Article pubs.acs.org/jnp
Delineation of the Role of Glycosylation in the Cytotoxic Properties of Quercetin using Novel Assays in Living Vertebrates Si-Hwan Park,† Hyun Jung Kim,‡ Soon-Ho Yim,§ Ah-Ra Kim,† Nisha Tyagi,† Haihong Shen,† Kyung Keun Kim,⊥ Boo Ahn Shin,⊥ Da-Woon Jung,*,† and Darren R. Williams*,† †
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Jeonnam 534-729, Republic of Korea § College of Public Health and Welfare, Dongshin University, Jeonnam 520-714, Republic of Korea ⊥ Chonnam National University Medical School, Gwangju 501-746, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Quercetin is a plant-derived flavonoid and its cytotoxic properties have been widely reported. However, in nature, quercetin predominantly occurs as various glycosides. Thus far the cytotoxic activity of these glycosides has not been investigated to the same extent as quercetin, especially in animal models. In this study, the cytotoxic properties of quercetin (1), hyperoside (quercetin 3-O-galactoside, 2), isoquercitrin (quercetin 3-O-glucoside, 3), quercitrin (quercetin 3-O-rhamnoside, 4), and spiraeoside (quercetin 4′-O-glucoside, 5) were directly compared in vitro using assays of cancer cell viability. To further characterize the influence of glycosylation in vivo, a novel zebrafish-based assay was developed that allows the rapid and experimentally convenient visualization of glycoside cleavage in the digestive tract. This assay was correlated with a novel human tumor xenograft assay in the same animal model. The results showed that 3 is as effective as 1 at inhibiting cancer cell proliferation in vivo. Moreover, it was observed that 3 can be effectively deglycosylated in the digestive tract. Collectively, these results indicate that 3 is a very promising drug candidate for cancer therapy, because glycosylation confers advantageous pharmacological changes compared with the aglycone, 1. Importantly, the development of a novel and convenient fluorescence-based assay for monitoring deglycosylation in living vertebrates provides a valuable platform for determining the metabolic fate of naturally occurring glycosides. he flavonol quercetin (1) is one of the major flavonoids present in fruits, vegetables, green tea, and red wine.1,2 Compound 1 has been shown to possess cytotoxic properties, which has attracted much research attention.2,3 For example, dietary 1 reduced the occurrence of cancer-linked chromosomal aberrations in murine bone marrow and suppressed the development of precancerous lesions in a rat colon cancer model.4,5 Moreover, it has recently been shown that 1 can inhibit proliferation in orthotopically transplanted pancreatic xenografts.6 However, plants generally store chemicals in an inactive form as glycosides, which can be subsequently activated by enzyme hydrolysis.1 For example, the onion (Allium cepa L.) contains 1.2−1.9 mol/100 g quercetin in the fresh edible part, compared with 92−178 mol/100 g quercetin glycosides.7 In green tea, 1 is only present as glycosides.7 However, the cytotoxic properties of quercetin glycosides have been investigated to a much lesser degree than those of 1.2 The glycoside rutin (quercetin-3-O-rutinoside) has been investigated with mixed results; there is no effect on carcinogenesis in a rat colon cancer model4 or some positive effect if combined with other herbal supplements.5 However, a recent study indicates that rutin inhibits human leukemia tumor growth in a
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© XXXX American Chemical Society and American Society of Pharmacognosy
murine xenograft model.6 More recently, enzymatically modified isoquercitrin (α-oligoglucosyl isoquercitrin) has been shown to suppress tumor suppression in rats.8,9 Overall, the cytotoxic properties of quercetin glycosides have not been directly compared in vivo. Thus, the effect of type and position of glycosylation on cytotoxic activity is currently uncertain. Over the past decade, the zebrafish (Danio rerio) has become established as an invaluable model for drug discovery research.10,11 This small, hardy teleost fish possesses considerable advantages as a research tool. For example, it is the only currently available vertebrate model that can be grown in 96 well plates. In addition, housing costs are low compared with other vertebrate models, which allows large numbers to be maintained in the laboratory. Zebrafish embryogenesis is rapid, optically transparent (allowing simple visualization of the major organ systems), and amenable to in vivo manipulation. Thus, zebrafish-based assays remove an important “bottleneck” in the drug characterization process: most of the hits generated by drug screening fail in animal tests due to issues such as Received: April 30, 2014
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RESULTS AND DISCUSSION Inhibition of Cancer Cell Proliferation by Quercetin (1) Is Influenced by Glycoside Modification. The cytotoxicity of 1 and its glycosides (2−5) against human colon cancer cells was measured using the MTT assay. The glycosides tested were 2, 3, 4, and 5. As shown in Figure 1A,
Figure 1. Cytotoxic effect of quercetin (1) and its glycosides (2−5) on colon cancer cells. (A) Effect of 1−5 on human HCT116 colon cancer cell viability. Data are representative of two independent assays (mean ± SD). *p < 0.05 compared with 2 at the same concentration. (B) Effect of 1, 3, and 5 on PARP cleavage in human HCT116 colon cancer cells. The vehicle (DMSO) served as a negative control. Cells were incubated with compound for 72 h.
