EXPERIMENTAL
PARASITOLOGY
71, 169-175 (1990)
Entamoeba histolytica:
Physiology of Multidrug Resistance
PATRICIA AYALA,* JOHN SAMUELSON,? DYANN WIRTH,~ AND ESTHER OROZCO**’ *Departamento de Genetica y Biologia Molecular, Centro de Investigation y de Estudios Avanzados de1 I.P.N., Apartado Postal 14-740, Mexico, 07000 D.F., and tDepartment of Tropical Public Health, Harvard School of Public Health, Harvard University, 665 Huntington Avenue, Boston, Massachusetts 02115, U.S.A. AYALA, P., SAMUELSON, J., WIRTH, D., AND OROZCO, E. 1990. Entamoeba histolytica: Physiology of multidrug resistance. Experimental Parasitology 71, 169-175. Crossresistance to unrelated drugs has been previously observed in multidrug-resistant carcinoma cells and the goal of this work was to determine whether a similar mechanism existed in Entamoeba histolytica. An emetine and a colchicine-resistant clone, C2(9O)(IC,, = 62 )LM, and 1.5 mM, respectively), and the parental clone, A (I&, = 5 )LM and 1 mM, respectively), were analyzed for resistance to other drugs and for the effect of verapamil. Both clones, C2(9O)and A, exhibited similar resistance to both daunomycin (IC,, = 50 (LM) and actinomycin D (IC,, = 13 ~LW).In the presence of verapamil, the I&, for emetine was reduced to 0.5 FM, while the IC, for colchicine was reduced to 0.3 mM. These results demonstrate that verapamil reverses both emetine and colchicine resistance in the mutant C2(9O). In uptake experiments with [3H]emetine, drug accumulation was lower in resistant trophozoites. However, in the presence of verapamil, drug accumulation was increased in clone C2(9O) to a level close to that of the parental strain, clone A. These results are consistent with observations made using malaria and multidrug-resistant tumor cells and suggest that a P-glycoprotein-like molecule may play a role in drug resistance in E. histolytica. o IWO Academic Press, Inc. INDEX DESCRIPTIONS
AND
Entamoeba
ABBREVIATIONS:
histolytica;
Multidrug-resis-
tance; Multidrug-resistance gene (mdr); Polymerase chain reaction (PCR). INTRODUCTION
In Entamoeba histolytica, the protozoan parasite responsible for human amebiasis, a multidrug-resistant phenotype, has been reported in mutants selected by the alkaloid emetine, after chemical mutagenesis (Orozco et al. 1985). The parasites resistant to emetine are cross-resistant to colchicine, an unrelated drug, both in terms of structure and mechanism of action. Thus, this phenomenon of cross-resistance resembles that originally observed in mammalian tumor cells selected for resistance to a single drug and was found to be cross-resistant to unrelated drugs. Multidrug resistance in eukaryotic cells was first described in mammalian-transformed cell lines (Dano 1973; Skovsgaard 1978). The physiology of multidrug resis’ To whom correspondence should be addressed.
tance has been defined in these systems. Drug resistance is due to the decreased accumulation of drug, mediated by an increased drug efflux. This efflux can be reversed by calcium channel blockers such as verapamil (Tsuruo et al. 1981, 1982). These characteristics are associated with the overexpression of the P-glycoprotein which is the gene product of the multidrugresistant locus (mdr) (Borst 1984; Roninson et al. 1984; Riordan and Ling 1985a, b; Scotto et al. 1986). The mammalian Pglycoprotein consists of two homologous halves which include potential channelforming transmembrane domains with two nucleotide-binding sites (Hamada and Tsuruo 1986; Gross et al. 1986; Higgins et al. 1986; Choi et al. 1988). The sequence similarity of P-glycoprotein to bacterial transport proteins has led to the model that the P-glycoprotein is an ATP-depending efflux pump responsible for decreasing drug accu169 OO14-4894190 $3.00 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
170
AYALAETAL.
