Retinoids display anti-tumour activity on various cancer cells and therefore have been used as important therapeutic agents. However, adverse side effects and RA (retinoic acid) resistance limit further development and clinical application of retinoid-based therapeutic agents. We report in the present paper the identification of a natural marine product that activates RARs (RA receptors) with a chemical structure distinct from retinoids by high-throughput compound library screening. Luffariellolide was uncovered as a novel RAR agonist by inducing co-activator binding to these receptors in vitro, further inhibiting cell growth and regulating RAR target genes in various cancer cells. Structural and molecular studies unravelled a unique binding mode of this natural ligand to RARs with an unexpected covalent modification on the RAR. Functional characterization further revealed that luffariellolide displays chemotherapeutic potentials for overcoming RA resistance in colon cancer cells, suggesting that luffariellolide may represent a unique template for designing novel non-retinoid compounds with advantages over current RA drugs.
- cancer chemoprevention
- crystal structure
- nuclear receptor
- retinoic acid receptor (RAR)
Retinoids are vitamin A and its natural or synthetic derivatives that are important regulators of cell proliferation and differentiation in a diverse array of tissues [1–3]. These small molecules exert their effects by activating retinoid receptors which are encoded by six different genes, RAR [RA (retinoic acid) receptor] α, β and γ and RXR (retinoid X receptor) α, β and γ, which act as ligand-dependent transcription factors [4–6]. In addition, numerous isoforms have been shown arising from differential promoter usage and alternative splicing . Although the RARs bind to both ATRA (all-trans-RA) and 9-cis-RA, the RXRs are activated exclusively by 9-cis-RA. The RARs form heterodimers with the RXRs and recruit a variety of nuclear receptor co-activators (or co-repressors) to regulate downstream target genes in response to various ligands  via RA response elements [8–11]. All three RARs share a high sequence similarity and thus possess overlapping ligand-binding properties [12,13].
Similar to many other nuclear receptors, the ligand-dependent recruitment of co-activators by RARs is primarily determined by the interaction of co-activator LXXLL motifs with their LBDs (ligand-binding domains). Crystal structures of various nuclear receptor LBDs reveal a conserved binding mode of co-activator LXXLL motifs by nuclear receptors [14–16]. Upon the binding of an agonist, nuclear receptors form a hydrophobic groove for binding of the LXXLL motif of the co-activators such as the SRCs (steroid receptor co-activators) and GRIP1 (glucocorticoid receptor interacting protein 1) . The binding of the co-activator LXXLL motifs was further stabilized by a charge clamp pocket, composed of residues from helix 5 and the C-terminal AF (activation function)-2 helix[18,19]. In addition, N-terminal AF-1 also co-ordinates with AF-2 in RAR activation .
RAR ligands slow or arrest the growth of many transformed cell lines and have been used as therapeutic agents for various cancers, such as APL (acute promyelocytic leukaemia) and breast cancer [21,22]. Unfortunately, adverse side effects may limit further development and clinical application of retinoids and retinoid-based RAR ligands . For instance, retinoids are extremely teratogenic and are one of the most potent human teratogens. RA-based chemotherapy is further hampered by the development of RA resistance, which arises in a variety of cancer cells . Consequently, a new drug design strategy for RAR ligands distinct from retinoids may yield more efficacious RAR-targeted drugs with less adverse effects.
The human RARα LBD (residues 176–411) was expressed as N-terminal His6 fusion protein from the expression vector pET24a (Novagen). BL21 (DE3) cells transformed with each expression plasmid were grown in LB (Luria–Bertani) broth at 25°C to an D600 of ~1.0 and induced with 0.1 mM IPTG (isopropyl β-D-thiogalactopyranoside) at 16°C. The cells were harvested, resuspended and sonicated [65% output (5 s on and 10 s off) for 10 min] in 200 ml of extract buffer [20 mM Tris/HCl (pH 8.0), 150 mM NaCl, 10% glycerol and 25 mM imadazole] per 6 l of cells. The lysate was centrifuged at 48384 g for 30 min at 4°C and the supernatant was loaded on a 5 ml of NiSO4-loaded HisTrap HP column (GE Healthcare). The column was washed with extract buffer and the protein eluted with a gradient of 25–500 mM imidazole. The RARα LBD was further purified with a gel-filtration column (GE Healthcare). For crystallization, the RARα LBD was complexed with 5-fold of luffariellolide and 2-fold of SRC1–4 peptide (AQQKSLLQQLLTE) followed by filter concentration.
