Reductive Activation of Mitomycin C by Thiols - American Chemical

30 Sep 2009 - with MC activated by other reducing agents (14). Scheme 1. Mechanism of the Reductive Activation of MCa a NuH represents a nucleophile...
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Chem. Res. Toxicol. 2009, 22, 1663–1668

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Communication Reductive Activation of Mitomycin C by Thiols: Kinetics, Mechanism, and Biological Implications Manuel M. Paz* Departmento de Quı´mica Orga´nica, UniVersidade de Santiago de Compostela, Facultade de Ciencias, Campus de Lugo, 27002 Lugo, Spain ReceiVed August 10, 2009

The clinically used antitumor antibiotic mitomycin C requires a reductive activation to be converted to a bis-electrophile that forms several covalent adducts with DNA, including an interstrand cross-link which is considered to be the lesion responsible for the cytotoxic effects of the drug. Enzymes such as cytochrome P450 reductase and DT-diaphorase have traditionally been implicated in the bioreduction of mitomycin C, but recent reports indicate that enzymes containing a dithiol active site are also involved in the metabolism of mitomycin C. The reductive activation can also be effected in vitro with chemical reductants, but until now, mitomycin C was considered to be inert to thiols. We report here that mitomycin C can, in fact, be reductively activated by thiols. We show that the reaction is autocatalytic and that the end product is a relatively stable aziridinomitosene that can be trapped by adding several nucleophiles after the activation reaction. Kinetic studies show that the reaction is highly sensitive to pH and does not proceed or proceeds very slowly at neutral pH, an observation that explains the unsuccessful results on previous attempts to activate mitomycin C with thiols. The optimum pH for the reactions is around the pKa values of the thiols used in the activation. A mechanism for the reaction is hypothesized, involving the initial formation of a thiolate-mitomycin adduct, that then evolves to give the hydroquinone of mitomycin C and disulfide. The results presented here provide a chemical mechanism to explain how some biological dithiols containing an unusually acidic thiol group (deprotonated at physiological pH) participate in the modulation of mitomycin C cytotoxicity. Introduction 1

Mitomycin C (MC, 1) (Figure 1) is a natural antitumor antibiotic used in anticancer therapy (1) that requires reductive activation to exert its biological activity (2). After bioreduction, MC generates a bifunctional electrophile (4, Scheme 1) that alkylates cellular nucleophiles, in particular the complementary strands of DNA (1, 3). A number of enzymes are known to activate MC in mammalian cells (4, 5), but only recently have proteins containing a dithiol active site been implicated in the activation of MC in cell cultures (6, 7). However, in Vitro studies have so far failed to show that simple thiols are able to activate MC (8). The results we present here reconcile this apparent paradox. We report that MC is indeed activated in Vitro by simple thiols; a mechanism for the reaction is proposed on the basis of kinetic data and the identification of mitosene metabolites, and the biological implications of these findings are discussed. The mechanism of activation of MC starts with a reduction to the hydroquinone form (3), followed by a spontaneous elimination of MeOH to generate a leucoaziridinomitosene (4), which then alkylates nucleophiles at its 1 and 10 positions * Corresponding author. E-mail: [email protected]. 1 Abbreviations: DHLA, dihydrolipoic acid; DTT, D,L-dithiothreitol; MA, mitomycin A; MC mitomycin C; MER, 2-mercaptoethanol; Trx, thioredoxin; TrxR, thioredoxin reductase; mitosene, structure 6, without substituents in the 1-, 2- and 7-positions.

