[CANCER RESEARCH 46, 4352-4356, September 1986]

Resistance to Anthrapyrazoles and Anthracyclines in Multidrug-resistant P388

Murine Leukemia Cells: Reversal by Calcium Blockers and CalmodulinAntagonistsWayne D. Klohs,1 Randall W. Steinkampf, Martha J. Havlick, and Robert C. Jackson

Department of Chemotherapy, Warner-Lambert IParke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105

ABSTRACT

A series of anthrapyrazoles was examined for their cytotoxic effect onP388 cells resistant (P388R) to anthracyclines, A-|4-(')-;nridiinlamino)-

3-methoxyphenyl|methanesulfonamide, trimetrexate, and vinblastine.

The degree of resistance of P388R cells to Adriamycin (ADR) anddaimom)oin was 50-fold and 38-fold, respectively, when compared to theparent cell line (P388S). The Adriamycin-resistant cells were highlycross-resistant to some anthrapyrazoles, but the degree of cross-resistance was not uniform and was less than 3-fold for one member of the

series. The lipophilicity of these compounds appeared to correlate tosome extent with the level of resistance. The calcium channel blockersverapamil (VER) and diltiazem and the calmodulin antagonist trifluoper-

azine potentiated the cytotoxicity of the anthrapyrazoles and ADR inP388R. This potentiating effect was concentration dependent with VERbeing the most efficacious. VER increased ADR cytotoxicity by greaterthan 10-fold and CI-937 by almost 40-fold. However, VER, diltiazem,

and trifluoperazine had no effect on ADR or anthrapyrazole activity inP388S cells. The antiarrhythmic drug, quinidine, and the detergent,Tween 80, also potentiated ADR activity in P388R cells to the sameextent as VER. Both the net accumulation and efflux of [3H]daunomycin

were altered in P388R cells by nontoxic concentrations of Tween 80 in afashion virtually identical to that demonstrated for VER. These datasuggest that agents which potentiate drug cytotoxicity in P388R cellsmay do so by their interaction with the lipid domain of the plasmamembrane. In addition, these results demonstrate that some members ofthe new series of DNA binding drugs, the anthrapyrazoles, may be activeagainst anthracycline-resistant tumors and that, where cross-resistance

to them occurs, it can be partially reversed by agents such as VER.

INTRODUCTION

The development of resistance by tumor cells to certainnatural product antitumor drugs is often associated with cross-resistance to other unrelated antineoplastic agents (1-8). Forexample, tumors that acquire resistance to ADR2 are also

frequently resistant to other DNA binding drugs (1, 2) as wellas plant alkaloids (3, 4) and other structurally dissimilar compounds (5-7). This multidrug resistance appears to be associated with altered membrane permeability, resulting in a decreased drug uptake and/or efflux (1, 3-5, 8-11), althoughother lesions have been described (12,13). Multidrug resistanceof tumors represents one of the critical limitations in thetreatment of cancer patients, and its circumvention is a majorchallenge in cancer chemotherapy. The recent reports describing the ability of calcium channel blockers (3, 14-17) and

Received 7/29/85; revised 1/13/86, 4/28/86; accepted 5/27/86.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.

1To whom requests for reprints should be addressed, at Department ofChemotherapy, Warner-Lambert/Parke-Davis Pharmaceutical Research, 2800Plymouth Road, Ann Arbor, MI 48105.

1The abbreviations used are: ADR, Adriamycin; P388R, pleiotropically resistant P388 leukemia cells; P388S, parental drug-sensitive P388 leukemia cells;DAU, daunomycin; VER, verapamil; m-AMSA, /V-[4-(9-acridinylamino)-3-meth-oxyphenyljmethanesulfonamide; HEPES, 4 12 11>drc>\UMli>1) 1-piperazineeth-anesulfonic acid; NIF, nifedipine; W-7, /V-(6-aminohexyl)-5-chloro-I-naphthal-ene-sulfonamide; EGTA, ethyleneglycol bis(2-aminoethyl ether)-Ar,Ar'-tetraacetic

acid.

calmodulin antagonists (15-18) to potentiate drug activity inresistant cell lines may represent one avenue for overcomingdrug resistance.

The anthrapyrazoles are a new class of DNA binding anti-cancer drugs (19) which will soon enter clinical trials. Thesedrugs have been demonstrated to have a high level of broadspectrum activity against a large number of /// vivo animaltumor models (20). In the present study, we evaluated severalof these new drugs against an ADR-resistant P388 leukemiccell line and determined the ability of calcium and calmodulinantagonists to potentiate their activity.

