J. Photochem
Photobiol. B: Biol., 15 (1992) 317-335
317
Photosensitization by anticancer agents 11. Mechanisms of photosensitization of human leukemic cells by diaminoanthraquinones: singlet oxygen and radical reactions Krzystzof
J. Reszka,
P. Bilski
and
Colin
F. Chignell
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (USA) John
A. Hartley,
Naem
Khan
and
Robert
L. Souhami
Department of Oncology, University College and Middlesex School of Medicine, 91 Riding House Street, London, WIP 8BT (UK) Aubrey
J. Mendonca
and J. William
Lownt
Department of Chemistry, Universi@ of Alberta, Edmonton, Alta., T6G 2G2 (Canada) (Received
December
3, 1991; accepted
March 13, 1992)
Abstract The synthesis of several aminoanthraquinone derivatives (AAQs), designed to suppress the dark toxicity and to promote more efficient cancer cell photosensitization for potential use in photodynamic therapy (PDT), is described. The following AAQs were synthesized: l-NH,-4,5-(MeO),-AQ (l),1,5-(NH&-4,8-(Me0)2-AQ (2), 1,8-(NH&-4,5-(MeO),-AQ (3), and 1,5-(NHPhMe),-4,8-(MeO),-AQ (8). The agents exhibit strong absorption in the region 480-620 nm. Possible mechanisms of photosensitization were studied by measuring ‘Ox phosphorescence at 1270 nm, detecting superoxide radicals employing an electron paramagnetic resonance (EPR)-spin trapping technique, and measuring oxygen consumption during the photo-oxidation of a representative biological electron donor, NADH. Strong phosphorescence from ‘02 was observed upon illumination of 2 and 3 in Cd-& (quantum yield of 0.25 and 0.5 respectively), and in EtOH (quantum yield of 0.23 and 0.34). The 1-amino-AQ (1)was the weakest ‘02 sensitizer, with quantum yield of 0.13 in benzene. No phosphorescence was observed in EtOH. A superoxide radical was detected as a spin adduct of SJ-dimethyl-1-pyrroline-N-oxide (DMPO) in irradiated benzene solutions of 1, 2 or 3 and DMPO. AAQs 2 and 3 sensitized photo-oxidation of NADH in H20/EtOH mixture with the intermediacy of singlet oxygen as judged by the effect of sodium azide on the photostimulated oqgen consumption. Evolution of O2 upon addition of catalase to the illuminated solution confirmed the ultimate formation of hydrogen peroxide. These tindings suggested that the (di)amino-dimethoxyanthraquinones might exert photosensitization via both Type I and Type II mechanisms. The AAQs were tested for their ability to photosensitize K562 human chronic myeloid leukemic cells in culture. Viability was measured using the 3,4,5-diethylthiazol-2,5-diphenyl tetrazolium blue assay, and DNA and possible membrane damage were assessed. The results from illuminating cells with light >475 nm show that for tbe l,S-compounds, the presence of methoxy substituents at 4,8 positions reduces the dark toxicity from IDS,, of 23 to 250 PM and for the l&compounds correspondingly from IDw, of 53 to > 300 PM. In the l&series ,this decrease of the dark ‘Author
to whom correspondence
loll-1344/92/$.5.00
should be addressed.
0 1992 - Elsevier Sequoia. All rights reserved
318
toxicity is accompanied by an increase in light-induced dose modification from 8.85 to 14.4.Differences exist in the mechanisms of cytotoxicity between the prototype phenolic AAQs and their methoxy counterparts. It appears that the cytotoxic action of the latter causes cell damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks. No evidence for membrane damage, as determined by transport of the model amino acid cycloleucine, could be observed even at supralethal doses.
Keywords: Aminoanthraquinones, photosensitization, superoxide radical, leukemic cells.
singlet oxygen, spin trapping,
1. Introduction
Photosensitization [l] has been demonstrated by several chemotherapeutic agents including a hematoporphyrin derivative [2,3], anthracyclines [4, 51 and anthrapyraxoles and related structures [6-121. In the case of the hematoporphyrin derivative, this has found a practical application in the photodynamic therapy of cancer [l, 21. With the intensely colored l+diamino-substituted antitumor anthraquinones mitoxantrone 6 [13, 141 and ametantrone 7 [14] (Fig. l), although drug-sensitized decarboxylation of peptides has been observed upon ultraviolet (W) (313 nm) irradiation [15], visible light does not initiate any photosensitized reaction with either compound [16]. In contrast, however, 1,5- and 1,8-diamino-substituted anthraquinones structurally related to ametantrone (AM1 and AM2, respectively, Fig. 1) have been shown to cause oxygen consumption and the formation of superoxide radicals and hydrogen peroxide upon visible light illumination of aerated samples in the presence of a representative biological electron donor, NADH [16]. In the presence of DNA these compounds caused the formation of single-strand breaks upon light exposure, and a correlation between the
RI
R2
R3
R4
1
MAMI
NHz
0CH3
0CH3
H
2
MAW.5
NH2
0CH3
NH;?
OCH3
3
MAMI.8
NH2
OCH3
OCH3
NH2
NH(CH~)~N(CHZCH~)~
H
Rl
H
NHKH2hNWWH3h
4
AM1
H
H
RI
6
Mitoxanaone NH(CHZ)ZNH(CH&OH
Rl
OH
OH
7
Ametantmne NH(CH2)2NH(CH2)20H
Rl
H
H
SAM2
Fig. 1. Structures of the anthracenediones
used in this study.
