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Carcinogenesis Advance Access originally published online on April 18, 2006
Carcinogenesis 2006 27(10):1991-2000; doi:10.1093/carcin/bgl046
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Hypericum sampsonii induces apoptosis and nuclear export of retinoid X receptor-alpha

Jin-Zhang Zeng1,*,{dagger}, De-Fu Sun1,{dagger}, Li Wang1, Xihua Cao3, Jian-Bin Qi2, Ting Yang1, Chang-Qi Hu2, Wen Liu3 and Xiao-Kun Zhang1,3

1 Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Shanghai, China
2 School of Pharmacy, Medical Center of Fudan University Shanghai, China
3 Cancer Center, Burnham Institute for Medical Research La Jolla, CA, USA

*To whom correspondence should be addressed at: The Samost Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 500 Caobao Road, Shanghai 200233, China. Tel: +86 21 54481877; Fax: +86 21 54971085; Email: jzzeng{at}sibs.ac.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Natural products derived from plants provide a rich source for development of new anticancer drugs. Recent studies suggest that modulation of subcellular localization of retinoid X receptor-alpha (RXR{alpha}) represents a potential approach for inducing cancer cell apoptosis. In this study, we screened a herbal library for inducing translocation of RXR{alpha} from the nucleus to the cytoplasm. Our results revealed that the extract of Hypericum sampsonii, a member of the genus Hypericum, had remarkable effect on RXR{alpha} subcellular localization in various cancer cells. Treatment of NIH-H460 human lung cancer cells with H.sampsonii extract resulted in relocalization of RXR{alpha} from the nucleus to the cytoplasm. Cytoplasmic RXR{alpha} induced by H.sampsonii was associated with mitochondria, accompanied with cytochrome c release and apoptosis. H.sampsonii extract effectively inhibited the growth of various cancer cell lines, including NIH-H460 lung cancer, MGC-803 stomach cancer and SMMC7721 liver cancer cells. The growth inhibitory effect of H.sampsonii extract depended on levels of RXR{alpha}, as it failed to inhibit the growth of CV-1 cells lacking detectable RXR{alpha}, whereas transfection of RXR{alpha} into CV-1 cells restored its apoptotic response to H.sampsonii. Furthermore, the apoptotic effect of H.sampsonii was significantly enhanced when RXR{alpha} was overexpressed in NIH-H460 cells. Together, our results demonstrate that H.sampsonii contains ingredient(s) that induce apoptosis of cancer cells by modulating subcellular localization of RXR{alpha}.

Abbreviations: CAT, chloramphenicol acetyltransferase; DAPI, 4'6'-diamidino-2-phenylindole; EtOH, ethanol; GFP, green fluorescence protein; Hsp60, heat shock protein 60; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NES, nuclear export sequence; NRs, Nuclear receptors; 9-cis-RA, 9-cis-retinoic acid; PARP, poly (ADP-ribose) polymerase; PBS, phosphate-buffered saline; RXR, retinoid X receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Herbal medications have been widely practiced for centuries by various cultures throughout history. In China, the prevalence of traditional Chinese medicine (TCM) can be dated back to two thousand years ago. Specific herbal extracts and their combinations have been devised to treat specific diseases including cancers (1,2). On the basis of well-documented efficacy in clinic, natural products of plant provide excellent and reliable sources for the development of new drugs (1–3). In Western medicine, one of the challenges in searching for an effective cancer treatment is that in vitro activity does not always lead to human efficacy. In contrast, TCM, which is effective in humans, is often without a known molecular target. Recent studies have provided various screening approaches for identifying novel leads from herbal extracts for drug discovery (4). Among them, mechanism-based screening is one of the most valuable methods, as it offers opportunity of optimizing the leads (5,6).

Nuclear receptors (NRs) represent the largest family of eukaryotic transcriptional factors, which plays a critical role in the regulation of cell growth, proliferation and differentiation, metabolism, immune response and apoptosis (7–11). They are activated by the binding of ligands such as vitamins, steroid hormones and fatty acids (7,8,12,13). Ligand-binding promotes a conformational change of the ligand-binding domain (LBD) that affects dimerization, binding of accessory proteins and cross-talk with other signaling pathways (7,8,14). NRs are frequent biological targets of active compounds contained in herbal remedies (5,9–11). Several diseases related to malfunction of NRs, such as cancers, osteoporosis, diabetes and obesity, are currently treated with NR-targeted drugs (10,11).

Among NRs, retinoid X receptor (RXR) subtypes ({alpha}, ß and {gamma}) are unique in both structure and diametric functions (12,13,15–19). They heterodimerize with many members of the NR superfamily, including retinoic acid receptor (RAR), vitamin D receptor, peroxisome proliferator-activated receptor and thyroid hormone receptor, as well as several orphan receptors (12,13,15–19). RXRs, therefore, play an essential role in the regulation of multiple nuclear hormone-signaling pathways. Genetic disruption of RXR{alpha} targeted at the prostatic epithelium results in intraepithelial neoplasia (20), whereas targeted disruption of RXR{alpha} in the skin leads to various skin dysfunctions (21,22). Diminished RXR{alpha} protein expression is frequently observed in cancer cells, suggesting its role in the development of human cancer (23). Owing to the promiscuity of the RXR ligand-binding pocket (LBP) (24), a number of natural and synthetic compounds with diverse structures, such as 9-cis-retinoic acid (9-cis-RA) (25), dietary fatty acids (26–28), Targretin/bexarotene (29), phytanic acid (30,31) and non-steroidal anti-inflammatory drug (NSAID)—R-etodolac (32), have been shown to bind RXRs and act as RXR ligands. Some of them are used or are being evaluated in various models for the prevention and treatment of cancers and diseases.

The mechanisms by which RXRs exert their biological effects have been the subject of intensive study. RXRs bind specific DNA response elements either as heterodimers or homodimers to positively or negatively regulate transcription of target genes (12,13,15–19). On binding ligands, the receptors undergo conformational changes to release corepressors and recruit coactivators, permitting the multiprotein transcriptional machinery to initiate transcription (7,8,14). Recent studies have revealed an RXR non-genotropic signal transduction pathway, which appears to play a role in development, differentiation and apoptosis. Like many nuclear proteins, RXR shuttles between the cytoplasm and the nucleus (33,34). Cytoplasmic localization of RXR may play a role in postnatal testicular development (35,36). In response to nerve growth factor (NGF) treatment in PC12 pheochromocytoma cells, RXR translocates from the nucleus to the cytoplasm, resulting in co-migration of orphan NR NGFI-B (also known as Nur77 and TR3) and differentiation (37), whereas RXR co-migrates with thyroid hormone receptor to mitochondria to regulate mitochondrial transcription (38). Similarly, translocation of RXR{alpha}/Nur77 heterodimer from the nucleus to the cytoplasm (34,39,40) leads to apoptosis of cancer cells, which is mediated by mitochondrial targeting of RXR{alpha}/Nur77 heterodimer and their regulation of Bcl-2 activity (39,41). Such a translocation of RXR{alpha}/Nur77 heterodimer between the nucleus and the cytoplasm can be regulated by RXR{alpha} ligand (34,39) and epidermal growth factor (42).