(Scheme 1) (1, 3). The proteins most frequently implicated in the in ViVo activation of MC are cytochrome P450 reductase (4) and DT-diaphorase (5). Nevertheless, many studies indicate that there is also a direct implication of biological dithiols in the modulation of MC activity in cell cultures, both as activators and inhibitors of the cytotoxic effects of the drug. Thioredoxin (Trx), a cellular dithiol, protected Fanconi anemia cell lines from the cytotoxic effects of MC (6). Several MC-resistant human bladder cancer cell lines showed much higher levels of thioredoxin than their MC-sensitive relatives, while transfectants of these resistant cell lines, containing lower levels of thioredoxin, presented increased MC-sensitivity compared to that of their wild-type counterparts (9). By contrast, the dithiol-containing domains of glucose regulatory protein (GRP58) have been shown to be involved in the activation of MC to form toxic metabolites in various tumor cell lines and to induce the formation of DNA-DNA cross-links (7, 10). Chemical reductants known to activate MC include sodium dithionite, catalytic hydrogenation, formate radicals, or sodium borohydride (2, 3, 11). Mitomycin A (MA, 2), a congener of

Figure 1. Structure of mitomycin C and mitomycin A.

10.1021/tx9002758 CCC: $40.75  2009 American Chemical Society Published on Web 09/30/2009

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Scheme 1. Mechanism of the Reductive Activation of MCa

Figure 2. UV assay of the reduction of 40 µM mitomycin C by 100 mM DTT in 500 mM phosphate buffer, pH 7.5. Reaction times are indicated on top of each spectrum.

a

NuH represents a nucleophile.

MC that differs only in the substituent in the 7 position (Figure 1), can be activated by the same reagents and enzymes that activate MC and also can be reduced efficiently by thiols (12, 13). However, MC was shown to be inert to reductive activation by monothiols and dithiols (8, 14),2 a result incongruous with the aforementioned in ViVo cell culture results. We decided to re-examine the reaction of MC with thiols, in particular after recent reports linked the thioredoxin-like domains of GRP58 to the cytotoxic effects of MC in cancer cells (7) and in the in Vitro generation of DNA cross-links (10), an explicit sign of the involvement of the dithiol group in the cellular activation of MC.

Materials and Methods General Procedures. Mitomycin C was a gift from Dr. Maria Tomasz. DTT, 1,3-propanedithiol, and mercaptoethanol were from Sigma-Aldrich. HPLC was performed with a Hewlett-Packard Series 1100 diode array system. LCMS was performed with a Hewlett-Packard Series 1100 diode array HPLC system connected to a Hewlett-Packard Series 1100 MSD mass spectrometer. Agilent (Zorbax C-18, 5 µm, 4.6 × 150 mm), Waters (X-terra MS C18, 2.5 µm, 2.1 × 50 mm), or Macherey Nagel (Nucleodur C18, 5 µm, 4.6 × 125 mm) columns were used. The elution system was 5% to 30% B in 20 min, 30% to 60% B in 9 min. (A ) 10 mM ammonium acetate, pH 5.5; B ) acetonitrile). UV spectra and kinetic studies were performed with a CARY 300 UV-visible spectrometer. Activation of MC with Thiols and Trapping of the Putative Aziridinomitosene 5 with Water, Methanol, or Ethanol. A solution of MC in water (100 mL of a 5 µM solution) was treated with a solution of DTT/DTT- (15 µL of a 10 mM solution adjusted to pH 9.5). After a short period (2-5 min), the blue solution veered in a matter of seconds into a deep purple solution. After 5 additional minutes, an aliquot (3 µL) was diluted with 50 mM sodium phosphate buffer (pH 7.0) and analyzed by UV (200-400 nm), showing a spectrum that corresponded to a mitosene. The solution was divided into 3 portions that were respectively diluted with H2O, MeOH, or EtOH (2 mL) containing 5 µL of 200 mM Tris at pH 2 While thiols themselves did not reduce MC, they reacted as nucleophiles with MC activated by other reducing agents (14).

