Chem. Res. Toxicol. 2007, 20, 1903–1912
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Quinone Formation as a Chemoprevention Strategy for Hybrid Drugs: Balancing Cytotoxicity and Cytoprotection Tareisha Dunlap, R. Esala P. Chandrasena, Zhiqiang Wang, Vaishali Sinha, Zhican Wang, and Gregory R. J. Thatcher* Department of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, UniVersity of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612 ReceiVed June 21, 2007
Cellular defense mechanisms that respond to damage from oxidative and electrophilic stress, such as from quinones, represent a target for chemopreventive agents. Drugs bioactivated to quinones have the potential to activate antioxidant/electrophile responsive element (ARE) transcription of genes for cytoprotective phase 2 enzymes such as NAD(P)H-dependent quinone oxidoreductase (NQO1) but can also cause cellular damage. Two isomeric families of compounds were prepared, including the NONSAIDs (NO-donating nonsteroidal anti-inflammatory drugs) NCX 4040 and NCX 4016; one family was postulated to release a quinone methide on esterase bioactivation. The study of reactivity and GSH conjugation in model and cell systems confirmed the postulate. The quinone-forming family, including NCX 4040 and conisogenic bromides and mesylate, was rapidly bioactivated to a quinone, which gave activation of ARE and consequent induction of NQO1 in liver cells. Although the control family, including NCX 4016 and conisogenic bromides and mesylates, cannot form a quinone, ARE activation and NQO1 induction were observed, compatible with slower SN2 reactions with thiol sensor proteins, and consequent ARE-luciferase and NQO1 induction. Using a Chemoprevention Index estimate, the quinone-forming compounds suffered because of high cytoxicity and were more compatible with cancer therapy than chemoprevention. In the Comet assay, NCX 4040 was highly genotoxic relative to NCX 4016. There was no evidence that NO contributes to the observed biological activity and no evidence that NCX 4040 is an NO donor, instead, rapidly releasing NO3- and quinone. These results indicate a strategy for studying the quinone biological activity and reinforce the therapeutic attributes of NO-ASA through structural elements other than NO and ASA. Introduction Cellular defense mechanisms that respond to damage from oxidative stress and electrophiles, key causes of malignant transformation, represent a target for chemopreventive agents (1). The concept that compounds which possess weak carcinogenic activity have the potential to protect against carcinogenesis has been recognized and discussed (1–4). Induction of cytoprotective enzymes, such as NAD(P)H-dependent quinone oxidoreductase (NQO1), is mediated primarily by activation of the ARE,1 the antioxidant/electrophile responsive element that promotes transcriptional activation of phase 2 genes [which encode for so-called phase 2 enzymes, such as NQO1 (5)] (6). It was research on induction by quinones that prompted the hypothesis that a thiol-rich sensor protein acts as a transducer of thiol modification, leading to ARE activation (7). Clearly, * To whom correspondence should be addressed. E-mail: thatcher@ uic.edu. 1 Abbreviations: ARE, antioxidant responsive element; ASA, acetylsalicylic acid; BF, 4′-bromoflavone; BSA, bovine serum albumin; CD, concentration required to double the specific activity of NQO1; CI, chemopreventive index; DCPIP, 2,6-dichlorophenolindophenol; EDTA, ethylenediaminetetraacetic acid; FAD, flavin adenine dinucleotide; GSMP, glutathionylmethyl phenol; GST, glutathione-S-transferase; HBA, hydroxybenzyl alcohol; Keap1, Kelch-like ECH-associated protein 1; NADP, β-nicotinamide adenine dinucleotide phosphate; NQO1, NAD(P)H-dependent quinone oxidoreductase; Nrf2, nuclear factor-eryhthroid 2 related factor 2; NSAID, nonsteroidal antiinflammatory drugs; NO-NSAIDs, NO-donating NSAIDs; NO-ASA, NO-donating ASA; PLE, pig liver esterase; QM, quinone methide; SERMs, selective estrogen receptor modulators; SSA, 5-sulfosalicylic acid.