mulation in resistant cells (Gross et al. 1986). The purpose of the experiments reported here was to determine if the physiology of multidrug resistance in E. histolytica was similar to multidrug resistance in eukaryotic cells. Recent work in Plasmodiumfalciparum has demonstrated that the physiology of chloroquine resistance is similar to that in mammalian multidrug resistance and further, that increased expression of a gene homologous to mammalian mdr is associated with this resistance (Krogstadt et al. 1987; Martin et al. 1987; Wilson et al. 1989; Foote et al. 1989). In this paper we report that drug resistance in E. histolytica can be reversed by verapamil and that the resistance is characterized by decreased drug accumulation. MATERIALSAND
METHODS
Materials. Chemicals were obtained from Sigma Chemical (St. Louis, MO) and radioactive products were from Amersham, England. E. histolytica cultures. Trophozoites of clones A and C2 (strain HMI:IMSS) were grown in TYl-S-33 medium and harvested during the logarithmic growth phase as described (Diamond et al. 1978). Clone A was directly isolated from semisolid agar (Gillin and Diamond 1978) and clone C2 was obtained by chemical mutagenesis of clone A and selected for its resistance to 22.5 w (Orozco et al. 1985). To produce subclones of C2 more resistant to emetine, ameba were cultured in 28 PM emetine until their growth was similar to those of parasites growing without drug. Subclone C2(28) was then isolated in soft agar containing 28 pM emetine. This process was repeated with 45 p,M emetine (subclone C2(45)) and finally with 90 pM emetine (subclone C2(90)). Parenthetic numbers mean the molar concentration of emetine that was added to the soft agar for the isolation of clones (Table I). Drug resistance and effect of verapamil. Trophozoites of clones A and C2(90) were inoculated in 11.5 ml of culture medium, in duplicate cultures. Trophozoites were incubated at 37°C without drug, with 18 to 90 pM emetine, with 0.5 to 1.5 mM colchicine, with 10 to 100 pM daunomycin, with 0.13 to 13 n&f actinomycin D, or with 10 to 100 p&f verapamil. To half the cultures 10 PM verapamil was added, a nontoxic concentration of the calcium-channel blocker. Numbers of trophozoites were counted in duplicate cultures every 24 hr, and the concentration that caused a 50% growth inhibition
Step Increasement
TABLE I of Emetine Resistance in Clone C2
Clone
Emetine resistance (PM)
Relative resistance Subclone/A
A c2 C2(28) C2(54) C2(90)
9.0 22.5 28.8 54.0 90.0
1.00 2.25 3.20 6.00 10.00
Note. To produce C2 subclones with increased resistance to emetine, trophozoites were cultured, step by step, first in 28 PM (subclone C2(28)), then in 54 PM (subclone C2(54)), and finally in 90 PM emetine (subclone C2(90)), until their growth was similar to those of parasites growing without drug. Subclones with different resistance to emetine were obtained in semisolid agar. (IC,,) was determined for each drug and clone (Martin et a/. 1987). Drug uptake in drug-resistant and drug-sensitive clones. Trophozoites were incubated at 37°C in 4 ml of amebic medium without bovine serum. Cultures were supplemented with 22.5 )IM emetine (12.5 &ml) and 2.5 ).&i/ml [3H]emetine (Amersham TRQ 5114, sp act = 12.9 Ci/mmol), with or without verapamil(l0 t&f). Emetine uptake was measured in duplicate tubes by taking duplicate 200~ul ahquots of each tube at different times after drugs were added (5 to 400 min). Ahquots were layered over 200 ~1 of n-butyl phthalate (Sigma) in order to take out most of the drug that was out of the cells; cells were pelleted by centrifugation according to the technique described by Phillips and Wang (1987). Cell pellets were washed once with 200 ~1 of amebic medium without serum. Aqueous supernatants were pooled and after removal of the oil phase, the cell pellets were lysed with 1% Triton X-100. 13H]Emetine was measured by liquid scintillation counting in the pellet, supematant, and oil fractions. Percentage uptake was calculated by dividing the counts per minute (cpm) in the pellet by the total cpm (pellet + supematants + oil phase). The amount of emetine accumulated per cell was determined by the following calculation: emetine/cell
= (percentage uptake) x 12.5 *g/ml X 1 ml/No. of cells. RESULTS
Drug sensitivity of clones A and C2(90).
Clones A and C2(90) were tested for sensitivity to five drugs: emetine, cholchicine,
171
E. histolytica: MULTIDRUG RESISTANCE daunomycin, actinomycin D, and verapamil. In the original work describing the isolation of the C2 clone, the resistant parasites were able to grow in 60 p,M emetine (Orozco et al. 1985). By stepwise selection of C2 in increasing concentrations of emetine, the IC,, for emetine was increased to 62 pJ4, and parasites grew well in 90 l&f. All the experiments reported here were done using the C2 clone with an IC,, of 62 ~.LM (C2(90)). As can be seen in Figs. 1 and 2 and Table II, clone A is more susceptible than C2(90) to both emetine and colchicine,
46
72
96
I20
46
72
96
120
TIME
4672%l20
4672%uo
while both clones show similar sensitivity to verapamil, daunomycin, and actinomytin D. The IC,, of emetine for clone A is 5 pJ4 and for clone C2 is 62 @4, demonstrating an approximately lo-fold difference in drug susceptibility between the sensitive and resistant parasites. In contrast, the I&, of colchicine for clone A is 1 mil4 and for clone C2(90) it is greater than 1.5 mM. This is similar to observations in mammalian cells in which the relative resistance to the selecting drug is often greater than for cross-
72
96
I20
46
72
96
120
72
96
120
46
72
96
120
6;
46
TIME (h ) FIG. 1. Sensitivity of E. histolytica to different drugs. Trophozoites (104) of clones A and C2(90) were incubated in TYl-S-33 medium with different drug concentrations. Emetine (EMT): (0) control without any drug, (A) 18 uhf, (A) 22.5 uM, (0) 54 pLM,and (X) 90 FM. Colchicine (COL): (0) control without any drug, (A) 0.5 mM, (Cl) 1.Omkf, and (X) 1.5 mM. Daunomycin (DAU): (0) control without any drug, (A) 10 t&f, (0) 50 pJ4, and (x) 100 FM. Actinomycin D (ACT D): (0) control without any drug, (A) 0.13 nM, (Cl) 1.3 nkf, and (x) 13 nkf.
172
AYALA
ET AL.
concentrations of verapamil, but cell viability was reduced at concentrations of verapamil greater than 20 p.M. A nontoxic verapamil concentration of 10 l&f was chosen for the drug reversal experiments in E. histolytica.