7.0. After 1 h, the solutions were admixed with 1 mL of water and concentrated in Vacuo to a volume of approximately 0.5 mL. The resulting solutions were analyzed by LCMS, obtaining the following results. 6a: Retention times 6.9 and 7.5 min. EI-MS (ES+) m/z: 260 (M - OCONH2-), 321 (M + H+), 343 (M + Na+), 359 (M + K+). UV (λmax, nm): 252, 310. 6c: Retention times 10.7 and 12.2 min. EI-MS (ES+) m/z: 274 (M - OCONH2-), 335 (M + H+), 357 (M + Na+), 373 (M + K+). UV (λmax, nm): 252, 310. 6d: Retention times 13.3 and 14.6 min. EI-MS (ES+) m/z: 288 (M OCONH2-), 371 (M + Na+), 387 (M + K+). UV (λmax, nm): 252, 310. Comparison with Authentic Samples. Samples of 1-hydroxyor 1-alkoxy-substituted mitosenes were prepared by acid hydrolysis of MC in water, methanol, or ethanol according to the procedure described by Taylor and Remers (18). This material was analyzed by HPLC, showing retention times analogous to those of the material obtained by activation of MC with DTT as described above. Each of the samples of 6a, 6c, and 6d prepared by acidic hydrolysis was admixed with the corresponding sample of 6a, 6c, and 6d, prepared from DTT-activated MC as described above. HPLC analysis of these mixtures (λ ) 315 nm, Agilent column) showed that the material coeluted in all cases. General Protocol for Kinetic Measurements of the Reactions between MC and Thiols. Reactions were started by adding an aqueous thiol solution to an aqueous mitomycin solution in a UV cuvette at 20 °C, using a total volume of 0.50 mL. The absorbance at 360 nm was recorded at 1-s intervals. The molar fraction of mitomycin C at time t (χMC(t)) was calculated from the absorbance readings using the following equation:

χMC(t) ) (At - A∞)/(A0 - A∞) where A0 is the absorbance of the solution at 360 nm before the addition of thiol (corrected for the final volume), At is the absorbance of the solution at a time t after the addition of thiol, and A∞ is the absorbance of the solution after all of the mitomycin C is consumed. Kinetics of the Reactions of MC with MER or DTT. For experiments performed at pH ) pKa(thiol) ( 1, the thiol per se was used as internal buffer. The thiolates were prepared by adding aqueous NaOH to aqueous solutions of thiol. For experiments at other pH values, phosphate, Tris, or carbonate buffers were used. Kinetics of the Reactions of MC with Propanedithiol and DHLA. A modified protocol was employed because of the low water solubility of the thiols. Propanedithiol and DHLA stock solutions in acetonitrile of at least 20 times the final concentration were used so that the final acetonitrile concentration never exceeded 5%. The pH of the reacion was controlled by an external buffer (0.1 M Tris, 0.1 M carbonate, or 0.1 M KCl-NaOH).

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Figure 3. Selected curves representing the autocatalytic decay of MC by reaction with the four thiols studied in this work. Thiol concentrations and pH were as follows: (a) 5 mM propanedithiol, pH 10.5; (b) 1 mM DHLA, pH 9.6; (c) 15 mM DTT, pH 9.5; (d) 400 mM mercaptoethanol, pH 9.3. [MC] ) 30 µM. See Matherials and Methods and Supporting Information for additional details.

Results and Discussion The first experiments in our re-evaluation of the reaction of MC with thiols were performed by UV spectroscopy, by tracking the conversion of the mitosane chromophore of MC (λmax 360 nm) to the mitosene chromophore (λmax 315 nm). UV analysis of a reaction containing MC and a large excess of DTT (0.1 M) at neutral pH clearly showed the gradual formation of the mitosene compounds at the expense of MC (Figure 2), the reaction reaching completion in about 120 min, as judged by constant UV spectra. In marked contrast to MC, MA was reduced in just a few minutes using only submilimolar DTT concentrations (12). The kinetic course of the reaction of MC with DTT was followed by measuring the decline of the UV absorbance at 360 nm (Figure 3, curve c). The decay of MC displayed the shape of a sigmoid curve, characteristic of autocatalytic reactions. The course of the reaction of MC with the other three thiols studied presented a similar shape. Dissimilarly, the kinetics of the reaction of MA with thiols presented an exponential decay (12). The autocatalytic activation of MC has been observed before, both during enzymatic (15) and chemical (11) reductions. This mechanism is based on the large redox potential difference between the mitosane (1) and mitosene (5) structures (16, 17). Thus, the leucomitosene species, e.g., 4, transfer their electrons to unreduced MC, creating an electron transfer chain reaction, provided the reducing agent is slow to reduce excess MC (3, 4, 12, 13). The identity of the mitosenes formed in the reaction of MC with DTT was studied by LCMS. MC was activated with substoichometric DTT at pH 9.5 until all MC was consumed, then quenched with diluted aqueous buffer at neutral pH. LCMS analysis showed the formation of the expected diastereomeric pair of hydroxymitosenes 6a (18). In our initial experiments, we quenched an aliquot of activated MC solution with acetate buffer prior to HPLC analysis. The chromatogram showed two Scheme 2. Hypothesis for the Mechanism of Reduction of MC by Thiols

