in our testsystem. The Cytotoxicity of these agents also depended onincubation time. For instance, for the 1-h exposure, the ID50ofADM for CHRC5 cells was 25 times as high as the ID50for the24-h exposure. CHRC5 cells showed no resistance to 5-FU or

CDDP. These agents also inhibited the colony formation ofboth cell lines in a time-dependent manner. Although CHRC5

cells were resistant to NCS, SMANCS, and ricin, their degreeof resistance was relatively small; only 5- to 10-fold higher thanAUXBI cells. Activity of both NCS and SMANCS in eithercell type was not affected by incubation time (Table 1).

Time Course of Cytotoxic Action of Drugs. According to theID50of the 1- and 24-h exposures of the drugs shown in Table1, the effect of all drugs except NCS and SMANCS seemed todepend greatly on the incubation time. As shown in Fig. 2, thefractions surviving after exposure to NCS and SMANCS wereimmediately reduced. About 90% of AUXBI and CHRC5 cells

were killed after only 6 and 10 min, respectively, by these drugs.In contrast, ADM required 100 and 140 min to kill 90% ofAUXBI and CHRC5 cells, respectively. In the case of 5-FU orCDDP, more than 60% of the cells survived after the 240-minincubation period.

Accumulation of Drugs in AUXBI and CHRC5 Cells. We

compared the accumulation of NCS, SMANCS, and ricin withthat of ADM in both cell lines. Fig. 3 shows the uptake profilesof ADM, F-NCS, F-SMANCS, and F-ricin by both AUXBIand CHRC5 cells at 37°Cor 4°C.ADM accumulated progressively in AUXBI cells at 37°Ceven after 3 h, whereas CHRC5

cells reached a plateau after 30 min. After 3 h of treatment, theuptake of ADM by AUXBI cells was about 2.2 times higherthan that by CHRC5 cells. The uptake of F-ricin by AUXBIcells was also about 2 times more than that of CHRC5 cells at37°C(Fig. 3). No significant differences in the uptake of F-

NCS or F-SMANCS were observed between AUXBI andCHRC5 cells.

Efflux of Drugs from AUXBI and CHRC5 Cells. Many investigators have reported that the decreased accumulation of an-ticancer drugs in MDR cell lines was due to increased effluxactivity in these cell lines (1). Therefore, we examined the effluxof the drugs from AUXBI and CHRC5 cells. The efflux of cell-associated ADM from CHRC5 cells was markedly increasedcompared with the parental AUXBI cells (Fig. 4/4). After a 30-min incubation, about 90% of cell-associated ADM was lostfrom CHRC5 cells, whereas more than 80% of the drug was

retained in AUXBI cells. In contrast, there was no significantdifference in the efflux of F-NCS, F-SMANCS, or F-ricin inthe two different cell lines (Fig. 4, B-D). Almost all F-NCSand F-SMANCS associated with the cells were retained in bothcell lines even after 120 min of incubation at 37°C.

DISCUSSION

The macromolecular agents NCS, SMANCS, and ricin effectively killed the MDR subline of Chinese hamster ovary cells(CHRC5) similarly to the sensitive line (AUXBI) in vitro. To

obtain 50% inhibition of the colony formation of AUXBI cells,2-4 nM NCS and SMANCS and 1.5 nM ricin, with 1 h ofincubation, were required, whereas for CHRC5 cells, about 30

nM of NCS and SMANCS and 5.5 nM of ricin were needed.1572

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Table 1 Drug cross-resistance: /D50 values in clonogenic assays after I- and 24-h incubations ofAUXBI and CH"C5 cells

DrugADMACMVBLMMC5-FUCDDPNCSSMANCSRicin1h1.06.0>1404.425.000.07.80.00390.00320.00156AUXB124h0.270.0220.0010.09033.00.870.00240.00260.000042ID«,

(MiTD"3.7270>70.000497609.01.61.237.1«)1

h720.0>210>140300.015.000.012.00.0310.0280.00549CH"C524h29.00.921.61.860.00.720.0250.0220.00045TD"25>220>8017(1250171.21.312.2«Tr»

.• r . ir» -rr. I D5O. 1 h

ID,».24 h

100

1000

120 240 0Time(min)

120 240

Fig. 2. Time-dependent cytotoxicity of CDDP (+). 5-FU (•).ADM (•).NCS(T). and SMANCS (A) in AUXB1 (A) and CH"C5 cells (fi). AUXB1 and CH"C5cells were exposed to a drug concentration that completely inhibited colonyformation in 24 h (based on Fig. I) for various times at 37'C. At the end of eachincubation period, the cells were washed with phosphate-buffered saline andgrow-in medium was added to the culture for 5-6 days.

These ID5»values were very low compared with those of theother low-molecular-weight anticancer drugs, which were be

tween 1 fiM and 25 IHM. For NCS, SMANCS, and ricin, theratios of the ID50values for resistant cells to values for parentalcells were relatively small (5- to 10-fold) compared with the

ratios of the anthracyclines (ADM and ACM), Vinca alkaloid(VBL), and MMC, which varied greatly (20- to 889-fold) (Table1). The sensitivity of CHRC5 cells to 5-FU and CDDP was not

very different from that of AUXB1 cells, and molar IDâ„¢valuesfor these two drugs were 2 x IQ-'to 6 x IO6 times higher than

those for NCS and SMANCS (Table 1). In addition, the cyto-cidal action of ADM, 5-FU, and CDDP required a much longerexposure time than that of NCS and SMANCS: more than 2 hversus less than 10 min (Fig. 2). As seen in Fig. 3, however, thecellular accumulation of ADM in both cell lines seemed to bemuch greater than that of NCS or SMANCS on a molar basis.Thus, these results indicate that the cells were killed by a muchsmaller amount of NCS or SMANCS, which was internalizedinto the cells very rapidly (13-16).