319
extent of DNA damage, oxygen consumption, and NADH oxidation was established in cell-free systems [12]. Recently AM1 and AM2 were shown to be capable of photosensitizing human leukemic cells in culture [17], and of producing efficient phototoxic effects when illuminated by laser light in an animal model system (unpublished observations). The cell study showed that the degree of photosensitized degradation of DNA correlated well with the viability of the cells, whereas no membrane damage could be detected even at supralethal doses. This was in contrast to the case with the more hydrophobic porphyrins that photosensitize mainly at sites of amino acid and nucleoside transport [3, 181. Although the photosensitization would presumably contribute to unnecessary phototoxic side effects during conventional chemotherapy, the magnitude of the effect suggested a possible application in the photodynamic therapy (PDT) of cancer. As a result, a study was initiated to explore structural variations from the lead compounds AM1 and AM2 in a systematic way with the aim of producing better candidate agents for use in PDT, i.e. water-soluble compounds with low toxicity in the dark, good photosensitization, and with a maximum visible absorbance around 700 run. As an initial step towards this goal the novel compounds MAMl, 1, MAMl,S, 2 and MAM1,8, 3 and 8 (Fig. 1) were synthesized to investigate the influence of different side chains on the photosensitizing properties of the anthracenediones. We report an investigation of the molecular mechanisms of photosensitization by compounds l-3 and 8. In particular we examine the participation of the AAQs in Type I (radical) and Type II (singlet oxygen) reactions, and seek a correlation between structural characteristics and photobiological-photochemical activities. We also report on the ability of these agents to photosensitize human leukemic cells in culture which was assessed by measuring dark and light toxicities, DNA and membrane damage. The results are compared directly with those obtained with the lead compounds AM1 and AM2.
2. Materials
and methods
The spin trap $5dimethyl-1-pyrroline-N-oxide (DMPO), sodium azide and strychnine were purchased from the Aldrich Chemical Company (Milwaukee, WI) and NADH from the Sigma Chemical Company (St. Louis, MO). Actinochrome N (475/ 610) was purchased from Photon Technology International, Inc. (Princeton, NJ). Dry solvents were prepared under an argon atmosphere [19] and distilled before use. Organic layers obtained from extractions were dried over anhydrous Na2S0+ The term in vacua refers to the removal of solvent on a rotary evaporator followed by evacuation ( 450 nm.
vs. NADH concentration. Samples contained AQ 2 (38 buffer pH 7.1 (1:l v/v) mixture and were illuminated with
3 (Table 2). This indicates that both anthraquinones sensitize NADH oxidation mainly via the singlet oxygen mechanism (Type II). Oxygen is consumed in reaction 1 followed by fast reaction 2, but it may be partially recovered via dismutation (eqn. 3). for AAQ
326 TABLE 2 Effect of azide anion on oxygen consumption rates during AAQ 2- and AAQ J-sensitized photooxidation of NADH in EtOH-phosphate buffer, pH 7.0 (1:l v/v) mixture
AAQ 2 (5 mM Na&) AAQ 3 (10 mM NaN3)
‘02+NADH-
O;-
NADH (mM)
Rate ( -NS-) (10m6 M min-‘)
Rate (+N,-) (10m6 M min-‘)
Rate (-)/rate
0.39 2.05 0.34 2.52
51.0 175.0 56.0 228.0
17.0 30.0 4.0 23.0
3.0 5.8 14.0 9.9
+NAD-+H+
(+)
(1)
k1 = 7.9 X 10’ M-’ s-’ (ref. 41) O,+NAD’-
o’-+NAD+
(2)
k2= 1.9 X lo9 M-’ s-’ (ref. 42) 202’- + 2H+ -
HzOz + O2
(3)
/c~=cu. lo6 M-’ s-l (ref. 43) Oxygen recovery from dismutation is quantitative since the ratio of the quantum yield of oxygen consumption to that of NADH depletion was close to one. We found that at [NADH] = 1.4 mM measured yields are: C#J( - 02) = 0.076 and C#J( - NADH) = 0.071 giving the ratio of 1.07. This, together with observation that the photo-oxidation process is initiated by ‘02 (Fig. 4), allowed us to formally ascribe oxygen consumption to reaction 1. At steady state, the reciprocal of the rate of oxygen consumption is described by eqn. 4 (straight line in Fig. 3(B). (- d[Oz]/dt)-’
=k,I(k&NADH])
+ l/(R,)
(where Rf is the rate of ‘02 production). From the plot in Fig. the kd/kl ratio to be 1.9X 10m3 M, which leads to $0,) 6.7 ps. performed assuming that kI in H,O/EtOH (1:l v/v) is the same as (k, =7.9x lo7 M-l s-l [41]). A similar value of 7 (8 ps) can be ‘02 lifetime additivity in 1:l ethanol-water mixture.
(4) 3(B) we estimated (Calculations were in aqueous solution obtained assuming
4.5. Cell viability studies The effect of the three anthracenedione compounds l-3 on the viability of human leukemic K562 cells in culture was investigated both in the dark and following illumination with visible light for 2 minutes. The band filter employed in the illumination (50% transmission at 475 nm) transmitted light being absorbed by all the drugs studied and the drugs were the only light-absorbing species. Light illumination up to 10 min was completely non-toxic to the cells in the absence of drug (data not shown). Viability curves for the three compounds are shown in Fig. 5. In the dark, all three compounds exhibited little toxicity; at a concentration of 250 PM only MAM1,5,2, (Fig. 5(A)) caused any significant loss of cell viability. Acquisition of accurate data above 250 PM was not possible due to problems of drug aggregation and solubility. Following illumination
327
1 1220
1240
1260
1260
1300
1320
1340
WAVELENGTH (nm)
emission observed during illumination (1:l v/v; pD 7.1); awe 2, in EtOH&O (1:l v/v; pH 7.1).