MATERIALS AND METHODS

Cell Lines. P388 murine leukemia cells (P388S) and an ADR-resistant subline (P388R) obtained from the Southern Research Institute (Birmingham, AL) were cultured from ascites fluids of tumor-bearing DBA/2 mice and maintained in Fischer's medium supple

mented with 10% horse serum, 10 /¿M2-mercaptoethanol, and genta-mycin (50 Mg/ml).

The effect of anticancer agents alone or in combination with calciumor calmodulin antagonists on the growth of both cell lines was determined as previously described (21). Cells were counted with a ModelZF Coulter Counter (Coulter Electronics, Inc., 11¡alcali.FL). Viabilitywas determined by trypan blue exclusion.

Chemicals. [3H]Daunomycin, specific activity, 5 Ci/mmol, was pur

chased from New England Nuclear, Boston, MA. Unlabeled daunomycin was purchased from Sigma Chemical Co., St. Louis, MO. Theanthrapyrazoles were obtained from the Warner-Lambert Co. Fischer's

medium and horse serum were purchased from K. C. Biological, Inc.,Lenexa, KS. Adriamycin, quinidine sulfate, verapamil, diltiazem, nifedipine, trifluoperazine, W-7, ethidium bromide, and Tween 80 werepurchased from Sigma Chemical Co., St. Louis, MO.

Transport Studies. The uptake of [3H]DAU (0.25 nM [3H]daunomycin

plus 9.5 ¿IMdaunomycin) into P388S and P388R cells in the presenceor absence of VER (5 Mg/ml) was measured at 37°Cin Fischer's medium

containing 10% horse serum and 25 IHMHEPES. Initial uptake rateswere determined as described by Skovsgaard (11). Cell pellets weredissolved in l N NaOH at 70"( ' for 1 h, neutralized, and counted in

Beckman Ready-Solv cocktail by liquid scintillation counting.For initial [3H]DAU efflux studies, cells were incubated for 10 min

at 37'C in 0.7 pM DAU for P388S cells or 9.5 MMDAU for P388R

cells in order to equalize the intracellular concentration of drug in bothcells. Efflux was measured as described by Kessel and Wheeler (9).

RESULTS

Characterization of Multidrug Resistance in P388R Cells. TheP388 subline selected in vivo for resistance to ADR when grownin tissue culture was found to be cross-resistant to other anthracyclines, such as DAU and Marcellomycin, as well as toother classes of untiluiiior agents, such as vinblastine, trimetrexate, and m-AMSA (Table 1). Resistance to ADR was thehighest (51-fold) of the drugs tested with m-AMSA being thelowest (17-fold). Five anthrapyrazoles (see Fig. 1 for structures)were also tested for their activity against P388R cells in comparison to the sensitive P388 cells (Table 2). The degree of

4352

on June 15, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ANTHRAPYRAZOLE RESISTANCE

Table 1 Sensitivity of cultured P38SS and P388R leukemia cellsto antitumor agents

Table 3 Relationship of the lipophilic and DNA binding properties of theanthrapyrazoles with their effect on P38SR cells

CompoundDaunomycin

AdriamycinMarcellomycinVinblastinem-AMSATrimetrexateID»,P388S1

10.0 ±4.0*

128.8 ±9.614.5 ±3.43.7 ±1.4

50.9 ±0.812.2 ±3.2(nM)*

»P388R4151.7

±555.56586.5 ±662.3

543.0 ±9.7161.5 ±5.7851.4 ±12.3269.5 ±2.7Degree

ofresistance'38

5137

441722

" IDso, concentration that inhibits 72-h growth by 50%.* Cells were incubated for 72 h with the indicated drug. Results are the

mean of three experiments, each performed in duplicate., . IDwofP388RDegree of resistance = -

' Mean ±SE.ID»of P388S '

Fig. 1.StructiCompoundPD

111,391PD 110,911PD 112,940PD 111,815

(CI-942)PD 113,309

(CI-937)\x-irai

features oftheR\^0anthrapyrazolesstuY•V^yNHYdied in thispaper.Z(CH2)2OH

(CH^NÕCH,), 7,1<HOH),(CH2)2N(C2H,)2 (CH2)2NH2 7,1<MOH)2(CH2),N(CH,)2 (CH2)2NH2 7,10-(OH)2(CH2)2NH(CH2)2OH (CH2)3NH27,10-(OH)2(CH2)NH(CH2)2OH

