A highly selective fluorescent probe for human NAD(P)H:quinone oxidoreductase 1 (hNQO1) detection and imaging in living tumor cells

Human NAD(P)H:quinone oxidoreductase (hNQO1) can be used as a biomarker for the early diagnosis of cancer. In this article, a novel fluorescent probe 1 for detecting hNQO1 was designed and synthesized by using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) derivative (TCF-OH) as a fluorophore and quinone propionic acid (QPA) as a recognition group. The probe, which has high selectivity for hNQO1 and a Stokes shift of about 117 nm, shows a linear relationship with hNQO1 concentrations in the range of 0.25–3 μg ml⁻¹ with the color changes from yellow to red and was successfully applied to intracellular hNQO1 imaging.

Human NAD(P)H:quinone oxidoreductase (hNQO1) can be used as a biomarker for the early diagnosis of cancer.     

A novel near-infrared fluorescent probe for highly selective recognition of hydrogen sulfide and imaging in living cells

A novel near-infrared fluorescent probe (L) based on a 1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano[2,3-g]quinoxalin-7-one scaffold has been synthesized and characterized. Probe L displays highly selective and sensitive recognition to H₂S over various anions and biological thiols with a large Stokes shift (125 nm) in THF/H₂O (6/4, v/v, Tris–HCl 10 mM, pH = 7.4). This probe exhibits turn-on fluorescence for H₂S through HS⁻ induced thiolysis of dinitrophenyl ether. Confocal laser scanning micrographs of MCF-7 cells incubated with L confirm that L is cell-permeable and can successfully detect H₂S in living cells.

A novel “off–on” fluorescent probe (L) for H₂S detection with NIR emission and imaging H₂S in living cells.     

A highly selective quinolizinium-based fluorescent probe for cysteine detection

A novel fluorescent quinolizinium-based turn-off probe has been developed for selective detection of cysteine. The probe showed high selectivity and sensitivity towards cysteine over other amino acids including the similarly structured homocysteine and glutathione with a detection limit of 0.18 μM (S/N = 3). It was successfully applied to cysteine detection in living cells with low cytotoxicity and quantitative analysis of spiked mouse serum samples with moderate to good recovery (96–109%).

A novel fluorescent quinolizinium-based turn-off probe for selective detection of cysteine has been developed.

Biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), are biomolecules that play important roles in a variety of biological processes, such as cellular growth, redox homeostasis and immune system regulations.^1–5 Among the three biothiols, Cys is the essential amino acid involved in various physiological processes, in which it serves as a biomarker for different dysfunctions and diseases.⁶ The deficiency of Cys can lead to adverse symptoms such as liver damage, psoriasis and lethargy, while high levels of Cys can cause a wide range of disorders such as Alzheimer''s and cardiovascular diseases.^7–10 Therefore, it is of importance to develop effective and selective approaches for Cys detection under physiological conditions.In the past decades, various techniques had been established for the detection of Cys, such as high performance liquid chromatography (HPLC),^11,12 capillary zone electrophoresis (CZE),^13–15 mass spectrometry (MS).^16,17 However, these methods require specialized equipment and sophisticated sample preparations, which restrict their applications on routine detection. In comparison, fluorescence spectroscopy is considered as a powerful technique for detection of Cys due to its high selectivity, operation simplicity, and non-invasiveness.^18–20 Nowadays, a variety of fluorescent probes have been developed based on the characteristic redox properties and strong nucleophilicity of the thiol group on Cys.^21–38 However, due to the structural similarity of Cys, Hcy and GSH, selective fluorescent detection of Cys in biological samples still remains a challenge.^39,40 Therefore, development of fluorescent probes for highly selective Cys detection is important.Cys-triggered addition–cyclization–cleavage reaction with acrylate, which was first reported by Yang and Strongin in 2011,⁴¹ is the most widely used response mechanism for the design of Cys-selective fluorescent probes.^5,18,20,21 Upon the addition of Cys, nucleophilic attack of Cys on acrylic ester followed by intramolecular cyclization releases the fluorophore''s hydroxyl and a seven-membered ring amide. The high selectivity of this reaction towards Cys over Hcy and GSH is attributed to the kinetic difference of the intramolecular cyclization.Various Cys-responsive fluorescent probes have been developed based on the incorporation of acrylate group on common fluorephores, such as BODIPY, rhodamine, coumarin and fluorescein.^42–50 However, the use of these dyes might suffer from low water solubility, which results in decreased sensitivity of detection and difficulty in biological applications.²² In comparison, quinoliziniums are cationic aromatic heterocycles with improved water solubility, which enable their applications in cell imaging with good biocompatibility.^51,52 Compared with these common fluorescent scaffolds, studies on the applicability of quinolizinium compounds as fluorescent chemosensors remain largely elusive (Scheme 1).Open in a separate windowScheme 1(a) Common fluorophores used for construction of thiol detection probes. (b) Novel fluorescent quinolizinium-based probe for cysteine detection.In 2017, we have developed a new series of fluorescent quinolizinium compounds with tunable emission properties in visible light region (λ_em = 450 to 640 nm) and large Stokes shifts (up to 6797 cm⁻¹).⁵³ The application of this class of fluorescent quinoliziniums in live cell imaging was demonstrated by incubation with HeLa cells, in which the subcellular localization of the quinoliziniums could be switched by modifying the substituents. Based on this work, we envision that the fluorescent quinoliziniums would be amenable for the design of fluorescent probes for Cys detection in biological samples.Herein we introduce a novel fluorescent quinolizinium-based turn-off probe 1 for highly selective detection of Cys over Hcy, GSH and other amino acids. The acrylate group was incorporated on the phenyl ring of the quinolizinium, which served as the moiety for the reaction with Cys. Cys triggered the change in fluorescence intensity of probe 1 due to the conjugated addition–cyclization reaction with the acrylate group. The probe exhibited highly selective detection for Cys and good biocompatibility, which could be successfully applied to detection of Cys in living cells and quantitative analysis of Cys concentrations in mouse serum samples.To verify the feasibility of probe 1 for Cys detection, the spectral properties of probe 1 towards Cys were firstly investigated in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS). As shown in Fig. 1, the free probe 1 showed absorption bands at 360 nm and 420 nm. Upon excitation at 420 nm, strong fluorescent signal was observed at 495 nm. After the addition of Cys (20 equiv.), the absorption at 360 nm increased with the decreased absorption band at 420 nm, while the fluorescence intensity of probe 1 significantly reduced. These results indicated that probe 1 displayed fluorescence signal response towards Cys.Open in a separate windowFig. 