cross … · 2006. 12. 19. · [cancerresearch53,5225-5232,november1,1993)...

[CANCER RESEARCH 53, 5225-5232, November 1, 1993)

Cross-Resistance to Diverse Drugs Is Associated with Primary Cisplatin Resistancein Ovarian Cancer Cell Lines1

Kinya Hamaguchi, Andrew K. Godwin, Michiaki Yakushiji, Peter J. O'Dwyer, Robert F. Ozols, andThomas C. Hamilton2

Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [K. H., A. K. G., P. J. O., R. F. O., T. C. H.Â¡,and Department of Obstetrics andGynecology, Kurume University School of Medicine, Fukuoka. Japan X30 Â¡M.Y./

ABSTRACT

We have previously obtained, by exposure to near continuous increasing concentrations of cisplatin, a panel of human ovarian cancer cell linesthat exhibit a wide range of primary resistance to the drug (9- to >400-fold). These cells had strikingly increased (4- to 50-fold) levels of gluta-thione (GSH) as compared with the drug-sensitive cells of origin (A. K.Godwin et al., Proc. Nati. Acad. Sci. USA, 89: 3070-3074, 1992). Utilizingthis panel of resistant cell lines, we evaluated cross-resistance to classical

alkylating agents, natural product drugs, and irradiation. We observedthat cross-resistance to carboplatin paralleled that of cisplatin, culminating in approximately 250-fold resistance. Similarly, melphalan cross-resistance continued to increase to >400-fold and again paralleled the pri

mary cisplatin resistance. Cell lines with low to very high levels ofresistance to cisplatin are 8- to 850-fold resistant to the epipodophyllotoxinderivative etoposide. Cross-resistance is also observed for other naturalproduct drugs, including Adriamycin (â€”80-fold), mitoxantrone (â€”440-fold), and taxol (~40-fold). Cross-resistance to irradiation is, however,

modest (<2-fold). The cells with the greatest primary resistance to cisplatinmost commonly had the highest cross-resistance to the other drugs examined. The cross-resistance to the natural product category drugs was found

not to be mediated by the products of either the multidrug resistance 1(MDR\) or multidrug resistance-associated protein (MRP) genes based on

lack of coordinate increased expression or amplification of these genes asassessed by Northern and Southern blot analyses. Furthermore, verapamilfailed to markedly increase drug sensitivity. Although there was no indication that these natural product drug efflux pumps were operative, weobserved decreased doxorubicin accumulation in these cell lines cross-resistant to natural products. In addition, alternations in DNA topoisom-

erase II mRNA levels, which have been observed in a variety of humantumor cell lines selected in vitro for resistance to etoposide or teniposide,were not detected. Only intracellular levels of GSH correlated with cross-

resistance to these diverse anticancer agents and partial loss of resistancewas associated with a marked decrease in glutathione levels. In the absence of alternative mechanisms, we speculate that the very broad clinically relevant cross-resistance seen in this model system may, at least inpart, be the direct result of GSH-mediated drug inactivation or may be

due to a combination of GSH conjugation to drug and conjugate effluxmediated by the putative ATP-dependent glutathione 5-conjugate export

pump.

INTRODUCTION

Treatment of ovarian cancer with cisplatin-based combination che

motherapy has yielded increased complete response rates, and cisplatin has become a cornerstone in the treatment of this disease duringthe last two decades (1, 2). Nevertheless, an improvement in long-term survival of cisplatin-treated patients with advanced stage ovarian

cancer has appeared to plateau over the last decade, since the majorityof these patients relapse after responding to initial chemotherapy anddie of their chemotherapy-refractory disease (3). Thus, emergence of

Received 5/6/93; accepted 8/23/93.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by Grants CA51228, CA06927, and CA5I175 from

the National Cancer Institute.2 To whom requests for reprints should be addressed, at Department of Medical

Oncology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111.

drug-resistant tumor cells leads to tumor progression and subsequent

death. The resistance observed clinically is quite complex in that, inaddition to resistance to the drugs used for primary chemotherapy,tumors are refractory to diverse unrelated drugs. Hence, second linechemotherapy has been of limited benefit in ovarian cancer (2). Theone potential exception to this complex pattern of clinical multidrugresistance is the recently observed utility of taxol as a salvage therapyin refractory ovarian cancer (4).

One approach, in addition to new drug discovery, with the potentialto improve the treatment for ovarian cancer is to identify the mechanisms responsible for the complex resistance phenotype observedclinically and then exploit pharmacological approaches to prevent theemergence of drug resistance or reverse the phenotype when it occurs.Resistance to the diverse drugs generally categorized as natural products has been most studied. Increased expression of the MDR\ genewhich encodes the p 170 glycoprotein is one mechanism by whichcells may become resistant to natural product drugs (5-7). Two other

proteins potentially involved in natural product resistance have morerecently been reported: the multidrug resistance-associated protein

(MRP); and a protein identified using an antibody that recognizes aconserved ATP-binding domain in mitoxantrone-resistant cell lines (8,

9). In addition, alternations in DNA topoisomerase II have been identified in a cell line selected for resistance to natural products includingetoposide, mitoxantrone, Adriamycin, or teniposide (10-14). It is of

note that the activity of classical alkylating agents and platinum drugs,which are major components in the chemotherapy of ovarian cancer,have been found not to be influenced by these mechanisms of resistance where studied. This is of particular interest since the failure ofprimary chemotherapy with these agents results in tumors refractoryto second line therapy with the diverse natural products as well asother DNA-reactive drugs. This suggests the existence of a mecha

nism or mechanisms which contribute to primary resistance and alsoto the broad cross-resistance typical of clinical ovarian cancer.

