Free radicals in anticancer drug pharmacology

Chem.-Biol Interactio~s, 69 (1989) 293--317

293

Elsevier Scientific Publishers Ireland Ltd.

Review Article

F R E E R A D I C A L S IN A N T I C A N C E R D R U G P H A R M A C O L O G Y

BIRANDRAK. SINHA Clinical Pharmacology Branch, Building 10, Room 6N-119, National Cancer Institute, National Institutes of Health, Bethesd~ MD ~089~ (U.S.A.)

(Received April 1st, 1988) (Revision received August 19th, 1988) (Accepted August 29th, 1988)

SUMMARY This review examines the formation of free radical intermediates from a number of clinically active antitumor agents inciuding quinone-containing antibiotics and eto~oside. An attempt is also made to relate the formation of these reactive intermediates to biochemical and pharmacological basis for tumor cell kill and resistance. The formation of these intermediates in some tumor cells has been detected by both direct ESR and spin-trapping technique. The detection of free radicals in biological systems, however, depends upon cellular bioenvironments, e.g. reducing conditions, and the presence and/or absence of activation and detoxification mechanisms. Evidence shows that certain antitumor drugs generate free radicals in vitro and in vivo and that these reactive species kill tumor cells by causing damage to DNA, membranes or enzymes. K e y words: F r e e radicals -

Antitumour drug -- Tumour cell kill -- Drug

resistance

INTRODUCTION The free radical metabolism of xenobiotics and subsequent toxic effects of their reactive intermediates in organs and tissues have become an intense area of research in recent years [1,2]. Antitumor drugs, like toxic xenobiotics, are metabolized in vitro and in vivo to reactive species including free radical intermediates. Although a large body of work has been carried out in the last decade, the significance of metabolism in the biochemical pharmacology of antitumor drugs has remained unexplored, and therefore disregarded as a potentially significant biochemical event for cytotoxicity. This 0009-2797/89~08.50 © 1989Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

294 misunderstanding stems, in part, from lack of regard for metabolism of the antitumor drugs as the basis for activity or toxicity in the clinic. Moreover, the role of free radicals in the mechanisms of action of antitumor drugs is not fully appreciated because of the inability to detect these reactive intermediates by electron spin resonance spectroscopy at clinically relevant drug concentrations. This review examines recent evidence for the free radical

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Fig. 1. Structures of anticaneer drugs which form free radical intermediates during enzymatic activation.

295

metabolism of the clinically active antitumor drugs, the relationship of free radicals in the mechanisms of action of these drugs in tumor cell kill, and their toxicological manifestations in vitro and in vivo. Antitumor drugs (Fig. 1) containing quinone/hydroquinone moieties, e.g. anthracyclines, mitomycin-C (MC), or streptonigrin, undergo one-electron reduction to the corresponding semiquinone radicals which, in the presence of molecular oxygen, produce superoxide anion radical, hydrogen peroxide, and ultimately hydroxyl radical [3-7]. Hydroxyl radicals ('OH) are shortlived and extremely reactive. In biological systems, "OH may react with: (a) unsaturated lipids causing peroxidation and destruction, (b) cellular DNA, thereby inducing DNA damage, or (c) tryptophan, tyrosine and thiol groups of functional proteins and enzymes, resulting in inactivation. These events ultimately may cause cell death. The one-electron reduction of the quinone-containing drugs can be catalyzed by enzymes such as NADPH-cytochrome P-450 reductase [3,8-11], cytochrome bs-reductase [9,10], xanthine oxidase [9], or reduced chemically by agents such as sodium borohydride [12,13]. Etoposide and teniposide, clinically active antitumor drugs for the treatment of small cell lung cancer, lymphomas, and testicular cancer, have also been shown to form free radical intermediates [14,15]. The chemistry of this activation, however, is dependent upon the presence of a phenolic hydroxyl group which is oxidized (via one-electron transfer) to a phenoxyl radical. Procarbazine, a hydrazine-containing antitumor drug used for the treatment of leukemia and Hodgkin's disease, forms a reactive carbon-centered radical with alkylating potential for DNA and proteins [16]. Bleomycin is a metal and oxygen-requiring antibiotic, shown to be active against a wide variety of neoplasms including Hodgkin's disease and testicular carcinomas; although formation of free radicals from bleomycin has not been demonstrated in vivo or in vitro, ample indirect evidence exists in in vitro systems to suggest that reduced oxygen species, either free [17] or bound to iron [18,19], are involved in its cytotoxicity and biochemical actions. ANTHRACYCLINES

The antitumor drugs, Adriamycin* and daunomycin, are the clinically most active and widely used anthracyclines. These drugs are active against a variety of human tumors including lung, breast and leukemia. However, one of the problems associated with Adriamycin has been dose-dependent irreversible and often fatal cardiac toxicity in man; more than 500 anthracyclines have been synthesized in attempts to improve therapeutic efficacy and simultaneously decrease the cardiotoxicity of this drug class. The results have been somewhat disappointing. This may be due, in part, to the difficulty in clearly defining and separating the mechanism of action of tumor cell kill from that of cardiotoxicity. A number of mechanisms (summarized in *Adriamycin is a registered trademark of Farmitalia Carlo Erba, Milan, Italy.