1(a) UV-Vis absorption and (b) fluorescence spectra of 1 (20 μM) with and without the addition of Cys (20 equiv.) in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS) after 100 min.To examine the sensitivity of the probe, fluorescence titration of probe 1 (20 μM) was carried out in the presence of Cys in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS) at 25 °C. The fluorescence quantum yield was evaluated to be 0.43 using coumarin 153 as a reference. Addition of 0.5 equiv. of Cys resulted in a decrease in fluorescence emission at 495 nm. The emission intensity was almost completely quenched upon addition of 20 equiv. of Cys, which showed a decrease in approximately 8-fold as compared with that of free probe 1. Furthermore, probe 1 exhibited a good linear relationship between the emission intensities at 495 nm and the concentration of Cys ranging from 0 to 100 μM with a R² value of 0.9904 (Fig. 2b). The detection limit was evaluated to be 0.18 μM based on the equation LOD (Cys) = 3σ/m, where σ is the standard deviation of blank measurements and m is the slope obtained from the calibration curve of probe 1 against Cys, indicating that probe 1 was highly sensitive to Cys.Open in a separate windowFig. 2(a) Fluorescence titration of 1 (20 μM) upon the addition of Cys (0, 10, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180, 200, 400, 600, 1000 μM). (b) Linear correlation between emission intensities at 495 nm and concentrations of Cys (0–100 μM).We next investigated the selectivity of probe 1 for Cys. Under the same reaction conditions, other amino acids including Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val caused almost no fluorescence intensity changes, which demonstrated the high selectivity of 1 to Cys over other amino acids even at high concentration (20 equiv., 400 μM). As shown in Fig. 3a, a distinct fluorescence ratio (F₀/F) induced by Cys could be observed in contrast to other amino acids, while Hcy and GSH showed only little effect to the fluorescence intensity changes. Besides, other potential biologically relevant cations and anions were investigated, including Na⁺, K⁺, Cu⁺, Zn²⁺, Cu²⁺, Ni²⁺, Mg²⁺, Ca²⁺, Fe³⁺, Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, HPO₄⁻, H₂PO₄⁻ and no significant fluorescence responses was observed (Fig. 3b).Open in a separate windowFig. 3Fluorescence changes F₀/F (λ_em = 495 nm) of 1 (20 μM) upon the addition of various (a) amino acids (20 equiv.) and (b) potential biologically-relevant ions in CH₃CN/H₂O solution (1 : 1, v/v, 50 mM pH 7.4 PBS) after 100 min.To study the effect of pH to the fluorescence of probe 1, the change in fluorescence emission intensity of probe 1 with and without Cys was investigated in a range of pH from 1 to 14, respectively. The fluorescence emission intensity of probe 1 at 495 nm was stable in the pH range of 6–9 (Fig. 4). Decrease in the fluorescence intensity was observed under basic conditions (pH > 9), which could be attributed to the hydrolysis of acrylate. The results suggested that probe 1 was capable of detecting Cys under physiological conditions.Open in a separate windowFig. 4Fluorescence intensity of 1 (20 μM) with and without the addition of Cys (20 equiv.) at different pH values.The response time was examined based on the change in fluorescence emission intensity of probe 1 upon reaction with 20 equiv. of Cys, Hcy, and GSH, respectively. As shown in Fig. 5, Cys caused a rapid fluorescence quenching than Hcy and GSH, and the fluorescence intensity remained stable after 100 min. However, the reaction rates of Hcy and GSH with probe 1 were significantly lower than that of Cys. This result indicated that probe 1 could selectively distinguish Cys from Hcy and GSH.Open in a separate windowFig. 5Time-dependent fluorescence changes of 1 (20 μM) upon the addition of Cys, Hcy, and GSH (20 equiv.).Align with literature reports,^54–59 we proposed the reaction mechanism of probe 1 with Cys was based on the nucleophilic addition reaction of Cys with C C bond of acrylate, followed by the cyclization–cleavage reaction and resulting in the formation of 2 with a hydroxyl group (Scheme 2). HRMS analysis of the crude reaction mixture showed the presence of peak with m/z 394.16, which revealed the formation of 2 after the reaction (Fig. S2^†). The high selectivity of probe 1 towards Cys over Hcy and GSH could be attributed to the difference in reaction rates of the intramolecular cyclization reaction. The intramolecular cyclization reaction for the formation of the seven-membered amide promoted by Cys was more kinetically favored than the formation of a strained eight or twelve-membered ring in the case of Hcy or GSH, respectively. As shown in the MS spectra (Fig. S3 and S4^†), the presence of peaks