Article pubs.acs.org/ac
Nitroreductase Detection and Hypoxic Tumor Cell Imaging by a Designed Sensitive and Selective Fluorescent Probe, 7‑[(5Nitrofuran-2-yl)methoxy]‑3H‑phenoxazin-3-one Zhao Li, Xiaohua Li,* Xinghui Gao, Yangyang Zhang, Wen Shi, and Huimin Ma* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: A highly selective and sensitive fluorescence probe, 7-[(5-nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one (1), is developed for imaging the hypoxic status of tumor cells via the indirect detection of nitroreductase. The detection mechanism is based on the fact that nitroreductase can selectively catalyze the reduction of the nitro group in 1 to a hydroxylamine or amino group in the presence of reduced nicotinamide adenine dinucleotide as an electron donor that is indispensable, followed by the 1,6-rearrangement−elimination and the release of resorufin. As a result, the reaction produces a distinct color and fluorescence change from almost colorless and nonfluorescent to pink and strong red fluorescence. The fluorescence increase of probe 1 at λ550/585 nm is directly proportional to the concentration of nitroreductase in the range of 15− 300 ng/mL, with a detection limit of 0.27 ng/mL. The ready reduction of the nitro group in 1 under hypoxic conditions leads to the establishment of a sensitive and selective fluorescence method for imaging the hypoxic status of tumor cells, and with this method Hela and A549 cells under normoxic and hypoxic conditions (even for different extents of hypoxia) can be differentiated successfully. This method is simple and may be useful for the imaging of disease-relevant hypoxia. et al. prepared a novel fluorescent probe by connecting a pnitrobenzyl moiety to naphthalimide via a carbamate group for hypoxia detection22 and Huang et al. synthesized an elegant fluorescence off−on probe by attaching 4-nitrobenzene to coumarin with a diamine linker for monitoring nitroreductase activities.27 Despite these progresses, fluorescent probes with superior properties such as long analytical wavelength (>500 nm), good water solubility, high selectivity, and sensitivity are still lacking for nitroreductase detection and in particular hypoxic tumor cell imaging. In this work, a new spectroscopic off−on probe, 7-[(5nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one (1; Scheme 1),
H
ypoxia, caused by an inadequate oxygen supply, is an important feature of many diseases, including cardiac ischemia,1−3 inflammatory diseases,4 and solid tumors.5,6 The hypoxic status of solid tumors has been considered to be an indicator of adverse prognosis because of tumor progression toward a more malignant phenotype with increased metastatic potential and resistance to treatment.5−8 Consequently, hypoxic cells usually provide a tumor-specific targeting strategy for therapy.9,10 Because of this, developing novel methods for hypoxia detection is of great importance. There have been a number of detection methods for hypoxia.11−15 Among them, fluorescence spectroscopy has attracted much attention because of its high spatiotemporal resolution. On the other hand, under hypoxic conditions, intracellular reductases that catalyze oneelectron reduction are involved in the selective activation of specific functional compounds, a well-known pathophysiological characteristic of solid tumors.16,17 Such an enzymecatalyzed one-electron reduction has been recognized to be a useful reaction for the design of a hypoxia targeting and imaging approach. Nitroaromatic compounds are well-characterized to be superior substrates for nitroreductase in the presence of reduced nicotinamide adenine dinucleotide (NADH) as an electron donor, and they can readily be metabolized in a stepwise reduction pathway by cellular nitroreductase under hypoxic conditions.18 Thus, nitroaromatic compounds have been employed to develop not only bioreductive prodrugs19,20 but recently also fluorescent probes for nitroreductase and tumor hypoxia.21−27 For example, Qian © 2013 American Chemical Society
Scheme 1. Synthesis of Probe 1
Received: November 20, 2012 Accepted: March 17, 2013 Published: March 17, 2013 3926
