Antitumor activity of the Cu(II)-mitoxantrone complex and its interaction with deoxyribonucleic acid

ELSEVIER

Antitumor Activity of the Cu(ll)-Mitoxantrone Complex and Its Interaction with Deoxyribonucleic Acid Pin Yang, Hongfei Wang, Fei Gao, and Bingsheng Yang PY. Department of Chemistry, Nanjing University, Nanjing, P. R~ China.--HW, FG, BY. Institute of Molecular Science, Shanxi University, Ta~uan, P. 1~ China

ABSTRACT The effect of the Cu(II)-mitoxantrone complex on the DNA synthesis of HL-60 human leukemia cells has been studied by the technique of isotopic liquid scintillation. The results indicated that the complex shows a stronger ability to inhibit DNA synthesis of the tumor cells, and thus it may become a better antitumor drug. The interaction of mitoxantrone and its Cu(II) complex was studied by the methods of electrochemistry and spectroscopy. The complex gives rise to more changes on the conformation and the double-helical structure of DNA; this is closely related to the antitumor mechanism of the complex.

INTRODUCTION A n t h r a c e n e d i o n e m i t o x a n t r o n e (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxethyl)amino]-ethyl]-amino]-9,10-anthracynedione dihydrochloride, MX), a new synthetic analog of the anthracycline antibiotics, has shown significant clinical effectiveness in the treatment of breast cancer, lymphoma, and acute leukemia [1]. In contrast to other anthracycline antitumor drugs, mitoxantrone produces fewer side effects such as cardiactoxicity [2]. The structure of mitoxantrone is shown in Scheme 1. Studies of cell biology and biochemistry have indicated that nucleic acids are the main cell target of mitoxantrone; it binds to D N A with high affinity, at least in part by intercalation, but via electrostatic interactions as well [3, 4]. Mitoxantrone has been shown to induce compaction of isolated chromatin [5], as well as protein-associated D N A cleavage [6], and to inhibit macromolecular biosynthesis in a number of tumor cell lines [7]. The most dominant molecular mechanism of antitumor action of mitoxantrone appears to be the induction of long-term D N A damage. Address reprint requests and correspondence tO: Dr. Hongfei Wang, Institute of Molecular Science, Shanxi University, Taiyuan, P.R. China.

Journalof InorganicBiochemistry,62, 137-145 (1996) © 1996Elsevier ScienceInc., 655 Avenue of the Americas, NY, NY 10010

0162-0134/96/$15.00 SSDI 0162-0134(95)00130-G

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HO

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NI ICH2CH~JHCH2CII.jOH

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NHCH2CH~NIICII2CII20H

SCHEME 1.

Structure of mitoxantrone.

The complexation of anthracycline drugs with metal ions may be developing a new class of antitumor compounds [8, 9]. This drug-to-metal complexation may have several effects; among the most important are: (i) the modification of the redox properties of the drug, which may be related to its cardiotoxicity; and (ii) the modification of the polarity of the drug, giving rise to a modification of its interaction with cell components such as membranes and nucleic acids. Copper is an important life element. Much research has indicated that the content of copper in the environment is related to tumor, and many compounds of copper with antitumor activity, such as Cu-DIP, Cu-ATP, Cu-Amino Acid, CuSO 4, Cu(AC) 2, etc., have been found. In this paper, the antitumor activity of the Cu(II) mitoxantrone complex was determined first, and then the different interaction models of mitoxantrone and Cu(II)-mitoxantrone complex were studied by electrochemical and UV, CD spectrophotometric methods. The action mechanism of the complex was analyzed.

EXPERIMENTAL Reagents and Apparatus Mitoxantrone was provided by the Zhenan Pharmaceutical Plant, China. C u C 1 2 • 2H20 is the product of Tianjin No. 3 Chemical Plant. Calf thymus DNA was the product of Sigma. 3H-labeled thymidine was purchased from the Institute of Atomic Energy, China. HL-60 cells were obtained from the Institute of Cancer Research, Shanxi. The other chemical reagents were of A. R. grade, and deionized water was used. The concentrations of DNA and MX solution were determined spectrophotometrically at 260 nm (~ = 6600 cm -1 .nucleotide-1) and 682 nm (e = 8360 cm- 1. M- 1), respectively. Absorption spectra were measured on a Shimadzu UV-365 spectrophotometer, the fluorescence spectra on a HITACHI RF-850 spectrofluorimeter. Circular dichroism spectra were recorded on a JASCO J-500C spectropolarimeter. The potentials were scanned on a JP-2 single-sweep oscillopolarograph (Chengdu Instrument Factory, China) with a saturated calomel electrode (SCE), and a BAS-100A electrochemical analytical instrument (U.S.) with saturated Ag/AgC1 electrode as reference electrode. All the measurements were carried out at room temperature. Radioactivity was determined on PACKARD-Tri-Carb 2200CA liquid scintillation detector. Procedure DNA synthesis were assessed by monitoring cellular incorporation of 3H-labeled thymidine. HL-60 human leukemia cells were diluted with PRMI 1640 medium to 5 × 105 cells/mL cell suspension. Comparing with the same concentration of

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FIGURE 1. Absorption spectra of different molar ratios of Cu(II)-MX. 1. 1.0xl0 -5 M MX, 2. 1:1, 3. 2:1, 4. 3:1,5.4:1.