corresponding to the reaction intermediates, m/z 583.21 for Hcy and m/z 755.26 for GSH, respectively, was observed. These results indicated that Hcy and GSH exhibited slower reaction rates with probe 1.Open in a separate windowScheme 2Proposed reaction mechanism of 1 with Cys.NMR analysis of the crude reaction mixture of probe 1 with Cys (3 equiv.) was performed to provide further evidence on this reaction mechanism. As shown in Fig. 6, the hydrogen atoms on the acrylate group were located at 6.12 ppm (1H), 6.40 ppm (1H) and 6.60 ppm (1H). After reaction with Cys, the peaks corresponding to the hydrogen atoms on the acrylate group disappeared, while the shift of two peaks at 7.28 ppm (2H) and 7.51 ppm (2H) to 6.90 ppm (2H) and 7.27 ppm (2H), respectively, which corresponding to the hydrogen atoms on the phenyl ring, was observed. By comparing the NMR spectrum of isolated 2 with that of the crude reaction mixture, the result indicated that Cys reacted with the acrylate group on probe 1, resulting in the formation of 2 with the hydroxyl group.Open in a separate windowFig. 6Study of reaction mechanism using ¹H NMR analysis. (a) ¹H NMR spectrum of isolated 1; (b) ¹H NMR spectrum of isolated 2; (c) ¹H NMR spectrum of crude reaction mixture of 1 with Cys.The fluorescence was proposed to be quenched by the presence of hydroxyl substituent on the phenyl ring (i.e. phenol moiety) of the quinolizinium via intramolecular photo-induced electron transfer (PET). According to our previous study on the structure–photophysical property relationship (SPPR) studies of the quinolizinium compounds, the HOMO is composed of a π orbital of the quinolizinium and phenyl ring while the LUMO is composed of a π* orbital of the quinolizinium ring. The O atom of the phenol moiety served as an electron-donating group that donated an electron from its HOMO to the half-filled HOMO of the quinolizinium upon excitation by light, resulting in the quenching of fluorescence.To demonstrate the practical applicability of probe 1 in biological systems, cytotoxicity test and cell imaging experiments were carried out. HeLa cell lines (American Type Culture Collection) were cultured with Dulbecco''s Modified Eagle''s Medium (DMEM) (Gibco) supplemented with 44 mM sodium bicarbonate (Sigma-Aldrich), 10% v/v fetal bovine serum (Gibco), and 100 U mL⁻¹ penicillin (Gibco), 100 μg mL⁻¹ streptomycin (Gibco) at 37 °C with 5% CO₂. The cells had over 50% cell viability for concentrations of probe 1 up to 20.51 μM, revealing that probe 1 is of low toxicity and good biocompatibility. The colocalization images of HeLa cells were observed after treating with probe 1 and MitoTracker™ Red FM. As shown in Fig. 7c, the green fluorescence from probe 1 overlaid well with the red fluorescence from MitoTracker™ Red FM, indicating that probe 1 could specifically localized in the mitochondria.Open in a separate windowFig. 7Confocal fluorescence microscopic images of HeLa cells. (a) Subcellular localization of 1. (b) Subcellular localization of MitoTracker™ Red FM. (c) Merged images of (a) and (b). (d) Control experiment of 1-treated cells; (e) 1-treated cells incubated with Cys (100 μM). (f) Relative fluorescence of cells measured by ImageJ.For Cys detection in living cells, HeLa cells were first treated with 100 μM of l-cysteine for 30 min, followed by incubation with probe 1 for 2 h. l-Cysteine was replaced by PBS as the control experiment. The fluorescence imaging was conducted with a confocal microscope Leica TCS SP8 MP (Fig. 7d and e). Green fluorescence emission was observed for the control experiment, which possibly revealed that the interfering effects of other intracellular thiol-containing molecules, including Hcy, GSH and H₂S, should be negligible. The fluorescence emission was quenched by the presence of Cys in cells. These results demonstrated that probe 1 could detect Cys in living cells with mitochondrial targeting capability.We further explored the application of probe 1 in quantitative analysis of biological samples. Probe 1 was applied to the detection of Cys in mouse serum samples with literature references.^60–62 The serum samples were obtained from C57BL/6 mouse (source from The Chinese University of Hong Kong). Whole blood collected was allowed to clot by leaving it undisturbed for an hour at room temperature. The clotted blood was centrifuged at 1000 g for 10 min to remove the clot. Sera were separated and stored at −80 °C prior to the assay. The standard addition method was used to detect Cys in mouse serum. Mouse serum samples were diluted 1000-fold with PBS and Cys at different concentrations were added to the samples, respectively. After the reaction was incubated with probe 1 at 25 °C for 100 min, the fluorescence signals of samples were measured. The Cys concentration of each spiked sample was calculated from the linear calibration curve (Fig. S8^†). As shown in

