Direct Fluorescent Detection of a Polymethoxyflavone in Cell Culture

Nov 30, 2015 - Fluorescence microscopic image of cross section of mouse colon: (a) colon section from mice fed diet without 5DN and not stained with D...
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Direct Fluorescent Detection of a Polymethoxyflavone in Cell Culture and Mouse Tissue Jingjing Chen,† Mingyue Song,† Xian Wu,† Jinkai Zheng,†,# Lili He,† David Julian McClements,† Eric Decker,† and Hang Xiao*,†,§ †

Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, People’s Republic of China # Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China §

ABSTRACT: Convenient detection methods for bioactive food compounds and their metabolites in biological samples are needed to better understand their mechanism of actions. Herein, we developed a novel approach to directly monitor and visualize the distribution of 5,3′,4′-tridemethylnobiletin (TDN), a unique polymethoxyflavone metabolite derived from citrus polymethoxyflavone, in biological samples such as cultured cells and mouse colonic tissues. Our results showed that a fluorescent conjugate could be formed between TDN and 2-aminoethyl diphenyl borate (DPBA) under simple reaction conditions, which was confirmed by both Raman spectroscopy and mass spectroscopy. We further demonstrated the application of DPBA-based conjugation reaction in the characterization of TDN in different biological samples including floating cells, adherent cells, and animal tissues. This is the first report demonstrating direct fluorescent detection of polymethoxyflavone in biological samples. KEYWORDS: polymethoxyflavone, fluorescence detection, colon cancer cell, mouse tissue, imaging

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cells and cancer cells.10 For compounds that do not produce strong fluorescence by themselves, there are several ways to visualize them in cells or tissues. One approach is to label compounds of interest with fluorescent dye before they are added to the test systems. For example, fluorescein isothiocyanate (FITC)-labeled insulin encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles was prepared before addition to Caco-2 cells to study the uptake of insulin by the cell.11 Rhodamine123 was encapsulated in dextran sulfate− PLGA hybrid nanoparticles to study the uptake of nanoparticles by breast cancer cells.12 Transferrin-conjugated paclitaxel nanoparticles were used to track the retention of bioactive compound inside the cell.13 One disadvantage of this type of method is that these conjugated compounds may cause toxicity to the cells due to the incorporation of toxic fluorophores.14 Another method of tracking the compounds of interests is to stain the compounds after they are localized in the test systems, such as in cultured cells and/or animal tissues. For example, adding 2-aminoethyl diphenyl borate (DPBA, or Naturstoff reagent A) to human keratinocytes after incubating the cells with cyanidin for 24 h can locate the distribution of cyanidin inside the cell.15 This type of approach is convenient and does not cause potential toxicity because fluorophore is added after the cells and animal tissues have been fixed. For both methods mentioned above, antifade medium is frequently used to

INTRODUCTION Polymethoxyflavones (PMFs) are a group of compounds found mostly in citrus fruits. There has been increasing interest in the study of PMFs in recent years, due to their multiple beneficial health effects reported in the literature, including anticancer, anti-inflammation, antioxidant activities1 and antiangiogenic activity.2 The water solubility of most PMFs is relatively low due to the presence of multiple methoxyl groups on the flavonoid skeleton. Some studies have reported the use of nanoemulsion or nanoparticle-based delivery systems to increase the bioavailability of hydrophobic PMFs.3 So far, the fate of these PMFs inside the cells as well as in the tissue has not been fully realized. To better understand the intracellular activities of these plant-derived PMFs inside cells or in animal tissue, it is necessary to directly detect the localization of PMFs in biological samples such as cultured cells and animal tissues. Fluorescent detection is one of the most promising approaches to track compounds of interests in biological samples. This method has been used to detect plant caffeic acid O-methyltransferases in switchgrass plants.4 The result was similar to that reported following a conventional method. The fluorescent detection method has also been used to track the translocation of insecticides in plants.5 It is ideal if the compound of interest itself can be fluorescent when excited under certain wavelengths, so that no fluorophore would be needed. Curcumin is one of these compounds that has broad band fluorescence in various solutions due to the phenolic group and conjugated double bonds of its chemical structure.6 This distinct feature has been used to study the transport of liposomal curcumin into living cells7 and the uptake of curcumin, curcumin microemulsion,8 and micelle9 by normal © XXXX American Chemical Society