Fig. 4. Singlet
oxygen
of AQ 2: curve 1, in EtQH/D@
the viability curve was not shifted in the case of MAMl, 1, (Fig. 5(B)) but was significantly shifted in the case of MAMl,S, 2, and MAMl,S, 3 (Figs. 5(B) and 5(C)). IDso values (the dose of drug required to give 50% loss of cell viability after one hour exposed to drug) were obtained where possible and can be seen in Table 3. MAMl,S gave the lowest IDSo values both in the dark and under the illumination conditions which resulted in a light-induced dose modification of 14.4. MAMl,S and 1,8 were much less toxic in the dark than the corresponding diamino-substituted compounds AM1 and AM2 (Fig. 1) and in the case of MAMl,S where it was possible to estimate a value for the light-induced dose modification, this was higher than for its corresponding compound AMl. Both the drug and light are necessary to produce the observed effect. Reversing the order and illuminating the cells prior to treatment with drug did not shift the dark viability curve (data not shown). 4.6. DNA damage DNA damage induced by MAMl,S, 2, and MAM1,8, 3 was assessed using the technique of alkaline elution. In the case of MAMl,S (Fig. 3) DNA single strand breaks were observed in the dark but only following a one h treatment at doses of 10 PM or above (upper panel). Following illumination, single strand breaks are easily detected at 1 PM and with concentrations of 20 PM and above essentially all the DNA is eluted in the first three h fraction (lower panel). A similar pattern was observed with MAM1,8 but in this case higher doses of drug were required to give the same extent of DNA damage. Both the increased damage following illumination and the difference between MAMl,S and MAM1,8 followed the pattern of cell viability of the drugs observed under identical conditions (Table 4). It is interesting to note, however, that the extent of DNA single strand breakage, both in the dark and following illumination, for a given decrease in viability is much greater for MAMl,S and MAM1,8
328
.li Cay
. 50
,
. 100
.
3
150
-
’
200
.
’ 250
, 50
100
150
200
250
Fig. 5. Cell viability curves for (A) MAMl, (B) MAMlJ and (C) MAMl,B either in the dark (filled symbols) or following 2 minute illumination (open symbols). Points represent the mean of eight individual cell wells; bars represent SD.
than for the diamino-substituted compound AM2. Strand breaks are observed when proteinase K is omitted from the alkaline elution suggesting the formation of frank breaks (Figs. 6 and 7). The amount of damage is less in the absence of proteinase K however, particularly in the dark, suggesting that a proportion of the single strand breaks are protein-concealed. A sensitive probe for membrane damage is the transport of the model amino acid cycloleucine [3]. Uptake of cycloleucine occurs through active transport [3]. Following treatment of cells with MAMl,S for one h no inhibition of [‘4C]-cycloleucine transport could be detected with or without illumination at doses up to 250 PM (data not shown), indicating that no significant membrane damage occurred. The results suggest that aminoanthraquinones plus light cause no damage to those regions of cell membranes that are responsible for transport of this amino acid. In contrast it has been reported that photosensitization by hematoporphyrin derivatives affects uptake of this model amino acid [3].
TABLE
3
Effect of illumination cenediones
Drug
on the viability of K562 leukemic
cells following treatment
Light-induced
IDm (PM) Dark
LigbP
>300 260 > 300
>300 18 67
1 2 3 4b 5
23 53
with anthra-
dose modification
14.4 > 4.5
2.6 2.4
8.85 22.0
“rwo minute Wunination. bData for 4 and 5 are from ref. 17. TABLE 4 Effect on cell viability and DNA damage of anthracenedione-induced cells
Dw
Dose (PM) 0
Viability’
photosensitization
of K562
DNA damageb
Dark
Light
Dark
Light
1
1 0.9 0.84 0.28 -z 0.01
0.96 0.88 0.85 0.37 0.05
0.95 0.50 0.07 0.02 N.D.
0.99 0.91 0.75
0.86 0.75 0.64
0.49 0.15 0.07
0.57 0.12 Bco.01
0.96 0.92 0.59
0.28 0.07 0.05
1 1 1 0.53
2
1 5 20 250
3
5 20 40
1 1 1
5C
2 10 50
1 0.84 0.53
‘Expressed as ccl1 viability fraction. bExpressed as fraction of label remaining ‘Data for 5 are from ref. 17. N.D. not determined.
on the filter after 12 h of alkaline elution.
5. Discussion We have demonstrated that 1,5- and l&diamino-substituted dimethoxyanthraquinones, 2 and 3, are efficient sensitizers of singlet oxygen. This is in agreement with earlier reports on photosensitizing properties of anthraquinones, substituted with amino and/or hydroxyl groups [16, 34, 44, 45].We attribute the photochemical reactivity of our anthraquinones to the excited triplet states, which follows the results of these earlier studies. The generation of singlet oxygen proceeds via transfer of the triplet energy from the excited dye molecule to the ground state oxygen.