Resistance to platinum drugs and alkylating agents is multifactorialincluding as components: decreased drug accumulation (non-MD/fl

mediated); increased cellular inactivation; and increased DNA repair.Of these mechanisms, increased drug inactivation, mediated by glutathione, is a mechanism with the potential to contribute to bothprimary resistance and the complex drug-resistant phenotype observed

in ovarian cancer and other solid tumors similarly treated. Glutathioneis the predominant intracellular nonprotein thiol in all mammaliancells and plays an important role in homeostatis and likely xenobioticdetoxification. The concept that the binding of GSH3 to anticancer

drugs could serve as a means of their inactivation and hence contributeto drug resistance may be credited to Meister (15). The experimentalsupport for the validity of this hypothesis, although not conclusive, isextensive. We and others have shown in numerous cell lines thatselection for cisplatin and alkylating agent resistance is accompaniedby elevations in GSH and that depletion of GSH by nutritional deprivation or treatment with buthionine sulfoximine, an irreversible in-

3 The abbreviations used are: GSH, glutathione; -y-GCS, y-glutamylcysteine synthe-tase; VP-16. etoposide; PBS. phosphate-buffered saline; kbp, kilobase pair; cDNA,

complementary DNA; \CW, concentration of drug resulting in 50% decrease in staining.

5225

on March 15, 2021. © 1993 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

MULTIDRUG RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED GSH

Table I Cross-resistance of CDDP"-resistant human ovarian cancer cell lines

RelativeresistanceDrugsCDDPCBDCAL-PAMADRMITVP-

16TaxolGSHIC5()

forA2780(JIM)0.347

Â±0.0004"6.033

Â±0.7630.767Â±0.0110.048Â±O.(XX)0.013+0.00010.054

Â±0.0020.0025Â±0.063(nmol/106

cells) forA27801.707*0.140CP205211232814CP701724146819112C302901411382235212113C50255166150302612965Relativeratio19C8041215423436346370525C100364199366764158333624C200424244447784468523842

" CDDP, cisplatin; CBDCA, carboplatin; L-PAM. melphalan; ADR, Adriamycin; MIT, mitoxantrone; VP-16, etoposide; MTT, 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide.h IC5(i determined by MTT assay continuously exposed for 72 h. Cross-resistance is defined as the ratio of the IC.in of the resistant variant to the relatively sensitive parental A2780

cell line. Values are means Â±SD of triplicate determinations obtained from at least 2 independent experiments.

hibitor of -/-CCS, the rate-limiting enzyme in GSH synthesis (16),partially reverses resistance in vitro and in vivo (17-19). These studies

have led to clinical trials of buthionine sulfoximine and melphalan indrug-refractory cancer patients (20). Another level of evidence supportive of a role for GSH in resistance to platinum-containing drugs

and alkylating agents comes from several studies, including our own,which have shown a strong correlation in cell lines (21-23) and

clinical samples (24, 25) between GSH amount and level of resistance.Although it is unlikely that GSH is the sole contributor to cisplatin andalkylating agent resistance, it potentially serves a pivotal role in somecases and could likely be a participant in producing the complexcross-resistance phenotype typical of ovarian cancer.

In the present work, we determined the cross-resistance phenotype

of a related series of human ovarian cancer cell lines selected in vitrofor a wide range of primary cisplatin resistance. Roles for MDR1,MRP, and topoisomerase II expression in manifestation of the complex cross-resistance phenotype were excluded.

MATERIALS AND METHODS

Chemicals and Reagents. Cisplatin, carboplatin. and VP-16 were obtained

from Bristol Myers (Evansville, IN). Melphalan was obtained from SigmaChemical Co. (St. Louis, MO). Adriamycin was obtained from Cetus Corp.(Emeryville, CA). Mitoxantrone was obtained from the American CyanamidCompany (Pearl River, NY). Taxol for clinical use was provided by theDivision of Cancer Treatment, National Cancer Institute, NIH, and was resus-

pended at a concentration of 6 mg/ml in 50% polyoxyethylated castor oil(Cremophor EL) and 50% dehydrated alcohol. Radiolabeled Adriamycin(['4C]doxorubicin hydrochloride; specific activity, 50-60 mCi/mmol) was ob

tained from Amersham (Arlington Heights, IL). Regular iletin II pork insulinwas obtained from Eli Lilly. Glutamine-free concentrated RPMI 1640, fetal

calf serum, penicillin/streptomycin, and glutamine were obtained from Gibco(Grand Island, NY). All other chemicals and reagents were obtained from

Sigma.Cell Lines. A2780 is an ovarian cancer cell line derived from an untreated

patient (26). A panel of platinum-resistant cell lines derived from A2780designated as A2780/CP20, CP70, C30, C50, C80, C100, C200 (i.e., C30 = 30

fjiM; C50 = 50 /AMwith regard to cisplatin exposure, etc.) was obtained by

intermittent exposure, in the case of CP20 and CP70, and to near continuousexposure to increasing concentrations of cisplatin in the case of the C series(21). All cell lines were maintained as monolayers in RPMI 1640 containing10% fetal calf serum 100 /xg/ml streptomycin, 100 units/ml penicillin, 0.3mg/ml glutamine, and 0.3 unit/ml insulin, designated as complete medium.Cells were grown at 37Â°Cin a humidified atmosphere of 5% CO2 (v/v) in air.

The C-series were exposed to cisplatin for 72 h once every 10-21 days. C30Rand C200R are partial revenants of the drug-resistant variants, obtained fol

lowing passage of C30 and C200 cells, respectively, in vitro in the absence of

drug for nearly 2 years.Cell Sizing. Cells were harvested with trypsin and resuspended at a con

centration of 2 x H)4 cells/ml. A Coulter Model ZM particle counter was used

to determine the mean cell volume for each cell line. A2780 was arbitrarily

assigned a value of 1.00 and all other lines were assigned a relative volume

compared to A2780.Cytotoxicity Assays. Cisplatin, carboplatin, melphalan, Adriamycin, mi

toxantrone, taxol, and etoposide cytotoxicity was determined by the tetra-

zolium assay (27). Cells (1000 to 8000) were plated in 150 fj.1of medium/wellin 96-well plates (Corning Co., Corning, NY). After incubation overnight, each

drug was added in varying concentrations. After 72 h, 40 fil of 5 mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were added perwell. Following a 2-h incubation, the cells were lysed with 100 /j.l/well ofextraction buffer (20% (w/v) sodium dodecyl sulfate-50% MJV-dimethylfor-

mamide, pH 4.7), and incubation overnight. The absorbance at 570 nm wasmeasured using wells without cells as blanks. IC50 was used as the measure ofrelative cytotoxicity. The fold resistance was considered to be the ratio of theIC5U of the cisplatin-resistant subline to the IC5U of the parental line. To

examine the effect of verapamil on drug resistance, cells were coincubated withverapamil (5 fÂ¿M)and with various concentrations of drugs as described above.