Correction: A highly selective ratiometric fluorescent probe for the cascade detection of Zn2+ and H2PO4− and its application in living cell imaging

Correction for ‘A highly selective ratiometric fluorescent probe for the cascade detection of Zn²⁺ and H₂PO₄⁻ and its application in living cell imaging’ by Kui Du et al., RSC Adv., 2017, 7, 40615–40620.

Affiliation c was incomplete in the original publication; the corrected version is shown below.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Kinetics of NAD(P)H:quinone oxidoreductase I (NQO1) inhibition by mitomycin C in vitro and in vivo

The bioreductive activation of the antitumor quinone mitomycin C (MMC) by NAD(P)H: quinone oxidoreductase 1 (NQO1) is complicated by the ability of MMC to also act as a mechanism-based inhibitor of NQO1 in a pH dependent manner. Inhibition of NQO1 by MMC has been studied in purified enzyme preparations and in cultured cells but has not determined in vivo. In the studies presented here, NQO1 activity was measured in mouse tissues following treatment with MMC or the potent mechanism-based human NQO1 inhibitor 5-methoxy-1,2-dimethyl-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936). NQO1 activity was significantly decreased at 1, 2, and 4 h following MMC (10 or 20 mg/kg) treatment in kidney and lung but was unchanged in brain, heart, liver, and bladder. ES936 (1 mg/kg) treatment led to a significant and much more potent inhibition of NQO1 in all murine tissues analyzed except for bladder. To extrapolate these in vivo results from mice to humans, the species-specific kinetics of NQO1 inactivation by MMC was determined in vitro using mouse, rat, and human recombinant NQO1 proteins. Results showed the inactivation kinetics of mouse and human proteins by MMC were similar. Treatment of human and murine endothelial cells with MMC or ES936 showed similar inhibition of NQO1 activity. The aforementioned results clearly demonstrate that MMC can serve as a substrate for NQO1 in vivo; however, the metabolism resulting in enzyme inactivation is possibly tissue-specific. Furthermore, the kinetic similarities for inactivation between murine and human forms of NQO1 show these results are apropos to clinical use of MMC.     

Correction: A colorimetric and far-red fluorescent probe for the highly sensitive detection of silver(i)

Correction for ‘A colorimetric and far-red fluorescent probe for the highly sensitive detection of silver(i)’ by Yong-jun Wang et al., RSC Adv., 2017, 7, 55567–55570.

The published article incorrectly indicates four corresponding authors. The two corresponding authors should be Jin-wu Yan and Lei Zhang. In addition, a link to the equal contribution footnote, confirming that Yong-jun Wang and Jing-gong Liu contributed equally to the work, is missing in the published article. These errors are corrected herein.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

帕金森病遗传易患性与依赖还原型辅酶Ⅰ/Ⅱ醌氧化还原酶基因多态性的关系

背景帕金森病的病因至今尚未阐明,遗传易患性学说是目前解释帕金森病的主要理论,但哪些遗传因素与此有关,还没有定论.目的探讨依赖还原型辅酶Ⅰ/Ⅱ醌氧化还原酶基因cDNA609位碱基C→T点突变所致的基因多态性与帕金森病遗传易患性的关系.设计以患者和健康人为研究对象,非随机同期化对照研究.单位两所大学医院的神经内科和一所大学医院的老年病研究所.对象1994-09/1997-09中山大学第一附属医院神经科门诊诊断为帕金森患者126例(帕金森病组),年龄46～73岁,其中男74例,女52例;136名健康成人(对照组),其中男66名,女70名,同期来自门诊健康查体,年龄40～72岁.方法采用聚合酶链反应限制性片段长度多态性(PCR-RFLP)的方法分析帕金森病人组与健康成人对照组NQO1基因多态性.主要观察指标NQO1基因cDNA609位碱基C→T点突变的频率及其基因型.结果帕金森病组的T等位基因频率为52%,而对照组为43%,两组差异有显著意义(P＜0.05);基因型分布在帕金森病和对照组之间差异有显著意义(P＜0.05),带T等位基因的个体患帕金森病的风险增加3.8倍.结论NQO1基因cDNA609突变T等位基因可能是帕金森病发生的危险性因素,与帕金森病的遗传易患性有关.     

A highly sensitive and selective fluorescent probe for quantitative detection of Al3+ in food,water, and living cells

Three novel β-pinene-based fluorescent probes 2a–2c were designed and synthesized for the selective detection of Al³⁺. Probe 2a showed higher fluorescence intensity toward Al³⁺ than the other two compounds. Probe 2a determined the concentration of Al³⁺ with a rapid response time (45 s), wide pH range (pH = 1–9), excellent sensitivity (LOD = 8.1 × 10⁻⁸ M) and good selectivity. The recognition mechanism of probe 2a toward Al³⁺ was confirmed by ¹H NMR, HRMS and DFT analysis. Probe 2a was successfully used as a signal tool to quantitatively detect Al³⁺ in food samples and environmental water samples. Furthermore, probe 2a was successfully utilized to label intracellular Al³⁺, indicating its promising applications in living cells.