(CH2)2NHCH,7,10-(OH)2Table

2 Potentiation by verapamil of anthrapyrazole and anthracycline activityin pleiotropically resistant P388 leukemiacellsID»

(nM)*'CompoundP388S

Verapamil—

+P388R

Verapamil D«»o;—

+ resistiree

Degreeof

mcepotentiation'PD

111,391 163 178 463 328 2.8 1.4PD 110,911 127 114 628 437 4.9 1.4PD 112,940 141 145 3,227 1,014 23 3.2CI-942 498 468 75,870 4,069 152 18CI-937 157 161 46,781 1,263 298 37Adriamycin 129 121 6,587 626 51 10.5Daunomycin 110 121 4,152 464 389.0"

IDjo, concentration that inhibits 72-h growth by 50%.* Cells were grown for 72 h in the presence of the indicated drug ±5 ng of

verapamil per ml. Results are the mean of 2 experiments, each performed intriplicate.

ID»of P388R - verapamilI '1.^,1VV VI |M M V.ll I 111! I«'11 _ _____ „ •

ID»of P388R + verapamil

resistance of P388R cells to these compounds ranged from 2.8-fold for PD 111,391 to almost 300-fold for CI-937. PD 111,391differs from the other members of the series in having an alcoholsubstitution at position 2, while the other analogues have a 2-amine substitution and a tertiary amine at position 5, whereasthe other anthrapyrazoles have a primary or secondary aminosubstitution at position 5 (Fig. 1).

Correlation of Anthrapyrazole Lipophilicity with Lack ofCross-Resistance. A comparison of the partition ratios of theanthrapyrazoles in chloroform-water or octanol-water with thedegree of resistance of P388R cells to these compounds demonstrated, in part, that the greater the lipophilicity, the lower

Anthra-pyrazolePD

111,391PD 110,911PD 112,940CI-942CI-937Degree

ofresistance2.8

5.023

152297Partition

ratio"Chloroform/

water0.79±0.06''

0.15 ±0.020.033 ±0.0010.030 ±0.0040.070 ±0.004Octanol/

water2.57

±0.170.33 ±0.020.14 ±0.010.13 ±0.010.13 ±0.01DNA

ethidiumdisplacement(C»,nM)»-c142

±8.7530 ±1.0027 ±1.4733 ±0.7525 ±1.70

" Partition ratios were performed with 10 fig of drug per ml at the emission

maximum following procedures described by Skovsgaard (33). Results are themean of two experiments, each performed in triplicate.

* DNA ethidium displacement assays were performed as described by

Baguley et al. (34).' ( 'M,,the concentration of drug required to displace 50% of DNA-bound

ethidium. Results are mean of two experiments, each performed in triplicate.* Mean ±SE.

300

200

fe 100a so

0 5 20 30 40DEGREEOFPOTENTIATION

Fig. 2. Relationship of P388R resistance to the anthrapyrazoles with theirpotentiation by VER.

the level of resistance (Table 3). P388R cells were the leastresistant to PD 111,391, which was the most lipophilic of theanthrapyrazoles, while these cells were the most cross-resistantto CI-937, which was one of the least lipophilic analogues.However, partition ratios of the anthrapyrazoles were not completely predictive for their activity in P388R cells. PD 112,940and CI-937 had virtually the same partition coefficients, butthere was a 10-fold difference in the resistance of P388R cellsto these compounds. While lipophilicity appears related toresistance, the affinity of the anthrapyrazoles for DNA appearsto have little influence on this phenomenon (Table 3). Four ofthe five anthrapyrazoles had the same affinity for DNA asmeasured by ethidium displacement, yet varied widely in theP388R activity. PD 111,391 had the lowest DNA affinity byalmost 6-fold in comparison to CI-937, yet was the best anthrapyrazole in overcoming drug resistance.