absorption, solubility, metabolic stability, and toxicology.10,12−15 Recently, the zebrafish has also become established as a valuable validated model for cancer research.16−19 In zebrafish cancer models, tumors develop at various organ sites and show striking histologic and genetic similarities with their human counterparts. For example, intestinal and liver tumors in zebrafish apc mutants, mouse apc mutants, and human patients all show constitutive activation of the Wnt signaling pathway.20 Recently, zebrafish has also been validated as a human tumor xenograft model for cytotoxic drug discovery.21−23 In this study, zebrafish-based experimental systems were used to address an important issue in natural products-based research for cancer drug discovery: how does the glycosylation of candidate cytotoxic natural compounds affect their metabolic fate and cytotoxic activity in vivo? Using the compound 1 as a well-known example, it was demonstrated that the influence of glycosylation on cytotoxic activity can be assessed using rapid and simple zebrafish-based assays. Moreover, this study presents a novel and experimentally convenient fluorescencebased assay for rapidly assessing the effect of glycosylation on the metabolic fate of candidate drugs. Using this approach, it was observed that quercetin 3-O-glucoside (isoquercitrin, 3) underwent deglycosylation more effectively in the zebrafish gut compared with quercetin 4′-O-glucoside (spiraeoside, 5) and exerted comparable cytotoxic activity to 1. These results indicate that compound 3 is a very promising drug candidate for cancer therapy. Overall, the findings presented herein provide a novel in vivo-based structure−activity relationship study for investigating the role of glycosylation in determining antitumor activity. Therefore, the experimental approaches described in this study can facilitate translational research for cancer drug discovery.
the aglycone 1 exhibited stronger inhibitory effects on cancer cell proliferation compared with its glycopyranosides. In addition, the glucosides 3 and 5 were significantly more effective than 2 and 4. This result suggested that both glycoside modifications of 1 significantly attenuate the cytotoxicity of 1 against cancer cells in vitro. Glycoside Modification of Quercetin (1) Influences the Induction of Apoptosis in Cancer Cells. When cells undergo programmed cell death (apoptosis), the enzyme poly(ADP-ribose) polymerase (PARP) is activated and cleaved.24 This cleavage can be used to detect apoptosis in cells.25 In order to determine whether 1 or its glycosides induce apoptosis of the cancer cells, PARP cleavage in HCT116 colon cancer cells was measured after treatment with 1 or the quercetin glucopyranosides 3 and 5. Treatment with 20 μM 1 for 72 h induced greater PARP cleavage compared with that with 3 and 5, consistent with the MTT assay results for cancer cell proliferation (shown in Figure 1A). Quercetin Glycosides Display Different Toxicological Profiles and Antitumor Effects in Vivo. The zebrafish is a very useful model system because it is a verified vertebrate platform for predicting toxicological effects in mammals.26,27 To determine whether 1, 3, or 5 produces teratogenic effects in vivo, developmental defects were assessed in zebrafish embryos. B
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It was observed that a dose of 160 μM 5 induced delayed hatching, skeletal deformities, abnormal heart, abnormal head, slow heartbeat, heart sac edema, and abnormal development of the yolk sac in 31.25% of the treated embryos (Figure 2A,B). In
Figure 2. Toxicological analysis of quercetin (1), quercetin 3-Oglucopyranoside (3), and quercetin 4′-O-glucopyranoside (5). (A) Representative pictures of 72 hpf zebrafish treated with 80 or 160 μM of each compound at the 8-cell stage. (B) Quantification of the survival rates for embryos treated with 1, 3, or 5. *p < 0.05 compared with the 1 or 3 treated groups.