Crystallization, data collection and structure determination
The crystals of the RARα–luffariellolide complex were grown at room temperature (20°C) in hanging drops containing 1.0 μl of the above protein–peptide solutions and 1.0 μl of well buffer containing 2% (v/v) Tacsimate (pH 7.0), 5% propan-2-ol, 0.1 M imadazole and 8% PEG [poly(ethylene glycol)] 3350. The crystals were directly flash-frozen in liquid nitrogen for data collection. Diffraction data were collected with a MAR300 CCD (charge-coupled device) detector at the ID line of sector-21 at the Advanced Photon Source (Argonne, IL, U.S.A.). The observed reflections were reduced, merged and scaled with DENZO and SCALEPACK in the HKL2000 package . The structures were determined by molecular replacement in the CCP4 suite (http://www.ccp4.ac.uk). Manual model building was carried out with Coot , followed by REFMAC refinement in the CCP4 suite. Co-ordinates for the structures of RARα–luffariellolide have been deposited in the PDB under the code 4DQM.
Co-factor binding assays
The binding of the various peptide motifs to RARs in response to ligands was determined by AlphaScreen™ assays using a His6 detection kit from PerkinElmer as described previously . The experiments were conducted with approximately 20–40 nM receptor LBDs and 20 nM biotinylated co-factor peptides (SRC1-2, SPSSHSSLTERHKILHRLLQEGSP) in the presence of 5 μg/ml donor and acceptor beads in a buffer containing 25 mM Hepes, 100 mM NaCl and 0.1 mg/ml BSA, all adjusted to a pH of 7.0.
Transient transfection assay
Cos7 cells were maintained in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (fetal bovine serum) and were transiently transfected using Lipofectamine™ 2000 (Invitrogen) . All of the mutant RARα plasmids were created using the QuikChange site-directed mutagenesis kit (Stratagene). At 24 h prior to transfection, 24-well plates were plated (5×104 cells per well). For Gal4 (Galectin-4)-driven reporter assays, the cells were transfected with 200 ng of Gal4 LBDs of various nuclear receptors and 200 ng of pG5Luc reporter (Promega). For native promoter reporter assays, the cells were co-transfected with plasmids encoding full-length nuclear receptors and their cognate luciferase reporters. Ligands were added 5 h after transfection. Cells were harvested 24 h later for the luciferase assays. Luciferase data were normalized to Renilla activity co-transfected as an internal control.
Gene expression analysis
Total RNA was extracted with TRizol reagent (Life Technologies). RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad Laboratories). Real-time quantitative PCR analysis was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems). The mRNA expression was normalized to β-actin.
MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS)
Purified RARα LBD (30 μM) in 25 mM Tris (pH 7.5), 100 mM NaCl and 5 mM DTT (dithiothreitol) was incubated with DMSO, ATRA or luffariellolide at 4°C for 24 h. MALDI–TOF-MS measurements were performed using a Reflex III spectrometer (Bruker Daltonics) operating in the positive linear ion mode. For all of the samples, a solution of supersaturated sinapinic acid in water/acetonitrile (50:50) with 0.1% TFA (trifluoroacetic acid) was used as the matrix. Equal volumes (0.8 μl) of the acidified sample solution and the matrix were spotted on to the target and air dried.
Flow cytometry analysis
To determine if growth inhibition was due to cell-cycle arrest, cells were seeded on to 60 mm dishes at a density of 5×105 cells per dish. The cells were incubated in DMEM with no FBS for 24 h and were treated with 2 mg/ml aphidicolin for another 24 h. Before harvesting, the cells were treated with 5 μM ATRA or luffariellolide for an additional 24 h. The cells were fixed in 70% ethanol at 4°C overnight, and were stained with propidium iodide/RNase A (Sigma–Aldrich) for 1 h. At least 20000 cells were analysed per sample using an Epics XL Flow Cytometer (Beckman Coulter). The DNA content was determined using Modfit software version 3.2 (Verity Software House).
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] proliferation assays
Cells were plated in 24-well culture plates at a density of 1×104 cells per well. After being incubated overnight, the cells were treated with ATRA or luffariellolide for 24 or 48 h. Separate stock solutions of MTT (Sigma–Aldrich) were prepared and added to the cultures to a final concentration of 0.1 mg/ml and the plates were incubated at 37°C for another 4 h. Then the mixture containing the medium, the drug and the unconverted MTT was removed. DMSO was added to each well to dissolve the formazan, and absorbance was read at 490 nm and 630 nm.