Probe 2a exhibiting high sensitivity, good selectivity, wide pH range, lower detection limit, and rapid detection for Al³⁺, probe 2a was applied for the successful detection of Al³⁺ in water samples, food samples and HeLa cells.     

A fast-responsive fluorescent turn-on probe for nitroreductase imaging in living cells

Nitroreductase (NTR) may be more active under the environment of hypoxic conditions, which are the distinctive features of the multiphase solid tumor. It is of great significance to effectively detect and monitor NTR in the living cells for the diagnosis of hypoxia in a tumor. Here, we synthesized a novel turn-on fluorescent probe NTR-NO₂ based on a fused four-ring quinoxaline skeleton for NTR detection. The highly efficient probe can be easily synthesized. The probe NTR-NO₂ showed satisfactory sensitivity and selectivity to NTR. Upon incubation with NTR, NTR-NO₂ could successively undergo a nitro reduction reaction and then generate NTR-NH₂ along with significant fluorescence enhancement (30 folds). Moreover, the fluorescent dye NTR-NH₂ exhibits a large Stokes shift (Δλ = 111 nm) due to the intramolecular charge transfer (ICT) process. As a result, NTR-NO₂ displayed a wide linear range (0–4.5 μg mL⁻¹) and low detection limit (LOD = 58 ng mL⁻¹) after responding to NTR. In addition, this probe was adopted for the detection of endogenous NTR within hypoxic HeLa cells.

Probe NTR-NO₂ was effectively reduced in the presence of NTR generating a highly fluorescent product.     

NAD(P)H:quinone oxidoreductase-1-dependent and -independent cytotoxicity of potent quinone Cdc25 phosphatase inhibitors

Cdc25 dual-specificity phosphatases coordinate cell cycle progression and cellular signaling. Consequently, Cdc25 inhibitors represent potential anticancer agents. We evaluated >10,000 compounds for inhibition of human Cdc25 phosphatases and identified many potent and selective inhibitors, which all contained a quinone. Bioreductive enzymes frequently detoxify or activate quinones. Therefore, we evaluated the effect of NAD(P)H:quinone oxidoreductase-1 (NQO1) and reductase-rich microsomes on the activity of three quinone-containing Cdc25 inhibitors: 2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone (Cpd 5, compound 5; NSC 672121), 2,3-bis-(2-hydroxyethylsulfanyl)-1,4-naphthoquinone (NSC 95397), and 6-chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione (NSC 663284). Each inhibitor was reduced by human NQO1 (K(m) of 0.3-0.5 microM) but none by microsomes. Compounds were evaluated with six cancer cell lines containing different amounts of NQO1: HT-29 (1056 nmol/mg/min), HCT116 (660 nmol/mg/min), sublines HCT116-R30A (28 nmol/mg/min) and HCT-116R30A/NQ5 (934 nmol/mg/min), MDA-MB-231/Q2 (null NQO1), and subline MDA-MB-231/Q6 (124 nmol/mg/min) but containing similar amounts of microsomal cytochrome P450 reductase and cytochrome b(5) reductase. Growth inhibition and G2/M arrest by Cpd 5 was proportional to NQO1 levels, requiring 4- to 5-fold more Cpd 5 to inhibit HCT-116 or HCT-116R30A/NQ5 compared with HCT-116R30A. In contrast, in all tested cell lines irrespective of NQO1 level, growth inhibition and G2/M arrest by NSC 95375 and NSC 663284 were similar (average IC(50) of 1.3 +/- 0.3 and 2.6 +/- 0.4 microM, respectively). NSC 95375 and NSC 663284 also caused similar Cdk1 hyperphosphorylation, indicating similar Cdc25 inhibition. However, lower Cpd 5 concentrations were needed to produce Cdk1 hyperphosphorylation in sublines with minimal NQO1. Thus, NQO1 detoxified Cpd 5, probably by reducing it to a less active hydroquinone, whereas NSC 95397- and NSC 663284-generated cytotoxicity was unaffected by NQO1.     