In order to determine irradiation cytotoxicity, cell suspensions receivedgraded doses of radiation 100 to 500 rads. Irradiated cells for each cell linewere plated in 35-mm Petri dishes as triplicates at cell densities of 100 to20,000 cells/dish. Dishes were incubated for 7 to 14 days at 37Â°C.The dishes

were fixed and stained with mÃ©thylÃ¨neblue solution in absolute methanol andcolony counts were determined. D,,, the slope of the exponential portion of thecurve (the dose required to reduce survival to 0.37 on any survival level of theexponential portion of the curve) was utilized as a parameter to evaluate

irradiation cytotoxicity on each cell line.Influx and Efflux of Doxorubicin. Cells were harvested with trypsin and

plated as a monolayer in 35-mm dishes at a concentration of 5 X IO5 cells/dish.Forty-eight h later. 0.25 JIM [IJC]doxorubicin was added to the medium and

dishes were incubated in tissue culture conditions. At 1, 2, and 4 h afteraddition of drug, the medium was removed and the dishes were washed (X3)with PBS (4Â°C).Trypsin/EDTA (1.0 ml) was used to remove cells from the

dish. Each dish was then washed with 0.5 ml of cold PBS and cell suspensionswere transferred to a microcentrifuge tube. Tubes were centrifuged at a speedof 10,000 rpm for 10 min at 4Â°C.The aqueous layer was discarded and the

pellet resuspended with 1 ml of 0.5% sodium dodecyl sulfate in PBS for lysis.The radioactivity of the lysate was then determined by liquid scintillation. Forefflux studies, cells were preloaded with the same concentration of doxorubicindescribed above for 4 h. The [l4C]doxorubicin-containing medium was then

removed and the cells were washed with PBS followed by incubation in a freshmedium at 37Â°Cfor the indicated times. Cells were harvested and the remain

ing radioactivity was determined as described above.RNA and DNA Analysis. Expression of the MDR\, MRP. topoisomerase

II, and -V-GCS genes in the cisplatin-resistant cell lines was evaluated by

Northern blot analysis. Total cellular RNA was extracted by a modified one-step guanidinium isothiocyanate-phenol-chloroform extraction procedure (28).

Total RNA (16 fig/lane) was denatured in 50% formamide containing 7.4%formaldehyde and separated by electrophoresis on an agarose (1% agarose-2.2

M formaldehyde) gel. RNA gels were stained with ethidium bromide andexamined to ascertain that equivalent amounts of RNA were analyzed. TheRNA was blotted by capillary action onto Magna NT membrane filters (MicronSeparations, Inc.. Westboro, MA) in 10X standard saline-citrate and hybridized with 32P-labeled DNA probes prepared using the Prime-It random primer

5226



MULTIDRUO RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED OSH

kit (Stratagene) to a specific activity of >5 X 10" dpm//ig. The following

cDNA probes used were kindly provided by the following investigators: A.Fojo, pSA^MDR [1.4-kbp human MDR\ fragment (29)]; G. Kruh (a 1.2-kbphuman MRP fragment: Fox Chase Cancer Center); L. Liu, pC15 [a 1.8-kbphuman pl7() topoisomerase II fragment (30)] and pGCS19-l [a 3.7-kbp humanfull-length -y-glutamylcysteine synthetase cDNA fragment; described by us,(21)]. Autoradiography was performed at -70Â°C for 1 to 4 days following

washing.Glutathione Determinations. Intracellular GSH levels were determined as

described previously (21).

RESULTS

Multidrug Sensitivity of Cisplatin-resistant Cells to VariousAnticancer Agents. The relative sensitivity to melphalan, a classicalalkylating agent, to carboplatin, the currently widely used cisplatinanalogue, to irradiation, and to the diverse natural products Adriamy-

cin, mitoxantrone, VP-16, and taxol, was evaluated in a panel of

related human ovarian cancer cell lines selected in vitro for a range ofprimary cisplatin resistance (Table 1). As shown in Fig. 1, cross-

resistance to carboplatin paralleled that of cisplatin culminating inapproximately 250-fold resistance in C200 cells; these cells are approximately 400-fold more resistant to cisplatin than is the parentalA2780 cell line. Similarly, melphalan cross-resistance continued toincrease to >400-fold and again paralleled the primary cisplatin re

sistance. Cell lines with low to very high levels of resistance tocisplatin (9- to >400-fold) are 8- to 850-fold resistant to VP-16. which

is usually included among the diverse drugs considered to be part ofthe classical multidrug resistance (MDR) phenotype. Cross-resistance

was also observed for other natural product category drugs, includingAdriamycin (nearly 80-fold), mitoxantrone (up to 440-fold), andtaxol. Cross-resistance to taxol, a unique antineoplastic agent which

120

CBDCA (uM)

10'

10 10' 10' 10> 10'

VP-16 (uM)

10

Taxol inMIFig. I. Resistance of cisplatin-resislant human ovarian cancer cell lines: A2780( + ), CP20 (A). CP70 (O), C30 ( + ), C50 (A). C80 (â€¢),C100 (V) and C2(X) (0) to various drugs.

Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-y!)-2,5-diphenyltetrazolium bromide assay after continuous exposure to a range of drug concentrations for 72 h. Points,mean of 3 independent experiments; CBDCA, carboplatin; I.-PAM, melphalan; ADR, Adriamycin; MIT, mitoxantrone.

5227



MULTIDRUG RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED OSH

appears to exert its cytotoxic action as a result of interference withmicrotuhular structure and function, had an interesting pattern. CP20,CP70, and C30 cells with 9-, 29-, and 250-fold resistance to cisplatin,respectively, were not cross-resistant to taxol (Table 1; Fig. 1). Themore highly platinum-resistant variants (C100 and C200 cells), however, displayed significant levels of cross-resistance to taxol (~40-fold). In contrast to marked cross-resistance to diverse drugs observedin these cell lines, cross-resistance to irradiation was not remarkable(~2-fold) as measured by D0 values (A2780, 155, CP70, 270, C100,

200, C200, 205).Expression of the MDRl, MRP, Topoisomerase II, and y-GCS

Genes. In order to determine whether known mechanisms of resistance to diverse natural products were operative in manifestation ofthe cross-resistance phenotype of these cells, we evaluated the expression of three genes, MDR\, MRP, and topoisomerase II. Overex-pression of the transmembrane transport protein P-glycoprotein hasbeen detected in many multidrug-resistant cell lines and in a variety of

tumors from cancer patients. This protein is encoded by the MDRlgene, and in vitro studies have shown that it confers resistance to arange of natural products that are used as chemotherapeutic drugs(29). Using Northern blot analysis, the drug-resistant cell lines wereexamined for the presence of mRNA coding for the P-glycoprotein. As

indicated in Fig. 2, none of the resistant cell lines expressed detectable

O00t-

o o o o(N C- o O O O OOHCL,co in oo --i INO O U O O O O

IOIâ€”IE-"

OE

MDRl

MRP

TopoII

y-GCS

â€¢¿�4.5 kb

â€”¿�8.0 kb

TotalRNA

-â€”6.2 kb

-_ 4.0 kbâ€”¿�3.2 kb

, 28S

18S

Fig. 2. Expression of MDRl. MRP. Topo Â¡I,and y-GCS genes by Northern blo!analysis. Total cellular RNA was isolated from the cell lines by a one-step guanidiniumisothiocyanale-phenol-chlorotbrm extraction procedure. Total RNA (16 fig/lane) was denatured in 50% formamidc containing 7.4c/c formaldehyde and separated by electropho-resis on an agarose (!*# agarose-2.2 M formaldehyde) gel and blotted. RNA gels were

routinely stained with elhidium bromide and visualized to ensure that equivalent amountsof total RNA were analyzed in each case. The position of the 28S and IKS rRNA isindicated.

levels of MDRl mRNA, while MDRl mRNA was readily detectablein the colon carcinoma cell line HCT-15 included as a positive control.

To evaluate alternative pathways involved in the observed multi-drug-resistant phenotype, we examined the cell lines listed in Table 1

for altered expression of the MRP and topoisomerase II genes. MRPis a newly identified member of the ATP-binding cassette transmem

brane transporter superfamily the overexpression of which is potentially associated with multidrug resistance in human lung cancer celllines and is distantly related to MDRl. As shown in Fig. 2, MRPmRNA levels varied only slightly in the drug-resistant variants as

compared to parental A2780 cells. In fact, most of the resistant variantdisplayed lower steady-state mRNA levels (~2-fold) as compared to

A2780 and HCT15 cells. Previous studies have shown that resistanceto cisplatin, doxorubicin, and VP-16 may be associated with alterations in topoisomerase II expression and activity. Since the platinum-resistant variants are highly cross-resistant to these anticancer agents,

topoisomerase II mRNA levels were also evaluated. As shown in Fig.2, there was no apparent difference in mRNA levels between drug-

sensitive A2780 cells and the resistant sublines. We next examinedmessage levels for y-glutamylcysteine synthetase, the rate-limiting

enzyme in GSH synthesis, where we observed dramatic increases inC100 and C200 cells, comparably lower levels were observed in celllines CP20, CP70, HCT15, and parental A2780. These cell lines werealso examined for amplification of the MDRl, MRP, topoisomerase II,and y-GCS genes by Southern blot analysis which revealed no change

in gene copy number.Doxorubicin Accumulation. In order to determine whether non-

MDR1-mediated decreased drug accumulation contributed to thebroad spectrum of cross-resistance to natural product drugs displayed

in these cell lines, the accumulation of doxorubicin was determined inA2780, CP70, C30, C100, and C200 cells. As shown in Fig. 3A, therewas an approximately 2-fold reduced accumulation of ['4C]doxoru-

bicin after 4 h of drug exposure in the most resistant variant (i.e., C200cells) as compared to drug-sensitive A2780 cells. The efflux of drugfrom A2780 and C200 cells loaded with [14C]doxorubicin by 4 h

exposure was also determined (Fig. 3ÃŸ).There was some loss ofintracellular radioactivity from C200 cells as compared to the parentalA2780 cells. Approximately 20% of radioactive doxorubicin effluxedfrom C200 cells after 90 min, while nearly all the radiolabeled drugremained in A2780 cells.

Effect of Verapamil on Drug Cytotoxicity. To further exclude thepossible involvement oÃMDRl and related genes in the natural product cross-resistance phenotype of these cells, we evaluated the effect

on cytotoxicity of blocking this pump with verapamil. Incubation ofA2780, C30, and C200 with 5 /XMverapamil did not significantly altertheir sensitivity mitoxantrone, VP-16, or melphalan (Table 2). In

contrast, verapamil had a noticeable effect on mitoxantrone cytotoxicity in 2780 ADÃ•Ocells, where an ~5-fold shift in ICS<)was observed. [ADÃ•Ocells were selected for primary resistance to Adriamy-

cin (31) and have previously been shown to overexpress the MDRlgene product].

