Retinol Esters Research Articles

Boosting of retinol activity using novel lecithin: Retinol acyltransferase inhibitors.

Lecithin:retinol acyltransferase (LRAT) is the main enzyme catalysing the esterification of retinol to retinyl esters and, hence, is of central importance for retinol homeostasis. As retinol, by its metabolite retinoic acid, stimulates fibroblasts to synthesize collagen fibres and inhibits collagen-degrading enzymes, the inhibition of LRAT presents an intriguing strategy for anti-ageing ingredients by increasing the available retinol in the skin. Here, we synthesized several derivatives mimicking natural lecithin substrates as potential LRAT inhibitors. By exploring various chemical modifications of the core scaffold consisting of a central amino acid and an N-terminal acylsulfone, we explored 10 different compounds in a biochemical assay, resulting in two compounds with IC50 values of 21.1 and 32.7 μM (compounds 1 and 2), along with a simpler arginine derivative with comparative inhibitory potency. Supported by computational methods, we investigated their structure-activity relationship, resulting in the identification of several structural features associated with high inhibition of LRAT. Ultimately, we conducted an exvivo study with human skin, demonstrating an increase of collagen III associated with a reduction of the skin ageing process. In conclusion, the reported compounds offer a promising approach to boost retinol abundance in human skin and might present a new generation of anti-ageing ingredients for cosmetic application.

Abstract 6971: The impact of lecithin retinol acyltransferase in squamous cell carcinoma: Tissue formation, cell mechanics, and gene transcription

Abstract Squamous cell carcinoma, along with several other cancers, has been found to lack expression of lecithin retinol acyltransferase (LRAT), a protein which catalyzes retinol esterification and thereby mediates retinoic acid biosynthesis. When expression of LRAT was reintroduced in squamous cell carcinoma, the protein promoted cell differentiation in addition to retinoid status normalization—whether this effect was dependent on the known enzymatic activity of LRAT in retinol esterification, versus another function, has not yet been established. Cell elasticity differences have also been reported between squamous cell carcinoma and normal keratinocytes. In this study, the role of LRAT expression and activity was evaluated in human squamous cell carcinoma cell line SCC13. LRAT was integrated into the genome of SCC13 via retroviral transduction, both as wild-type LRAT and LRAT with a cysteine-to-serine mutation at the active site for retinol esterification (C161-S). In these transformed cell lines, LRAT promoted formation of a cohesive sheet of tissue which remained intact after its removal from the tissue culture plastic surface, as evaluated at two weeks post-confluence. SCC13 alone produced tissue which easily fragmented during this scraping assay. In an evaluation of cell elasticity with shear modulation force microscopy, relative cell modulus measurements of pre-confluent SCC13 with LRAT expression were found to be significantly greater and consistent with the cell modulus of human keratinocytes (DO33). These effects persisted with mutation of the retinol esterification active site, suggesting that LRAT possesses an additional function which has yet to be described. Variation in transcription levels of multiple genes—urokinase-type plasminogen activator, plasminogen activator inhibitor 1, tissue inhibitor of metalloproteinases 1, and kallikrein 5—was also found based on LRAT expression and level of confluence. These findings suggest that the presence of LRAT influences tissue formation, cell elasticity, and gene transcription in SCC13. Citation Format: Emily Peterson, Kuan-Che Feng, Jay Gao, Miriam Rafailovich, Marcia Simon. The impact of lecithin retinol acyltransferase in squamous cell carcinoma: Tissue formation, cell mechanics, and gene transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6971.

Super-Antioxidant Vitamin A Derivatives with Improved Stability and Efficacy Using Skin-Permeable Chitosan Nanocapsules.