RXR{alpha} translocation to the cytoplasm is regulated by its dimerization and ligand binding (39). Previous studies (39) demonstrated that certain RXR{alpha} transcription agonists, such as 9-cis-RA, inhibited the RXR{alpha} translocation. However, RXR{alpha} ligands that induce the translocation remain to be identified. In this study, we hypothesized that certain Chinese herbs could inhibit cancer cell growth through their modulation of RXR{alpha} subcellular localization. After screening >500 kinds of crude extracts from a herbal library, we found that extract of Hypericum sampsonii (also referred to as Yuan Bao Cao in China) (43–45) contains active components that induce RXR{alpha} nuclear export and apoptosis in cancer cells. H.sampsoniiis a member of the genus Hypericum, which also includes Hypericum perforatum (commonly known as St John's wort). St John's wort is widely used for the treatment of depression and a range of other ailments, including bacterial and viral infection, peptic ulcers and inflammation, burns and skin disease (46,47). Lipophilic extracts of St John's wort also show anti-neoplastic activity in vitro and in vivo (48,49). H.sampsonii has been traditionally used in China for the treatment of hematemesis, epistaxis, menstrual irregularity, external traumatic injury and swellings (44). It also exhibits anticancer activity (44,45), and has been used as a promising anticancer herb in Taiwan (44). Our present results demonstrated that H.sampsonii extract was able to induce the translocation of RXR{alpha} from the nucleus to the cytoplasm where it targeted mitochondria. The effect of H.sampsonii on RXR{alpha} translocation was associated with extensive RXR{alpha}-dependent apoptosis of cancer cells. This finding demonstrates for the first time that a Chinese herb exerts its anticancer activity through its modulation of RXR{alpha} subcellular localization. Our results provide a mechanistic rationale for the identification of an active component(s) in H.sampsonii and the development of RXR{alpha}-based herbal medicine.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
Lipofectamine PLUS reagents from Invitrogen (Carlsbad, CA), enhanced chemiluminescence (ECL) reagents and anti-mouse IgG conjugated with Cy3 from Amersham Pharmacia Biotech (Piscataway, NJ), polyclonal anti-RXR{alpha} (D20), anti-Hsp60, anti-PARP (sc-7150), anti-Flag (mouse), and goat anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal anti-ß-actin antibody and fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG from Sigma (St Louis, MO), monoclonal anti-cytochrome c antibody from Pharmingen (San Diego, CA) were used in this study. All other chemicals used were commercial products of analytical grade obtained from Sigma.

Herb material and extraction
H.sampsonii was collected from Nan-ning, Guangxi Province, China, in June, 2004, and authenticated by Professor Changqi Hu (School of Pharmacy, Medical Center of Fudan University, Shanghai, China). The dried whole herb was finely ground and macerated for 5 h twice at 50°C with a 5-fold amount of 95% ethanol (EtOH). The combined EtOH extracts were evaporated under reduced pressure to give a residue. Usually, extracting by this way generated some 5% solid product. The residue was suspended in water and extracted successively with petroleum ether, chloroform and ethyl acetate. The chloroform partition was purified by flash chromatography on a silica gel using EtOH gradient (0–100%). The elute was concentrated to dryness. Water extracts (control) were obtained by refluxing at 100°C for 3 h and were freeze-dried. All dried extracts were stored at –80°C until use.

Cell lines and culture
NIH-H460 lung cancer cells [American Type Culture Collection (ATCC)] were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), SMMC-7721 liver cancer (ATCC) and MGC-803 gastric cancer cells (41), and CV-1 African green monkey kidney cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. All cultured cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Cell lines were sub-cultured according to their individual growth profiles in order to ensure exponential growth throughout the experiments.

MTT assay
Cells were seeded in a 96-well plate and treated the following day with H.sampsonii extracts dissolved in 0.2% dimethyl sulfoxide (DMSO) at serial concentrations (5, 10, 20, 40, 60 and 80 µg/ml). Control group received 0.2% DMSO. After 3 days, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (4.14 mg/ml) was added to each well and incubated at 37°C for 4 h. The formed formazan crystals were dissolved in 100 µl DMSO for 10 min with shaking. Each plate was read immediately on a microplate reader (Bio-Rad, USA) at a wavelength of 490 nm. Three independent experiments were performed in triplicate. Results are expressed as the concentration of the extract required to inhibit cellular growth 50% (IC50) ± standard deviation (SD).

Apoptosis assays
Cells cultured in six-well plates in 0.5% FBS medium were incubated with vehicle or different amounts of extract of H.sampsonii for 24 h. After incubation, detached and attached cells were collected and centrifuged. The cells were resuspended in phosphate-buffered saline (PBS) containing 50 µg/ml 4'6'-diamidino-2-phenylindole (DAPI) and 100 µg/ml DNase-free RNase A, and incubated for 20 min at 37°C with protection from light. Fluorescence microscope (Olympus) was used to visualize the nuclei. Apoptotic cells were identified as typical morphology of shrinkage of the cytoplasm, membrane blebbing and nuclear condensation and/or fragmentation (24,41). At least 1000 cells from >10 random microscopic fields were counted by two investigators.

Cell lysis and fraction
Control and treated cells were rinsed with ice-cold PBS and harvested in a lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 1 mg/ml aprotinin, 2 µg/ml pepstatin A and 2 µg/ml leupeptin] for 20 min on ice. The whole cell lysates were purified by centrifuging at 12 000x g for 10 min. Subcellular fractionation was performed as described with minor modifications (39). Briefly, cells were suspended for 5 min on ice in 0.5 ml of hypotonic buffer [250 mM sucrose, 20 mM HEPES–KOH (pH 7.4), 10 mM KCl, 10 mM MgCl2, 0.5 mM EGTA, 1.5 mM EDTA (pH 8.0), 1 mM dithiothreitol (DTT)] with proteinase inhibitors and homogenized. The cell extracts were centrifuged at 800x g for 10 min. The pellet containing nuclei was resuspended in 200 µl of 1.6 M sucrose in hypotonic buffer plus protease inhibitors and laid over 1 ml of 2.0 M sucrose in the same buffer and then centrifuged at 150 000x g for 90 min at 4°C to obtain the nuclear fraction. The supernatant was purified by centrifuging at 10 000x g for 30 min at 4°C to obtain cytosolic fractions. Nuclear fractions were resuspended in 100 µl of lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100 and 5 mM EDTA (pH 8.0)] with a cocktail of proteinase inhibitors. Protein concentrations were determined using the Bradford method according to the manufacturer's instruction (Bio-Rad).

