- Email: [email protected]
Binding mode of chemically activated semiquinone free radicals from quinone anticancer agents to DNA
Chem.-Biol. Interactions, 28 (1979) 301--308 @ Elsevier]North-Holland Scientific Publishers Ltd.
301
BINDING MODE OF CHEMICALLY ACTIVATED SEMIQUINONE FREE RADICALS FROM QUINONE ANTICANCER AGENTS TO DNA
B.K. SINHA* and C.F. CHIGNELL Laboratory of Environmental Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S,A.) (Received February 14th, 1979) (Revision received July l l t h , 1979) (Accepted July 20th, 1979)
SUMMARY
Chemical reduction of the highly active quinone-containing antitumor drugs, adriamycin and daunorubicin formed the same partially reduced free radical previously reported [9] by microsomal activation. In vitro incubation of the chemically activated free radical intermediates with DNA resulted in covalent binding of these drugs to DNA. The adriamycin semiquinone radical has a greater affinity for DNA and covalent complexes up to one adriamycin per 12 nucleotides were obtained. The daunorubicin semiquinone radical, on the other hand, showed a lesser binding affinity and gave rise to complexes in which one drug molecule was covalently bound per 135 nucleotides. The stronger covalent binding of adriamycin to DNA may account for more severe DNA damage induced by this drug.
INTRODUCTION
The anthracycline antitumor drugs, adriamycin and danorubicin, both of which contain the quinone moiety, are currently being used in the treatment of acute leukemia and solid tumors in man [1,2]. In addition, these agents induce damage in DNA as evidenced by an increase both in chromosomal aberrations and in the frequency of sister chromatid exchange (SCE) [3]. The biological activities of these agents seem to result from their ability to intercalate into DNA base pairs by virtue of their planar ring structure [4,5]. Such tight binding to nucleic acids results in the inhibition of both DNA and RNA synthesis [6] and DNA- and RNA
302 microsomal incubations containing NADPH. The formation of semiquinone metabolites of anthracycline antibiotics has been demonstrated using electron spin resonance (ESR) spectroscopy in both anaerobic NADPH-microsomal incubations [9--11] and Ehrlich ascites t u m o r cells [9]. Bachur et al. [11] have suggested that the free radicals may either be sufficiently stable to bind to nuclear DNA and RNA through intercalation, or may generate superoxide which then reacts with DNA. In this paper, we report on the binding mode of the chemically generated semiquinone radicals from adriamycin and daunorubicin. We present, for the first time, evidence for the covalent binding of semiquinone free radicals from adriamycin and daunorubicin to DNA. We have included in this study mitomycin-C semiquinone, which has also been reported to bind covalently to DNA [12]. MATERIALS Adriamycin HC1 (NSC-123, 127) and daunorubicin (NSC-82, 152) were gifts from the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute. Calf thymus DNA was purchased from Sigma Biochemicals. Mitomycin-C was purchased from Aldrich Chemicals. METHODS Adriamycin (1 mg/ml),daunorubicin (1 mg/ml)and mitomycin-C (2 mg/ml) were dissolved in 5 mM phosphate buffer (pH 7.4) and were deaerated by bubbling nitrogen for 10 min. Chemical activation was carried out by adding in 5 equal portions, at 5-min intervals, sodium borohydride solution (2 mg/ml in phosphate buffer, 0.75 equivalent of NaBH4 added) to adriamycin and daunorubicin solutions. Calf thymus DNA (1 mg/ml), dissolved in 5 mM phosphate buffer (pH 7.4) containing 50 mM NaCl, was deaerated by bubbling nitrogen for 10 rain. The DNA solution was added (1 mg DNA/1 mg drug) to the activated adriamycin and daunorubicin and the mixtures were incubated in the dark for 18 h at room temperature. The DNA-drug complexes were isolated by ethanol precipitation and the unbound drugs were removed. The complexes were washed 3 times with ethanol (assays for total bound drugs were carried out on these samples). The DNA-drug complexes were dissolved in 2 M NaC1 solution to dissociate intercalated drugs, stirred for 1 h and reprecipitated in ethanol. After the dissociated drugs were removed, the complexes were redissolved in 2 M NaC1 and dialyzed extensively against 5 mM phosphate buffer containing 1 M NaC1. The DNA-drug complexes were then chromatographed in Sephadex G-100 column (2.0 × 30 cm) using 5 mM phosphate containing 100 mM NaC1 as eluent. Activation of mitomycin-C, was achieved b y mixing DNA (2 mg DNA/1 mg drug) with mitomycin-C, and adding the reducing agent in 5 equal portions at 5min intervals as described by Tomasz et al. [12]. The DNA-mitomycin-C complex was isolated as described for adriamycin and daunorubicin.