Retinyl palmitate (RP) is a retinol ester with strong antioxidant and anti-inflammatory properties as an antiwrinkle agent. However, it has poor aqueous solubility and easily degrades into inactive forms for topical applications. Therefore, we developed chitosan-coated nanocapsules (ChiNCs) to encapsulate RP using a simple nanoprecipitation method for protection against physiological conditions and to enable deep skin penetration. The as-prepared RP-loaded nanocapsules (RP@ChiNCs) loaded with approximately 5 wt.% RP exhibited a hydrodynamic diameter of 86 nm and surface charge of 24 mV. They had adequate stability to maintain their physicochemical properties after lyophilization in a biological buffer. Notably, ChiNCs provided RP with remarkable protection against degradation for 4 weeks at 37 °C. Thus, RP@ChiNCs exhibited good antioxidant activity in situ for sufficiently long periods without considerable changes in their efficacy. Furthermore, ChiNCs enhanced the skin penetration of lipophilic RP based on the inherent nature of chitosan. RP@ChiNCs exhibited good in vitro antioxidant and anti-inflammatory effects without causing any cytotoxicity in dermal fibroblasts. Accordingly, they promoted cell proliferation in a wound-scratch test and enhanced collagen synthesis. These results suggest that RP@ChiNCs are promising candidates for cosmetic and biomedical applications.

Cellular retinol-binding protein 1: a therapeutic and diagnostic tumor marker.

Cellular Retinol Binding Protein 1 (CRBP1) gene is a protein coding gene located on human chromosome 3q21, which codifies a protein named CRBP1. CRBP1 is widely expressed in many tissues as a chaperone protein to regulate the uptake, subsequent esterification and bioavailability of retinol. CRBP1 combines retinol and retinaldehyde with high affinity to protect retinoids from non-specific oxidation, and transports retinoids to specific enzymes to promote the biosynthesis of retinoic acid. The vital role of CRBP1 in retinoids metabolism has been gradually discovered, which has been implicated in tumorigenesis. However, the precise functions of CRBP1 in different diseases are still poorly understood. The purpose of this review is to provide an overview of the role of CRBP1 in various diseases, especially in both the promotion and inhibition of cancers, which may also offer a novel biomarker and potential therapeutic target for human diseases.

Hydrophobically modified inulin based nanoemulsions for enhanced stability and transdermal delivery of retinyl propionate

Nanovesicles are attractive for cosmetics application owing to their enhanced stability and transdermal delivery of active ingredients. Retinyl propionate (RP) is a promising anti-aging ingredient but it is usually unstable in formulations. In this work, we developed RP-loaded nanoemulsions using polymeric hydrophobically modified inulin (HMI), small-molecular polysorbate 20 (PS-20) as the main emusifiers and positively-charged behentrimonium chloride (BC) as a zeta-potential adjuster. Systematically optimizing the composition of the three emulsifiers led to small droplet size (<100 nm) and high physical stability of the nanoemulsions. Importantly, RP loaded in the nanoemulsions exhibited a high retention rate exceeding 80% after storage at 50 ℃ for 30 days. The superior RP stabilization and anti-coalescence property of the nanoemulsion could be attributed to the densely-packed, sterically-hindered and highly-charged interfacical layers composed of multiple emulsifiers at the oil-water interface. In addition, in vitro skin permeation experiments in a Franz diffusion cell suggested that the as-prepared nanoemulsions exhibited highly enhanced transdermal delivery of RP into the epidermis and dermis compared to the conventional emulsions. Therefore, the present work provides a feasible approach for more effective application of retinol esters in cosmetic formulations.

KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.

The role of local retinoids in eliminating signs of skin aging

Skin aging is a complex process involving both internal (chronological aging) and external (biological aging) factors. Slowing down the proliferative and immune processes in the epidermis, reducing the activity of fibroblasts and vascularization of the dermis during chronological aging lead to thinning, dryness, hypersensitivity, vulnerability and superficial wrinkles. Exposure to ultraviolet rays, pollutants, climate, and thermal factors cause keratinocyte disorganization, enhanced melanogenesis, collagen dystrophy, solar elastosis, and disorder of microcirculation. The main signs of external skin aging are deep wrinkles, sagging, pigmentation, telangiectasia, skin neoplasms.&#x0D; Among the local anti-aging agents, retinoids occupy a leading place, as they eliminate the main signs of skin aging. Of the entire group of retinoids, retinoic acids are the most active. However, the possibility of skin irritation limits their use. Therapeutic and cosmetic products with retinol esters (retinol palmitate) have a minimal irritating effect and can be used both for the prevention of skin aging and the elimination of its signs. Oral use of isotretinoin as an anti-aging agent is undesirable due to the many side effects and contraindications.