Received: September 14, 2015 Revised: November 21, 2015 Accepted: November 30, 2015

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DOI: 10.1021/acs.jafc.5b04484 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry prevent fluorescence quenching during observation under a fluorescence or confocal microscope.16 Among PMFs, recently 5-demethylnobiletin (5DN) was reported as one of most potent inhibitors of cancer cell growth.17 Furthermore, 5,3′,4′-tridemethylnobiletin (TDN) was identified as a major metabolite of 5DN and may contribute significantly to the biological effects of 5DN.18 The goal of this study was to develop a method for direct fluorescent detection of TDN by using DPBA. DPBA has been used to visualize flavonoids in plants.19 Our results showed that DPBA could form fluorescent conjugates with TDN. In this study, we used fluorescence spectroscopy, mass spectroscopy, and Raman spectroscopy to characterize and validate this direct fluorescent detection method for visualization of TDN in biological samples such as cultured human cancer cells and mouse colonic tissues. It may provide a new perspective of PMF properties and may have some implications for the study of the biological metabolism of PMFs when used as functional food ingredients.

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cells were incubated with 0.25% Triton X-100 solution. After incubation, the cells were washed with PBS three times in the dark. The PBS solution of the last wash was kept in the tube, protected from light. The samples were analyzed with a BD LSR II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Settings for detection of Alexa fluor 488 were used to collect the signals of our sample. Fluorescent Detection of TDN in Adherent Cells. Caco-2 cells were seeded on pretreated and UV light sterilized 25 mm cover glasses placed inside a 6-well plate at the seeding density of 50 × 104. When the cell reached 80% confluence, cell culture medium was removed and then incubated with culture media or HBSS (pH 7.4) containing 10 μM TDN. After incubation for 2 h, medium was removed and the cells were washed three times with ice-cold PBS and the supernatant was discarded. Cells incubated with TDN for different times (0.5, 1, 1.5, 2 h) were obtained. Then the cells were fixed with formaldehyde (2%, w/w) for 10 min. The cells were washed with PBS again and then incubated with dye solution (DPBA dissolved in Triton X-100) in the dark for 15 min. The slides were then washed with PBS three times (5 min each). After rinsing with PBS, the cover glasses were mounted with antifade mounting medium on a slide, sealed with nail polish, and dried in the dark. The slides were observed under a Nikon E600 fluorescent microscope (Tokyo, Japan) with a mercury lamp as the excitation source. The images were collected through an FITC filter. The same instrument parameters were used for analyzing cells treated with TDN for different times. Fluorescence Detection of PMF in Mouse Colon Tissue. The animal experiment procedure was approved by the Institutional Animal Care and Use Committee of the University of Massachusetts (permission 2011-0066). Male CD-1 mice (5 weeks old) were fed basal AIN93G diet supplemented with 0.05% (w/w) of 5demethylnobiletin for 1 week. Orally administered 5-demethylnobiletin was transformed to TDN in the colon of the mice. The mice were sacrificed, and colon tissues were harvested, quickly washed with icecold PBS, and fixed in formaldehyde. Formalin-fixed colon tissues were processed for paraffin-embedding and sectioning as we previously described.20 The paraffin on the slide was removed by immersing in xylene three times, each for 5 min. The tissues were then rehydrated by being immersed in 100, 95, 70, and 50% of ethanol, each for 5 min. Finally, the slide was washed with double-distilled water. The slide was incubated with staining solution (DPBA dissolved in 0.25% Triton X100) in a humidified chamber in the dark for 20 min. Then the slide was rinsed with PBS buffer. A cover glass was mounted on top of the tissue section with freshly made antifade mounting medium. The cover glass was sealed with nail polish and then dried in the dark. The slide was observed with a Zessis confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany). A 488 nm laser was used as the excitation source, and an FITC filter was used to collect the images. The same instrument parameters were used for different samples. Statistical Analysis. All experiments were conducted at least three times. One-way ANOVA was used to test significant differences of two or more groups. Statistical significance was at the level of p < 0.05.

