The retinoid X receptor has a critical role in synthetic rexinoid-induced increase in cellular all-trans-retinoic acid.

Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.

Retinoid X Receptor agonists as selective modulators of the immune system for the treatment of cancer.

Upon heterodimerizing with other nuclear receptors, retinoid X receptors (RXR) act as ligand-dependent transcription factors, regulating transcription of critical signaling pathways that impact numerous hallmarks of cancer. By controlling both inflammation and immune responses, ligands that activate RXR can modulate the tumor microenvironment. Several small molecule agonists of these essential receptors have been synthesized. Historically, RXR agonists were tested for inhibition of growth in cancer cells, but more recent drug discovery programs screen new molecules for inhibition of inflammation or activation of immune cells. Bexarotene is the first successful example of an effective therapeutic that molecularly targets RXR; this drug was approved to treat cutaneous T cell lymphoma and is still used as a standard of care treatment for this disease. No additional RXR agonists have yet achieved FDA approval, but several promising novel compounds are being developed. In this review, we provide an overview of the multiple mechanisms by which RXR signaling regulates inflammation and tumor immunity. We also discuss the potential of RXR-dependent immune cell modulation for the treatment or prevention of cancer and concomitant challenges and opportunities.

RXR Agonists Enhance Lenalidomide Anti-Myeloma Activity and T Cell Functions while Retaining Glucose-Lowering Effect.

Retinoid X receptor (RXR) heterodimerizes with the PPAR nuclear hormone receptor and regulates its downstream events. We investigated the effects of RXR agonists (LG100754, bexarotene, AGN194204, and LG101506) on lenalidomide's anti-myeloma activity, T cell functions, and the level of glucose and lipids in vivo. Genetic overexpression and CRISPR/Cas9 knockout experiments were conducted in multiple myeloma (MM) cell lines and Jurkat T cell lines to determine the roles of CRBN in RXR-agonist mediated effects. A xenograft mouse model of MM was established to determine the combination effect of LG100754 and lenalidomide. The combination of RXR agonists and lenalidomide demonstrated synergistic activity in increasing CRBN expression and killing myeloma cells. Mechanistically, the RXR agonists reduced the binding of PPARs to the CRBN promoter, thereby relieving the repressor effect of PPARs on CRBN transcription. RXR agonists downregulated the exhaustion markers and increased the activation markers of Jurkat T cells and primary human T cells. Co-administration of LG100754 and lenalidomide showed enhanced anti-tumor activity in vivo. LG100754 retained its glucose- and lipid-lowering effects. RXR agonists demonstrate potential utility in enhancing drug sensitivity and T-cell function in the treatment of myeloma.

The novel function of bexarotene for neurological diseases.

Bexarotene, a retinoid X receptor (RXR) agonist, is approved by FDA to treat cutaneous T-cell lymphoma. However, it has also demonstrated promising therapeutic potential for neurological diseases such as stroke, traumatic brain injury, Parkinson's disease, and particularly Alzheimer's disease(AD). In AD, bexarotene inhibits the production and aggregation of amyloid ß (Aß), activates Liver X Receptor/RXR heterodimers to increase lipidated apolipoprotein E to remove Aß, mitigates the negative impact of Aß, regulates neuroinflammation, and ultimately improves cognitive function. For other neurological diseases, its mechanisms of action include inhibiting inflammatory responses, up-regulating microglial phagocytosis, and reducing misfolded protein aggregation, all of which aid in alleviating neurological damage. Here, we briefly discuss the characteristics, applications, and adverse effects of bexarotene, summarize its pharmacological mechanisms and therapeutic results in various neurological diseases, and elaborate on the problems encountered in preclinical research, with the aim of providing help for the further application of bexarotene in central nervous system diseases.

The Novel RXR Agonist MSU-42011 Differentially Regulates Gene Expression in Mammary Tumors of MMTV-Neu Mice.

