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Effects of biochemical modulation of drug combinations directed at the ribonucleotide reductase site on leukemia L1210 cell growth in culture
EFFECTS OF BIOCHEMICAL MODULATION OF DRUG COMBINATIONS DIRECTED AT THE RIBONUCLEOTIDE REDUCTASE SITE ON LEUKEMIA L1210 CELL GROWTH IN CULTURE ATSUSHi SATO, GAY L. CARTER, PATRICIA E. BACON and JOSEPH G. CORY Department of Biochemistry, College of Medicine, University of South Florida, Tampa, Florida 33612
INTRODUCTION
During the S-phase of the cell cycle when DNA synthesis is occurring, a balanced supply of the four dNTP's (dATP, dGTP, dCTP and dTTP) is required if the replication of DNA is to be completed and cell division is to follow. The pathway by which the dNTP's are formed is shown in Figure 1. The reaction from N D P to 2 ' - d N D P is the rate-limiting step in the process leading to t h e formation of dNTP's. This step is catalyzed by the enzyme, ribonucleotide reductase (EC 1.17.4.1, nucleoside diphosphate reductase). The activity of ribonucleotide reductase has been shown to be extremely low in normal liver (1) or essentially unmeasurable (2). The level of activity of reductase has been shown to correlate with the growth rate of chemicallyinduced hepatomas (1, 2). This enzyme also showed the greatest increase in enzymatic activity in the neoplastic process (!). Measurements of the intracellular concentrations of the substrates (ribonucleoside diphosphates) demonstrated that they were not limiting for this reaction (3). Since ribonucleotide reductase catalyzes such a key metabolic reaction and of the enzymes involved in nucleotide metabolism has the lowest cellular activity (4), this metabolic site offers an excellent target for antineoplastic drugs. Further, the enzyme from mammalian cells has been shown to consist of two nonidentical components (5-8). The two components making up the active enzyme in Ehrlich tumor cells were specifically and independently inhibited/ inactivated (8). Utilizing highly-purified ribonucleotide reductase, it was shown that combinations of ribonucleotide reductase inhibitors directed at the individual components resulted in synergistic inhibition of enzyme activity
(10). In the current studies, the effects of combinations of ribonucleotide reductase inhibitors (e.g. deoxyadenosine/hydroxyurea or deoxyadenosine/ IMPY) on the growth of L I210 cells in culture are reported. To potentiate the 259
260
ATSUSHISATO, etaL de novo NUCLEOTIDE SYNTHESIS
a
,NMP
b
"NDP
c
'- dNDP
d
-dNTP
FIG. 1. Pathway for formation of deoxyribonucleosidetriphosphates. The enzymescatalyzing these reactions are: (a) enzymes for de novo synthesis of purine and pyrimidine nucleoside monophosphates; (b) nucleoside monophosphate kinase; (c) ribonucleotide reductase; and (d) nucleoside diphosphate kinase. effects of each of these agents, other drugs were used to modulate the effects of deoxyadenosine and I M P Y or hydroxyurea. E H N A was used to inhibit the deamination of deoxyadenosine (11) while Desferal was used to potentiate the effects of hydroxyurea (12, 13) or IMPY (13). M A T E R I A L S AND METHODS G r o w t h o f l e u k e m i a L1210 cells. L1210 cells were grown in suspension
culture in R P M I 1640 culture medium which was supplemented with 10% horse serum, NaHCO3 (2 g/l) and antibiotic (Gentamicin sulfate, 50 mg/l). Aliquots of the cell suspensions ( l ml) were removed. The cells were collected by centrifugation, washed with phosphate-buffered saline and counted in a Biophysics Cytograph. The cultures were initially set up on day-zero with an initial cell concentration of 1.2 to 2 x 105/ml. The drugs, alone or in combination, were added only at this time. Dr. Richard G. Moran, Children's Hospital of I.os Angeles, CA, kindly supplied the L 1210 cell line used in these studies. The data presented in the Table l were calculated from the cell numbers at day 4 of the growth curve. The data in Tables 2-7 were calculated from the cell numbers at day 5 of the growth curve. The % of control values were calculated according to the equation: % of Control = Cells/ml (treated) - Cells/ml (seeded) x 100 Cells/ml (control) - CeUs/ml (seeded) The values listed as "calculated" were determined according to the methods of Webb (14) to describe whether the combinations of drugs yielded synergistic inhibition of cell growth. When the "calculated" value was larger than the fraction of cell growth observed, the combination resulted in synergistic inhibition; when the observed and calculated values were the same the effects were additive; if the "calculated" value was smaller than the observed fraction of cell growth, then the combination resulted in antagonistic inhibition of cell growth. Materials. Pyrazolo-imidazole (IMPY) was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute, through the assistance of Dr. V. L Narayanan. E r y t h r o - 9 - ( 2 - h y d r o x y - 3 - n o n y l ) adenine (EHNA) was a gift from Dr. G. Elion, Burroughs-Wellcome, Research
MODULATION OF DRUG COMBINATIONS Triangle,
N.
C.
Hydroxyurea,
deoxyadenosine
and
261
Gentamicin
were
p u r c h a s e d f r o m S i g m a C h e m i c a l C o . , St. L o u i s , M O . R P M I 1640 c u l t u r e medium, sodium bicarbonate and horse serum were purchased from Grand Island B i o l o g i c a l C o . , G r a n d Island, NY. D e s f e r a l was a gift f r o m C I B A P h a r m a c e u t i c a l C o . , S u m m i t , N. d. RESULTS
Modulation of Deoxyadenosine Inhibition of Leukemia L1210 Cell Growth in Culture by EHNA D e o x y a d e n o s i n e is r a p i d l y d e a m i n a t e d by a d e n o s i n e d e a m i n a s e w h i c h p r e v e n t s the b u i l d u p o f the i n t r a c e l l u l a r c o n c e n t r a t i o n o f d A T P , a n e g a t i v e e f f e c t o r o f r i b o n u c l e o t i d e r e d u c t a s e activity. In the p r e s e n c e o f E H N A , a p o t e n t i n h i b i t o r o f a d e n o s i n e d e a m i n a s e , d e o x y a d e n o s i n e is s h u n t e d t o w a r d d A M P f o r m a t i o n a n d s u b s e q u e n t l y to d A T P . T h e d a t a in T a b l e i s h o w the effects o f v a r i o u s c o n c e n t r a t i o n s of E H N A o n d e o x y a d e n o s i n e t o x i c i t y o f L I 2 1 0 g r o w t h in culture. C o n c e n t r a t i o n s o f d e o x y a d e n o s i n e (0.025 mM o r 0.05 mM) had essentially n o effect on L I 2 1 0 cell g r o w t h w h e n a d d e d to the c u l t u r e m e d i u m . In fact, d e o x y a d e n o s i n e at a c o n c e n t r a t i o n o f 0.150 mM had e s s e n t i a l l y n o effect on L I210 cell g r o w t h ( d a t a n o t shown). E H N A , a l o n e , at c o n c e n t r a t i o n s o f 0.005 mM o r 0.010 mM had o n l y a slight i n h i b i t o r y effect o n the g r o w t h o f L I210 cells. H o w e v e r , w h e n c o m b i n a t i o n s o f d e o x y a d e n o s i n e a n d E H N A were a d d e d , a m a r k e d s y n e r g i s m o f the i n h i b i t i o n o f L I 2 1 0 cell TABLE I. MODULATION OF DEOXYADENOS1NE INHIBITION OF LEUKEMIA LI210 CELL GROWTH BY EHNA Drug treatment Control dAdo, 0.025 mM 0.050 mM EHNA, 0.001 mM 0.0025 mM 0.005 mM 0.010 mM dAdo, 0.025 mM + EHNA, 0.0025 mM 0.005 mM 0.010 mM dAdo, 0.050 mM + EHNA, 0.001 mM 0.0025 mM 0.005 mM
Cell growth (% of control)*
Calculated (al x a2 x 100)1
100 93 86 I00 86 72 66 79 48 5 72 59 0
80 67 61 86 74 62
*The cell number used in this Table is for day 4; the cell number in the control was 1.6 x l06 cells/ml. The % of control value was calculated as described in the 'Methods' section. 1"a~x a2 = (I-i~) (I-i2); where it and i2 are the fractional inhibitions caused by the presence of Ii or 12 alone (14).