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ments were performed on a Zeiss LSM 780 confocal laser scanning microscope (Carl Zeiss, Germany) with excitation at 488 nm. Optical sections were acquired at 0.8 μm. Synthesis of Probe 1. To a suspension of resorufin sodium salt (0.24 g, 1.0 mmol) in anhydrous DMF (10 mL), K2CO3 (0.21 g, 1.5 mmol) was added, followed by stirring at 40 °C for 10 min under an Ar atmosphere. Then, a solution of 2(bromomethyl)-5-nitrofuran (0.21 g, 1.0 mmol) in DMF (2 mL) was added dropwise. The resulting mixture was stirred at 40 °C for 2 h and then diluted with dichloromethane (50 mL). The organic layer was separated, washed three times with water (50 mL × 3) and brine (50 mL × 3), and then dried over Na2SO4. The solvent was removed by evaporation, and the residue was subjected to silica gel chromatography, eluted with petroleum ether (bp 60−90 °C)/ethyl acetate (v/v, 1:1), affording 1 as an orange solid (0.16 g, 46%). The 1H NMR and 13 C NMR spectra of 1 are given in Figures S1 and S2 in the Supporting Information, respectively. 1H NMR (600 MHz, 298 K, CF3COOD): δ 8.54 (m, 2 H), 7.83 (m, 2 H), 7.78 (s, 1 H), 7.64 (s, 1 H), 7.54 (d, 1 H, J = 3.6 Hz), 7.00 (d, 1 H, J = 3.6 Hz), 5.62 (s, 2 H). 13C NMR (150 MHz, 298 K, CF3COOD): δ 175.6, 171.3, 151.1, 150.9, 149.9, 139.0, 136.7, 136.6, 135.2, 125.6, 123.7, 114.9, 114.5, 113.0, 102.2, 99.6, 63.6. Elemental analysis, calcd for 1 (C17H10N2O6): C 60.36, H 2.98, N 8.28%; found, C 60.18, H 3.11, N 8.19%. Modeling of Binding Affinity between Probe 1 and Nitroreductase. Surflex-dock module, which is available on SYBYL version 1.1 (Tripos Inc.), was used to evaluate the binding affinity between 1 and nitroreductase. The crystal structure of nitroreductase complex was collected from PDB under code 4DN2. General Procedure for Nitroreductase Detection. Unless otherwise stated, all the fluorescence measurements were made in 10 mM PBS (pH 7.4), according to the following procedure. In a 10 mL tube, 5 mL of PBS and 50 μL of 1 mM 1 were mixed, followed by addition of NADH (final concentration, 500 μM), and an appropriate volume of nitroreductase sample solution. The final volume was adjusted to 10 mL with PBS, and the reaction solution was mixed rapidly. After incubation at 37 °C for 30 min in a thermostat, a 3 mL portion of the reaction solution was transferred to a quartz cell of 1 cm optical length to measure the absorbance or fluorescence with λex/em = 550/585 nm and both excitation and emission slit widths of 10 nm. In the meantime, a blank solution containing no nitroreductase (control) was prepared and measured under the same conditions for comparison. Fluorescence Imaging of Hypoxia in Hela and A549 Cells. Hela and A549 cells were grown on glass-bottom culture dishes (MatTek Company) at 37 °C using Dulbecco’s modified eagle media (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin−streptomycin in a humidified incubator under normoxic [95% air and 5% CO2 (i.e., 20% O2)] and different hypoxic (80% N2, 15% O2, and 5% CO2; 85% N2, 10% O2, and 5% CO2; 90% N2, 5% O2, and 5% CO2; or 94% N2, 1% O2, and 5% CO2)35,36 conditions, respectively. Before use, the adherent cells were washed with FBS-free DMEM. For fluorescence imaging, the cells were further incubated with 5 μM of 1 in FBS-free DMEM at 37 °C for 30 min under the respective conditions and then washed three times with the PBS buffer (pH 7.4) to remove the free probe. Cytotoxicity Assay. The cytotoxicity of 1 to Hela and A549 cells was evaluated following the approach reported
with the above desired properties is presented for imaging the hypoxic status of tumor cells via its reaction with the endogenous nitroreductase. The probe was designed by introducing 5-nitrofuran as a masking and reacting moiety to resorufin through an ether bond. We chose resorufin as a signaling unit because of its long analytical wavelength (λ550/585 nm), good water solubility, and efficient fluorescence quenching via alkylation of the 7-hydroxy group. By such design, a latent fluorescent probe28−32 with an extremely low background signal has been prepared, which is rather favorable to affording high detection sensitivity for nitroreductase. Reaction of 1 with nitroreductase in the presence of NADH causes the reduction of the 5-nitrofuran moiety, followed by the 1,6-rearrangement−elimination reaction and thereby the release of resorufin. As a result, both color and fluorescence of resorufin are recovered, which leads to the development of a highly sensitive and selective method for monitoring nitroreductase activity, as well as for imaging the hypoxic status of tumor cells. Most notably, by using this method, Hela and A549 cells under normoxic and hypoxic conditions (even for different extents of hypoxia) can be differentiated successfully, indicating its great potential for studying hypoxia-related diseases.
