(CANCER RKSKARCII 49. 690I-61«)?. December 15. 1989]

Reversal by Cefoperazone of Resistance to Etoposide, Doxorubicin, and Vinblastinein Multidrug Resistant Human Sarcoma Cells'

Michael P. Gosland, Bert L. Lum, and Branimir I. Sikic2

Oncology Division. Department of Medicine, Stanford I'niversity School of Medicine, Stanford, California 94305

ABSTRACT

The cephalosporins are a family of semisynthetic antibiotics, someof which have structural features associated with substrates for themultidrug transporter, P-glycoprotein. The activity of a series of sixcephalosporins in reversing multidrug resistance (MDK) was examinedin MDK variants (Do cells) of the human sarcoma line MES-SA. DxScells express high levels of the mdr\ gene product P-glycoprotein andare 25- to 30-fold resistant to doxorubicin (DOX), etoposide (VP-16),and vinblastine (VBL). Cytotoxicity was measured by the M'lT assay.

Cefoperazone (1.0 HIM) was the most effective modulator of MDK,lowering the IC«for VP-16 by 29-fold (29x), for VBL by 16x, and forDOX by 14x. Ceftriaxone at 1.0 HIMproduced lOx modulation of VP-16cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of resistancewas concentration dependent, decreasing to 4x and 5x, respectively, forDOX with 0.25 HIMcefopcra/.one and ceftriaxone. No modulation ofcytotoxicity was observed in the parental MES-SA cells, which do notexpress mdr\. Cefazolin, cefotetan, ccphradine, and ceftazidime wereineffective, producing less than 5x modulation of DOX at 1.0 HIM.Amongthese cephalosporins, cefopcra/.one and ceftriaxone were the most highlyprotein bound in the media (30 and 52%), and the most lipid soluble,with octanol/water partitioning coefficients of —¿�0.49and —¿�0.60.Varyingthe serum concentration in medium from 5 to 50% had less than a twofold effect on the modulation of MDK by ceftriaxone. The ability toreverse MDK among these agents is associated with lipid solubility, highprotein binding, a polycyclic planar geometry, and the presence of thepiperà/ine group in ccfoperazone. These data and the potential forachieving high tissue concentrations indicate that Cefoperazone meritsfurther study as a modulator of MDR.

INTRODUCTIONThe typical MDR' phenotype includes cross-resistance to

anthracyclines. Vinca alkaloids, podophyllotoxins, and othercytotoxic compounds, and increased expression of a membraneprotein termed P-gp (1-3). The proposed mechanism of resistance is a decrease in the accumulation and retention of thexenobiotics by P-gp, functioning as an active efflux pump.Evidence supporting this mechanism includes the associationof MDR with increased expression of the mdr\ gene whichencodes P-gp, the demonstration of direct binding of transportsubstrates to P-gp by photoaffinity labelling, and transfer of theMDR phenotype by transfection of the mdr\ gene (1-6). Thedemonstration of increased mdr\ expression in some normaltissues and human cancers supports the hypotheses that P-gpis involved both in normal detoxification of various cytotoxinsand in resistance of some cancers to chemotherapy (7).

Received 6/16/89: revised 9/7/89: accepted 9/18/89.The cosls of publication of this article were defrayed in part b\ the payment

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

1Supported by American Cancer Society Grant CH-41 1 and NIH (¡rantCA-

49605.! Recipient of a Faculty Development Award in Clinical Pharmacology from

the Pharmaceutical Manufacturers Association Foundation. To whom requestsfor reprints should be addressed, at Oncology Division. Room M-211. Department of Medicine. Stanford l'ni\ersity Medical Center. Stanford. C'A 94305-

5306."The abbreviations used are: MDR. multidrug resistance; DOX, doxorubicin;

ICW, the drug concentration resulting in 50'< inhibition of MIT dye formation:MTT. 3-(4.5-dimethylthiaTOl-2-yD-2.5-diphenyltetra/olium bromide: log,,,?, thepartition coefficient or log ratio of the concentration of drug in octanol versanaqueous medium: P-gp. P-glycoprotein; VBL, \inblastine; VP-16. etoposide.