330
1
0 UM
5 UM 10 uM
L
0 = ii
. 20 uM
6
9
Time
12
15
(h)
0
uM
1 UM
2.5 uM
5 uM lo uM
20 uM 6
9 Time
Fig. 6. Alkaline
12
15
(h)
elution profiles of DNA from KS62 cells treated with MAMl,S following a one h exposure to drug; cells were either kept in the dark (top) or illuminated for 2 min (bottom). The assays were performed in the presence of proteinase K.
z
;
VI-
Iu-
(D-
m-
w-
0
.
!
Fraction
Retalned
on
Fllter
Fraction
Retained
on
Filter
332 %AQ+02tl-AAQ+‘02
(3
Dye triplets are quenched by molecular oxygen with rate constants near the difhtsioncontrolled limit, and for l,S- and 1,8-(NH&AQ the estimated values of k5 were 2.1 and 2.9~10~ M-l s-l in methanol [34]. Similarly, the triplets of our AAQs must be efficiently quenched by oxygen. Both the position and the number of the amino substituents in the AAQs chromophore influence the yield of ‘Oz generation, which increases in the order: l< l,S- < l,&AAQ (Table 1). These differences in +((‘Oz) may well be due to differences in intersystem crossing yields, c#+ Rembold and Kramer [34] reported +r for 1,4(NI&)-AQ to be 300 PM. In the l,S-series this decrease of the dark toxicity is accompanied by an increase in the light-induced dose modification from 8.85 to 14.4. Similar comparison of the, 1,8-series is difficult but it is apparent that a corresponding improvement in the ’ light-induced dose modification was not achieved because of an increase in IDso for MAM1,8 in the presence of light. Differences exist in the mechanisms of cytotoxicity between the prototype aminoanthraquinones
333
and their methoxy counterparts. Thus it appears that the cytotoxic action of the latter involves cell DNA damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks.
6. Conclusions
We have shown that certain l-, 1,5- and 1,8-amino-substituted dimethoxyanthraquinones are photochemically reactive. In particular they are able to sensitize ‘Oz and superoxide radical production in benzene solutions. The 1,5- and 1,8-diaminodimethoxyanthraquinones sensitize photo-oxidation of a representative biological electron donor, NADH, with concomitant hydrogen peroxide formation. Singlet oxygen is the predominant oxidizing species in this system. The high yields of ‘Oz formation in benzene and in ethanol suggest that 2 and 3 might be efficient sensitizers in less polar environments such as cell membranes. Because only those compounds that showed high yield of singlet oxygen generation and free radical production, that is AAQ 2 and 3, appeared to be phototoxic both Type I and Type II processes may be pertinent to the toxicity exerted by these and related aminoanthraquinones towards leukemic cells in vitro [17]. However, the phototoxicity did not correlate with the ability to generate ‘Oz as the more phototoxic agent 1 is characterized by a lower N(‘Oz). Compounds of the type MAh41,5 meet several important criteria for development as photodynamic therapy agents namely: stability, ease of cellular uptake, extremely low dark toxicity, and relatively efficient photoexcitation. New structural types based on these lead compounds and designed to address the remaining criterion, Le. of strong absorption in the region 65&700 run will be the subject of future reports.
Acknowledgment
This research was supported Cancer Institute of Canada.
in part by a grant
to J. W. L. from the National
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335 27 A. A. Krasnovsky Jr., S. Yu. Egorov, 0. V. Nasarova, E. J. Yartsev and G. V. Ponomarev, Photosensitized formation of singlet molecular oxygen in solutions of water-soluble porphyrins: direct luminescence measurements, Shcdia Biophysicu, 124 (1988) 123-142. 28 R. Schmidt and H-D. Brauer, Self-sensitized photo-oxidation of aromatic compounds and photocycloconversion of endoperoxides: applications in chemical actinometty, J. Photochem., 25 (1984) 489-499. 29 J. Tokunaga, Solubilities of oxygen, nitrogen and carbon dioxide in aqueous alcohol solutions, J. Chem. Erg. Data, 20 (1975) 41-46. 30 J. Carmichael, W. G. De Graff, A. F. Gazdar, J. D. Minna and J. B. Mitchell, Evaluation of a tetrazolium-based semi-automated calorimetric assay: Assessment of chemosensitivity testing, Cancer Res., 47 (1987) 936. 31 K. W. Kohn, L. C. Erickson, R. A. G. Ewig and D. A. Friedman, Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15 (1976) 4629-4637. 32 K. W. Kohn, R. A. G. Ewig, L. C. Erickson and L. A. Zwelling, Measurements of strandbreaks and cross-links by alkaline elution, in E. C. Friedberg and P. C. Hanawalt (eds.), DNA Repair: A Loboratory Manual of Research Procedures, Vol. 1, Part B, Marcel Dekker, New York, 1981, pp. 379-401. 33 W. Luck and H. Sand, The phenomenon of phototropy, Angew. Chem. Intern. Ed., 3 (1964) 570-580. 34 M. W. Rembold and H. E. A. Kramer, The role of anthraquinonoid dyes in the “catalytic fading” of dye mixtures - substituent-dependent triplet yield of diaminoanthraquinones, J. Sot. L$erz and Colorists, 96 (1980) 122-126. 35 R. H. Peters and H. H. Sumner, Spectra of anthraquinone derivatives, J. Chem. Sot., (1953) 2101-2110. 36 Z. Yoshida and F. Takabayashi, Electronic spectra of mono-substituted anthraquinones and solvent effects, Tetrahedron, 24 (1968) 933-943. 