Analysis of Vitamin A in Multivitamin Products

Vitamin A is present in multivitamin products mainly in the form of retinol esters: retinyl acetate, retinyl palmitate, and beta carotene—retinol precursor (dimer) found in plants, which is capable of converting into retinol in liver cells. Retinol is determined in medicinal products primarily by high performance liquid chromatography (HPLC), with preliminary purification and vitamin isolation by liquid-liquid extraction. However, scientific literature also describes other methods of sample preparation and analysis of such compounds. An important issue is differentiation of vitamin A from other fat-soluble vitamins often included as components in multivitamin products. The aim of the study was to analyse and summarise data on current methods used for determination of vitamin A and its derivatives in medicinal products. The authors analysed the range of vitamin A products authorised in the Russian Federation, and the test methods described in their product specification files. The study demonstrated that the test method most often used for determination of retinol esters was HPLC with isocratic elution mode using octadecylsilyl packing in the reverse-phase mode, and, less frequently, aminopropylsilyl packing in the normal phase mode. Determination of beta carotene in medicinal products is most often performed using spectrophotometry.

Zymbilan®-PSO cream with nano-encapsulated RetileX-A®-PRO mitigates the clinical severity and relieves the symptoms of chronic plaque psoriasis: an interventional clinical study

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Skin psoriasis is a serious inflammatory disorder with increased risk of rheumatic, cardiometabolic and psychosocial complications. At present, &amp;gt;50% of patients are dissatisfied with their treatment; thus, novel approaches are in high clinical demand. Retinoids are a major group of anti-psoriatic compounds; nonetheless, dose-dependent topical side effects have limited their effective topical application. RetileX-A&lt;sup&gt;®&lt;/sup&gt;-PRO (Pharma Medico, Aarhus, Denmark) is a novel nano-encapsulated retinol ester designed to minimise the risk of skin irritation. The proprietary nano-encapsulation technology used in this compound can protect retinoid molecules from degradation, and thereby prolong and stabilise its release profile. The safety and efficacy of RetileX-A&lt;sup&gt;®&lt;/sup&gt;-PRO were evaluated in this study.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt; 45 patients (58% female, aged 18-80 years) with mild to moderate skin psoriasis were enrolled in a 4-week interventional study. Participants were treated once daily with Zymbilan&lt;sup&gt;®&lt;/sup&gt;-PSO cream with RetileX-A&lt;sup&gt;®&lt;/sup&gt;-PRO and evaluated both objectively and subjectively at baseline, after 8 hours and at endpoint.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; Our observations demonstrated that Zymbilan&lt;sup&gt;®&lt;/sup&gt;-PSO cream possesses both short-term and long-term anti-psoriatic effects. Shortly after application, objective skin characteristics were improved, and itching reduced in &amp;gt;84% of subjects. At endpoint, 87% of skin lesions improved, 9% did not change and 4% progressed as assessed by a dermatologist. Patients’ self-evaluation yielded similar results: 78% judged Zymbilan&lt;sup&gt;®&lt;/sup&gt;-PSO cream as a ‘good’ to ‘very good’ skin care product with suitable cosmetic parameters. No side effects recorded in 95% of participants.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; In conclusion, Zymbilan&lt;sup&gt;®&lt;/sup&gt;-PSO cream is a well-tolerated treatment that can mitigate both clinical severity and subjective symptoms of chronic skin psoriasis.&lt;/p&gt;

Carotenoid Extraction from Locally and Organically Produced Cereals Using Saponification Method