peaks corresponding to the expected hydroxymitosenes 6a and also two additional peaks that were identified as acetylated mitosenes (6b and an N-acetyl regioisomer), on the basis of their MS and UV (18).3 This outcome indicated that the mixture of DTT-activated MC contained intact aziridinomitosene 5 and that the opening of the aziridine ring occurred only after the solution was treated with acetate buffer, prior to HPLC injection. The aziridinomitosene 5 has been isolated previously from a disproportionation reaction in pyridine (19) and as a precipitate in the electrolytic reduction of MC in MeOH (20), but it has never been detected as a stable entity in water. To demonstrate that the reaction of MC with DTT generates long-lived 5 in aqueous media, a solution of activated MC was quenched with neutral aqueous buffer, methanol, or ethanol (also at neutral pH). HPLC analysis of the resulting mixtures showed that the hydroxy, methoxy, and ethoxy substituted mitosenes (6a, 6c, and 6d, respectively) are formed. In the reactions with MeOH and EtOH, minimal amounts of the hydroxymitosene 6a are observed,4 proving that the end product of MC activation is 5 and, at the same time, reflecting the remarkable stability of 5 in aqueous alkaline media. The half-life of 5 is about 100 min at pH 10, as measured by the exponential decay in the competency of an aged solution of activated MC to form trans-6b (and its cis-N-acetyl regioisomer) when treated with acetate buffer.4 The hydroxymitosenes 6a were also the major metabolites observed after the reduction of MC with propanedithiol and dihydrolipoic acid.4 The activation with MER did not proceed with substoichiometric concentrations of thiol. Larger concentrations of MER (40-fold excess) resulted in full conversion of MC to mitosenes, and the metabolites formed were identified by their MS as disubstituted mitosenes (7, NuH ) MER). Analogous compounds were observed earlier in the reaction of thiols with MC activated by other reducing agents (14). The kinetics of activation of MC with thiols was studied in detail varying the thiol, the concentration of thiol, and the pH. The rate of reduction (measured as t1/2-1) was proportional to the concentration of thiolate, as observed either by increasing the concentration of thiol at constant pH or by increasing the pH at constant concentration of thiol (until a certain pH was reached, as will be discussed below).4 The observed dependence on thiolate concentration suggests that the reaction may occur by a mechanism analogous to the one we previously proposed for the activation of MA by thiols (12): an initial conjugate addition of thiolate to the quinone of MC, followed by an internal redox reaction to give the hydroquinone of MC and the corresponding disulfide (Scheme 2). The reaction of MC with thiols is an autocatalytic reaction, while the reaction of MA with thiols follows an exponential decay, impeding us from quantitatively comparing the reactivity of MC and MA with thiols. To gain access to truly comparable rate constants, we estimated pseudofirst order constants (kobs) for the reaction of MC with thiols using the initial data points from some slow reactions before the autocatalytic reaction started. Second order rate constants were obtained by plotting kobs versus the concentration of thiolate.4 The k values (in min-1 M-1) thus obtained were 20 for kMC-DTT and 0.5 for kMC-MER. The values for MA were 2 × 106 for kMA-DTT and 500 for kMA-MER (12). These numbers 3 It has been reported that the solvolysis of MC with acetic acid gives as the major product cis-acetylaminomitosene, resulting from the O- to N-acetyl migration from cis-6b, and trans-acetoxymitosene (trans-6b) as the minor product (18). The fragmentation pattern observed in the EI-MS of the two compounds formed after treating activated MC with acetate corroborates this assignation (see Supporting Information for details). 4 See Supporting Information for details.