Role for NAD(P)H:quinone oxidoreductase 1 and manganese-dependent superoxide dismutase in 17-(allylamino)-17-demethoxygeldanamycin-induced heat shock protein 90 inhibition in pancreatic cancer cells

Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH?). The formation of 17AAGH? by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH? were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH? could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH?), reduction of 17AAG to 17AAGH? by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH?. In addition, oxidation of 17AAGH? could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH? was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH? with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH?, the active Hsp90 inhibitor derived from 17AAG.     

RH1 induces cellular damage in an NAD(P)H:quinone oxidoreductase 1-dependent manner: relationship between DNA cross-linking, cell cycle perturbations, and apoptosis

Structure-based development of NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones resulted in development of RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], a methyl-substituted diaziridinyl quinone. We conducted experiments to evaluate the mechanism of RH1-induced cytotoxicity and the inter-relationship between DNA cross-linking, cell cycle changes, and apoptosis using an isogenic cell line pair developed from the human breast cancer cell line MDA-MB-468 differing only in expression of wtNQO1 (NQ16 cells). Statistically significant DNA cross-linking was detected using a modified comet assay in cells with wtNQO1 within 1 h of dosing, whereas in parental cells, only marginal DNA cross-linking was observed and required a concentration up to 50 times higher. Cross-linking in NQ16 cells could be abrogated with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NQO1. RH1 prolonged S phase and caused a G(2)/M block. Cell cycle changes were observed up to 10-fold lower in RH1 concentrations in NQ16 cells relative to parental cells. Apoptosis was similarly observed morphologically in both cell lines after RH1 treatment but was induced preferentially in NQ16 cells at lower concentrations and earlier time points. Marked cleavage of caspase-3 was observed in NQ16 cells relative to parental cells using lower concentrations of RH1. Temporally, low doses of RH1-induced rapid DNA cross-linking in NQ16 cells followed by induction of apoptosis at times when a G(2)/M block was not observed. This suggests that cell cycle arrest is not required for RH1-induced apoptosis and that DNA damage may directly initiate apoptotic events. In summary, RH1-induced preferential DNA cross-linking, cell cycle changes, and apoptosis in an NQO1-dependent manner.     

A naphthalimide-based turn-on fluorescence probe for peroxynitrite detection and imaging in living cells

Peroxynitrite (ONOO⁻) is a potent biological oxidant that plays a significant role in diverse physiological and pathological processes. A novel fluorescent probe HCA-OH was developed for specific and sensitive detection of peroxynitrite, and displayed a significant fluorescence turn-on signal. With low cytotoxicity and good photostability, the probe HCA-OH could be applied in imaging ONOO⁻ distribution in HepG2 cells and live C. elegans in real time. Therefore, the probe can be a promising tool for imaging in vivo.

A novel fluorescent probe HCA-OH was designed for selective detection of peroxynitrite and imaging in HepG2 cells and C. elegans.     

A selective and sensitive near-infrared fluorescent probe for real-time detection of Cu(i)

The disruption of copper homeostasis (Cu⁺/Cu²⁺) may cause neurodegenerative disorders. Thus, the need for understanding the role of Cu⁺ in physiological and pathological processes prompted the development of improved methods of Cu⁺ analysis. Herein, a new near-infrared (NIR) fluorescent turn-on probe (NPCu) for the detection of Cu⁺ was developed based on a Cu⁺-mediated benzylic ether bond cleavage mechanism. The probe showed high selectivity and sensitivity toward Cu⁺, and was successfully applied for bioimaging of Cu⁺ in living cells.

The disruption of copper homeostasis (Cu⁺/Cu²⁺) may cause neurodegenerative disorders.     

A highly selective TPE-based AIE fluorescent probe is developed for the detection of Ag+

The detection of Ag⁺ in the environment is very important to determine the level of pollution from silver complexes, which have caused various human health problems. Herein, an aggregation-induced emission (AIE) chromophore (tetraphenylethane, TPE) attached to a benzimidazole group (tetra-benzimidazole, TBI–TPE) is synthesized and utilized to detect Ag⁺ in the environment. The strong chelating effect between the benzimidazole group and Ag⁺ leads to the formation of aggregates, and strong yellow fluorescence signals were observed after adding Ag⁺ into a TBI–TPE solution. The stoichiometry of the complex of TBI–TPE and Ag⁺ was established to be 1 : 2 using photochemical and mass spectra measurements. The detection limit of the Ag⁺ assay is 90 nM with a linear range from 100 nM to 6 μM. This study provides a facile method to determine Ag⁺ in real environmental samples with satisfactory results.