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EXPERIMENTAL SECTION Reagents. Resorufin sodium salt, 2-(bromomethyl)-5-nitrofuran, nitroreductase (≥100 units/mg) from Escherichia coli, and NADH were purchased from Sigma-Aldrich. The lyophilized powder of nitroreductase was dissolved in pure water, and the solution was divided into 20 parts as suitable amounts for daily experiments. All these enzyme solutions were frozen immediately at −20 °C for storage and allowed to thaw before use according to the known procedure,33 which results in no change of the enzyme activity. A phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) solution was obtained from Invitrogen Company. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Serva Electrophoresis GmbH. All other chemicals used were local products of analytical grade. A stock solution (1 mM) of 1 was prepared by dissolving an appropriate amount of 1 in DMSO. Ultrapure water (over 18 MΩ cm) from a Milli-Q reference system (Millipore) was used throughout. Apparatus. 1H NMR and 13C NMR spectra were measured on a Bruker DMX-600 spectrometer in CF3COOD. Electrospray ionization mass spectra (ESI-MS) were recorded in negative mode with a Shimadzu liquid chromatography−mass spectrometry (LC−MS) 2010A instrument (Kyoto, Japan). Elemental analyses were performed on a Flash EA 1112 instrument. High-performance liquid chromatography (HPLC) analyses were carried out with LC-20AT solvent delivery unit, SPD-20A UV−vis detector (Shimadzu, Japan), and Inertsil ODS-SP column (5 μm, 4.6 mm × 250 mm, GL Sciences Inc.). A model HI-98128 pH meter (Hanna Instruments Inc.) was employed for pH measurements. Absorption spectra were recorded in 1 cm quartz cells with a TU-1900 spectrophotometer (Beijing, China). Fluorescence measurements were performed on a Hitachi F-2500 spectrofluorimeter in 10 × 10 mm quartz cells (Tokyo, Japan), with a 400 V PMT voltage. Fluorescence quantum yield (Φ) was determined by using resorufin (Φ = 0.75 in aqueous solutions)34 as a standard. The absorbance for MTT analysis was recorded on a microplate reader (BIO-TEK Synergy HT). Fluorescence imaging experi3927
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Figure 1. (A) Absorption and (B) fluorescence emission (λex = 550 nm) spectra of 1 (5 μM) (a) before and (b) after reaction with nitroreductase (0.20 μg/mL), in the presence of 500 μM NADH at 37 °C for 30 min. The color and fluorescence changes of 1 before and after the reaction are shown in the insets of the corresponding figures.
previously.37 In brief, the cells were seeded in 96-well U-bottom plates at a density of 7000 cells per well and incubated with 1 at varied concentrations (1−20 μM) at 37 °C for 24 h. Then, the culture media were discarded, and 0.1 mL of the MTT solution (0.5 mg/mL in DMEM) was added to each well, followed by incubation at 37 °C for 4 h. The supernatant was abandoned, and 110 μL of DMSO was added to each well to dissolve the formed formazan. After shaking the plates for 10 min, absorbance values of the wells were read with a microplate reader at 490 nm. The cell viability rate (VR) was calculated according to the equation: VR = A/A0 × 100%, where A is the absorbance of the experimental group (i.e., the cells treated with probe 1) and A0 is the absorbance of the control group (i.e., the cells untreated with probe 1). The cell survival rate from the control group was considered to be 100%.