Figure 3. Antitumor activity of quercetin (1), quercetin 3-Oglucopyranoside (3), and quercetin 4′-O-glucopyranoside (5) in vivo. (A) Schematic diagram of the cancer cell proliferation assay using the zebrafish human tumor xenograft model. (B) Representative phase contrast, fluorescent, and merged images of cancer cells extracted from enzymatically dissociated embryos. Xenografted embryos were treated with 80 μM of 1, 3, or 5, or 10 μM API-H7 for 4 d (8 animals/group). Arrows indicate HCT116 cancer cells. (C) Inhibition of cancer cell proliferation by 1, 3, or 5 in vivo. Data are representative of two independent assays. *p < 0.05 compared with the negative control group. DMSO and compound API-H7 were used as negative and positive controls, respectively.
contrast, 1 and 3 were not toxic at the same concentration, demonstrating that the nature of the glycoside modification of 1 significantly influences its effect on developmental toxicity. However, it was observed that 1, 3, and 5 did not induce developmental toxicity in the embryos at lower concentrations, such as 40 and 80 μM (Figure 2A,B). Therefore, a dose of 80 μM 1, 3, or 5 was selected for characterizing the antitumor effects of quercetin glycosides using the in vivo zebrafish human tumor xenograft model, which has become established as a validated assay for testing cytotoxic drug candidates in vivo.17,21,22,28 For this analysis, 48 h postfertilization (hpf) zebrafish received xenografts of HCT116 human colon cancer cells stained with a fluorescent dye, DiI (shown schematically in Figure 3A). Xenografted embryos were incubated with 80 μM 1, 3, or 5 for 96 h. The previously reported triazine-based cytotoxic agent API-H722 was used as a positive control. It was observed that cancer cell proliferation was significantly decreased in embryos treated with 1 or 3 (Figure 3B,C). The inhibitory effect of 3 on cancer cell proliferation in vivo was confirmed using a dose dependent study (Supporting Information, Figure S3). However, 5 did not significantly affect cancer cell proliferation, indicating that the position of the sugar moiety markedly affects this activity, which cannot be detected using in vitro assays (comparison with Figure 1A). As a further test, we also analyzed compounds 2 and 4 in the
zebrafish human tumor xenograft model (Supporting Information, Figure S2). It was observed that 2 and 4 did not inhibit cancer cell proliferation in vivo. Deglycosylation of Quercetin Glycosides Can Be Monitored Using a Novel Assay for Zebrafish Intestinal β-Glucosidase Activity. As shown in Figure 3, the glucoside 3 showed significant inhibitory effects, comparable to 1, on cancer cell proliferation in vivo, even though 3 exhibited much weaker cytotoxicity than that of 1 in vitro. Therefore, it was speculated that 3 readily undergoes deglycosylation by βglucosidase in the zebrafish, producing its aglycone. To test this hypothesis, a novel zebrafish model was established for testing the deglycosylation of drug candidates using a pro-fluorescent probe, resorufin-β-D-glucopyranoside (Res-β-Glc).29 This probe is a fluorogenic substrate for β-glucosidase, which is expressed C
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Figure 4. Detection of β-glucosidase activity in the zebrafish embryonic intestine using Res-β-Glc. (A) Schematic diagram of our novel, in vivo deglycosylation assay. (B) DIC and fluorescent images of 7 dpf larvae treated with increasing concentrations of the Res-β-Glc probe. Increased fluorescence indicates cleavage of the probe by glucosidase in the digestive tract. (C) Quantification of the concentration-dependent increase in Resβ-Glc probe fluorescence from the digestive tract by ImageJ software (National Institutes of Health). (D) Representative images of 7 dpf larvae treated with 400 μM Res-β-Glc for 30 min or 1, 2, or 3 h. (E) Detection of the time-dependent increase in fluorescence derived from Res-β-Glc. The embryos treated with the pro-fluorescent probe (4 embryos/group) were lysed and dispensed into a 96 well plate (error = standard error of the mean; n = 3). The fluorescent signal was detected by a fluorescent microplate reader.