xenobiotics that are metabolically bioactivated to quinones have this capacity. Quinones are redox active electrophiles that may be formed on oxidative metabolism of drugs, such as selective estrogen receptor modulators (SERMs), and endogenous biomolecules, such as dopamine and estrogens. (8) SERMs are used as chemopreventive agents: There is evidence that these and other compounds can be chemopreventive via quinone formation (9) and that they can also be cytotoxic and genotoxic via oxidative metabolism to quinones (10, 11). The balance between the deleterious effects and the chemopreventive potential of compounds that are able to form quinone metabolites is determined by their metabolic bioactivation and ultimately their chemical structure. Understanding of the balance between cytotoxicity and cytoprotection is crucial not only in recognizing cytotoxic drugs but also in avoiding discard of good drug candidates early in preclinical development. Paradoxically, natural products are being pursued as chemopreventive agents, which are bioactivated to reactive metabolites that induce phase 2 enzymes such as glutathione-S-transferse (GST), whereas the pharmaceutical industry has considered bioactivation and enzyme induction as red flags for drug development (12–14). The NO-NSAID (NO-releasing nonsteroidal anti-inflammatory drug) class contains a subset structurally poised for bioactivation to an electrophilic quinone methide metabolite, which can potentially trigger either chemopreventive or cytotoxic mechanisms. A number of NO-NSAIDs continue to be explored in clinical trials in a range of indications, including
10.1021/tx7002257 CCC: $37.00 2007 American Chemical Society Published on Web 11/01/2007
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of the ASA derivatives in this study group (1a,b) are drugs in clinical trials in man.
Experimental Procedures
Figure 1. Chemical structures for NO-ASA compounds developed by NicOx SA (NCX; two of which, 1a and 1b, are studied in this paper), together with a typical schematic used to represent the general mechanisms of action of NO-NSAIDs. Esterase-mediated cleavage of the acyl linker is proposed to liberate the NSAID, which functions via COX inhibition and liberates NO from the aliphatic nitrate by an unknown mechanism; biological activity is proposed to be mediated through NO release and COX inhibition.
Scheme 1. Comparison of Formation of Quinone Methide from Enzyme-Catalyzed Reactions of Acyl and Phosphoryl Benzyl Fluorides (4) and Putative Formation from Acyl Benzyl Nitrates (1)
cancer chemoprevention. NO-NSAIDs are hybrid nitrate drugs; aliphatic nitrates conjugated via a labile ester linkage to an NSAID (15). Figure 1 shows the structure of NO-ASA drugs and the drug design rationale provided by their developers. A search of PubMed reveals over 70 publications on the NO-ASA isomers NCX 4040 (1b) and NCX 4016 (1a). The structure of 1b allows formation of a quinone methide in simile with benzyl compounds (4) developed by Widlanski and co-workers specifically to liberate a quinone methide at an enzyme active site (16, 17) (Scheme 1). The ability of 1b to form a quinone has recently been confirmed (18). The structure of the isomeric NOASA, 1a, precludes bioactivation to a quinone methide (Figure 1). It is therefore hypothesized that comparative study of 1a and 1b and conisogeners will reveal the cytoprotective vs cytotoxic impact of intracellular quinone methide release. Nitrates are NO mimetic drugs that have been in clinical use for over 130 years in cardiovascular therapy (19, 20). The biological activity of NO-NSAIDs is attributed in part to the cleavage of the linker releasing NO bioactivity (Figure 1); therefore, the conisogenic mesylates (2a,b; Ms-ASA) and bromides (3a–c; Br-ASA) were prepared, to define bioactivity deriving from NO release. The expectation was that comparison of the o- or p-substituted “caged quinone” isomers (1b, 2b, and 3b,c) with the m-isomers (1a, 2a, and 3a), which cannot form quinones, would reveal the cytoprotective vs cytotoxic effects resulting from rapid intracellular release of the simplest quinone methides. Obviously, of additional interest is the fact that two