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Figure 4. pH dependence of the rates of reaction of MC with the three thiols evaluated in this work: DTT (red); 1,3-propanedithiol (blue); MER (green); and DHLA (black). The relative rates are defined as the ratio t1/2-1/(t1/2-1)max, where t1/2, is the time required for 50% conversion of MC at a given pH determined from the curves of decay of MC, and (t1/2-1)max is the maximum rate observed at the pH values assayed for each thiol. See Matherials and Methods and Supporting Information for additional details.

Scheme 3. Hypothesis for the Mechanism of the Internal Redox Reaction Step in the Reduction of MC by Dithiols

support our previous conclusion that the high toxicity of MA compared to MC can be attributed to an efficient activation of MA by intracellular thiols (12, 13). A striking difference emerges when comparing the four rate constants: MA is activated with DTT 4000 faster than that with MER, while MC reacts with DTT only 40 times faster than that with MER.5 The reason for the large disparity (2 orders of magnitude) in the relative activation rates is unclear. One possible explanation is that the rate-limiting step for the reduction of both mitomycins by DTT is the first step (conjugate addition of thiolate), while for the reactions with MER, the rate-limiting step is the second step (internal redox reaction). In this scenario, the experimentally observed rates are justified if the rate deceleration of the first step is 2 orders of magnitude higher than the rate deceleration of the second step. We consider this a convincing hypothesis, as it implies that the most significant differences in the rates of activation of MC and MA originate in the conjugate addition of thiolate to the quinone: the Michael addition to a vinylogous amide (MC) will certainly be less favored than the addition to a vinylogous ester (MA) (21),6 while the second step, the formation of a hydroquinone and disulfide from the thiolate-mitomycin adduct, should plausibly be less affected by the substituent at the 7-position. When the pH dependence was studied, it was found that the reaction rate increases with higher pH values until it reaches an optimum pH and then decreases. The optimum pH values for the activation of MC with the four thiols used in this work 5 According to the mechanism shown is Scheme 1, the observed differences in the autocatalytic reduction of MC (Figure 3) must be proportional to the rate of reduction of MC by thiols (1 to 3). Using this reasoning, a ratio of reduction rates calculated from more accurate data obtained from plots of t1/2-1 vs the concentration of thiolate matches the ratio of reduction rates derived from the first minutes of exponential decay: MC reacts 60 times faster with DTT than with MER (see Supporting Information for details). 6 The reaction rate of nucleophiles with amides in water is about 3 orders of magnitude slower than that with esters for the hydroxide anion. The equilibrium constants show similar relative values (21).

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Figure 5. Comparison of the thiol pKa values for biological dithiols and simple dithiols (21, 22, 25). Trx, thioredoxin; TrxR, thioredoxin reductase.