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297 Fig. 2) have been postulated to account for antitumor activity: intercalation into DNA bases [20,21], covalent binding of reactive metabolites [22-26], interactions with membrane [27-29], topoisomerase II-mediated DNA strand breaks [30--31] and free radical formation [4,5]. Although several of these

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Fig. 3. Electron spin resonance spectrum obtained from Adriamycin (AI in MCF-7 human breast tumor cell mierosomes under anaerobic conditions, (B) multi-line semiquinone radical spectrum obtained in the presence of xanthine oxidase, and (C) computer simulation of (B). For detail see Ref. 37.

298 events have been shown to occur in tumor cells, no single mechanism has emerged as 'the mechanism' for the tumor cell kill. The formation of free radicals from anthracyclines through reductive activation {either chemically or in an isolated biological milieu) has been well characterized. It is now recognized that there are two mechanisms by which Adriamycin can generate reactive free radicals. The best described mechanism involves one-electron reduction of the drug to the semiquinone free radical intermediate by flavin reductase [4]. The semiquinone radical reacts rapidly with oxygen (k = 108 M -1 s -~) to produce superoxide anion radical (0"~), hydrogen peroxide (H202) and "OH. Therefore, it has been proposed that the antitumor activity of Adriamycin is due to the formation and reaction of •OH with cellular macromolecules [3,32]. The role of oxygen in Adriamycin-induced tumor cell kill in vitro and in vivo is very confusing and appears to depend upon the cell type used. For instance, Chinese hamster V79 cell is differentially more sensitive to Adriamycin under aerobic conditions [33] whereas EMT6 mouse mammary tumor cells displayed greater sensitivity under hypoxic conditions [34]. In a recent report [35], no difference in the cytotoxicity of Adriamycin in Ehrlich ascites tumor cells could be detected under oxic or hypoxic conditions. However, using a direct electron spin resonance technique, Sato et al. [36] were able to detect a symmetrical single line ESR signal (g = 2.004) when Adriamycin was incubated with Ehrlich tumor cells. The drug-dependent ESR signal has been shown to result from the one-electron reduction of Adriamycin to its semiquinone free radical intermediate (Fig. 3) [37]. Baehur et al. [38] have also detected this Adriamycin semiquinone free radical intermediate in the isolated nuclei from leukemia L1210 cells. Doroshow has used quenchers of oxy-radicals to demonstrate the formation of Adriamycin-dependent superoxide anion radical and hydrogen peroxide in Ehrlich tumor cells [32]. Okamato and Ogura [39] have shown that Adriamycin caused stimulation of lipid peroxidation in Ehrlich tumor cells, which was inhibited by vitamin E and coenzyme Q10" Furthermore, Doroshow has shown that superoxide dismutase and catalase, when added exogenously to Ehrlich tumor cells and human breast tumor cells are capable of protecting these cells against Adriamycin-induced tumor cell kill. This protection was also observed in the presence of quenchers of hydroxyl radicals. These observations therefore suggest that oxy-radicals formed from Adriamycin were responsible for the Ehrlich tumor cell kill [32,40]. Because "OH radical is short-lived, extremely reactive, and has no detectable ESR spectrum at room temperature, we utilized the spin-trapping technique to detect Adriamycin-induced "OH formation in human breast tumor (MCF-7) cells [41,42]. Since "OH may be involved in the cell killing of these cells by Adriamycin, we also compared the formation of the "OH in drug sensitive (WT) and resistant (ADR ® ) cells. In the presence of 5,5-dimethyl-1pyrroline oxide (DMPO), a spin-trapping agent, Adriamycin stimulated the formation of "OH in the WT cells, detected as DMPO-OH adducts by ESR (Fig. 4). The adduct spectrum consisted of a quartet ( 1 : 2 : 2 : 1 ) with hyper-

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300 fine splitting constants of a N ---- a H ---- 14.9 G and was characteristic of DMPO-OH (Fig. 4). This was further confirmed by adding either ethanol or DMSO which reacted with free "OH resulting in the formation of the carboncentered radical (Fig. 4). The resistant ADR ® cells, under identical conditions, did not stimulate DMPO-OH formation from Adriamycin [41,42]. Addition of either NADPH or NADH, markedly enhanced Adriamycin-stimulated "OH formation in both cell lines, however, NADPH was a better reducing cofactor. Furthermore, while there was an increase in "OH production in ADR ® cells, WT cells generated significantly more "OH in the presence of Adriamycin and NADPH. This difference in "OH formation was not due to differences in Adriamycin transport or activation, but resulted from enhanced activities of glutathione-dependent peroxidase and transferase [43,44]. Our results show that the relative activities of both peroxidase and transferase were significantly increased in ADR ® cells, 12- and 44-fold, respectively. Our observation that NADPH stimulated "OH formation in intact cells is interesting because NADPH does not cross the cell membrane while Adriamycin is believed to be reduced intracellularly. We, therefore, examined the site of formation/location of DMPO-OH adducts using spin-broadening techniques. This technique is based on the fact that large paramagnetic ions cannot penetrate cell membranes because of their size and charge, but that they will broaden the ESR spectrum of nitroxides by dipole-dipole interactions; thus results in a decrease in intensity of the nitroxide signal. Addition O f C r 3÷ to incubation mixtures containing Adriamycin decreased the intensity of the DMPO-OH signal by 60--65% indicating that DMPO-OH was located extracellularly (Fig. 4). Furthermore, in the presence of high molecular weight polyethylene glycol which also does not penetrate cell membrane, a decrease in DMPO-OH intensity was observed due to quenching of extracellular "OH. In addition, DMPO-polyethylene glycol adducts were also formed (Fig. 4). These observations suggest that some Adriamycin-stimulated "OH is located extracellularly in MCF-7 cells [42]. We have also found that addition of superoxide dismutase (SOD) or catalase to the incubation mixtures containing cells and Adriamycin inhibited DMPO-OH formation, thereby suggesting that superoxide and hydrogen peroxide were the intermediates for "OH formation [41,42]. Desferal, an iron chelator, partially inhibited DMPO-OH formation indicating that the formation of "OH was also metal ion-dependent. Inclusion of SOD or catalase significantly modulated cytotoxicity of Adriamycin in both cells and offered complete protection against cell toxicity. Denatured proteins offered no protection [42]. These studies clearly indicate that (a) Adriamycin forms "OH in human breast tumor cells, (b) peroxidases and tranferases present in resistant cells protect these cells by rapidly detoxifying peroxides formed and (c) Adriamycin-induced cell killing of the MCF-7 breast tumor cells is mediated by the formation of these toxic oxygen radicals. Although MCF-7 appears to be an excellent human tumor cell line for activation of Adriamycin to its free radical state, the formation of "OH, as