Carotenoids are important phytochemicals contributing nutritional health benefits in the human diet, with a significant contribution from cereals as one of the major food component around the world. Different methods have been described and adopted for the extraction and isolation of carotenoid compounds. Saponification can be seen as an option for carotenoid extraction from cereals as it converts retinol esters to retinol and removes other abundant compounds such as triglycerides. Extraction of carotenoids content of locally adapted and organic cereals have been limitedly investigated and was, therefore, evaluated in the present study, with a specific aim to understand genotypic and local cultivation effects and interactions. Therefore, 17 diverse cereal genotypes of local origin were grown organically in four localities and evaluated for carotenoid content and composition by HPLC. The results showed a large variation in content and composition of carotenoids in locally adapted and organically grown cereal genotypes, with lutein as the dominating type in wheat and rye, while zeaxanthin was the dominating type in barley. High-level genotypes showed values (9.9 mg/kg of total carotenoids) similar to the highest values previously reported in specific types of wheat. The barley genotypes showed relatively high stability in carotenoids content within and between cultivation locations, while large interactions were found with the cultivation location for the rest of the genotypes, indicating their local adaptation. The local adaptation of the cereal genotypes evaluated contributes large opportunities for local production of high value, highly nutritious food products, while the direct value of these genotypes for conventional plant breeding for varieties performing similar over broad environmental ranges, are more limited.

Usage of unsaturated esters of retinol to defeat Hopf’s acrokeratosis verruciformis in hands and feet of a youg man

Usage of unsaturated esters of retinol to defeat Hopf’s acrokeratosis verruciformis in hands and feet of a youg man

Cultivation of Hepatic Stellate Cells in 3D ECM to Simulate Physiological and Fibrotic Conditions

BackgroundHepatic stellate cells, the major providers of hepatic retinoids and extracellular matrix (ECM), reside in the space of Disse filled with loose basement like ECM, and flanked by hepatocytes and sinusoidal endothelial cells. Upon hepatic injury, stellate cells are activated to express multiple matrix metalloproteinases (MMPs) and to mobilize retinoids for wound healing. In chronic injury, the quiescent stellate cells undergo trans‐differentiation into myofibroblast like cells, marked by loss of retinoids, expression interstitial ECM as well as smooth muscle actin (alpha‐SMA), and gain of contractibility. Although knowledge has been evolved substantially, two fundamental questions remain largely unknown as to how, in normal liver, HSCs maintain quiescence, and how, by injury, HSCs are activated within the ECM milieu.ResultsTo recapitulate the sub‐sinusoid microenvironment we embedded rat primary stellate cells in three‐dimensional ECM (3D ECM). Cultured on plastic, the stellate cells activated spontaneously, showing loss of vitamin A droplets and expression of alpha‐SMA. However, in 3D Matrigel, stellate cells retained retinoid droplets, in association with elevated level of lecithin retinoid acyltransferase (LRAT), a key enzyme for retinol esterification. In 3D ECM, stellate cells were arrested at G0/G1 phase, while on plastic the cells underwent cell cycling and stopped at G2/M phase. We then reconstituted the microenvironment of sinusoids by introducing Kupffer cells, the major hepatic inflammatory cells, into 3D ECM with HSCs. Activated Kupffer cells triggered subsequent activation of stellate cells, evidenced by expression of multiple MMPs, buildup of stress fibers, and loss of retinoids. The Kupffer cell‐mediated activation of stellate cells was blocked by interleukin‐1 receptor antagonist, demonstrating interleukin‐1 as a major injury signal from Kupffer cells conducting stellate cell activation and trans‐differentiation. Furthermore, within 3D ECM, but not on the plastic, interleukin‐1 signal was greatly amplified by stellate cells in an autocrine manner, which may exacerbate liver injury by expressing MMPs in the space of Disse. The myofibroblastic cells derived from the 3D ECM culture were contractile in response to endothelin.ConclusionThe 3D ECM culture provides a model to simulate key physiological functions of stellate cells in normal liver, and also to recapitulate patho‐physiological responses in liver injury. The method may also be used in cell‐based drug screening to identify components targeting liver failure and fibrosis.Support or Funding InformationNational Institutes of Health (NIH) Grants R01s, DK069418‐05 and AR051558, and the Natural Science Foundation of ChinaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of supplemental tocopherol level (0, 250 and 500 IU RRR-α-tocopherol/d/steer) and injectable retinol form (retinyl propionate vs retinyl palmitate) on growth performance, carcass characteristics and plasma concentration in calf-fed Holstein steers