Reagents and Synthesis. All chemicals and reagents were purchased from Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI) unless stated otherwise. NCX 4040 and NCX 4016 were purchased from Cayman Chemicals (Ann Arbor, MI). The CometAssay Kit was purchased from Trevigen Inc. (Gaithersburg, MD). The synthetic scheme is summarized in Scheme 1, and further details are provided in the Supporting Information. Briefly, the acyl halide 5 was condensed with hydroxybenzyl alcohol (HBA; 6) or o-hydroxybenzaldehyde (8) in 1 N sodium hydroxide solution to obtain either the appropriate isomer of 7 or 9. The aldehyde function of 9 was reduced with H2/Pd/C in ethyl acetate to provide 10. Compounds 7 and 10 were converted to the appropriate bromomethyl isomers, 3a, 3b, or 3c, using PPh3/CBr4. Treatment of 7a or 7b with methylsulfonyl chloride at low temperature gave the mesylates 2a or 2b, respectively, in good yield. Porcine Liver Esterase Incubation. A solution containing substrates (1–3; 500 µM), porcine liver esterase (0.15 mg of enzyme/0.4 mL), and GSH (1 mM) in 50 mM phosphate buffer (pH 7.0, 0.4 mL total volume) was incubated for 10 min at 37 °C. The reaction was quenched by the addition of acetonitrile (0.6 mL) and centrifugation for 10 min. The solution was filtered and analyzed by reverse phase HPLC (4.6 mm × 150 mm C18 column; flow rate, 1.0 mL/min; mobile phase, 10–90% acetonitrile in water over 15 min, held at 90% for 10 min. The GSH conjugate (glutathionylmethyl phenol; GSMP) was identified by LC-ES-MS in positive ion detection as [M + H]+ (m/z 414). The identities of hydroxybenzyl alcohol (HBA), ASA, and SA were confirmed using authentic samples. Control reactions were carried out in the absence of enzyme; the GSH substitution product resulting from reaction with 3 was identified by LC-ES-MS in positive ion detection as [M + H]+ (m/z 576). NOx Assay. Further analysis of esterase incubations was carried out to identify NOx reaction products. A chemiluminescence detector (Sievers Research Inc., model 280i) attached to a computer was used to measure NO production by injections of reaction headspace gas at 5 and 10 min of reaction. The detector was calibrated using DEA/NO for which the rate of NO release at pH 7.4 is known (21). Production of nitrite was assayed using the Griess reagent [0.5% sulfanilamide and 0.05% (N-1-naphthyl)ethylenediamine dihydrochloride] in 2.5% w/w H3PO4. Griess reagent (150 µL) and reaction mixtures (50 µL) were mixed and incubated in a 96 well plate; absorbance was measured at 530 nm in a microplate reader and compared to a NaNO2 standard curve. Nitrate production was measured by extension of the Griess method, using a fresh solution of VCl3 (80 mg in 1 N HCl 10 mL) to reduce inorganic nitrate to nitrite. Construction of calibration curves in the presence and absence of GSH and esterase for both measurement of NO2and NO3- showed that the thiol diminished the response of the assay to NO3-, possibly by trapping of an intermediate in V-mediated reduction of NO3- (Supporting Information). The difference between the absorbance values in the Griess/VCl3 assay and the Griess assay indicates the absorbance due to reduced nitrate, when a KNO3 standard calibration curve is used. Cell Culture. Hepa 1c1c7 murine hepatoma cells were supplied by Dr. J. P. Whitlock, Jr. (Stanford University, Stanford, CA) and were cultured in R-MEM with 1% penicillin–streptomycin and 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA). HepG2 cells stably transfected with ARE-luciferase reporter were kindly provided by Dr. A.N. Kong (Rutgers University, Piscataway, NJ) and cultured in DMEM/F-12 medium with 10% fetal bovine serum, 1% penicillin–streptomycin, 1% nonessential amino acids, and 0.2 mg/mL insulin (22). NQO1 Activity Assay. For cultured Heap 1c1c7 liver cells, the induction of NQO1 activity was assayed as described previously with minor modifications (23). Briefly, cells were seeded in 96 well plates with proper density in 190 µL of media. After 24 h of