301 detected by spin trapping experiments in the presence of Adriamcyin has been reported in other cell lines, e.g. murine leukemia P388 and L1210 cells (P. Gutierrez, pers. commun.I. While the in vivo formation of "OH from Adriamycin has not been detected directly using spin-trapping experiments, a number of recent publications support the involvement of free radicals in Adriamycin toxicity. Thayer [45] has shown that the serum from rats chronically treated with Adriamycin has elevated serum endoperoxides and hydroperoxides, indicative of lipid peroxidation resulting from Adriamycin-induced free radical formation. Floyd et al. [46] have demonstrated that co-administration of Adriamycin and salicylate in rats caused 100-fold increases in dihydroxybenzoic acids, formed from the reaction of "OH with salicylate, in rat heart but not in brain or blood. This would suggest that "OH may be involved in Adriamycin-induced cardiotoxicity. The second mechanism of free radical formation is dependent upon chelation of metal ions, iron in particular, by Adriamycin. In this case the free radical chemistry appears to be independent of the reductive activation and semiquinone radical chemistry described above. Nakano et al. [47--49] have reported that Adriamycin forms an iron coordination complex with dinucleotides, and is detected by an increase in the absorbance spectra band over 550 nm. Furthermore, they have also shown that such a complex and not free Adriamycin in the presence of oxygen and reducing enzymes, catalyzes peroxidation of lipids. Formation and participation of ferryl-iron species have been proposed in this peroxidation [47,48]. Myers et al. [50--52] and Zweier [53] have shown that drug-iron complexes can catalyze three separate redox chemistry: (1) Adriamycin can directly reduce Fe(III) to Fe(II) which then reacts with molecular oxygen or hydrogen peroxide; (2) chelated complex can react with reduced glutathione and molecular 02 to form hydrogen peroxide; (3) it can reduce hydrogen peroxide to form reactive "OH which causes nicking of the DNA [54]. Studies to investigate the structural requirements for iron binding to the drug and the ability of the resulting complex to catalyze "OH formation and DNA damage have shown that the presence of C-11-hydroxyl group is essential for the iron binding, thiol-dependent oxygen consumption, "OH formation, and DNA destruction [55]. The significance of the metal-dependent free radical chemistry is the correlation with the known cardiotoxicity of anthracyclines. For example, Adriamycin is reported to be significantly more cardiotoxic than carminomycin which is in turn, more cardiotoxic than daunomycin and 4demethoxydaunomycin. These in vitro studies, based on the ability of these anthracyclines to form iron complexes to produce peroxide-dependent "OH and to cleave DNA, correlated with their clinical cardiotoxic potential. Furthermore, this peroxide-promoted metal-dependent "OH formation has been suggested to be complementary and could potentiate and utilize peroxide formed during enzymatic reduction of antracyclines in cardiac tissue. Recently, Myers et al. [56] used 5-iminodaunomycin to confirm this metalcatalyzed, free radical-dependent damage hypothesis as basis for anthracyc-