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A T S U S H I S A T O , et aL
growth was observed. This was especially noticeable with the combinations dAdo, 0.025 m M / E H N A , 0.010 mM and dAdo, 0.050 m M / E H N A , 0.005 mM. From the inhibition of cell growth caused by each drug alone, the calculated inhibition to be expected in the combinations was 39 and 38% and yet the observed inhibition of cell growth was 95 and 100% for these combinations of d A d o / E H N A . These data indicated that not only could synergistic combinations be generated, but that the concentrations of both components were important since only additive effects were seen at the lower concentrations of each drug.
Modulation of Hydroxyurea Inhibition of L1210 Cell Growth by Desferal It has been shown that the inhibitions of ribonucleotide reductase activity (12, 13) and incorporation of thymidine into D N A by hydroxyurea were increased by the presence of Desferal (12). The effect of various concentrations of hydroxyurea on L1210 cell growth was studied. As seen in Table 2, 0.20 mM hydroxyurea caused a decrease in cell growth of 96%. However, the concentration of hydroxyurea required to inhibit cell growth to this extent could be decreased to 0.05 mM when Desferal (0.10 mM) was added in combination. The combinations of hydroxyurea and Desferal gave synergistic effects at the concentrations studied. The same extent of inhibition of cell growth could be achieved at 0.05 mM hydroxyurea, 0.10 M Desferal as with the higher concentration of hydroxyurea (0.10 mM) and 0.10 mM Desferal. There was essentially complete inhibition (99%) of LI210 cell growth at these lower hydroxyurea concentrations if Desferal was present.
T A B L E 2. M O D U L A T I O N O F H Y D R O X Y U R E A I N H I B I T I O N O F L E U K E M I A L I 2 1 0 C E L L G R O W T H BY D E S F E R A L
Drug t r e a t m e n t Control H y d r o x y u r e a , 0.05 mM 0.10 mM 0.20 mM Desferal, 0.02 mM 0.10 mM HU, 0.05 mM + Df, 0.02 mM HU, 0.05 mM + Df, 0.10 mM HU, 0.10 mM + Df, 0.10 mM
Cell g r o w t h (% of control)*
Calculated (al x a2 x 100)~"
100 80 5l 4 87 54 49 I 1
70 43 28
*The cell n u m b e r used in this Table is for day 5; the cell n u m b e r in the control was 1.8 x 106 c e l l s / m l . The % of control value was calculated as described in the " M e t h o d s " section. 1-al x a2 -- (1-i~) ( I-i2); whe re il and i2 are the fractional inhibitions caused by the presence of I~ or 12 alone (14).
MODULATION OF DRUG COMBINATIONS
263
Modulation of I M P Y Inhibition of L1210 Cell Growth in Culture by Desferal Earlier studies had shown that the i n h i b i t i o n of highly-purified ribonucleotide reductase by 1MPY could be m a r k e d l y potentiated by ironchelating agents such as Desferal, E D T A or 8 - h y d r o x y q u i n o l i n e (13). Studies were carried out to determine whether Desferal could potentiate the effects of I M P Y on the growth of Ll210 cells in culture. The data in Table 3 show the effects of I M P Y and Desferal alone and in c o m b i n a t i o n . At the c o n c e n t r a tions of I M P Y (0.25 mM) a n d Desferal (0. l0 M) used 46 and 39% i n h i b i t i o n of cell growth, respectively, were observed. When these agents were used in c o m b i n a t i o n and at these c o n c e n t r a t i o n s a marked synergistic effect was observed (96% inhibition). W h e n the c o n c e n t r a t i o n of Desferal was lowered from 0. l0 mM to 0.02 m i the i n h i b i t i o n observed was only additive.
TABLE 3. MODULATION OF IMPY INHIBITION OF LEUKEMIA LI210 CELL GROWTH BY DESFERAL Drug treatment Control IMPY, 0.10 mM 0.25 mM Desferal, 0.02 mM 0. I0 mM IMPY, 0.25 mM + Df, 0.02 mM IMPY, 0.10 mM + Df, 0.10 mM IMPY, 0.25 mm + Df, 0.10 mM
Cell growth (% of control)*
Calculated (az × a2 × 100)~"
100 87 54 87 61 48 46 4
47 53 33
*The cell number used in this Table is for day 5; the cell number in the control was 1.7 × 106 cells/ml. The % of control value was calculated as described in the Methods section. ~al × a2 = ( I-ij ) ( 1-i2);where it and i2 are the fractional inhibitions caused by the presence of Ii or 12 alone (14).
Effect of Modulators on the Inhibition of L1210 Cell Growth by the Combination of I M P Y Plus Deoxyadenosine The effects of the c o m b i n a t i o n s of d e o x y a d e n o s i n e and I M P Y on LI210 cell growth were studied. Because of the rapid d e a m i n a t i o n of deoxya d e n o s i n e by a d e n o s i n e deaminase, E H N A was utilized to protect deoxyadenosine. Further, the i n h i b i t i o n of cell growth by 1M PY was potentiated by Desferal (Table 3). As seen from the data in Table 4, at c o n c e n t r a t i o n s of d e o x y a d e n o s i n e (0.015 raM) and I M P Y (0.19 mM), this c o m b i n a t i o n did not result in the synergistic i n h i b i t i o n of cell growth. If a n y t h i n g , it appeared to be slightly antagonistic. The c o m b i n a t i o n of d e o x y a d e n o s i n e / I M P Y / D e s f e r a l also was slightly antagonistic. On the other hand, the c o m b i n a t i o n of d e o x y a d e n o s i n e / E H N A / I M P Y resulted in a slight synergistic i n h i b i t i o n of
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ATSUSHI SATO, et al. TABLE 4. E F F E C T OF M O D U L A T O R S ON THE INHIBITION OF L E U K E M I A LI210 CELL G R O W T H BY THE C O M B I N A T I O N OF IMPY PLUS DEOXYADENOSINE
Drug treatment* Control dAdo I M PY dAdo + IMPY dAdo/EHNA IMPY/Df dAdo + I M P Y / D f d A d o / E H N A + IMPY d A d o / E H N A 4- I M P Y / D f
Cell growth (% of control)~"
Calculated (at x a2 x 100)*
100 94 59 71 77 71 71 33 0
55 67 45 55
*The concentrations of deoxyadenosine (dAdo), IMPY, EHNA and Desferal (Df)were 0.015 raM, 0.19 mM, 0.005 mM, respectively. tThe cell number used in this Table is for day 5; the cell number in the control was 1.9 x 106 cells/ml. The % of control value was calculated as described in the Methods section. *al × a2 --- (I-i~) ( I-i2); whe re il and i2 are the fractional inhibitions caused by the presence of II or 12 alone (14).
Li210 cell growth. However, when all four drugs were used in combination ( d e o x y a d e n o s i n e / E H N A 4- IMPY/Desferal) a strong synergism which resulted in the complete inhibition of cell growth was obtained. From a comparison of the three-drug combinations, it appeared that E H N A was more important as a modulator than was Desferal. As shown in Table 5, the concentration of deoxyadenosine could be lowered still further from 0.015 mM to 0.005 mM if the concentration of one of the other components were increased (in this case, Desferal) and the synergistic combination still be TABLE 5. E F F E C T OF C O M B I N A T I O N S OF D E O X Y A D E N O S I N E , EH N A A N D IMPY ON L E U K E M I A LI210 CELL G R O W T H
Drug combinations Control A. IMPY, 0.19 mM/Df, 0.020 mM B. 1MPY, 0.19 mM/Df, 0.040 mM C. dAdo, 0.005 raM/EHNA, 0.005 mM D. dAdo, 0.015 mM/EHNA, 0.005 m i A+ C A+ D B+C B+ D
Cell growth (% of control)*
Calculated (al x a2 × 100)~
100 71 71 77 77 21 0 0 0
55 55 55 55
*The cell number used in this Table is for day 5; the cell number in the control was 1.9 × 106 cells/ml. The % of control value was calculated as described in the Methods section. ~al x a2 = ( l - i 0 ( 1-i2); where il and i2 are the fractional inhibitions caused by the presence of I~ or 12 alone (14).