Correlation between Drug Resistance and Intracellular GSHLevels. We have previously reported a strong correlation betweenglutathione, the most abundant nonprotein thiol in cells, and theprimary cisplatin resistance of these cell lines (21). Based on thesestudies and the studies of others showing a role for glutathione inresistance to several drugs, we sought to determine if a linear relationship was present between cross-resistance to the diverse drugs

reported here and cellular glutathione. The concentration of GSH inthese cell lines is shown in Table 1. A strong to moderate correlationwas observed between intracellular levels of GSH and ICjo obtainedafter 72 h continuous exposure to cisplatin (r = 0.94), carboplatin(/â€¢= 0.83), melphalan (r = 0.89), Adriamycin (r = 0.80), mitoxan-

5228



MULTIDRUG RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED GSM

Fig. 3. Drug influx (A ) and efflux (ÃŸ)in A2780(D), CP70 (A), C30 (O), C100 ( + ). and C2(X) (A)cells. (A ) Exponentially growing above cells (5 x10s cells/dish) incubated with 0.25 fiM [l4C]Adria-mycin at 37Â°Cfor the indicated time. Dishes were

washed with ice-cold PBS after removal of themedium at the indicated time. Cells were lysed with0.5% sodium dodecyl sulfate/l X PBS. and theradioactivity was determined by scintillation counting, (ÃŸ)Following 4 h incubation with the sameconcentration of drug, the medium was replacedwith drug-free medium and the cells were incubated at 37Â°Cfor the indicated time. Cells were

harvested in the same manner as described above,and the remaining radioactivity was determined.The values represent the means Â±SD (bars) obtained from triplicate determinations. The cell sizeof A2780 was arbitrarily assigned a value of l.(M)and relative volume ratios for all other cell lineswere calculated as described in "Materials andMethods." Values shown are normalized for cell

size.

30001

2500-

o

il

OCo

1500-

500-

60 120 180

Time (min)

240 15 30 45 60 75 90

Time(min)

Table 2 Effect of vempamil on drug cyloloxicity in CDDP"-resistant Inumiti

ovarian cancer cell lines

CelllinesA2780

C30C2(K)

AD,,,#ADR0.97

Â±0.21c

1.24 Â±0.171.74 Â±0.34MIT1.13

Â±0.131.23 Â±0.192.07 Â±0.055.21 Â±0.90VP-

161.80

Â±0.331.11 Â±0.091.09 Â±0.08CDDP1.39

Â±0.131.09 Â±0.041.06 Â±0.03L-PAM1.09

Â±0.160.97 Â±0.061.00 Â±0.05

" CDDP, cisplatin: ADR. Adriamycin: MIT. mitoxantronc; VP-16, etoposide; L-PAM.melphalan: DMF. dose-modifying factor.

6 DMF. represents the ratio of ICjÂ«(each drug) to IC5n (each drug plus 5 JÃŒMvcrap-amil). ADm*. a variant of A2780 selected for resistance to ADR. is 446-fold cross-resistant to MIT. as compared to A2780.

r Mean Â±SD.

tronÃ©(r = 0.81), and etoposide (r = 0.78) (Fig. 4). Furthermore,highly drug-resistant variants maintained in a drug-free environmenl

(i.e., without cisplatin) for long periods of time (i.e., at least 2 years)showed increase sensitivity to cisplatin. The levels of GSH in C30Rand C200R, partial revenants, were one-half and one-fourth of that

found in C30 and C200 cells (Table 3), which further substantiates therole of increased cellular glutathione in the drug resistance phenotype.

DISCUSSION

We have exposed the A2780 human ovarian cancer cell line toincreasing concentrations of cisplatin to isolate a scries of related celllines which vary widely in their primary cisplatin resistance. Even inthis relatively simple model system, it is apparent that primary cisplatin resistance is associated with the development of numerousresistance mechanisms including: decreased cisplatin accumulation:increased potential for inactivation by increased amounts of glutathione, increased ability to repair total platinum-DNA lesions; alterations

in the categories of lesions formed at the DNA sequence level; andincreased ability to repair cisplatin interstrand cross-links in specificDNA sequences (32).4 The cell lines described in our study will

complement those developed by other investigators with lower levelsof cisplatin resistance (22, 23, 33, 34). It is evident that our cell linesare well suited to defining the fullest possible repertoire of mechanisms which can contribute to cisplatin resistance. The use of theseand of other related cell lines which vary widely in resistance willallow determination of the relative significance of individual mecha-

4 S. W. Johnson. R. P. Perez.A. K. Godwin, A. T. Yeung. L. M. Handel. R. F. Ozols.

and T. C. Hamilton. Biochem. Pharmacol.. in press.

nisms as cisplatin resistance increases. It will then be necessary todetermine the relative significance of these individual mechanisms inclinical cancer specimens.

We have now examined the cell lines for cross-resistance to diverse

chemotherapeutic agents to determine whether the characteristics ofthese cells are consistent with the drug resistance phenotype of clinicalovarian cancer. It is well accepted that ovarian tumors that becomerefractory to aggressive cisplatin-based chemotherapy are unlikely to

respond to the diverse other drugs in the arsenal of chemotherapeuticagents. The emerging exception to this rule are data on responses totaxol (4, 35, 36). In our model system we observed strikingly highlevels of cross-resistance to carboplatin, melphalan, etoposide, Adriamycin, and mitoxantrone culminating in 250-, 400-, 850-, 80-, and440-fold changes in sensitivity, respectively. These changes paralleled

the primary cisplatin resistance. The exception to these findings waswith regard to taxol cross-resistance which was apparent only in Ihemore highly resistant variants and only reached 38-fold. This infor

mation is consistent with emerging clinical data on taxol as notedabove. Hence, the cross-resistance profile of these cell lines mimicscisplatin-refractory clinical ovarian cancer and suggests that this

model system should yield important leads as to the mechanismsresponsible for the clinical cross-resistance which is observed after the