302 line-mediated cardiotoxicity. 5-Iminodaunomycin, an anthracycline with an altered quinone group, neither forms a semiquinone radical nor produces superoxide/hydroxyl radicals. Preclinical studies have shown that 5-iminodaunomycin has little, if any, cardiotoxicity. Furthermore, this analog is less mutagenic than Adriamycin while retaining its antitumor activity. These studies show that 5-iminodaunomycin forms a complex with Fe(III), however, this complex formation did not result in the reduction of of Fe(III) to Fe(II) as in the case of Adriamycin. Although, 5-iminodaunomycin-Fe(III) complex supported H202-dependent "OH formation which was maximal at a 2-to-1 drug-to-iron ratio, the yield of the "OH formed was significantly less than that obtained with Adriamycin-Fe [56]. Furthermore, increasing the drug-toiron ratio, decreased "OH production such that it could not be detected. In addition, the presence of DNA did not stimulate "OH formation, nor was there any significant nicking or destruction of DNA observed with 5-iminodaunomycin. These behaviors are in marked contrast with Adriamycin-iron complexes, where "OH production increased with increasing drug-to-iron ratio and is stimulated by DNA addition. Interestingly, 5-iminoduanomycin is a chelator of iron with much higher affinity than Adriamycin; this may lessen the availability of iron to water or peroxide. These findings provide an explanation for less cardiotoxicity and mutagenicity of 5-iminodaunomycin and confirm the hypothesis that anthracycline-iron-dependent radical chemistry may be responsible for cardiotoxicity of this class of drugs [56]. It is not known at this time how the anthracycline-Fe complexes are involved in the tumor cell killing in vivo. Since the complexes are not expected to penetrate cellular membranes due to charge, the biological activity of such a complex remains unknown. At the present time, it is also not clear whether such complexes are formed in vivo. However, recent works by Demant and Norskov-Lauritsen [57] and Thomas and Aust [58] would suggest that Adriamycin can mobilize iron from the iron pools (Ferritin) during active redox-cycling and thus can form a complex with intrinsic cytotoxic properties. In this regard, quelamycin, a triferric-Adriamycin complex first proposed by Gosalvez et al. [59], was claimed to have decreasing toxicity while maintaining high antitumor activity. More recently Beraldo et al. [60] have shown that (Adrimaycin)3 - Fe complex binds to DNA by intercalation, however, iron was released despite its strong metal chelation with the drug. Moreover, the drug-metal complex displayed an antitumor activity against leukemia (P388) cells comparable to that of the free drug; it is not known whether the complex remained stable in the t u m o r cells since the ironAdriamycin complex has been shown to undergo chemical modification in the presence of artificial and natural membranes [61]. More work is needed to establish the involvement of the iron-dependent chemistry in tumor cell kill. Work from our laboratories has shown that Adriamycin-dependent free radical intermediates may also participate in covalent binding to cellular macromolecules [22--25]. This binding requires reductive activation of the drug by cytochrome P-450 NADPH reductase/NADPH system. The binding in the absence of oxygen has been detected in microsomal proteins [23,26],

303 exogenously added DNA [22,23], rat liver nuclear NDA [24] and in vivo [25], and is unaffected by a-tocopherol but is significantly decreased by GSH and ethylxanthate, a negatively charged trapping agent [22]. While the chemical identity of the alkylating species is not established, we proposed that a C7radical (Fig. 2), formed from the semiquinone radical in the absence of oxygen, may alkylate both proteins and nucleic acids [22--24]. A number of observations support this proposal. First, 7-deoxyaglycone, probably formed as a result of a hydrogen abstraction by the CT-radical, has been detected in in vitro and in vivo metabolism of the drug, and second, formations of dimeric aglycones, linked at C~ to C7, have been isolated by Oki et ah [62] during reductive activation. In addition to the CT-radical, the C7 quinone methide derived from the two-electron reduction product of Adriamycin, which was first described by Moore [63,64], may also alkylate cellular macromolecules [22--24,63--67]. The fully reduced hydroquinone derivative can be formed by a two-electron reduction catalyzed by DT-diaphorase, or through cytochrome P-450 reductase by a sequential one-electron transfer. Alternatively, the semiquinone free radical can undergo a comproportionation-disproportionation to produce the alkylating species. The rate of the reaction has been proposed to be greater than 10SflV1-1 s -I, indicating that comproportionation-disproportionation reaction may be a viable mechanism for alkylation even in the presence of oxygen [68]. It is not clear how this Adriamycin-induced covalent binding relates to tumor cell kill, however, it is possible that the covalently bound DNA adduct, if not repaired properly, may inhibit DNA and protein synthesis. It is also likely that the DNA-bound drug can remain active generating oxyradicals at the site of the DNA molecules and causing strand-breaks, inhibiting DNA template functions, and ultimately causing cell death. MITOMYCIN C AND ANALOGS

Mitomycin-C (MC), a clinically active drug against certain solid tumors, has also been shown to be activated to a bioalkylating species; both monoalkylated DNA and cross-linked DNA have been isolated in vitro and in vivo [69-72]. Although several recent publications [73,74] have described the formation of oxygen radicals from MC and its analogs and an excellent correlation has been found between MC-induced tumor cell kill and the formation of oxy-radicals [7,32,73], many investigators believe that alkylation and subsequent cross-linking of DNA is the major mechanism of action of this drug. This may be due to MCs significant activity against solid tumors [75] which are hypoxic due to poor blood flow and in which the formation of oxy-radicals by MC may therefore be minimal. While the formation of the cross-linking species is believed to result from a two-electron reduction of MC, oxy-radical formation is catalyzed by the one-electron reduction to the semiquinone free radical. Recently, Pritsos et al. [7] have examined the formation of oxy-radicals from mitomycin C and its analogs using tumor cell sonicates, and have