Retinoid X receptor (RXR) agonists, which activate the RXR nuclear receptor, are effective in multiple preclinical cancer models for both treatment and prevention. While RXR is the direct target of these compounds, the downstream changes in gene expression differ between compounds. RNA sequencing was used to elucidate the effects of the novel RXRα agonist MSU-42011 on the transcriptome in mammary tumors of HER2+ mouse mammary tumor virus (MMTV)-Neu mice. For comparison, mammary tumors treated with the FDA approved RXR agonist bexarotene were also analyzed. Each treatment differentially regulated cancer-relevant gene categories, including focal adhesion, extracellular matrix, and immune pathways. The most prominent genes altered by RXR agonists positively correlate with survival in breast cancer patients. While MSU-42011 and bexarotene act on many common pathways, these experiments highlight the differences in gene expression between these two RXR agonists. MSU-42011 targets immune regulatory and biosynthetic pathways, while bexarotene acts on several proteoglycan and matrix metalloproteinase pathways. Exploration of these differential effects on gene transcription may lead to an increased understanding of the complex biology behind RXR agonists and how the activities of this diverse class of compounds can be utilized to treat cancer.

Bexarotene enhances astrocyte phagocytosis via ABCA1-mediated pathways in a mouse model of subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Enhancing phagocytosis can facilitate the removal of inflammatory molecules, limit the toxicity of dead cells and debris, and promote recovery after brain injury. In this study, we aimed to explore the role of bexarotene (Bex), a retinoid X receptor (RXR) agonist, in promoting astrocyte phagocytosis and neurobehavioral recovery after subarachnoid hemorrhage (SAH). METHODS: Mice SAH model was induced by pre-chiasmatic injection of blood. Modified Garcia score, novel object recognition, rotarod test, and Morris water maze were performed to assess neurological function. Immunofluorescence and electron microscopy were used to evaluate astrocyte phagocytosis in vivo. In addition， ABCA1/MEGF10&GULP1, the primary astrocyte phagocytosis pathway, were stimulated by Bex or suppressed by HX531 (a RXR antagonist) to evaluate their impacts on astrocyte phagocytosis and neurological recovery. RESULTS: Astrocytes phagocytosis of blood components were observed in mice after SAH induction, which is further increased by Bex treatment. Bex dramatically attenuated neuroinflammation, reduced brain edema, improved early neurological performance and promoted neurocognitive recovery. Meanwhile, Bex decreased neurotoxic reactive astrocytes and preserved neurogenesis after SAH. Bex increased the expression of astrocyte phagocytosis-related proteins ABCA1, MEGF10, and GULP1. Bex also increased the lysosomal processing of engulfed blood components in astrocytes. Moreover, Bex significantly promoted astrocytes to phagocytize debris in vitro by increasing the expression of ABCA1, MEGF10 and GULP1, while HX531 inhibited astrocyte phagocytosis and decreased these protein levels. CONCLUSIONS: Bex enhanced astrocyte phagocytosis through the ABCA1-mediated pathways, and promoted neurobehavior recovery in mice after SAH induction.

Remyelination in humans due to a retinoid-X receptor agonist is age-dependent.

Remyelination efficiency declines with advancing age in animal models, but this has been harder to demonstrate in people with multiple sclerosis. We show that bexarotene, a putatively remyelinating retinoid-X receptor agonist, shortened the visual evoked potential latency in patients with chronic optic neuropathy aged under 42 years only (with the effect diminishing by 0.45 ms per year of age); and increased the magnetization transfer ratio of deep gray matter lesions in those under 43 years only. Addressing this age-related decline in human remyelination capacity will be an important step in the development of remyelinating therapies that work across the lifespan.

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

Development of Scaled-Up Synthetic Method for Retinoid X Receptor Agonist NEt-3IB Contributing to Sustainable Development Goals.