P’M FIG. 2. Sensitivity of clones A and C2(90) to verapamil. Relative growth of clones (0) A and (a) C2(90) was obtained after 96 hr of incubation of trophozoites in the presence of different concentrations of vera-
resistant drugs (Choi et al. 1988). Clone A and C2(90) have similar sensitivity to both daunomycin and actinomycin D. These are two drugs which are often associated with the multidrug-resistant phenotype in mammalian cells. Reversal of drug resistance by verapamil. Verapamil has been shown to reverse the
multidrug-resistant phenotype in mammalian tumor cells and chloroquine resistance in P. falciparum. In order to know whether verapamil was able to reverse the drug resistance in E. histolytica, trophozoites were incubated with the drugs in the presence of verapamil. Verapamil is toxic for most cells at high concentrations and the sensitivity of E. histolytica to verapamil was determined. As can be seen in Fig. 2, the viability of clones A and C2(90) was similar at various
I&
TABLE II for Different Drugs on Clones A and C2(90) EMT
COL (m.W
WO
DAU (IL-W
ACT-D Wfl
Clone
-v
+v
-v
+v
-v
+v
-v
+v
:2,90,
625
0.50 0.30
1.5 1.0
0.45 0.28
50
11.0 6.6
13
13
Nofe. I&,, is the drug concentration which inhibited 50% growth of clones A and C2(90). EMT, emetine; COL, colchitine; DAU, daunomycin; ACT-D, actinomycin D; -V, without verapamil, +V, with 10 pLMverapamil.
Incubation of clones A and C2(90) with emetine in the presence of verapamil decreased the ICso for emetine approximately lo-fold for clone A and loo-fold for clone C2(90) (Fig. 3 and Table II). A decrease in the IC,, for colchicine for both clones was also observed, but it was not as dramatic as it was for emetine. Although there was no difference in the IC,, for daunomycin for clones A and C2(90) without verapamil, incubation in the presence of verapamil reduced the IC,, approximately 5-fold for each clone. The IC,, for actinomycin D was not changed by the addition of verapamil. Emetine accumulation in drug-resistant and drug-sensitive trophozoites. The multi-
drug-resistant phenotype in mammalian cells is characterized by a decreased accumulation of drug in resistant cells. To determine if a similar decreased accumulation of emetine occurred in clone C2(90) of E. histolytica, parasites were incubated in the presence of [3H]emetine. Clone C2(90) exhibited a lower accumulation of the drug compared with clone A (Fig. 4). In multidrug-resistant mammalian cells, differences in accumulation between sensitive and resistant cells are diminished by the addition of verapamil. This was also observed in E. histofytica as can be seen in Fig. 4. After 5 hr, clone C2(90) incubated with emetine alone accumulated 290 fg/cell and with the addition of verapamil accumulated 540 fg/cell. Clone A accumulated 650 fg/cell of emetine in the presence and absence of verapamil after 5 hr incubation. Femtograms of emetine taken up by trophozoites were calculated from the percentage of drug uptake (Fig. 4). DISCUSSION
In this paper we demonstrated that the
E.
histdytica:
MULTIDRUG
TIME
48
72
96
48
72
96
173
RESISTANCE
I
,
I
46
72
96
72
96
(h)
46
40
72
96
TIME 1 h) FIG. 3. Effect of verapamil on drug resistance of E. hisrolytica. Trophozoites (104)of clones A and C2(90) were incubated with different drug concentrations and 10 @I verapamil. Emetine (EMT): (0) no emetine, (A) 0.18 @4, and (m) 1.8 WM. Colchicine (COL): (0) no colchicine, (A) 0.5 mM, and (m) 1.Omk4. Daunomycin (DAU): (0) no daunomycin, (A) 10 PM, and (m) 50 (LM. Actinomycin D (ACT D): (0) no actinomycin, (A) 0.13 nM, and (W) 1.3 nM.
emetine-resistant clone C2(90) of E. histolytica has several characteristics similar to those described for multidrug-resistant mammalian tumors. E. histolytica mutants selected by their resistance to the alkaloid emetine showed cross-resistance to an unrelated drug, colchicine. The C2(90) clone was not cross-resistant to either daunomytin or actinomycin D. Both emetine and colchicine resistance could be reversed by the addition of the calcium channel blocker, verapamil. This is similar to observations in mammalian tumor cells. In the resistant clone C2(90), there was a decreased accu-
mulation of [3H]emetine which was reversed by the addition of verapamil. Both clones A and C2(90) were resistant to killing by actinomycin D at concentrations of 13 ti, which is IOOO-fold higher than that necessary to kill mammalian cells. Resistance was not reversed by the addition of verapamil. Our experiments cannot distinguish whether the lack of sensitivity to actinomycin D was due to impermeability of the cells to the drug or to the lack of target inside the parasite. In this regard, other workers (Vanik et al. 1986) have demonstrated that E. histolytica RNA polymer-
AYALAETAL.
174
%zo
N !m 120
270
sx
Time (min)
FIG. 4. Emetine accumulation in drug-resistant and drug-sensitive trophozoites. Trophozoites of clones A and C2(90) were incubated with 22.5 uM cold emetine and 2.5 &i/ml tritiated emetine with (0, A) or without (0, A) 10 @f verapamil at 37°C. Tritiated emetine accumulation was measured as described under Materials and Methods. Clone A (0,O) clone C2(90)(A, A).