Results are expressed as means±S.E.M. Differences among groups were tested with Student's t test. P<0.05 was considered statistically significant.
RESULTS AND DISCUSSION
In searching for novel ligands that activate RARs, we used the RARα LBD as bait to screen chemical libraries based on the AlphaScreen biochemical assay, which is widely used for detecting ligand-dependent interaction between the nuclear receptors and their co-activators [28,29]. Results from Enzo Natural Compound library revealed positive RARα activators which are all known retinoids (Supplementary Figure S1 at http://www.BiochemJ.org/bj/446/bj4460079add.htm). The continual screen on the Enzo Rare Natural Products library revealed luffariellolide as a positive RARα activator. The marine natural product luffariellolide, a sesterterpene, is a hexane extract isolated from sponges of Luffariella sp. and Fascaplysinopsis . Similar to ATRA, luffariellolide also possesses a hydrophobic chain and a trimethylcyclohexene group (Figure 1A). Notably, the chemical structure of luffariellolide shows a unique γ-hydroxybutenolide ring terminus instead of a carboxylic acid moiety for retinoids, thus representing a novel approach for an RAR ligand design strategy distinct from the retinoid scaffold.
To further attain characteristics of luffariellolide in activating nuclear receptors, Cos7 cells were co-transfected with a Gal4-driven reporter together with plasmids encoding various nuclear receptor LBDs fused with the Gal4 DNA-binding domain. Consistent with initial library screen results, luffariellolide showed agonist property on RARα by inducing its transcriptional activity (Figure 1B). In addition, luffariellolide had no impact on a variety of other orphan nuclear receptors tested, including RXRα, PPARγ (peroxisome-proliferator-activated receptor γ), GR (glucocorticoid receptor), LXRα (liver X receptor α), ERRα (oestrogen-related receptor α), ROR (RAR-related orphan receptor) α, β and γ, TR3 and RTR (retinoid receptor-related testis-specific receptor) (Figure 1B). Moreover, luffariellolide also activated the reporter activity of RARβ and RARγ, but not RXRα, suggesting luffariellolide is a pan-RAR agonist (Figure 2). Similar to ATRA, luffariellolide-mediated activity of all three RARs was inhibited by the pan-RAR inverse agonist BMS 493 that stabilizes the inactive conformation of RARs in cell-based reporter assays [8,31], whereas the treatment of BMS 493 had no effects on the control nuclear receptors RXRα, PPARγ and GR (Figure 2). All of these results reaffirm that luffariellolide functions through directly binding to RARs. To unravel the biochemical mechanism of RAR activation by luffariellolide, we determined the ability of luffariellolide in promoting recruitment of co-activator motifs by RARs using the AlphaScreen biochemical assay. As shown in Supplementary Figure S2(A) (at http://www.BiochemJ.org/bj/446/bj4460079add.htm), luffariellolide strongly enhanced the interaction of the co-activator LXXLL motif with all three RARs, but not RXRα. Furthermore, the effects of luffariellolide on RAR activity were lost when tested with the RAR inverse agonist BMS 493 (Supplementary Figure S2A), suggesting that luffariellolide is a bona fide pan-RAR ligand.
To determine the molecular basis for the binding of luffariellolide by RARs, we solved the crystal structure of RARα complexed with luffariellolide (Table 1). The structure reveals that the luffariellolide-bound RARα LBD displays a dimer fold and adopts a canonical active conformation that resembles the ATRA-bound RAR structures , with both ligands occupying the same binding site in the RARα pocket (Figure 3A, and Supplementary Figure S3 at http://www.BiochemJ.org/bj/446/bj4460079add.htm). The RARα–luffariellolide crystal structure reveals that the AF-2, together with helices H3, H4 and H5, forms a charge-clamp pocket (Lys244 from H3 and Glu412 from AF-2) to interact with the SRC1 LXXLL motif (Figure 3B and Supplementary Figure S3), which is a conserved mode for nuclear receptors to interact with co-activators [18,32]. To test the significance of the charge clamp in RARα activation in response to luffariellolide, we mutated these two residues and tested them in cell-based reporter assays. Accordingly, the mutations of either charge residue (K244E and E412K) substantially reduced RARα activity in response to both ATRA and luffariellolide in cell-based reporter assays (Figure 3E), supporting a critical conserved mechanism for ligand-mediated activation of RARs.