Western blotting
The total lysates or fractions were electrophoresed on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. Blots were blocked in 5% non-fat milk in TBST [20 mM Tris–HCl (pH 7.4), 137 mM NaCl and 0.05% Tween-20] overnight at 4°C. After two additional washes in TBST, the blots were incubated with various primary antibodies overnight at 4°C, followed by peroxidase-conjugated secondary antibody for another 1 h at room temperature (24,41). The blots were developed by using ECL system according to proposed protocol. The blots were reprobed with anti-ß-actin antibody to confirm equal loading of proteins in each lane.

Immunohistochemistry
The immunostaining method was described previously (24,39,41) and was adopted with modification. For initial herbal screening, cells were cultured on 96-well plates or glass slides in 24-well plates. Control and treated cells were fixed with cold 4% polyformaldehyde in PBS for 30 min. The fixed cells were washed twice in PBS, and then incubated in cold permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 10 min. Cells were stained with polyclonal anti-RXR{alpha} antibody (1 : 500) followed by anti-goat IgG conjugated with Cy3 (1 : 1000), and then re-stained with DAPI. For confocal study, NIH-H460 cells were co-stained with anti-RXR{alpha} antibody and anti-Hsp60 goat IgG (1 : 500) to determine whether RXR{alpha} targeted at mitochondria. FITC-labeled anti-rabbit IgG (1 : 500) and anti-goat IgG conjugated with Cy3 (1 : 1000) were used to recognize RXR{alpha} and Hsp60, respectively. To determine whether RXR{alpha} nuclear export was associated with cytochrome c release and apoptosis, cells were immunostained with anti-cytochrome c (mouse) and anti-RXR{alpha} (rabbit) followed by FITC-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG. Cells were stained with DAPI. Alternatively, CV-1 cells transfected with or without Flag-tagged RXR{alpha} were exposed to H.sampsonii. Cells were then immunostained with anti-Flag (mouse) followed by Cy3-conjugated anti-mouse IgG and co-stained with DAPI. The images were taken under a fluorescent microscopy (Olympus) or an LSM-510 confocal laser scanning microscope system (Carl Zeiss, Oberkochen, Germany).

Reporter gene assay
CV-1 cells were seeded at a concentration of 5.0 x 104 cells per well in 48-well plates. Lipofectamine transfection reagent was used for transient transfection according to manufacture's instruction. Reporter constructs, (TREpal)2-tk-CAT and ßRARE-tk-CAT, have been described previously (15,16). To assay RXR{alpha} homodimer transactivation, (TREpal)2-tk-CAT (50 ng) and RXR{alpha} expression vector (10 ng) were co-transfected into cells. To assay RXR{alpha}/Nur77 heterodimer transactivation, ßRARE-tk-CAT (100 ng), RXR{alpha}(5 ng) and Nur77 (20 ng) were co-transfected. Each transfection also contained 50 ng pCMV-ß-gal plasmid along with carrier DNA pBluescript to give 1000 ng of total DNA/well. Twenty-four hours after transfection, cells were treated with SR11237 and/or various concentrations of H.sampsonii extracts for another 20 h. Cells were then harvested for measuring ß-galactosidase activity and CAT activity as described (15,16). Relative CAT activity was normalized to ß-galactosidase value to correct transfection efficiency. Values are the means ± SD of three independent experiments.

Statistical analysis
Statistical significance of differences between groups was analyzed by using the Student's t-test. Values of P< 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
H.sampsonii induces RXR{alpha} nuclear export
It has been demonstrated that translocation of RXR{alpha} from the nucleus to the cytoplasm represents a unique pathway in the inhibition of cell growth in various cancer cells (34,39,40,42). To identify RXR{alpha} candidate ligands that induce RXR{alpha} nuclear export and apoptosis, we screened a herbal library by immunostaining using anti-RXR{alpha} antibody (39). NIH-H460 lung cancer cells were chosen for this study because RXR{alpha} is highly expressed in these cells and is known to translocate from the nucleus to the cytoplasm in response to certain apoptotic stimuli (39). Cells were treated for 3 h with vehicle or various herbal extracts at concentrations that induced apoptosis on the basis of MTT assays (data not shown). In some experiments, time-course was also determined. After treatment, cells were fixed with 4% polyformaldehyde and subjected to immunostaining using anti-RXR{alpha} antibody. The subcellular distribution of RXR{alpha} was visualized via fluorescent microscopy. Analysis of the effects of extracts from our herbal library revealed that a crude alcoholic extract of H.sampsonii, one of the genus Hypericum (43–45), could significantly induce RXR{alpha} nuclear export. Endogenous RXR{alpha} in NIH-H460 cells was mainly found in the nucleus before treatment. Treatment of cells with vehicle control did not show any effect on RXR{alpha} nuclear localization. However, when cells were treated with H.sampsonii extract, a majority of RXR{alpha} was found in the cytoplasm (Figure 1A). Induction of RXR{alpha} nuclear export by H.sampsonii was confirmed by our cellular fractionation approach (39). Immunoblotting showed that accumulation of RXR{alpha} in the cytosolic fraction was significantly enhanced when cells were treated with H.sampsonii, whereas RXR{alpha} remained in the nuclear fraction in vehicle control (Figure 1B). To further explore the effect of H.sampsonii extract on RXR{alpha} subcellular localization, an expression vector encoding RXR{alpha} fused with green fluorescence protein (GFP) was transfected into NIH-H460 cells. Transfected GFP–RXR{alpha} mainly resided in the nucleus. Upon treatment of cells with H.sampsonii extract, GFP–RXR{alpha} was found predominantly in the cytoplasm in a significant amount of transfected cells (Figure 1C).


Figure 1
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  		Fig. 1 Induction of RXR{alpha} nuclear export by H.sampsonii. (A) Immunohistochemistry showed extensively cytoplasmic staining of RXR{alpha} induced by H.sampsonii. NIH-H460 cells were cultured in 96-well plates and exposed for 10 h to vehicle or the indicated concentrations of H.sampsonii extract. Endogenous RXR{alpha} was detected by anti-RXR{alpha} antibody. (B) Immunoblotting showed that H.sampsonii induced RXR{alpha} translocation. NIH-H460 cells were exposed to vehicle or 40 µg/ml of H.sampsonii for 12 h. Cell lysates were fractioned and subjected to western blotting analysis. RXR{alpha} was significantly detected in the cytosol (C) of H.sampsonii-treated cells, but in the nucleus (N) only in vehicle control. The purity of cellular fraction was confirmed by anti-PARP. (C) H.sampsonii induces migration of transfected RXR{alpha}. GFP–RXR{alpha} expression vector was transfected into NIH-H460 cells. Twenty-four hours after transfection, cells were treated with H.sampsonii extract (40 µg/ml) for 6 h. Subcellular localization of GFP–RXR{alpha} was visualized by fluorescence microscopy. (D) Effect of H.sampsonii extract and fractions on RXR{alpha} nuclear export. NIH-H460 cells were cultured in the presence of 40 µg/ml of H.sampsonii extract or its different fractions, chloroform fractions (Chlor Fr) and chloroform extraction plus gradient EtOH and water (H2O) elution, for 12 h. Cells were co-stained with anti-RXR{alpha}. and DAPI. (E) H.sampsonii induces RXR{alpha} nuclear export in various cancer cells. SMMC-7721 liver cancer and MGC-803 gastric cancer cells were treated with H.sampsonii extract (40 µg/ml) for 6 h and detected with anti-RXR{alpha}. (F) RXR{alpha} transcriptional agonist inhibits the effect of H.sampsonii on RXR{alpha} nuclear export. NIH-H460 cells were treated with or without 1 µM of SR11237 for 90 min, followed by treatment with 40 µg/ml of H.sampsonii for 6 h. RXR{alpha} subcellular localization was determined by immunostaining as described.