Quinones: Balancing Cytotoxicity and Cytoprotection incubation, 10 µL test samples and DMSO or 4-bromoflavone (BF) as a positive control were added to each well, and the cells were incubated for an additional 48 h. The medium was decanted, and the cells were incubated at 37 °C for 10 min with 50 µL of 0.8% digitonin and 2 mM EDTA solution (pH 7.8). The plates were then agitated on an orbital shaker (100 rpm) for 10 min at room temperature, and 200 µL of reaction mixture {0.7 mg/mL bovine serum albumin (BSA), 0.3 mg/mL [3-(4,5-dimethylthiazo-2-yl)2,5-diphenyltetrazolium bromide] (MTT), 25 mM Tris-HCl, 0.01% Tween 20, 5 µM FAD, 1 mM glucose-6-phosphate, 30 µM NADP, 2 U/mL glucose-6-phosphate dehydrogenase, and 50 µM menadione} was added to each well. After agitation on a shaker for 5 min, the plates were scanned at 595 nm. The specific activity of NQO1 was determined by the standard procedure of measuring NADPH-dependent menadiol-mediated reduction of MTT to blue formazan, correcting for cell number determined by the crystal violet (CV) staining. Induction of NQO1 activity was calculated by comparing the NQO1 specific activity of sample-treated cells with that of solvent-treated cells. CD values represent the concentration required to double NQO1 induction. The chemopreventive index (CI ) IC50/CD) is calculated from IC50 for inhibition of cell growth (24). Cell Growth Determination. Cells were plated and treated as described for the NQO1 assay. After the cells were treated with test samples for 48 h, the medium was decanted, and 200 µL of 0.2 % CV solution in 2% ethanol was added. After incubation at 37 °C for 10 min, the plates were rinsed for 2 min with water and dried. The bound dye was then solubilized by incubation at 37 °C for 10–20 min with 200 µL of 0.5% SDS in 50% ethanol and agitation for 20 min. The absorption of CV was measured at 595 nm, and the IC50 values were determined using GraphPad Prism 4.00. ARE-Luciferase Reporter Assay. HepG2-ARE-Luc cells were plated in a six well plates for 24 or 48 h (25). Cells were treated with DMSO, test samples, or positive control (BF) and incubated for an additional 8 h. The luciferase activity was determined according to the protocol provided by the manufacturer (Promega, Madison, WI). Briefly, cells were washed with cold PBS twice and lysed with 500 µL of reporter lysis buffer. After centrifugation, 20 µL of the supernatant was used for determination of the luciferase activity by a luminometer (FLUOstar OPTIMA, BMG Labtechnologies, Germany). The luciferase activity was normalized to protein concentration using the BCA assay. The experiment was repeated on three separate cell passages (representative data are shown) and expressed as fold induction over control (treated cells/ DMSO-treated cells ( SD). GSH Levels. The total cellular glutathione (GSH + GSSG) levels were measured by an enzymatic recycling procedure with some modifications. Briefly, HepG2 cells were seeded in six well plates. The cells were treated with DMSO and sample compounds. The cells were washed twice with PBS (50 mM, pH 7.4) and homogenized with 200 µL of ice cold 5% SSA (5-sulfosalicylic acid). Lysates were centrifuged (8000 rpm), and the supernatant was assayed for total glutathione levels by adding the reaction mixture [NADPH (0.3 mM), 5,5′-dithiobis-(2-nitrobenzoic acid) (0.6 mM), and GSH reductase (0.5 units/mL) in PBS (125 mM) with EDTA (6.3 mM, pH 7.5)]. The absorbance was measured at 412 nm at 25 °C. The mean GSH concentration in DMSO vehicletreated cells measured at 10 min was 145 µM. GSH concentrations in the samples were calculated based on a standard curve. DNA Damage in Hepa 1c1c7 Cells Using Single-Cell Gel Electrophoresis Assay (Comet Assay). This assay was performed according to the manufacturer’s recommendations (Trevigen, Inc. Gaithersburg, MD). Briefly, Hepa 1c1c7 cells (3 × 105 cells/mL) were incubated with DMSO, 1a, or 1b (10 and 25 µM) for 1 h. After incubation, the cells were collected and centrifuged (1500 rpm, 5 min). The supernatant was discarded, and the cells were suspended in PBS (2 mL, Ca2+ and Mg2+ free). The cell suspension (30 µL) was added to 300 µL of 1% LMAgarose (37 °C), and 50 µL of the resulting solution was added to the CometAssay slide. Slides were placed at 4 °C in the dark for 10 min and thereafter
Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1905 placed in lysis solution (2.5 M sodium chloride, 100 mM EDTA pH 10, 10 mM tris base, 1% sodium lauryl sarcosinate, and 1% Triton X-100) for 30 min. The slides were removed from the lysis solution; the slides were transferred in the dark to alkali solution (pH > 13, 300 mM NaOH, 1 mM EDTA) for 40 min. After electrophoresis, slides were stained with SYBR Green (50 µL), viewed with a fluorescence microscope, and analyzed using CometScore Freeware (http://www.autocomet.com), and the data were presented as the percent DNA in the tail. Each sample contained at least 80 cells.