We develop a highly selective TPE-based AIE fluorescent probe containing a benzimidazole group for the detection of Ag⁺.     

A novel ratiometric fluorescent probe for rapid detection of hydrogen peroxide in living cells

In this work, we present a new ratiometric fluorescent probe JNY-1 for rapid and convenient detection of H₂O₂. The probe could selectively and sensitively respond to H₂O₂ within 10 min. In addition, this probe was successfully applied for monitoring and imaging of H₂O₂ in liver cancer HepG2 cells under physiological conditions.

A new ratiometric fluorescent probe JNY-1 for sensitive detection of H₂O₂ is presented with selectivity over other reactive oxygen species, reactive nitrogen species, and biologically relevant species. Imaging of H₂O₂ in liver cancer HepG2 cells was achieved.     

A novel hydrophilic fluorescent probe for Cu2+ detection and imaging in HeLa cells

Copper is an essential element in living systems and plays an important role in human physiology; therefore, methods to detect the concentration of copper ions in living organisms are important. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe that can be used for the detection of Cu²⁺. The probe, BNQ, has high selectivity and sensitivity. The fluorescence intensity of the probe at 520 nm was visible to the naked eye under a UV lamp; upon the gradual addition of Cu²⁺, there was a colour change from green to nearly colourless. Furthermore, the detection limit of BNQ for Cu²⁺ was 45.5 nM. The detection mechanism was investigated using a Job''s plot and density functional theory (DFT) calculations. In addition, owing to great biocompatibility, we were able to successfully use BNQ to detect Cu²⁺ in living HeLa cells with low toxicity.

Probe BNQ was successfully used for detection of exogenous Cu²⁺ in cells using a rare ESDPT sensing mechanism.     

A real-time ratiometric fluorescent probe for imaging of SO2 derivatives in mitochondria of living cells

A real-time ratiometric fluorescent probe (IN-CZ) for highly selective detection of sulfite was designed and synthesized, which is based on modulating the intramolecular charge transfer (ICT) of the hemicyanine dye platform. The mechanism of using the probe is mainly through the Michael addition that occurs between IN-CZ and sulfite with a detection limit of 2.99 × 10⁻⁵ M. IN-CZ displays a fast response (within 1 minute) and is highly selective for SO₃²⁻/HSO₃⁻ over ROS, biologically relevant ions, biological mercaptans and other reactive species. More importantly, IN-CZ was suitable for ratiometric fluorescence imaging in living cells, by real-time monitoring of SO₃²⁻/HSO₃⁻ changes in mitochondria targeted in living cells.

A real-time ratiometric fluorescent probe (IN-CZ) for highly selective detection of sulfite was designed and synthesized, which is based on modulating the intramolecular charge transfer of the hemicyanine dye platform.     

A methylene blue-based near-infrared fluorescent probe for rapid detection of hypochlorite in tap water and living cells

A methylene blue-based near-infrared fluorescent probe was designed for the selective determination of hypochlorite (ClO⁻), over other reactive oxygen species or interfering agents. Acetylated methylene blue was synthesized by introducing the acetyl group into the methylene blue framework, which can specifically recognize exogenous and endogenous ClO⁻. The acetylated methylene blue fluorescent probe was characterized by ¹H NMR, ¹³C NMR and HRMS. The response process and possible mechanism were studied using products of the probe. The emission response of the probe to ClO⁻ presented good linear relationship in the 0–60 μM concentration range, with the detection limit of 0.1 μM (measured at 660 nm and 690 nm). The absorption and emission wavelengths of acetylated methylene blue are both in the near-infrared region; in addition, the probe itself and the degradation products were well-dissolved in water and have almost no toxicity. The probe was used for intracellular ClO⁻ imaging and showed a large fluorescence enhancement (about 200-fold increase).

We developed a MB-based probe to detect OCl⁻, whose product is almost non-toxic. The fluorescence enhancement times are large.