303 The assays of the binding ratios in the DNA-drug complexes were carried out spectrophotometrically. The binding ratio is defined here as the molar ratio of mononucleotide unit to the drug. The nucleotide concentration was determined using e260 = 7000 for native DNA [12]. Bound mitomycin-C was determined at 310 nm (e = 11 000) [12]. Bound adriamycin and daunorubicin were determined at 505 nm (e = 6900) [13,14]. The ESR measurements were carried out on a Varian E-109 spectrometer operating at 100 krIz. The samples were introduced into an E-238 Tml~o cavity in quartz fiat cells provided with a syringe in the b o t t o m for mixing. The g-values for adriamycin and daunorubicin semiquinone radicals were measured using Fremy's salt by a m e t h o d similar to one described by Mason et al. [15]. Thermal denaturation studies were carried out in 5 mM phosphate buffer at 260 nm by means of a Gilford 250 recording spectrophotometer with a thermoprogrammer 2527 programmed for a temperature rise of l ° C / m i n according to previously published methods [16]. Control sample contained equivalent amounts of DNA. The stability of the DNA-drug complexes was demonstrated by treating the complex with sodium dodecyl sulfate (SDS) and urea, agents that are known to dissociate noncovalent complexes, and monitoring loss of any bound drug spectrophotometrically (see discussion). RESULTS AND DISCUSSION The formation of semiquinone radical intermediates from quinone-containing antitumor drugs in microsomal incubation is well established [11]. Lown et al. [17] have shown that chemical reduction of the mitomycins leads to the formation of the semiquinone radical intermediate and Tomasz et al. [12] have obtained evidence that this radical binds covalently to DNA. The ESR spectra obtained by chemical activation of the anthracycline antitumor drugs are shown in Fig. 1 (daunorubicin) and Fig. 2 (adriamycin). The signal position (g-value) and peak to peak width of observed free radical is characteristic of the drug. Sato et al. [9] have reported g-values of 2.004 for both adriamycin and daunorubicin semiquinone radicals from microsomal activation. Under our conditions of chemical activation and using Fremy's salt as standard, the g-values for adriamycin and daunorubicin semiquinone radicals were found to be 2.0035 and 2.0037 respectively. These g-values suggest that the same free radical intermediates are formed from either microsomal or chemical activation. The partially resolved ESR spectra of daunorubicin (Fig. 1A) and adriamycin (Fig. 2A) result from the hyperfine splittings of aromatic hydrogens and a detailed analysis will be published elsewhere (B.K. Sinha et al., unpublished). Addition of DNA to daunorubicin and adriamycin semiquinone radicals resulted in loss of the hyperfine structure (Fig. 2), suggesting that these semiquinone radicals become immobilized upon binding to DNA. When the DNA-complexes, isolated by ethanol precipitation (from which u n b o u n d drugs were removed), were activated with the reducing agent no semiquinone radical could be detected by ESR. Sato et al.
<
lOG
log
>
<
lOG
B
Fig. 2. The electron spin resonance spectra of adriamycin (1 mg/ml) semiquinone radical in the absence (A) and presence (B) of calf thymus DNA (1 mg/ml) in 5 mM phosphate buffer (pH 7.4).
Fig. 1. The electron spin resonance spectra of daunorubicin (1 mg/ml) semiquinone radical in the absence (A) and presence (B) of calf thymus DNA (1 mg/ml) in 5 mM phosphate buffer (pH 7.4).
<
A
O
305 [9] have also reported that intercalated daunorubicin is not substrate for enzymatic activation suggesting that the quinone part of the intercalated drug is sterically hindered from enzymatic activation. Tomasz et al. [12] have shown that chemical reduction of mitomycin-C leads to the formation of an active species which binds covalently to DNA. In the present study, we demonstrate that similar reduction of adriamycin and daunorubicin also results in covalent binding to DNA. Table I shows the binding ratios for chemically activated semiquinone free radicals from adriamycin and daunorubicin. We have included the binding ratio for mitomycin-C obtained under our experimental conditions for comparative purposes and it is similar to the one obtained by Tomasz [12]. The results in Table I clearly show that adriamycin and daunorubicin, in addition to intercalating into DNA, are bound covalently to the nucleic acid. The striking difference in both the total binding ratios and the covalent binding ratios (Table I) between two similar compounds, adriamycin and daunorubicin, is very surprising. While the reason for this behavior is not clear at this time, the strong covalent binding of adriamycin to DNA may account for the ability of this drug to induce extensive chromosal aberration and increase the frequency of SCE. It is interesting to note that mitomycin-C, another strong inducer of SCE also exhibited a binding ratio similar to that observed with adriamycin (Table I). The covalent binding of adriamycin and daunorubicin to DNA could then explain why there is an absolute requirement for cellular activation before in vivo DNA strand breaks are observed. Furthermore, a higher covalent binding of adriamycin to DNA could also explain its more severe and progressively increased damage to DNA [18].
TABLE I BINDING OF SEMIQUINONE RADICALS TO DNA Drug
Total binding ratioa
Covalent binding ratiob
SCEd/Cell
Adriamycin Daunorubicin Mitomycin-C AdriamycinC
8 30 10 10
12 135 12 75
33 18 99.5
a Total binding ratios (tool nueleotide/mol drug bound) were obtained by analyzing drug-DNA complexes after ethanol precipitation. b Covalent binding (tool nucleotide/mol drug bound) w e r e obtained by dissolving the drug-DNA complexes in 2 M NaC1 and extensive dialysis followed by chromatographic isolation using Sephadex G-100. c Adriamycin was reduced to hydroquinone in one step by adding the reducing agent. d Sister chromatid exchange;the data is taken from ref. 21. Adriamycin and daunorubicin at doses 12 mg/kg and mitomycin C at 5 mg/kg, in bone marrow cells in vivo. Controls had SCE of 5.