ase activity is inhibited by actinomycin D at a concentration of 40 )&ml. There was no difference in the sensitivity of clones A and C2(90) to daunomycin. However, sensitivity to this drug is increased in the presence of verapamil in both clones. These results are consistent with the existence of a multidrug-resistant phenotype in E. histolytica which is similar to that demonstrated in mammalian tumor cells and recently reported in drug-resistant P. fulciparum (Wilson et al. 1989). Recently, we have reported the identification of multiple 344-bp segments of amebic DNA similar to the human multidrug-resistant gene using primers to conserved regions of the P-glycoprotein and the polymerase chain reaction (PCR) (Samuelson et al. 1990). The amino acid sequences of amebic mdrlike PCR products were 46-5 1% identical to human mdr-1 sequences and 30 to 35% identical to P. fulcipurum (Wilson et al. 1989). The E. histolytica mdr-like gene is
expressed at an increased level in the resistant clone, C2, compared with its expression in clone A. This evidence, together with the physiology presented in this paper, argue for the involvement of a P-glycoprotein-like molecule in drug resistance in E. histolyticu. Drug resistance in clinical isolates of E. histolytica has not yet been described as a major problem, but with increased drug usage it may well emerge as an important health problem. Previous work has shown that laboratory strains of E. histolyticu have differential sensitivity to drugs and drug-resistant parasites can be easily isolated in vitro. If a mechanism of multidrug resistance exists in E. histolyticu, resistance to multiple drugs may arise simultaneously in clinical isolates as has been observed in mammalian tumor cells. ACKNOWLEDGMENTS This work was partially supported by grants from The MacArthur Foundation (U.S.A.) and CONACYT (Mexico). Dr. Orzco was the recipient of a J. Guggenheim award.
REFERENCES BORST, P. 1984. DNA amplification, multidrugresistance and cancer chemotherapy. Nature (Lon-
don)309,580. CHOI, K., CHEN, C., KIUEGLER,M., AND RONINSON, I. B. 1988. An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdrl (P-glycoprotein) gene. Cell 53, 51%529. DANO, K. 1973. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochemical and Biophysics Acta 323, 466-483. DIAMOND, L. S., HARLOW, R., AND CUNNICK, C. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Transactions of the Royal Society for Tropical Medicine and Hygiene 12, 431-432. FOOTE,S. J., THOMPSON,J. K., COWMAN,A. F. AND KEMP, D. J. 1989. Amplification of the multidrug resistant gene in some chloroquine-resistant isolates of P. falciparum. Cell 51, 921-930. GILLIN, F. D., AND DIAMOND, L. S. 1978. Clonal growth of Entamoeba histolytica and other species of Entamoeba in agar. Journal of Protozoology 25,
539-543.
E. histolytica: MULTIDRUG RESISTANCE GROSS,P., CROOP,J., AND HOIJSMAN,D. 1986. Mammalian multidrug resistance gene: Complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell 41, 371-380. HAMADA, H., AND TSURUO,T. 1986. Purification of the P-glycoprotein associated with multidrug resistance. Journal of Biological Chemistry 263, 1454 1458. HIGGINS, C. F., HILES, I. D., SALMOND, G. P. C., GILL, D. R., D~WNIE, J. A., EVANS, I. J., HOLLAND, I. B., GRAY, L., BUCKEL, D. D., BELL, A. W., AND HERMODSON,M. A. 1986. A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature (London) 323, 448-450.
KROGSTADT,D. J., GLUZMAN, I. Y., KYLE, D. E., ODUOLA, A. M., MARTIN, S. K., MILHOUS, W. K., AND SCHELINGER,P. H. 1987.Efflux of chloroquine from Plasmodium falciparum: Mechanism of chloroquine resistance. Science 238, 1283-1285. MARTIN, S. K., ODUOLA, A. M. J., AND MILHOUS, W. K. 1987. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235, 899-901. OROZCO, E., HERNANDEZ, F., AND RODRIGUEZ, M. A. 1985. Isolation and characterization of Entamoeba histolytica mutants resistant to emetine. Molecular and Biochemical Parasitology 15, 49-59. PHILLIPS, M. A., AND WANG, C. C. 1987.A Trypanosoma brucei mutant resistant to alfa-difluoromethylomithine. Molecular and Biochemical Parasitology 22, P-17. RIORDAN, J. R., DEUCHARS, K., KARTNER, N., ALON, N., TRENT, J., AND LING, V. 1985a. Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. Nature (London) 316, 817-819. RIORDAN, J. R., AND LING, V. 1985b. Genetic and biochemical characterization of multidrug resistance. Pharmacology Therapy 28, 51-57.
175
RONINSON, I. B., ABELSOON, H. T., HOUSMAN, D. E., HOWELL, AND VARSHARSKY,A. 1984. Amplification of specific DNA sequences correlates with multidmg resistance in Chinese hamster cells. Nature (London)
309, 626-628.
SAMUELSON, J., AYALA, P., OROZCO, E., AND WIRTH, D. F. 1990. Emetine resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance. Molecular and Biochemical in press. Parasitology, SCOTTO, K. W., BLIEDLER, J. L., AND MELERA, P. W. 1986. Amplification and expression of genes associated with multidrug resistance in mammalian cells. Science 232, 751-755. SKOVSGAARD,T. (1978). Mechanisms of resistance to daunorubicin in Ehrlich ascites tumor cells. Cancer Research 38, 1785-1791. TSURUO,T., IIDA, H., TSUKOGOSHI,S., AND SAKURAI, Y. 1981. Overcoming the vincristine resistance of P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Research 41, 19761979. TSURUO,T., IIDA, H., TSUKAGOSHI,S., AND SAKURAI, Y. 1982. Increased accumulation of vincristine and adriamycin in drug resistant P399 tumor cells following incubation with calcium antagonists and calmodulin inhibitors. Cancer Research 42, 47304733. VANIK, J. M., DETKE, S., AND ALBACH, R. A. 1986. Partial characterization of DNA-dependent RNA polymerases from Entamoeba histolytica. Archives de Investigation Medica 17, Suppl. 1, 101-106. WILSON, C. M., SERRANO,A. E., WASLEY, A., BoGENSCHUTZ,M. P., SHANKAR, A. H., AND WIRTH, D. F. 1989. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 244, 1184-l 186. Received 26 October 1989; accepted with revision 6 February 1990.