MDR can be reversed or modulated by many drugs, includingverapamil, trifluoperazine, quinidine. reserpine, and cyclospor-ine A (8-18). These agents share some or all of the features ofknown substrates of P-gp, including lipophilicity, a planarpolycyclic stereochemistry, and weak basicity. The mechanismof modulation in most cases is thought to be competitiveinhibition of drug efflux.

Verapamil and trifluoperazine have been used in clinical trialsto reverse MDR (19-24). A major problem with these trialshas been toxicity of the modulators (heart block by verapamiland depression of the central nervous system by trifluoperazine), at drug concentrations below those which reverse MDRin vitro. The ability of these agents to modulate MDR isunrelated to their primary pharmacological action, since theirability to reverse drug resistance does not correlate with theirproperties of calcium channel blocking or calmodulin inhibition(25-31).

Some of the cephalosporin antibiotics (Fig. 1) share structuralcharacteristics with agents known to modulate MDR, and areconsiderably less toxic in vivo. In this study, we assessed theability of a series of cephalosporin antibiotics to modulate MDRand enhance drug accumulation in human sarcoma cells, andidentified important physicochemical characteristics for modulating activity.

MATERIALS AND METHODS

Reagents. The sources of the drugs used in these experiments are asfollows: DOX (Adriamycin), Adria Laboratories, Columbus. OH; VBL(Velban), Eli Lilly Co., Indianapolis, IN; VP-16 (Vepesid), BristolMyers Co., Wallingford. CT: verapamil (Isoptin). Knoll Pharmaceuticals. Whippany, NJ; cephradine (Velosef), E. R. Squibb and Sons Inc.,Princeton. NJ: Cefoperazone (Cefobid), Roerig, New V'ork, NY; cefo

tetan (Cefotan), Stuart Pharmaceuticals, Wilmington. DE; ceftriaxone(Rocephin), Roche Laboratories, Nutley, NJ; ceftazidime (Tazidime)and ccfazolin (Kefzol). Eli Lilly Co.. Indianapolis, IN. The MTT saltand other chemicals unless specified were obtained from Sigma Chemical Co. (St. Louis, MO). All reagents were initially reconstituted inDulbecco's phosphate-buffered saline (GIBCO, Grand Island, NY) andfinal dilutions were made in McCoy's 5A medium (GIBCO) supple

mented with penicillin (100 fjg/ml), streptomycin (100 ^g/ml), insulin(5 fig/ml), and I0rr newborn calf serum (GIBCO). High-performanceliquid Chromatograph) grade I-octanol was purchased from AldrichChemical Co., Milwaukee, WI.

Cell Culture. The cells used in these experiments were the humansarcoma cell line MES-SA and its doxorubicin-selected MDR variantDx5 (32, 33). Monolayer cultures of MES-SA and DxS cells weregrown in tissue culture flasks (Corning Glass Works, Corning, NY)containing McCoy's 5A medium and 10rc newborn calf serum. Cellswere maintained at 37°Cin a humidified atmosphere containing 5%

CO2 and subcultured every 5 to 7 days.MIT Cytotoxicity Assay. Cells grown in 75-cnr tissue flasks were

harvested with 0.06 M EDTA, and cell number and viability wasdetermined by a hemocytometer and trypan blue dye exclusion. Cellswere seeded in 96-well plates (Falcon, Becton Dickinson Co., LincolnPark, NJ) at 8 x IO1 cells per well and allowed to grow for 24 h at37°C.The cells were then treated with drugs. DOX. VBL. and VP-16were used at concentrations from 3.0 x IO'1*to 3.0 x IO"6 M and the

cephalosporin antibiotics at concentrations ranging from 0.25 to 1.0

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MULTIDRUG RESISTANCE AND CEFOPERAZONE

CONII C---CONII--,CEFOPERA20N1-:

NH

Å’I-TRIAXONK

CEFOTETAN

CEFAZOLIN

Fig. 1. Chemical structures of the ccphalosporins.