failure of cisplatin therapy.In order to gain insight into the issue ol whether the cross-resistance

to the natural product drugs we observed could be mediated by previously described mechanisms, we examined the expression of threegenes, MDR\, MRP, and topoisomcrase II. Functional assays in theform of drug accumulation, and pharmacological manipulation ofnatural product cytotoxicity were also performed in order to provideadditional evidence of whether the classical p 170 glycoprotein drugefflux pump, coded by the MDRl gene, was operative as a mechanismof natural product resistance in this model system. There was noevidence of increased steady-state expression of the MDRl gene or the

distantly related MRP gene in these cells. This coupled to the inabilityof verapamil to pharmacologically alter the sensitivity of mitoxantrone and etoposide in these cells strongly suggests that classicalmechanisms of natural product resistance are not operative in thismodel. It was. however, noteworthy that there was a modest decreasein doxorubicin accumulation in these cell lines, as well as some abilityof the cross-resistant cells to efflux doxorubicin. It is tempting to

speculate as to whether the same, as yet to be identified, mechanism(s)responsible for decreased cisplatin accumulation'1 could function to

decrease Adriamycin accumulation in this model system.

5229



MULTIDRUO RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED GSH

i 40IMO

r-0.827

60 120 IK) 240

CBDCA ( fold-resistance)

aS 40

ina

r-0.891

L-PAM ( fold-r.sitt.ncÂ»)

IinO

r-0.797

IB 32 41

ADR ( fold-rÂ»sisUncÂ»)

o

ÃŒ.

ina

r-0.812

100 200 300 400

MIT ( fold-resistance)

aÂ£ 40

XIOo

r-0.786

mo

r-0.785

VP-16 ( fold-rÂ»iistÂ«ncÂ») TAXOL ( fold-resistance)

Fig. 4. Correlalion between intraccllulur GSH and fold resistance of cisplatin-resistant ovarian cancer cell lines to 6 drugs. Fold resistance was defined as the ratio of the IC50 ofthe resistant variant to the relatively sensitive parental A2780 cell line. Intracellular GSH levels were determined as described in "Materials and Methods."

5230



MULTIDRUO RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED OSH

Table 3 GSH levels in CDDP"-resistant and revenant human ovarian cancer cell lines

(fold-resistant)'' GSH (fold GSH increase)'

Cell line Parental Revertan! Parental Revertan!

ACKNOWLEDGMENTS

10.

A2780 0.37 Â±0.01 (\)d 0.34 Â±0.07 (1) 1.35 Â±0.02 (1) 1.68 Â±0.13 (1)

C30 102.7 Â±11.6(278) 43.7 Â±5.1 (129) 27.8 Â±5.5 (21) 16.5 Â±1.5 (10)C200 153.3 Â±5.8(414) 47.0 Â±3.0 (136) 63.1 Â±3.2(47) 19.1 Â±1.7(11) 5

"CDDP. cisplatin: MTT, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bro

mide. ICso. concentration of drug which results in a 50% decrease in staining.* Cytoloxicity was determined by MTT assay continuously exposed for 72 h.c Total GSH (nmol/106 cells). 6.'' Mean Â±SD of triplicate determinations.

Cross-resistance to etoposide was remarkable and, in fact, substantially exceeded primary cisplatin resistance. In the absence of MDR1-mediated cross-resistance to this natural product, we evaluated the

possibility of altered expression of topoisomerase II, the target foretoposide. Reports by others have shown that etoposide resistance canbe associated with similar or decreased levels of its target enzyme (11,37). We, however, observed no relationship between topoisomerase IIexpression and etoposide sensitivity. This does not exclude the possibility that etoposide resistance results from a difference in activity ofthe topoisomerase II enzyme in the resistant variants relative to theparental cell line.

We and others have reported a close association between platinumand classical alkylating agent resistance and cellular glutathione invarious model systems. We have shown that the increased glutathionelevels are associated with increased expression of â€¢¿�y-glutamylcysteinesynthetase and â€¢¿�y-glutamyltranspeptidase (Fig. 2; Ref. 21). Further

more, reversion to cisplatin, Adriamycin, and VP-16 sensitivity was

associated with decreased levels of intracellular glutathione (Table 3and other data not included). We evaluated whether a linear relationship exists between GSH and the sensitivity to the diverse drugsdescribed here. As would be anticipated based on the strong relationship between the level of primary cisplatin resistance and cross-

resistance to individual drugs, we found a moderate to strong correlation (/â€¢= 0.785 to 0.891) between cross-resistance to individual

drugs and glutathione. It is possible that the conjugation of GSH todrug is sufficient to abrogate cytotoxicity. The tantalizing proposal,however, has been put forward by others (38) that these conjugatescould act in some cases as cytotoxic intermediates. Therefore, resistance would require the excretion of the conjugate by a molecularpump (e.g., the putative ATP-dependent glutathione S-conjugate ex

port pump). Ongoing investigations include transfection of the parental A2780 cells with cDNAs for -y-glutamylcysteine synthetase andâ€¢¿�y-glutamyltranspeptidase under the control of strong promoters in

order to markedly increase glutathione levels in the absence of otherpotential contributors to resistance. Evaluation of sensitivity to thediverse drugs examined here in these transfectants will clarify thelevel to which glutathione contributes to the complex clinically relevant cross-resistance we have observed. Furthermore, application of

multiple molecular biological approaches to this model has the potential to uncover additional mechanisms responsible for the pheno-

type.

12.

13.

14.

15.

16.

17.

18.

19

20.

21

22

We thank Terry Halbherr and Laura Handel for their skillful technicalassistance and Drs. Steven Johnson and Donald Chapman for their thoughtful 23comments. We also thank Catherine Thompson for careful preparation of themanuscript.

24.

REFERENCES

1. Advanced Ovarian Cancer Trialists Group. Chemotherapy in advanced ovarian can- 25cer: an overview of randomized clinical trials. Br. Mcd. J.. 303: 884-893, 1991.

