Xanthohumol Uptake and Intracellular Kinetics in Hepatocytes

ARTICLE pubs.acs.org/JAFC

Xanthohumol Uptake and Intracellular Kinetics in Hepatocytes, Hepatic Stellate Cells, and Intestinal Cells Horst Wolff,† Magdalena Motyl,§ Claus Hellerbrand,# J€org Heilmann,§ and Birgit Kraus*,§ †

Institute of Virology, Helmholtz Zentrum M€unchen
German Research Center for Environmental Health, Ingolst€adter Landstrasse 1, Neuherberg, Germany § Institute of Pharmaceutical Biology, University of Regensburg, Universit€atsstrasse 31, Regensburg, Germany # Department of Internal Medicine I, University Hospital, Regensburg, Germany

bS Supporting Information ABSTRACT: Xanthohumol (XN) is the major prenylated chalcone of hops and hence an ingredient of beer. Despite many advances in understanding of the pharmacology of XN, one largely unresolved issue is its low bioavailability in the human organism. Also, not much is known about its actual concentrations and pharmacokinetics in liver and intestinal cells. Therefore, the uptake, intracellular distribution, and kinetics of XN were studied in various cell types, namely, hepatocellular carcinoma cells (HuH-7), hepatic stellate cells (HSC), primary cultured hepatocytes, and colorectal adenocarcinoma cells (Caco-2). Fluorescent microscopy allowed for the first time visualization and tracing of the uptake and intracellular distribution of XN. A rapid accumulation of XN concentrations that were up to >60-fold higher than the concentration present in the ambient culture medium was observed. Fluorescence recovery after photobleaching experiments revealed that most XN molecules are bound to cellular proteins, which may alter properties of cellular factors. KEYWORDS: FRAP, hepatic stellate cells, intracellular concentration, liver cells, xanthohumol

’ INTRODUCTION Xanthohumol (XN; 20 ,40 ,4-trihydroxy-60 -methoxy-30 -prenylchalcone) is the most abundant chalcone derivative in the female inflorescences of hop (Humulus lupulus L.). It has been characterized as an anticarcinogenic,1
4 chemopreventive,2 antiinflammatory 2 and antiangiogenic5 agent. In addition, a broad anti-infective potential was described,6 and even anti-HIV-1 activity was attributed to XN.7 Recent progress in elucidating the underlying mechanisms led to a variety of modes of action for XN, such as inhibition of nuclear factor kB,8,9 suppression of monocyte chemoattractant protein-1 and tumor necrosis factorα,10 up-regulation of heme oxygenase-1,11 and influence on the binding behavior and lateral mobility of GABAA receptors,12 to mention only a few. However, in many cases the precise mechanisms are not clarified yet. Despite the discovery of so many favorable biological properties and the great progress in understanding how XN acts, the poor bioavailability,13 especially after oral uptake, is still not well understood and poses problems. Indeed, sufficient bioavailability is in general a prerequisite for a controlled and efficient therapeutic application. Furthermore, bioavailability, metabolism, and cellular reservoirs in humans need to be understood to accurately assess the chemopreventive impact of XN or, in a more common approach, the biological activity of polyphenols present in food. Metabolism of XN by microorganisms of the gut microflora may be one mechanism that influences bioavailability in general,14,15 and passage through intestinal cells as well as uptake into and the fate inside cells of the liver is another. The chemopreventive properties of XN16
18 are very attractive, especially for therapeutic perspectives regarding the liver r 2011 American Chemical Society

and intestine.19
22 In this study, we therefore aimed at determining the cellular pharmacokinetics of XN in different intestinal and hepatic cell culture models. The goals were to quantify uptake, intracellular distribution, and fate of XN in intestinal and liver cells to resolve XN bioavailability issues.