Small-molecular drugs, which are generally inexpensive compared with biopharmaceuticals and can often be taken orally, may contribute to the Sustainable Development Goals (SDGs) adopted by the United Nations. We previously reported the retinoid X receptor (RXR) agonist 4-(ethyl(3-isobutoxy-4-isopropylphenyl)amino)benzoic acid (NEt-3IB, 1) as a small-molecular drug candidate to replace biopharmaceuticals for the treatment of inflammatory bowel disease. The previous synthetic method to 1 required a large amount of organic solvent and extensive purification. In line with the SDGs, we aimed to develop an environmentally friendly, inexpensive method for the large-scale synthesis of 1. The developed method requires only a hydrophobic ether and EtOH as reaction and extraction solvents. The product was purified by recrystallization twice to afford 99% pure 1 at 100 mmol scale in about 30% yield. The optimized process showed a 35-fold improvement of the E-factor (an index of environmental impact) compared to the original method. This work, which changes the solvent used to environmentally preferable ones based on the existing synthetic method for 1, illustrates how synthetic methods for small-molecular drugs can be adapted and improved to contribute to the SDGs.

The rexinoid V-125 reduces tumor growth in preclinical models of breast and lung cancer.

Rexinoids are ligands which activate retinoid X receptors (RXRs), regulating transcription of genes involved in cancer-relevant processes. Rexinoids have anti-neoplastic activity in multiple preclinical studies. Bexarotene, used to treat cutaneous T cell lymphoma, is the only FDA-approved rexinoid. Bexarotene has also been evaluated in clinical trials for lung and metastatic breast cancer, wherein subsets of patients responded despite advanced disease. By modifying structures of known rexinoids, we can improve potency and toxicity. We previously screened a series of novel rexinoids and selected V-125 as the lead based on performance in optimized in vitro assays. To validate our screening paradigm, we tested V-125 in clinically relevant mouse models of breast and lung cancer. V-125 significantly (p < 0.001) increased time to tumor development in the MMTV-Neu breast cancer model. Treatment of established mammary tumors with V-125 significantly (p < 0.05) increased overall survival. In the A/J lung cancer model, V-125 significantly (p < 0.01) decreased number, size, and burden of lung tumors. Although bexarotene elevated triglycerides and cholesterol in these models, V-125 demonstrated an improved safety profile. These studies provide evidence that our screening paradigm predicts novel rexinoid efficacy and suggest that V-125 could be developed into a new cancer therapeutic.

A2E-induced inflammation and angiogenesis in RPE cells in vitro are modulated by PPAR-α, -ß/δ, -γ, and RXR antagonists and by norbixin.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9'-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -ß/δ and -γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E's deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

The MS Remyelinating Drug Bexarotene (an RXR Agonist) Promotes Induction of Human Tregs and Suppresses Th17 Differentiation In Vitro.

The retinoid X receptor agonist bexarotene promotes remyelination in patients with multiple sclerosis. Murine studies have also demonstrated that RXR agonists have anti-inflammatory effects by enhancing the ability of all-trans-retinoic acid (atRA) to promote T-regulatory cell (Treg) induction and reduce Th17 differentiation in vitro. By stimulating human naïve CD4 T-cells in the presence of Treg or Th17 skewing cytokines, we show that bexarotene also tips the human Treg/Th17 axis in favor of Treg induction, but unlike murine cells this occurs independently of atRA and retinoic acid receptor signaling. Tregs induced in the presence of bexarotene express canonical markers of T-regulation and are functionally suppressive in vitro. Circulating Treg numbers did not increase in the blood of trial patients receiving bexarotene; we believe this is because Treg induction is likely to occur within tissues. These findings lend support to developing RXR agonists as treatments of autoimmune diseases, in particular multiple sclerosis.

Safety and efficacy of bexarotene in patients with relapsing-remitting multiple sclerosis (CCMR One): a randomised, double-blind, placebo-controlled, parallel-group, phase 2a study.