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Next, we determined the kinetic parameters for the enzymatic cleavage reaction of 1. Figure S4 of the Supporting Information shows the Lineweaver−Burk plot of 1/V (here, V is the initial reaction rate) versus the reciprocal of the probe concentration. By fitting the data with the Michaelis−Menten equation,40,41 the corresponding Michaelis constant (Km) and maximum of initial reaction rate (Vmax) for the enzymeactivated reaction were determined to be 50.5 μM and 0.09 μM/s, respectively, which are comparable to the values reported previously.27 Moreover, a docking study was performed to evaluate the binding ability of nitroreductase with 1. The docking score (−log Kd) is found to be 9.15, indicating that the binding affinity between probe 1 and nitroreductase is very strong.42 This is also supported by the result of the ribbon model created by Pymol. As is seen from Figure S5 of the Supporting Information, there are eleven potential hydrogen bonds in the resulting complex, which may contribute to the strong binding affinity as well. To study the selectivity of the reaction, various potential interfering species, such as inorganic salts (KCl, CaCl2, MgCl2), glucose, vitamin C, vitamin B6, human serum albumin (HSA), reactive oxygen species (H2O2, ·OH), glutamic acid, arginine, serine, biothiols (glutathione, cysteine, homocysteine, and dithiothreitol), and some enzymes (carboxylesterase, DTdiaphorase, and glutathione transferase) were examined in parallel under the same conditions. As shown in Figure 2, the probe shows high selectivity for nitroreductase over the other species tested, even including reductive biothiols at a high concentration, which may be ascribed to the specific reduction of the substrate (5-nitrofuran) by the enzyme. The effects of pH and temperature on the reaction system were explored (Figure S6 of the Supporting Information), which reveals that probe 1 functions well under physiological conditions (about pH 7 and 37 °C). Moreover, the effect of NADH on the reaction was also studied, and the result showed that the fluorescence increase reached a plateau in 30 min when the concentration of NADH was not less than 500 μM. Under the optimized conditions (reaction at 37 °C for 30 min in 10 mM PBS of pH 7.4 in the presence of 500 μM NADH), the fluorescence response of 1 to nitroreductase at varied concentrations is shown in Figure 3. As can be seen, the fluorescence intensity is increased with an increase in the nitroreductase concentration, and a good linearity is obtained in the concentration range of 15−300 ng/mL nitroreductase, with a linear equation of ΔF = 3.12 × 103C (μg/mL) − 37.8 (R = 0.998), where ΔF is the difference of fluorescence intensity of 1 in the presence and absence of nitroreductase. The detection limit (3S/m, in which S is the standard deviation of blank
RESULTS AND DISCUSSION
Spectroscopic Response of 1 to Nitroreductase. The absorption and fluorescence spectra of 1 before and after reaction with nitroreductase are shown in Figure 1. Probe 1 exhibits a very weak absorption in the long-wavelength region (Figure 1A), but its reaction solution with nitroreductase produces a strong one at about 550 nm, with a distinct color change from nearly colorless to pink (see the inset of Figure 1A). Moreover, the latent fluorescent probe itself has almost no emission at 585 nm (Figure 1B), with a quantum yield of Φ ≈ 0.04. This low background signal is due to the alkylation of the 7-hydroxy group of resorufin and is rather desirable for sensitive detection. However, reaction of 1 with nitroreductase leads to a 100-fold fluorescence enhancement, accompanying a great fluorescence color change (inset of Figure 1B). Interestingly, both the absorption and fluorescence spectra from the reaction system resemble those38,39 of resorufin, supporting the fact that the enzyme-triggered cleavage reaction causes the release of free resorufin. Moreover, the long analytical wavelength feature (λ550/585 nm) of the reaction system is favorable for cell imaging studies. Fluorescence kinetic curves of 1 reacting with nitroreductase at varied concentrations are depicted in Figure S3 of the Supporting Information, from which it can be seen that higher concentrations of nitroreductase result in faster cleavage reaction and stronger fluorescence intensity. For nitroreductase of no more than 1 μg/mL, this fluorescence increase could reach a plateau in about 30 min. In contrast, the fluorescence of 1 without nitroreductase (control) hardly changes during the same period of time, also indicating that the probe is stable in the detection system. 3928