 
Induction of RXR{alpha} nuclear export was H.sampsonii dose-dependent and again seen in certain fractions of the herb. We observed that about 30% of NIH-H460 cells displayed RXR{alpha} cytoplasmic localization when cells were treated with 20 µg/ml H.sampsonii extract, while treatment with 40 µg/ml of the extract caused RXR{alpha} export in >90% cells. When H.sampsonii alcoholic extract was partitioned with petroleum ether, chloroform and ethyl acetate, the active components that induced RXR{alpha} nuclear export were mainly retained in the chloroform fraction. RXR{alpha} translocation was recognized again in gradient EtOH elute from the chloroform fractions, strongly in 70% aqueous EtOH, weakly in 30% aqueous EtOH and undetectably in water elute (Figure 1D).

H.sampsonii-induced RXR{alpha} nuclear export was also observed in several other human cancer cell lines, including SMMC-7721 liver cancer and MGC-803 gastric cancer cells. Treatment with H.sampsonii extract (40 µg/ml) resulted in RXR{alpha} nuclear export in ~62% SMMC-7721 and 55% MGC-803 cells (Figure 1E). Time-course analysis demonstrated that induction of RXR{alpha} translocation by H.sampsonii extract in different cancer cells occurred as early as 3 h post-treatment and lasted until 16 h when extensive apoptosis occurred (data not shown).

Previous studies showed that RXR{alpha} transactivation agonist SR11237 inhibited RXR{alpha} nuclear export by silencing RXR{alpha} nuclear export sequence (NES) (39). We, therefore, examined whether SR11237 could modulate the effect of H.sampsonii on RXR{alpha} subcellular localization. Treatment of NIH-H460 cells with SR11237 did not significantly alter RXR{alpha} nuclear localization (Figure 1F). However, when cells were treated with SR11237 and H.sampsonii, H.sampsonii-induced RXR{alpha} migration was significantly inhibited by SR11237 (Figure 1F). Thus, H.sampsonii-induced RXR{alpha} nuclear export is probably regulated by RXR{alpha} ligand binding.

H.sampsonii inhibits RXR{alpha} transactivation
Our observation that H.sampsonii induced RXR{alpha} nuclear export suggested that it might inhibit RXR{alpha} transcriptional function. We, therefore, examined whether H.sampsonii regulated transactivation of RXR{alpha} homodimers and heterodimers. To determine its effect on RXR{alpha} homodimer activity, a plasmid containing two copies of RXR{alpha} homodimer-responsive element (TREpal) (16) fused with the thymidine kinase (tk) minimal promoter and chloramphenicol acetyltransferase (CAT) reporter gene, (TREpal)2-tk-CAT, was co-transfected with RXR{alpha} expression vector into CV-1 cells. Transfected cells were exposed to RXR{alpha} ligand SR11237 in the presence or absence of H.sampsonii for 20 h. As shown in Figure 2A, SR11237 strongly induced reporter gene activity, whereas H.sampsonii did not. When cells were treated with SR11237 and H.sampsonii, SR11237-induced RXR{alpha} homodimer transactivation was significantly reduced in a H.sampsonii concentration-dependent manner.


Figure 2
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  		Fig. 2 H.sampsonii dose-dependently suppresses RXR{alpha} transactivation activity. (A) Inhibition of RXR{alpha} homodimer transactivation by H.sampsonii. CV-1 cells were transfected with (TREpal)2-tk-CAT reporter gene and RXR{alpha} expression vector for 24 h. Transfected cells were treated with 1 µM and/or various concentrations of H.sampsonii (10, 30, 60, 90 and 120 µg/ml) for 20 h. Control cells were treated with vehicle. Collected cells were lysed and subjected to CAT assay. (B) Inhibition of RXR{alpha}/Nur77 heterodimer transactivation by H.sampsonii. CV-1 cells were transfected with ßRARE-tk-CAT reporter plasmid and RXR{alpha} and/or Nur77 expression vector. Twenty-four hours after transfection, cells were treated with or without SR11237 (1 µM) and/or various concentrations of H.sampsonii (10, 30, 60, 90 and 120 µg/ml) for 20 h. The CAT reporter activities were then determined. In both (A) and (B) experiments, the relative CAT values were normalized to ß-galactosidase activity. Data were expressed as mean ± SD. Each assay was repeated in triplicate in three independent experiments.

 
To determine whether H.sampsonii regulated RXR{alpha} heterodimer transactivation, we evaluated its effect on transactivation of RXR{alpha}/Nur77 heterodimer on ßRARE (ß retinoic acid responsive element) derived from the RARß promoter (50). RXR{alpha}/Nur77 heterodimer is known to bind to the ßRARE and activate the response element in response to RXR{alpha} ligands (51). Co-transfection of RXR{alpha} and Nur77 expression vectors strongly activated a reporter containing the ßRARE (ßRARE-tk-CAT) (51) when CV-1 cells were treated with SR11237. Similar to its inhibitory effect on RXR{alpha} homodimer, H.sampsonii dose-dependently suppressed SR11237-induced RXR{alpha}/Nur77 transactivation (Figure 2B). Together, H.sampsonii inhibits RXR{alpha} transactivation probably by inducing its nuclear export.

H.sampsonii induces RXR{alpha} mitochondrial targeting, cytochrome c release and poly (ADP-ribose) polymerase (PARP) cleavage
Once in the cytoplasm, RXR{alpha} may associate with mitochondria, an event that is known to induce cytochrome c release and apoptosis (39). To determine whether cytoplasmic RXR{alpha} induced by H.sampsonii associated with mitochondria, NIH-H460 cells were treated with or without H.sampsonii, and immunostained with anti-RXR{alpha} antibody and an antibody against heat shock protein 60 (Hsp60), a mitochondria-specific protein (24). Confocal microscopy analysis showed that the distribution of RXR{alpha} overlapped extensively with that of Hsp60 when cells were treated with H.sampsonii (Figure 3A), suggesting an association of cytoplasmic RXR{alpha} with mitochondria. Thus, in response to H.sampsonii, RXR{alpha} migrated from the nucleus to the cytoplasm where it targeted mitochondria.