Results Synthesis. Synthetic procedures represent adaptations of standard methods (Scheme 2). Esterase Catalysis As a Model for Drug Metabolism. In the presence of esterase to catalyze cleavage of the benzoate linker, metabolic degradation of ASA derivatives was modeled by studying the reaction in the presence and absence of GSH. The reaction products were the isomeric HBA, isomeric GSH adducts (GSMP), and salicylic acids. GSMP and HBA were quantified by HPLC-UV and expressed as a percentage of the total benzyl-containing compounds, using LC-MS to confirm the identity of metabolites (Scheme 3); a 100% yield of either GSMP or HBA would correspond to quantitative conversion of substrate to product. Degradation was relatively rapid, as shown by the 80–90% conversion of the o- and p-substituted ASA derivatives to the GSH conjugate after only 10 min of reaction (Figure 2a); the balance of the benzyl compounds was HBA isomers. The mnitrooxybenzyl (1a) and m-mesyloxybenzyl (2a) derivatives yielded negligible amounts of GSH conjugates, whereas the m-bromobenzyl derivative (3a) underwent approximately 60% conversion to the corresponding GSH conjugate. In the reaction mixture of 3a, the balance of the benzyl compounds at 10 min was made up almost entirely of m-hydroxybenzyl bromide. The observed results for formation of HBA isomers in the absence of GSH were entirely consistent with this reactivity pattern. All o- and p-substituted ASA derivatives (1b, 2b, and 3b) were converted quantitatively to the corresponding HBA product after only 10 min of reaction (Figure 2b). The m-nitrooxybenzyl (2a) and m-mesyloxybenzyl (1a) derivatives again yielded small or negligible quantities of HBA, and in this experiment, the m-bromobenzyl derivative (3a) again showed increased reactivity, with a yield of HBA reaching almost 35%. For the nitrate esters, NOx species were measured in the esterase incubation mixtures at 10 min under identical conditions to those used in the measurement of GSH conjugates. Production of NO, NO2-, and NO3- was not measurable within detection limits for 1a (100 µM). Similarly, NO and NO2- were not detectable for 1b (100 µM), but release of NO3- was detected. Corrected calibration curves for NO3- revealed the formation of 96.6 ( 3.51 µM inorganic nitrate, corresponding to quantitative conversion from 1b. Further studies were conducted at higher concentrations of 1a,b (2.2 mM) expressly to facilitate detection of minor NOx products. Under these conditions, increased formation of NO3- and measurable NO2- were detected from breakdown of 1b, but NO remained below detection limits; for 1a, both NO2- (3 µM) and NO (0.26 ( 0.02 µM) were measured at the higher substrate concentration, but NO3- was not detected. GSH Depletion in Liver Cells. Depletion of total GSH was measured in HepG2 cells, to provide a link from the study of reactivity towards GSH in model systems to biological activity in cell lines. Total GSH was measured, which includes free thiol and GSH disulfides, which are reduced in the assay procedure.
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Scheme 2. Synthesis of NO-ASA Analoguesa
a
Key: (i) 1 N NaOH, toluene. (ii) CBr4, PPh3, CH2Cl2. (iii) CH3SO2Cl, Et3N, THF. (iv) NaBH4, CH3OH.