MODULATION OF DRUG COMBINATIONS
265
m a i n t a i n e d . These data in c o n j u n c t i o n with the data presented in Table 4 show the i m p o r t a n c e not only of the agents, d e o x y a d e n o s i n e a n d I M P Y , but also the i m p o r t a n c e of the m o d u l a t o r s in the c o m b i n a t i o n s .
Effect of Modulators on the Inhibition of LI210 Cell Growth by the Combinations of Deoxyadenosine and H vdroxyurea As shown in Table 6 d e o x y a d e n o s i n e a n d h y d r o x y u r e a in c o m b i n a t i o n do not result in the synergistic i n h i b i t i o n of cell growth. However, if E H N A is added to protect d e o x y a d e n o s i n e from d e a m i n a t i o n a synergistic c o m b i n a tion, consisting of d e o x y a d e n o s i n e / E H N A / h y d r o x y u r e a can be generated. O n the other hand, if E H N A is used to protect d e o x y a d e n o s i n e from d e a m i n a t i o n and Desferal is used to potentiate the effects of h y d r o x y u r e a , not only is a synergistic i n h i b i t i o n of cell growth achieved, but the c o n c e n t r a t i o n s of the agents required can be markedly reduced. In c o m p a r i s o n with the data in Table 6, the d e o x y a d e n o s i n e c o n c e n t r a t i o n could be lowered from 0.055 mM to 0.015 raM. Again as seen with the c o m b i n a t i o n s which included I M P Y , the three-drug c o m b i n a t i o n d e o x y a d e n o s i n e / E H N A / h y d r o x y u r e a was a better c o m b i n a t i o n t h a n d e o x y a d e n o s i n e / h y d r o x y u r e a / D e s f e r a l in t e r m s of i n h i b i t i n g L 1210 cell growth (Table 7). At these low c o n c e n t r a t i o n s of d e o x y a d e n o s i n e (0.015 mM) it appears very critical that E H N A be present.
TABLE 6. EFFECT OF COMBINATIONS OF DEOXYADENOSINE, EHNA AND HYDROXYUREA ON LEUKEMIA LI210 CELL GROWTH Drug treatment Control dAdo, 0.055 mM H U, 0.055 mM HU, 0.110 mM EHNA, 0.002 mM dAdo + HU, 0.055 mM dAdo + HU, 0.110 mM dAdo/EHNA dAdo/EHNA + HU, 0.055 mM dAdo/EHNA + HU, 0.110 mM
Cell growth (% of control)
Calculated (at × a2 x 100)I
100 78 62 37 95 56 40 45 7 1
48 29 74 46 17
*The cell number used in this Table is for day 5; the cell number in the control was 2.0 × 106 cells/ml. The % of control value was calculated as described in the Methods section. 1"al × a2 = ~(l-i j)(I-i2); where ij and i2 are the fractional inhibitions caused by the presenceof 11 or 12 alone (14).
DISCUSSION The m a m m a l i a n ribonucleotide reductase enzyme has been shown to consist of two n o n - i d e n t i c a l c o m p o n e n t s (5-8). The properties of the n o n -
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ATSUSHI SATO, et al.
TABLE 7. EFFECT OF MODULATORS ON THE INHIBITION OF LEUKEMIA LI210 CELL GROWTH BY THE COMBINATION OF HYDROXYUREA PLUS DEOXYADENOSINE Drug treatment* Control dAdo HU dAdo + HU dAdo/EHNA HU/Df dAdo + .HU/Df dAdo/EHNA + HU dAdo/EHNA + HU/Df
Cell growth (% of control)l
Calculated (al × a2 × 100}:~
100 93 88 69 69 68 69 38 0
63 61 47
*The concentrations of deoxyadenosine (dAdo), hydroxyurea (H U), EHNA and Desferal (Df) were 0.015 mM, 0.05 mM, 0.005 mM and 0.02 mM, respectively. ~The cell number used in this Table is for day 5; the cell number in the control was 1.9 × I 06 cells/ml. The % of control value was calculated as described in the Methods section. ~:al × a2 = (I-i2) ( l-iv); where il and i2 are the fractional inhibitions caused by the presence of Ii or 12 alone (14). heme iron and e f f e c t o r - b i n d i n g c o m p o n e n t s were significantly different such that each could be specifically and i n d e p e n d e n t l y i n h i b i t e d / i n a c t i v a t e d (9). The n o n - h e m e iron c o m p o n e n t of r i b o n u c l e o t i d e reductase was i n h i b i t e d / inactivated by h y d r o x y u r e a , guanazole, I M P Y , M A I Q a n d IQ, while the e f f e c t o r - b i n d i n g c o m p o n e n t was i n h i b i t e d / i n a c t i v a t e d by l n o x , 5 ' - d l n o x , d A T P , d G T P a n d d T T P (15). Studies with highly-purified r i b o n u c l e o t i d e r e d u c t a s e indicated that c o m b i n a t i o n s of agents directed at the individual c o m p o n e n t s resulted in synergistic inhibition of r i b o n u c l e o t i d e reductase activity (10). H y d r o x y u r e a , g u a n a z o l e , I M P Y , M A I Q a n d IQ a p p e a r to be relatively specific for the r i b o n u c l e o t i d e reductase site. On the other hand, while I n o x and 5 ' - d l n o x are specific for the effector-binding c o m p o n e n t of reductase, there are multiple other sites such as R N A p o l y m e r a s e , etc. which are blocked by these dialdehydes. All of these drugs, with the e x c e p t i o n of 5'd l n o x have been studied as a n t i t u m o u r agents in h u m a n phase I and phase I1 trials. The lack of a p p r o p r i a t e t u m o r response or drug toxicity has precluded further use of these agents (with the e x c e p t i o n of h y d r o x y u r e a , which is still in clinical use). H y d r o x y u r e a , guanazole, I M P Y , M A I Q and IQ are specifically directed at the n o n - h e m e iron c o m p o n e n t of r i b o n u c l e o t i d e reductase (15). Recent studies in o u r l a b o r a t o r y have shown that the non-heine iron and e f f e c t o r - b i n d i n g subunits are not c o o r d i n a t e l y regulated in either t u m o r cells or in regenerating rat liver (16). The n o n - h e m e iron c o m p o n e n t is in excess d u r i n g the periods of p e a k r i b o n u c l e o t i d e reductase activity in the cell. It would follow that if m o r e effective a n d selective inhibitors of the effectorb i n d i n g c o m p o n e n t (which a p p e a r s to be limiting) were available, these
MODULATION OF DRUG COMBINATIONS
267
compounds would be better drugs for this site. On the other hand, since these agents are not available, it might be possible to utilize combinations of drugs which would serve to inhibit both components and result in the additive or synergistic inhibition of the ribonucleotide reductase site. The inhibition of ribonucleotide reductase by hydroxyurea and IMPY was shown to be increased by Desferal and other iron-chelating agents (13). The inhibition of DNA synthesis of hydroxyurea was also shown to be stimulated by Desferal (12). In the studies presented here, the effects of combinations of agents directed at the individual components of the ribonucleotide reductase site in L I210 cells was determined. IMPY and hydroxyurea were utilized as the specific inhibitors of the non-heme iron component while d A T P was directed at the effector-binding components. Desferal was added to modulate the effects of IMPY or hydroxyurea while EHNA was included to decrease the deamination of deoxyadenosine. As others have observed (17, 18), the toxicity of deoxyadenosine could be significantly increased by the presence of EHNA. In this L1210 cell line, deoxyadenosine as high as 0.150 mM was not toxic to the growth of these cells (data not shown). When EHNA was added in combination with lower concentrations of deoxyadenosine (0.025 or 0.050 mM) synergistic inhibition of L1210 cell growth was observed (Table 1). The inhibition of LI210 cell growth by hydroxyurea was potentiated by the presence of Desferal. The concentration of hydroxyurea alone required to give essentially complete inhibition of cell growth was 0.200 mM; in the presence of Desferal, the hydroxyurea concentration could be reduced at least 4-fold (Table 2) to achieve the same degree of inhibition of cell growth. Clearly, the combination of hydroxyurea and Desferal gave synergistic inhibition when appropriate concentrations of agents were used. The concentration of the modulator was as critical as the concentration of hydroxyurea in providing the synergistic response. L1210 cell growth in culture was inhibited approximately 46% by IMPY (0.25 mM). The combination of Desferal (0.10 mM) and IMPY (0.25 mM) gave strong synergistic inhibition of cell growth (Table 3). Lowering the concentration of IMPY decreased the extent of synergism while lowering the Desferal concentration led to only additive effects. These studies indicated the critical balance which had to be achieved between an agent and its modulator if synergism were to be achieved. These studies also indicated that the effects of hydroxyurea or IMPY, and deoxyadenosine could be synergistically potentiated by Desferal and EHNA, respectively. When these agents (deoxyadenosine and IMPY or hydroxyurea) were combined with their respective modulator, strong synergistic combinations were generated. With the use of a four-drug combination (deoxyadenosine/EHNA plus IMPY/Desferal, or deoxyadenosine/EHNA plus hydroxy-
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ATSUSHI SATO, et
al.