Figure 3
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  		Fig. 3 H.sampsonii induces RXR{alpha} mitochondrial targeting and apoptosis. (A) RXR{alpha} targeting at mitochondria by H.sampsonii treatment. NIH-H460 cells plated on slides were treated with vehicle or H.sampsonii (40 µg/ml) for 6 h. Distribution of RXR{alpha} was examined by immunostaining using anti-RXR{alpha} antibody. RXR{alpha} images overlaid with those of mitochondria protein Hsp60 by co-staining with anti-Hsp60 antibody were visualized using confocal microscopy. (B) Association of cytochrome c release with translocation of RXR{alpha}. NIH-H460 cells were treated with or without H.sampsonii (20 µg/ml) for 12 h. Cells were immunostained with anti-cytochrome c (mouse) and anti-RXR{alpha} (rabbit) followed by FITC-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG. Cells were stained with DAPI. (C) Induction of PARP cleavage by H.sampsonii. NIH-H460 cells were treated with 40 µg/ml of H.sampsonii for the indicated time. Control cells were collected before drug administration (C0) and 24 h post-treatment with vehicle (C24). PARP and its fragment were detected by immunoblotting using anti-PARP antibody.

 
It has been widely accepted that mitochondria plays an important role in many critical apoptotic pathways (24,52). We previously reported that mitochondrial localization of RXR{alpha} resulted in extensive apoptosis (39). Induction of cytochrome c release from mitochondria is a key step to initiate apoptosis, which normally occurs before nuclear fragmentation and represents an early event in apoptosis. To determine whether H.sampsonii-induced RXR{alpha} mitochondrial targeting was associated with cytochrome c release, NIH-H460 cells were treated with or without H.sampsonii for 12 h. Cells were immunostained with anti-cytochrome c and anti-RXR{alpha}. In the absence of H.sampsonii treatment, cytochrome c showed punctate distribution in NIH-H460 cells, demonstrating its localization in mitochondria. However, cytochrome c was diffusely distributed in a majority of NIH-H460 cells when they were treated with H.sampsonii, indicating cytochrome c release from mitochondria (Figure 3B). As PARP cleavage is an another sensitive apoptotic marker, which occurs early in the apoptotic response as a result of caspase-3 activity, we further analyzed the cleavage of PARP in response to H.sampsonii in NIH-H460 cells. H.sampsonii treatment resulted in cleavage of PARP, producing an 85 kDa fragment, which was visible at 12 h post-treatment. The amount of the 85 kDa PARP fragment increased when cells were treated with H.sampsonii for prolonged times (Figure 3C).

H.sampsonii-induced apoptosis and growth inhibition is associated with levels of RXR{alpha} protein
We next evaluated the effect of H.sampsonii on growth and apoptosis in a number of cancer cell lines. Using MTT assays, we found that NIH-H460 lung cancer cells were very sensitive to H.sampsonii with an IC50 of 38 µg/mg, followed by SMMC7721 liver cancer and MGC-803 gastric cancer cells, with an IC50 of 49 and 52 µg/mg, respectively (Table I). Consistently, DAPI staining demonstrated that H.sampsonii induced apoptosis in 45% NIH-H460, followed by SMMC7721 (31%) and MGC-803 (24%) (Figure 4A and B). Together, these results demonstrate that H.sampsonii is a potent apoptosis inducer in cancer cells.


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  		Table I Growth inhibitory effect of H.sampsonii crude extracts and fractions in different cell lines

 

Figure 4
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  		Fig. 4 H.sampsonii induces apoptosis in RXR{alpha}-dependent manner. (A and B) Apoptotic effect of H.sampsonii on various cancer cells. NIH-H460 lung cancer, SMMC-7721 liver cancer and MGC-803 stomach cancer cells were cultured in 6-well plates in 0.5% serum medium in the presence of 40 µg/mlx H.sampsonii for 24 h. Detached and attached cells were collected and subjected to DAPI staining as indicated in Materials and methods. Representative apoptotic morphology for different cancer cells was indicated in (A). The number of apoptotic cells (B) were independently counted by two observers from at least 1000 cells in 10 random microscopic fields. The data were expressed as mean ± SD from three independent experiments. Each concentration was conducted in triplicate. (C, D and E) The effect of RXR{alpha} expression levels on H.sampsonii-induced growth inhibition and apoptosis. NIH-H460 and its RXR{alpha} stable line (NIH-H460/RXR{alpha}, see Materials and methods) were used to determine the effect of RXR{alpha} expression levels on H.sampsonii activities. NIH-H460/RXR{alpha} showed more sensitivity than its parental NIH-H460 to H.sampsonii, as demonstrated by MTT methods during a time-course of 72-h treatment (C) and by DAPI staining when cells were treated with vehicle or various concentrations of H.sampsonii in 0.5% serum medium for 24 h (D). The number of apoptotic cells (E) were independently counted by two observers from at least 1000 cells in 10 random microscopic fields. Results are expressed as mean ± SD. Each assay was repeated in triplicate in three independent experiments. (F) The role of RXR{alpha} in the apoptosis induced by H.sampsonii. CV-1 cells were transfected with or without Flag-tagged RXR{alpha} before treatment of H.sampsonii (10 µg/ml) for 24 h. Cells were immunostained with anti-Flag (mouse) followed by Cy3-conjugated anti-mouse IgG and co-stained with DAPI.

 
To determine whether H.sampsonii-induced growth inhibition was associated with RXR{alpha} protein expression levels, NIH-H460 cells were stably transfected with RXR{alpha}. The resulting stable clones (NIH-H460/RXR{alpha}) expressed significant higher levels of RXR{alpha} protein, as compared with their parental NIH-H460 cell line (data not shown). An RXR{alpha} stable clone and its parental cells were subjected to H.sampsonii treatment. Treatment of H.sampsonii dose-dependently inhibited the growth of both cell lines. However, the RXR{alpha} stable cells were more sensitive to growth inhibition by H.sampsonii than its parental cells (Figure 4C). DAPI staining revealed that H.sampsonii caused significant morphological changes in nuclear chromatin that represented typical apoptosis in both parental and the RXR{alpha} stable lines in a dose-dependent manner (Figure 4D). However, H.sampsonii was much more effective in the RXR{alpha} stable line than in its parental line, when two concentrations (20 and 40 µg/ml) of H.sampsonii were used (Figure 4E). In response to 40 µg/ml H.sampsonii, 92% RXR{alpha} stable cells underwent apoptosis, while only 42% parental NIH-H460 cells were apoptotic. At 20 µg/ml, H.sampsonii induced ~54% cell death in the RXR{alpha} stable line, whereas it had much reduced effect (~14%) in the NIH-H460 parental cells.