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clearly support the fact that the fluorescence response arises from the generation of resorufin. The effect of a common inhibitor (e.g., dicoumarin)43 of nitroreductase on the activity of the enzyme was also investigated. As shown in Figure S9 of the Supporting Information, the fluorescence intensity in the presence of 0.1 mM dicoumarin (curve D) is much less than that (curve C) in the absence of the inhibitor, and more dicoumarin (0.2 mM) can result in a greater decrease in fluorescence intensity (curve E). This indicates that the enzyme activity can be effectively inhibited by dicoumarin, and thus the fluorescence off−on response of 1 to nitroreductase indeed arises from the enzymecatalyzed cleavage reaction. To evaluate the potential toxicity of 1 to cells, a standard MTT assay was performed (Figure S10 of the Supporting Information). The results showed that cell viability was not significantly changed upon treatment, even with 5 μM 1 at 37 °C for 24 h, indicating the low cytotoxicity and good biocompatibility of the probe. Fluorescence Imaging of Hypoxia in Hela and A549 Cells by Probe 1. As mentioned above, nitroaromatic compounds can readily be transferred into nitro-anion free radicals via a one-electron reduction pathway by cellular nitroreductase under hypoxic conditions.16−18 Thus, probe 1 is anticipated to be capable of imaging hypoxia in tumor cells via its reaction with the endogenous nitroreductase, and the reaction might proceed through the route depicted in Scheme 2: nitroreductase catalyzes the reduction of the nitrofuran
Figure 2. Fluorescence responses of 1 (5 μM) in the presence of NADH (500 μM) to various species: control (probe 1 + NADH), KCl (150 mM), CaCl2 (2.5 mM), MgCl2 (2.5 mM), glucose (10 mM), vitamin C (1 mM), vitamin B6 (1 mM), HSA (100 μM), H2O2 (10 μM), ·OH (10 μM), glutamic acid (1 mM), arginine (1 mM), serine (1 mM), glutathione (5 mM), cysteine (1 mM), homocysteine (1 mM), dithiothreitol (1 mM), carboxylesterase (1 units/mL), DTdiaphorase (1 μg/mL), glutathione transferase (1 μg/mL), and nitroreductase (0.5 μg/mL). The results are the mean ± standard deviation of three separate measurements. λex/em = 550/585 nm.
Scheme 2. Proposed Reaction Mechanism of 1 with Nitroreductase
Figure 3. Fluorescence response of 1 (5 μM) to nitroreductase at varied concentrations (0, 0.015, 0.025, 0.05, 0.1, 0.15, 0.2, 0.3, 0.6, 0.8, and 1 μg/mL). λex = 550 nm.
measurements, n = 11, and m is the slope of the linear equation) is determined to be 0.27 ng/mL nitroreductase, which is the lowest detection limit to the best of our knowledge.22,27 The reaction products of 1 with nitroreductase were subjected to ESI-MS and HPLC analyses to explore the spectroscopic response mechanism. The ESI-MS spectrum of the reaction solution of 1 with nitroreductase shows a major peak at m/z = 212 [M − H]− (Figure S7 of the Supporting Information), which is characterized as resorufin; the peak at m/z = 95 may be ascribed to the formation of 5methylenefuran-2(5H)-imine. Moreover, resorufin was further verified as a major final product by HPLC analysis. As shown in Figure S8 of the Supporting Information, upon reaction with nitroreductase for 10 min, the peak at 24.26 min representing probe 1 decreases markedly, concomitant with the emergence of the one at 16.24 min for resorufin (curve E). All these results
moiety in the presence of endogenous NADH, followed by the 1,6-rearrangement−elimination and the subsequent release of the fluorescent resorufin. Obviously, according to Scheme 2, the fluorescence in cells would increase with the decrease of O2 level and/or the increase of endogenous nitroreductase concentration. To perform the potential of probe 1 for imaging hypoxia in tumor cells, Hela and A549 cells were selected because they are known to express nitroreductase.17,22,44 In our experiments, these two kinds of cells were first grown at 37 °C under normoxic (20% O2) and different hypoxic (15%, 10%, 5%, and 1% O2) conditions for 4 or 8 h and then incubated with probe 1 for 30 min under the respective conditions. As shown in Figure 4A, Hela cells treated with 1 under 4 h normoxic conditions show negligible intracellular background fluores3929
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Figure 4. Confocal fluorescence images of Hela cells under normoxic (20% O2) and different hypoxic (15%, 10%, 5%, and 1% O2) conditions for (A) 4 h and (B) 8 h. Hela cells pretreated at various O2 levels were incubated with 5 μM probe 1 for 30 min. The differential interference contrast (DIC) images of the corresponding samples are shown below. The scale bar is 20 μm. The fluorescence images from the corresponding control experiments in the absence of probe 1 are shown in Figure S11 of the Supporting Information.