were all around the sulfhydryl pKa value of each thiol (Figure 4) (22). The graphic data of pH dependence presented in Figure 4 clearly illustrates the reason why previous attempts to activate MC at pH around neutral have failed: as the reaction approaches neutrality, the half-time for the conversion of MC tends to be infinite, which explains why the experimental conditions employed in the earlier assays (that used relatively low concentrations of thiols) resulted in no appreciable reaction (8, 13, 14). The pH dependence data concords with a mechanism analogous to the one we previously proposed for the reduction of MA by dithiols. The formation of the intermediate requires a thiolate anion, while its conversion to hydroquinone requires a protonated thiol (Scheme 3). The fact that reductions of MC by dithiols are favored at a pH bound by the pKa1 and pKa2 of the dithiol lends support to the proposed mechanism, as it is precisely at those pH values where higher concentrations of both thiol and thiolate will be present. Are the results presented here relevant in ViVo? In principle, it would appear not to be so, as the pH required for an efficient reaction is distant from regular physiological values. We consider improbable the involvement of glutathione (a cellular monothiol) in the activation of MC in ViVo, as the concentrations of thiolate required for activation are several orders of magnitude higher than those present in cells. Likewise, free lipoate coenzymes are unlikely to contribute significantly to the cellular activation of MC, as the optimum pH for the activation with DHLA is 4 units above physiological pH values. However, the rationale changes if we consider the exceptional acid-base properties of other biological dithiols. Proteins of the thioredoxin superfamily (thioredoxin, glutaredoxin, protein disulfide-isomerases, and disulfide bond formation protein A) contain a dithiol in the active site, where the pKa of the N-terminal cysteines in the -Cys-X-X-Cys- motif is abnormally low (23-25), while thioredoxin reductases contain an even more acidic selenol group (Figure 4) (26). At physiological pH, both the thiol from Trx and the selenol from TrxR are deprotonated, and in the proposed mechanisms for their reactions, they behave as strong nucleophiles (27-29).7 In the mechanism we postulate (Scheme 3), the optimum pH for the activation of MC by dithiols is predicted to present a value flanked by the pKa values of their sulfhydryl residues. At such pH values, the most acidic thiol (or selenol) group will be deprotonated, and the nucleophilic addition (first step in Scheme 7 Of special interest, the mutation of 498-Sec to Cys in mammalian TrxR shifted the optimum pH from 7 to 9 (29).

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3) will be favored. The second sulfhydryl group of the dithiol presents pKa values above 8 both for Trx and TrxR, and will be mostly protonated at neutral or acidic pH (Figure 5), therefore favoring the second step in the proposed mechanism (Scheme 3). As a result, our model predicts that the optimum pH for the reduction of MC by Trx (pKa1 ) 7.1) will be around neutral and that for TrxR (pKa1 ) 5.8), the optimum pH will be above 6 and could, therefore, be capable of activating MC even at acidic pH values.8

Conclusions In summary, we have shown that thiols activate MC by reduction to form aziridinomitosene 5, a known DNA monoalkylating agent (2). The formation of 5 constitutes proof of the formation of leucoaziridinomitosene 4, a species implicated in the formation of DNA-DNA cross-links by reductively activated MC (2). Dithiols are more efficient than monothiols by about 2 orders of magnitude. The pH is crucial for a successful reaction, with an optimum pH located between the pKa values of the sulfhydryl groups of the dithiol. We conclude that the activation of MC with DTT presented here can possibly be reproduced in living organisms by biological dithiols, by virtue of the low pKa1 of their dithiol group (30).9 The results presented here allowed us to propose a reaction mechanism that serves as a chemical validation to the reported involvement of biological dithiols in the modulation of mitomycin C cytotoxicity: dithiols could detoxify MC by activating the drug in the cytosol, where it would be hydrolyzed to inactive mitosenes (6, 9). Conversely, dithiols could generate cytotoxic metabolites by reducing MC in close proximity to the nucleus (7, 31).10 The results of this work warrant further research to ascertain whether Trx and other dithiol-containing proteins are indeed capable of reducing MC. Acknowledgment. We thank Dr. Maria Tomasz for a gift of mitomycin C and for helpful discussions. We thank Dr. Arne Holmgren for useful suggestions. We also thank Dr. Eugenio Va´zquez Sentı´s and Dr. Jose´ Va´zquez Tato for the use of their facilities. Some expenses of this work were defrayed from grants from the Spanish Ministry of Education and Science (Grant CTQ200607854/BQU) and Xunta de Galicia (PGIDT06PXIB209109PR). Supporting Information Available: HPLC chromatograms showing the formation of 6a-d and 7 (NuH ) MER); UV and ESIMS of 6a-d and 7 (NuH ) MER); experimental data showing the decay of 5 by trapping with acetate; detailed kinetic data of the of the reaction of MC with thiols; and experimental procedures for the calculation of the rate constants of the reaction of MC with thiols. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Paz, M. M. (2008) Antitumor Antibiotics, in Anticancer Therapeutics (Missailidis, S., Ed.) pp 112-115, John Wiley and Sons, Chichester, U.K. 8 The optimum pH for the activation of MC by GRP58 to form interstrand DNA-DNA cross-links is around 6 (10). However, the pKa values for the thiols in the active site of the enzyme are unknown, impeding us from judging whether it fits our hypothesis. 9 It has been reported that TrxR reduced other antitumor quinones at pH 7.0 with an efficacy similar to that of Trx, its natural substrate (30). 10 It has been shown that the cellular localization of MC-activating enzymes influences the toxicity of the drug. MC citotoxicity was enhanced when activating enzymes were located close to the nucleus (31).