Reversal of Resistance to Anthrapyrazoles and Anthracyclinesby Calcium Channel Blockers and Calmodulin Antagonists. Thelevel of resistance of P388R cells to the anthrapyrazoles oranthracyclines could be partially reversed if the calcium channelblocker, VER (5 Mg/ml), was included in the culture medium(Table 2). The degree of potentiation by VER was linearlycorrelated with the degree of resistance (Fig. 2). The activity ofthe anthrapyrazoles and anthracyclines in P388S cells wasunaffected by the addition of verapamil to the medium (Table2). Other calcium channel blockers as well as calmodulin antagonists were also tested for their ability to potentiate ADR oranthrapyrazole activity in P388R cells (Fig. 3). At the maximally tolerated nontoxic dose (1.0 tig/ml), the calmodulinantagonist W-7 had only a slight effect on ADR cytotoxicity inP388R cells and had no effect on anthrapyrazole cytotoxicity(Fig. 3). Trifluoperazine, the only other calmodulin antagonisttested in this study, was a potent potentiator of ADR andanthrapyrazole cytotoxicity in P388R cells at the same concen-

4353

on June 15, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ANTHRAPYRAZOLE RESISTANCE

Table 5 Influx and efflux ofdaunomycin in P388S and P388R cells

Fig. 3. Comparison of the effect of calcium channel blockers and calmodulinantagonists on the potentiation of ADR, PD 112,940, and CI-937 activity inP388R cells. Cells were incubated for 72 h in the presence of drug ±the indicatedpotentiating agent. Columns, mean of 3 experiments, each performed in duplicate;bars, SE. Ill', trifluoperazine; /'//. diltiazem; in.,,,, concentration that inhibits72-h growth by 50%.

Table 4 Potentiation by quinidine and Tween 80 of ADR activity in P388R celts

CompoundNoneQuinidineTween

80Concentrationl.O^g/ml5.0

»iu'ml0.005%

0.010%0.025%ID»(MM)**6.59

±0.48*1.84

±0.180.94 ±0.081.63

±0.060.86 ±0.160.64 ±0.15Degree

ofpotentiation3.6

7.04.0

7.710.3

•'ID,,,, concentration that inhibits 72-h growth by 50%.* Conditions were as described in Table 3.c Mean ±SE.

trations as W-7 (Fig. 3). Of the calcium channel blockers, NIFwas completely ineffective in potentiating ADR or anthrapyr-azole activity in P388R cells, while diltiazem and VER reversedthe resistance of P388R cells to ADR and the anthrapyrazolesin a concentration-dependent manner (Fig. 3). Of the compounds tested, VER, at nontoxic doses, appeared to be the mostactive in reversing multidrug resistance in the P388R cells.Concentrations of NIF, trifluoperazine, or W-7 greater than1.0 fig/ml were too toxic by themselves to determine any furtherpotentiating activity. None of the compounds altered the cyto-toxicity of ADR or anthrapyrazoles in P388S cells (data notshown).

The antiarrhythmic drug, quinidine, was also found to potentiate ADR activity in P388R cells (Table 4). In addition, atnontoxic concentrations, the detergent Tween 80 potentiatedADR activity in P388R cells (Table 4). At 0.025%, Tween 80was as effective as VER in overcoming ADR resistance.

Transport Studies. The uptake and net accumulation of l'M |-

DAU were examined in P388S and P388R cells in the presenceor absence of VER (10 Mg/ml) (Table 5; Fig. 4). The initial rateof uptake (Table 5), as well as the net accumulation of DAU(Fig. 4), was significantly reduced in P388R cells when compared to P388S cells. The addition of VER to the medium hadno effect on the rate of uptake over the first min in either cellline, but it did partially restore DAU levels over a 4-h periodin P388R cells to those observed for P388S cells. VER had no

Celltype

±Verapamil(lUMg/ml)

Daunomycin(pmol/min/lO4 cells)"

Influx Efflux

P388S

P388R

45.5 ±4.1*

43.9 ±4.8

28.0 ±1.926.6 ±2.2

2.40 ±0.312.1 5 ±0.23

16.5 ±1.51.96 ±0.27

" Influx of [3H]daunomycin was measured over the first min. Incubations

were terminated by rapid chilling and centrifugation. For efflux experiments,cells were first incubated with daunomycin for 10 min, after which outwardtransport was measured over the initial min of efflux as described by Kesseland Wheeler (9). Influx and efflux were determined by linear regressionanalysis. Results are the mean of three experiments, each performed induplicate.

1 Mean ±SE.

30 60 120

INCUBATION TIME (MIN.)