2. Ozols, R. F. Chemotherapy for advanced epithelial ovarian cancer. Hematol. Oncol.

5231

Clin. North Am., 6: 879-894, 1992.

Ozols, R. F., and Young. R. C. Chemotherapy of ovarian cancer. Semin. Oncol., II:251-263, 1984.

Mcguire, W. P.. Rowinsky, E. K., Rosenshein. N. B., Grumbine, F. C.. Ettinger, D. S.,Armstrong, D. K., and Donehowcr, R. C. Taxol: a unique antincoplastic agent withactivity in advanced ovarian epithelial neoplasms. Ann. Intern. Med.. Ill: 273-279,

1989.Chen, C.. Chin, J. E., Ueda, L., Clark, D. P., Pastan, I., Gottesman, M. M., andRoninson. I. B. Internal duplication and homology with bacterial transport proteins inthe mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell, 47: 381-

389, 1986.Gerlach, J. H.. Endicotte. J. A., Juranka. P. F.. Henderson. G.. Sarangi. F.. Deuchars.K. L., and Ling, V. Homology between P-glycoprotein and a bacterial hemolysin

transport protein suggests a model for multidrug resistance. Nature (Lond.), 324:485^*89, 1986.Roninson, I., Clin, J. E., Choi, K., Gros. P.. Housman. D. E., Fojo, A.. Shen. D. W.,Gottesman, M. M., and Pastan, I. Isolation of human mdr DNA sequence amplifiedin multidrug resistant KB carcinoma cells. Proc. Nail. Acad. Sci. USA, 83: 4538-

4542, 1986.Cole, S. P. C., Bhardwaj, J. H.. Gerlach. J. E., Mackie, J. E., Grant, C. E.. Almquist.A. J., Stewart. Kurz, E. U.. Duncan, A. M. V. and Deeley. R. G. Overexprcssion ofa transporter gene in a multidrug-resistant human lung cancer cell line. Science(Washington DC), 25Â«:1650-1654, 1992.

Nakagawa. M.. Schneider, E.. Dixon. K. H.. Horton, J.. Kelley, K.. Morrow, C., andCowan. K. H. Reduced intraccllular drug accumulation in the absence of P-glyco-protcin (mdrl) overcxpression in mitoxantrone-resistant human MCF-7 breast cancercells. Cancer Res., 52: 6175-6181, 1992.Ferguson, P. J., Fisher, M. H., Stephcnson, J., Li, D., Zhou, B.. and Cheng. Y.Combined modalities of resistance in etoposide-resistant human KB cell lines. CancerRes., 50: 5819-5824, 1990.Harker, W. G., Slade, D. L.. Drake. F. H., and Parr. R. L. Mitoxantrone resistance inHL-6II leukemia cells: reduced nuclear lopoisomerase II catalytic activity and drug-

induced DNA cleavage in association with reduced expression of the topoisomera.seIlÃŸisoform. Biochemistry, 30: 9953-9961, 1991.

De Jong, S., Zijstra, J. G., de Vries, E. G. E., and Mulder. N. H. Reduced DNAtopoisomerase II activity and drug-induced cleavage activity in an Adriamycin resistant human small cell lung carcinoma cell line. Cancer Res.. 50: 304-309. 1990.

Matsuo, K., Kohno, K.. Takano. H., Sato. S.. Kiue, A., and Kuwano. M. Reduction ofdrug accumulation and DNA topoisomerase H activity in acquired teniposide-resislanthuman cancer KB cell lines. Cancer Res., 50: 5819-5824, 1990.

Binaschi. M.. Capranico. G., De Isabella, P., Mariani. M.. Supino. R.. Tinelli, S., andZunino, F. Comparison of DNA cleavage induced by ctoposide and doxorubicin intwo human small cell lung cancer lines with different sensitivities to topoisomerase IIinhibitors. Int. J. Cancer, 45: 347-352, 1990.

Meister, A., and Griffith. O. W. Effects of methionine sulfoximine analogs on thesynthesis of glutamine and glutathione: possible chemotherapeutic implications. Cancer Treat. Rep., 6.?: 1115-1121, 1979.

Griffin, D. W. Mechanism of action, metabolism, and toxicity of bulhionine sulfoximine and its higher homologies. potent inhibitors of glutathione synthesis. J. Biol.Chem., 257: 13704-13712, 1982.

Louie. K. G.. Behrens, B. C., Kinsella, T. J.. Hamilton, T. C., Grotzingcr. K. R.,Mckoy. W. M.. Winker, M. A., Young, R. C.. and Ozols, R. F. Radiation survivalparameters of antineoplastic drug-sensitive and resistant human ovarian cancer celllines and their modification by buthioninc sulfoximine. Cancer Res., 45: 2110-2115,

1985.Hamilton, T. C.. Winker, M. A., Louie, K. G., Batist, G., Behrens, B. C., Tsuruo, T.,Grotzinger, K. R., Mckoy, W. M., Young, R. C., and Ozols, R. F. Augmentation ofAdriamycin, mclphalan and cisplatin cytotoxicity in drug resistant and sensitivehuman ovarian cancer cell lines by buthionine sulfoximine mediated glutathionedepletion. Biochcm. Pharmacol., 34: 2583-2586, 1985.Ozols. R. F., U)uie, K. G.. Plowman. J.. Behrens, B. C., Fine, R. L.. Dykes. D., andHamilton, T. C. Enhanced mclphalan cytotoxicity in human ovarian cancer in vitroand in tumor hearing nude mice by buthionine sulfoximine depletion of glutathione.Biochem. Pharmacol., 36: 147-153, 1987.O'Dwyer, P. J.. Hamilton, T. C., Young, R. C., LaCreta, F. P., Carp. N., Tew. K. D.,

Padavic. K.. Comis. R. L.. and Ozols. R. F. Depletion of glutathione in normal andmalignant human cells in vivo by buthionine sulfoximine: clinical and biochemicaleffects. J. Nati. Cancer Inst., 84: 264-267, 1992.Godwin, A. K.. Meister, A., O'Dwyer, P. J.. Huang. C. S.. Hamilton, T. C., and

Anderson. M. E. High resistance to eisplatin in human ovarian cancer cell lines isassociated with marked increase of glutathione synthesis. Proc. Nati. Acad. Sci. USA,89: 3070-3074. 1992.