ABSTRACTThe influence of supplemental tocopherol level (0, 250 and 500 IU RRR-α-tocopherol/d/steer) and injectable retinol form (retinyl propionate vs retinyl palmitate) on growth performance, carcass characteristics and plasma tocopherol and retinol concentrations were evaluated in 108 Holstein steers fed a steam flaked corn-based finishing diet during 314-d feeding period. There were no treatment interactions (P > 0.10). During the initial 112-d period, dietary supplemental tocopherol tended to increase ADG (linear, P = 0.07) and DMI (linear, P = 0.06). Overall 314-d ADG, DMI, gain efficiency and carcass characteristics were not affected (P > 0.10) by dietary supplemental tocopherol. Overall DMI tended to be greater (3%, P ≤ 0.10) for steers injected with retinyl palmitate vs retinyl propionate. It is concluded that vitamin E supplementation above basal requirements may enhance growth performance during the initial 112-d phase. However, the overall effect on growth performance and carcass characteristics was not appreciable. Injectable retinol ester form did not affect overall ADG, gain efficiency, or dietary NE. Based on plasma retinol concentrations, the bioavailability of retinyl palmitate is greater than that of retinyl propionate.

English

Carotenoids are a class of yellow-orange-red pigments distributed in various fruits, spices, herbs and especially in vegetables. These pigments are bioactive components essential to human health. Among the numerous classes of carotenoids, a smaller number is known for having provitamin A activity. This vitamin is essential to the organism in the proper functioning of the vision and the immune system. On the other hand, the deficiency in this vitamin is related to a great number of diseases, which can in severe cases, lead to death. This makes essential the proper supplying of this vitamin to human nutrition. There are two sources from which vitamin A can be derived: (a) vitamin A preformed with retinol esters from animal food sources and (b) from carotenoid found in plant foods. Thus, food fortification is presented as an alternative to reduce vitamin A deficiency. Currently, plant breeding has contributed to the development and introduction of biofortified products. Improved varieties obtained from biofortification have a higher content of nutrients and vitamins, leading to the improvement of human diet. The genetic breeding of vegetables aiming nutritional biofortification is a promising strategy for the increasing of carotenoid concentration in agricultural products and prevention of vitamin A deficiency. Besides the crops that have commonly been used in the biofortification programs, a series of horticulture crops are also promising for insertion in the programs of biofortification in pro vitamin A. Thus, the objective of this review is addressing the problems resulting from vitamin A deficiency and the adoption potential of horticulture species by genetic breeding programs aiming the biofortification in carotenoids. Key words: Horticulture, hypovitaminosis A, pro vitamin A.

Molecular Basis for Vitamin A Uptake and Storage in Vertebrates.

The ability to store and distribute vitamin A inside the body is the main evolutionary adaptation that allows vertebrates to maintain retinoid functions during nutritional deficiencies and to acquire new metabolic pathways enabling light-independent production of 11-cis retinoids. These processes greatly depend on enzymes that esterify vitamin A as well as associated retinoid binding proteins. Although the significance of retinyl esters for vitamin A homeostasis is well established, until recently, the molecular basis for the retinol esterification enzymatic activity was unknown. In this review, we will look at retinoid absorption through the prism of current biochemical and structural studies on vitamin A esterifying enzymes. We describe molecular adaptations that enable retinoid storage and delineate mechanisms in which mutations found in selective proteins might influence vitamin A homeostasis in affected patients.

Synthesis of lipase-catalyzed retinol ester derivative

Synthesis of lipase-catalyzed retinol ester derivative

Effect of retinol and retinol esters on performance, egg quality, and blood and egg vitamin A levels in laying quails

Effect of retinol and retinol esters on performance, egg quality, and blood and egg vitamin A levels in laying quails