BACKGROUND: Progressive disability in multiple sclerosis occurs because CNS axons degenerate as a late consequence of demyelination. In animals, retinoic acid receptor RXR-gamma agonists promote remyelination. We aimed to assess the safety and efficacy of a non-selective retinoid X receptor agonist in promoting remyelination in people with multiple sclerosis. METHODS: This randomised, double-blind, placebo-controlled, parallel-group, phase 2a trial (CCMR One) recruited patients with relapsing-remitting multiple sclerosis from two centres in the UK. Eligible participants were aged 18-50 years and had been receiving dimethyl fumarate for at least 6 months. Via a web-based system run by an independent statistician, participants were randomly assigned (1:1), by probability-weighted minimisation using four binary factors, to receive 300 mg/m2 of body surface area per day of oral bexarotene or oral placebo for 6 months. Participants, investigators, and outcome assessors were masked to treatment allocation. MRI scans were done at baseline and at 6 months. The primary safety outcome was the number of adverse events and withdrawals attributable to bexarotene. The primary efficacy outcome was the patient-level change in mean lesional magnetisation transfer ratio between baseline and month 6 for lesions that had a baseline magnetisation transfer ratio less than the within-patient median. We analysed the primary safety outcome in the safety population, which comprised participants who received at least one dose of their allocated treatment. We analysed the primary efficacy outcome in the intention-to-treat population, which comprised all patients who completed the study. This study is registered in the ISRCTN Registry, 14265371, and has been completed. FINDINGS: Between Jan 17, 2017, and May 17, 2019, 52 participants were randomly assigned to receive either bexarotene (n=26) or placebo (n=26). Participants who received bexarotene had a higher mean number of adverse events (6·12 [SD 3·09]; 159 events in total) than did participants who received placebo (1·63 [SD 1·50]; 39 events in total). All bexarotene-treated participants had at least one adverse event, which included central hypothyroidism (n=26 vs none on placebo), hypertriglyceridaemia (n=24 vs none on placebo), rash (n=13 vs one on placebo), and neutropenia (n=10 vs none on placebo). Five (19%) participants on bexarotene and two (8%) on placebo discontinued the study drug due to adverse events. One episode of cholecystitis in a placebo-treated participant was the only serious adverse event. The change in mean lesional magnetisation transfer ratio was not different between the bexarotene group (0·25 percentage units [pu; SD 0·98]) and the placebo group (0·09 pu [0·84]; adjusted bexarotene-placebo difference 0·16 pu, 95% CI -0·39 to 0·71; p=0·55). INTERPRETATION: We do not recommend the use of bexarotene to treat patients with multiple sclerosis because of its poor tolerability and negative primary efficacy outcome. However, statistically significant effects were seen in some exploratory MRI and electrophysiological analyses, suggesting that other retinoid X receptor agonists might have small biological effects that could be investigated in further studies. FUNDING: Multiple Sclerosis Society of the United Kingdom.

Retinoids promote penis development in sequentially hermaphroditic snails.

Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.

Oxaprozin Analogues as Selective RXR Agonists with Superior Properties and Pharmacokinetics.

The retinoid X receptors (RXR) are ligand-activated transcription factors involved in multiple regulatory networks as universal heterodimer partners for nuclear receptors. Despite their high therapeutic potential in many pathologies, targeting of RXR has only been exploited in cancer treatment as the currently available RXR agonists suffer from exceptional lipophilicity, poor pharmacokinetics (PK), and adverse effects. Aiming to overcome the limitations and to provide improved RXR ligands, we developed a new potent RXR ligand chemotype based on the nonsteroidal anti-inflammatory drug oxaprozin. Systematic structure-activity relationship analysis enabled structural optimization toward low nanomolar potency similar to the well-established rexinoids. Cocrystal structures of the most active derivatives demonstrated orthosteric binding, and in vivo profiling revealed superior PK properties compared to current RXR agonists. The optimized compounds were highly selective for RXR activation and induced RXR-regulated gene expression in native cellular and in vivo settings suggesting them as excellent chemical tools to further explore the therapeutic potential of RXR.

DHA and Its Metabolites Have a Protective Role against Methylmercury-Induced Neurotoxicity in Mouse Primary Neuron and SH-SY5Y Cells.