mM. Verapamil was used as a positive control for modulation at 6.0/iM. Cells were incubated for 72 h with the cytotoxic agent (DOX, VBL,or VP-16) and the modulating drug. Then the medium was removedfrom each well, and 100 p\ of fresh medium and 10 /¿Iof MTT dye (5mg/ml in phosphate-buffered saline stock solution) were added to eachwell. Cells were incubated with MTT for 6 h, after which blue formazancrystals were dissolved in 0.1 N HCI in 2-propanol (100 ^1 per well).Within 30 min after dissolving the crystals, each plate was read on aDynatech MR580 Microelisa reader with a test wavelength of 570 nmand a reference wavelength of 630 nm (34-36). Each drug concentrationwas assayed in quadruplicate. The ICW(the drug concentration resultingin 50% inhibition of MTT dye formation, compared to untreatedcontrols) was determined directly from semilogarithmic dose-responsecurves.

Octanol-VVater Partitioning Studies. Five of the cephalosporin antibiotics (cefoperazone, ceftriaxone, cephradine, cefazolin, and cefotetan)were initially reconstituted in phosphate-buffered saline (pH 7.4) to aconcentration of I x IO"* M. Then 3 ml of this solution was added to

3 ml of high-performance liquid chromatography grade I-octanol andvigorously mixed, and the phases were allowed to separate. Drugconcentrations were determined using an LKB Biochrom spectropho-tometer to measure optical density at a wavelength of 272 nm in theaqueous phase before and after octanol-water partitioning. Referencesolutions were also prepared for each antibiotic at concentrations from1 x 10 6 to 1 x 10~4 M, which provided a linear standard curve. The

partition coefficient. log10P,was determined by dividing the concentration of drug in the octanol phase by that in the aqueous phase (37).

Protein Binding Studies. Three ml of each cephalosporin (at a finalconcentration of 1.0 x 10~5 in McCoy's 5A medium, pH 7.4) were

placed in Centricon-30 microconcentrator tubes (Amicon, Danvers,MA) and centrifuged at 1500 x g for 45 min at room temperature.Medium without drug was used as a blank for reading the opticaldensity of the ultrafiltrate in each tube. Samples were read at 272 nm

on a LKB Biochrom spectrophotometer and compared to standardcurves. The percentage of drug protein bound was determined by theformula: 100 x (total drug concentration-free drug concentration)/totaldrug concentration.

Accumulation Studies with |'I I]Vinblast inc. MES-SA and Dx5 cellswere harvested with 0.06 M EDTA, washed twice in Hank's balanced

salt solution with 10% newborn calf serum, and resuspended at a cellconcentration of 2.0 x IO6cells/ml in McCoy's 5A medium with 10%

newborn calf serum and 20 mm of HEPES buffer (Sigma ChemicalCompany, St. Louis. MO). Both the MES-SA and Dx5 cells wereexposed to ['Hjvinblastine (specific activity, 23 Ci/mmol; Amersham

International, UK) at a concentration of 1.25 u\\ alone and in thepresence of 1.0 mivi of cefoperazone. Cells were incubated for 60 minin a water bath at 37°Cwith shaking. Then 0.5-ml aliquots (containingIO6cells) were removed layered on silicone oil (Versilube F50; General

Electric Co., Waterford, NY) in microcentrifuge tubes, and centrifugedat 12,000 x g for 1 min to remove the cells from drug-containingmedium (2). Following the aspiration of the medium and oil, the tubetips containing the cell pellets were cut off and the cells were solubilizedin 0.2 N NaOH. The tube contents were then neutralized, scintillationfluor was added, and radioactivity was counted.

Statistical Analyses. Correlations of lipid solubility and protein binding with MDR modulation were performed by linear regression. Acoefficient of variation was determined for each drug concentration oneach plate (four wells per drug concentration) and standard deviationswere determined from each IC50of DOX with the various modulators.Each experiment was performed at least three times.