Scheme 3. Organic Products of Reaction of NO-ASA and Conisogeners with PLE and GSH in Aqueous Solution
The observed GSH depletion will therefore largely reflect alkylation of GSH by electrophiles generated from the added ASA derivatives. Depletion of GSH was observed to be rapid, compatible with rapid intracellular esterolysis. Significant depletion of GSH was observed by p- and o-derivatives (1b and 3b,c) (Figure 3a); more than 25% of total intracellular GSH was lost after only 10 min of incubation. The results also indicated that the reactivity of the methanesulfonyl derivatives in the intracellular milieu was lower than that of nitrooxy and bromo derivatives. In addition, the enhanced reactivity in the esterase model system of 3a as compared to the conisogenic m-isomers was not mirrored by increased GSH depletion for 3a in the cellular milieu. The efficiency with which 1b was able to deplete intracellular stores of GSH was demonstrated by varying concentration and comparison with the m-derivative, 1a (Figure 3b); depletion of GSH correlated linearly with added 1b, whereas 1a had no significant effect on measured GSH levels after 10 min of reaction. The amount of GSH detected in any individual well in a cell culture experiment is a function of the cell density in that well, meaning that concentrations of GSH vary across wells. The mean concentration of GSH in the DMSO control incubations was approximately 145 µM; therefore, in these experiments, (i) the depletion of GSH by the quinoneforming derivatives (1b, 3b,c; 25 µM) was estimated as 30 (
10 µM, and (ii) the addition of 1b (50 µM) led to loss of approximately 50% of available and reducible intracellular GSH, that is, depletion of approximately 60 ( 15 µM GSH. In summary, the depletion of GSH by 1b and 3b,c was observed to be highly efficient. Induction of NQO1 in Liver Cells. To investigate the ability of putative quinone-forming ASA derivatives to induce phase 2 enzymes, compounds were administrated to Hepa 1c1c7 cells at different concentrations and the NQO1 activity was measured. The Hepa 1c1c7 cell system is widely used to screen chemopreventive agents and potential anticarcinogens, since NQO1 is readily induced to measurable levels that allow comparison of agents (26). Induction of NQO1 activity was compared to solvent and to 4-bromoflavone (BF) as a positive control (Table 1) (24). The data, presented in Figure 4, showed that all ASA derivatives can induce NQO1 in Hepa 1c1c7 cells, with the p-substituted derivatives being the most efficacious and most potent inducers within each of the three families. Induction of NQO1 has previously been reported for NCX 4016 both in vivo and in vitro (27). The nitrooxy derivatives (1) induced NQO1 up to 1.7-fold at the concentrations studied; the cytotoxicity of 1b caused problems with assay at higher concentrations. The m-substituted methanesulfonyl derivative, 2a, was equally a weak inducer. The concentration at which a doubling of NQO1
Quinones: Balancing Cytotoxicity and Cytoprotection
Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1907 Table 1. Parameters for Inhibition of Cell Proliferation and Induction of NQO1 in Hepa 1c1c7 Cells Indicating Chemopreventive Potential 1a 1b 2a 2b 3a 3b 3c
IC50a (µM)
CDb (µM)
C1.5c (µM)
CI
CI1.5d
65 12 >100 50 43 13 19
>100 >25 >100 70 20 8 8
100 7 90 40 10 3 3
2b. The biological activity of the ASA derivatives, predicted on the basis of chemical reactivity towards GSH, was therefore largely borne out: (i) The reactive quinone-forming derivatives (1b, 2b, and 3b,c) rapidly activated ARE, with the mesylate the least reactive and least potent of these, whereas the less reactive m-derivatives (1a, 2a, and 3a) led to lower activation but still induced NQO1, with the bromide the most reactive and potent (potency followed the order 3a > 1a > 2a). These data are entirely compatible with the intermediacy of a protein thiol sensor, such as Keap1, and the known Nrf2/ARE pathway for phase 2 enzyme induction. These data provide a correlation of reactivity towards thiols with activity towards phase 2 enzyme induction. Correlations of Reactivity with Toxicity of ASA Derivatives. The reactive o- and p-substituted ASA derivatives were observed to inhibit Hepa 1c1c7 cell growth more potently than the corresponding m-derivatives as shown by measurements of IC50 and subsequent calculation of chemoprevention indices (Table 1). All quinone-forming derivatives were cytotoxic at higher concentrations, with the nitrate, 1b, and bromide, 3b, the most potent agents. The potential genotoxicity of the quinone-forming derivatives was assessed by measuring DNA damage using a Comet assay on incubation of HepG2 cells with 1a,b. As anticipated for a quinone methide-forming compound, 1b was observed to cause DNA single strand breaks in a concentration-dependent manner (Figure 6). The assay was performed after incubation of test compounds with cells for only 60 min, emphasizing the high reactivity of the quinone-forming compounds.