306 The c o n c e p t of covalent binding to DNA was established by carrying o u t studies under a variety of conditions known to dissociate noncovalent DNAdrug complexes. Muller and Crothers [19] have shown that SDS dissociates actinomycin-DNA complexes. F u r t h e r m o r e , SDS (0.1--2%) has also been used to dissociate daunorubicin-DNA complexes [13, 14]. Urea dissociates actinomycin from DNA [20] and high ionic strength is also known to break noncovalent drug-DNA complexes [20]. The drug-DNA complexes of adriamycin and daunorubicin isolated in this investigation were found to be stable to the following conditions (a) 5% SDS, 3 h, (b) 7 M urea, 40 min, (c) 2 M NaC1; 1 h and (d) pH 12.0, 15 min. Similar stabilities have been shown for covalent complexes of mitomycin-C with DNA [12]. Thermal denaturation data with adriamycin- and daunorubicin-DNA complexes are presented in Table II. The melting profiles of these complexes were broad. An increase in T m associated with a broader melting profile is characteristic of covalent binding [12]. The small increase of I°C obtained with the daunorubicin-DNA complex may be due to the small a m o u n t of this drug that is covalently b o u n d to DNA. Since adriamycin- and daunorubicin-DNA complexes remained stable u n d er conditions know n to dissociate intercalated complexes and exhibited a characteristic broad melting profile only seen with covalently bound drugs, we conclude that in the presence of a reducing agent adriamycin and daunorubicin bind covalently to DNA. In addition to covalent binding, intercalation of these intermediates into DNA base pairs is also involved as 40% o f adriamycin and 80% of daunorubicin were f o u n d to be intercalated. Covalent binding must follow the intercalation o f the drug semiquinone radicals into DNA. Bachur et al. [11] have suggested that the anthracycline semiquinone radicals f o r m e d by microsomal incubation may interact selectively with nucleic acids. Previously, Tomasz et al. [12] have suggested that the semiquinone f or m of mitomycin-C, rather than the fully reduced form is the biologically active species and this form was subsequently detected by
TABLE II T m DETERMINATION a
DNA DNA-adriamycin b DNA-daunorubicin c
Tin, (°C)
A T m , (°C)
68.5 83.5 69.5
-15.0 1.0
a Tm determinations were carried out in 5 mM phosphate buffer (pH 7.4) at 260 nm by
means of a Gilford 250 recording spectrophotometer with a thermoprogrammer 2527 programmed for a temperature rise of 1.0°C rain. b Binding ratio 1 mol drug/12 mol nucleotide. c Binding ratio 1 tool drug/135 tool nucleotide.
307 E S R [ 1 7 ] . H o w e v e r , d u e t o lack o f a n y d i r e c t E S R evidence, it s e e m s uncertain w h e t h e r t h e fully r e d u c e d or t h e half r e d u c e d f o r m is t h e active f o r m . In this p r e s e n t s t u d y , t h e f o r m a t i o n o f the s e m i q u i n o n e radical interm e d i a t e b y c h e m i c a l a c t i v a t i o n is u n e q u i v o c a l l y p r o v e n t h r o u g h E S R detect i o n a n d c h a r a c t e r i z a t i o n . F u r t h e r m o r e , o u r p r e s e n t E S R studies, suggest t h a t t h e s e s e m i q u i n o n e radical i n t e r m e d i a t e s i n t e r a c t w i t h D N A , as t h e i r h y p e r f i n e s t r u c t u r e is lost u p o n t h e a d d i t i o n o f D N A . This i n t e r a c t i o n p r o b a b l y involves i n t e r c a l a t i v e b i n d i n g m e c h a n i s m as it is highly u n l i k e l y t h a t b i n d i n g at the p h o s p h a t e g r o u p s o f the D N A helix w o u l d r e d u c e the m o t i o n o f the radical so drastically. In o u r a c t i v a t i o n p r o c e d u r e s w h i c h are similar to t h o s e o f T o m a s z et al. [ 1 2 ] , o n l y 0 . 7 5 equivalents o f NaBH4 w e r e a d d e d and h e n c e an excess o f the u n r e d u c e d drugs was always present. In a d d i t i o n , the r e d u c i n g a g e n t was n o t a d d e d at o n c e b u t in five p o r t i o n s at 5-min intervals. This a l l o w e d a build u p o f the s e m i q u i n o n e i n t e r m e d i a t e s at t h e e x p e n s e o f the h y d r o q u i n o n e and resulted in c o v a l e n t binding o f o n e a d r i a m y c i n p e r 12 n u c l e o t i d e s . In c o n t r a s t , w h e n a d r i a m y c i n was fully r e d u c e d t o h y d r o q u i n o n e b y adding t w o equivalents o f t h e r e d u c i n g a g e n t in a single step, a significantly d e c r e a s e d c o v a l e n t binding ratio ( o n e adriam y c i n per 75 n u c l e o t i d e s ) was o b t a i n e d (Table