PARASITOLOGY
71, 169-175 (1990)
Entamoeba histolytica:
Physiology of Multidrug Resistance
PATRICIA AYALA,* JOHN SAMUELSON,? DYANN WIRTH,~ AND ESTHER OROZCO**’ *Departamento de Genetica y Biologia Molecular, Centro de Investigation y de Estudios Avanzados de1 I.P.N., Apartado Postal 14-740, Mexico, 07000 D.F., and tDepartment of Tropical Public Health, Harvard School of Public Health, Harvard University, 665 Huntington Avenue, Boston, Massachusetts 02115, U.S.A. AYALA, P., SAMUELSON, J., WIRTH, D., AND OROZCO, E. 1990. Entamoeba histolytica: Physiology of multidrug resistance. Experimental Parasitology 71, 169-175. Crossresistance to unrelated drugs has been previously observed in multidrug-resistant carcinoma cells and the goal of this work was to determine whether a similar mechanism existed in Entamoeba histolytica. An emetine and a colchicine-resistant clone, C2(9O)(IC,, = 62 )LM, and 1.5 mM, respectively), and the parental clone, A (I&, = 5 )LM and 1 mM, respectively), were analyzed for resistance to other drugs and for the effect of verapamil. Both clones, C2(9O)and A, exhibited similar resistance to both daunomycin (IC,, = 50 (LM) and actinomycin D (IC,, = 13 ~LW).In the presence of verapamil, the I&, for emetine was reduced to 0.5 FM, while the IC, for colchicine was reduced to 0.3 mM. These results demonstrate that verapamil reverses both emetine and colchicine resistance in the mutant C2(9O). In uptake experiments with [3H]emetine, drug accumulation was lower in resistant trophozoites. However, in the presence of verapamil, drug accumulation was increased in clone C2(9O) to a level close to that of the parental strain, clone A. These results are consistent with observations made using malaria and multidrug-resistant tumor cells and suggest that a P-glycoprotein-like molecule may play a role in drug resistance in E. histolytica. o IWO Academic Press, Inc. INDEX DESCRIPTIONS
AND
Entamoeba
ABBREVIATIONS:
histolytica;
Multidrug-resis-
tance; Multidrug-resistance gene (mdr); Polymerase chain reaction (PCR). INTRODUCTION
In Entamoeba histolytica, the protozoan parasite responsible for human amebiasis, a multidrug-resistant phenotype, has been reported in mutants selected by the alkaloid emetine, after chemical mutagenesis (Orozco et al. 1985). The parasites resistant to emetine are cross-resistant to colchicine, an unrelated drug, both in terms of structure and mechanism of action. Thus, this phenomenon of cross-resistance resembles that originally observed in mammalian tumor cells selected for resistance to a single drug and was found to be cross-resistant to unrelated drugs. Multidrug resistance in eukaryotic cells was first described in mammalian-transformed cell lines (Dano 1973; Skovsgaard 1978). The physiology of multidrug resis’ To whom correspondence should be addressed.
tance has been defined in these systems. Drug resistance is due to the decreased accumulation of drug, mediated by an increased drug efflux. This efflux can be reversed by calcium channel blockers such as verapamil (Tsuruo et al. 1981, 1982). These characteristics are associated with the overexpression of the P-glycoprotein which is the gene product of the multidrugresistant locus (mdr) (Borst 1984; Roninson et al. 1984; Riordan and Ling 1985a, b; Scotto et al. 1986). The mammalian Pglycoprotein consists of two homologous halves which include potential channelforming transmembrane domains with two nucleotide-binding sites (Hamada and Tsuruo 1986; Gross et al. 1986; Higgins et al. 1986; Choi et al. 1988). The sequence similarity of P-glycoprotein to bacterial transport proteins has led to the model that the P-glycoprotein is an ATP-depending efflux pump responsible for decreasing drug accu169 OO14-4894190 $3.00 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
170
AYALAETAL.
mulation in resistant cells (Gross et al. 1986). The purpose of the experiments reported here was to determine if the physiology of multidrug resistance in E. histolytica was similar to multidrug resistance in eukaryotic cells. Recent work in Plasmodiumfalciparum has demonstrated that the physiology of chloroquine resistance is similar to that in mammalian multidrug resistance and further, that increased expression of a gene homologous to mammalian mdr is associated with this resistance (Krogstadt et al. 1987; Martin et al. 1987; Wilson et al. 1989; Foote et al. 1989). In this paper we report that drug resistance in E. histolytica can be reversed by verapamil and that the resistance is characterized by decreased drug accumulation. MATERIALSAND
METHODS