304 reported that those analogs which produce significantly more oxy-radicals are also significantly more cytotoxic to aerobic EMT6 cells than to the hypoxic EMT6 cells. More recently, these authors have shown that the inhibition of SOD by diethyldithiocarbamate exascerbated the cytotoxicity of oxy-radical producing MC analogs without significantly increasing DNA cross-linking (C. Pritsos, pers. commun.). These observations indicate that the intracellular production of potentially toxic oxy-radicals from MC and its analogs may contribute to tumor cell kill. This conclusion is further confirmed by the studies of Doroshow who has shown that the toxicity of mitomycin C against Ehrlich ascites carcinoma is abolished by superoxide dismutase and catalase enzymes responsible for the detoxification of superoxide and hydrogen peroxide [32]. Furthermore, the inhibitors of "OH were also effective modulators of MC toxicity. Recently, we examined the cytotoxicity, DNA interstrand crosslinking and formation of "OH in human normal (IMR-90) and viraly transformed (VA-13) fibroblast cell lines by MC [74]. Our studies show that although the IMR-90 cells were ~fold more resistant to MC, the interstrand DNA cross-linking was similar in both cell lines. However, the formation of DMPO-OH was significantly higher (4--5-fold) in the VA-13 cells, the cell line more sensitive to MC-induced killing [76]. This difference in the "OH production resulted from enhanced removal of H202 as there was a 3-fold increase in the catalase activity in the resistant IMR-90 cells; this suggests free radical-dependent cell killing [76]. Based on limited studies in tumor cells, these results suggest that MC and certain analogs are capable of producing oxy-radicals when reduced intracellularly in the presence of oxygen and that the formation of these potentially toxic species may cause cell damage and cell death. Furthermore, covalently DNA bound MC has been reported to remain redox active and produce oxyradical [77]. It is possible a similar reaction may also operate in tumor cells and cause disruption of cellular functions. DIAZIQUONE

Diaziquone (AZQ), like mitomycin C, contains both an alkylating aziridine moiety and a quinone group and can be reductively activated to an alkylating species and to a semiquinone free radical intermediate. Due to the presence of two aziridines in the para-position of the diaziquone molecule, the reductive activation has been shown to lead to interstrand cross-linking of the DNA molecule in the Chinese hamster ovary cells and in other tumor cell lines [78,79]. The structure-activity relationship with AZQ analogs shows that the presence of the aziridine ring is essential for antitumor activity and it has been proposed to involve formation of DNA cross-links [78,79]. Like other quinone-containing drugs, AZQ has been reported to form a semiquinone radical by the autooxidation of the fully reduced AZQ in the presence of liver microsomal proteins [80]. This activation is catalyzed by cytochrome P-450 reductase and requires NADPH [81]. The semiquinone free radical intermediate of AZQ has also been detected

305 in a number of tumor cell lines without adding any co-factors [82], e.g. human leukemic cell lines K562, HL60 and L1210. It is interesting to note that the semiquinone radical of AZQ is stable in the presence of oxygen, indicating the chemistry is somewhat different than that of the Adriamycin semiquinone which is extremely sensitive to oxygen. It is also interesting that "OH has been directly detected by spin-trapping techniques in murine P388 and Hep II cell lines (P. Gutierrez, pets. commun.) and during photochemical activation of AZQ [83]. The formation of oxy-radical from AZQ during reductive activation implies that the direct reaction of oxygen with the semiquinone (since it is detected by EPR in the presence of oxygen) is not involved and must therefore arise from the reaction of oxygen with the dihydro-AZQ during its autooxidation. It has been reported that toxic radicals may be involved in DNA strand breaks and in the cytotoxicity of AZQ. For example, Szmigiero et al. [78,79] have shown that AZQ-induced strand breaks in both intact cells and in isolated nuclei were partially protected by SOD and catalase, while SOD completely inhibited single strand DNA breaks in isolated nuclei. Doroshow [32] has also reported that the scavengers of oxy-radicals, SOD and catalase significantly reduce AZQ toxicity to Ehrlich tumor cells. These observations suggest that free radicals may also have a role in AZQ-dependent tumor cell kill. ETOPOSIDE AND TENIPOSIDE

The semisynthetic podophyllotoxin derivative, VP-16 (Fig. 1) is the most active single agent in the treatment of small cell lung cancer. In addition, VP-16 has shown considerable activity against testicular cancer and malignant lymphoma. VP-16 has been shown to induce DNA strand breaks in tumor cells and it is believed that topoisomerase II is the likely intracellular target for this DNA damage [84,85]. Although the topoisomerase-mediated DNA strand breaking activity of VP-16 has been implicated in its cytotoxicity, the molecular basis is not clearly established. For DNA damage and the biological activity to occur, Loike and Horwitz found that the presence of both cellular components and a free hydroxyl group in the C-4' is essential [86], indicating that other factors also contribute in the biochemical pharmacology of these drugs. Enzymatic activations, catalyzed by peroxidase (prostaglandin synthetase or myeloperoxidase) activate phenolic compounds to free radical intermediates which have been implicated in the toxicity of this class of compounds. Since VP-16 and the related VM-26 are phenoxyl-containing drugs, i.e. containing a free OH group in the 4'-position, it is possible that this OH group is involved in free radical formation and in tumor cells kill. Using horseradish peroxidase, a model enzyme for peroxidases, we have shown that either drug rapidly formed the phenoxy radical in the presence of hydrogen peroxide [14,15] (Fig. 5). Unlike the semiquinone radical intermediate derived from Adriamycin, the phenoxy radical of VP-16 or VM-26 was found to be stable at neutral pH and did not react with oxygen to generate toxic oxygen radi-

306

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Fig. 5. ESR spectrum obtained from (A) VP-16 during peroxidase activationand its reaction with {B)glutathione,and (C) cystein in the presence of DMPO.

cals; thus indicating that neither 0"2 nor "OH directly contributed to the cytotoxicity or DNA breaking activities of VP-16/VM-26. However, the peroxidative oxidation of VP-16NM-26 formed a number of metabolites including o-quinone derivatives through oxidative o-demethylation [15,87]. Prostaglandin synthetase (PES), an enzyme present in most mammalian cells, also catalyzed activation of the drugs to the phenoxy radicals with subse-