I). O u r findings s t r o n g l y suggest t h a t s e m i q u i n o n e i n t e r m e d i a t e s f r o m a d r i a m y c i n and d a u n o r u b i c i n are the active species w h i c h b i n d c o v a l e n t l y t o D N A . T h e s e findings are i n t e r e s t i n g in c o n n e c t i o n w i t h t h e p o s t u l a t e d m e c h a n i s m o f r e d u c t i v e a c t i v a t i o n o f a d r i a m y c i n and d a u n o r u b i c i n to c o v a l e n t binding agents [ 2 2 ] . ACKNOWLEDGEMENTS T h e a u t h o r s wish to a c k n o w l e d g e the D r u g D e v e l o p m e n t Branch, Division o f Cancer T r e a t m e n t , N a t i o n a l Cancer I n s t i t u t e f o r a s u p p l y o f a d r i a m y c i n and d a u n o r u b i c i n . T h e a u t h o r s also wish to t h a n k Drs. R.P. Mason a n d B. K a l y a n a r a m a n for h e l p f u l discussions. REFERENCES 1 C. Tan, H. Tasaka, K. Yu, M.L. Murphey and D.A. Karnofsky, Daunomycin, an antitumor antibiotic in the treatment of neoplastic disease, Cancer, 20 (1967) 333. 2 M. Boiron, C. Jacquillan, M. Well, J. Tanzer, D. Levy, G. Sultan and J. Bernard, Daunomycin in the treatment of acute myelocytic leukemia, Lancet (1969) 330. 3 S.M. Sieber and R.H. Adamson, Toxicity of antineoplastic agents in man; chromosomal aberrations, antifertility effects, congenital malformations, and carcinogenic potential, Adv. Cancer Res., 22 (1975) 57. 4 A. Dimarco, N. Gaetani, P. Orezzi, B. Scarpinato, R. Silverstrini, M. Soldati, T. Dasdia and L. Valentine, Daunomycin, a new antibiotic of the rhodomycin group, Nature, 201 (1964) 706. 5 A. DiMarco, F. Arcamore and F. Zunino, in: J. Cockran and F.H. Hahn (Eds.), Mechanism of action of antimicrobial and antitumor agents, Springer-Verlag, Berlin, 1975, pp. 101--128. 6 A. Theologides, J. Yarbro and B.J. Kennedy, Daunomycin, inhibition of DNA and RNA synthesis, Cancer, 21 (1968) 16.
308 7 F. Zunino, A. DiMarco, A. Zaccara and G. Juoni, The inhibition of RNA polymerase by daunomycin, Chem-Biol. Interact., 9 (1974) 25. 8 K. Handa and S. Sato, Generation of free radicals of quinone group-containing anticancer chemicals in NADPH-microsome system as evidenced by initiation of sulfite oxidation, Gann, 66 (1975) 43. 9 S. Sato, M. Lwaizumi, K. Handa and Y. Tamura, Electron spin resonance study on the mode of generation of free radicals of daunomycin, adriamycin and carboquinone in NADPH-microsome system, Gann, 68 (1977) 603. 10 N.R. Bachur, S.L. Gordon and M.V. Gee, Antlxacycline antibiotic augmentation of microsomal electron transport and free radical formation, Mol. Pharmacol., 13 (1977 ) 901. 11 N.R. Bachur, S.L. Gordon and M.V. Gee, A general mechanism for microsomal activation of quinone anticancer agents to free radicals, Cancer Res., 38 (1978) 1745. 12 M. Tomasz, C.M. Mercado, J. Olson and N. Chatterjie, The mode of interaction of mitomycin C with deoxyribonucleic acid and other polynucleotides in vitro, Biochemistry, 13 (1974) 9878. 13 W.D. Wilson, D. Grier, R. Reimer, J.D. Bauman, J.F. Preston and E.J. Gabbay, Structure-activity relationship of Daunorubicin and its peptide derivatives, J. Med. Chem. 19, (1976) 381. 14 E.J. Gabbay, D. Grief, R.E. Fingerle, R. Reimer, R. Levy, S.W. Pearce and W.D. Wilson, Interaction specificity of the anthracyclines with deoxyribonucleic acid, Biochemistry, 15 (1976) 2062. 15 R.P. Mason, F.J. Peterson and J.L. Holtzman, The formation of an azo anion free radical metabolite during the microsomal azo reduction of sulfonazo III., Biochem. Biophys. Res. Commun., 75 (1977) 532. 16 B.K. Sinha, P.M. Philen, R. Sato and R. Cysyk, Synthesis and antitumor properties of bis (quinaldine) derivatives, J. Med. Chem., 20 (1977) 1528. 17. J.W. Lown, S.K. Sim and H.H. Chen, Hydroxyl radical production by free and DNAbound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of h y d r o x y l radicals b y spin trapping, Can. J. Biochem. 56 (1978) 1042. 18 H.S. Schwartz, DNA breaks in P-288 t u m o r cells in mice after treatment with daunorubicin and adriamycin, Res. Commun. Chem. Pathol. Pharmacol., 10 (1975) 51. 19 W. Muller and D.M. Crothers, Studies of binding of antinomycin and related compounds to DNA, J. MoL Biol., 35 (1968) 251. 20 H.L. White and J.R. White, Hedamycin and Rubiflavin complexes with deoxyribonucleic acid and other polynucleotides, Biochemistry, 8 (1969) 1030. 21 Y. Nakanishi and E.L. Schneider, in vivo sister chromatid exchange: a sensitive measure of DNA damage, Mutat. Res., 60 (1979) 329. 22 H.W. Moore, Bioactivation as a model for drug design bioreductive alkylation, Science, 197 (1977) 527.