Materials. Chemicals were obtained from Sigma Chemical (St. Louis, MO) and radioactive products were from Amersham, England. E. histolytica cultures. Trophozoites of clones A and C2 (strain HMI:IMSS) were grown in TYl-S-33 medium and harvested during the logarithmic growth phase as described (Diamond et al. 1978). Clone A was directly isolated from semisolid agar (Gillin and Diamond 1978) and clone C2 was obtained by chemical mutagenesis of clone A and selected for its resistance to 22.5 w (Orozco et al. 1985). To produce subclones of C2 more resistant to emetine, ameba were cultured in 28 PM emetine until their growth was similar to those of parasites growing without drug. Subclone C2(28) was then isolated in soft agar containing 28 pM emetine. This process was repeated with 45 p,M emetine (subclone C2(45)) and finally with 90 pM emetine (subclone C2(90)). Parenthetic numbers mean the molar concentration of emetine that was added to the soft agar for the isolation of clones (Table I). Drug resistance and effect of verapamil. Trophozoites of clones A and C2(90) were inoculated in 11.5 ml of culture medium, in duplicate cultures. Trophozoites were incubated at 37°C without drug, with 18 to 90 pM emetine, with 0.5 to 1.5 mM colchicine, with 10 to 100 pM daunomycin, with 0.13 to 13 n&f actinomycin D, or with 10 to 100 p&f verapamil. To half the cultures 10 PM verapamil was added, a nontoxic concentration of the calcium-channel blocker. Numbers of trophozoites were counted in duplicate cultures every 24 hr, and the concentration that caused a 50% growth inhibition
Step Increasement
TABLE I of Emetine Resistance in Clone C2
Clone
Emetine resistance (PM)
Relative resistance Subclone/A
A c2 C2(28) C2(54) C2(90)
9.0 22.5 28.8 54.0 90.0
1.00 2.25 3.20 6.00 10.00
Note. To produce C2 subclones with increased resistance to emetine, trophozoites were cultured, step by step, first in 28 PM (subclone C2(28)), then in 54 PM (subclone C2(54)), and finally in 90 PM emetine (subclone C2(90)), until their growth was similar to those of parasites growing without drug. Subclones with different resistance to emetine were obtained in semisolid agar. (IC,,) was determined for each drug and clone (Martin et a/. 1987). Drug uptake in drug-resistant and drug-sensitive clones. Trophozoites were incubated at 37°C in 4 ml of amebic medium without bovine serum. Cultures were supplemented with 22.5 )IM emetine (12.5 &ml) and 2.5 ).&i/ml [3H]emetine (Amersham TRQ 5114, sp act = 12.9 Ci/mmol), with or without verapamil(l0 t&f). Emetine uptake was measured in duplicate tubes by taking duplicate 200~ul ahquots of each tube at different times after drugs were added (5 to 400 min). Ahquots were layered over 200 ~1 of n-butyl phthalate (Sigma) in order to take out most of the drug that was out of the cells; cells were pelleted by centrifugation according to the technique described by Phillips and Wang (1987). Cell pellets were washed once with 200 ~1 of amebic medium without serum. Aqueous supernatants were pooled and after removal of the oil phase, the cell pellets were lysed with 1% Triton X-100. 13H]Emetine was measured by liquid scintillation counting in the pellet, supematant, and oil fractions. Percentage uptake was calculated by dividing the counts per minute (cpm) in the pellet by the total cpm (pellet + supematants + oil phase). The amount of emetine accumulated per cell was determined by the following calculation: emetine/cell
= (percentage uptake) x 12.5 *g/ml X 1 ml/No. of cells. RESULTS
Drug sensitivity of clones A and C2(90).
Clones A and C2(90) were tested for sensitivity to five drugs: emetine, cholchicine,
171
E. histolytica: MULTIDRUG RESISTANCE daunomycin, actinomycin D, and verapamil. In the original work describing the isolation of the C2 clone, the resistant parasites were able to grow in 60 p,M emetine (Orozco et al. 1985). By stepwise selection of C2 in increasing concentrations of emetine, the IC,, for emetine was increased to 62 pJ4, and parasites grew well in 90 l&f. All the experiments reported here were done using the C2 clone with an IC,, of 62 ~.LM (C2(90)). As can be seen in Figs. 1 and 2 and Table II, clone A is more susceptible than C2(90) to both emetine and colchicine,
46
72
96
I20
46
72
96
120
TIME
4672%l20
4672%uo
while both clones show similar sensitivity to verapamil, daunomycin, and actinomytin D. The IC,, of emetine for clone A is 5 pJ4 and for clone C2 is 62 @4, demonstrating an approximately lo-fold difference in drug susceptibility between the sensitive and resistant parasites. In contrast, the I&, of colchicine for clone A is 1 mil4 and for clone C2(90) it is greater than 1.5 mM. This is similar to observations in mammalian cells in which the relative resistance to the selecting drug is often greater than for cross-
72
96
I20
46
72
96
120
72
96
120
46
72
96
120
6;
46
TIME (h ) FIG. 1. Sensitivity of E. histolytica to different drugs. Trophozoites (104) of clones A and C2(90) were incubated in TYl-S-33 medium with different drug concentrations. Emetine (EMT): (0) control without any drug, (A) 18 uhf, (A) 22.5 uM, (0) 54 pLM,and (X) 90 FM. Colchicine (COL): (0) control without any drug, (A) 0.5 mM, (Cl) 1.Omkf, and (X) 1.5 mM. Daunomycin (DAU): (0) control without any drug, (A) 10 t&f, (0) 50 pJ4, and (x) 100 FM. Actinomycin D (ACT D): (0) control without any drug, (A) 0.13 nM, (Cl) 1.3 nkf, and (x) 13 nkf.
172
AYALA
ET AL.
concentrations of verapamil, but cell viability was reduced at concentrations of verapamil greater than 20 p.M. A nontoxic verapamil concentration of 10 l&f was chosen for the drug reversal experiments in E. histolytica.