37 A. A. Gorman, I. Hamblett, C. Lambert, B. Spencer and M. C. Standen, Identification of both pre-equilibrium and dsusion limits for reaction of singlet oxygen, O,(‘A,), with both physical and chemical quenchers: variable-temperature, time-resolved infrared luminescence studies, J. Am Chem. Sot., 110 (1988) 8053-8059. 38 P. Murasecco-Suardi, E. Gassmann, A. M. Braun and E. Oliveros, Determination of the quantum yield of intersystem crossing of rose bengal, Helv. Chim. Actu, 70 (1987) 1760-1722. 39 E. Gandin, Y. Lion and A. Van de Vorst, Quantum yield of singlet oxygen production by xanthine derivatives, Photo&m. Photobiol., 37 (1983) 271-278. 40 K. Reszka and C. F. Chignell, Spin-trapping of the superoxide radical in aprotic solvents, Free Rad. Res. Commun., 14 (1991) 97-106. 41 G. Peters and M. A. J. Rodgers, Single-electron transfer from NADH analogues to singlet oxygen, Biochim. Biophys. Actu, 637 (1981) 43-52. 42 R. L. Wilson, Pulse radiolysis studies of electron transfer reactions in aerobic solution, J. Chem. Sot., Chem. Commun., (1970) 1005. 43 B. H. J. Bielski and D. E. Cabelli, Highlights of current research involving superoxide and perhydroxyl radicals in aqueous solutions, ht. J. Radiut. BioL, 590 (1991) 291-319. 44 I. M. Byteva, G. P. Gurinovich, 0. L. Golomb and V. V. Karpov, Anthraquinone derivatives as sensitizers of the formation of singlet oxygen, Zhumal Prihladnoi Spectroskopii, 44 (1986) 356-358 (English translation from Russian). 45 R. Dabestani, K. J. Reszka, D. G. Davis, R. H. Sik and C. F. Chignell, Spectroscopic studies of cutaneous photosensitizing agents: XVI. Disperse Blue-35, Photochem. Photobiol., 54 (1991) 37-42. 46 J. Ritter, H.-U. Borst, T. Lindner, M. Hauser, S. Brosig, K. Bredereck, U. E. Steiner, D. Kuhn, J. Kelemen and H. E. A. Kramer, Substituent effects on triplet yields in aminoanthraquinones: radiationless deactivation via intermolecular and intramolecular hydrogen bonding, 1. Photochem. Photobiol. A: Chem., 41 (1988) 227-244. 47 E. D. Owen and R. T. Allen, the photochemistry of triphenylmethane dyes in films of poly(methylmethacrylate), I. Appl. Chem. Biorechnol., 22 (1972) 799-810.
Photobiol. B: Biol., 15 (1992) 317-335
317
Photosensitization by anticancer agents 11. Mechanisms of photosensitization of human leukemic cells by diaminoanthraquinones: singlet oxygen and radical reactions Krzystzof
J. Reszka,
P. Bilski
and
Colin
F. Chignell
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (USA) John
A. Hartley,
Naem
Khan
and
Robert
L. Souhami
Department of Oncology, University College and Middlesex School of Medicine, 91 Riding House Street, London, WIP 8BT (UK) Aubrey
J. Mendonca
and J. William
Lownt
Department of Chemistry, Universi@ of Alberta, Edmonton, Alta., T6G 2G2 (Canada) (Received
December
3, 1991; accepted
March 13, 1992)
Abstract The synthesis of several aminoanthraquinone derivatives (AAQs), designed to suppress the dark toxicity and to promote more efficient cancer cell photosensitization for potential use in photodynamic therapy (PDT), is described. The following AAQs were synthesized: l-NH,-4,5-(MeO),-AQ (l),1,5-(NH&-4,8-(Me0)2-AQ (2), 1,8-(NH&-4,5-(MeO),-AQ (3), and 1,5-(NHPhMe),-4,8-(MeO),-AQ (8). The agents exhibit strong absorption in the region 480-620 nm. Possible mechanisms of photosensitization were studied by measuring ‘Ox phosphorescence at 1270 nm, detecting superoxide radicals employing an electron paramagnetic resonance (EPR)-spin trapping technique, and measuring oxygen consumption during the photo-oxidation of a representative biological electron donor, NADH. Strong phosphorescence from ‘02 was observed upon illumination of 2 and 3 in Cd-& (quantum yield of 0.25 and 0.5 respectively), and in EtOH (quantum yield of 0.23 and 0.34). The 1-amino-AQ (1)was the weakest ‘02 sensitizer, with quantum yield of 0.13 in benzene. No phosphorescence was observed in EtOH. A superoxide radical was detected as a spin adduct of SJ-dimethyl-1-pyrroline-N-oxide (DMPO) in irradiated benzene solutions of 1, 2 or 3 and DMPO. AAQs 2 and 3 sensitized photo-oxidation of NADH in H20/EtOH mixture with the intermediacy of singlet oxygen as judged by the effect of sodium azide on the photostimulated oqgen consumption. Evolution of O2 upon addition of catalase to the illuminated solution confirmed the ultimate formation of hydrogen peroxide. These tindings suggested that the (di)amino-dimethoxyanthraquinones might exert photosensitization via both Type I and Type II mechanisms. The AAQs were tested for their ability to photosensitize K562 human chronic myeloid leukemic cells in culture. Viability was measured using the 3,4,5-diethylthiazol-2,5-diphenyl tetrazolium blue assay, and DNA and possible membrane damage were assessed. The results from illuminating cells with light >475 nm show that for tbe l,S-compounds, the presence of methoxy substituents at 4,8 positions reduces the dark toxicity from IDS,, of 23 to 250 PM and for the l&compounds correspondingly from IDw, of 53 to > 300 PM. In the l&series ,this decrease of the dark ‘Author
to whom correspondence
loll-1344/92/$.5.00
should be addressed.