’ MATERIALS AND METHODS Cells and Cell Culture. HuH-7 hepatocellular carcinoma cells23 were obtained from the Japanese Collection of Research Bioresources (JCRB), and Caco-2 colorectal adenocarcinoma cells24 were from ATCC. The generation of immortal activated human hepatic stellate cells (HSC) has been described before.25 HuH-7 and HSC were kept under standard cell culture conditions using Dulbecco’s Modified Eagle Medium (Invitrogen, Karlsruhe, Germany) with 10% heat-inactivated fetal calf serum (FCS) and 2 mM glutamine. Caco-2 cells were grown in Minimum Essential Medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 1 mM sodium pyruvate, and 1% nonessential amino acids (Biochrom AG, Berlin, Germany). Human liver tissue samples were obtained and experimental procedures were performed according to the guidelines of the charitable state controlled foundation HTCR (Human Tissue and Cell Research), with the informed patient’s consent. Isolation of primary human hepatocytes was carried out at the Center of Liver Cell Research, University Hospital of Regensburg. Received: September 18, 2011 Revised: November 7, 2011 Accepted: November 16, 2011 Published: November 16, 2011 12893

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Journal of Agricultural and Food Chemistry Quantification of XN Uptake via HPLC. Cells were incubated for 3 h with 10 μM XN (98.0% determined with HPLC, Nookandeh Institut, Hamburg, Germany) in culture medium. Subsequently, cells were washed with phosphate-buffered saline (PBS), homogenized with glass beads (0.75
1.0 mm), and extracted with an equivalent amount (v/v) of acetone. Prior to HPLC analysis, supernatants were further purified by centrifugation (20000g; 4 °C; 20 min) using NanoSep centrifugal devices (catalog no. 5168502, VWR, Darmstadt, Germany). For quantification of XN we used a LaChrom Elite system (Hitachi) with a Purospher STAR Hibar RP-18 (5 μm, 250  4 mm) column equipped with a precolumn (LiChroCART 4-4). The mobile phase consisted of water with 0.1% formic acid and pure acetonitrile. A flow rate of 1 mL/min and a column temperature of 30 °C were used. The gradient was as follows: 0
9 min, 35
65% acetonitrile; 9
11 min, 65% acetonitrile isocratic; followed by washing. A calibration curve was generated with XN at 368 nm over a concentration range of 0.1
100.0 μM. Typical retention time for XN was 11.0 min. The amount of XN per cell was calculated by dividing the total XN by the cell number. The intracellular concentrations attained were estimated by combining the cellular amount of XN with the averaged cell volume determined by fluorescence microscopy (see Determination of Cell Volumes). Uptake of XN into HuH-7, HSC, Caco-2 Cells and Primary Hepatocytes. The uptake kinetics of XN was determined by fluorescence imaging, exploiting the fluorescent properties of this compound. XN fluorescence properties were determined with an LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). For this, 5 μL of a 10 mM XN solution was placed on top of a coverslip and excited with a 458 nm laser, and the resulting emission fingerprint was recorded (Supporting Information, Figure 1). Subsequently, it was confirmed that XN fluorescence characteristics (especially the main excitation and emission ranges) do not change greatly inside mammalian cells. The obtained information about the spectral properties of XN provided the basis for the selection of appropriate filters for imaging of living cells in uptake experiments. Upon excitation around 460 nm (with a bandwidth of 80 nm), still a major emission signal from XN was detectable at wavelengths >570 nm, making it possible to design filters that avoid cellular autofluorescence in the blue and green range (570 nm, making it possible to design filters that avoid cellular autofluorescence in the blue and green range (90% of nuclear fluorescence has recovered, indicating nucleocytoplasmic transfer of free or bound XN. For HuH-7 cells three individual experiments have been carried out, and for Caco-2 and HSC one experiment has been carried out to confirm the findings in HuH-7. Please also see the video available as Supporting Information.