(2) Suresh Kumar, G., Lipman, R., Cummings, J., and Tomasz, M. (1997) Mitomycin C-DNA adducts generated by DT-diaphorase. Revised mechanism of the enzymatic reductive activation of mitomycin C. Biochemistry 36, 14128–14136. (3) Tomasz, M., Lipman, R., Chowdary, D., Pawlak, J., Verdine, G. L., and Nakanishi, K. (1987) Isolation and structure of a covalent crosslink adduct between mitomycin C and DNA. Science 235, 1204–1208. (4) Sartorelli, A. C., Hodnick, W. F., Belcourt, M. F., Tomasz, M., Haffty, B., Fischer, J. J., Rockwell, S., and Mitomycin, C. (1994) A prototype bioreductive agent. Oncol. Res. 10/11, 501–508. (5) Siegel, D., Beall, H., Senekowitsch, C., Kasai, M., Arai, H., Gibson, N. W., and Ross, D. (1992) Bioreductive activation of mitomycin C by DT-diaphorase. Biochemistry 31, 7879–7885. (6) Kontou, M., Adelfalk, C., Ramirez, M. H., Ruppitsch, W., HirschKauffmann, M., and Schweiger, M. (2002) Overexpressed thioredoxin compensates Fanconi anemia related chromosomal instability. Oncogene 21, 2406–2412. (7) Adikesavan, A. K., and Jaiswal, A. K. (2007) Thioredoxin-like domains required for glucose regulatory protein 58 mediated reductive activation of mitomycin C leading to DNA cross-linking. Mol. Cancer Ther. 6, 2719–2727. (8) He, Q.-Y., Maruenda, H., and Tomasz, M. (1994) Novel bioreductive activation mechanism of mitomycin C derivatives bearing a disulfide substituent in their quinone. J. Am. Chem. Soc. 116, 9349–9350. (9) Yokomizo, A., Ono, M., Nanri, H., Makino, Y., Ohga, T., Wada, M., Okamoto, T., Yodoi, J., Kuwano, M., and Kohno, K. (1995) Cellular levels of thioredoxin associated with drug sensitivity to cisplatin, mitomycin C, doxorubicin, and etoposide. Cancer Res. 55, 4293–4296. (10) Celli, M., and Jaiswal, A. K. (2003) Role of GRP58 in mitomycin C-induced DNA cross-linking. Cancer Res. 63, 6016–6025. (11) Hoey, B. M., Butler, J., and Swallow, A. J. (1988) Reductive activation of mitomycin C. Biochemistry 27, 2608–2614. (12) Paz, M. M., and Tomasz, M. (2001) Reductive activation of mitomycin A by thiols. Org. Lett. 3, 2789–2792. (13) Paz, M. M., Das, A., Palom, Y., He, Q.-Y., and Tomasz, M. (2001) Selective activation of mitomycin A by thiols to form DNA crosslinks and monoadducts: biochemical basis for the modulation of mitomycin cytotoxicity by the quinone redox potential. J. Med. Chem. 44, 2834–2842. (14) Sharma, M., and Tomasz, M. (1994) Effects of glutathione on alkylation and cross-linking of DNA by mitomycin C. Isolation of a ternary glutathione-mitomycin-DNA adduct. Chem. Res. Toxicol. 6, 390–400. (15) Peterson, D. M., and Fisher, J. (1986) Autocatalytic quinone methide formation from mitomycin C. Biochemistry 25, 4077–4084. (16) Kinoshita, S., Uzu, K., Nakano, K., Shimizu, M., and Takahashi, T. (1971) Mitomycin derivatives. 