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308 quent formation of VP-16 metabolites in the presence of either hydrogen peroxide or arachidonic acid [15,87]. Thus, it appears that co-oxidation of VP16 and VM-26 to the phenoxy radical intermediate during prostaglandin biosynthesis represents an important pathway for the metabolism of VP-16 and VM-26 (Fig. 6). The peroxidase-catalyzed activation of VP-16 also formed reactive species [87]. This was confirmed by the trapping of these species with either heatdenatured proteins or DNA. Binding required the co-factors, H202 or arachidonic acid, and increased with time, moreover, the PES-dependent binding was inhibited by indomethacin, an inhibitor of the cyclooxygenase. This suggested that the peroxidative arm of the prostaglandin synthetase was responsible for binding and involved quinone-methides formed from disproportionation-comproportionation of the phenoxy radical intermediate [87]. The metabolism and the binding of VP-16 required the presence of 4'-0H group since O-methyl derivatives did not form the phenoxy radical when activated by the peroxidase systems. It appears then that the phenoxy radical is the key intermediate for metabolism and formation of the reactive intermediates for binding to cellular macromolecules, however, this crucial intermediate does not bind to either DNA or proteins. VP-16 also formed reactive intermediates when activated by cytochrome P-450 systems that bound covalently to proteins and DNA [88--90], but no phenoxy radical was detected in this system. Since o-quinones are extremely reactive, the binding species was postulated to be VP-16-quinone. We also suggested that the oquinones of VP-16 and VM-26 may thus represent cytotoxic derivatives of the parent drugs which has been subsequently confirmed in a number of tumor cell lines. Phenoxy radicals react with sulfhydryl-containing compounds and form thiyl radicals [91]. This implication of such reactions is that glutathione or soluble sulfhydryl will be depleted. Since glutathione is essential for the protection of tumor cells and detoxifications of the reactive intermediates (free radicals and electrophils), such depletion induced by antitumor drugs may result in enhanced tumor cell killing. Because VP-16 forms both a phenoxy radical and a reactive bioalkylating species, it is reasonable to expect that VP-16 administration could deplete tissue or cellular glutathione. We therefore examined the reaction of the VP-16 radical with soluble thiols in vitro and in vivo. We found that both glutathione and cysteine rapidly reduce the VP-16 radical resulting in the regeneration of the parent drug and the oxidation of the thiols. Using electron spin resonance and DMPO as a spin trap, we have shown that this one-electron reduction/hydrogen donation by thiols formed thiyl radicals [92]. The hyperfine coupling constants for the DMPO adducts were: a N = 15.3 G, a H = 17.0 G for DMPO-cysteine and a N = 15.0 G, a H = 16.3 G for DMPO-glutathione, respectively (Fig. 5). We also reasoned that a similar reaction may exist in vivo and administration of VP-16 would therefore deplete protective thiols. A single dose of VP-16 (40 mg/kg, i.p.) in mice caused a significant decrease in total thiols in the liver and concomitantly increased the formation of the oxidized thiols [92]. In

309 contrast, VP-16 caused a marked increase in both forms of thiols (reduced and oxidized) in mouse kidney; this level remained high for 48 h. Large increases in thiols in kidney result from thiol depletion in liver and other tissues due to unidirectional release of GSH into blood plasma which is carried to kidney for GSH catabolism and the reutilization of the amino acids. The significance of this finding is that free radical-dependent metabolism of VP16 may deplete soluble thiols in tumor cells resulting in enhanced sensitivity to other antitumor drugs such as cis-platinum or alternately, cause oxidation of important enzymes containing sulfhydryl groups, e.g. topoisomerase II. We have found that the dihydroxy derivative of VP-16, a metabolite of VP-16, is cytotoxic to human tumor cells. This dihydroxy derivative also chelates iron and the resulting complex effectively catalyzes the formation of •OH in the presence of either H202 or reduced glutathione. Moreover, under the conditions of free radical formation from the drug-Fe complex, significant DNA damage was also observed in SV40 viral DNA. Again, this suggests that "OH may be involved in the antitumor activity of the metabolite [93]. MISCELLANEOUS

Dependency on free radical formation has been reported in the cytotoxicity of a number of other antitumor drugs, e.g. procarbazine, streptonigrin and bleomycin. The chemistry and the metabolism of procarbazine, a hydrazine-containing antitumor drug has been recently reviewed [94]. Cytochrome P-450-dependent oxidative metabolism of procarbazine [95,96] results in the formation of reactive methylating species which have been reported to be essential for its activity. Two reactive methylating agents have been postulated: a methyl carbonium ion (CH~) and methyl radical ('CH 8) [97,98]. In support of a free radical-dependent metabolic pathway, methane [98] and N-isopropyl-/~toluamide [99] have been detected from procarbazine during its metabolism. We have confirmed the formation of methyl radical by using spin-trapping technique during the metabolism catalyzed by peroxidases and the P-450 systems [16]. It is not clear at this time which of the reactive methylating species is formed in tumor cells leading to cell death. Streptonigrin, a quinone-containing antibiotic, is reduced via a one-electron reduction pathway to form the semiquinone radical intermediate and ultimately to "OH in the presence of oxygen [100]. The presence of certain metal ions and oxygen enhances cytotoxic effects of the drug [101], and an iron chelator, desferal, is inhibitory [102] therefore indicating the role of oxygen radicals in the mechanism of toxicity. It has been proposed that the cytotoxic and the antibacterial effects of streptonigrin arise from "0Hinduced DNA damage [103]. Enhancement of drug toxicity in the presence of metal ions could occur through two different mechanisms: chelation of metal ions with streptonigrin bringing about a conformational change in the drug molecule causing intercalation into DNA and metal-drug complex dependent site specific, free radical generation, alternatively, the drug metal complexes