MX, when the molar ratio of [Cu(II)]/[MX] is 1 : 1 or 2 : 1, six concentrations: 5 x 1 0 -5 M, 1 x 1 0 -5 M, 5 x 1 0 -6 M, 1 x l 0 -6 M, 5)<10 -7 M, l x l 0 -7 M were selected, respectively. 100 /zL drug and 100 /zL cell suspension were transferred in each well of a 96-well plate. The plates were incubated at 37°C under CO 2 for 1 hr, and another 1 hr after addition of 3H-thymidine (15.0 /~Ci/nmol). The cells were washed with salt water and then filtered through Millipore AP filters. The filters were dried by vacuum at 80°C, and associated radioactivity was determined after addition of a scintillation mixture. Every sample was triplicated in a parallel manner, and average values were used. The rates of incorporation were calculated by the value of CPM, shown by T / C % (CPM of drug/CPM of drug free). We added Cu(II) to the solution of DNA-MX, adjusted to pH 7.0, 0.02 M NH3-NH4CL as electrolyte. When the ratio of concentration [Cu(II)]/[MX] was 1 : 1 or 1 : 2, the potentials were scanned in a negative direction over the range of 0.0 to - 1.0 V; absorption spectra and circular dichroism spectra were measured, respectively. Spectra of CD are expressed in terms of A e = ~/~ - ~R (molar CD coefficient). RESULTS AND DISCUSSION Characteristics of Complexation By adding Cu(II) to a 1 × 10 -5 M MX solution, absorption spectra and fluorescent spectra changed obviously, as shown in Figures 1 and 2. When the molar ratio of [Cu(II)]/[MX] increased to 2:1, absorption spectra do not change obviously, and the fluorescent peak of emission was quenched completely. By adding Cu(II) to a 1 x 10 -5 M MX solution, a new reduction peak appeared at -0.275 V (vs. Ag/Agcl), which corresponds to the reduction potential of the Cu(II) ion of the complex, and the peak of - 0.817 V (vs. Ag/AgCI) corresponds to the reduction potentials of the 9,10-quinone of MX [10], as shown in Figure 3. Adding MX to a 1 × 10 -5 M Cu(II) solution determined the height of peak current at -0.275 V; when [MX]/[Cu(II)] equaled 1:2, the height did not change, and then leveled off. This indicated that one molecule of drug can bind

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FIGURE 2. Fluorescence spectra of different molar ratios of Cu(II)-MX. 1. 1.0× 10 -5 M MX, 2. 1 : 1 , 3 . 2 : 1 , 4 . 3 : 1 , 5.4:1.

with two Cu(II) ions. The height of peak current at -0.275 V increased by adding an amount of the Cu(II), and then leveled off; however, the peak potential and peak current of -0.817 V mostly do not change. This showed that Cu(II) ions complex with nitrogens of the side-chain of MX, but not complex with the oxygen atoms of the hydroxyl groups at the 1,4 position and oxygen atoms of the carbonyl function on C-9 and C-10. The Pd(II)-MX complex has been studied by analyzing the absorption spectra of MX and ametantron (a drug without 1,4-hydroxyal compared with MX) [9]; the results showed that Pd(II) ions are only bound to the nitrogens of the side-chain. These studies are similar to ours, and support our points as well. The nitrogen atoms of the drug molecule