MATERIALS AND METHODS

Chemicals. PMFs such as 5DN, 5,3′,4′-tridemethylnobiletin, 5,3′didemethylnobiletin, and 5,4′-didemethylnobiletin were synthesized and purified in our laboratory as previously reported.18 Each compound has a purity of >98%. Formaldehyde, methanol, ethanol, glycerol, and phosphate buffer saline (PBS) were purchased from Fischer Scientific (Fair Lawn, NJ, USA), Dulbecco’s modified Eagle medium (DMEM) was obtained from Mediatech (Manassas, VA, USA), and DPBA and Hanks balanced salt (HBSS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caco-2 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Double-distilled water was prepared with a model D1403 Nanopure water purification system (Thermo Scientific, Waltham, MA, USA). Preparation and Characterization of Sample Compounds. Fluorescence Spectroscopy. TDN was dissolved in methanol at the concentration of 20 μM. It was then mixed with DPBA solution (20 μM dissolved in double-distilled water) at the same volume. Sample was then analyzed in a PTI fluorescence spectrometer (PTI Inc., Edison, NJ, USA). Both excitation and emission spectra were recorded. Raman Spectroscopy. TDN powder and DPBA powder were analyzed by Raman microscopy, respectively. Conjugate was obtained by mixing TDN solution (dissolved in methanol) and DPBA solution (dissolved in double-distilled water) at the same molar ratio. Then 200 μL of mixture was transferred onto a glass slide and dried in the dark. A DXR/SERS (surface-enhanced Raman scattering) Raman spectrometer was used in this experiment (Thermo Scientific). The laser power was 24 mV, and the exposure time was 2 s. The excitation wavelength was 780 nm, and all of the spectra were measured in the range between 0 and 2000 cm−1. Mass Spectroscopy. TDN, DPBA, and TDN−DPBA conjugate were dissolved in HPLC grade methanol and analyzed on a JEOL-700 MStation (JEOL Ltd., Tokyo, Japan). Fluorescence Detection of TDN in Floating Cells. Caco-2 cells were seeded in 6-well plates at the seeding density of 50 × 104 24 h before the experiment. The cells were incubated with culture media containing 10 μM TDN for 0.5, 1, 1.5, and 2 h. Then the cells were trypsinized for 5 min and transferred to a plastic flow cytometry tube. Cell pellet was obtained after centrifugation (1000g, 5 min). The cells were then washed with ice-cold PBS by gentle vortexing and centrifuged at 1000g for 5 min. The cells were fixed with 2% of formaldehyde for 10 min. Then the supernatant was discarded after centrifugation for 5 min (1000g, 4 °C). The cells were washed with PBS and then centrifuged, and the supernatant was discarded. Then the cells were incubated with staining solution (DPBA dissolved in 0.25% Triton X-100) in the dark for 15 min. For the control group,

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RESULTS AND DISCUSSION Characterization of TDN−DPBA Conjugates. Raman Spectroscopy. As can be seen in Figure 1, TDN, DPBA, and TDN−DPBA conjugates showed different Raman patterns. For TDN, 1550 and 1600 cm−1 peaks were from the ring stretch of benzene derivatives. Peaks at 790 and 1210 cm−1 were from the para-disubstituted benzene ring vibration. The peaks at 520 and 590 cm−1 represent the ring stretch (ring B−C, see Figure 1).21 For DPBA, the peak at 615 cm−1 was from the monosubstituted benzene. The peak around 1590 cm−1 was from the NH2 scissors of primary amines. Boronic acid (H3BO3) had the peak at about 1166 cm−1 when excited at 514.5 nm.22 The peak around 1050 cm−1 was the stretching of C−B for diphenyl boronate.23 The broad and short peak near 1330 cm−1 was the asymmetric stretching of B−O.24 For TDN−DPBA, after conjugation with TDN, the peak for B−O became sharper and B

DOI: 10.1021/acs.jafc.5b04484 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Raman spectra of DPBA, TDN, and TDN−DPBA conjugate. Figure 3. Excitation and emission spectra of TDN−DPBA conjugate.