CV-1 cells lacking detectable RXR{alpha} protein showed significant resistance to H.sampsonii. Their growth was not clearly affected even when high concentration of H.sampsonii (80 µg/ml) was used (Table I). To further determine whether levels of RXR{alpha} protein regulated the apoptotic effect of H.sampsonii, CV-1 cells were transfected with Flag-RXR{alpha} and subjected to vehicle or H.sampsonii treatment. Flag-RXR{alpha} transfection did not have any effect on apoptosis of CV-1 cells. However, when cells were treated with H.sampsonii, cells transfected with Flag-RXR{alpha} underwent extensive apoptosis indicated by nuclear condensation, whereas non-transfected CV-1 cells were not apoptotic even though they were treated with H.sampsonii (Figure 4F). Collectively, these results demonstrate that the apoptotic effect of H.sampsonii depends on RXR{alpha} levels.


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Since St John's wort is widely used for the treatment of mild and moderate depression (38,39), attention has also been attracted to determine pharmacological value of several other species of this genus. H.sampsonii is the closely related species of St John's wort in China, which has been of scientific interest for many years owing to its widespread use in folk medicine for a range of ailments (44,45). Recent studies have shown that H.sampsonii possesses anticancer activities (44,45). However, how H.sampsonii exerts its various biological effects remains virtually unknown. Here, we report that H.sampsonii contains components that modulate transactivation and subcellular localization of RXR{alpha}. In addition, our results demonstrated that the effect of H.sampsonii was associated with growth inhibition and apoptosis of cancer cells in an RXR{alpha}-dependent manner. Given the fact that RXR{alpha} plays a role in the regulation of diverse endocrine signal transduction pathways (12,13,15–19), our findings suggest that RXR{alpha} may be an important mediator of the biological activities of H.sampsonii.

An important finding reported here is that H.sampsonii extract could induce rapid migration of RXR{alpha} from the nucleus to the cytoplasm. RXR{alpha} immunostaining revealed that RXR{alpha} resided mainly in the cytoplasm when cells were treated with H.sampsonii extract (Figure 1A–E). Confocal microscopy analysis showed that the cytoplasmic RXR{alpha} was associated with mitochondria (Figure 3A). Consistently, H.sampsonii extract antagonized SR11237-induced RXR{alpha} transactivation (Figure 2A and B), presumably owing to its induction of RXR{alpha} nuclear export. Evidence has been accumulating to demonstrate that non-genotropic action commonly exists for a number of different NRs (44,53–56). The redistribution of NRs between nucleus and cytoplasm is an important event for the regulation of their activities and the execution of their functions. Translocation of RXR{alpha} was previously observed in cells treated with a number of structurally different agents, such as NGF (37), 12-O-tetradecanoylphorbol-13-acetate (TPA) and those related to retinoid-derived compound AHPN/CD437 (39). To our knowledge, our finding is the first report that a natural Chinese herbal medicine modulates RXR{alpha} subcellular distribution. Since the cytoplasmic localization of RXR{alpha} is associated with the regulation of important biological processes, such as development (35,36), differentiation (37) and apoptosis (34,39,40,42), our results will provide impetus for further characterization of bioactive components in H.sampsonii, which modulate RXR{alpha} subcellular localization. The identification and characterization of such bioactive components will certainly add to our knowledge on the mechanism of H.sampsonii action and may provide important leads for developing new RXR{alpha}-based modern medicine. In this regard, St John's wort is known to bind pregnane X receptor (also known as steroid X receptor) (53,54), a finding that provides an important explanation for its interaction with a variety of drugs.

How H.sampsonii induces RXR{alpha} nuclear export remains to be determined. Induction of RXR{alpha} nuclear export by AHPN/CD437 did not require its binding to RXR{alpha} (39). Instead, the effect of AHPN/CD437 and TPA was mediated by its induction of RXR{alpha} heterodimerization partner, Nur77, and their post-translational modifications (39,55,57). H.sampsonii extract may indirectly induce RXR{alpha} nuclear export in a fashion similar to that utilized by AHPN/CD437. We recently reported that RXR{alpha} nuclear export was mediated by an NES, which was highly regulated by RXR{alpha} conformation induced by RXR{alpha} dimerization and ligand binding (39). RXR{alpha} transcriptional agonists such as SR11237 and 9-cis-RA have been demonstrated to silence the RXR{alpha} NES activity and retain RXR{alpha} in the nucleus (39). It is not impossible that certain agents induce RXR{alpha} nuclear export by directly binding to RXR{alpha}., resulting in an RXR{alpha} conformation that activates its NES. It remains to be seen if an active component in H.sampsonii induces RXR{alpha} migration by directly binding to RXR{alpha}. In support of this hypothesis, RXR{alpha} has been shown to bind to natural compounds with diverse structures (25–28,30–32), such as 9-cis-RA and various fatty acids, owing to the promiscuity of its LBP (56,58,59).

Another interesting finding reported here is that H.sampsonii potently induced apoptosis in various cancer cells, which was associated with its induction of RXR{alpha} nuclear export. The apoptotic effect of H.sampsonii was demonstrated by several independent assays, including DAPI staining (Figure 4F), cytochrome c release (Figure 3B) and PARP cleavage (Figure 3C). The ability of H.sampsonii to induce apoptosis of tumor cells may explain the potential anti-neoplastic activity of this herb. Interestingly, recent studies have also demonstrated that St John's wort exerts anti-neoplastic effect in vitro and in vivo, owing to the potent apoptotic effect of its ingredients, hypericin and hyperforin, in cancer cells (48,49). However, when examining the effect of both compounds on inducing RXR{alpha} translocation in different cancer cells such as NIH-H460 lung cancer cells and MGC-803 gastric cancer cells, we did not observe any effect of these compounds at concentrations between ~0.01 and 1.0 µM on RXR{alpha} subcellular localization examined by either immunostaining or immunoblotting (data not shown). These results suggest that ingredients other than hypericin and hyperforin function to modulate RXR{alpha} cellular distribution.

The apoptotic effect of H.sampsonii could be observed in several cancer cell lines derived from lung, stomach and liver (Figure 4A). Among different cancer cell lines, NIH-H460 lung cancer cells were the most sensitive to H.sampsonii, probably reflecting different levels of RXR{alpha} expressed in these cells (data not shown), different cellular environment or different levels of factors, such as Nur77. Under the same cellular environment, levels of RXR{alpha} determined the efficacy of H.sampsonii, as overexpression of RXR{alpha} in NIH-H460 cells enhanced the apoptotic effect of H.sampsonii (Figure 4C–E), whereas H.sampsonii exhibited limited activity in CV-1 cells that express undetectable levels of RXR{alpha} (Table I). When CV-1 cells were transfected with Flag-RXR{alpha}, cells expressing transfected Flag-RXR{alpha} were highly responsive to H.sampsonii, displaying extensive apoptosis (Figure 4F). Thus, RXR{alpha} is an important mediator of the apoptotic effect of H.sampsonii. The apoptotic effect of H.sampsonii appears to be resulting from its induction of RXR{alpha} cytoplasmic localization, as both events are closely associated (Figure 3B). This is consistent with previous observations that cytoplasmic localization of RXR{alpha} induced by several apoptotic stimuli resulted in apoptosis (34,39,40,42). Our observation that cytoplasmic RXR{alpha} was able to target mitochondria (Figure 3A) suggests that RXR{alpha} mitochondrial targeting may be responsible for its apoptosis induction.

