Natural β-apocarotenoid; RXRα.

In rat intestinal mucosal homogenates, β-apo-13-carotenone was found to be the enzymatic cleavage product of β-carotene. At concentrations as low as 1 nM, β-apo-13-carotene was found to be effective in opposing 9-cis-retinoic acid's activation of RXRα. β-apo-13-carotene has been demonstrated in molecular modeling studies to generate molecular interactions akin to an RXRα antagonist [1]. The binding affinity of β-apo-13-carotene (7–8 nM) for 9cRA to RXRα is the same as that of 9cRA. β-apo-13-carotenone has an antagonistic effect on 9cRA activation of full-length hRXRα that is comparable to that of the well-known UVI-3003. β-apo-13-carotene does not prevent coactivator recruitment to the isolated LBD, but it does cause the formation of transcriptionally silent tetramers of RXRα [2]. These beta-carotene metabolites cannot build up in cells due to the absorption and/or metabolism of beta-apo-13-carotene. Treatment with β-apo-carotenoid induces the expression of the 3T3-L1 adipocyte marker gene [3].

RXRα Transactivation Assay[1]
Cos-1 cells were plated at 1.5 × 105 cells/35-mm2 tissue-culture plate in DMEM with 10% FBS. The cells were grown overnight at 37°C with 5% CO2. The next day, the cells were transfected in serum-free medium with three plasmids mixed in the following amounts per well, 0.05 µg of pRL-TK, 2 µg of pRXRE-luciferase, 2.5 µg of pSG5-RXRα in triplicates using Lipofectamine 2000 according to the manufacturer's protocol. Following transfection, the plates were incubated at 37°C in 5 % CO2 for 4 h. The medium was then changed to complete DMEM. Note that complete DMEM contains 10% charcoal stripped FBS instead of 10% FBS. Charcoal stripped FBS has been treated with activated carbon to adsorb lipophilic compounds including retinoids. Twenty hours after transfection, cells were treated with test compounds that were dissolved in ethanol or 0.1% ethanol alone for an additional 24 h. Cells were washed once with PBS and lysed by incubation with 500 µl passive lysis buffer for 15 min at room temperature. A 20-µl aliquot of cell lysate was then assayed for luciferase activities using a GloMax 96 Microplate Luminometer (Promega) and the Dual Luciferase Reporter (DLR) assay system, according to the supplier's recommendations. For each experiment, the firefly luciferase activity (experimental reporter) was normalized to Renilla luciferase (control reporter) activity. The change in normalized firefly luciferase activity was calculated relative to that for cells that were transfected with vehicle (ethanol), which was set as 1. Fold Activation = Average (Firefly/Renilla) from test compound / Average (Firefly/Renilla) from vehicle (Ethanol treated cells)

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Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that β-Apo-13-carotenone, an eccentric cleavage product of β-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of β-Apo-13-carotenone's modulation on the transcriptional activity of RXRα is not understood and is the subject of this report. We performed transactivation assays using full-length RXRα and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. β-Apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both β-Apo-13-carotenone and UVI3003 shifted the dose-dependent RXRα activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but β-apo-13-carotenone did not. However, both β-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of β-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. β-Apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that β-apo-13-carotenone regulates RXRα transcriptional activity by inducing the formation of the "transcriptionally silent" RXRα tetramer.[2]

Nuclear Receptor Reporter Cell Assay with Full-length hRXRα[2]
COS-7 cells were cultured in 96-well plates overnight. cDNA of full-length human RXRα in pSV sport vector (Addgene) was cotransfected with Renilla (pRL-tk) and Firefly luciferase (RXRE-Luc) reporter constructs into COS-7 cells in serum-free DMEM with X-tremeGENE 9 DNA. Twenty four hours after transfection, COS-7 cells in DMEM with 10% charcoal-stripped FBS were then treated with 9cRA in the presence or absence of β-Apo-13-carotenone or UVI3003 for an additional 24 h. Cell lysates were used in the dual-luciferase assay to determine the activation of hRXRα by 9cRA and the inhibition by β-Apo-13-carotenone and UVI3003. For each experiment, the firefly luciferase (experimental reporter) activity was normalized to Renilla luciferase (control reporter).

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Human RXRαLBD Reporter Cell Assay[2]
Reporter cells expressing human RXRαLBD fused to the GAL-4 DBD were treated according to the manufacturer's protocol. Reporter cells were incubated with 0, 0.32, 1.6, 8, 40, 200, 1000, and 5000 nm 9cRA for 24 h at 37 °C in the presence or absence of fixed concentrations of either β-Apo-13-carotenone or UVI3003. Luminescence was detected with Glomax96 luminometer.