P’M FIG. 2. Sensitivity of clones A and C2(90) to verapamil. Relative growth of clones (0) A and (a) C2(90) was obtained after 96 hr of incubation of trophozoites in the presence of different concentrations of vera-
resistant drugs (Choi et al. 1988). Clone A and C2(90) have similar sensitivity to both daunomycin and actinomycin D. These are two drugs which are often associated with the multidrug-resistant phenotype in mammalian cells. Reversal of drug resistance by verapamil. Verapamil has been shown to reverse the
multidrug-resistant phenotype in mammalian tumor cells and chloroquine resistance in P. falciparum. In order to know whether verapamil was able to reverse the drug resistance in E. histolytica, trophozoites were incubated with the drugs in the presence of verapamil. Verapamil is toxic for most cells at high concentrations and the sensitivity of E. histolytica to verapamil was determined. As can be seen in Fig. 2, the viability of clones A and C2(90) was similar at various
I&
TABLE II for Different Drugs on Clones A and C2(90) EMT
COL (m.W
WO
DAU (IL-W
ACT-D Wfl
Clone
-v
+v
-v
+v
-v
+v
-v
+v
:2,90,
625
0.50 0.30
1.5 1.0
0.45 0.28
50
11.0 6.6
13
13
Nofe. I&,, is the drug concentration which inhibited 50% growth of clones A and C2(90). EMT, emetine; COL, colchitine; DAU, daunomycin; ACT-D, actinomycin D; -V, without verapamil, +V, with 10 pLMverapamil.
Incubation of clones A and C2(90) with emetine in the presence of verapamil decreased the ICso for emetine approximately lo-fold for clone A and loo-fold for clone C2(90) (Fig. 3 and Table II). A decrease in the IC,, for colchicine for both clones was also observed, but it was not as dramatic as it was for emetine. Although there was no difference in the IC,, for daunomycin for clones A and C2(90) without verapamil, incubation in the presence of verapamil reduced the IC,, approximately 5-fold for each clone. The IC,, for actinomycin D was not changed by the addition of verapamil. Emetine accumulation in drug-resistant and drug-sensitive trophozoites. The multi-
drug-resistant phenotype in mammalian cells is characterized by a decreased accumulation of drug in resistant cells. To determine if a similar decreased accumulation of emetine occurred in clone C2(90) of E. histolytica, parasites were incubated in the presence of [3H]emetine. Clone C2(90) exhibited a lower accumulation of the drug compared with clone A (Fig. 4). In multidrug-resistant mammalian cells, differences in accumulation between sensitive and resistant cells are diminished by the addition of verapamil. This was also observed in E. histofytica as can be seen in Fig. 4. After 5 hr, clone C2(90) incubated with emetine alone accumulated 290 fg/cell and with the addition of verapamil accumulated 540 fg/cell. Clone A accumulated 650 fg/cell of emetine in the presence and absence of verapamil after 5 hr incubation. Femtograms of emetine taken up by trophozoites were calculated from the percentage of drug uptake (Fig. 4). DISCUSSION
In this paper we demonstrated that the
E.
histdytica:
MULTIDRUG
TIME
48
72
96
48
72
96
173
RESISTANCE
I
,
I
46
72
96
72
96
(h)
46
40
72
96
TIME 1 h) FIG. 3. Effect of verapamil on drug resistance of E. hisrolytica. Trophozoites (104)of clones A and C2(90) were incubated with different drug concentrations and 10 @I verapamil. Emetine (EMT): (0) no emetine, (A) 0.18 @4, and (m) 1.8 WM. Colchicine (COL): (0) no colchicine, (A) 0.5 mM, and (m) 1.Omk4. Daunomycin (DAU): (0) no daunomycin, (A) 10 PM, and (m) 50 (LM. Actinomycin D (ACT D): (0) no actinomycin, (A) 0.13 nM, and (W) 1.3 nM.
emetine-resistant clone C2(90) of E. histolytica has several characteristics similar to those described for multidrug-resistant mammalian tumors. E. histolytica mutants selected by their resistance to the alkaloid emetine showed cross-resistance to an unrelated drug, colchicine. The C2(90) clone was not cross-resistant to either daunomytin or actinomycin D. Both emetine and colchicine resistance could be reversed by the addition of the calcium channel blocker, verapamil. This is similar to observations in mammalian tumor cells. In the resistant clone C2(90), there was a decreased accu-
mulation of [3H]emetine which was reversed by the addition of verapamil. Both clones A and C2(90) were resistant to killing by actinomycin D at concentrations of 13 ti, which is IOOO-fold higher than that necessary to kill mammalian cells. Resistance was not reversed by the addition of verapamil. Our experiments cannot distinguish whether the lack of sensitivity to actinomycin D was due to impermeability of the cells to the drug or to the lack of target inside the parasite. In this regard, other workers (Vanik et al. 1986) have demonstrated that E. histolytica RNA polymer-
AYALAETAL.
174
%zo
N !m 120
270
sx
Time (min)
FIG. 4. Emetine accumulation in drug-resistant and drug-sensitive trophozoites. Trophozoites of clones A and C2(90) were incubated with 22.5 uM cold emetine and 2.5 &i/ml tritiated emetine with (0, A) or without (0, A) 10 @f verapamil at 37°C. Tritiated emetine accumulation was measured as described under Materials and Methods. Clone A (0,O) clone C2(90)(A, A).