Scientific investigations of herbal and alternative therapies represent a potentially important source for developing new modern medicine (1–3). Integrating ancient knowledge and modern technology may facilitate the process. The central role that RXR{alpha} plays in the regulation of diverse endocrine signal transduction pathways through its exceptional dimerization function makes it an attractive molecular target for drug development (19). The complexity in RXR signaling also offers an excellent opportunity to develop RXR ligands that selectively regulate a specific RXR-signaling pathway, which is clearly therapeutically advantageous by reducing adverse effects caused by interaction with other receptors. The results presented here demonstrate that H.sampsonii contains component(s) that induce migration of RXR{alpha} from the nucleus to the cytoplasm, an important RXR{alpha} pathway that regulates apoptosis and differentiation (34,37,39,40,42). It remains to be seen whether other Chinese herbal medicines exert their effects, such as modulation of growth, differentiation, apoptosis, immune response, by targeting various RXR{alpha} pathways.


   Notes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
This work was supported by grants from National Natural Science Foundation of China (No. 30471939), Shanghai Key Project (04DZ19115), Shanghai KREX Pharmaceuticals, the National Institutes of Health (R01CA87000 and R01CA109345) and Susan G. Komen Breast Cancer Foundation (BCTR0403351).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. da Rocha A.B., Lopes R.M., Schwartsmann G. (2001) Natural products in anticancer therapy. Curr. Opin. Pharmacol. 1:364–369.[CrossRef][Medline]
  2. Newman D.J., Cragg G.M., Snader K.M. (2003) Natural products as sources of new drugs over the period 1981–2002. J. Nat. Prod. 66:1022–1037.[CrossRef][Medline]
  3. Koehn F.E. and Carter G.T. (2005) The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. 4:206–220.[CrossRef][ISI][Medline]
  4. Archer J.R. (2004) History, evolution, and trends in compound management for high throughput screening. Assay Drug Dev. Technol. 2:675–681.[CrossRef][ISI][Medline]
  5. Lazar M.A. (2004) East meets West: an herbal tea finds a receptor. J. Clin. Invest. 113:23–25.[CrossRef][ISI][Medline]
  6. Nagle D.G., Zhou Y.D., Mora F.D., Mohammed K.A., Kim Y.P. (2004) Mechanism targeted discovery of antitumor marine natural products. Curr. Med. Chem. 11:1725–1756.[ISI][Medline]
  7. Novac N. and Heinzel T. (2004) Nuclear receptors: overview and classification. Curr. Drug Targets Inflamm Allergy 3:335–346.[CrossRef][Medline]
  8. Nagy L. and Schwabe J.W. (2004) Mechanism of the nuclear receptor molecular switch. Trends Biochem. Sci. 29:317–324.[CrossRef][ISI][Medline]
  9. Chawla A., Repa J.J., Evans R.M., Mangelsdorf D.J. (2001) Nuclear receptors and lipid physiology: opening the X-files. Science 294:1866–1870.[Abstract/Free Full Text]
  10. Schapira M. (2002) Pharmacogenomics opportunities in nuclear receptor targeted cancer therapy. Curr. Cancer Drug Targets 2:243–256.[CrossRef][Medline]
  11. Gronemeyer H., Gustafsson J.A., Laudet V. (2004) Principles for modulation of the nuclear receptor superfamily. Nat. Rev. Drug Discov. 3:950–964.[CrossRef][ISI][Medline]
  12. Kastner P., Mark M., Chambon P. (1995) Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 83:859–869.[CrossRef][ISI][Medline]
  13. Mangelsdorf D.J., Thummel C., Beato M., et al. (1995) The nuclear receptor superfamily: the second decade. Cell 83:835–839.[CrossRef][ISI][Medline]
  14. Xu L., Glass C.K., Rosenfeld M.G. (1999) Coactivator and corepressor complexes in nuclear receptor function. Curr. Opin. Genet. Dev. 9:140–147.[CrossRef][ISI][Medline]
  15. Zhang X.K., Hoffmann B., Tran P.B., Graupner G., Pfahl M. (1992) Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature 355:441–446.[CrossRef][Medline]
  16. Zhang X.K., Lehmann J., Hoffmann B., Dawson M.I., Cameron J., Graupner G., Hermann T., Tran P., Pfahl M. (1992) Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid. Nature 358:587–591.[CrossRef][Medline]
  17. Zhang X.K. and Pfahl M. (1993) Hetero- and homodimeric receptors in thyroid hormone and vitamin A action. Receptor 3:183–191.[ISI][Medline]
  18. Love J.D., Gooch J.T., Benko S., Li C., Nagy L., Chatterjee V.K., Evans R.M., Schwabe J.W. (2002) The structural basis for the specificity of retinoid-X receptor-selective agonists: new insights into the role of helix H12. J. Biol. Chem. 277:11385–11391.[Abstract/Free Full Text]
  19. Szanto A., Narkar V., Shen Q., Uray I.P., Davies P.J., Nagy L. (2004) Retinoid X receptors: X-ploring their (patho)physiological functions. Cell Death Differ. 11:Suppl. 2, S126–S143.[CrossRef][Medline]
  20. Huang J., Powell W.C., Khodavirdi A.C., Wu J., Makita T., Cardiff R.D., Cohen M.B., Sucov H.M., Roy-Burman P. (2002) Prostatic intraepithelial neoplasia in mice with conditional disruption of the retinoid X receptor alpha allele in the prostate epithelium. Cancer Res. 62:4812–4819.[Abstract/Free Full Text]
  21. Li M., Indra A.K., Warot X., Brocard J., Messaddeq N., Kato S., Metzger D., Chambon P. (2000) Skin abnormalities generated by temporally controlled RXRalpha mutations in mouse epidermis. Nature 407:633–636.[CrossRef][Medline]
  22. Li M., Chiba H., Warot X., Messaddeq N., Gerard C., Chambon P., Metzger D. (2001) RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations. Development 128:675–688.[Abstract]
  23. Mao G.E., Reuter V.E., Cordon-Cardo C., Dalbagni G., Scher H.I., DeKernion J.B., Zhang Z.F., Rao J. (2004) Decreased retinoid X receptor-alpha protein expression in basal cells occurs in the early stage of human prostate cancer development. Cancer Epidemiol. Biomarkers Prev. 13:383–390.[Abstract/Free Full Text]
  24. Li H., Kolluri S.K., Gu J., et al. (2000) Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 289:1159–1164.[Abstract/Free Full Text]
