J Cancer Res Clin Oncol (1995) 121:407-412
9
Springer-Verlag 1995
K. Schwabe 9 G. Steinheider 9 A. Lawen R. Traber 9 A. Hildebrandt
Reversal of multidrug resistance by novel cyclosporinA analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro
Received: 29 December 1994/Accepted: 14 March 1995
Abstract It was shown that cyclopeptolide SDZ 214-103 (10 ~tM) is more active in rhodamine-123 accumulation in actinomycin-D-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 gM), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 gM) and cyclosporin A (10 gM) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporinA was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used. Key words Multidrug resistance 9 Chemosensitizer Cyclosporins 9 Rhodamine-123 Abbreviations M D R multidrug resistance 9 Pgp-170 P-glycoprotein with a molecular mass of 170 kDa " D-Hiv D-2-hydroxyisovaleric acid
K. Schwabe ( ~ ) 9G. Steinheider 9A. Hildebrandt Bundesinstitut fiir Arzneimittel und Medizinprodukte, Seestr. 10-11, D-13353 Berlin, Federal Republic of Germany A. Lawen Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia R. Traber Sandoz Pharma Ltd., Pr/iklinische Forschung, CH-4002 Basel, Switzerland
Introduction
The resistance to antineoplastic drugs is mediated by diverse mechanisms which can function alone as well as synergically. The main mechanisms recognised are (a) the overexpression of the mdrl gene product Pgp170, (b) the overexpression of the gene encoding the multidrug-resistance-associated protein, (c) the overexpression of the detoxifying enzymes glutathione S-transferase and glutathione peroxidase and (d) the alteration of the DNA-associated nuclear enzyme topoisomerase II. In this report we will deal with the mechanism mentioned first. In multidrug resistance (MDR) the tumor cells become cross-resistant to a variety of chemotherapeutics such as anthracyclines, Vinca alkaloids, podophyllotoxin derivatives, actinomycin D, colchicine and mitoxantrone. This MDR phenomenon has been correlated to the overexpression of the mdrl gene, which encodes a transmembrane glycoprotein called P-glycoprotein (P = permeability) with a molecular mass of 170 kDa (Pgp-170). This protein acts as an ATP-dependent efflux pump, transporting a number of apparently unrelated organic compounds. Several classes of agents have been demonstrated to overcome Pgp-170-mediated multidrug resistance in experimental tumor systems (for review see Ford and Hair 1990); among them are calcium channel blockers such as verapamil, nifedipine, and dexniguldipine, the acridonecarboxamide derivative GF120918, the immunosuppressive drugs FK 506 and cyclosporin A, the non-immunosuppressive cyclosporin D derivative SDZ PSC 833, and the hydrophobic cyclopeptolide SDZ 280-446. These compounds are able to restore, partly or in some cases completely, the normal sensitivity of tumor cells to antineoplastic agents that are substrates for P-glycoprotein. It is assumed that these chemosensitizers act by interfering with Pgp-170, inhibiting the drug efflux (Tamai and Safa 1990; Ryffel et al. 1991).
408
The cyclosporin derivative SDZ PSC 833, the cyclopeptolide SDZ 280-446, and the acridonecarboxamide derivative GF120918 are reported to be the most potent resistance-modulating compounds known so far (Gave'riaux et al. 1991; Pourtier-Manzanedo et al. 1992; Loor et al, 1992; Hyafil et al. 1993; Emmer et al. 1994). They are in vitro about two orders of magnitude more active than other chemosensitizers. Because of the high capacity of cyclosporin analogues and cyclopeptolides to reverse MDR we were interested in screening novel compounds of this chemical class with respect to their chemosensitizing activity. In 1988, a novel cyclosporin-related peptolide, with several substitutions compared with cyclosporin A, called SDZ 214-103, was discovered. It is produced by the fungus Cylindrotrichum oligospermum (Corda) bonorden (Dreyfuss et al. 1988). The main structural difference is a 2-hydroxy acid instead of an amino acid in position 8. It can be designated as [Thr 2, Leu s, D-Hiv s, Leul~ This novel drug exhibits properties similar to those of cyclosporin A, including immunosuppressive, anti-inflammatory, anti-fungal and anti-parasitic activities, SDZ 214-103 is like cyclosporinA (Lawen and Zocher 1990) produced by a multifunctional enzyme, called peptolide SDZ 214 103 synthetase (Lawen et al. 1991). Here we report on the ability of the peptolide SDZ 214-103 and some novel biosynthetic cyclosporins (Lawen et al. 1989; Lawen and Traber 1993) to reverse MDR using doxorubicin(ADR)-resistant F4-6/ADR Friend erythroleukemia cells and actinomycin-D-resistant CCRF/ACTD400 human lymphoma cells. The effects of the cyclosporins and the SDZ 214-103 were tested by the rhodamine-123 uptake assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay.