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are candidates for binding to Cu(II); taking into the steric hindrace and Cu(II)-amine complexes are usually tetra-coordinate. We suggest that one Cu(II) ion is bound to the two nitrogens of the side-chain on C-5 of the drug molecule, the second Cu(II) ion to the two nitrogen atoms of the side-chain on C-8 of the drug molecule; the other two coordination positions of Cu(II) are NH 3 or H20. A tetrahedral coordination geometry is preferable. Antitumor Activity of Cu(II)-MX Complex 3H-labeled thymidine is the precursor of DNA synthesis. HL-60 cells were incubated with an incubation solution, and by adding certain concentrations of drugs, respectively; the effect of the drug on the DNA biosynthesis can be determined by the incorporation rate of thymidine into cells [11]. The results of scintillation determination are shown in Table 1; it indicates that the incorporation of 3H-TdR is inhibited further by complex than MX was, namely, the Cu(II)-MX complex showed stronger inhibition ability on the DNA synthesis. Thus, the Cu(II)-MX complex may become a better antitumor drug, and the activity of Cu(II)-MX(I:I) complex is more potent than that of the Cu(II)MX(2 : 1) complex. When the sample of Cu(II)-MX(2: 1) is laid away for several hours, the precipitate of fine powder can be found; it may be unable to transport through the membrane, which will bring about a reduction of the pharmaceutical effect. Study on the Interaction Model of Cu(I/)-MX Complex with DNA Similar to other anthracycline-type drugs such as daunorubicin and adriamycin, MX possesses a planar geometry, the drug molecular intercalates between the base pairs of DNA, with the positively charged amine groups involved in an electrostatic interaction with neighboring phosphate groups [4]. The complexation of Cu(II) is equated to the structure modification of the drug; analysis of the different action models between them will be helpful to elucidate the mechanism of antitumor as well.

LSV Study. Native DNA is not reducible at the mercury electrode because the stability of the intact double helix makes the reducible bases inaccessible to the electrode. In NH3-NH4CI electrolyte, pH 7.0, only one reduction peak of MX appeared at - 0.870 V (vs. SCE) over the range of 0 to - 1.0 V. When adding MX to the solution of DNA, the peak of the -0.870 V decreased to one very low in height, shown in Figure 4. We think that MX intercalates into the base pairs of DNA by the anthracenedione planar, and cannot be accessible to the electrode easily, thus causing the peak to decrease greatly. Adding Cu(II) ions to

TABLE 1. The Effect of MX and Cu(II)-MX Complex on the Incorporation of 3H-TdR into HL-60 Cell, Expressed as T/C% Concentration of MX/M Compound MX Cu(II)-MX(1 : 1) Cu(II)-MX(2 : 1)

5.0x10 -5 1.0×10 -5 11.1% 5.7% 5.9%

24.7% 19.7% 21.5%

5.0)<10 -6 35.5 % 29.7% 32.1%

1.0xl0 64.7% 46.8% 58.8%

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87.9% 87.4% 88.8%

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E(VOLT) F I G U R E 4. Linear sweep voltammogram of system of MX-Cu(II)-DNA. I. 1.0 X 10 -5 M M X + 1.0 × 10 -4 M D N A . 2. 1.0 x 10 -5 M M X + 1.0 x 10-4 M D N A + 1.0 x 10 -5 M Cu(II). 3. 1.0× 10 -5 M M X + 1.0× 10 -4 M D N A + 2.0 × 10 -5 M Cu(II). 4. 1.0x 10 -5 M MX.

solution of MX-DNA, the peak of -0.870 V increased gradually. This indicated that the binding strength of intercalation was weakened due to the complexation of Cu(II). Part of the plane of MX may be dragged from the bases of DNA.

UVStudy. When MX binding with DNA by intercalation, the absorption peak of MX red shifted [4], as shown in Figure 5. There is no absorption band of DNA in the visible region; the change of absorption spectra in the visible region

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~,(nm) F I G U R E 5. Absorption spectra of system of MX-Cu(II)-DNA. I. 1.0 × 10 -5 M M X . 2. 1 . 0 × 10 -5 M M X + 1 . 0 × 10 -4 M D N A . 3. 1 . 0 × 10 - s M M X + 1 . 0 × 10 -4 M D N A + 1.0 x 10 -5 M Cu(II). 4. 1.0 × 10 -5 M M X + 1.0 × 10 -4 M D N A + 2.0 x 10 -5 M Cu(II). 5. 1.0 × 10 -5 M M X + 2.0 × 10 -5 M Cu(II) + 1.0 × 10 -4 M D N A .

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reflects the effect of the binding environment on the drug molecular. By adding Cu(II) to the solution of MX-DNA, the absorption spectra of this system decreased gradually. These indicates that the two side-chains were free from the bone of DNA phosphate to some degree; after MX intercalated into the base pairs by the anthracenedion planar, they can still bind with Cu(II) ions. The Cu(II) ion was combined with nitrogens of the side-chain; this affected the chromophore on MX, leading to the decrease of the absorption peak. The absorption spectra of MX first complexed with Cu(II), then binding with DNA, curve 5 in Figure 5, and that of MX first binding with DNA, then adding Cu(II), curve 4 in Figure 5, are very dose. We can deduce that Cu(II) only complexes with nitrogen atoms of the side-chain, but does not complex with oxygen atoms of hydroxyl group and carbonyl function by this because if MX binds with DNA first, oxygen atoms on the planar ring were packed; then adding Cu(II), it cannot complex with oxygen atom; the change of spectra will be different from that of the MX complex with Cu(II) first and then adding DNA. Some hard acid ions, such as Fe(III) and Ce(IV) ions, bind with oxygen atoms of the aryl ring of MX; if MX complexed with metal ions first and then was added to the solution of DNA, the spectra should be obviously different from that of MX-DNA, but if MX bound with DNA first and then added metal ions, the spectra of the system did not change.