0 1992 - Elsevier Sequoia. All rights reserved
318
toxicity is accompanied by an increase in light-induced dose modification from 8.85 to 14.4.Differences exist in the mechanisms of cytotoxicity between the prototype phenolic AAQs and their methoxy counterparts. It appears that the cytotoxic action of the latter causes cell damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks. No evidence for membrane damage, as determined by transport of the model amino acid cycloleucine, could be observed even at supralethal doses.
Keywords: Aminoanthraquinones, photosensitization, superoxide radical, leukemic cells.
singlet oxygen, spin trapping,
1. Introduction
Photosensitization [l] has been demonstrated by several chemotherapeutic agents including a hematoporphyrin derivative [2,3], anthracyclines [4, 51 and anthrapyraxoles and related structures [6-121. In the case of the hematoporphyrin derivative, this has found a practical application in the photodynamic therapy of cancer [l, 21. With the intensely colored l+diamino-substituted antitumor anthraquinones mitoxantrone 6 [13, 141 and ametantrone 7 [14] (Fig. l), although drug-sensitized decarboxylation of peptides has been observed upon ultraviolet (W) (313 nm) irradiation [15], visible light does not initiate any photosensitized reaction with either compound [16]. In contrast, however, 1,5- and 1,8-diamino-substituted anthraquinones structurally related to ametantrone (AM1 and AM2, respectively, Fig. 1) have been shown to cause oxygen consumption and the formation of superoxide radicals and hydrogen peroxide upon visible light illumination of aerated samples in the presence of a representative biological electron donor, NADH [16]. In the presence of DNA these compounds caused the formation of single-strand breaks upon light exposure, and a correlation between the
RI
R2
R3
R4
1
MAMI
NHz
0CH3
0CH3
H
2
MAW.5
NH2
0CH3
NH;?
OCH3
3
MAMI.8
NH2
OCH3
OCH3
NH2
NH(CH~)~N(CHZCH~)~
H
Rl
H
NHKH2hNWWH3h
4
AM1
H
H
RI
6
Mitoxanaone NH(CHZ)ZNH(CH&OH
Rl
OH
OH
7
Ametantmne NH(CH2)2NH(CH2)20H
Rl
H
H
SAM2
Fig. 1. Structures of the anthracenediones
used in this study.
319
extent of DNA damage, oxygen consumption, and NADH oxidation was established in cell-free systems [12]. Recently AM1 and AM2 were shown to be capable of photosensitizing human leukemic cells in culture [17], and of producing efficient phototoxic effects when illuminated by laser light in an animal model system (unpublished observations). The cell study showed that the degree of photosensitized degradation of DNA correlated well with the viability of the cells, whereas no membrane damage could be detected even at supralethal doses. This was in contrast to the case with the more hydrophobic porphyrins that photosensitize mainly at sites of amino acid and nucleoside transport [3, 181. Although the photosensitization would presumably contribute to unnecessary phototoxic side effects during conventional chemotherapy, the magnitude of the effect suggested a possible application in the photodynamic therapy (PDT) of cancer. As a result, a study was initiated to explore structural variations from the lead compounds AM1 and AM2 in a systematic way with the aim of producing better candidate agents for use in PDT, i.e. water-soluble compounds with low toxicity in the dark, good photosensitization, and with a maximum visible absorbance around 700 run. As an initial step towards this goal the novel compounds MAMl, 1, MAMl,S, 2 and MAM1,8, 3 and 8 (Fig. 1) were synthesized to investigate the influence of different side chains on the photosensitizing properties of the anthracenediones. We report an investigation of the molecular mechanisms of photosensitization by compounds l-3 and 8. In particular we examine the participation of the AAQs in Type I (radical) and Type II (singlet oxygen) reactions, and seek a correlation between structural characteristics and photobiological-photochemical activities. We also report on the ability of these agents to photosensitize human leukemic cells in culture which was assessed by measuring dark and light toxicities, DNA and membrane damage. The results are compared directly with those obtained with the lead compounds AM1 and AM2.
2. Materials
and methods
The spin trap $5dimethyl-1-pyrroline-N-oxide (DMPO), sodium azide and strychnine were purchased from the Aldrich Chemical Company (Milwaukee, WI) and NADH from the Sigma Chemical Company (St. Louis, MO). Actinochrome N (475/ 610) was purchased from Photon Technology International, Inc. (Princeton, NJ). Dry solvents were prepared under an argon atmosphere [19] and distilled before use. Organic layers obtained from extractions were dried over anhydrous Na2S0+ The term in vacua refers to the removal of solvent on a rotary evaporator followed by evacuation ( 450 nm.