RESULTS

Modulating of Cytotoxicity. The strain of Dx5 cells in theseexperiments was 30-fold resistant to DOX compared to theparental drug-sensitive MES-SA cell line. They were also approximately 30-fold cross-resistant to both etoposide and vin-blastine. As a positive control for modulation of MDR, vera-pamil (6 //M) lowered the 1C5(,of DOX from 3 x IO'7 M to 1.3x 10'* M in Dx5 cells, nearly complete reversal of resistance.The IC«,of DOX in MES-SA cells was 1.0 x IO"8 M.

The structures of the six cephalosporins used in these experiments are shown in Fig. 1. Among these, cefoperazone (1.0mivi) was the most effective modulator of MDR, lowering theIC50 for VP-16 from 10 x 10~" M to 3.4 x 10~7M (modulationratio of 29x); the 1C«,for DOX from 3 x 10~7M to 2.2 x 10~8M (14x); and the 1C,,, for VBL from 5.3 x IO'8 M to 3.3 x 10~9

M (16x), Tables 1-3. Cefoperazone completely reversed resistance to VP-16 (Table 2), and reduced the IC5(Iof VBL andDOX in Dx5 cells to a level only two-fold higher than thesensitive parental line.

Ceftriaxone at 1.0 mivi produced lOx modulation of VP-16cytotoxicity, 8x for DOX, and 2x for VBL. The reversal of

Table I Modulation of resistance to f>O\ in DxS cells by cephalosporinantibiotics

Cephalosporin(mM)Control.

DOXaloneCefoperazone( 1.0)Cefoperazone

(0.5)Cefoperazone(0.25)Ceftriaxone(I.O)Ceftriaxone

(0.5)Ceftriaxone(0.25)Cefazolin(1.0)Ceftazidime(1.0)Cephradine(1.0)Cefotetan

(1.0)ICM.

DOX.(nM)°300

±8222±950

±1275±738

±1045±1055

±2085±101

10 ±15120±20130

±30Modulation

ratio*U6487S4332

" Each value represents the mean ±standard deviation of at least three

determinations.h The modulation ratio is defined as the ratio of the IC5Ufor DOX alone versus

the IC50in the presence of the modulating agent.

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MU.IÕDRl'C; RESISTANCE AND CEFOPERAZONE

Table 2 Modulation of resistance lo VP-16 in D.\5 cells by cefoperazone andceftriaxone

100

Cephalosporin(IHM)Control,

VP- 16aloneCefopera/one( 1.0)Cefoperazone

(0.5)Cefopera/one(0.25)Ceftriaxone

( 1.0)Ceftriaxone0.5Ccftriaxone

0.25I*

-n. (nM)10.00.340.862.401.042.157.85l.Ox0.050.040.140.090.070.49Modulationratio*291241051

" Each value represents the mean ±standard deviation of at least three

determinations.*The modulation ratio is defined as the ratio of the IC50 for VP-16 alone

versus the IC50in the presence of the modulating agent.

Table 3 Modulation of resistance to V'BLin I)x5 cells by cefopera:one and

ceftriaxone

Cephalosporin(m\i)Control.

VBLaloneCefoperazone( 1.0)Cefoperazone

(0.5)C'efopera/one(0.25)Ceftriaxone

(1.0)Ceftriaxone0.5Ceftriaxone

0.251C*,,

(nM)°53

±4.33.3±0.27.9

±0.215±7.026

±1.429±1.434±1.5Modulation

ratio*1C74222

0 Each value represents the mean ±standard deviation of at least three

determinations.* The modulation ratio is defined as the ratio of the IC»for VBL alone versus

the 1Câ„¢in the presence of the modulating agent.

100

¡OT

80 -

60 -

40 -

20

001 01 10 100

DOXORUBICIN (uM)Fig. 2. Modulation by cefoperazone of the cytoloxicity of doxorubicin in Dx5

cells. CFP. cefoperazonc. D. no CFP: ».1.0 BM CFP; I 0.5 mM CFP; O. 0.25HIMCFP.

resistance was dependent on the concentrations of cefoperazoneand ceftriaxone over the range of 0.25-1.0 mM, Tables 1-3,and Figs. 2-4. No modulation of cytotoxicity was observed inthe parental MES-SA cells, which do not express mdr\ (datanot shown). Cefazolin, cephradine, cefotetan, and ceftazidimewere relatively ineffective as modulators for DOX cytotoxicity,and were not tested with VBL and VP-16 (Table 1).