Materials and methods
Chemosensitizers The cyclosporin A anaIogues were prepared, purified and characterized as described in the literature (Lawen et al 1989; Lawen and Traber 1993). Cyclopeptolide SDZ 214-103 was produced using the multifunctionaI peptolide SDZ 214-103 synthetase (Lawen et al. 1991). The other chemosensitizers were commercially available. The chemosensitizers were dissolved in dimethylsulfoxide and diluted 100-fold by addition to the test mixture.
Chemicals 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) was used as a 5 mg/ml solution in RPMI-1640 medium without phenol red. The M T T solution was kept in the clark at 4 ~ Rhodamine-123 (Eastman Kodak Co, Rochester, N.Y., USA) was prepared as a stock solution of 1 mg/ml in distilled water.
M T T cytotoxicity assay The ability of various chemosensitizers to overcome M D R were compared in the doxorubicin-resistant F4-6/ADR cells by the MTT cytotoxicity assay. This assay was used as described by P a g e t al. (1988). The method relies on the ability of mitochondrial dehydrogenase enzymes in the residual living cells to convert MTT to a purple formazan precipitate. Cell growth was measured in triplicate cultures. The cells (10000) were grown in 200 gl medium in round-bottomed microtiter plates for 24 h in the presence of various concentrations of the drugs employed. Samples (20 gl) of MTT solution were then added to each well and the plate was incubated at 37~ for 4 h in a humidified atmosphere. The supernatant was removed by pipetting, 200 gl dimethylsulfoxide was added and the plate was agitated for a minimum of 15 min to solubilize formazan crystals. The absorbance at 540 nm was measured after 60 min in a Dynatech MR 7000 plate reader. The cyt0toxic effects of various concentrations of doxorubicin in the presence of a chemosensitizer (10 gM) were calculated using the formula cytotoxicity (%) = (Achemo --
Acombi/Achemo)•
100
where Achemo is the absorbance in the presence of a chemosensitizer alone and Acombi is the absorbance in the presence of the chemosensitizer and doxorubicin. Cultivation of the cells in the presence of doxorubicin alone was used as control for potential cytotoxicity. No cytotoxicity was found in the experimental range of concentrations.
Tumor cell lines Rhodamine-123 uptake assay Two pairs of parental and multidrug resistant (MDR) tumor cell lines were used. (a) The Friend erythroleukemia cell line F4-6 was kindly provided by Dr. W. Ostertag, (Heinrich-Pette-Institut ffir Experimentelle Virologic, Hamburg, Germany) (Ostertag et al. 1972) and the M D R subline F4-6/ADR was generated by inducing multidrug resistance in a subclone of the parent line with doxorubicin as the selecting agent (resistance factor = 100). The cell line F4-6/ADR expresses Pgp-170 as demonstrated by immunocytochemistry (unpublished). (b)Actinomycin-D-resistant human lymphoma cells (CCRF/ACTD400) (resistance factor = 400) and their sensitive counterparts (CCRF/CEM) were kindly provided by Dr. V. Gekeler (Gekeler et al. 1990). The cells were propagated in suspension cultures in Eagle's minimal essential medium (MEM alpha, Gibco) supplemented with 10 % (v/v) fetal calf serum, 2 mM L-glutamine, and penicillin/streptomycin at 37~ inhumidified 5 % CO 2 atmosphere.
The fluorescent vital dye rhodamine-123 specifically stains sensitive cells, whereas it is effluxed from MDR cells as a P-glycoprotein substrate (Neyfakh 1988; Bucana et al. 1990). Chemosensitizers are able to restore rhodamine-123 retention within M D R cells (Lampidis et al. 1985). Samples containing (1-2) • 1 0 6 cells in a total volume of 1 ml were incubated in the presence of the chemosensitizer (10 gM) for 90 min at 37 ~ in phosphate-buffered saline (PBS). Then 10 gl rhodamine123 was added and the cells were incubated for a further 30 rain. Cell sediments were then washed twice with PBS. Rhodamine-123 retention was examined by fluorescence microscopy ()~,~ = 515 nm, 2~x = 488 nm; Mikroskop-Photometer MPM200, Zeiss, Germany). The adaptation of the microscope for measurement of cell-associated fluorescence was done by using a single arbitrarily selected bright cell from the sensitive cell line as a calibration standard. The fluorescene intensity of 20 cells from each sample was measured.
409 The data are expressed as a percentage of the fluorescence intensity of resistant cells in the presence of cyclosporin A, which was arbitrarily defined as 100 %. The experiments with each chemosensitizer were done at least three times.
I
Statistical analysis Statistical significance was analyzed by the two-tailed t-test for unpaired samples. When P was less than 0.05 the means were considered significantly different.