amounts in the different cell types, already suspected because of differences in XN fluorescence intensity. XN amounts in Caco-2 cells after 1 h of incubation with XN have been quantified previously by biochemical methods and HPLC13 and are in accordance with our data after 3 h of incubation with XN (Table 1). Notably, the combination of HPLC quantification data with the different cell volumes determined by fluorescence microscopy enabled us to estimate actual XN concentrations inside the different cell types. Intracellular concentrations were, depending on the cell type, up to >60-fold higher than the concentration present in the ambient culture medium. This indicates a strong accumulation of XN inside the cells, at which HSC and Caco-2 cells showed the highest concentrations after 3 h. There are a number of possible reasons why the maximum XN concentration in the HuH-7 liver cell line is much lower than in primary hepatocytes and this in turn lower than in Caco-2 intestinal cells and HSC. Enhanced export by ABC-transporters, decreased uptake, or increased metabolism all would be able to explain the observation of lower XN concentration, but will each have different implications. In preliminary experiments, we found no effect of a co-incubation with verapamil on the final accumulation of XN in HuH-7 cells (data not shown). Although there are a number of other transporters, these preliminary data may point to a negligible effect of an enhanced XN efflux mediated by the verapamil-sensitive ABC-transporter, P-glycoprotein, in these cells. Notably, it has also been described that XN alone is already an inhibitor of P-glycoprotein expression33 and function,34 potentially masking less potent effects by additional inhibitors. Further experiments will be conducted to exclude contributions of other transporters, as it was reported that, at least in Caco-2

Figure 6. Distribution of XN fluorescence revealed by confocal imaging. (A
D) Cells were incubated with 10 μM XN for 60 min and then imaged in medium without XN. (E) HSC were incubated with 100 μM XN for 60 min. Scale bars = 50 μm.

cells, they might contribute to XN clearance from the cells.13 However, we have found that the behavior of Caco-2 cells in terms of XN uptake markedly deviates from the other three cell types, as the plateau of XN fluorescence at a given XN concentration in the medium is reached later than in all other cells (Figures 1 and 2). It is also likely that metabolism and efflux play different roles in liver and intestinal cell lines. Pohjala et al. have reported that HuH-7 cells show approximately 2-fold higher IC50 values for polymyxin B and camptothecin than Caco-2 cells, probably due to a higher rate of oxidative metabolism.35 Cell- and tissue-specific differences in the metabolism of periodically ingested polyphenolic compounds seem to be an important topic to explain the effects of bioactive, dietary secondary compounds. Profiling of the intracellular XN metabolites in different cell lines is currently under investigation via UPLC-MS/MS. Indeed, in HuH-7 cells we have observed a slow but constant decrease of cellular XN fluorescence over 24 h if XN was removed from the culture medium (data not shown). This provides evidence that if XN is present in the surrounding environment, the uptake of XN into cells is simply more efficient than the combined effects of metabolism and removal of XN. When using a concentration of 10 μM XN in culture medium, intracellular XN fluorescence exhibited neither a prominent 12898

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Figure 7. Intracellular fate of XN. XN enters the cell and is bound to cellular proteins, by forming a thiol adduct. Whereas both free and bound XN can be metabolized and degraded, only the free proportion of XN can be transported efficiently out of the cell, but not the XN
protein complexes. XN
protein complexes consisting of XN and factors with nuclear functions can be imported into the nucleus and may there exhibit lower or higher activity or changed specificity.