Octanol-Water Partitioning Studies. Cefoperazone was themost lipid soluble of the cephalosporins, with a partition coefficient (logioP) of —¿�0.49at pH 7.4, Table 4. Ceftriaxone wasthe next most lipid soluble agent with a log,0P of—0.60. There

was a highly significant correlation between lipid solubility andMDR modulation of these five agents (H = 0.881).

Protein Binding and Effects of Serum Concentration on Modulation of MDR. Cefoperazone and ceftriaxone were also themost highly protein bound agents in medium with 10% newborn

oc3

80-

60 -

40 -

20-

.01 10 100 1000

ETOPOSIDE (uM)

Fig. 3. Modulation by cefoperazone of the cytotoxicity of etoposide in Dx5cells. CFP, cefoperazone. G, no CFP; »,1.0 HIMCFP: •¿�0.5 mM CFP; O, 0.25mM CFP.

100

OC3en

60 -

40 -

20 -

.0001 001 .01

VINBLASTINE (uM)

Fig. 4. Modulation by cefoperazone of the cytotoxicity of vinblastine in Dx5cells. CFP, cefoperazone. D. no CFP; »,1.0 mM CFP; (•).0.5 mM CFP; 0.0.25mM CFP.

Table 4 Physicochemical properties of Cephalosporin antibiotics

DrugCefoperazone

CeftriaxoneCefotetanCefazolinCephradineLog,„P°-0.49

-0.60—¿�1 2-OÃŒ90-1.5îî

Proteinbound*30%52%

22%15%8%

°As determined by oclanol-water partitioning (see "Materials and Methods").* Determined in McCoy's 5A medium containing 10rínewborn calf serum.

calf serum. Table 4. Ceftriaxone was 52% protein bound,whereas cefoperazone was 30% bound. The correlation of percentage protein binding in medium by the various agents andtheir ability to modulate MDR was poor (r- = 0.181).

Changing the protein concentration in the medium did notmarkedly alter modulating activity. Increasing the protein concentration in the medium from 10% to 50% only slightlydecreased modulation of DOX by ceftriaxone, with the IC5oincreasing from 3 x 10~" to 5 x 10~" M.

Cellular Accumulation of Vinblastine. The effect of 1.0 mMcefoperazone on the accumulation of ['H]vinblastine in MES

SA and Dx5 cells is presented in Table 5. Cefoperazone did not6903

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MULTIDRUG RESISTANCE AND CEFOPERAZONE

Table 5 Effect of cefoperazone (CFP) on vinblastine accumulation in drug-sensilive MES-SA versus multidrug resistant Dx5 cells

Cells were incubated for 60 min at 37"C with 2 >IM[3H]vinblastine, with and

without the presence of 1.0 ITIMCFP.

Cell line (-) CFP (+) CFP

MES-SADx5

43.0 ±1.4"

20.1 ±0.542.0 ±0.743.2 ±3.5*

°Expressed as nanomolesof [3H|vinblastine per IO6cells. Values represent themean ±standard deviation, n = 3.

" P< 0.05, (-) CFP versus (+) CFP.

affect drug accumulation in the MES-SA cells, which do notexpress detectable levels of P-gp. However, it restored vinblastine accumulation in the Dx5 cells to the same level as in thesensitive control cells.

DISCUSSION

The search for safe and effective modulators of MDR is ofimportance for several reasons. First, there is increasing evidence that the mdr\ gene is expressed in many human cancers,and is likely to contribute to failure of chemotherapy in thosecancers (1, 3, 7). Second, P-gp is a multidrug transporter whosesubstrates are not restricted to cytotoxins, with many differentkinds of agents capable of reversing resistance in preclinicalmodels (1-3, 8-18, 25-31, 37-42). Structure-activity relationships for modulation of MDR have been studied in severalclasses of drugs, and some general principles governing thebinding and export of compounds by P-gp are emerging (10,12, 27-31, 37-41). Finally, initial clinical trials with two of themost potent modulators, verapamil and trifluoperazine, havebeen limited by severe toxicities of the modulators at plasmaconcentrations below those necessary to reverse resistance basedon preclinical experiments (19-24).