Hribisko, M.. Mcgown. A. T., and Fox. B. W. The role of metallolhionein. glutathione.glutathione 5-transferases and DNA repair in resistance to platinum drugs in a seriesof L1210 cell lines made resistant to anticancer platinum agents. Biochem. Pharmacol., 45: 253-256, 1993.

Mistry, P., Kelland, L. R.. Abel. G.. Sidhar, S., and Harrap, K. R. The relationshipsbetween glutathione. glutathione S-transferase and cytotoxicity of platinum drugs andmelphalan in eight human ovarian carcinoma cell lines. Br. J. Cancer. 64: 215-220,1991.Britten, R. A., Green, J. A., and Warenius, H. M. Cellular glutathionine (GSH) andglutalhionc 5-transferase (GST) activity in human ovarian tumor biopsies followingexposure to alkylating agents. Int. J. Radial. Oncol. Biol. Phys.. 24: 527-531, 1992.Lee, F. Y. F., Siemann. D. W., and Sutherland, R. M. Changes in cellular glutathionecontent during Adriamycin treatment in human ovarian cancer-possible indicator ofchemosensitivity. Br. J. Cancer, 60: 291-298. 1989.



MULTIDRUG RESISTANCE IN OVARIAN CANCER CELLS WITH ELEVATED GSH

26. Eva, A., Robbins, K.. Anderson, P., Srinivasan, A., Tronick, S., Reddy, E., ZJImore.N., Gallen, A., Laulenberger, J., Papas, T., Westin, E., Wong-Staal, F., Gallo, R., and

Aaronson, S. Cellular genes analogous to retroviral one genes are transcribed inhuman tumor cells. Nature (Lond.), 295: 116-119, 1982.

27. Hansen, M. B., Nielsen, S. E., and Berg, K. J. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Immunol.Methods, 119: 203-210, 1989.

28. Chomczynski, P.. and Sacchi. N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 762.' 156-

159, 1987.29. Fojo, A. T., Veda, K., Slamon, D. J., Poplack, D. G., Gottesman, M. M., and Pastan,

I. Expression of a multidrug resistance gene in human tumors and tissues. Proc. Nati.Acad. Sci. USA. 84: 265-269, 1987.

30. Tsai-Pflugfelder, M., Liu, L. F., Liu, A. A., Tewey. K. M., Whang-Peng, J., Knutsen,

T., Huebner, K., Croce, C. M., and Wang, J. C. Cloning and sequencing of cDNAencoding human DNA topoisomerase II and localization of the gene to chromosomeregion 17q21-22. Proc. Nati. Acad. Sci. USA, 85: 7177-7181, 1988.

31. Rogan, A. M., Hamilton, T. C., Young, R. C, Klecker, R. W., and Ozols, R. F.Reversal of Adriamycin resistance by verapamil in human ovarian cancer. Science(Washington DC), 224: 994-996, 1984.

32. Zhen, W., Link, C. J., O'Connor, P. M., Reed, E., Parker, R., Howell, S. B., and Bohr,

V. A. Increased gene-specific repair of cisplatin interstrand cross-links in cis-

platin-resistant human ovarian cancer cell lines. Mol. Cell. Biol., 12: 3689-3698,

1992.33. Andrews, P. A., Murphy. H. P.. and Howell, S. B. Differential potentiation of alkyl-

ating and platinating agent toxicity in human ovarian carcinoma cells by glutathionedepletion. Cancer Res., 45: 6250-6253, 1985.

34. Hospers, G. A. P.. Mulder, N. H., deJong, B., deLey, L., Uges, D. R. A., Fiichtinger-

Schepman, A. M. J., Scheper, R. J., and de Vries, E. G. E. Characterization of a humansmall cell lung carcinoma cell line with acquired resistance to cis-diamminedichlo-roplatinum(II) in vitro. Cancer Res., 48: 6803-6807, 1988.

35. Thigpen. T., Blessing, J.. Ball, H.. Hummel, R., and Bartet, R. Phase II trial of taxolas second-line therapy for ovarian carcinoma: a gynecologic oncology group study.

Proc. Am. Soc. Clin. Oncol., 9: 604, 1990.36. Einzig, A. I., Wiernik, P., Sasoff, J., Runowicz, C. D., and Goldberg. G. L. Phase II

study and long-term follow-up of patients treated with taxol for advanced ovarianadenocarcinoma. J. Clin. Oncol., 10: 1748-1753, 1992.

37. Sullivan. D. M., Zsildsen, L. A., Groom, K. R., Webb, C. D., Latham, M. D., Martin,A. W., Wellhausen, S. R., Kroeger. P. E., and Rowe, T. C. Topoisomerase II activityinvolved in cleaving DNA into topological domains is altered in a multiple drug-resistant Chinese hamster ovary cell line. Mol. Pharmacol., 43: 207-216, 1993.

38. Ishikawa, T. The ATP-dependent glutathione 5-conjugate export pump. Trends Biochem. Sci., 17: 463-468, 1992.

5232



1993;53:5225-5232. Cancer Res Kinya Hamaguchi, Andrew K. Godwin, Michiaki Yakushiji, et al. Cisplatin Resistance in Ovarian Cancer Cell LinesCross-Resistance to Diverse Drugs Is Associated with Primary

Updated version

http://cancerres.aacrjournals.org/content/53/21/5225

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/53/21/5225To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



cross … · 2006. 12. 19. · [cancerresearch53,5225-5232,november1,1993)...

Documents