Figure 5. Confocal fluorescence images of A549 cells under normoxic (20% O2) and different hypoxic (15%, 10%, 5%, and 1% O2) conditions for (A) 4 h and (B) 8 h. A549 cells pretreated at various O2 levels were incubated with 5 μM probe 1 for 30 min. The differential interference contrast (DIC) images of the corresponding samples are shown below. The scale bar is 20 μm. The fluorescence images from the corresponding control experiments in the absence of probe 1 are shown in Figure S12 of the Supporting Information.
the fluorescence becomes brighter under the same hypoxic conditions for 8 h (Figure 4B), which can be ascribed to the generation of more resorufin. For A549 cells, similar results were obtained (Figure 5). In addition, HEK293 cells that do not express nitroreductase were also studied as a control for
cence. However, Hela cells treated with the probe under hypoxic conditions produce noticeable fluorescence, and most importantly, the fluorescence increases with the decease of the O2 concentration from 20% to 1%, implying that probe 1 can indicate the extent of relative hypoxia in the cells. Moreover, 3930
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comparison. As expected, no considerable fluorescence was observed from either normoxic or hypoxic (even for 8 h) HEK293 cells (Figure S13 of the Supporting Information). The above results clearly indicate that probe 1 is cell membrane permeable and can be used to visualize the hypoxic status of a wide variety of tumor cell lines via its reaction with the endogenous nitroreductase. To further prove that the fluorescence in the 1-loaded cells was caused by nitroreductase-catalyzed reduction, we also investigated the effect of dicoumarin as an inhibitor of endogenous nitroreductase. As can be seen from Figures S14 and S15 of the Supporting Information, the fluorescence from either Hela or A549 cells that were pretreated with dicoumarin is much weaker than that from the untreated cells, clearly indicating that dicoumarin can effectively inhibit the activity of nitroreductase in the cells. In other words, the fluorescence of the 1-loaded cells indeed arises from the action of endogenous nitroreductase. To compare quantitatively the hypoxic status of the above tumor cells, the pixel intensity of the cells was analyzed using Image J (version 1.37c, NIH). In doing so, the pixel intensity of at least 5 cells was averaged, and the obtained results are shown in Figure 6. As can be seen, the fluorescence intensity from
of the nitrofuran moiety, followed by the 1,6-rearrangement− elimination and the release of resorufin. The detection limit of the fluorescence method for nitroreductase is 0.27 ng/mL, which is the lowest detection limit to the best of our knowledge. More importantly, the probe can be used to monitor the hypoxic status of tumor cells via the detection of endogenous nitroreductase, and such an application has successfully been demonstrated by imaging Hela and A549 cells with different extents of hypoxia. This method is simple and may be of potential for tumor diagnosis via hypoxia imaging.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: H.M.: e-mail,
[email protected]. X.L.: e-mail, lixh@ iccas.ac.cn. Tel: +86-10-62554673. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the financial support from the NSF of China (Grants 21275146, 21275147, 20935005, and 21105104), the Ministry of Science and Technology (Grant 2011CB935800), and the Chinese Academy of Sciences (Grant KJCX2-EW-N0601).
