Concentrations Of All-trans Retinoic Acid Research Articles

GSK-126 Enhances All-Trans-Retinoic Acid (ATRA) Response in Hepatocellular Carcinoma (HCC) by Upregulating RARG Expression.

Hepatocellular carcinoma (HCC) stands out as one of the most prevalent malignant tumors globally. The combination of all-trans-retinoic acid (ATRA) with FOLFOX chemotherapy has shown promise in enhancing the prognosis of HCC patients. ATRA, serving as a chemosensitizing agent, presents novel possibilities for therapeutic applications. Nevertheless, the responsiveness of HCC cells to ATRA varies. The epigenetic modifier-GSK-126 is currently under investigation as a potential antitumor drug. Our aim is to explore the molecular mechanisms underlying the diverse sensitivity of HCC patients to ATRA, and to propose a new combination regimen. This research aims to lay the groundwork for personalized medication approaches for individuals with HCC. A cell model with low expression of retinoic acid receptor Alfa (RARA), retinoic acid receptor belta (RARB), and retinoic acid receptor gamma (RARG) was established through siRNA interference. The impact of reduced expression of RARA, RARB, and RARG on the half maximal inhibitory concentration (IC50) of ATRA in Hep3B cells was assessed using the 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT) cytotoxicity assay. Flow cytometry revealed that RARG emerged as the key receptor influencing the combination's sensitivity. Conducting ChIP-qPCR analysis on genomic DNA from HCC cells through relevant websites demonstrated enrichment of the trimethylation modification of lysine 27 on histone H3 (H3K27me3) upstream of the RARG promoter. ChIP-PCR assay confirmed that GSK-126 could diminish H3K27me3 levels on the RARG promoter, subsequently elevating RARG expression. The synergistic efficacy of GSK-126 and ATRA was validated through MTT assay, flow cytometry apoptosis assay, cell cycle assay, and cell scratch assay. Our study unveiled that the insensitivity of HCC cells to ATRA could be linked to the low expression of RARG. ChIP-qPCR analysis illuminated that GSK-126 activated RARG expression by diminishing H3K27me3 enrichment in the RARG promoter region. Consequently, the concurrent administration of ATRA and GSK-126 to hepatoma cells exhibited a synergistic effect, inhibiting cell proliferation, inducing cell apoptosis, and reducing the proportion of cells in the S-phase. Our findings emphasize that the synergistic action of GSK-126 and ATRA enhances the sensitivity of HCC cells by upregulating the expression of RARG. This presents a potential foundation for personalized HCC treatment.

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.

Levels of retinol and retinoic acid in pancreatic cancer, type-2 diabetes and chronic pancreatitis.

Retinoids participate in multiple key processes in the human body e.g., vision, cell differentiation and embryonic development. There is growing evidence of the relationship between retinol, its active metabolite- all-trans retinoic acid (ATRA) - and several pancreatic disorders. Although low levels of ATRA in pancreatic ductal adenocarcinoma (PDAC) tissue have been reported, data on serum levels of ATRA in PDAC is still limited. The aim of our work was to determine serum concentrations of retinol and ATRA in patients with PDAC, type-2 diabetes mellitus (T2DM), chronic pancreatitis (CHP) and healthy controls. High performance liquid chromatography with UV detection (HPLC) was used to measure serum levels of retinol and ATRA in 246 patients with different stages of PDAC, T2DM, CHP and healthy controls. We found a significant decrease in the retinol concentration in PDAC (0.44+/-0.18 mg/L) compared to T2DM (0.65+/-0.19 mg/L, P<0.001), CHP (0.60+/-0.18 mg/L, P< 0.001) and healthy controls (0.61+/-0.15 mg/L, P<0.001), significant decrease of ATRA levels in PDAC (1.14+/-0.49 ug/L) compared to T2DM (1.37+/-0.56 ug/L, P<0.001) and healthy controls(1.43+/-0.55 ug/L, P<0.001). Differences between early stages (I+II) of PDAC and non-carcinoma groups were not significant. We describe correlations between retinol, prealbumin and transferrin, and correlation of ATRA and IGFBP-2. Significant decrease in retinol and ATRA levels in PDAC compared to T2DM, healthy individuals and/or CHP supports existing evidence of the role of retinoids in PDAC. However, neither ATRA nor retinol are suitable for detection of early PDAC. Correlation of ATRA levels and IGFBP-2 provides new information about a possible IGF and retinol relationship.

Trinary Fusion with Ligand Binding Domain Castration Is Crucial in Rarg-Driven APL and Render Unresponsive to ATRA Via Allosteric Disability Mechanism