staining of cell or nuclear membrane nor a marked cytoskeletal localization pattern, assuming that XN is not preferably binding to specific cellular structures or organelles. FRAP experiments revealed high mobile fractions with values for cytoplasmic XN of around 60% and even higher mobile fractions between 90 and 92% for nucleoplasmic XN for all cell types. Inside living cells XN behaves as if it were a much heavier molecule, with a t1/2 that is comparable to or even longer than that of GFP. A plausible explanation for this fact would be that XN binds to cytosolic or nucleosolic cellular factors with a mean size of GFP (27 kDa) or larger, by either noncovalent or covalent interactions. Pang et al. also reported on binding of XN to cytosolic proteins larger than 10 kDa, which was assessed by an affinity binding assay.13 As XN acts as a Michael acceptor, one mechanism could be the formation of a thiol adduct between XN and a sulfhydryl group of cysteine residues of cellular proteins9 (Figure 7). The specificity of binding of XN to proteins by a Michael-type addition is probably very low as many cellular proteins contain accessible cysteine residues. However, and most notably, important transcription factors, such as nuclear factor kB, possess cysteine residues that are involved in mediating the interaction with target DNA. Of note, FRAP experiments revealed a prominent exchange between cytosolic and nucleoplasmic XN. This means that XN is able to take effect in the whole cell, including the nucleus. This may provide explanations for many of the biological functions that XN exhibits in terms of cellular signaling and gene expression. It can be speculated that at least a certain percentage of the total XN that is imported into the nucleus from the cytoplasmic compartment is bound to transcription factors and proteins involved in cellular signaling. Examples for such interactions are the binding of XN to NF-kB9 and the binding of XN to Keap1,16 which both have been shown to have a direct influence on transcriptional regulation by these factors. In fact, the observation that the mobile fraction of XN is higher in the nucleus than in the cytoplasm supports the hypothesis that most of the XN that gets imported into the nucleus is already bound to a mobile protein and can, therefore, not bind to

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immobile nuclear structures, such as chromatin-bound histones or nucleolar proteins. Only when a great excess of XN (in our case, 100 μM) is added to the culture medium does this stepwise process of XN saturation in the cytoplasm and later import into the nucleus in its protein-bound form not seem to function anymore, leading to a brighter nucleus in general and nucleoli in particular. Our data provide evidence that the binding of XN primarily to cytosolic proteins leads to the high accumulation of XN in intestinal and liver cells. This may be an important clue to understanding the low abundance of XN in other tissues and body fluids. XN is trapped at high concentrations inside the cells, largely bound to cellular proteins, until it is degraded by the cellular metabolism. To conclude, our findings shed light on what likely can happen to XN as soon as it enters a number of cell types after oral uptake of XN-containing food. The described specific characteristic of XN very likely contributes to the poor oral bioavailability observed previously in vivo and limits its use and importance as a therapeutic, but also as a toxicant, in distant parts and tissues of the body. In addition, the here-presented techniques and methods open possibilities to further investigate XN and its derivatives in terms of their cellular benchmarks, such as maximum amount, concentration, or uptake rate. Derivatives of XN and other prenylated chalcones already exist.36,37 Due to different substituents and stereochemistry they will vary in their potential Michael-acceptor activity and their cellular behavior. Some of these derivatives may possess favorable properties and could develop into more powerful chemopreventive agents than XN.38,39 These, and newly generated ones, could be used to investigate their biological profile in respect to influencing the activity of a variety of important transcription factors and signal transducers. Of special interest would be the affinity of XN for binding to serum proteins, such as albumin. It is likely that in a living organism, specific serum constituents also play an important role in modulating the bioavailability and bioactivity of XN.

’ ASSOCIATED CONTENT

bS

Supporting Information. Further information (Figures 1
4 and video) on XN fluorescence properties, cell volume determination, and FRAP experiments. This information is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Postal address: Institute of Pharmaceutical Biology, University of Regensburg, Universit€atsstrasse 31, D-93053 Regensburg, Germany. Phone: +49 (0)941 943-4494. Fax: +49 (0)941 9434990. E-mail: [email protected]. Funding Sources

The study was supported by the charitable state controlled foundation HTCR supplying human tissue for research purposes. We thank the Hopfenverwertungsgesellschaft, Hallertau, for financial support.

’ ACKNOWLEDGMENT We thank PD Dr. Thomas Weiss, Center for Liver Cell Research, Department of Surgery, University of Regensburg, Germany, for providing primary hepatocytes 12899

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