Conclusions Quinoid Cytotoxicity vs Cytoprotection. The results presented herein on ASA derivatives demonstrate that quinones are released upon rapid esterolysis in the intracellular environment when caged in an aroyl ester precursor. This observation provides a platform for further study of the effect of quinone structure on reactivity and biological activity via use of appropriately designed aroyl esters. In the present study, the quinone-forming ASA derivatives generated the simplest pquinone and o-quinone methides, which were seen to rapidly react with GSH and to activate ARE and induce NQO1, probably via reaction with thiol-rich sensor proteins. Although generally similar characteristics were observed, the identity of the leaving group (nitrate 1b, mesylate 2b, or bromide 3b,c) did influence the observed potency for induction and cytotox-
Quinones: Balancing Cytotoxicity and Cytoprotection
icity, possibly through compartmentalization of the mesylate ester or hindrance to esterase hydrolysis. For the quinoneforming derivatives, the chemoprevention index was relatively low, arguing that the balance between cytotoxicity and cytoprotection for the simplest quinone methides is unsatisfactory for a chemopreventive agent. Conversely, such compounds may be exploited for their cytotoxicity in cancer therapy, if quinone release can be targeted to tumor cells. From literature reports, 1b is known to be cytotoxic and pro-apoptotic (43), and other quinoid drugs have been reported to inhibit cell growth and induce apoptosis via reaction with GSH and protein thiols (58, 59). The potential genotoxicity of 1b was confirmed in comparison to 1a in the Comet assay. NO-ASA Drug Action. There is ample data in the literature to indicate that the NO-ASA derivatives, NCX 4040 (1b), NCX 4016 (1a), and NCX 4215, have very different properties and, because these drugs differ only in the “linker” moiety, that this structural element is a major contributor to biological activity. The present data demonstrate that the activity of the NO-ASA 1a (i) is common with the conisogenic ASA derivatives 2a and 3a, which are obviously not able to donate NO, and (ii) is different from the activity of the isomeric nitrate 1b. The observed activity of 1b is common with the caged quinone ASA derivatives 2b and 3b,c, and results from quinone methide formation leading both to induction of cytoprotective enzymes and to cytotoxicity. The ASA moiety is not expected to contribute significantly to ARE activation and NQO1 induction, and in the current study, there is no evidence that requires a contribution from NO. Indeed, there is no evidence herein that 1b is an NO donor, since esterase activity leads to rapid expulsion of NO3- and quinone formation. It is noted that hybrid and related nitrates have provided positive results in vivo (60), and importantly, nitrates require reductive bioactivation to liberate NO bioactivity. The capacity of in vitro systems to replicate in vivo bioactivation is problematic (19). In vitro studies of hybrid nitrates remain of value in assessing activity that might be provided by ancillary structural elements; however, in vivo, NO bioactivity is expected to contribute to therapeutic activity for most nitrates. The cytotoxic and cytoprotective characteristics observed in liver cells herein are common for NO-ASA and conisogenic agents without NO-donating properties. Acknowledgment. This work was supported by NIH Grant CA102590. Birgit Deitz and Ghenet Hagos are thanked for technical assistance in the study. Supporting Information Available: Figures for induction of NQO1 activity in hepa1c1c7 cells by 1a and 1b, figure of calibration curves for [NO3–], and synthetic experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
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