301
BINDING MODE OF CHEMICALLY ACTIVATED SEMIQUINONE FREE RADICALS FROM QUINONE ANTICANCER AGENTS TO DNA
B.K. SINHA* and C.F. CHIGNELL Laboratory of Environmental Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S,A.) (Received February 14th, 1979) (Revision received July l l t h , 1979) (Accepted July 20th, 1979)
SUMMARY
Chemical reduction of the highly active quinone-containing antitumor drugs, adriamycin and daunorubicin formed the same partially reduced free radical previously reported [9] by microsomal activation. In vitro incubation of the chemically activated free radical intermediates with DNA resulted in covalent binding of these drugs to DNA. The adriamycin semiquinone radical has a greater affinity for DNA and covalent complexes up to one adriamycin per 12 nucleotides were obtained. The daunorubicin semiquinone radical, on the other hand, showed a lesser binding affinity and gave rise to complexes in which one drug molecule was covalently bound per 135 nucleotides. The stronger covalent binding of adriamycin to DNA may account for more severe DNA damage induced by this drug.
INTRODUCTION
The anthracycline antitumor drugs, adriamycin and danorubicin, both of which contain the quinone moiety, are currently being used in the treatment of acute leukemia and solid tumors in man [1,2]. In addition, these agents induce damage in DNA as evidenced by an increase both in chromosomal aberrations and in the frequency of sister chromatid exchange (SCE) [3]. The biological activities of these agents seem to result from their ability to intercalate into DNA base pairs by virtue of their planar ring structure [4,5]. Such tight binding to nucleic acids results in the inhibition of both DNA and RNA synthesis [6] and DNA- and RNA
302 microsomal incubations containing NADPH. The formation of semiquinone metabolites of anthracycline antibiotics has been demonstrated using electron spin resonance (ESR) spectroscopy in both anaerobic NADPH-microsomal incubations [9--11] and Ehrlich ascites t u m o r cells [9]. Bachur et al. [11] have suggested that the free radicals may either be sufficiently stable to bind to nuclear DNA and RNA through intercalation, or may generate superoxide which then reacts with DNA. In this paper, we report on the binding mode of the chemically generated semiquinone radicals from adriamycin and daunorubicin. We present, for the first time, evidence for the covalent binding of semiquinone free radicals from adriamycin and daunorubicin to DNA. We have included in this study mitomycin-C semiquinone, which has also been reported to bind covalently to DNA [12]. MATERIALS Adriamycin HC1 (NSC-123, 127) and daunorubicin (NSC-82, 152) were gifts from the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute. Calf thymus DNA was purchased from Sigma Biochemicals. Mitomycin-C was purchased from Aldrich Chemicals. METHODS Adriamycin (1 mg/ml),daunorubicin (1 mg/ml)and mitomycin-C (2 mg/ml) were dissolved in 5 mM phosphate buffer (pH 7.4) and were deaerated by bubbling nitrogen for 10 min. Chemical activation was carried out by adding in 5 equal portions, at 5-min intervals, sodium borohydride solution (2 mg/ml in phosphate buffer, 0.75 equivalent of NaBH4 added) to adriamycin and daunorubicin solutions. Calf thymus DNA (1 mg/ml), dissolved in 5 mM phosphate buffer (pH 7.4) containing 50 mM NaCl, was deaerated by bubbling nitrogen for 10 rain. The DNA solution was added (1 mg DNA/1 mg drug) to the activated adriamycin and daunorubicin and the mixtures were incubated in the dark for 18 h at room temperature. The DNA-drug complexes were isolated by ethanol precipitation and the unbound drugs were removed. The complexes were washed 3 times with ethanol (assays for total bound drugs were carried out on these samples). The DNA-drug complexes were dissolved in 2 M NaC1 solution to dissociate intercalated drugs, stirred for 1 h and reprecipitated in ethanol. After the dissociated drugs were removed, the complexes were redissolved in 2 M NaC1 and dialyzed extensively against 5 mM phosphate buffer containing 1 M NaC1. The DNA-drug complexes were then chromatographed in Sephadex G-100 column (2.0 × 30 cm) using 5 mM phosphate containing 100 mM NaC1 as eluent. Activation of mitomycin-C, was achieved b y mixing DNA (2 mg DNA/1 mg drug) with mitomycin-C, and adding the reducing agent in 5 equal portions at 5min intervals as described by Tomasz et al. [12]. The DNA-mitomycin-C complex was isolated as described for adriamycin and daunorubicin.