Interestingly, superimposition of both ligand-bound structures clearly revealed the differential binding modes of luffariellolide and retinoids. Compared with ATRA, the larger luffariellolide ligand expanded the RARα pocket, resulting in part of helix 10 shifting 1.2 Å (1 Å=0.1 nm) outside (Figure 3A). Otherwise, Val397 of RARα bound to ATRA would have steric clashes with the luffariellolide ligand. In addition to a larger ligand-binding pocket, there are also several unique features observed for the luffariellolide-bound RARα structure. Luffariellolide initiated a novel Van der Waals contact with Trp225 from helix 3 (Figure 3C), which facilitates the binding of this larger ligand. Indeed, the W225F mutation that disrupts this interaction decreased the activation of RARα by luffariellolide, but had no effect on ATRA in cell-based assays using a RAR response reporter (Figure 3E).
Remarkably, the electron density map reveals that Cys235 from RARα forms a covalent bond with the ketone group of the γ-hydroxybutenolide ring terminus from luffariellolide (Figure 3D and Supplementary Figure S4A at http://www.BiochemJ.org/bj/446/bj4460079add.htm), which is different from a carboxylate group for retinoids at the same position. The existence of a covalent binding of luffariellolide with RARα was validated by MS analysis as shown in Figure 4. RARα LBDs showed similar MS results when complexed with DMSO and ATRA. In contrast, incorporation of luffariellolide into the RARα LBD yielded a mass addition corresponding to its molecular mass. To further assess the roles of this covalent modification on luffariellolide binding and RARα activation, we mutated Cys235 from RARα and tested the transcriptional activity of these mutated RARα in response to luffariellolide in cell-based reporter assays. Both the C235A and C235L mutations abolished or substantially reduced the activation of RARα by luffariellolide, but not by ATRA (Figure 3E). Unlike ATRA, luffariellolide failed to induce the interaction of co-activator LXXLL motif with both C235A and C235L mutations by Alphascreen assays (Supplementary Figure S4B). The loss of a covalent binding of luffariellolide with the RARα C235A mutant was also validated by MS analysis as shown in Supplementary Figure S4(C), whereas the RARα C235A mutant had no effect on the luffariellolide binding (Supplementary Figure S4D). Interestingly, pre-treatment of RARα with luffariellolide prevents the effects of both the RAR agonist ATRA and the RAR inverse agonist BMS 493, whereas luffariellolide and BMS 493 affected the co-factor binding by RARα pre-treated with ATRA, suggesting that the covalent modification on RARα by luffariellolide is irreversible (Supplementary Figure S2B). As shown in Supplementary Figure S5(A) (at http://www.BiochemJ.org/bj/446/bj4460079add.htm), sequence alignment indicates that Cys235 of RARα is conserved for all three of the RAR subtypes. Indeed, MS results validated the covalent modifications on RARβ and RARγ by luffariellolide (Supplementary Figures S5B and S5C). All of these results reaffirm the importance of this unique binding mode of luffariellolide to RARs.
To further assess the roles of luffariellolide in the physiological function of RARs, we studied the effects of luffariellolide on the growth of the monocytic leukaemia cell line THP-1 by MTT and flow cytometry assays. Similar to ATRA, luffariellolide inhibited the growth of THP-1 cells and also increased the population of cells in G1-phase (Figures 5A and 5B). We further performed RAR-target gene analysis on the THP-1 cancer cells treated with luffariellolide. As shown in Figure 5(C), the treatment of luffariellolide substantially induced mRNA of several known RAR target genes including RARα, RARβ  and IRF-1 (interferon regulatory factor-1) . In addition, the expression of RAR target genes was also induced by luffariellolide in the promyeloid leukaemic cell line HL-60 and the breast carcinoma line MCF-7 (Supplementary Figure S6 at http://www.BiochemJ.org/bj/446/bj4460079add.htm), reaffirming that luffariellolide is a potent activator of RAR-responsive genes. Taken together, the gene expression analysis is consistent with cell reporter assays and biochemical binding studies, further supporting luffariellolide as an RAR agonist.