1. Preparation of mitosane and mitosene compounds and their biological activities. J. Med. Chem. 14, 103– 109. (17) Iyengar, B. S., Remers, W. A., and Bradner, W. T. (1986) Preparation and antitumor activity of 7-substituted 1,2-aziridinomitosenes. J. Med. Chem. 29, 1864–1868. (18) Taylor, W. G., and Remers, W. A. (1975) Structure and stereochemistry of some 1,2-disubstituted mitosenes from solvolysis of mitomycin C and mitomycin A. J. Med. Chem. 18, 307–311. (19) Danishefsky, S. J., and Egbertson, M. (1986) The characterization of intermediates in the mitomycin activation cascade: a practical synthesis of an aziridinomitosene. J. Am. Chem. Soc. 108, 4684–4650. (20) Han, I., and Kohn, H. (1991) 7-Aminoaziridinomitosenes: synthesis, structure, and chemistry. J. Org. Chem. 56, 4648–4653. (21) Carey, F. A., and Sundberg, R. J. (2007) AdVanced Organic Chemistry, Part A: Structure and Mechanisms, 5th ed., pp 328-329, Springer, New York. (22) Lamoureux, G. V., and Whitesides, G. M. (1993) Synthesis of dithiols as reducing agents for disulfides in neutral aqueous solution and comparison of reduction potentials. J. Org. Chem. 58, 633–641. (23) Dyson, H. J., Jeng, M. F., Tennant, L. L., Slaby, I., Lindell, M., Cui, D. S., Kuprin, S., and Holmgren, A. (1997) Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: Structural and functional characterization of mutants of Asp 26 and Lys 57. Biochemistry 36, 2622–2636. (24) Hawkins, H. C., and Freedman, R. B. (1991) The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase. Biochem. J. 275, 335–339. (25) Foloppel, N., and Nilsson, L. (2004) The glutaredoxin -C-P-Y-C- motif: influence of peripheral residues. Structure 12, 289–300. (26) Johansson, L., Gafvelin, G., and Arne´r, E. S. J. (2005) Selenocysteine in proteins: Properties and biotechnological use. Biochim. Biophys. Acta 1726, 1–13. (27) Kallis, G. B., and Holmgren, A. (1980) Differential reactivity of the functional sulfhydryl groups of cysteine-32 and cysteine-35 present

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in the reduced form of thioredoxin from Escherichia coli. J. Biol. Chem. 255, 10261–10265. (28) Zhong, L., Arne´r, E. S. J., and Holmgren, A. (2000) Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence. Proc. Natl. Acad. Sci. U.S.A. 97, 5854–5859. (29) Zhong, L., and Holmgren, A. (2000) Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. J. Biol. Chem. 275, 18121–18128.

Communications (30) Cenas, N., Nivinskas, H., Anusevicius, Z., Sarlauskas, J., Lederer, F., and Arne´r, E. S. J. (2004) Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein. J. Biol. Chem. 279, 2583–2592. (31) Seow, H. A., Belcourt, M. F., Penketh, P. G., Hodnick, W. F., Tomasz, M., Rockwell, S., and Sartorelli, A. C. (2005) Nuclear localization of NADPH:cytochrome c (P450) reductase enhances the cytotoxicity of mitomycin C to Chinese hamster ovary cells. Mol. Pharmacol. 67, 417–423.

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