303 The assays of the binding ratios in the DNA-drug complexes were carried out spectrophotometrically. The binding ratio is defined here as the molar ratio of mononucleotide unit to the drug. The nucleotide concentration was determined using e260 = 7000 for native DNA [12]. Bound mitomycin-C was determined at 310 nm (e = 11 000) [12]. Bound adriamycin and daunorubicin were determined at 505 nm (e = 6900) [13,14]. The ESR measurements were carried out on a Varian E-109 spectrometer operating at 100 krIz. The samples were introduced into an E-238 Tml~o cavity in quartz fiat cells provided with a syringe in the b o t t o m for mixing. The g-values for adriamycin and daunorubicin semiquinone radicals were measured using Fremy's salt by a m e t h o d similar to one described by Mason et al. [15]. Thermal denaturation studies were carried out in 5 mM phosphate buffer at 260 nm by means of a Gilford 250 recording spectrophotometer with a thermoprogrammer 2527 programmed for a temperature rise of l ° C / m i n according to previously published methods [16]. Control sample contained equivalent amounts of DNA. The stability of the DNA-drug complexes was demonstrated by treating the complex with sodium dodecyl sulfate (SDS) and urea, agents that are known to dissociate noncovalent complexes, and monitoring loss of any bound drug spectrophotometrically (see discussion). RESULTS AND DISCUSSION The formation of semiquinone radical intermediates from quinone-containing antitumor drugs in microsomal incubation is well established [11]. Lown et al. [17] have shown that chemical reduction of the mitomycins leads to the formation of the semiquinone radical intermediate and Tomasz et al. [12] have obtained evidence that this radical binds covalently to DNA. The ESR spectra obtained by chemical activation of the anthracycline antitumor drugs are shown in Fig. 1 (daunorubicin) and Fig. 2 (adriamycin). The signal position (g-value) and peak to peak width of observed free radical is characteristic of the drug. Sato et al. [9] have reported g-values of 2.004 for both adriamycin and daunorubicin semiquinone radicals from microsomal activation. Under our conditions of chemical activation and using Fremy's salt as standard, the g-values for adriamycin and daunorubicin semiquinone radicals were found to be 2.0035 and 2.0037 respectively. These g-values suggest that the same free radical intermediates are formed from either microsomal or chemical activation. The partially resolved ESR spectra of daunorubicin (Fig. 1A) and adriamycin (Fig. 2A) result from the hyperfine splittings of aromatic hydrogens and a detailed analysis will be published elsewhere (B.K. Sinha et al., unpublished). Addition of DNA to daunorubicin and adriamycin semiquinone radicals resulted in loss of the hyperfine structure (Fig. 2), suggesting that these semiquinone radicals become immobilized upon binding to DNA. When the DNA-complexes, isolated by ethanol precipitation (from which u n b o u n d drugs were removed), were activated with the reducing agent no semiquinone radical could be detected by ESR. Sato et al.
<
lOG
log
>
<
lOG
B
Fig. 2. The electron spin resonance spectra of adriamycin (1 mg/ml) semiquinone radical in the absence (A) and presence (B) of calf thymus DNA (1 mg/ml) in 5 mM phosphate buffer (pH 7.4).
Fig. 1. The electron spin resonance spectra of daunorubicin (1 mg/ml) semiquinone radical in the absence (A) and presence (B) of calf thymus DNA (1 mg/ml) in 5 mM phosphate buffer (pH 7.4).
<
A
O
305 [9] have also reported that intercalated daunorubicin is not substrate for enzymatic activation suggesting that the quinone part of the intercalated drug is sterically hindered from enzymatic activation. Tomasz et al. [12] have shown that chemical reduction of mitomycin-C leads to the formation of an active species which binds covalently to DNA. In the present study, we demonstrate that similar reduction of adriamycin and daunorubicin also results in covalent binding to DNA. Table I shows the binding ratios for chemically activated semiquinone free radicals from adriamycin and daunorubicin. We have included the binding ratio for mitomycin-C obtained under our experimental conditions for comparative purposes and it is similar to the one obtained by Tomasz [12]. The results in Table I clearly show that adriamycin and daunorubicin, in addition to intercalating into DNA, are bound covalently to the nucleic acid. The striking difference in both the total binding ratios and the covalent binding ratios (Table I) between two similar compounds, adriamycin and daunorubicin, is very surprising. While the reason for this behavior is not clear at this time, the strong covalent binding of adriamycin to DNA may account for the ability of this drug to induce extensive chromosal aberration and increase the frequency of SCE. It is interesting to note that mitomycin-C, another strong inducer of SCE also exhibited a binding ratio similar to that observed with adriamycin (Table I). The covalent binding of adriamycin and daunorubicin to DNA could then explain why there is an absolute requirement for cellular activation before in vivo DNA strand breaks are observed. Furthermore, a higher covalent binding of adriamycin to DNA could also explain its more severe and progressively increased damage to DNA [18].
TABLE I BINDING OF SEMIQUINONE RADICALS TO DNA Drug
Total binding ratioa
Covalent binding ratiob
SCEd/Cell
Adriamycin Daunorubicin Mitomycin-C AdriamycinC
8 30 10 10
12 135 12 75
33 18 99.5
a Total binding ratios (tool nueleotide/mol drug bound) were obtained by analyzing drug-DNA complexes after ethanol precipitation. b Covalent binding (tool nucleotide/mol drug bound) w e r e obtained by dissolving the drug-DNA complexes in 2 M NaC1 and extensive dialysis followed by chromatographic isolation using Sephadex G-100. c Adriamycin was reduced to hydroquinone in one step by adding the reducing agent. d Sister chromatid exchange;the data is taken from ref. 21. Adriamycin and daunorubicin at doses 12 mg/kg and mitomycin C at 5 mg/kg, in bone marrow cells in vivo. Controls had SCE of 5.