is deglycosylated by β-glucosidase. In addition, it was observed that the fluorescence signal from cleaved Res-β-Glc could be quantified using the ImageJ software (National Institutes of Health, Figure 4C) or a fluorescence microplate reader (Figure 4E). Based on this result, a treatment concentration of 400 μM Res-β-Glc was selected for the deglycosylation assay. A time course study for Res-β-Glc deglycosylation was also carried out in the zebrafish (Figure 4D,E). It was observed that the 2−3 h time point was optimal for detecting Res-β-Glc deglycosylation. Compound 1, 3, or 5 at a dose of 80 μM was tested for deglycosylation in the presence of Res-β-Glc. Compounds were added to zebrafish embryos in the fish media and incubated for 2.5 h with the pro-fluorescent probe. Among them, 3 greatly reduced the level of fluorescence compared with control
in the small intestine.30 The emission peak for Res-β-Glc is 590 nm, which reduces interference from natural product-based fluorescent compounds. Moreover, this probe does not require a “stop” solution.29 To optimize and establish an in vivo assay for deglycosylation, increasing concentrations of Res-β-Glc were added to 7 days postfertilization (dpf) zebrafish embryos, followed by incubation for 2.5 h at 31 °C. Fluorescent signal from the ingested probe was captured by microscopy (Figure 4A). Treatment with 100, 200, 400, and 1600 μM Res-β-Glc produced increased fluorescent signals in the recipient embryos in a dose-dependent manner (Figure 4B). This result shows that the zebrafish embryos exhibit β-glucosidase activity in their intestine, because this probe does not emit fluorescence until it D
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Figure 5. Detection of quercetin glycoside deglycosylation in vivo using Res-β-Glc. (A) Zebrafish larvae, 7 dpf, were incubated with Res-β-Glc in the presence or absence of 80 μM 1, 3, or 5. DMSO served as a control. Fluorescent microscopic analysis indicated that the deglycosylation of the profluorescent probe was inhibited competitively in the presence of 3 and 5. The aglycone, 1, however, did not quench probe fluorescence because it is not a substrate for glucosidase. (B) Numbers of larvae (24 lavae/group, n = 3) showing reduced intensity were counted under the fluorescent microscope. Compound 3 treatment inhibited the deglycosylation of the pro-fluorescent probe in larger numbers of zebrafish larvae, compared with compound 5 treatment. (C−E) Compound 3 inhibited the fluorescent signals produced from the probe in a dose-dependent manner. Representative fluorescent images are shown in panel C. This could be quantified by counting the number of zebrafish larvae exhibiting reduced fluorescence (D) or measuring Res-β-Glc fluorescent signal in lysed larvae (E). Error = SD; *p < 0.05 compared with the control group [for panels B and D] or the probe only group [for panel E].
which generates relatively greater quantities of the bioactive aglycone, 1. Our study reported herein compares cytotoxic activities of 1 and 2−5 using both in vitro and in vivo assays and demonstrates that the types and positions of glycosylation are important determinants of bioactivity and bioavailability. Compounds 1 and 2−5 comprise one of the most common dietary flavonoids, with an intake level of 30−40 mg/day.31 Given the widely reported biological activities of these flavonoids, it is important to develop new and rapid in vivobased assays that characterize their beneficial effects and help us to understand the influence of glycosylation on their activity
(Figure 5A,B), indicating that the quercetin glycoside competitively inhibited deglycosylation of the Res-β-Glc. Treatment with 5 also reduced probe fluorescence (45% reduction), but this reduction was less than what was observed after treatment with 3 (57% reduction, Figure 5B), which also correlated with the cytotoxic activity of 3 and 5 in vivo (compare with Figure 3C). Moreover, the aglycone, 1, could not decrease probe fluorescence (Figure 5A,B). Further analysis using a 40, 80, or 160 μM dose of 3 showed that the degree of 3 deglycosylation in the digestive tract was dose-dependent (Figure 5). These results indicate that 3 undergoes deglycosylation more effectively than 5 in the digestive tract, E
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The zebrafish-based assays described herein allow rapid evaluation of deglycosylation, cytotoxic activity, and toxicity. Moreover, the novel zebrafish-based assay for deglycosylation presented herein should be amenable to screening large collections of compounds, because the experimental steps can be carried-out in the 96 well plate format. It is known that glycosides of natural origin are promising as drug leads, based on the general rules of drug design.35 In summary, these findings provide a novel, convenient in vivo-based structure−activity relationship protocol for investigating the role of glycosylation in determining biological activity, by showing that “simple” changes in the position of the sugar moiety can produce dramatic effects on toxicity and cytotoxic potential. To illustrate the potential of this protocol, the quercetin monoglycoside, 3, was identified as a promising cytotoxic drug candidate.