stronger. This was due to the reaction between the hydroxyl group in TDN and B in DPBA, which formed new B−O linkage. The Raman spectrum further confirmed that the linkage between TDN and DPBA was formed between O in one of hydroxyl groups of the TDN and B in DPBA. As can be seen from Figure 1, the spectrum of TDN−DPBA was not simply an overlay of individual compounds, which indicated the formation of a new conjugate. In addition, the characteristic peak of TDN had a blue shift after reaction with DPBA (Figure 1, shaded area), indicating the introduction of a new functional group with higher energy. In conclusion, Raman analysis proved the formation of a conjugate between TDN and DPBA. Mass Spectroscopy. Negative mode was used for mass spectroscopy analysis in this study. This mode of mass spectroscopy can provide high sensitivity in terms of molecular

structure.25 The molecular weights of TDN and DPBA are 360.31 and 225.09, respectively. The peak at 358.9 was the molecular weight of TDN after loss of hydrogen (TDN−) (Figure 2). The fragment at m/z 328.8 was produced by removing the OCH3 group from the A ring of TDN. m/z 523.2 was the sum of TDN− and 164.3, and 164.3 was the molecular weight of two benzene rings and one boron. This suggested the conjugate was formed by covalent bond between the −O− group of TDN and the −B− (benzene)2 part of DPBA. Moreover, m/z 523.2 showed the strongest signal, suggesting the high abundance of the conjugate. The fat m/z 687.3 was the sum of TDN− and two units of 164.3, suggesting that a small amount of conjugate was formed between one molecule of

Figure 2. Mass spectra of DPBA, TDN, and TDN−DPBA conjugate. C

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Figure 4. Cytometric analysis of Caco-2 cells treated with TDN and DPBA. The laser used for dye Alexa fluor 488-A was used to excite TDN− DPBA fluorescent conjugate: (a) control cells stained with DPBA; (b) cells treated with TDN and not stained with DPBA; (c) cells treated with TDN and stained with DPBA. All experiments were repeated three times. Representative dot plots are shown (p < 0.05).

Figure 5. Cytometric analysis of Caco-2 cells treated with TDN and DPBA. Cells were incubated with TDN for (a) 0.5 h, (b) 1 h, (c) 1.5 h, or (d) 2 h and then stained with DPBA. Left gate was constructed to include all cells that were treated with TDN and not stained with DPBA. All experiments were repeated three times. Representative dot plots are shown (p < 0.05).

TDN and two molecules of DPBA by the B−O bond. Overall, the results from mass spectroscopy analysis demonstrated that DPBA could form a conjugate with TDN through the B−O bond. Fluorescence Spectroscopy. The fluorescence property of the conjugate was characterized by florescence spectroscopy. As seen in Figure 3, the spectra showed that the conjugate had the

maximum excitation peak around a wavelength of 490 nm and an emission peak around a wavelength of 570 nm. Fluorescent Detection of TDN in Floating Cells. Direct detection of bioactive compounds in mammalian cells can enable characterization of the distribution of these compounds among different cells. Flow cytometry is widely used to detect specific physical and chemical features of individual cells.26 The D

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Figure 6. Fluorescent detection of TDN in Caco-2 cells after 2 h of incubation with TDN: (a) optical microscope image of cells treated with TDN but not stained with DPBA; (b) optical microscope image of cells treated with TDN and also stained with DPBA; (c) fluorescent microscope image of cells treated with TDN but not stained with DPBA; (d) fluorescent microscope image of cells treated with TDN and also stained with DPBA. All experiments were repeated three times. Representative images are shown.

Figure 8. Fluorescent detection of (a) TDN, (b) 5-demethylnobiletin, (c) 5,3′-didemethylnobiletin, and (d) 5,4′-didemethylnobiletin inside Caco-2 cells with fluorescent microscope. All experiments were repeated three times. Representative images are shown.

and increased the fluorescence of TDN, which can facilitate its direct detection by flow cytometry. To further demonstrate the usefulness of this detection method, we monitored the time-dependent uptake of TDN by human intestinal cancer Caco-2 cells. The Caco-2 cells were treated with TDN for different time periods, 0.5, 1, 1.5, or 2 h, and then stained with DPBA before flow cytometry analysis. As seen in Figure 5, as the incubation time increased from 0.5 to 2 h, the cell population migrated from the low fluorescence intensity side to the high fluorescence intensity side. As a result, the majority of cells migrated from the left-gated area to the right-gated area within 2 h. These results showed that the uptake of TDN by Caco-2 cells was time dependent; that is, the longer the incubation time, the higher the amount of TDN absorbed by the cells. Our results also demonstrated that this flow cytometry-based method could be used to quantify the relative abundance of TDN in different cell populations. As we know, bulk cancer cells consist of many different subpopulations that have distinct cellular properties, such as different levels of resistance against cancer treatments. One potential application of the flow cytometry-based detection method for TDN is to identify and isolate the cell populations that have high absorption rates of TDN and determine their cellular response to the TDN treatment. These subpopulations of cells may be more sensitive to TDN treatment. Furthermore, this fluorescence detection of TDN can also be combined with detection of other fluorescent markers (such as protein markers for cancer stem cells) to identify potential correlation between different cell types and cellular responses to TDN treatment. Overall, our results successfully demonstrated direct fluorescence detection of TDN by flow cytometry, and this method has high potential for multiple applications in detecting a wide range of cellular responses to TDN treatment among different subpopulations of cells.