CD Study. Calf thymus DNA is a B-form helical conformation, while MX is not chiral; no characteristic of circular dichroism is displayed. The measurements of CD spectra are shown in Figure 6; the change of CD spectra reflects the effect of drugs on the conformation of DNA. When MX binds with DNA, the positive band at 275 nm of DNA increases greatly, and the negative band at 245 nm +2 0

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X (nm) FIGURE 6. Circular dichroism spectra of system of MX-Cu(II)-DNA. 1. 1.0 x 10-5 M MX. 2. 1.0 x 10-4 M DNA. 3. 1.0 × 10-5 M MX + 1.0 × 10-4 M DNA. 4. 1.0 x 10-5 M MX+ 1.0x 10-5 M Cu(II) + 1.0x 10-4 M DNA. 5. 1.0× 10-5 M MX+ 2.0x 10-5 M Cu(II) + 1.0 × 10 - 4 M DNA.

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decreases obviously; this indicates that binding with MX leads to the conformation transition of DNA from B to A [12]. When adding Cu(II) to the solution of MX and DNA, there is a red shift from 275 to 280 nm for positive CD band and 237 to 247 nm for negative CD band, both losing intensity seriously; this suggests a decrease in helicity and denaturation of DNA [13]. So, the complexation of Cu(II) leads to further denaturation of DNA. The transition metal ions interacted preferentially with GC sites by chelation to the N-7 of guanine and to the phosphate residue [14], so we think that a binding model of ternary complex, DNA (base or phosphate)--Cu(II)-MX (sidechain) is formed. Complexation of Cu(II) with MX increased the ability of MX to change the conformation of DNA and to cause denaturation of DNA; this is a plausible action mechanism for the complex. APPENDIX Blank(cells and culture medium) CPM of drug free: 3032 3068 2876 CPM of different concentration and molar ratio 5 x 10 -5 M MX 332 364 (11.1%) 304

MX-Cu (0.5 : 1) 212 324 (8.7%) 248

MX-Cu (1 : 1) 190 182 (5.7%) 144

MX-Cu (1.5 : 1) 152 184 (5.9%) 192

MX-Cu (2 : 1) 156 175 (5.8%) 192

1 x 10 -5 M MX 750 782 (24.7%) 690

MX-Cu (0.5: 1) MX-Cu (1 : 1) 528 590 588 (19.2%) 620 (19.7%) 616 562

MX-Cu (1.5: 1) MX-Cu (2: 1) 586 692 642 (21.3%) 602 (21.5%) 680 638

5 x 10 -6 M MX 1150 1096 (35.5%) 944

MX-Cu (0.5: 1) MX-Cu (1 : 1) 992 928 892 (29.9%) 842 (29.7%) 798 896

MX-Cu (1.5 : 1) 812 980 (30.8%) 972

MX-Cu (2: 1) 906 1040 (32.1%) 936

1 x 10 -6 M MX 2028 1802 (64.8%) 1982

MX-Cu (0.5 : 1) 1068 1472 (46.5%) 1632

MX-Cu (1 : 1) 1312 1408 (46.8%) 1476

MX-Cu (1.5 : 1) 1700 1804 (57.8%) 1682

MX-Cu (2 : 1) 1612 1808 (58.8%) 1846

5 X 10 -7 M MX 2194 2124 (73.2%) 2256

MX-Cu (0.5 : 1) 2140 2102 (70.5%) 2086

MX-Cu(1 : 1) 2190 2224 (72.0%) 2046

MX-Cu (1.5 : 1) 2280 2224 (74.1%) 2150

MX-Cu (2 : 1) 2230 2302 (75.0%) 2206

1 X 10 -7 M MX 2458 2604 (87.9%) 2828

MX-Cu (0.5 : 1) 2336 2498 (83.0%) 2620

MX-Cu (1 : 1) 2536 2602 (87.4%) 2708

MX-Cu (1.5 : 1) 2684 2712 (88.2%) 2620

MX-Cu (2 : 1) 2506 2818 (88.8%) 2646

This work was supported by the National Science Foundation of China and Key Lab of Coordination Chemistry at Nanjing University. We are thankful for the help of Prof. Yong~en Zhou, Beijing University, who helped us finished the determination of CD spectra.

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Received April 27, 1995; accepted June 11, 1995