310 (Zn 2÷) have been reported to enhance the drug-induced irreversible binding

to DNA during reductive activation [104]. Actinomycin D, a quinone-imine containing pentapeptide antibiotic, is active against Wilm's tumor and gestational choriocarcinoma. The mechanism of action of this drug is believed to result from its interactions with double-stranded DNA and the subsequent inhibition of protein synthesis [105,106]. However, studies reported from a number of laboratories have shown that actinomycin D undergoes one-electron reductive activation, in a microsomal system or in the presence of nuclei, to a free radical intermediate which rapidly consumes oxygen to produce superoxide anion radical [107 --109]. The structure-activity analysis has shown that analogs which generate more oxygen-radicals are also superior antitumor drugs in vivo [108,110], suggesting that reductive activation and free radical formation may be important in the cytotoxicity of this class of drugs. ROLE OF FREE RADICALS IN DRUG-RESISTANCE

One of the problems commonly faced in the clinic is that drug treatment often leads to the development of resistance to one or a wide variety of drugs of different structures and possibly different mechanism of actions. This cross-resistant or more commonly referred to as multi-drug resistant cells, show an increased expression of a p-170 glycoprotein [111], and decrease in drug accumulation as a result of increased removal of the drug [112]. Although very little is known about the role of free radical formation in drug-resistance, recent studies suggest that the formation of these reactive species and their detoxification may be important in the development of resistance in certain tumor cells. Studies from our laboratories, as discussed before, have shown that human breast tumor (MCF-7) cells selected for resistance against Adriamycin showed a decrease in the formation of "OH in the presence of Adriamycin when compared to drug sensitive cells [41,42]. The decrease in "OH formation was not due to decreased activation of Adriamycin in the resistant cells, but appeared to correlate with increased activities of detoxification enzymes, glutathione peroxidase and glutathione Stransferase, in the resistant cells. No increase in either SOD or catalase was detected. We have recently also shown that depletion of glutathione with buthionine sulfoximine enhanced "OH formation 2-fold in the MCF-7 resistant cells without increasing intracellular Adriamycin, indicating that GSH and glutathione peroxidase were responsible for the detoxification of hydrogen peroxide [113]. Moreover, GSH depletion in this cell line also caused a 4 - 5 fold increase in Adriamycin cytotoxicity. Increased glutathione peroxidase and glutathione transferase activities in Adriamycin-resistant MCF-7 cells appear to be unique for this cell line since this phenomenon is absent in other Adriamycin-resistant cell lines. For example, Ramu et al. [114] have reported no significant differences in the activities of SOD, catalase or glutathione peroxidase activity in an Adriamycin-resistant P388 murine leukemia cell line. Recently, Meijer et al. [115] have demonstrated that Adriamycin-

311 resistant sublines, derived from a human small cell lung carcinoma line, also became resistant to radiation and H202. This indicates that there is a common mechanism, i.e. free radical-dependent cell kill, for these diverse agents. Although there was no increase in the activity of free radical detoxifying enzymes such as SOD, catalase or glutathione peroxidase, the resistance to Adriamycin and free radicals in this cell line appeared to be due to increased repair of the DNA lesions caused by the production of oxy-radicals (by Adriamycin, hydrogen peroxide or X-ray) and not by decreasing the damage through increased detoxification of oxy-radicals. CONCLUSIONS The evidence presented in this review indicates that anticancer drugs of different chemical structures generate free radical intermediates during reductive or oxidative activation. The formation of the intermediates has been detected in biological systems and, in some cases, in human tumor cells using ESR and spin-stabilization techniques. Although, free radical intermediates have not been detected from these agents in vivo, evidence is now available which indicates that free radicals are also formed in vivo. While the involvement of free radicals in tumor cell kill remains controversial, there appears to be more than a casual relationship between free radical formation and tumor cell kill for certain drugs, i.e. anthracyclines and mitomycin C. However, activation of these drugs to free radical species in all tumor cell lines has not been an universal finding. The biochemistry of the tumor cell, i.e. the presence of activating and detoxification enzymes would dictate whether the free radical is formed and subsequently detected by ESR. This was clearly evident in the case of the MCF-7 breast tumor cell line in the presence of Adriamycin or IMR-90 cells in the presence of mitomycin C, indicating that a generalization should not be made for all tumor cells. Thus, before a free radical-dependent mechanism is discarded, an examination and characterization of the enzymes present in tumor cells is warranted. It is also advisable that a generalization and extrapolation from chemically activating system should not be made to tumor cells. Furthermore, tumor cells in tissue culture conditions, an artificial system at best, cannot and should not be compared to in vivo conditions. A tumor growing in an animal or human is a mixture of many different cell types and it is rather difficult to predict if such a heterogeneous system can or cannot cause drug activation. Furthermore, the stability of primary radical intermediates will depend upon cellular biological environments, i.e. concentrations of sulfhydryl compound, GSH and the oxygen. Under normal oxygen conditions, oxy-radical-dependent cellular damage, e.g. peroxidation and oxidation of critical enzymes and DNA may occur. On the other hand, under hypoxic conditions, primary radicals such as semiquinone or the phenoxy radical may undergo further metabolism to form species with alkylation potential for critical cellular macromolecules. Thus, it is concluded that some of the clinically useful anticancer drugs form free radical species (primary or oxy-radi-