Results g
The effects of chemosensitizers on the uptake of rhodamine-123
g
Sensitive CCRF/CEM and actinomycin-D-resistant (CCRF/ACTD400) human lymphoma cells The MDR-reversing effects of the various chemosensitizers were examined in the paired cell lines C C R F / C E M and CCRF/ACTD400, which are sensitive and resistant to actinomycin D on the basis of the rhodamine-123-uptake assay. In the absence of rhodamine-123 exposure, neither cell line showed any intrinsic fluorescence. After incubation with rhodamine-123, 90 % - 9 5 % of the sensitive C C R F / C E M cells were fluorescent, whereas only 1 % - 5 % of the resistant CCRF/ACTD400 cells were fluorescent in the absence of a chemosensitizer. The ability of the various chemosensitizers to restore the fluorescence was examined using 10 gM of each (Fig. 1). There was strong fluorescence after incubation with both cyclosporinA and peptolide SDZ 214 103, whereas all other drugs tested had a much smaller effect (Fig. 1). The fluorescence uptake with peptolide SDZ 214-103 was significantly greater than that for cyclosporin A. Surprisingly, nifedipine and verapamil, at the concentration of 10 gM, were unable to increase the uptake of rhodamine-123 in this resistant cell type (Fig. 1).
Sensitive Friend erythroleukemia cell line F4-6 and doxorubicin-resistant F4-6/ADR cells The MDR-reversing effects of the various chemosensitizers were examined in the paired cell lines F4-6 and F4-6/ADR, which are sensitive and resistant to doxorubicin according to the rhodamine-123-uptake assay. In the absence of rhodamine-123 exposure, neither cell line showed any intrinsic fluorescence. After incubation with the dye, 96 % 99 % of the sensitive F4-6 cells were fluorescent, whereas only 1 % - 5 % of the resistant F4-6/ADR cells were fluorescent in the absence of a chemosensitizer.
~
~
Fig. 1 Uptake of rhodamine-123 into actinomycin-D-resistant human lymphoma cells (CCRF/ACTD400) under the influence of multidrug-resistance (MDR) modulators (10 gM). Fluorescence of rhodamine-123 in the cells in the presence of cyclosporin A (10 gM) = 100 %. Data are shown as the mean _+ standard deviation (n = 3-8). *Statistically significant (P < 0.01). Derivative 1 EaIles, MeaIle 11] cyclosporinA, derivative 2 [D-Abu 8] cyclosporin A, derivative 3 [Nva s, MeNva 11] cyclosporin A, derivative 4 FGly8] cyclosporin A, cyclosporin H [D-MeVal 1~] cyc!osporin A (non-immunosuppressive), cyclosporin V [Abu 7] cyclosporin A (the immunosuppressive activity is one order of magnitude lower than that of cyclosporin A)
Surprisingly, the strong fluorescence of sensitive F46 cells can be further increased in the presence of cyclosporin A. The fluorescence of individual cells in the presence and in the absence of 10 gM cyclosporin A showed a broad range of intensity. Interestingly, in a few cells cyclosporin A seemed to be unable to increase the rhodamine-123 uptake. The ability of the various chemosensitizers to restore the fluorescence in F4-6/ADR cells was examined using 10 gM of each (Fig. 2). When tested on F4-6/ A D R cells, SDZ 214-103 was shown to be comparatively active with cyclosporin A in rhodamine-123 accumulation. The non-immunosuppressive substances cyclosporin H and SDZ 90-215 are comparatively weak M D R modulators. Surprisingly, nifedipine shows only marginal effects and verapamil was totally inactive (Fig. 2).
410
Fig. 2 Uptake of rhodamine-123 into doxorubicin(ADR)-resistant F4-6/ADR cells under the influence of M D R modulators (10 gM). Fluorescence of rhodamine-123 in the presence of cyclosporin A (10gM = 100%. Data are shown as the mean + standard deviation (n = 3-8). CyclosporinH [D-MeVal ~1] cyclosporin A (non-immunosuppressive), SDZ 90 215 cyclic decapeptide (nonimmunosuppressive) (Loor et al. 1992; Lee and Lawen 1993; Emmer et al. 1994)
MTT cytotoxicity assay The MDR-reversing effects of various chemosensitizers were compared in the doxorubicin-resistant F4-6/ADR cells by the MTT cytotoxicity assay (Fig. 3). At the lowest concentration of doxorubicin (25 ng/ml) cyclosporin A has no MDR-modulating effects while SDZ 214-103 shows a small effect. At a concentration of 50 ng/ml doxorubicin about 30 % of the cells were killed in the presence of SDZ 214-103. At higher concentrations (250 ng/ml) doxorubicin Can reduce the number of living cells through the influence of cyclosporinA or SDZ 214-103 by about 80%. Verapamil is nearly equipotent and SDZ 90-215 has only marginal effects. Cyclosporin H is without any effect. At the highest concentration (1000ng/ml) of doxorubicin in the presence of SDZ 214 103, cyclosporin A or verapamil the cells were killed to the same extent (about 95 % killing). The effects of SDZ 90-215 and cyclosporin H are considerably lower (about 60 % and 20 % killing resp.). In summary, the MDR-modulating effect of SDZ 214-103 is comparable with that of cyclosporin A. The MDR-reversing effect of verapamil is comparable with SDZ 214-103 and cyclosporin A at the higher concentrations of doxorubicin (250-1000 ng/ml).