vs. NADH concentration. Samples contained AQ 2 (38 buffer pH 7.1 (1:l v/v) mixture and were illuminated with
3 (Table 2). This indicates that both anthraquinones sensitize NADH oxidation mainly via the singlet oxygen mechanism (Type II). Oxygen is consumed in reaction 1 followed by fast reaction 2, but it may be partially recovered via dismutation (eqn. 3). for AAQ
326 TABLE 2 Effect of azide anion on oxygen consumption rates during AAQ 2- and AAQ J-sensitized photooxidation of NADH in EtOH-phosphate buffer, pH 7.0 (1:l v/v) mixture
AAQ 2 (5 mM Na&) AAQ 3 (10 mM NaN3)
‘02+NADH-
O;-
NADH (mM)
Rate ( -NS-) (10m6 M min-‘)
Rate (+N,-) (10m6 M min-‘)
Rate (-)/rate
0.39 2.05 0.34 2.52
51.0 175.0 56.0 228.0
17.0 30.0 4.0 23.0
3.0 5.8 14.0 9.9
+NAD-+H+
(+)
(1)
k1 = 7.9 X 10’ M-’ s-’ (ref. 41) O,+NAD’-
o’-+NAD+
(2)
k2= 1.9 X lo9 M-’ s-’ (ref. 42) 202’- + 2H+ -
HzOz + O2
(3)
/c~=cu. lo6 M-’ s-l (ref. 43) Oxygen recovery from dismutation is quantitative since the ratio of the quantum yield of oxygen consumption to that of NADH depletion was close to one. We found that at [NADH] = 1.4 mM measured yields are: C#J( - 02) = 0.076 and C#J( - NADH) = 0.071 giving the ratio of 1.07. This, together with observation that the photo-oxidation process is initiated by ‘02 (Fig. 4), allowed us to formally ascribe oxygen consumption to reaction 1. At steady state, the reciprocal of the rate of oxygen consumption is described by eqn. 4 (straight line in Fig. 3(B). (- d[Oz]/dt)-’
=k,I(k&NADH])
+ l/(R,)
(where Rf is the rate of ‘02 production). From the plot in Fig. the kd/kl ratio to be 1.9X 10m3 M, which leads to $0,) 6.7 ps. performed assuming that kI in H,O/EtOH (1:l v/v) is the same as (k, =7.9x lo7 M-l s-l [41]). A similar value of 7 (8 ps) can be ‘02 lifetime additivity in 1:l ethanol-water mixture.
(4) 3(B) we estimated (Calculations were in aqueous solution obtained assuming
4.5. Cell viability studies The effect of the three anthracenedione compounds l-3 on the viability of human leukemic K562 cells in culture was investigated both in the dark and following illumination with visible light for 2 minutes. The band filter employed in the illumination (50% transmission at 475 nm) transmitted light being absorbed by all the drugs studied and the drugs were the only light-absorbing species. Light illumination up to 10 min was completely non-toxic to the cells in the absence of drug (data not shown). Viability curves for the three compounds are shown in Fig. 5. In the dark, all three compounds exhibited little toxicity; at a concentration of 250 PM only MAM1,5,2, (Fig. 5(A)) caused any significant loss of cell viability. Acquisition of accurate data above 250 PM was not possible due to problems of drug aggregation and solubility. Following illumination
327
1 1220
1240
1260
1260
1300
1320
1340
WAVELENGTH (nm)
emission observed during illumination (1:l v/v; pD 7.1); awe 2, in EtOH&O (1:l v/v; pH 7.1).
Fig. 4. Singlet
oxygen
of AQ 2: curve 1, in EtQH/D@
the viability curve was not shifted in the case of MAMl, 1, (Fig. 5(B)) but was significantly shifted in the case of MAMl,S, 2, and MAMl,S, 3 (Figs. 5(B) and 5(C)). IDso values (the dose of drug required to give 50% loss of cell viability after one hour exposed to drug) were obtained where possible and can be seen in Table 3. MAMl,S gave the lowest IDSo values both in the dark and under the illumination conditions which resulted in a light-induced dose modification of 14.4. MAMl,S and 1,8 were much less toxic in the dark than the corresponding diamino-substituted compounds AM1 and AM2 (Fig. 1) and in the case of MAMl,S where it was possible to estimate a value for the light-induced dose modification, this was higher than for its corresponding compound AMl. Both the drug and light are necessary to produce the observed effect. Reversing the order and illuminating the cells prior to treatment with drug did not shift the dark viability curve (data not shown). 4.6. DNA damage DNA damage induced by MAMl,S, 2, and MAM1,8, 3 was assessed using the technique of alkaline elution. In the case of MAMl,S (Fig. 3) DNA single strand breaks were observed in the dark but only following a one h treatment at doses of 10 PM or above (upper panel). Following illumination, single strand breaks are easily detected at 1 PM and with concentrations of 20 PM and above essentially all the DNA is eluted in the first three h fraction (lower panel). A similar pattern was observed with MAM1,8 but in this case higher doses of drug were required to give the same extent of DNA damage. Both the increased damage following illumination and the difference between MAMl,S and MAM1,8 followed the pattern of cell viability of the drugs observed under identical conditions (Table 4). It is interesting to note, however, that the extent of DNA single strand breakage, both in the dark and following illumination, for a given decrease in viability is much greater for MAMl,S and MAM1,8
328
.li Cay
. 50
,
. 100
.
3
150
-
’
200
.
’ 250
, 50
100
150
200
250
Fig. 5. Cell viability curves for (A) MAMl, (B) MAMlJ and (C) MAMl,B either in the dark (filled symbols) or following 2 minute illumination (open symbols). Points represent the mean of eight individual cell wells; bars represent SD.
than for the diamino-substituted compound AM2. Strand breaks are observed when proteinase K is omitted from the alkaline elution suggesting the formation of frank breaks (Figs. 6 and 7). The amount of damage is less in the absence of proteinase K however, particularly in the dark, suggesting that a proportion of the single strand breaks are protein-concealed. A sensitive probe for membrane damage is the transport of the model amino acid cycloleucine [3]. Uptake of cycloleucine occurs through active transport [3]. Following treatment of cells with MAMl,S for one h no inhibition of [‘4C]-cycloleucine transport could be detected with or without illumination at doses up to 250 PM (data not shown), indicating that no significant membrane damage occurred. The results suggest that aminoanthraquinones plus light cause no damage to those regions of cell membranes that are responsible for transport of this amino acid. In contrast it has been reported that photosensitization by hematoporphyrin derivatives affects uptake of this model amino acid [3].