Figure 7. Fluorescence of TDN in Caco-2 cell after incubation with TDN for different time intervals: (a) 0.5 h; (b) 1 h; (c) 1.5 h; (d) 2 h. Scale bar = 100 μm. All experiments were repeated three times. Representative images are shown.

purpose of this experiment is to demonstrate that TDN absorbed into individual human cells can be detected by a flow cytometer. When treated with TDN, human intestinal cancer cells (Caco-2) can absorb TDN. After detachment and fixation of the cells, the cells were incubated with DPBA. The cellular TDN can react with DPBA to form a fluorescent conjugate that can be detected by flow cytometry. Indeed, as shown in Figure 4, the cells treated without TDN or DPBA showed low intensity of fluorescence (Figure 4a), whereas cells treated with TDN and without reacting with DPBA showed higher fluorescence intensity (Figure 4b). This suggested that TDN itself had a certain level of fluorescence under the detection conditions. Most interestingly, as shown in Figure 4c, cells treated with TDN and stained with DPBA produced the highest fluorescence intensity compared with cells in Figure 4a,b. These results demonstrated that DPBA reacted with TDN E

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Figure 9. Fluorescence microscopic image of cross section of mouse colon: (a) colon section from mice fed diet without 5DN and not stained with DPBA; (b) colon section from mice fed 5DN and stained with DPBA; (c) colon section from mice fed diet without 5DN and stained with DPBA; (d) colon section from mice fed 5DN and not stained with DPBA. These images were taken with identical fluorescent microscopic parameters so the relative fluorescence intensity can be compared. Scale bar = 200 μm. Three sections from three different mice from each group were analyzed. (e) Detailed image of colon crypt. The picture was taken with a 63× lens. Scale bar = 20 μm. Representative images are shown.

Fluorescent Detection of PMF in Attached Cells. To further explore the application of direct fluorescence detection of TDN, in this section we investigate the possibility of direct visualization of TDN in mammalian cells using fluorescence microscope. Caco-2 cells growing on the glass slides were treated with TDN for different time periods, 0.5, 1, 1.5, and 2 h. After fixation, the cells were treated with DPBA to form fluorescence conjugate and observed under a fluorescence microscope. As seen in Figure 6, only cells treated with TDN and stained with DPBA showed fluorescence. Cells treated with TDN but were not stained with DPBA dye did not show any fluorescence when observed under a fluorescent microscope. As shown in Figure 7, the intensity of fluorescence in the cells increased as treatment time extended. In the first 0.5 h, only a small portion of TDN was taken up by the cells. The fluorescence intensity at 1 h increased by a small extent in comparison with that at 0.5 h. In contrast, the fluorescence intensity significantly increased at 1.5 h, and the intensity further increased at 2 h. The results suggested that the uptake of TDN occurs mostly after 0.5 h of incubation. It could be observed that TDN was more localized within the cell membrane region, which may be due to the high lipophilicity of TDN. This method could be further developed to provide more detailed information on the localization of TDN in the cells by utilization of a confocal fluorescence microscope. Detailed information on the localization of bioactive compounds in the cells can provide insights into their molecular mechanisms of action. Distribution of TDN in Mouse Colon Tissue. The in vivo fate and action of bioactive food compounds are of ultimate importance to realization of their health effects. We hypothesized that DPBA can be used to form a fluorescent conjugate with TDN that can be used to visualize the accumulation of TDN in the tissues of mice fed 5DN. In this section, we aim to determine the potential of direct fluorescent