Q .-- .~
~.1 7 6 ~§
+
~+~
"|8 §
Fig. 3 MTT cytotoxicity assay: cytotoxic effects of various concentrations of doxorubicin (ADR: 25, 50, 250 and 1000 ng/ml) on doxorubicin-resistant F4-6/ADR cells under the influence of various M D R modulators (10 gM). The cytotoxicity of the modulator alone was taken into account (see Materials and methods). No cytotoxicity was observed with doxorubicin alone in concentrations up to 1000 ng/ml. Data are shown as the mean _+ standard deviation
(n = 3-8)
Discussion We made an attempt to compare various MDR-modulating agents by two well-known methods in parallel. The rhodamine-123-uptake assay and the MTT cytotoxicity assay have shown that the newly biosynthesized cyclopeptolide SDZ 214-103 has a high MDR-modulating capacity comparable to that of cyclosporin A. In the actinomycin-D-resistant human lymphoma cell system (CCRF/ACTD400) the influence of SDZ 214-103 on rhodamine-123 accumulation is significantly higher than that of cyclosporin A. From this finding it can be concluded that changes in the four amino acids at positions 2, 5, 8 and 10 of the cyclosporin A ring did not lead to a loss of the MDRmodulating effect. It is conceivable that further substitutions in the immunosuppressivity-determining positions of this ring system (Sadeg et al. 1993) may uncouple the immunosuppressive properties to give novel interesting MDR-modulating agents. The effects
411 of the other tested novel cyclosporin derivatives are negligible in the cell systems studied. The a m i n o acid substitutions in these non-active derivatives m a y also be considered, however, in the further design of new chemosensitizers. There was a difference in the results between the applied m e t h o d s when one of our cell lines was used (F4-6/ADR): at equipotent m o d u l a t i n g doses of v e r a p a m i l (10 btM) and cyclosporin A (10 gM) in the M T T assay for d o x o r u b i c i n cytotoxicity, cyclosporin A was efficient in the r h o d a m i n e - 1 2 3 - u p t a k e assay while v e r a p a m i l was not active when identical incubation times were used. In this context it is of interest that a lack of activity of 10 p M v e r a p a m i l on r h o d a m i n e 123 a c c u m u l a t i o n but a significant effect of cyclosporin A in M D R - P 3 8 8 cells has also been seen by others ( P o u r t i e r - M a n z a n e d o et al. 1992). The controversal results o b t a i n e d m a y be due to the fact that the M T T cytotoxicity assay reflects accumulating toxic effects in the cell, whereas the r h o d a m i n e - 1 2 3 - u p t a k e assay shows the actual effectiveness of a m o d u l a t o r in inhibiting the t r a n s p o r t function of Pgp-170 for r h o d a m i n e 123. It is k n o w n f r o m the literature that the molarities o f c y c l o s p o r i n A and v e r a p a m i l required to allow a tenfold sensitization of the Pgp-170-expressing M D R P388 cells to d o x o r u b i c i n are 0.3 p M and 3.9 p M respectively (Boesch et al. 1991). These differences in the molarities of a b o u t one order of m a g n i t u d e might reflect differences in the affinities to Pgp-170 between cyclosporin A and v e r a p a m i l and m i g h t explain the differences observed in the actual activity of b o t h m o d u l a t o r s in inhibiting the t r a n s p o r t function of Pgp170 for rhodamine-123. Interestingly, cyclosporin A was able to increase the high u p t a k e of rhodamine-123 in the sensitive F4-6 cells also. This effect has also been observed b y others ( C h a m b e r s et al. 1989; G a v e ' r i a u x et al. 1989; Boer et al. 1994). However, in a few cells cyclosporin A seems to be unable to increase the rhodamine-123 uptake. The reason for the influence of cyclosporin A on the m a j o r i t y of wild-type cells is still u n k n o w n . Acknowledgements We are indebted to Dr. M. Rudolph and Prof. V. Wunderlich, Berlin-Buch, for their immunocytochemical studies with the F4-6/ADR cells. We thank Mrs. G. Oelze for her excellent technical assistance and Dr. Merrill Rowley (Monash University) for critically reading the manuscript.
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