TABLE
3
Effect of illumination cenediones
Drug
on the viability of K562 leukemic
cells following treatment
Light-induced
IDm (PM) Dark
LigbP
>300 260 > 300
>300 18 67
1 2 3 4b 5
23 53
with anthra-
dose modification
14.4 > 4.5
2.6 2.4
8.85 22.0
“rwo minute Wunination. bData for 4 and 5 are from ref. 17. TABLE 4 Effect on cell viability and DNA damage of anthracenedione-induced cells
Dw
Dose (PM) 0
Viability’
photosensitization
of K562
DNA damageb
Dark
Light
Dark
Light
1
1 0.9 0.84 0.28 -z 0.01
0.96 0.88 0.85 0.37 0.05
0.95 0.50 0.07 0.02 N.D.
0.99 0.91 0.75
0.86 0.75 0.64
0.49 0.15 0.07
0.57 0.12 Bco.01
0.96 0.92 0.59
0.28 0.07 0.05
1 1 1 0.53
2
1 5 20 250
3
5 20 40
1 1 1
5C
2 10 50
1 0.84 0.53
‘Expressed as ccl1 viability fraction. bExpressed as fraction of label remaining ‘Data for 5 are from ref. 17. N.D. not determined.
on the filter after 12 h of alkaline elution.
5. Discussion We have demonstrated that 1,5- and l&diamino-substituted dimethoxyanthraquinones, 2 and 3, are efficient sensitizers of singlet oxygen. This is in agreement with earlier reports on photosensitizing properties of anthraquinones, substituted with amino and/or hydroxyl groups [16, 34, 44, 45].We attribute the photochemical reactivity of our anthraquinones to the excited triplet states, which follows the results of these earlier studies. The generation of singlet oxygen proceeds via transfer of the triplet energy from the excited dye molecule to the ground state oxygen.
330
1
0 UM
5 UM 10 uM
L
0 = ii
. 20 uM
6
9
Time
12
15
(h)
0
uM
1 UM
2.5 uM
5 uM lo uM
20 uM 6
9 Time
Fig. 6. Alkaline
12
15
(h)
elution profiles of DNA from KS62 cells treated with MAMl,S following a one h exposure to drug; cells were either kept in the dark (top) or illuminated for 2 min (bottom). The assays were performed in the presence of proteinase K.
z
;
VI-
Iu-
(D-
m-
w-
0
.
!
Fraction
Retalned
on
Fllter
Fraction
Retained
on
Filter
332 %AQ+02tl-AAQ+‘02
(3
Dye triplets are quenched by molecular oxygen with rate constants near the difhtsioncontrolled limit, and for l,S- and 1,8-(NH&AQ the estimated values of k5 were 2.1 and 2.9~10~ M-l s-l in methanol [34]. Similarly, the triplets of our AAQs must be efficiently quenched by oxygen. Both the position and the number of the amino substituents in the AAQs chromophore influence the yield of ‘Oz generation, which increases in the order: l< l,S- < l,&AAQ (Table 1). These differences in +((‘Oz) may well be due to differences in intersystem crossing yields, c#+ Rembold and Kramer [34] reported +r for 1,4(NI&)-AQ to be 300 PM. In the l,S-series this decrease of the dark toxicity is accompanied by an increase in the light-induced dose modification from 8.85 to 14.4. Similar comparison of the, 1,8-series is difficult but it is apparent that a corresponding improvement in the ’ light-induced dose modification was not achieved because of an increase in IDso for MAM1,8 in the presence of light. Differences exist in the mechanisms of cytotoxicity between the prototype aminoanthraquinones
333
and their methoxy counterparts. Thus it appears that the cytotoxic action of the latter involves cell DNA damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks.
6. Conclusions
We have shown that certain l-, 1,5- and 1,8-amino-substituted dimethoxyanthraquinones are photochemically reactive. In particular they are able to sensitize ‘Oz and superoxide radical production in benzene solutions. The 1,5- and 1,8-diaminodimethoxyanthraquinones sensitize photo-oxidation of a representative biological electron donor, NADH, with concomitant hydrogen peroxide formation. Singlet oxygen is the predominant oxidizing species in this system. The high yields of ‘Oz formation in benzene and in ethanol suggest that 2 and 3 might be efficient sensitizers in less polar environments such as cell membranes. Because only those compounds that showed high yield of singlet oxygen generation and free radical production, that is AAQ 2 and 3, appeared to be phototoxic both Type I and Type II processes may be pertinent to the toxicity exerted by these and related aminoanthraquinones towards leukemic cells in vitro [17]. However, the phototoxicity did not correlate with the ability to generate ‘Oz as the more phototoxic agent 1 is characterized by a lower N(‘Oz). Compounds of the type MAh41,5 meet several important criteria for development as photodynamic therapy agents namely: stability, ease of cellular uptake, extremely low dark toxicity, and relatively efficient photoexcitation. New structural types based on these lead compounds and designed to address the remaining criterion, Le. of strong absorption in the region 65&700 run will be the subject of future reports.
Acknowledgment
This research was supported Cancer Institute of Canada.
in part by a grant
to J. W. L. from the National
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