One big challenge that limits the use of RAR ligands for cancer therapy is the development of RA resistance in various types of cancer cell lines. We next investigated the ability of luffariellolide to inhibit cell growth on the RA-resistant colon cancer cell line HCT-116 . As shown in Figure 6(A) and Supplementary Figure S7 (at http://www.BiochemJ.org/bj/446/bj4460079add.htm), both ATRA and luffariellolide were able to inhibit the growth of the RA-sensitive colon cancer cell line HCT-15. As expected, ATRA failed to have effects on the growth of HCT-116 cells. Surprisingly, the growth of HCT-116 cells was substantially inhibited by treatment with luffariellolide (Figure 6A), suggesting a potential advantage of luffariellolide over ATRA in treating cancer cells. Although the precise mechanisms for causing RA resistance is not yet clear, several factors in RA signalling have been shown to play important roles, such as poor expression of CRABPII (cellular RA-binding protein 2)  and loss of RARβ induction in response to RA . Interestingly, both RARβ and CRABPII have been suggested as tumour suppressors that are key to the anti-tumour action of RA [38–40]. Since the defects of gene induction of RARβ and CRABPII by ATRA may contribute to RA resistance [36,41,42], we next examined the effects of luffariellolide on the expression of these two genes. As shown in Figures 6(B) and 6(C), the expression of RARβ and CRABPII was induced by both ATRA and luffariellolide in HCT-15 cells. Conversely, ATRA treatment had no effect on the expression of these two genes in RA-resistant HCT-116 cells. However, luffariellolide was able to induce the expression of RARβ and CRABPII in HCT-116 cells, consistent with its ability to affect the growth of this cell line. Furthermore, the effects of luffariellolide on cell growth and gene expression was abolished or alleviated by the pan-RAR inverse agonist BMS 493, suggesting that luffariellolide affects cancer cells through binding to RARs. The advantage of luffariellolide on RA-resistant colon cancer cells may be related to its unique covalent binding mode with RARs, which strengthens the downstream RA signalling that overcomes the RA resistance.
In summary, we have shown in the present study the identification of a natural marine product that activates RARs with a unique anti-proliferative and pro-apoptotic activity. Given the importance of RAR ligands as therapeutic agents, especially anti-tumour drugs, the development of novel small molecules that regulate RAR activity has received tremendous attention [43,44]. Various strategies have been reported for drug design targeting retinoid receptors, like agonists specific for one receptor subtype and inverse agonists for RARs. However, many RAR ligands so far are structurally related to retinoids, which have been associated with many undesired side effects and also drug resistance. As a novel natural RAR agonist, luffariellolide displayed several unique features distinct from retinoids and other synthetic RAR ligands. First, luffariellolide represents an alternative scaffold for RAR ligands characterized by a distinctive γ-hydroxybutenolide ring terminus compared with a carboxylic acid for retinoids. Secondly, luffariellolide adopts a distinct binding mode with RARα using a covalent modification with its γ-hydroxybutenolide ring. This covalent binding, which has not been observed for any other RAR ligands, facilitates the stable interactions of RARs with its ligands. Thus the unique characteristics of the γ-hydroxybutenolide ring may represent a new pharmacophore that can be optimized for selectively targeting RARs. More importantly, this natural RAR agonist also showed activity in RA-resistant cancer cells. Taken together, the identification of luffariellolide as a novel RAR agonist may provide an alternative drug design strategy for non-retinoid compounds with advantages over current RA drugs.
Shanshan Wang and Zhao Wang designed and conducted the experiments, and contributed to writing the paper. Shengchen Lin, Weili Zheng, Rui Wang, Shikai Jin, Jinan Chen and Lihua Jin conducted the experiments. Yong Li designed the experiments and wrote the paper.
This work was supported by the National Basic Research Program of China [grant number 2012CB910104], the National Natural Science Foundation of China [grant number 31070646], the Ministry of Education of China [grant number B06016] and the National Science Foundation of Fujian Province [grant number 2010J01230].
We thank the staff at BL17U of Shanghai Synchrotron Radiation Source and the Advanced Photon Source for assistance in data collection.
The atomic co-ordinates and structure factors for RARα–luffariellolide have been deposited in the Protein Data Bank under code 4DQM.
Abbreviations: AF, activation function; ATRA, all-trans-retinoic acid; CRABPII, cellular retinoic acid-binding protein 2; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Gal4, Galectin-4; GR, glucocorticoid receptor; LBD, ligand-binding domain; LXRα, liver X receptor α; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PPARγ, peroxisome-proliferator-activated receptor γ; RA, retinoic acid; RAR, RA receptor; RXR, retinoid X receptor; SRC, steroid receptor co-activator
- © The Authors Journal compilation © 2012 Biochemical Society