240

Fig. 4. Effect of VER on the net accumulation of | 'I I]I)AI into P388S andP388R cells. Cells (2.5 x IO6 cells/ml) were incubated with 9.5 MM DAU(containing 0.25 MM[3H]DAU) in Fischer's medium supplemented with 10%

horse serum and 25 mm HEPES ±VER (10 Mg/ml) for up to 4 h. Points, meanof 2 experiments, each performed in triplicate; bars, SE.

60INCUBATION TIME (MIN.)

Fig. 5. Effect of VER on DAU efflux from P388S and P388R cells. Cellswere preloaded with DAU using conditions described above. Efflux was measuredin the presence or absence of VER (10 «nmil. Points, mean of 2 experiments,each performed in triplicate; bars, SE.

effect on either the initial uptake rate or total drug accumulationin P388S cells. In addition, no accumulation of DAU in P388S(Fig. 4) or P388R (data not shown) was observed at 4°C.

DAU efflux from P388R cells was considerably greater thanthat observed for P388S cells (Table 5), but unlike uptake, theaddition of VER to the drug-free medium completely reversedthis rate in P388R to that observed for P388S cells. VER hadno effect on the efflux in P388S cells. DAU efflux from P388Scells over a I-h incubation was also unaffected by VER, but aswith the initial rate of exodus, VER completely reversed DAUefflux from P388R cells over the 1-h incubation to the level

4354

on June 15, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ANTHRAPYRAZOLE RESISTANCE

600

'400

-$P388R*TW£EN80

-8P388S»TWEEN805P388S-TWEEN80

5P388R-TWEEN80

U0 30 60

INCUBATION TIME (MIN.)

Fig. 6. Effect of 0.025% Tween 80 on the net accumulation of [3H]DAU into

P388S and P388R cells. Conditions were as described in Fig. 4.

P388S »TWEEN80— ..^.^-4 P388R - TWEEN80

*P388S- TWEEN80

30 60INCUBATION TIME (MIN.)

Fig. 7. Effect of 0.025% Tween 80 on DAU efflux from P388S and P388Rcells. Methods are as described for Fig. 5.

found for P388S cells (Fig. 5). As with uptake, incubation ofcells in an ice bath completely blocked DAU efflux (Fig. 5).

Tween 80 (0.025%), like VER, also increased the net accumulation of DAU in P388R cells (Fig. 6). The pattern of thisincrease over 60 min was remarkably similar to that demonstrated for VER. The detergent had only a slight effect on DAUlevels in P388S cells. Also consistent with VER, Tween 80inhibited the efflux of DAU from P388R cells to the level ofthe sensitive cells (Fig. 7). The detergent had no effect onP388S efflux.

DISCUSSION

In the present study, we have characterized a P388 sublineselected in vivo for its resistance to ADR with respect to itscross-resistance to other anticancer drugs and in terms of thereversal of resistance, particularly to the anthrapyrazoles, bycalcium channel blockers and calmodulin antagonists.

The P388R cell line we isolated shares many of the samecharacteristics of pleiotropically resistant cells previously reported (1-8). There is an accelerated efflux of DAU which iscompletely blocked by VER (Fig. 5; Table 5) as well as adecrease in drug uptake which is unaffected by VER (Table 5).Both of these defects have been previously reported for othermodels of pleiotropic drug resistance (10, 11). In our P388Rcells, the failure of VER to completely reverse drug resistance(Table 2) or the net accumulation of DAU over time (Fig. 4)may be due to its inability to alter the decreased rate of druguptake as demonstrated for DAU (Table 5), particularly sinceVER reduced DAU efflux in P388R cells to the level observedin P388S cells (Table 5). We also observed that, as with previous

studies (10, 22), there were no differences in nuclear binding ofdrug between P388S and P388R cells (data not shown). Moreover, VER did not alter this binding. Thus, it is possible thatthe effect of VER on inward and outward drug transport inP388R could account for the partial rather than completerestoration of drug sensitivity. However, it is more likely thatthe mechanisms that contribute to total drug resistance aremultiple in nature and more complex than merely transportdefects. Chou and Yost (13), for instance, recently observedthat P388/ADR cells have a faster rate of repair for ADR-induced DNA strand breaks than do normal P388 cells. Totaldrug resistance could be the sum of many defects, such as thoseobserved in transport, DNA and membrane binding, and freeradical formation.