detection of TDN in visualization of TDN in animal tissues. Our previous study showed that 5DN was transformed to three metabolites, those being TDN, 5,3′-didemethylnobiletin, and 5,4′-didemethylnobiletin, in mice after oral consumption.16 These metabolites could be found in the tissues of mice fed 5DN. However, to visualize TDN in mouse tissue, first we need to ensure that 5DN and other metabolites, namely, 5,3′didemethylnobiletin and 5,4′-didemethylnobiletin, would not react with DPBA to produce strong fluorescence and interfere with the fluorescence produced by the TDN conjugate. We treated Caco-2 cells with TDN, 5DN, 5,3′-didemethylnobiletin, or 5,4′-didemethylnobiletin at the same concentration for 2 h and then stained the cells with DPBA. Figure 8 shows the images of these cells under a fluorescence microscope. These indicated that only TDN produced strong fluorescence after staining with DPBA, which showed the specificity of the DPBAbased fluorescent detection on TDN. In the animal experiment, 5DN was fed to mice for a week, and then colonic tissues were collected and processed to produce sections for fluorescent detection of TDN. The results showed that colon tissues of control mice that were fed diet without 5DN showed only a very low intensity of fluorescence when stained with DPBA (Figure 9c), whereas no fluorescence was observed for tissue sections not stained with DPBA (Figure 9a). The colon tissues of mice that were fed the diet containing 5DN showed very low intensity of fluorescence when not stained with DPBA (Figure 9d), whereas these tissues showed high intensity of fluorescence when stained with DPBA. On the basis of the results from Figure 8, it can be concluded that the fluorescence in Figure 9b was likely due to the presence of TDN in the mouse colonic tissues. It can be observed that high fluorescence was widely distributed throughout the entire colon tissue, including the mucosa and muscular layer. This indicated that TDN migrated across the whole colon without high accumulation in any particular tissues. Figure 9e shows a colonic tissue section of F

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(6) (a) Chignell, C. F.; Bilski, P.; Reszka, K. J.; Motten, A. G.; Sik, R. H.; Dahl, T. A. Spectral and photochemical properties of curcumin. Photochem. Photobiol. 1994, 59 (3), 295−302. (b) Sahu, A.; Kasoju, N.; Bora, U. Fluorescence study of the curcumin−casein micelle complexation and its application as a drug nanocarrier to cancer cells. Biomacromolecules 2008, 9 (10), 2905−2912. (7) Kunwar, A.; Barik, A.; Pandey, R.; Priyadarsini, K. I. Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760 (10), 1513−1520. (8) Lin, C.-C.; Lin, H.-Y.; Chi, M.-H.; Shen, C.-M.; Chen, H.-W.; Yang, W.-J.; Lee, M.-H. Preparation of curcumin microemulsions with food-grade soybean oil/lecithin and their cytotoxicity on the HepG2 cell line. Food Chem. 2014, 154, 282−290. (9) Tripodo, G.; Chlapanidas, T.; Perteghella, S.; Vigani, B.; Mandracchia, D.; Trapani, A.; Galuzzi, M.; Tosca, M. C.; Antonioli, B.; Gaetani, P.; Marazzi, M.; Torre, M. L. Mesenchymal stromal cells loading curcumin-INVITE-micelles: a drug delivery system for neurodegenerative diseases. Colloids Surf., B 2015, 125, 300−308. (10) Kunwar, A.; Barik, A.; Mishra, B.; Rathinasamy, K.; Pandey, R.; Priyadarsini, K. I. Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells. Biochim. Biophys. Acta, Gen. Subj. 2008, 1780 (4), 673−679. (11) Reix, N.; Parat, A.; Seyfritz, E.; Van Der Werf, R.; Epure, V.; Ebel, N.; Danicher, L.; Marchioni, E.; Jeandidier, N.; Pinget, M.; Frère, Y.; Sigrist, S. In vitro uptake evaluation in Caco-2 cells and in vivo results in diabetic rats of insulin-loaded PLGA nanoparticles. Int. J. Pharm. 2012, 437 (1−2), 213−220. (12) Ling, G.; Zhang, P.; Zhang, W.; Sun, J.; Meng, X.; Qin, Y.; Deng, Y.; He, Z. Development of novel self-assembled DS-PLGA hybrid nanoparticles for improving oral bioavailability of vincristine sulfate by P-gp inhibition. J. Controlled Release 2010, 148 (2), 241−248. (13) Sahoo, S. K.; Labhasetwar, V. Enhanced antiproliferative activity of transferrin-conjugated paclitaxel-loaded nanoparticles is mediated via sustained intracellular drug retention. Mol. Pharmaceutics 2005, 2 (5), 373−383. (14) Szwed, M.; Matusiak, A.; Laroche-Clary, A.; Robert, J.; Marszalek, I.; Jozwiak, Z. Transferrin as a drug carrier: cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells. Toxicol. In Vitro 2014, 28 (2), 187−197. (15) Ernst, I. M. A.; Wagner, A. E.; Lipinski, S.; Skrbek, S.; Ruefer, C. E.; Desel, C.; Rimbach, G. Cellular uptake, stability, visualization by ‘Naturstoff reagent A’, and multidrug resistance protein 1 generegulatory activity of cyanidin in human keratinocytes. Pharmacol. Res. 2010, 61 (3), 253−258. (16) Longin, A.; Souchier, C.; Ffrench, M.; Bryon, P. A. Comparison of anti-fading agents used in fluorescence microscopy-image-analysis and laser confocal microscopy study. J. Histochem. Cytochem. 1993, 41 (12), 1833−1840. (17) Qiu, P.; Dong, P.; Guan, H.; Li, S.; Ho, C.-T.; Pan, M.-H.; McClements, D. J.; Xiao, H. Inhibitory effects of 5-hydroxy polymethoxyflavones on colon cancer cells. Mol. Nutr. Food Res. 2010, 54 (S2), S244−S252. (18) Zheng, J.; Song, M.; Dong, P.; Qiu, P.; Guo, S.; Zhong, Z.; Li, S.; Ho, C. T.; Xiao, H. Identification of novel bioactive metabolites of 5-demethylnobiletin in mice. Mol. Nutr. Food Res. 2013, 57 (11), 1999−2007. (19) (a) Buer, C. S.; Imin, N.; Djordjevic, M. A. Flavonoids: new roles for old molecules. J. Integr. Plant Biol. 2010, 52 (1), 98−111. (b) Buer, C. S.; Muday, G. K.; Djordjevic, M. A. Implications of longdistance flavonoid movement in Arabidopsis thaliana. Plant Signaling Behav. 2008, 3 (6), 415−417. (c) Gavin, N. M.; Durako, M. J. Localization and antioxidant capacity of flavonoids in Halophila johnsonii in response to experimental light and salinity variation. J. Exp. Mar. Biol. Ecol. 2012, 416−417, 32−40. (d) Peer, W. A.; Brown, D. E.; Tague, B. W.; Muday, G. K.; Taiz, L.; Murphy, A. S. Flavonoid accumulation patterns of transparent testa mutants of arabidopsis. Plant Physiol. 2001, 126 (2), 536−548.