urea/Desferal), very effective synergistic combinations were generated. The concentrations of the individual drugs could be markedly reduced in the fourdrug combinations. Complete inhibition of cell growth was accomplished by concentrations of deoxyadenosine (0.015 mM), hydroxyurea (0.050 mM) or IMPY (0.19 mM), E H N A (0.005 mM) and Desferal (0.02 mM). Each of the drugs, alone at these concentrations, had minimal effects on L 1210 cell growth (Tables 4 and 7). Synergistic responses were obtained with some of the twodrug or three-drug combinations but higher drug concentrations were required. I M P Y or hydroxyurea in combination with Desferal gave synergistic inhibition (Tables 2 and 3) as did deoxyadenosine and E H N A (Table 1). At the lower drug concentrations (Table 4 and Table 7), significant synergism was achieved only when all four drugs were present. In three-drug combinations with deoxyadenosine and hydroxyurea or IMPY, E H N A was more important as a modulator than was Desferal in obtaining significant inhibition of tumor cell growth. This indicated that the protection of deoxyadenosine, presumably leading to increased d A T P pools, was an essential component of these combinations. While these data are supportive of the concept that combination chemotherapy directed at the individual components of ribonucleotide reductase can result in strong synergistic inhibition of t u m o r cell growth, it is possible that the observed synergism was not due, exclusively, to effects at the ribonucleotide reductase site. There are data to indicate that each of the compounds used (deoxyadenosine, IMPY, hydroxyurea and E H N A ) has other sites of action. Deoxyadenosine has been reported to be toxic to nondividing cells by an unknown mechanism which must be independent of the ribonucleotide reductase site (19). Hemolysis was a major side effect in patients treated with IMPY suggesting that other metabolic sites were involved (20). Hydroxyurea or its metabolites cause degradation of D N A (21) or inhibition of other enzymes (22, 23). E H N A has been shown to alter the metabolism of purine precursors (24). However, in each of these cases, the concentrations of the agents required to cause these effects were relatively higher than the concentrations reported here. Experiments are in progress to determine specifically the effects of these combinations on the deoxyribonucleoside triphosphate pools and on ribonucleotide reductase activity in intact cells. Our studies involving the combination chemotherapy directed at the individual components of ribonucleotide reductase have indicated that this approach can be extended beyond the highly-purified reductase, to studies in intact tumor cells in culture. It is felt that this is an important new concept (combination chemotherapy directed at the subunits of a multicomponent enzyme) which should be fully explored in the generation of drug combinations. Other metabolic sites may be found which will offer a situation very similar to the ribonucleotide reductase site. Clearly, other enzymes have
MODULATION OF DRUG COMBINATIONS
269
distinct substrate-binding and effector-binding sites which might be exploited. This research will not only provide the biochemical basis for selecting drug combinations for the ribonucleotide reductase site, but should provide the approaches to be used in future studies dealing with other key multicomponent enzymes. It is possible that with the use of an approach such as this in which each of the components of the combinations can be "titrated" down to its lowest effective concentration, useful protocols will emerge in which the toxicity of the individual components is minimal but the antitumor properties of the combinations are maximal. Agents such as 1MPY, hydroxyurea, guanazole, M A I Q and IQ, which have been utilized in some human studies, may be "rediscovered" as successful drugs in the treatment of human neoplasia if appropriately used in combinations directed at the ribonucleotide reductase site.
SUMMARY Ribonucleotide reductase from tumor cells consists of two non-identical components which can be specifically and independently inhibited. Combinations of agents directed at the individual components gave synergistic inhibition of LI210 cell growth in culture. Utilizing hydroxyurea and deoxyadenosine or IMPY and deoxyadenosine as the parent combinations, modulators were used to potentiate the effects of each of these drugs. E H N A was used to prevent the deamination of deoxyadenosine while Desferal was utilized to increase the effects of hydroxyurea and IMPY. Combinations consisting of d e o x y a d e n o s i n e / E H N A plus IMPY/Desferal and deoxya d e n o s i n e / E H N A plus hydroxyurea/Desferal gave synergistic inhibition of LI210 cell growth. Utilizing these combination chemotherapies, the concentrations of each of the agents could be kept to minimal, essentially noninhibitory levels and yet still achieve complete inhibition of L I210 cell growth with the specifically generated four-drug combinations.
ACKNOWLEDGEMENTS This work was supported by grants from the USPHS, National Cancer Institute, CA 27398, the C. T. Meyer Estate and the Phi Beta Psi Sorority.
REFERENCES E. TAKEDA and G. WEBER, Role of ribonucleotide reductase in expression of the neoplastic program, Life Sci. 28, 1007-1014 (1981). 2. H. L. ELFORD, M. FREESE, E. PASSAMANI and H. P. MORRIS, Ribonucleotide reductase and cell proliferation, i. Variations of ribonucleotide reductase acitivity with tumor growth rate in a series of rat hepatomas, Z Biol. Chem. 245, 5228-5233 (1970). I.
270 3.
4.
5. 6.
7.
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9.
10. 11.
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13. 14. 15.
16. 17.
18. 19. 20. 21. 22. 23. 24.