and potential for drug development. This is especially relevant for 1, because the glycoside form occurs predominantly in nature.1,7 Our cell-based in vitro assays showed that cytotoxic activity of 1 was attenuated by glycosidic modification. In addition, 3 and 5 have stronger cytotoxic activity than 2 or 4 (Figure 1A). Moreover, treatment with 3 and 5 weakly induced the expression of cleaved PARP in cancer cells, whereas 1 strongly induced the expression of cleaved PARP at 20 μM treatment (Figure 1B). These results show that the presence and types of sugar moiety alter the biological activity of the aglycone 1. The toxicology data presented herein further demonstrates the pivotal role of the sugar moiety in determining the biological activity of quercetin glycosides, because it was observed that 5 has a toxic effect on embryonic development, while 3 exhibited a nontoxic effect up to a 160 μM dose (Figure 2A,B). To our knowledge, this is the first report showing that the types or position of glycosidic modification of a naturally occurring compound significantly influence toxicological effects on vertebrate development. The zebrafish has become established as a powerful system for studying cancer biology, allowing pivotal studies to be carried out in this animal model.32−34 In this study, analysis of the cytotoxic effects of 3 and 5 in vivo, utilizing the validated human tumor xenograft model,22 indicates that 3 has the greater antitumor activity (Figure 3). This finding was in contrast to the in vitro cytotoxic analysis (Figure 1). Collectively, these data reiterate the need to follow-up cellbased assays of natural product activity with animal-based studies. Although the data presented herein suggests that 3 is a superior cytotoxic agent in vivo compared with 5, this result did not provide any information about why this occurs. Therefore, a novel, simple, economical, and rapid test for monitoring the deglycosylation of drug candidates in vivo was established (Figure 4). Assessing the effect of glycosylation on in vivo activity of the compounds is a pivotal component of the drug discovery process, because it may confer advantageous pharmacological changes and enhance drug formulation.35−37 The assay presented herein is based on the resorufinglucopyranoside probe, which is commercially available and was originally developed for screening compounds that overcome mutations in glucosidase, which causes Pompe disease.29 This new assay for deglycosylation is the first report that this probe can be used to rapidly assess glucosidase activity in an animal model. Importantly, the zebrafish digestive tract possesses marked similarities to that of humans, such as anatomical structures and highly homologous absorptive and secretory functions that allow human gastrointestinal diseases to be studied in this animal model.38 The results of the deglycosylation assay indicated that 3 undergoes deglycosylation more effectively than 5 (Figure 5A,B). Importantly, it was shown that the degree of deglycosylation in vivo also correlated with the cytotoxic effect of these glycosides in vivo (compare with Figure 4). Compounds 2 and 4 were also tested for comparison with the active compound, 3, using both the zebrafish tumor xenograft system and the deglycosylation assay (Supporting Information, Figures S2 and S4). Compounds 2 and 4 did not show significant effects in either assay system. Based on this data, it was concluded that no additional assay systems for measuring the deglycosylation of nonglucose glycosides was required.
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EXPERIMENTAL SECTION
Chemicals and Reagents. Quercetin (1, ≥95% purity), anti-actin antibodies, and CelLytic M Cell Lysis Reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-cleaved-PARP (polyADP ribose polymerase) antibody was obtained from Cell Signaling (Danvers, MA, USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) was purchased from Invitrogen (Carlsbad, CA, USA). Resorufin β-D-glucopyranoside (Res-β-Glc) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Test Compounds. Four quercetin glycosides (2−5) were isolated from the flowers of Hamamelis japonica (Hamamelidaceae). Extraction, isolation, and structural characterization for glycosides was described in detail in the Supporting Information. The structures of all the compounds were assigned by 1H, 13C, HSQC, and HMBC NMR and MS spectroscopic data. The purities of the compounds were estimated to be higher than 97% by HPLC and spectral data. Cancer Cell Culture. The HCT116 colon carcinoma cell line was obtained from the American Type Culture Collection, USA, and maintained in Dulbecco’s modified Eagles medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were cultured at 37 °C in an atmosphere containing 5% CO2. Cell Viability Test. HCT116 colon cancer cells (4 × 103) were seeded in 96 well plates and incubated at 37 °C overnight. The cells were