  25. Heyman R.A., Mangelsdorf D.J., Dyck J.A., Stein R.B., Eichele G., Evans R.M., Thaller C. (1992) 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68:397–406.[CrossRef][ISI][Medline]
  26. de Urquiza A.M., Liu S., Sjoberg M., Zetterstrom R.H., Griffiths W., Sjovall J., Perlmann T. (2000) Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain. Science 290:2140–2144.[Abstract/Free Full Text]
  27. Lengqvist J., Mata De Urquiza A., Bergman A.C., Willson T.M., Sjovall J., Perlmann T., Griffiths W.J. (2004) Polyunsaturated fatty acids including docosahexaenoic and arachidonic acid bind to the retinoid X receptor alpha ligand-binding domain. Mol. Cell. Proteomics 3:692–703.[Abstract/Free Full Text]
  28. Goldstein J.T., Dobrzyn A., Clagett-Dame M., Pike J.W., DeLuca H.F. (2003) Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor. Arch Biochem. Biophys 420:185–193.[CrossRef][ISI][Medline]
  29. Khuri F.R., Rigas J.R., Figlin R.A., et al. (2001) Multi-institutional phase I/II trial of oral bexarotene in combination with cisplatin and vinorelbine in previously untreated patients with advanced non-small-cell lung cancer. J. Clin. Oncol. 19:2626–2637.[Abstract/Free Full Text]
  30. Lemotte P.K., Keidel S., Apfel C.M. (1996) Phytanic acid is a retinoid X receptor ligand. Eur. J. Biochem. 236:328–333.[ISI][Medline]
  31. Kitareewan S., Burka L.T., Tomer K.B. (1996) Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR. Mol. Biol. Cell 7:1153–1166.[Abstract]
  32. Kolluri S.K., Corr M., James S.Y., Bernasconi M., Lu D., Liu W., Cottam H.B., Leoni L.M., Carson D.A., Zhang X.K. (2005) The R-enantiomer of the nonsteroidal antiinflammatory drug etodolac binds retinoid X receptor and induces tumor-selective apoptosis. Proc. Natl Acad. Sci. USA 102:2525–2530.[Abstract/Free Full Text]
  33. Prüfer K. and Barsony J. (2002) Retinoid X receptor dominates the nuclear import and export of the unliganded vitamin D receptor. Mol. Endocrinol. 16:1738–1751.[Abstract/Free Full Text]
  34. Lin X.F., Zhao B.X., Chen H.Z., Ye X.F., Yang C.Y., Zhou H.Y., Zhang M.Q., Lin S.C., Wu Q. (2004) RXR (acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells. J. Cell Sci. 117:5609–5621.[Abstract/Free Full Text]
  35. Dufour J.M. and Kim K.H. (1999) Cellular and subcellular localization of six retinoid receptors in rat testis during postnatal development: identification of potential heterodimeric receptors. Biol. Reprod. 61:1300–1308.[Abstract/Free Full Text]
  36. Boulogne B., Levacher C., Durand P., Habert R. (1999) Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes. Biol. Reprod. 61:1548–1557.[Abstract/Free Full Text]
  37. Katagiri Y., Takeda K., Yu Z.X., Ferrans V.J., Ozato K., Guroff G. (2000) Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B. Nat. Cell Biol. 2:435–440.[CrossRef][ISI][Medline]
  38. Casas F., Daury L., Grandemange S., Busson M., Seyer P., Hatier R., Carazo A., Cabello G., Wrutniak-Cabello C. (2003) Endocrine regulation of mitochondrial activity: involvement of truncated RXRalpha and c-ErbA alpha1 proteins. FASEB J. 17:426–436.[Abstract/Free Full Text]
  39. Cao X., Liu W., Lin F., Li H., Kolluri S.K., Lin B., Han Y.H., Dawson M.I., Zhang X.K. (2004) Retinoid X receptor regulates Nur77/TR3-dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting. Mol. Cell. Biol. 24:9705–9725.[Abstract/Free Full Text]
  40. Lee K.W., Ma L., Yan X., Liu B., Zhang X.K., Cohen P. (2005) Rapid apoptosis induction by IGFBP-3 involves an insulin-like growth factor-independent nucleomitochondrial translocation of RXRalpha/Nur77. J. Biol. Chem. 280:16942–16948.[Abstract/Free Full Text]
  41. Lin B., Kolluri S.K., Lin F., Liu W., Han Y.H., Cao X., Dawson M.I., Reed J.C., Zhang X.K. (2004) Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell 116:527–540.[CrossRef][ISI][Medline]
  42. Jacobs C.M. and Paulsen R.E. (2005) Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B. Biochem. Biophys. Res. Commun. 336:646–652.[CrossRef][ISI][Medline]
  43. Kirakosyan A., Sirvent T.M., Gibson D.M., Kaufman P.B. (2004) The production of hypericins and hyperforin by in vitro cultures of St John's wort (Hypericum perforatum). Biotechnol. Appl. Biochem. 39:71–81.[ISI][Medline]
  44. Don M.J., Huang Y.J., Huang R.L., Lin Y.L. (2004) New phenolic principles from Hypericum sampsonii. Chem. Pharm. Bull. (Tokyo) 52:866–869.[Medline]
  45. Hong D., Yin F., Hu L.H., Lu P. (2004) Sulfonated xanthones from Hypericum sampsonii. Phytochemistry 65:2595–2598.[CrossRef][ISI][Medline]
  46. Di Carlo G., Borrelli F., Ernst E., Izzo A.A. (2001) St John's wort: prozac from the plant kingdom. Trends Pharmacol. Sci. 22:292–297.[CrossRef][Medline]
  47. Muller W.E. (2003) Current St John's wort research from mode of action to clinical efficacy. Pharmacol Res. 47:101–109.[CrossRef][ISI][Medline]
  48. Agostinis P., Vantieghem A., Merlevede W., de Witte P.A. (2002) Hypericin in cancer treatment: more light on the way. Int. J. Biochem. Cell Biol. 34:221–241.[CrossRef][ISI][Medline]
  49. Schempp C.M., Kirkin V., Simon-Haarhaus B., et al. (2002) Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St John's wort that acts by induction of apoptosis. Oncogene 21:1242–1250.[CrossRef][ISI][Medline]
  50. Wu Q., Li Y., Liu R., Agadir A., Lee M.O., Liu Y., Zhang X. (1997) Modulation of retinoic acid sensitivity in lung cancer cells through dynamic balance of orphan receptors nur77 and COUP-TF and their heterodimerization. EMBO J. 16:1656–1669.[CrossRef][ISI][Medline]
  51. Wu Q., Dawson M.I., Zheng Y., Hobbs P.D., Agadir A., Jong L., Li Y., Liu R., Lin B., Zhang X.K. (1997) Inhibition of trans-retinoic acid-resistant human breast cancer cell growth by retinoid X receptor-selective retinoids. Mol. Cell. Biol. 17:6598–6608.[Abstract]
  52. Kroemer G. and Reed J.C. (2000) Mitochondrial control of cell death. Nat. Med. 6:513–519.[Cro