ase activity is inhibited by actinomycin D at a concentration of 40 )&ml. There was no difference in the sensitivity of clones A and C2(90) to daunomycin. However, sensitivity to this drug is increased in the presence of verapamil in both clones. These results are consistent with the existence of a multidrug-resistant phenotype in E. histolytica which is similar to that demonstrated in mammalian tumor cells and recently reported in drug-resistant P. fulciparum (Wilson et al. 1989). Recently, we have reported the identification of multiple 344-bp segments of amebic DNA similar to the human multidrug-resistant gene using primers to conserved regions of the P-glycoprotein and the polymerase chain reaction (PCR) (Samuelson et al. 1990). The amino acid sequences of amebic mdrlike PCR products were 46-5 1% identical to human mdr-1 sequences and 30 to 35% identical to P. fulcipurum (Wilson et al. 1989). The E. histolytica mdr-like gene is
expressed at an increased level in the resistant clone, C2, compared with its expression in clone A. This evidence, together with the physiology presented in this paper, argue for the involvement of a P-glycoprotein-like molecule in drug resistance in E. histolyticu. Drug resistance in clinical isolates of E. histolytica has not yet been described as a major problem, but with increased drug usage it may well emerge as an important health problem. Previous work has shown that laboratory strains of E. histolyticu have differential sensitivity to drugs and drug-resistant parasites can be easily isolated in vitro. If a mechanism of multidrug resistance exists in E. histolyticu, resistance to multiple drugs may arise simultaneously in clinical isolates as has been observed in mammalian tumor cells. ACKNOWLEDGMENTS This work was partially supported by grants from The MacArthur Foundation (U.S.A.) and CONACYT (Mexico). Dr. Orzco was the recipient of a J. Guggenheim award.
REFERENCES BORST, P. 1984. DNA amplification, multidrugresistance and cancer chemotherapy. Nature (Lon-
don)309,580. CHOI, K., CHEN, C., KIUEGLER,M., AND RONINSON, I. B. 1988. An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdrl (P-glycoprotein) gene. Cell 53, 51%529. DANO, K. 1973. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochemical and Biophysics Acta 323, 466-483. DIAMOND, L. S., HARLOW, R., AND CUNNICK, C. C. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Transactions of the Royal Society for Tropical Medicine and Hygiene 12, 431-432. FOOTE,S. J., THOMPSON,J. K., COWMAN,A. F. AND KEMP, D. J. 1989. Amplification of the multidrug resistant gene in some chloroquine-resistant isolates of P. falciparum. Cell 51, 921-930. GILLIN, F. D., AND DIAMOND, L. S. 1978. Clonal growth of Entamoeba histolytica and other species of Entamoeba in agar. Journal of Protozoology 25,
539-543.
E. histolytica: MULTIDRUG RESISTANCE GROSS,P., CROOP,J., AND HOIJSMAN,D. 1986. Mammalian multidrug resistance gene: Complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell 41, 371-380. HAMADA, H., AND TSURUO,T. 1986. Purification of the P-glycoprotein associated with multidrug resistance. Journal of Biological Chemistry 263, 1454 1458. HIGGINS, C. F., HILES, I. D., SALMOND, G. P. C., GILL, D. R., D~WNIE, J. A., EVANS, I. J., HOLLAND, I. B., GRAY, L., BUCKEL, D. D., BELL, A. W., AND HERMODSON,M. A. 1986. A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature (London) 323, 448-450.
KROGSTADT,D. J., GLUZMAN, I. Y., KYLE, D. E., ODUOLA, A. M., MARTIN, S. K., MILHOUS, W. K., AND SCHELINGER,P. H. 1987.Efflux of chloroquine from Plasmodium falciparum: Mechanism of chloroquine resistance. Science 238, 1283-1285. MARTIN, S. K., ODUOLA, A. M. J., AND MILHOUS, W. K. 1987. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235, 899-901. OROZCO, E., HERNANDEZ, F., AND RODRIGUEZ, M. A. 1985. Isolation and characterization of Entamoeba histolytica mutants resistant to emetine. Molecular and Biochemical Parasitology 15, 49-59. PHILLIPS, M. A., AND WANG, C. C. 1987.A Trypanosoma brucei mutant resistant to alfa-difluoromethylomithine. Molecular and Biochemical Parasitology 22, P-17. RIORDAN, J. R., DEUCHARS, K., KARTNER, N., ALON, N., TRENT, J., AND LING, V. 1985a. Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines. Nature (London) 316, 817-819. RIORDAN, J. R., AND LING, V. 1985b. Genetic and biochemical characterization of multidrug resistance. Pharmacology Therapy 28, 51-57.
175
RONINSON, I. B., ABELSOON, H. T., HOUSMAN, D. E., HOWELL, AND VARSHARSKY,A. 1984. Amplification of specific DNA sequences correlates with multidmg resistance in Chinese hamster cells. Nature (London)
309, 626-628.
SAMUELSON, J., AYALA, P., OROZCO, E., AND WIRTH, D. F. 1990. Emetine resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance. Molecular and Biochemical in press. Parasitology, SCOTTO, K. W., BLIEDLER, J. L., AND MELERA, P. W. 1986. Amplification and expression of genes associated with multidrug resistance in mammalian cells. Science 232, 751-755. SKOVSGAARD,T. (1978). Mechanisms of resistance to daunorubicin in Ehrlich ascites tumor cells. Cancer Research 38, 1785-1791. TSURUO,T., IIDA, H., TSUKOGOSHI,S., AND SAKURAI, Y. 1981. Overcoming the vincristine resistance of P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Research 41, 19761979. TSURUO,T., IIDA, H., TSUKAGOSHI,S., AND SAKURAI, Y. 1982. Increased accumulation of vincristine and adriamycin in drug resistant P399 tumor cells following incubation with calcium antagonists and calmodulin inhibitors. Cancer Research 42, 47304733. VANIK, J. M., DETKE, S., AND ALBACH, R. A. 1986. Partial characterization of DNA-dependent RNA polymerases from Entamoeba histolytica. Archives de Investigation Medica 17, Suppl. 1, 101-106. WILSON, C. M., SERRANO,A. E., WASLEY, A., BoGENSCHUTZ,M. P., SHANKAR, A. H., AND WIRTH, D. F. 1989. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 244, 1184-l 186. Received 26 October 1989; accepted with revision 6 February 1990.