then treated with various concentrations of drugs in 1% FBS− DMEM culture media. After 96 h incubation, media was removed, and cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow tetrazole solution, for 3 h. DMSO (50 μL) was then added to each well to dissolve the purple formazan produced by the MTT. Absorbance was measured using a spectrophotometer at a wavelength of 570 nm. Western Blotting. The treated HCT116 colon cancer cells were homogenized in ice-cold buffer (1 mL of cell lysis buffer (SigmaAldrich, St. Louis, MO, USA), 1 mM phenylmethanesulfonylfluoride (PMSF), and protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA)) and centrifuged at 10000 rpm for 10 min at 4 °C. The supernatant was separated and used as a protein fraction. The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein samples were loaded in 8% polyacrylamide gels, subjected to SDS-PAGE, transferred to nitrocellulose membranes, and visualized with an ECL detection kit (Santa Cruz, CA, USA). Teratogenicity Test. Care and treatment of zebrafish were conducted in accordance with guidelines established by the Animal Care and Ethics Committees of the Gwangju Institute of Science and Technology, Republic of Korea. Fertilized eggs at the eight-cell stage were incubated with each compound in a 24-well plate (8 eggs/well). Embryos were maintained at 31 °C. After 72 h incubation, the development and survival of embryos were recorded. Morphological features were examined by microscopy (Leica DMRBE, upright, Wetzlar, Germany). F
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Zebrafish Human Tumor Cell Xenograft. Zebrafish embryos were obtained using standard mating conditions and staged for cell xenoplantation at 48 h postfertilization. Embryos were dechorionized using microforceps, anesthetized with 0.0016% tricaine, and positioned on their right side on a wet 1.0% agarose pad. Cancer cells were detached from culture dishes using 0.05% trypsin−EDTA and washed twice with PBS at room temperature. Cells were stained with 2 μg/mL DiI diluted in PBS and washed four times, once with FBS, twice with PBS, and then once with 10% FBS diluted in PBS. Cells were kept on ice before injection. Cancer cells were counted by microscopy, suspended in 10% FBS, and injected into the center of the yolk sac using an injector (PV820 pneumatic picopump, World Precision Instruments, Sarasota, FL, USA) equipped with borosilicate glass capillaries (World Precision Instruments, Sarasota, FL, USA). After injection, embryos were transferred into 96-well plates (1 embryo per 200 μL per well) containing 80 μM concentration of each compound diluted in E3 media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2,0.33 mM MgSO4.7H2O) without methylene blue. Injected embryos were maintained at 31 °C for 96 h and analyzed for cancer cell proliferation. Quantification of Xenografted Colon Cancer Cells. At 4 days postinjection (dpi), larvae were incubated in 50 μL of protease solution (2 units/mL collagenase (Roche, Switzerland) and 60 units/ mL dispase (Roche, Switzerland) in DMEM medium (Life Technologies, Carlsbad, CA, USA)) for 2 h at 37 °C. Gentle dispersion of cells was performed with a pipet to dissociate xenotransplanted embryos into single cells. For fixation of cells, 50 μL of 8% paraformaldehyde solution was added directly to the wells. The cells were counted and imaged using fluorescence microscopy (Leica DMI3000B, inverted, Wetzlar, Germany). Res-β-Glc GC Enzyme Assay in Zebrafish. Zebrafish embryos were obtained using standard mating conditions and staged for the GC enzyme assay at 7 days postfertilization (dpf). Embryos were transferred into 24 well plates (24 embryos per 1 mL per well) containing Res-β-Glc diluted in E3 media without methylene blue with or without drugs of interest. At 2.5 h post-treatment, embryos were washed five times with E3 media without methylene blue and then placed on a 3% methyl cellulose plate for microscopic observation. The number of embryos exhibiting reduced fluorescent signal, compared with embryos incubated with Res-β-Glc alone, was counted under upright microscopy (Leica DM2500, Wetzlar, Germany). Representative pictures were also captured using upright microscopy. The fluorescence signal in the embryos was also quantified using a fluorescent microplate reader, based on a previous report that measured fluorescence from the glucose probe, 2-NBDG.39 Briefly, embryos were dissolved in CelLytic M cell lysis reagent (24 larvae per 120 μL per tube) by sonication for 10 min at 4 °C, and the supernatants were transferred to a 96 well plate, Fluorescent signal was detected at an excitation of 570 ± 10 nm and emission of 610 ± 10 nm, which yielded the optimal signal-to-basal ratio, using a fluorescent microplate reader (SpectraMAX Gemini XS). Statistical Analysis. The student’s t-test was used for comparison between experimental groups (Microsoft Excel, 2010 version); p values of