The consumption of fish now involves a risk of methylmercury (MeHg) exposure but also provides the benefit of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) such as docosahexaenoic acid (DHA). Some epidemiological studies have suggested that the intake of DHA can alleviate the neurotoxicity of MeHg, but the underlying mechanism is not known. Herein, we observed that pretreatment with 0.1-1 µM DHA suppressed MeHg-induced cytotoxicity in human neuroblastoma (SH-SY5Y) cells and mouse primary neuronal cells. These effects of DHA were canceled in the presence of the retinoid X receptor (RXR) antagonist UVI3003. An RXR agonist, bexarotene, suppressed the cytotoxicity of MeHg. DHA also suppressed the MeHg-induced production of reactive oxygen species (ROS) via an induction of antioxidant genes (catalase and SOD1). Pretreatment with DHA did not change the incorporation of MeHg. We showed previously that in the brain, the intake of DHA increased the level of 19,20-DHDP, which is the metabolite produced by cytochrome P450 and soluble epoxide hydrolase from DHA. In the present study, we observed that 19,20-DHDP also suppressed neurotoxicity from MeHg. These results indicate that DHA and its metabolites have a protective role in MeHg-induced neurotoxicity.

Retinoid X receptor activation promotes re-myelination in a very old triple transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the main cause of dementia in the world. Studies of human AD brains show abnormalities in the white matter and reduction of myelin and oligodendrocyte markers. It has been proposed that oligodendrocyte progenitor cells (OPCs) present in the adult brain are a potential source for re-myelination, through proliferation and differentiation into mature oligodendrocytes. Bexarotene, a Retinoid X Receptor agonist, has been demonstrated to reverse behavioral deficits and to improved synaptic transmission and plasticity in murine models of AD, which was associated with the reduction of soluble Aß peptides. In the present study, we analyzed changes in the expression of oligodendrocyte lineage markers following oral administration of Bexarotene in a very old (24-month-old) triple transgenic mouse model of AD (3xTg-AD), for which early demyelination changes have been previously described. Bexarotene increased the expression of OPCs and intermediate oligodendrocyte progenitors (Olig2+ and O4+), and increased the number of mitotic (O4+) and myelinating mature (MBP+) oligodendrocytes. We clearly show that Bexarotene promotes re-myelination which might be important for the previously observed cognitive improvement of 3xTg-AD mice treated with this drug.

Bexarotene inhibits cell proliferation by inducing oxidative stress, DNA damage and apoptosis via PPARγ/ NF-κB signaling pathway in C6 glioma cells.

Gliomas are one of the most aggressive brain tumors with a poor prognosis in the central nervous system. Bexarotene is a third-generation retinoid X receptor agonist that is promising in the treatment of both cancer and neurodegenerative diseases. In this study, we aimed to investigate the cytotoxic and anti-proliferative effects of bexarotene in C6 glioma cells through the PPARγ/NF-κB pathway. In the study, first cytotoxic bexarotene concentrations for C6 cells were detected, and then apoptosis profile, reactive oxygen species (ROS), total antioxidant (TAS), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor-κB (NF-κB) levels in the cells were determined. In addition, peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression analysis was carried out. As a result, we detected concentration- and time-dependent antiproliferative effects of bexarotene on C6 cells. We found that bexarotene treatment decreased NF-κB and TAS levels and increased PPARγ and 8-OHdG levels in C6 cells. Bexarotene enhanced PPARγ expression in a dose-dependent manner when compared to the control group (P < 0.01). Furthermore, we determined that bexarotene-induced apoptotic C6 cells enhanced through Annexin V-FITC/PI staining and caspase-3/-7 activation analyses since phosphatidylserine level on the outer surface of the cell membrane and caspase-3/-7 activities were increased in the cells treated with bexarotene. In conclusion, bexarotene treatment in C6 glioma cells could modulate apoptosis profile, DNA damage, ROS production, and reduction of TAS levels through inhibition of NF-κB by enhancing PPARγ expression.