Efeitos da Hipervitaminose A sobre o Disco Epifisário de Fêmures de Ratos

A vitamina A (C20H30O) é um álcool (retinol) isoprenóide, insaturado, termoestável, lipossolúvel e insolúvel na água. Pode ser encontrado na forma precursora de β-caroteno, sendo transformada posteriormente nas suas formas ativas (ou pré-formadas) denominadas retinol, ácido retinóico e éster retilínicos. A vitamina A exerce diversas funções no desenvolvimento do ser humano, entretanto, o seu excesso causa várias manifestações, como sonolência, irritabilidade, dermatite escamativa, unhas frágeis, gengivite, fadiga, hipertensão, cefaléias intensas, fraqueza generalizada e fragilidade óssea. Sabendo que a vitamina A influencia no crescimento ósseo, o presente estudo propôs avaliar eventuais alterações produzidas no disco epifisário do fêmur de ratos em crescimento, submetidos a uma hipervitaminose A. Foram administradas, em ratos Wistar, doses na concentração de 150.000 UI (0,5 ml / 100g de peso) por via intraperitoneal e comparados com um grupo controle. Cada grupo composto por 8 animais foi sacrificado com 10, 20, 30 e 40 dias após a injeção e os fêmures foram removidos para obtenção de cortes histológicos. A morfometria dos discos epifisários demonstrou que os animais que receberam as doses elevadas de vitamina A apresentaram uma diminuição na espessura dos discos epifisários, condizente com uma maturação mais acelerada. Dessa forma, os resultados sugerem que excesso de vitamina A pode levar a um aumento no amadurecimento dos discos epifisários.

Transgenic reporter mice with promoter region of murine LRAT specifically marks lens and meiosis spermatocytes.

Lecithin:retinol acyltransferase (LRAT) is the major enzyme responsible for retinol esterification in the mammalian body. LRAT exhibits specific activity in the cells with active retinol metabolism where it converts retinols into retinyl esters, which represents the major storage form of retinol. Besides hepatic stellate cells in the liver, LRAT appears to have a key physiologic role in several other tissues. In this study, we generated a transgenic reporter mouse expressing green fluorescence protein (EGFP) under the control of region containing -1166 bps from promoter upstream from the putative transcriptional start site and 262 bps downstream of this start. Transgenic reporter mice exhibited specific expression in eyes and testes. In eyes, expression of EGFP-reporter is found in lens and lens epithelium and fibers from embryo to adulthood. In testes, LRAT-EGFP reporter is expressed both in Sertoli and in spermatocytes marking initiation of spermatogenesis in prepubertal mice. Our data show that the examined LRAT regulatory region is sufficient to achieve strong and selective expression in the eye and testes but not in liver and other organs.

LRAT Overexpression Diminishes Intracellular Levels of Biologically Active Retinoids and Reduces Retinoid Antitumor Efficacy in the Murine Melanoma B16F10 Cell Line

Background/Aim: Vitamin A (all-trans-retinol, ATRol) serves as a precursor for all-trans-retinoic acid (ATRA), a ligand for the retinoic acid receptor (RAR), representing a potent regulator for many physiological processes. While murine melanoma cells are highly sensitive to retinoid treatment, human melanoma cells have developed still unidentified mechanisms that mediate cellular retinoid resistance. One of the key retinoid metabolizing enzymes is lecithin retinol acyltransferase (LRAT), which catalyzes the transformation of ATRol into inactive retinyl esters. LRAT is highly expressed in human melanoma cells. The aim of this study was to identify the mechanisms in retinol metabolism that are responsible for cellular retinoid sensitivity in the murine melanoma cell line B16F10. Methods: mRNA expression analysis, cell viability assessment and determination of intracellular retinoid levels using HPLC analysis of a generated LRAT-overexpressing B16F10 cell line compared to the control B16F10 cell line. Results: We found that the murine retinoid-sensitive B16F10 cell line does not express the enzyme LRAT. LRAT overexpression decreased the antiproliferative effects of retinoid treatment in these melanoma cells. The RAR-regulated enzyme Cyp26a1 showed a significantly lower expression in LRAT-overexpressing B16F10 cells. Cyp26a1 expression was restored after ATRA incubation. HPLC analysis revealed that the level of inactive retinyl ester increased after ATRol treatment, and levels of the substrate ATRol and biologically active ATRA significantly decreased in LRAT-overexpressing murine melanoma. Consistently with this, levels of 4-oxo-retinoic acid, an ATRA metabolite and Cyp26a1 product, were also decreased in LRAT-overexpressing cells. Conclusion: Our results revealed a direct link between LRAT expression and regulation of ATRA levels indicating that the absence of LRAT-catalyzed retinol esterification is important for mediating retinoid sensitivity in murine melanoma cells. Thus, our data suggest that LRAT overexpression represents a novel mechanism by which tumor cells can escape high supplementary ATRA levels that mediate tumor-suppressive RAR signaling.