ATSUSHI SATO, et aL R. C. J A C K S O N , M. S. LUI, T. J. BORITZKI, H. P. M O R R I S and G. WEBER, Purine and pyrimidine nucleotide pattern of normal, differentiating, regenerating and neoplastic liver, Cancer Res. 40, 1286-1291 (1980). G. WEBER, E. O L A H , J. E. DENTON, M. S. LUI, E. T A K E D A , D. Y. T Z E N G and J. BAN, Dynamics of modulation of biochemical programs in cancer cells, Advances in Enzyme Regulation 19, 87-101 (1981). E . C . MOORE, Components and control of ribonucleotide reductase system in the rat, Advances in Enzyme Regulation 15, 101-114 (1977). C.-H. C H A N G and Y.-C. CHENG, Demonstration of two components and association of adenosine diphosphate-cytidine diphosphate reductase from cultured lymphoblast cells (Molt-4F), Cancer Res. 39, 436-442 (1979). J. G. CORY, A. E. F L E I S C H E R and J. B. M U N R O , II1, Reconstitution of the ribonucleotide reductase enzyme from Ehrlich tumor cells, J. Biol. Chem. 253, 2898-2901 (1978). Y. E N G S T R O M , S. ERIKSSON, L. T H E L A N D E R a n d M . A K E R M A N , Ribonucleotide reductase from calf thymus. Purification and properties, Biochemistry 14, 2941-2948 (1979). J. G. CORY and A. E. FLEISCHER, Specific inhibitors directed at the individual components of ribonucleotide reductase as an approach to combination chemotherapy, Cancer Res.39, 4600-4604 (1979). A. S A T O and J. G. CORY, Evaluation of combinations of drugs that inhibit Ehrlich tumor cell ribonucleotide reductase, Cancer Res. 41, 1637-1641 (1981). R . P . A G A R W A L , T. S P E C T O R and R. E. P A R K S , JR., Tight-binding inhibitors IV. Inhibition of adenosine deaminases by various inhibitors, Biochem. Pharmaco126,359-367 (I 977). R . W . B R O C K M A N , S. S H A D D I X , V. S T R I N G E R and D. A D A M S O N , Enhancement by deferoxamine of inhibition of D N A synthesis by ribonucleotide reductase inhibitors, Proc. Am. Assoc. Cancer Res. 13, 88 (1972). J . G . CORY, L. L A S A T E R and A. SATO, Effect of iron-chelating agents on inhibitors of ribonucleotide red uctase, Biochem. Pharmacol. 30, 979-984 (1981). J . L . WEBB, Effects of more than one inhibitor, In: Enz~'me and Metabolic lnhibitors Vol. 1, pp. 507-512, New York, Academic Press (1963). J. G. CORY, A. S A T O and L. LASATER, Specific inhibition of the subunits of ribonucleotide reductase as a new approach to combination chemotherapy, Advances in Enzyme Regulation 19, 139-150 (1981). J, G. CORY and A. E. F L E I S C H E R , Non-coordinate changes in the components of ribonucleotide reductase in mammalian cells, J. Biol. Chem. 257, 1263-1266 (1982). B. U L L M A N , L. J. G U D A S , A. COHEN and D. W. M A R T I N , JR., Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma ceils: a model for immunodeficiency disease, Cell 14, 365-375 (1978). A. L. LIN and H. L. E L F O R D , Adenosine deaminase impairment and ribonucleotide reductase activity and levels in HeLa cells, J. Biol. Chem. 255, 8523-8528 (1980). R . F . K E F F O R D and R. M. FOX, Purine deoxyribonucleoside toxicity in non-dividing h u m a n lymphoid cells, Cancer Res. 42, 324-330 (1982). J. N E I D H A R T and A. SAGONE, IMPY and its/3-ribosyl metabolite show differential in vitro effects, Proc. Am. Assoc. Cancer Res. 20, 177 (1979). S . J . JACOBS and H. S. R O S E N K R A N Z , Detection of a reactive intermediate in the reaction between DNA and hydroxyurea, Cancer Res. 30, 1084-1094 (1974). H.S. ROSEN K R A N Z and S. J. JACOBS, Inhibition of DNA synthesis by hydroxyurea, Gann Monograph 6, 15-41 (1968). T . L . BOEHM, W. KREIS and D. D R A H O V S K Y , Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells, Biochim. Biophys. Acta 696, 52-56 (1982). J . F . - H E N D E R S O N , L. BROX, G. Z O M B O R , D. HUNTING and C. LOMAX, Specificity of adenosine deaminase inhibitors, Biochem. Pharmacol. 26, 1967-1972 (1977).
INTRODUCTION
During the S-phase of the cell cycle when DNA synthesis is occurring, a balanced supply of the four dNTP's (dATP, dGTP, dCTP and dTTP) is required if the replication of DNA is to be completed and cell division is to follow. The pathway by which the dNTP's are formed is shown in Figure 1. The reaction from N D P to 2 ' - d N D P is the rate-limiting step in the process leading to t h e formation of dNTP's. This step is catalyzed by the enzyme, ribonucleotide reductase (EC 1.17.4.1, nucleoside diphosphate reductase). The activity of ribonucleotide reductase has been shown to be extremely low in normal liver (1) or essentially unmeasurable (2). The level of activity of reductase has been shown to correlate with the growth rate of chemicallyinduced hepatomas (1, 2). This enzyme also showed the greatest increase in enzymatic activity in the neoplastic process (!). Measurements of the intracellular concentrations of the substrates (ribonucleoside diphosphates) demonstrated that they were not limiting for this reaction (3). Since ribonucleotide reductase catalyzes such a key metabolic reaction and of the enzymes involved in nucleotide metabolism has the lowest cellular activity (4), this metabolic site offers an excellent target for antineoplastic drugs. Further, the enzyme from mammalian cells has been shown to consist of two nonidentical components (5-8). The two components making up the active enzyme in Ehrlich tumor cells were specifically and independently inhibited/ inactivated (8). Utilizing highly-purified ribonucleotide reductase, it was shown that combinations of ribonucleotide reductase inhibitors directed at the individual components resulted in synergistic inhibition of enzyme activity
(10). In the current studies, the effects of combinations of ribonucleotide reductase inhibitors (e.g. deoxyadenosine/hydroxyurea or deoxyadenosine/ IMPY) on the growth of L I210 cells in culture are reported. To potentiate the 259
260
ATSUSHISATO, etaL de novo NUCLEOTIDE SYNTHESIS
a
,NMP
b
"NDP
c
'- dNDP
d
-dNTP
FIG. 1. Pathway for formation of deoxyribonucleosidetriphosphates. The enzymescatalyzing these reactions are: (a) enzymes for de novo synthesis of purine and pyrimidine nucleoside monophosphates; (b) nucleoside monophosphate kinase; (c) ribonucleotide reductase; and (d) nucleoside diphosphate kinase. effects of each of these agents, other drugs were used to modulate the effects of deoxyadenosine and I M P Y or hydroxyurea. E H N A was used to inhibit the deamination of deoxyadenosine (11) while Desferal was used to potentiate the effects of hydroxyurea (12, 13) or IMPY (13). M A T E R I A L S AND METHODS G r o w t h o f l e u k e m i a L1210 cells. L1210 cells were grown in suspension
culture in R P M I 1640 culture medium which was supplemented with 10% horse serum, NaHCO3 (2 g/l) and antibiotic (Gentamicin sulfate, 50 mg/l). Aliquots of the cell suspensions ( l ml) were removed. The cells were collected by centrifugation, washed with phosphate-buffered saline and counted in a Biophysics Cytograph. The cultures were initially set up on day-zero with an initial cell concentration of 1.2 to 2 x 105/ml. The drugs, alone or in combination, were added only at this time. Dr. Richard G. Moran, Children's Hospital of I.os Angeles, CA, kindly supplied the L 1210 cell line used in these studies. The data presented in the Table l were calculated from the cell numbers at day 4 of the growth curve. The data in Tables 2-7 were calculated from the cell numbers at day 5 of the growth curve. The % of control values were calculated according to the equation: % of Control = Cells/ml (treated) - Cells/ml (seeded) x 100 Cells/ml (control) - CeUs/ml (seeded) The values listed as "calculated" were determined according to the methods of Webb (14) to describe whether the combinations of drugs yielded synergistic inhibition of cell growth. When the "calculated" value was larger than the fraction of cell growth observed, the combination resulted in synergistic inhibition; when the observed and calculated values were the same the effects were additive; if the "calculated" value was smaller than the observed fraction of cell growth, then the combination resulted in antagonistic inhibition of cell growth. Materials. Pyrazolo-imidazole (IMPY) was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute, through the assistance of Dr. V. L Narayanan. E r y t h r o - 9 - ( 2 - h y d r o x y - 3 - n o n y l ) adenine (EHNA) was a gift from Dr. G. Elion, Burroughs-Wellcome, Research
MODULATION OF DRUG COMBINATIONS Triangle,
N.
C.