Recent studies have suggested that the mechanism by whichcalcium channel blockers or calmodulin antagonists promotedrug retention is unrelated to cellular calcium or calmodulinlevels (23) or to altered calcium ion fluxes (16, 24). Severalreports have suggested, instead, that the effect of VER as wellas other calcium channel blockers and calmodulin antagonistsmay be related to their ability to interact with the plasmamembrane (4, 23, 25, 26). Ramu and coworkers reported thatthere were differences in the membrane lipid composition (27)and membrane fluidity (28) between sensitive and multidrug-resistant P388 cells. They also noted that agents which inducealterations in the lipid domain of the plasma membrane canpotentiate drug activity in resistant cells (4, 25). Tsuruo et al.(26) observed that quinidine, an agent that can alter the organization of membrane lipids, could potentiate drug activity inmultidrug-resistant P388 cells. In our resistant cells, quinidinealso potentiated ADR activity to almost the same degree as didVER (Table 4). Moreover, in accord with a previous study byRiehm and Biedler (29), nontoxic concentrations of the detergent Tween 80 potentiated ADR activity in P388R cells (Table4). Tween 80 increased the net accumulation of DAU (Fig. 6)and inhibited its efflux in a manner virtually identical to thatobserved for VER (Fig. 7). The similarity in potentiation between Tween 80 and VER suggests that, at least for our resistantcells, calcium channel blockers and calmodulin antagonists mayalter drug resistance by their interaction with the plasma membrane, perhaps in a fashion similar to that of Tween 80. Previous studies (30, 31) have demonstrated that calcium channelblockers and calmodulin antagonists can interact with theplasma membrane. The differences in potentiating activity ofthese agents may, in fact, be related to their membrane interactive capacity. In this regard, it is interesting to note that, ofthe Fiveanthrapyrazoles tested in P388S and P388R, those withthe highest lipophilicity (PD 111,391 and PD 110,911) werethe most active in P388R cells, while those with the lowestpartition coefficients (CI-942 and CI-937) were the least active(Table 3). This is in spite of the greater than 5-fold differencein the binding affinity for DNA between the most effectiveanthrapyrazole (PD 111,391) and the least effective (CI-937).Given the complexity of multidrug resistance, it is not surprising, however, that the relationship of anthrapyrazole lipophilicity to its activity in P388R cells is less than perfect. P388Rcells showed a 10-fold difference in cross-resistance to PD112,940 versus CI-937, yet there was no significant differencein either drug's coefficients or DNA binding affinities (Table

3). While it is unclear why the level of resistance of P388R cellsto these two anthrapyrazoles is so great, it does, perhaps, serveto reinforce the multiple nature of drug resistance.

Although the P388R cells described in the present studyshare many of the same properties of other multidrug-resistant

4355

on June 15, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ANTHRAPYRAZOLE RESISTANCE

cells described previously, differences in their response to several known potentiating agents are one notable exception. Inabaand Johnson, for example, were unable to demonstrate anydrug potentiation in their resistant P388 cells by the detergentTween 80 (1, 22). Using this same cell line, Kessel and Wil-berding (32) concluded from their study that VER potentiatedADR activity in these cells by competing with ADR for exodus.While our study does not examine this possibility, the evidencewith quinidine and Tween 80 supports the contention that, inP388R cells, VER and other agents potentiate drug activity bytheir interaction with the lipid domain of the plasma membrane.Also, unlike previous studies (14, 16, 17), NIF had no effect onthe potentiation of activity of ADR or two anthrapyrazoles inP388R cells (Fig. 3). Similar discrepancies were evident for W-7. Ganapathi et al. (18) demonstrated that the calmodulinantagonist, W-13, potentiated ADR activity in ADR-resistantcells, yet W-7 in our studies had almost no drug-potentiatingactivity in P388R cells. It is possible that both NIF and W-7are less potent modifiers of plasma membrane lipids than theother agents tested, or that, in the case of W-13 versus W-7,structural differences between the two compounds render thelatter less effective. The differences between our cells and thoseof others may 'also serve notice to the chemotherapist that,

along with the presumption of multiple defects within a giventumor cell, the circumvention of resistance is further complicated by the diversity of mechanisms which may exist betweendifferent tumors.

Finally, the present study suggests that, in the planned clinical trials with the anthrapyrazoles, patients with tumors resistant to ADR may also be resistant to the anthrapyrazoles,depending on the analogue being tested, but that this resistancemay be at least partially reversed by potentiating agents similarto VER. The finding that the increase in ADR cytotoxicity wasnot observed in drug-sensitive cells indicates that the synergismmay be limited to multidrug-resistant tumors.