The unacceptable toxicity of verapamil and trifluoperazine atconcentrations required to modulate MDR in patients is notsurprising, since these drugs are primarily used for their actionsin cardiac conduction and the central nervous system, respectively. The search for modulators with a safer therapeutic ratiomight rationally focus on agents which are not targeted atmammalian physiology per se. The antibiotics represent such agroup of drugs of diverse chemical classes, targeted primarilyat microbial metabolism, but which may also be substrates formammalian P-glycoproteins.

We chose to study the cephalosporins because they containstructural features associated with some modulators of MDR(Fig. 1), and they are relatively nontoxic to humans even atconcentrations on the order of 1 m\i, which is the highestconcentration used in our experiments (43-45). Cefoperazonewas the most effective modulator of MDR, reducing resistanceto DOX in Dx5 cells from 30-fold to 1.8-fold when comparedto sensitive MES-SA cells. Modulation was most marked forVP-16. with complete reversal of resistance to this agent. Theconcentration dependence of the modulation, and the restoration of intracellular vinblastine levels at high cefoperazoneconcentrations, is consistent with competitive inhibition oftransport of the chemotherapeutic agents. Experiments to confirm this hypothesis regarding the nature of the interaction ofcefoperazone with P-gp are currently in progress.

Ceftriaxone was also capable of modulating MDR, but to alesser degree than cefoperazone for all three of the cytotoxins.These two agents share the physicochemical properties ofhigher protein binding and greater lipid solubility compared tothe other antibiotics tested. Lipid solubility correlated betterwith modulation of MDR than did protein binding. Lipid

solubility has also been shown to be important in the modulation of MDR by other classes of drugs such as analogues ofreserpine, quinoline, and dipyridamole (29-31). Because ofconcern that plasma protein binding in vivo may lessen theefficacy of modulation, we varied the serum concentration from5 to 50%. Only a modest (two-fold) decrease in modulationwas seen with the 10-fold increase in protein concentration.

The relative efficacy of cefoperazone as a modulator of MDRmay be related to its /V-ethylpiperazine group, which is uniqueamong these cephalosporin antibiotics (Fig. 1). This structuralcharacteristic is shared with trifluoperazine and prochlorpera-zine, two phenothiazines which modulate MDR (15, 25, 31,38). Other phenothiazones which lack this chemical moiety,such as thioridazine, were not effective modulators (25, 31).The importance of this piperazine group is also suggested byits presence in dilazep, a verapamil analogue, which is a morepotent modulator than its parent compound (40). In addition,the antihistamine prenylamine does not modulate MDR, unlikecinnarzine, a compound different only in the addition of apiperazine group (30). These examples support an importantrole for the piperazine moiety in binding to P-gp.

In summary, we have shown that the cephalosporin antibioticcefoperazone is an effective modulator of MDR in vitro. Thismodulation occurs at drug concentrations which may be achievable without major toxicity in patients (43-45). P-gp has beenshown to be present at high levels in several normal tissues,and is likely to be important in the normal disposition of MDR-related cytotoxins (7). Further studies should be performed onthe efficacy of cefoperazone in vivo in preclinical models as wellas its effects on the pharmacokinetics and toxicity of anticanceragents involved in MDR.

ACKNOWLEDGMENTS

We thank our colleagues, in particular D. H-M. Lau, J. M. Berry,J-P. Marie, K. L. Ross, N. A. Brophy, A. D. Lewis, and M. N. Ehsan,for their discussions and review of the manuscript.

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1989;49:6901-6905. Cancer Res   Michael P. Gosland, Bert L. Lum and Branimir I. Sikic  Sarcoma CellsDoxorubicin, and Vinblastine in Multidrug Resistant Human Reversal by Cefoperazone of Resistance to Etoposide,

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