Hydroxyurea,
deoxyadenosine
and
261
Gentamicin
were
p u r c h a s e d f r o m S i g m a C h e m i c a l C o . , St. L o u i s , M O . R P M I 1640 c u l t u r e medium, sodium bicarbonate and horse serum were purchased from Grand Island B i o l o g i c a l C o . , G r a n d Island, NY. D e s f e r a l was a gift f r o m C I B A P h a r m a c e u t i c a l C o . , S u m m i t , N. d. RESULTS
Modulation of Deoxyadenosine Inhibition of Leukemia L1210 Cell Growth in Culture by EHNA D e o x y a d e n o s i n e is r a p i d l y d e a m i n a t e d by a d e n o s i n e d e a m i n a s e w h i c h p r e v e n t s the b u i l d u p o f the i n t r a c e l l u l a r c o n c e n t r a t i o n o f d A T P , a n e g a t i v e e f f e c t o r o f r i b o n u c l e o t i d e r e d u c t a s e activity. In the p r e s e n c e o f E H N A , a p o t e n t i n h i b i t o r o f a d e n o s i n e d e a m i n a s e , d e o x y a d e n o s i n e is s h u n t e d t o w a r d d A M P f o r m a t i o n a n d s u b s e q u e n t l y to d A T P . T h e d a t a in T a b l e i s h o w the effects o f v a r i o u s c o n c e n t r a t i o n s of E H N A o n d e o x y a d e n o s i n e t o x i c i t y o f L I 2 1 0 g r o w t h in culture. C o n c e n t r a t i o n s o f d e o x y a d e n o s i n e (0.025 mM o r 0.05 mM) had essentially n o effect on L I 2 1 0 cell g r o w t h w h e n a d d e d to the c u l t u r e m e d i u m . In fact, d e o x y a d e n o s i n e at a c o n c e n t r a t i o n o f 0.150 mM had e s s e n t i a l l y n o effect on L I210 cell g r o w t h ( d a t a n o t shown). E H N A , a l o n e , at c o n c e n t r a t i o n s o f 0.005 mM o r 0.010 mM had o n l y a slight i n h i b i t o r y effect o n the g r o w t h o f L I210 cells. H o w e v e r , w h e n c o m b i n a t i o n s o f d e o x y a d e n o s i n e a n d E H N A were a d d e d , a m a r k e d s y n e r g i s m o f the i n h i b i t i o n o f L I 2 1 0 cell TABLE I. MODULATION OF DEOXYADENOS1NE INHIBITION OF LEUKEMIA LI210 CELL GROWTH BY EHNA Drug treatment Control dAdo, 0.025 mM 0.050 mM EHNA, 0.001 mM 0.0025 mM 0.005 mM 0.010 mM dAdo, 0.025 mM + EHNA, 0.0025 mM 0.005 mM 0.010 mM dAdo, 0.050 mM + EHNA, 0.001 mM 0.0025 mM 0.005 mM
Cell growth (% of control)*
Calculated (al x a2 x 100)1
100 93 86 I00 86 72 66 79 48 5 72 59 0
80 67 61 86 74 62
*The cell number used in this Table is for day 4; the cell number in the control was 1.6 x l06 cells/ml. The % of control value was calculated as described in the 'Methods' section. 1"a~x a2 = (I-i~) (I-i2); where it and i2 are the fractional inhibitions caused by the presence of Ii or 12 alone (14).
262
A T S U S H I S A T O , et aL
growth was observed. This was especially noticeable with the combinations dAdo, 0.025 m M / E H N A , 0.010 mM and dAdo, 0.050 m M / E H N A , 0.005 mM. From the inhibition of cell growth caused by each drug alone, the calculated inhibition to be expected in the combinations was 39 and 38% and yet the observed inhibition of cell growth was 95 and 100% for these combinations of d A d o / E H N A . These data indicated that not only could synergistic combinations be generated, but that the concentrations of both components were important since only additive effects were seen at the lower concentrations of each drug.
Modulation of Hydroxyurea Inhibition of L1210 Cell Growth by Desferal It has been shown that the inhibitions of ribonucleotide reductase activity (12, 13) and incorporation of thymidine into D N A by hydroxyurea were increased by the presence of Desferal (12). The effect of various concentrations of hydroxyurea on L1210 cell growth was studied. As seen in Table 2, 0.20 mM hydroxyurea caused a decrease in cell growth of 96%. However, the concentration of hydroxyurea required to inhibit cell growth to this extent could be decreased to 0.05 mM when Desferal (0.10 mM) was added in combination. The combinations of hydroxyurea and Desferal gave synergistic effects at the concentrations studied. The same extent of inhibition of cell growth could be achieved at 0.05 mM hydroxyurea, 0.10 M Desferal as with the higher concentration of hydroxyurea (0.10 mM) and 0.10 mM Desferal. There was essentially complete inhibition (99%) of LI210 cell growth at these lower hydroxyurea concentrations if Desferal was present.
T A B L E 2. M O D U L A T I O N O F H Y D R O X Y U R E A I N H I B I T I O N O F L E U K E M I A L I 2 1 0 C E L L G R O W T H BY D E S F E R A L
Drug t r e a t m e n t Control H y d r o x y u r e a , 0.05 mM 0.10 mM 0.20 mM Desferal, 0.02 mM 0.10 mM HU, 0.05 mM + Df, 0.02 mM HU, 0.05 mM + Df, 0.10 mM HU, 0.10 mM + Df, 0.10 mM
Cell g r o w t h (% of control)*
Calculated (al x a2 x 100)~"
100 80 5l 4 87 54 49 I 1
70 43 28
*The cell n u m b e r used in this Table is for day 5; the cell n u m b e r in the control was 1.8 x 106 c e l l s / m l . The % of control value was calculated as described in the " M e t h o d s " section. 1-al x a2 -- (1-i~) ( I-i2); whe re il and i2 are the fractional inhibitions caused by the presence of I~ or 12 alone (14).
MODULATION OF DRUG COMBINATIONS
263
Modulation of I M P Y Inhibition of L1210 Cell Growth in Culture by Desferal Earlier studies had shown that the i n h i b i t i o n of highly-purified ribonucleotide reductase by 1MPY could be m a r k e d l y potentiated by ironchelating agents such as Desferal, E D T A or 8 - h y d r o x y q u i n o l i n e (13). Studies were carried out to determine whether Desferal could potentiate the effects of I M P Y on the growth of Ll210 cells in culture. The data in Table 3 show the effects of I M P Y and Desferal alone and in c o m b i n a t i o n . At the c o n c e n t r a tions of I M P Y (0.25 mM) a n d Desferal (0. l0 M) used 46 and 39% i n h i b i t i o n of cell growth, respectively, were observed. When these agents were used in c o m b i n a t i o n and at these c o n c e n t r a t i o n s a marked synergistic effect was observed (96% inhibition). W h e n the c o n c e n t r a t i o n of Desferal was lowered from 0. l0 mM to 0.02 m i the i n h i b i t i o n observed was only additive.
TABLE 3. MODULATION OF IMPY INHIBITION OF LEUKEMIA LI210 CELL GROWTH BY DESFERAL Drug treatment Control IMPY, 0.10 mM 0.25 mM Desferal, 0.02 mM 0. I0 mM IMPY, 0.25 mM + Df, 0.02 mM IMPY, 0.10 mM + Df, 0.10 mM IMPY, 0.25 mm + Df, 0.10 mM
Cell growth (% of control)*
Calculated (az × a2 × 100)~"
100 87 54 87 61 48 46 4
47 53 33
*The cell number used in this Table is for day 5; the cell number in the control was 1.7 × 106 cells/ml. The % of control value was calculated as described in the Methods section. ~al × a2 = ( I-ij ) ( 1-i2);where it and i2 are the fractional inhibitions caused by the presence of Ii or 12 alone (14).