Evaluating Novel RXR Agonists That Induce ApoE and Tyrosine Hydroxylase in Cultured Human Glioblastoma Cells.

There is considerable interest in identifying effective and safe drugs for neurodegenerative disorders. Cell culture and animal model work have demonstrated that modulating gene expression through RXR-mediated pathways may mitigate or reverse cognitive decline. However, because RXR is a dimeric partner for several transcription factors, activating off-target transcription is a concern with RXR ligands (rexinoids). This off-target gene modulation leads to unwanted side effects that can include low thyroid function and significant hyperlipidemia. There is a need to develop rexinoids that have binding specificity for subsets of RXR heterodimers, to drive desired gene modulation, but that do not induce spurious effects. Herein, we describe experiments in which we analyze a series of novel and previously reported rexinoids for their ability to modulate specific gene pathways implicated in neurodegenerative disorders employing a U87 cell culture model. We demonstrate that, compared to the FDA-approved rexinoid bexarotene (1), several of these compounds are equally or more effective at stimulating gene expression via LXREs or Nurr1/NBREs and are superior at inducing ApoE and/or tyrosine hydroxylase (TH) gene and protein expression, including analogs 8, 9, 13, 14, 20, 23, and 24, suggesting a possible therapeutic role for these compounds in Alzheimer's or Parkinson's disease (PD). A subset of these potent RXR agonists can synergize with a presumed Nurr1 ligand and antimalarial drug (amodiaquine) to further enhance Nurr1/NBREs-directed transcription. This novel discovery has potential clinical implications for treatment of PD since it suggests that the combination of an RXR agonist and a Nurr1 ligand can significantly enhance RXR-Nurr1 heterodimer activity and drive enhanced therapeutic expression of the TH gene to increase endogenous synthesis of dopamine. These data indicate that is it possible and prudent to develop novel rexinoids for testing of gene expression and side effect profiles for use in potential treatment of neurodegenerative disorders, as individual rexinoids can have markedly different gene expression profiles but similar structures.

Suppression of TLR4/MyD88/TAK1/NF-κB/COX-2 Signaling Pathway in the Central Nervous System by Bexarotene, a Selective RXR Agonist, Prevents Hyperalgesia in the Lipopolysaccharide-Induced Pain Mouse Model.

A selective RXR agonist, bexarotene, has been shown to have anti-inflammatory, anti-nociceptive, and neuroprotective effects in several models of numerous neurological diseases characterized by systemic inflammation. The mechanisms underlying these effects remains unknown. To elucidate these mechanisms, we investigated whether the TLR4/MyD88/TAK1/NF-κB/COX-2 signaling pathway in the CNS mediates the effect of bexarotene to prevent hyperalgesia in the LPS-induced inflammatory pain mouse model. The reaction time to thermal stimuli within 30 s was evaluated by the hot plate test in male mice treated with saline, LPS (10 mg/kg), DMSO, and/or bexarotene (0.1, 1, 3, or 10 mg/kg) after 6 h. The latency to the thermal stimulus (18.11 ± 1.36 s) in the LPS-treated mice was significantly decreased by 30% compared with saline-treated mice (25.84 ± 1.99 s). Treatment with bexarotene only at a dose of 10 mg/kg showed a significant increase in the latency by 22.49 ± 1.00 s compared with LPS-treated mice. Bexarotene also prevented the reduction in RXRα protein expression associated with a rise in the expression of TLR4, MyD88, phosphorylated TAK1, NF-κB p65, phosphorylated NF-κB p65, COX-2, and IL-1ß proteins, in addition to COX-2 activity and levels of PGE2 and IL-1ß in the brains and spinal cords of the LPS-treated animals. Likely, decreased activity of TLR4/MyD88/TAK1/NF-κB/COX-2 signaling pathway in addition to increased pro-inflammatory cytokine formation in the CNS of mice participates in the protective effect of bexarotene against hyperalgesia induced by LPS.