306 The c o n c e p t of covalent binding to DNA was established by carrying o u t studies under a variety of conditions known to dissociate noncovalent DNAdrug complexes. Muller and Crothers [19] have shown that SDS dissociates actinomycin-DNA complexes. F u r t h e r m o r e , SDS (0.1--2%) has also been used to dissociate daunorubicin-DNA complexes [13, 14]. Urea dissociates actinomycin from DNA [20] and high ionic strength is also known to break noncovalent drug-DNA complexes [20]. The drug-DNA complexes of adriamycin and daunorubicin isolated in this investigation were found to be stable to the following conditions (a) 5% SDS, 3 h, (b) 7 M urea, 40 min, (c) 2 M NaC1; 1 h and (d) pH 12.0, 15 min. Similar stabilities have been shown for covalent complexes of mitomycin-C with DNA [12]. Thermal denaturation data with adriamycin- and daunorubicin-DNA complexes are presented in Table II. The melting profiles of these complexes were broad. An increase in T m associated with a broader melting profile is characteristic of covalent binding [12]. The small increase of I°C obtained with the daunorubicin-DNA complex may be due to the small a m o u n t of this drug that is covalently b o u n d to DNA. Since adriamycin- and daunorubicin-DNA complexes remained stable u n d er conditions know n to dissociate intercalated complexes and exhibited a characteristic broad melting profile only seen with covalently bound drugs, we conclude that in the presence of a reducing agent adriamycin and daunorubicin bind covalently to DNA. In addition to covalent binding, intercalation of these intermediates into DNA base pairs is also involved as 40% o f adriamycin and 80% of daunorubicin were f o u n d to be intercalated. Covalent binding must follow the intercalation o f the drug semiquinone radicals into DNA. Bachur et al. [11] have suggested that the anthracycline semiquinone radicals f o r m e d by microsomal incubation may interact selectively with nucleic acids. Previously, Tomasz et al. [12] have suggested that the semiquinone f or m of mitomycin-C, rather than the fully reduced form is the biologically active species and this form was subsequently detected by
TABLE II T m DETERMINATION a
DNA DNA-adriamycin b DNA-daunorubicin c
Tin, (°C)
A T m , (°C)
68.5 83.5 69.5
-15.0 1.0
a Tm determinations were carried out in 5 mM phosphate buffer (pH 7.4) at 260 nm by
means of a Gilford 250 recording spectrophotometer with a thermoprogrammer 2527 programmed for a temperature rise of 1.0°C rain. b Binding ratio 1 mol drug/12 mol nucleotide. c Binding ratio 1 tool drug/135 tool nucleotide.
307 E S R [ 1 7 ] . H o w e v e r , d u e t o lack o f a n y d i r e c t E S R evidence, it s e e m s uncertain w h e t h e r t h e fully r e d u c e d or t h e half r e d u c e d f o r m is t h e active f o r m . In this p r e s e n t s t u d y , t h e f o r m a t i o n o f the s e m i q u i n o n e radical interm e d i a t e b y c h e m i c a l a c t i v a t i o n is u n e q u i v o c a l l y p r o v e n t h r o u g h E S R detect i o n a n d c h a r a c t e r i z a t i o n . F u r t h e r m o r e , o u r p r e s e n t E S R studies, suggest t h a t t h e s e s e m i q u i n o n e radical i n t e r m e d i a t e s i n t e r a c t w i t h D N A , as t h e i r h y p e r f i n e s t r u c t u r e is lost u p o n t h e a d d i t i o n o f D N A . This i n t e r a c t i o n p r o b a b l y involves i n t e r c a l a t i v e b i n d i n g m e c h a n i s m as it is highly u n l i k e l y t h a t b i n d i n g at the p h o s p h a t e g r o u p s o f the D N A helix w o u l d r e d u c e the m o t i o n o f the radical so drastically. In o u r a c t i v a t i o n p r o c e d u r e s w h i c h are similar to t h o s e o f T o m a s z et al. [ 1 2 ] , o n l y 0 . 7 5 equivalents o f NaBH4 w e r e a d d e d and h e n c e an excess o f the u n r e d u c e d drugs was always present. In a d d i t i o n , the r e d u c i n g a g e n t was n o t a d d e d at o n c e b u t in five p o r t i o n s at 5-min intervals. This a l l o w e d a build u p o f the s e m i q u i n o n e i n t e r m e d i a t e s at t h e e x p e n s e o f the h y d r o q u i n o n e and resulted in c o v a l e n t binding o f o n e a d r i a m y c i n p e r 12 n u c l e o t i d e s . In c o n t r a s t , w h e n a d r i a m y c i n was fully r e d u c e d t o h y d r o q u i n o n e b y adding t w o equivalents o f t h e r e d u c i n g a g e n t in a single step, a significantly d e c r e a s e d c o v a l e n t binding ratio ( o n e adriam y c i n per 75 n u c l e o t i d e s ) was o b t a i n e d (Table