312 cals) which are involved in the tumor cell killing via modification of membranes, proteins, enzymes or DNA molecules. ACKNOWLEDGEMENT

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314 41 B.K. Sinha, A.G. Katki, G. Batist, K.H. Cowan and C.E. Myers, Adriamycin-stimulated hydroxyl radical formation in human tumor cells, Biochem. Pharmacol., 36 (1987) 793. 42 B.K. Sinha, A.G. Katki, G. Batist, K.H. Cowan and C.E. Myers, Differential formation of hydroxyl radicals by Adriamycin in sensitive and resistant MCF-7 human breast tumor cells: implication for the mechanism of action, Biochemistry, 26 (1987) 3776. 43 G. Batist, A. Tulpule, B.K. Sinha, A.G. Katki, C.E. Myers and K.H. Cowan, Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells, J. Biol. Chem., 261 (1986) 15544. 44 K.H. Cowan, G. Batist, A. Tulpule, B.K. Sinha and C.E. Myers, Similar biochemical changes associated with multidrug resistance in human breast cancer cells and carcinogeninduced resistance to xenobiotics in rats, Proc. Natl. Acad. Sci., 83 (1986) 9324. 45 W.S. Thayer, Serum lipid peroxides in rats treated chronically with Adriamycin, Biochem. Pharmacol., 33 (1984) 2259. 46 R.A. Floyd, R. Henderson, J.J. Watson and P.K. Wong, Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCED) as a sensitive measure of hydroxyl radicals in Adriamycin treated rats, J. Free-Rad. Biol. Med., 2 (1986) 13. 47 K. Sugoka, H. Nakano, T. Noguchi, J. Tsuchiya and M. Nakano, Decomposition of unsaturated phosphollpids by iron-ADP-Adriamycin co-ordination complex, Biochem. Biophys. Res. Commun., 100 (1981) 1251. 48 K. Sugoka and M. Nakano, Mechanism of phospholipid peroxidation induced by ferric ionADP-Adriamycin-co-ordination complex, Biochim. Biophys. Acta, 713 (1982) 333. 49 H. Nakano, K. Ogita, J.M.C. Gutterridge and M. Nakano, Inhibition by the protein ceruloplasmin of lipid peroxidation stimulated by an FeS'-ADP-Adriamycin complex, FEBS Lett., 166 (1984) 232. 50 H. Eliot, L. Gianni and C. Myers, Oxidative destruction of DNA by the Adriamycin-iron complex. Biochemistry, 23 (1984) 928. 51 L. Gianni, J.L. Zweir, A. Levy and C.E. Myers, Characterization of the cycle of ironmediated electron transfer from Adriamycin to molecular oxygen, J. Biol. Chem., 260 (1985) 6820. 52 L. Gianni, J.L. Zweir, A. Levy and C.E. Myers, Characterization of the cycle of ironmediated electron transfer from Adriamycin to molecular oxygen, J. Biol. Chem., 260 (1985) 6820. 53 J.L. Zweir, Reduction of 02 by iron-Adriamycin,J. Biol. Chem., 259 (1984) 6056. 54 J.R.F. Muindi, B.K. Sinha, L. Gianni and C.E. Myers, Hydroxyl radical production and DNA damage by anthracycline-iron complex, FEBS Lett., 172 (1984) 226. 55 J.R.F. Muindi, B.K. Sinha, L. Gianni and C.E. Myers, Thiol dependent DNA damage pr~ duced by anthracycline-iron complex: the structure activity relationships and molecular mechanisms, Mol. Pharmacol., 27 (1985) 356. 56 C.E. Myers, J.R. Muindi, J. Zweier and B.K. Sinha, 5-Iminodaunomycin: an anthracyclin~ with unique properties, J. Biol. Chem., 262 (1987) 11571. 57 E.J~F. Demant and N. Norskov-Lauritsen, Binding of transferrin-iron by Adriamycin at acidic pH, FEBS Lett., 196 (1986) 321. 58 C.E. Thomas and S.D. Aust, Release of iron from ferritin by cardiotoxic anthracycline antibiotics, Arch. Biochem. Biophys., 248 (1986) 686. 59 M. Gosalvez, M.F. Blanco, C. Vivero and F. Valles, Quelamycin, a new derivative of Adriamycin with several possible therapeutic advantages, Eur. J. Cancer, 14 (1978) 1185. 60 H. Beraldo, A. Garnier-Suillerot, L. Tosi and F. Lavelle, Iron-Adriamycin and iron-daunomycin complexes: physicochemical characteristics, interactions with DNA, and antitumor activity, Biochemistry, 24 (1985) 284. 61 A. Samuni, L.-G. Chong, Y. Barenholz and T.E. Thompson, Physical and chemical modifications of Adriamycin : iron complexes by phospholipid bilayers, Cancer Res., 46 (1986) 594. 62 T. Oki, T. Komiyama, H. Tone, T. Inui, T. Takeuchi and H. Umezawa, Reductive cleavage of anthracycline glycoside by microsomal NADPH-cytochrome C reductase, J. Antibiot., 30 (1977) 613.

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