REFERENCES

1. Inaba, M., and Johnson, R. K. Decreased retention of actinomycin D as thebasis for cross-resistance in anthracycline-resistant sublines of P388 leukemia. Cancer Res., 37:4629-4634, 1977.

2. Johnson, R. K., Chitnis, M. P., Embrey, W. M., and Gregory, E. B. Characteristics in vivoof resistance and cross-resistance of an Adriamycin-resistantsubline of P388 leukemia. Cancer Treat. Rep., 62: 1535-1547, 1978.

3. I sumo. T., lida, II.. Yamashiro, M., Tsukagoshi, S., and Sakurai, Y. Enhancement of vincristine- and Adriamycin-induced cytotoxicity by verapamilin P388 leukemia and its sublines resistant to vincristine and Adriamycin.Biochem. Pharmacol., 31: 3138-3140, 1982.

4. Ramu, A., Fuks, Z., Gatt, S., and Glaubiger, D. Reversal of acquiredresistance to doxorubicin in P388 murine leukemia cells by perhexilinemaléate.Cancer Res., 44:144-148, 1984.

5. Kartner, N., Shales, M., Riordan, J. R., and Ling, V. Daunorubicin-resistantChinese hamster ovary cells expressing multidrug resistance and a cell-surfaceP-glycoprotein. Cancer Res., 43:4413-4419, 1983.

6. Tapiero, H., Munck, J. N., Fourcade, A., and Lampidis, T. J. Cross-resistanceto rhodamine 123 in Adriamycin- and daunomycin-resistant Friend leukemiacell variants. Cancer Res., 44: 5544-5549, 1984.

7. Biedler, J. L., Chang, T., Meyers, M. B., Peterson, R. H. F., and Spengler,B. A. Drug resistance in Chinese hamster lung and mouse tumor cells. CancerTreat. Rep., 67: 859-867, 1983.

8. Beck, W. T. Vinca alkaloid-resistant phenotype in cultured human leukemiclymphoblasts. Cancer Treat. Rep., 67: 875-882, 1983.

9. Kessel, D., and Wheeler, C. m-AMSA as a probe for transport phenomenaassociated with anthracycline resistance. Biochem. Pharmacol., 33:991-993,1984.

10. Daño,K. Active outward transport of daunomycin in resistant Ehrlich ascitestumor cells. Biodi im. Biophys. Acta, 323:446-483, 1973.

11. Skovsgaard, T. Mechanism of cross-resistance between vincristine and dau-norubicin in Ehrlich ascites tumor cells. Cancer Res., 38: 4722-4727, 1978.

12. Beck, W. T., Cirtain, M. C., and Lefko, J. L. Energy-dependent reduced drugbinding as a mechanism of Vinca alkaloid resistance in human leukemiclymphoblasts. Mol. Pharmacol., 24:485-492, 1983.

13. Chou, T. H., and Yost, C. Adriamycin resistance in murine leukemia P388cells and increased DNA repair. Proc. Am. Assoc. Cancer Res., 26: 217,1985.

14. Tsuruo, T., lida, H., Nojiri, M., Tsukagoshi, S., and Sakurai, Y. Circumvention of vincristine and Adriamycin resistance in vitro and in vivo by calciuminflux blockers. Cancer Res., 43: 2905-2910, 1983.

15. Tsuruo, T., lida, H., Tsukagoshi, S., and Sakurai, Y. Increased accumulationof vincristine and Adriamycin in drug-resistant P388 tumor cells followingincubation with calcium antagonists and calmodulin inhibitors. Cancer Res.,«.•4730-4733,1982.

16. Kessel, D., and Wilberding, C. Anthracycline resistance in P388 murineleukemia and its circumvention by calcium antagonists. Cancer Res., 45:1687-1691,1985.

17. Ramu, A., Spanier, R., Rahamimoff, H., and Fuks, Z. Restoration of doxorubicin responsiveness in doxorubicin-resistant P388 murine leukemia cells.Br. J. Cancer, 50:501-507, 1984.

18. Ganapathi, R., Grabowski, I).. I uri nie. R., and Valenzuela, R. Correlationbetween potency of calmodulin inhibitors and effects on cellular levels andcytotoxic activity of doxorubicin (Adriamycin) in resistant P388 mouseleukemia cells. Eur. J. Cancer Clin. Oncol., 20: 799-806, 1981.