Effect of Modulators on the Inhibition of L1210 Cell Growth by the Combination of I M P Y Plus Deoxyadenosine The effects of the c o m b i n a t i o n s of d e o x y a d e n o s i n e and I M P Y on LI210 cell growth were studied. Because of the rapid d e a m i n a t i o n of deoxya d e n o s i n e by a d e n o s i n e deaminase, E H N A was utilized to protect deoxyadenosine. Further, the i n h i b i t i o n of cell growth by 1M PY was potentiated by Desferal (Table 3). As seen from the data in Table 4, at c o n c e n t r a t i o n s of d e o x y a d e n o s i n e (0.015 raM) and I M P Y (0.19 mM), this c o m b i n a t i o n did not result in the synergistic i n h i b i t i o n of cell growth. If a n y t h i n g , it appeared to be slightly antagonistic. The c o m b i n a t i o n of d e o x y a d e n o s i n e / I M P Y / D e s f e r a l also was slightly antagonistic. On the other hand, the c o m b i n a t i o n of d e o x y a d e n o s i n e / E H N A / I M P Y resulted in a slight synergistic i n h i b i t i o n of
264
ATSUSHI SATO, et al. TABLE 4. E F F E C T OF M O D U L A T O R S ON THE INHIBITION OF L E U K E M I A LI210 CELL G R O W T H BY THE C O M B I N A T I O N OF IMPY PLUS DEOXYADENOSINE
Drug treatment* Control dAdo I M PY dAdo + IMPY dAdo/EHNA IMPY/Df dAdo + I M P Y / D f d A d o / E H N A + IMPY d A d o / E H N A 4- I M P Y / D f
Cell growth (% of control)~"
Calculated (at x a2 x 100)*
100 94 59 71 77 71 71 33 0
55 67 45 55
*The concentrations of deoxyadenosine (dAdo), IMPY, EHNA and Desferal (Df)were 0.015 raM, 0.19 mM, 0.005 mM, respectively. tThe cell number used in this Table is for day 5; the cell number in the control was 1.9 x 106 cells/ml. The % of control value was calculated as described in the Methods section. *al × a2 --- (I-i~) ( I-i2); whe re il and i2 are the fractional inhibitions caused by the presence of II or 12 alone (14).
Li210 cell growth. However, when all four drugs were used in combination ( d e o x y a d e n o s i n e / E H N A 4- IMPY/Desferal) a strong synergism which resulted in the complete inhibition of cell growth was obtained. From a comparison of the three-drug combinations, it appeared that E H N A was more important as a modulator than was Desferal. As shown in Table 5, the concentration of deoxyadenosine could be lowered still further from 0.015 mM to 0.005 mM if the concentration of one of the other components were increased (in this case, Desferal) and the synergistic combination still be TABLE 5. E F F E C T OF C O M B I N A T I O N S OF D E O X Y A D E N O S I N E , EH N A A N D IMPY ON L E U K E M I A LI210 CELL G R O W T H
Drug combinations Control A. IMPY, 0.19 mM/Df, 0.020 mM B. 1MPY, 0.19 mM/Df, 0.040 mM C. dAdo, 0.005 raM/EHNA, 0.005 mM D. dAdo, 0.015 mM/EHNA, 0.005 m i A+ C A+ D B+C B+ D
Cell growth (% of control)*
Calculated (al x a2 × 100)~
100 71 71 77 77 21 0 0 0
55 55 55 55
*The cell number used in this Table is for day 5; the cell number in the control was 1.9 × 106 cells/ml. The % of control value was calculated as described in the Methods section. ~al x a2 = ( l - i 0 ( 1-i2); where il and i2 are the fractional inhibitions caused by the presence of I~ or 12 alone (14).
MODULATION OF DRUG COMBINATIONS
265
m a i n t a i n e d . These data in c o n j u n c t i o n with the data presented in Table 4 show the i m p o r t a n c e not only of the agents, d e o x y a d e n o s i n e a n d I M P Y , but also the i m p o r t a n c e of the m o d u l a t o r s in the c o m b i n a t i o n s .
Effect of Modulators on the Inhibition of LI210 Cell Growth by the Combinations of Deoxyadenosine and H vdroxyurea As shown in Table 6 d e o x y a d e n o s i n e a n d h y d r o x y u r e a in c o m b i n a t i o n do not result in the synergistic i n h i b i t i o n of cell growth. However, if E H N A is added to protect d e o x y a d e n o s i n e from d e a m i n a t i o n a synergistic c o m b i n a tion, consisting of d e o x y a d e n o s i n e / E H N A / h y d r o x y u r e a can be generated. O n the other hand, if E H N A is used to protect d e o x y a d e n o s i n e from d e a m i n a t i o n and Desferal is used to potentiate the effects of h y d r o x y u r e a , not only is a synergistic i n h i b i t i o n of cell growth achieved, but the c o n c e n t r a t i o n s of the agents required can be markedly reduced. In c o m p a r i s o n with the data in Table 6, the d e o x y a d e n o s i n e c o n c e n t r a t i o n could be lowered from 0.055 mM to 0.015 raM. Again as seen with the c o m b i n a t i o n s which included I M P Y , the three-drug c o m b i n a t i o n d e o x y a d e n o s i n e / E H N A / h y d r o x y u r e a was a better c o m b i n a t i o n t h a n d e o x y a d e n o s i n e / h y d r o x y u r e a / D e s f e r a l in t e r m s of i n h i b i t i n g L 1210 cell growth (Table 7). At these low c o n c e n t r a t i o n s of d e o x y a d e n o s i n e (0.015 mM) it appears very critical that E H N A be present.
TABLE 6. EFFECT OF COMBINATIONS OF DEOXYADENOSINE, EHNA AND HYDROXYUREA ON LEUKEMIA LI210 CELL GROWTH Drug treatment Control dAdo, 0.055 mM H U, 0.055 mM HU, 0.110 mM EHNA, 0.002 mM dAdo + HU, 0.055 mM dAdo + HU, 0.110 mM dAdo/EHNA dAdo/EHNA + HU, 0.055 mM dAdo/EHNA + HU, 0.110 mM
Cell growth (% of control)
Calculated (at × a2 x 100)I
100 78 62 37 95 56 40 45 7 1
48 29 74 46 17
*The cell number used in this Table is for day 5; the cell number in the control was 2.0 × 106 cells/ml. The % of control value was calculated as described in the Methods section. 1"al × a2 = ~(l-i j)(I-i2); where ij and i2 are the fractional inhibitions caused by the presenceof 11 or 12 alone (14).
DISCUSSION The m a m m a l i a n ribonucleotide reductase enzyme has been shown to consist of two n o n - i d e n t i c a l c o m p o n e n t s (5-8). The properties of the n o n -
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TABLE 7. EFFECT OF MODULATORS ON THE INHIBITION OF LEUKEMIA LI210 CELL GROWTH BY THE COMBINATION OF HYDROXYUREA PLUS DEOXYADENOSINE Drug treatment* Control dAdo HU dAdo + HU dAdo/EHNA HU/Df dAdo + .HU/Df dAdo/EHNA + HU dAdo/EHNA + HU/Df
Cell growth (% of control)l
Calculated (al × a2 × 100}:~
100 93 88 69 69 68 69 38 0
63 61 47
*The concentrations of deoxyadenosine (dAdo), hydroxyurea (H U), EHNA and Desferal (Df) were 0.015 mM, 0.05 mM, 0.005 mM and 0.02 mM, respectively. ~The cell number used in this Table is for day 5; the cell number in the control was 1.9 × I 06 cells/ml. The % of control value was calculated as described in the Methods section. ~:al × a2 = (I-i2) ( l-iv); where il and i2 are the fractional inhibitions caused by the presence of Ii or 12 alone (14). heme iron and e f f e c t o r - b i n d i n g c o m p o n e n t s were significantly different such that each could be specifically and i n d e p e n d e n t l y i n h i b i t e d / i n a c t i v a t e d (9). The n o n - h e m e iron c o m p o n e n t of r i b o n u c l e o t i d e reductase was i n h i b i t e d / inactivated by h y d r o x y u r e a , guanazole, I M P Y , M A I Q a n d IQ, while the e f f e c t o r - b i n d i n g c o m p o n e n t was i n h i b i t e d / i n a c t i v a t e d by l n o x , 5 ' - d l n o x , d A T P , d G T P a n d d T T P (15). Studies with highly-purified r i b o n u c l e o t i d e r e d u c t a s e indicated that c o m b i n a t i o n s of agents directed at the individual c o m p o n e n t s resulted in synergistic inhibition of r i b o n u c l e o t i d e reductase activity (10). H y d r o x y u r e a , g u a n a z o l e , I M P Y , M A I Q a n d IQ a p p e a r to be relatively specific for the r i b o n u c l e o t i d e reductase site. On the other hand, while I n o x and 5 ' - d l n o x are specific for the effector-binding c o m p o n e n t of reductase, there are multiple other sites such as R N A p o l y m e r a s e , etc. which are blocked by these dialdehydes. All of these drugs, with the e x c e p t i o n of 5'd l n o x have been studied as a n t i t u m o u r agents in h u m a n phase I and phase I1 trials. The lack of a p p r o p r i a t e t u m o r response or drug toxicity has precluded further use of these agents (with the e x c e p t i o n of h y d r o x y u r e a , which is still in clinical use). H y d r o x y u r e a , guanazole, I M P Y , M A I Q and IQ are specifically directed at the n o n - h e m e iron c o m p o n e n t of r i b o n u c l e o t i d e reductase (15). Recent studies in o u r l a b o r a t o r y have shown that the non-heine iron and e f f e c t o r - b i n d i n g subunits are not c o o r d i n a t e l y regulated in either t u m o r cells or in regenerating rat liver (16). The n o n - h e m e iron c o m p o n e n t is in excess d u r i n g the periods of p e a k r i b o n u c l e o t i d e reductase activity in the cell. It would follow that if m o r e effective a n d selective inhibitors of the effectorb i n d i n g c o m p o n e n t (which a p p e a r s to be limiting) were available, these