The subcellular mechanism of MDR in the CHRC5 subline

of Chinese hamster ovary cells, which were first obtained byLing's group, is attributed to reduced intracellular accumulation

of anticancer drugs (27, 28). In our studies reported here,CHRC5 cells released 90% of cell-associated ADM in 30 min.

120 180

Time (min)Fig. 3. Binding kinetics of ADM (A). F-NCS (A). F-SMANCS (C). and F-

ricin (D) for AUXBI cells (O) and CH"C5 cells (•).Cell monolayers wereincubated with 3.3 ¡¡MADM. 10 »¿MF-NCS, 10 MMF-SMANCS, or 15.6 ^M F-ricin for various times at 4*C ( ) and at 37'C ( ). After incubation, theamounts of cell-bound drugs were determined as described in the text. Points,mean values of duplicate samples.

whereas almost all F-NCS, F-SMANCS, and F-ricin remainedwithin the cells in both resistant and sensitive cell lines, evenafter 120 min of incubation (Fig. 4). Ling's group (24, 29) and

Luk et al. (28) also demonstrated that the content of P-glyco-protein correlated directly with both a decrease in intracellulardrug concentration and the degree of drug resistance of thecells. In their proposed mechanism of MDR, P-glycoproteinfunctions as an energy-dependent export pump to reduce intracellular levels of anticancer drugs (30). It was found previouslythat NCS, SMANCS, and ricin were taken up into cells byendocytosis (13-16, 17). Therefore, the intracellular site of

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' 1200 60 120

Time (min)Fig. 4. Efflux of ADM (A). F-NCS (B), F-SMANCS (C). and F-ricin (fl)

from At'XBl cells (O) and CH"C5 cells (•).AUXBI cells were exposed to 3.3MMADM. 10 MMF-NCS, 10 UMF-SMANCS. or 15.6 MMF-ricin for l h al 37'C.CH"C5 cells were exposed to 5 MMADM. 10 MMF-NCS. 10 MMF-SMANCS. or15.6 MMF-ricin for l h at 37'C. Retention of cell-associated drugs was determined

as described in the text. Points, mean values of duplicate determinations. AUXBIcells initially bound 362 pmol ADM. 4.7 pmol F-NCS. 63 pmol F-SMANCS.and 22.0 pmol F-ricin/mg cell protein, and CH*C5 cells initially bound 175 pmolADM. 6.1 pmol F-NCS. 66 pmol F-SMANCS. and 15.1 pmol F-ricin/mg cellprutein.

localization of NCS, SMANCS, or ricin (phagolysosome) isdifferent from that of the other low-molecular-weight antican-cer drugs, which incorporated into cytosol by passive transport(31-33). A rapid efflux of anticancer drugs by P-glycoproteinmay depend on appropriate intracellular localization (accessibility to P-glycoprotein) of the drugs which is not the case withendocytosis-dependent macromolecules. Thus, a method toovercome M DR may be possible by using macromoleculardrugs.

CHRC5 cells were 5- to 10-fold more resistant than AUXBI

cells to NCS, SMANCS, or ricin. Cytotoxicity of these macromolecular drugs are known to depend on their incorporationinto the cells (13-16, 34). The present study revealed that littledifference in the uptake of F-NCS or F-SMANCS by sensitiveand resistant cells existed (Fig. 3, B and C). However, theuptake of F-ricin by CHRC5 cells was about one-half of that by

AUXBI cells (Fig. 3D); therefore, a relatively low resistance toricin in CHRC5 cells may be attributed to the reduced uptake

of the toxin. Contrary to the present finding, Sehested et al.(35, 36) showed that fluid-phase endocytosis was increased inmultidrug-resistant cells compared to the sensitive parents. Wehave, however, not observed any significant increase in theuptake velocity in CHRC5 cells (Fig. 3).

Hamilton et al. (37) reported that ADM-resistant cells be

came high in cytoplasmic GSH content. It was reported previously that the presence of GSH or other thiols in cell or cell-free systems augment the ability of NCS and SMANCS (38-40). We determined the content of GSH and nonprotein sulfhy-dryls. However, their contents were almost the same in AUXBIand CHRC5 cells (data not shown).

Therefore, a small difference (less than 10-fold) in cytotox-icity described above of NCS or SMANCS against these sensitive and resistant cell lines needs to be explained by otherfactors, such as DNA topoisomerase II (41) and/or DNA repairenzymes (42), for which further studies are needed.

The results presented here show that NCS and SMANCSwere still effective at the 30 nM range against the M DR cancercells because of their potent cytocidal action and independencefrom the P-glycoprotein-dependent efflux process in the resistant cells. It is suggested that the use of macromolecular anti-cancer agents is a new approach to circumvent MDR in additionto their other advantages such as higher tumoritropism andimproved pharmacological properties (18-22).

ACKNOWLEDGMENTS

We thank Dr. Victor Ling for his generous supply of two cell lines,AUXBI and CHRC5.

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1990;50:1571-1575. Cancer Res   Yoichi Miyamoto, Tatsuya Oda and Hiroshi Maeda 

in VitroChinese Hamster Ovary Cells Multidrug-resistantLow-Molecular-Weight Anticancer Agents on

Comparison of the Cytotoxic Effects of the High- and

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