I). O u r findings s t r o n g l y suggest t h a t s e m i q u i n o n e i n t e r m e d i a t e s f r o m a d r i a m y c i n and d a u n o r u b i c i n are the active species w h i c h b i n d c o v a l e n t l y t o D N A . T h e s e findings are i n t e r e s t i n g in c o n n e c t i o n w i t h t h e p o s t u l a t e d m e c h a n i s m o f r e d u c t i v e a c t i v a t i o n o f a d r i a m y c i n and d a u n o r u b i c i n to c o v a l e n t binding agents [ 2 2 ] . ACKNOWLEDGEMENTS T h e a u t h o r s wish to a c k n o w l e d g e the D r u g D e v e l o p m e n t Branch, Division o f Cancer T r e a t m e n t , N a t i o n a l Cancer I n s t i t u t e f o r a s u p p l y o f a d r i a m y c i n and d a u n o r u b i c i n . T h e a u t h o r s also wish to t h a n k Drs. R.P. Mason a n d B. K a l y a n a r a m a n for h e l p f u l discussions. REFERENCES 1 C. Tan, H. Tasaka, K. Yu, M.L. Murphey and D.A. Karnofsky, Daunomycin, an antitumor antibiotic in the treatment of neoplastic disease, Cancer, 20 (1967) 333. 2 M. Boiron, C. Jacquillan, M. Well, J. Tanzer, D. Levy, G. Sultan and J. Bernard, Daunomycin in the treatment of acute myelocytic leukemia, Lancet (1969) 330. 3 S.M. Sieber and R.H. Adamson, Toxicity of antineoplastic agents in man; chromosomal aberrations, antifertility effects, congenital malformations, and carcinogenic potential, Adv. Cancer Res., 22 (1975) 57. 4 A. Dimarco, N. Gaetani, P. Orezzi, B. Scarpinato, R. Silverstrini, M. Soldati, T. Dasdia and L. Valentine, Daunomycin, a new antibiotic of the rhodomycin group, Nature, 201 (1964) 706. 5 A. DiMarco, F. Arcamore and F. Zunino, in: J. Cockran and F.H. Hahn (Eds.), Mechanism of action of antimicrobial and antitumor agents, Springer-Verlag, Berlin, 1975, pp. 101--128. 6 A. Theologides, J. Yarbro and B.J. Kennedy, Daunomycin, inhibition of DNA and RNA synthesis, Cancer, 21 (1968) 16.
308 7 F. Zunino, A. DiMarco, A. Zaccara and G. Juoni, The inhibition of RNA polymerase by daunomycin, Chem-Biol. Interact., 9 (1974) 25. 8 K. Handa and S. Sato, Generation of free radicals of quinone group-containing anticancer chemicals in NADPH-microsome system as evidenced by initiation of sulfite oxidation, Gann, 66 (1975) 43. 9 S. Sato, M. Lwaizumi, K. Handa and Y. Tamura, Electron spin resonance study on the mode of generation of free radicals of daunomycin, adriamycin and carboquinone in NADPH-microsome system, Gann, 68 (1977) 603. 10 N.R. Bachur, S.L. Gordon and M.V. Gee, Antlxacycline antibiotic augmentation of microsomal electron transport and free radical formation, Mol. Pharmacol., 13 (1977 ) 901. 11 N.R. Bachur, S.L. Gordon and M.V. Gee, A general mechanism for microsomal activation of quinone anticancer agents to free radicals, Cancer Res., 38 (1978) 1745. 12 M. Tomasz, C.M. Mercado, J. Olson and N. Chatterjie, The mode of interaction of mitomycin C with deoxyribonucleic acid and other polynucleotides in vitro, Biochemistry, 13 (1974) 9878. 13 W.D. Wilson, D. Grier, R. Reimer, J.D. Bauman, J.F. Preston and E.J. Gabbay, Structure-activity relationship of Daunorubicin and its peptide derivatives, J. Med. Chem. 19, (1976) 381. 14 E.J. Gabbay, D. Grief, R.E. Fingerle, R. Reimer, R. Levy, S.W. Pearce and W.D. Wilson, Interaction specificity of the anthracyclines with deoxyribonucleic acid, Biochemistry, 15 (1976) 2062. 15 R.P. Mason, F.J. Peterson and J.L. Holtzman, The formation of an azo anion free radical metabolite during the microsomal azo reduction of sulfonazo III., Biochem. Biophys. Res. Commun., 75 (1977) 532. 16 B.K. Sinha, P.M. Philen, R. Sato and R. Cysyk, Synthesis and antitumor properties of bis (quinaldine) derivatives, J. Med. Chem., 20 (1977) 1528. 17. J.W. Lown, S.K. Sim and H.H. Chen, Hydroxyl radical production by free and DNAbound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of h y d r o x y l radicals b y spin trapping, Can. J. Biochem. 56 (1978) 1042. 18 H.S. Schwartz, DNA breaks in P-288 t u m o r cells in mice after treatment with daunorubicin and adriamycin, Res. Commun. Chem. Pathol. Pharmacol., 10 (1975) 51. 19 W. Muller and D.M. Crothers, Studies of binding of antinomycin and related compounds to DNA, J. MoL Biol., 35 (1968) 251. 20 H.L. White and J.R. White, Hedamycin and Rubiflavin complexes with deoxyribonucleic acid and other polynucleotides, Biochemistry, 8 (1969) 1030. 21 Y. Nakanishi and E.L. Schneider, in vivo sister chromatid exchange: a sensitive measure of DNA damage, Mutat. Res., 60 (1979) 329. 22 H.W. Moore, Bioactivation as a model for drug design bioreductive alkylation, Science, 197 (1977) 527.