MODULATION OF DRUG COMBINATIONS
267
compounds would be better drugs for this site. On the other hand, since these agents are not available, it might be possible to utilize combinations of drugs which would serve to inhibit both components and result in the additive or synergistic inhibition of the ribonucleotide reductase site. The inhibition of ribonucleotide reductase by hydroxyurea and IMPY was shown to be increased by Desferal and other iron-chelating agents (13). The inhibition of DNA synthesis of hydroxyurea was also shown to be stimulated by Desferal (12). In the studies presented here, the effects of combinations of agents directed at the individual components of the ribonucleotide reductase site in L I210 cells was determined. IMPY and hydroxyurea were utilized as the specific inhibitors of the non-heme iron component while d A T P was directed at the effector-binding components. Desferal was added to modulate the effects of IMPY or hydroxyurea while EHNA was included to decrease the deamination of deoxyadenosine. As others have observed (17, 18), the toxicity of deoxyadenosine could be significantly increased by the presence of EHNA. In this L1210 cell line, deoxyadenosine as high as 0.150 mM was not toxic to the growth of these cells (data not shown). When EHNA was added in combination with lower concentrations of deoxyadenosine (0.025 or 0.050 mM) synergistic inhibition of L1210 cell growth was observed (Table 1). The inhibition of LI210 cell growth by hydroxyurea was potentiated by the presence of Desferal. The concentration of hydroxyurea alone required to give essentially complete inhibition of cell growth was 0.200 mM; in the presence of Desferal, the hydroxyurea concentration could be reduced at least 4-fold (Table 2) to achieve the same degree of inhibition of cell growth. Clearly, the combination of hydroxyurea and Desferal gave synergistic inhibition when appropriate concentrations of agents were used. The concentration of the modulator was as critical as the concentration of hydroxyurea in providing the synergistic response. L1210 cell growth in culture was inhibited approximately 46% by IMPY (0.25 mM). The combination of Desferal (0.10 mM) and IMPY (0.25 mM) gave strong synergistic inhibition of cell growth (Table 3). Lowering the concentration of IMPY decreased the extent of synergism while lowering the Desferal concentration led to only additive effects. These studies indicated the critical balance which had to be achieved between an agent and its modulator if synergism were to be achieved. These studies also indicated that the effects of hydroxyurea or IMPY, and deoxyadenosine could be synergistically potentiated by Desferal and EHNA, respectively. When these agents (deoxyadenosine and IMPY or hydroxyurea) were combined with their respective modulator, strong synergistic combinations were generated. With the use of a four-drug combination (deoxyadenosine/EHNA plus IMPY/Desferal, or deoxyadenosine/EHNA plus hydroxy-
268
ATSUSHI SATO, et
al.
urea/Desferal), very effective synergistic combinations were generated. The concentrations of the individual drugs could be markedly reduced in the fourdrug combinations. Complete inhibition of cell growth was accomplished by concentrations of deoxyadenosine (0.015 mM), hydroxyurea (0.050 mM) or IMPY (0.19 mM), E H N A (0.005 mM) and Desferal (0.02 mM). Each of the drugs, alone at these concentrations, had minimal effects on L 1210 cell growth (Tables 4 and 7). Synergistic responses were obtained with some of the twodrug or three-drug combinations but higher drug concentrations were required. I M P Y or hydroxyurea in combination with Desferal gave synergistic inhibition (Tables 2 and 3) as did deoxyadenosine and E H N A (Table 1). At the lower drug concentrations (Table 4 and Table 7), significant synergism was achieved only when all four drugs were present. In three-drug combinations with deoxyadenosine and hydroxyurea or IMPY, E H N A was more important as a modulator than was Desferal in obtaining significant inhibition of tumor cell growth. This indicated that the protection of deoxyadenosine, presumably leading to increased d A T P pools, was an essential component of these combinations. While these data are supportive of the concept that combination chemotherapy directed at the individual components of ribonucleotide reductase can result in strong synergistic inhibition of t u m o r cell growth, it is possible that the observed synergism was not due, exclusively, to effects at the ribonucleotide reductase site. There are data to indicate that each of the compounds used (deoxyadenosine, IMPY, hydroxyurea and E H N A ) has other sites of action. Deoxyadenosine has been reported to be toxic to nondividing cells by an unknown mechanism which must be independent of the ribonucleotide reductase site (19). Hemolysis was a major side effect in patients treated with IMPY suggesting that other metabolic sites were involved (20). Hydroxyurea or its metabolites cause degradation of D N A (21) or inhibition of other enzymes (22, 23). E H N A has been shown to alter the metabolism of purine precursors (24). However, in each of these cases, the concentrations of the agents required to cause these effects were relatively higher than the concentrations reported here. Experiments are in progress to determine specifically the effects of these combinations on the deoxyribonucleoside triphosphate pools and on ribonucleotide reductase activity in intact cells. Our studies involving the combination chemotherapy directed at the individual components of ribonucleotide reductase have indicated that this approach can be extended beyond the highly-purified reductase, to studies in intact tumor cells in culture. It is felt that this is an important new concept (combination chemotherapy directed at the subunits of a multicomponent enzyme) which should be fully explored in the generation of drug combinations. Other metabolic sites may be found which will offer a situation very similar to the ribonucleotide reductase site. Clearly, other enzymes have
MODULATION OF DRUG COMBINATIONS
269
distinct substrate-binding and effector-binding sites which might be exploited. This research will not only provide the biochemical basis for selecting drug combinations for the ribonucleotide reductase site, but should provide the approaches to be used in future studies dealing with other key multicomponent enzymes. It is possible that with the use of an approach such as this in which each of the components of the combinations can be "titrated" down to its lowest effective concentration, useful protocols will emerge in which the toxicity of the individual components is minimal but the antitumor properties of the combinations are maximal. Agents such as 1MPY, hydroxyurea, guanazole, M A I Q and IQ, which have been utilized in some human studies, may be "rediscovered" as successful drugs in the treatment of human neoplasia if appropriately used in combinations directed at the ribonucleotide reductase site.
SUMMARY Ribonucleotide reductase from tumor cells consists of two non-identical components which can be specifically and independently inhibited. Combinations of agents directed at the individual components gave synergistic inhibition of LI210 cell growth in culture. Utilizing hydroxyurea and deoxyadenosine or IMPY and deoxyadenosine as the parent combinations, modulators were used to potentiate the effects of each of these drugs. E H N A was used to prevent the deamination of deoxyadenosine while Desferal was utilized to increase the effects of hydroxyurea and IMPY. Combinations consisting of d e o x y a d e n o s i n e / E H N A plus IMPY/Desferal and deoxya d e n o s i n e / E H N A plus hydroxyurea/Desferal gave synergistic inhibition of LI210 cell growth. Utilizing these combination chemotherapies, the concentrations of each of the agents could be kept to minimal, essentially noninhibitory levels and yet still achieve complete inhibition of L I210 cell growth with the specifically generated four-drug combinations.
ACKNOWLEDGEMENTS This work was supported by grants from the USPHS, National Cancer Institute, CA 27398, the C. T. Meyer Estate and the Phi Beta Psi Sorority.
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