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REFERENCES
(1) Semenza, G. L. Annu. Rev. Med. 2003, 54, 17−28. (2) Huang, Z.; Shiva, S.; Kim-Shapiro, D. B.; Patel, R. P.; Ringwood, L. A.; Irby, C. E.; Huang, K. T.; Ho, C.; Hogg, N.; Schecher, A. N.; Gladwin, M. T. J. Clin. Invest. 2005, 115, 2099−2107. (3) Crawford, J. H.; Isbell, T. S.; Huang, Z.; Shiva, S.; Chacko, B. K.; Schechter, A. N.; Darley-Usmar, V. M.; Kerby, J. D.; Lang, J. D., Jr; Kraus, D.; Ho, C.; Gladwin, M. T.; Patel, R. P. Blood 2006, 107, 566− 574. (4) Murdoch, C.; Muthana, M.; Lewis, C. E. J. Immunol. 2005, 175, 6257−6263. (5) Kondoh, S. K.; Harada, M.; Hiraoka, H. M. Cancer Sci. 2003, 94, 1021−1028. (6) Harris, A. L. Nat. Rev. Cancer 2002, 2, 38−47. (7) He, F.; Deng, X.; Wen, B.; Liu, Y.; Sun, X.; Xing, L.; Minami, A.; Huang, Y.; Chen, Q.; Zanzonico, P. B.; Ling, C. C.; Li, G. C. Cancer Res. 2008, 68, 8597−8606. (8) Wilson, W. R.; Hay, M. P. Nat. Rev. Cancer 2011, 11, 393−410. (9) Shinohara, E. T.; Maity, A. Curr. Mol. Med. 2009, 9, 1034−1045. (10) Williams, K. J.; Albertella, M. R.; Fitzpatrick, B.; Loadman, P. M.; Shnyder, S. D.; Chinje, E. C.; Telfer, B. A.; Dunk, C. R.; Harris, P. A.; Stratford, I. J. Mol. Cancer Ther. 2009, 8, 3266−3275. (11) Dasu, A.; Denekamp, J. Int. J. Radiat. Oncol., Biol. Phys. 1999, 43, 1083−1094. (12) Stern, S.; Guichard, M. Radiother. Oncol. 1996, 41, 143−149. (13) Zhu, W.; Dai, M.; Xu, Y.; Qian, X. Bioorg. Med. Chem. 2008, 16, 3255−3260. (14) Dai, M.; Zhu, W. P.; Xu, Y. F.; Qian, X. H.; Liu, Y.; Xiao, Y.; You, Y. J. Fluoresc. 2008, 18, 591−597. (15) Tanabe, K.; Hirata, N.; Harada, H.; Hiraoka, M.; Nishimoto, S. ChemBioChem 2008, 9, 426−432. (16) Hodgkiss, R. J.; Parrick, J.; Porssa, M.; Stratford, M. R. J. Med. Chem. 1994, 37, 4352−4356.
Figure 6. Relative pixel intensities obtained from the images of Hela and A549 cells. The cells were grown under normoxic (20% O2) and different hypoxic (15%, 10%, 5%, and 1% O2) conditions for 4 or 8 h, and then incubated with 5 μM probe 1 for 30 min under the respective conditions. The strongest fluorescence intensity from the image of Hela cells under the hypoxic condition of 1% O2 for 8 h is defined as 1.0. The results are the mean ± standard deviation of three separate measurements.
either Hela or A549 cells increases with the decease of O2 concentration from 20% to 1%. For example, the fluorescence under the hypoxic condition of 10% O2 for 4 h rises by ca. 30%, with respect to that under the normoxic condition, whereas that under the hypoxic condition of 1% O2 for 4 h increases dramatically by ca. 4 times; for the hypoxic time of 8 h, the fluorescence at 10% and 1% O2 increases by ca. 0.75 and 6 times, respectively, compared to that under the normoxic condition. Interestingly, under either 4 or 8 h hypoxic conditions, a nonlinear fluorescence increase in the cells is observed with the decrease of the O2 level.
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CONCLUSIONS In summary, we have developed a highly sensitive and selective method for convenient detection of nitroreductase as well as the hypoxic status of tumor cells by designing probe 1. The probe displays a distinct color and fluorescence off−on response to nitroreductase via the enzyme-catalyzed reduction 3931
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Analytical Chemistry
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dx.doi.org/10.1021/ac400750r | Anal. Chem. 2013, 85, 3926−3932