19. Showalter, H. D. H., Johnson, J. L., Werbel, L. M., Leopold, W. R., Jackson,R. C., and Elslager, E. F. 5-[(Aminoalkyl)amino]-substituted anthra[l,9-cd]-nvra/oi h(2//) ones as novel anticancer agents. Synthesis and biologicalevaluation. J. Med. Chem., 27: 253-255, 1984.

20. Leopold, W. R., Nelson, J. M., Plowman, J., and Jackson, R. C. Theanthrapyrazoles, a new class of intercalating agents with high level, broadspectrum activity against murine tumors. Cancer Res., 45:5532-5539,1985.

21. Klohs, W. D., Steinkampf, R. W., Wicha, M. S., Menus, A. E., Tunac, 3.B., and Leopold, W. R. J. Nati. Cancer Inst., 75: 353-359, 1985.

22. Inaba, M., and Johnson, R. K. Uptake and retention of Adriamycin anddaunorubicin by sensitive and anthracycline-resistant sublines of P388 leukemia. Biochem. Pharmacol., 27: 2123-2130, 1978.

23. Nair, S., Sunn. T. S. A., and Krishan, A. Calcium, calmodulin, and proteincontent of Adriamycin-resistant and -sensitive murine leukemic cells. CancerRes., 46: 229-232, 1986.

24. Kessel, D., and Wilberding, C. Interaction between calcium antagonists,calcium fluxes, and anthracycline transport. Cancer Lett., 25:97-101, 1984.

25. Ramu, A., Shan, T., and Glaubiger, D. Enhancement of doxorubicin andvinblastine sensitivity in anthracycline-resistant P388 cells. Cancer Treat.Rep., 67: 895-899, 1983.

26. Tsuruo, T., lida, H., Kitatani, Y., Keiko, Y., Tsukagoshi, S., and Sakurai, Y.Effects of quinidine and related compounds on cytotoxicity and cellularaccumulation of vincristine and Adriamycin in drug-resistant tumor cells.Cancer Res., 44:4303-4307, 1984.

27. Ramu, A., and Weentraub, H. Differences in lipid composition of doxorubi-cin-sensitive and -resistant P388 cells. Cancer Treat. Rep., 68: 637-641,1984.

28. Ramu, A., Glaubiger, D., Magrath, I. T., and Joshi, A. Plasma membranelipid structural order in doxorubicin-sensitive and -resistant P388 cells.Cancer Res., 43: 5533-5537, 1983.

29. Riehm, H., and Biedler J. L. Potentiation of drug effect by Tween 80 inChinese hamster cells resistant to actinomycin D and daunomycin. CancerRes., 32: 1195-1200, 1972.

30. Triggle, D. J. Calcium antagonists: basic chemical and pharmacologicalaspects. In: G. B. Weiss (ed.), New Perspectives on Calcium Antagonists, pp.1-18. Bethesda: American Physiological Society, 1981.

31. Kanno, K., and Susaki, Y. Interaction of psychotropic drugs with phospho-lipids. Biochem. Pharmacol., 31: 2977-2981, 1982.

32. Kessel, D., and Wilberding, C. Mode of action of calcium antagonists whichalter anthracycline resistance. Biochem. Pharmacol., 33: 1157-1160, 1984.

33. Skovsgaard, T. Carrier-mediated transport of daunorubicin, Adriamycin, andrubidazone in Ehrlich ascites tumour cells. Biochem. Pharmacol., 27:1221-1227, 1978.

34. Baguley, B. C., Denny, W. A., Atwell, G. J., and Cain, B. F. Potentialantitumor agents. 34. Quantitative relationships between DNA binding andmolecular structure for 9-anilinoacridines substituted in the anilino ring. J.Med. Chem., 24:170-177, 1981.

4356

on June 15, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1986;46:4352-4356. Cancer Res   Wayne D. Klohs, Randall W. Steinkampf, Martha J. Havlick, et al.   Calcium Blockers and Calmodulin AntagonistsMultidrug-resistant P388 Murine Leukemia Cells: Reversal by Resistance to Anthrapyrazoles and Anthracyclines in

  Updated version

  http://cancerres.aacrjournals.org/content/46/9/4352

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

  .pubs@aacr.orgDepartment 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/46/9/4352To request permission to re-use all or part of this article, use this link

on June 15, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from