mice fed 5DN after being stained with DPBA at higher magnification. It can be observed that higher fluorescence intensity was found in the cell membrane region of epithelial cells. This may be due to the lipophilicity of TDN associated with multiple methoxyl groups in its chemical structure. On the basis of our results, it can be expected that the fluorescence method could be used to visualize TDN in other vital organs such as the liver and brain. Moreover, this method can be combined with other fluorescent dyes such as alcian blue for goblet cells,27 cytokeratin A1/A3 for cytokeratin, and Hoechst 33342 for nuclear28 to provide more detailed information on the localization of TDN in cell and tissue samples. In summary, a novel fluorescence-based method for visualization of TDN distribution in cultured cells and mouse colonic tissue has been successfully developed and validated in this study. This method does not require fluorescent labeling of TDN before the compound is applied to the biological systems, which not only eliminates the potential toxicity caused by fluorophore but also produces no interference with the uptake and distribution of the compound itself by the cells. This method could be used to analyze individual floating cells as well as adherent cells. In addition, when using this method, fluorescence-activated cell sorting could be utilized to further distinguish and enrich cells with different responses to TDN; hence, other biomarkers of interest could be studied within different cell populations. This method will be a useful tool to facilitate the better understanding of molecular mechanisms of PMFs.

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AUTHOR INFORMATION

Corresponding Author

*(H.X.) Phone: (413) 545-2281. Fax: (413) 545-1262. E-mail: [email protected]. Funding

This material was partly based upon work supported by the USDA, NRI Grants (2011-67021 and 2014-67021), and the N a t i o na l N a t u r a l Sc i e n c e F o u nd a t i o n o f C h i n a (NSFC31428017). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.5b04484 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b04484 J. Agric. Food Chem. XXXX, XXX, XXX−XXX