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β-Apo-carotenoids, including β-Apo-13-carotenone and β-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of β-apo-13-carotenone and β-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these β-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of β-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two β-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either β-apo-13-carotenone or β-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some β-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.[3]

[1]. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα. Arch Biochem Biophys. 2010 Dec 1;504(1):11-6.

[2]. β-Apo-13-carotenone regulates retinoid X receptor transcriptional activity through tetramerization of the receptor. J Biol Chem. 2014 Nov 28;289(48):33118-24.

[3]. Actions of β-apo-carotenoids in differentiating cells: differential effects in P19 cells and 3T3-L1 adipocytes. Arch Biochem Biophys. 2015 Apr 15;572:2-10.

13-apo-beta-carotenone is an apo carotenoid compound arising from oxidative degradation of the beta,beta-carotene skeleton at the 13-position. It is an apo carotenoid and an enone.

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In this study, we investigated the effects of eccentric cleavage products of β-carotene, i.e. β-apocarotenoids (BACs), on retinoid X receptor alpha (RXRα) signaling. Transactivation assays were performed to test whether BACs activate or antagonize RXRα. Reporter gene constructs (RXRE-Luc, pRL-tk) and RXRα were transfected into Cos-1 cells and used to perform these assays. None of the BACs tested activated RXRα. Among the compounds tested, β-apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis-retinoic acid and was effective at concentrations as low as 1 nM. Molecular modeling studies revealed that β-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling.[1]

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Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that β-apo-13-carotenone, an eccentric cleavage product of β-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of β-apo-13-carotenone's modulation on the transcriptional activity of RXRα is not understood and is the subject of this report. We performed transactivation assays using full-length RXRα and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. β-Apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both β-apo-13-carotenone and UVI3003 shifted the dose-dependent RXRα activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but β-apo-13-carotenone did not. However, both β-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of β-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. β-Apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that β-apo-13-carotenone regulates RXRα transcriptional activity by inducing the formation of the "transcriptionally silent" RXRα tetramer.[2]

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In summary, this study revealed the mechanism of ligand-dependent regulation of RXRα transcriptional activity by the antagonist β-apo-13-carotenone. The findings imply that tetramerization of RXR and factors that modulate the oligomer state may contribute to regulation of cellular signaling. β-Apo-13-carotenone-induced tetramerization could conserve RXRα as an inactive nuclear receptor pool that can rapidly supply dimeric or monomeric RXRα upon 9cRA generation. This may also suggest a ligand-dependent modulation controlling the availability of RXRα for the heterodimerization with other nuclear receptor partners engaged in multiple signaling pathways.[2]

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In summary, our studies in P19 cells indicate that the uptake and/or metabolism of β-apo-13-carotenone and β-apo-14′-carotenal does not allow for accumulation of these β-carotene metabolites in cells. This suggests that the RAR antagonist activities of each of these two β-apo-carotenoids will be limited by the intrinsic capacities of cells to obtain/retain sufficient quantities of each to allow for transcriptional regulatory actions to occur. Our data also suggest that β-apo-carotenoids, such as apo-13-carotenone and apo-10′-carotenoic acid, may have important modulatory effects on various cellular processes, and that some of these observed effects may be mediated through additional mechanisms other than RAR antagonism. Both of these conclusions will need to be explored in much more depth before the biological actions of the enzymatic and non-enzymatic β-apo-carotenoid by-products of β-carotene can be understood.[3]

C₁₈H₂₆O

258.40

258.198

17974-57-1

5363697

Light yellow to yellow ointment

5.16

0

1

4

19

456

0

CC(/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)=O

UBTNVRPIHJRBCI-LUXGDSJYSA-N

InChI=1S/C18H26O/c1-14(8-6-10-16(3)19)11-12-17-15(2)9-7-13-18(17,4)5/h6,8,10-12H,7,9,13H2,1-5H3/b10-6+,12-11+,14-8+

(3E,5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohexen-1-yl)octa-3,5,7-trien-2-one

beta-Apo-13-carotenone; 17974-57-1; 13-apo-beta-carotenone; (beta)-Apo-13-carotenone; 13-apo-beta-caroten-12-one; D'Orenone; (3E,5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohexen-1-yl)octa-3,5,7-trien-2-one; CHEBI:53175;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~110 mg/mL (~425.70 mM)

Solubility in Formulation 1: 2.75 mg/mL (10.64 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.67 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)