Introduction All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases that manifest as APL, and they often carry FGs involved in RARA, RARB, or RARG. X::RARG positive APL ( RARG-APL) cases have been increasingly reported in recent years, thanks to the utilization of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all evaluable RARG-APL cases were resistant to ATRA, and no mechanism has been elucidated. Intriguingly, cells transformed by artificial X::RARG were extremely sensitive to ATRA (PMID: 25510432), indicating intricate mechanisms unrevealed. This study first and systematically unveils the distinctive features and pivotal molecular mechanism of RARG-driven APL. Methods A total of 22 RARG-APL cases with RNA-seq and WGS data from 11 centers were enrolled (Fig. 1A). We used Arriba for routine FG calling and manually investigated the non-coding fusion sequence. Reverse transcription PCR (RT-PCR) and Sanger sequencing were used to validate the FGs and to determine whether the RARG 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 15 males and 7 females, aged 0.9-69 (median 38) years. Morphological and immunophenotype analyses all showed features of APL. Routine FGs calling reported RARG fusion to a 5′ partner in each case, with variable RARG splicing sites (Fig. 1A). There were 7 RARG 5' partner genes, with CPSF6, HNRNPC, and NUP98 observed in multiple cases. In the 3 index cases, we identified that they all had RARG 3‘ fusion events besides the 5‘ one (Fig. 1A). RT-PCR confirmed that the 5′ and 3′ RARG fusion events were in the same cistron in each case. Such as, the fusion transcript is the insertion of RARG-e4_9 (e refers to exon) between NPM1-e4 and e11 in case #G1, rather than two separate transcripts of NPM1-e4:: RARG-e4 and RARG-9:: NPM1-e11. We named this novel form of tandem splicingtrinary fusion. Analysis of the 19 validation cases further confirmed that all RARG-APL cases had RARG 3′ fusion events (Fig. 1A). We also validated that the 5′ and 3′ RARG fusion events were in the same cistron in 3 more cases, of which archived cDNA were available. Remarkably, the 3' splicing sits of all cases were consistently at the end of RARG-e9, leading to RARG-e10 truncation. The RARG 3' partner gene was the same as its 5' partner in 11 cases. But in the other 11 cases, the RARG 3' partner was a transposon element (TE) sequence, the most common being a LINE-L2a (8 cases). Sequences analysis indicated that the committed locus of the involved LINE-L2a was at 4.2 Kbp upstream of the RARG gene. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARG 3' fusions confer a poly_A signal sequence to the fusion transcript, which was essential for a mRNA. In-depth analysis indicated that TEs participate in the formation of RARG-FGs through a transposition mechanism rather than a translocation mechanism. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in allosteric response to ATRA (PMID: 31178221). The trinary fusion protein with RARG LBD-H11_12 castration will lose responsiveness to ATRA via an allosteric disability mechanism (Fig. 1B, 1C). Experiments confirmed that cells transfected with X::RARG fusion with intact LBD responded well to ATRA, while fusion with LBD-H11_12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1D, 1E). Conclusions This study clearly revealed that trinary fusion and LBD castration are crucial molecular etiologies in RARG-APL. The trinary fusion orchestrates the aberrant activation of RARG (5‘ splicing) and the complete allosteric disability in responding to ATRA (3‘ splicing). Thus, this study elegantly explains the mechanism of leukemogenesis and the extensive ATRA resistance of RARG-APL. The formation of trinary fusion requires two steps of genome splicing or transposition events, which explains the rarity of RARG-APL. This also clarifies that RARG-APL has a significant disparity in molecular mechanism from PML::RARA-positive APL, and we suggest that it should be considered as a separate entity of acute myeloid leukemia.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells.

To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells. After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 µmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR. Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 µmol/L and compared with the normal control group (P=0.027 and P=0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA (P<0.001) and promote the expression of MMP-2 and TGF-β2 mRNA (P=0.03 and P<0.001, respectively) in a dose-dependent manner, especially when treated with 5 µmol/L ATRA. The knockdown efficiency of RDH5 siRNA varies with different targets, among which RDH5 siRNA-435 had the highest knockdown efficiency, i.e., more than 50% lower than that of the negative control group (P=0.02). When RDH5 was knocked down for 48h, the results of qRT-PCR showed that the expressions of MMP-2 and TGF-β2 mRNA were significantly up-regulated (P<0.001). ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA.

All-Trans Retinoic Acid Promotes M2 Macrophage Polarization in Vitro by Activating the p38MAPK/STAT6 Signaling Pathway

ABSTRACT Background M2-type macrophages are inflammation-suppressing cells that are differentiated after induction by cytokines such as IL-4 or IL-13, which play an important regulatory role in inflammation and influence the regression of inflammation-related diseases. All-trans retinoic acid (ATRA) has an important role in suppressing immune-mediated inflammatory responses but the effect and underlying mechanism of ATRA on the polarization of M2 macrophages remains unclear. Methods Macrophages were isolated from peritoneal wash fluid, and IL-4 (20 ng/mL) was used to construct a m2-type macrophage polarization model. The model was incubated with different concentrations of ATRA (15 µg/ml, 30 µg/ml, 45 µg/ml) for 24 h, and pretreated macrophages with p38MAPKα inhibitor SB202190 (20 μM). MTT, Trypan blue staining, Annexin V-PE/7-AAD staining, flow cytometry, real-time PCR and western blotting were used to investigate the effect and mechanism of ATRA on the polarization of M2 macrophages. Results Compared with the IL-4 group, the proportion of F4/80+CD206+ M2-type macrophages was significantly higher in the ATRA group (P < 0.01). mRNA and protein expression levels of Arg-1, IL-10 and TGF-β1 were as significantly higher (P < 0.01) in the ATRA group as phosphorylation levels of STAT6 and p38MAPK (P < 0.01). After pretreatment with the addition of the inhibitor SB202190, M2-type macrophages proportion and their associated factors expression were significantly (P < 0.01) reduced, as compared with those in the ATRA group, but they were comparable (P > 0.05) with the IL-4 group. Conclusion The combination of ATRA and IL-4 activated the p38MAPK/STAT6-signaling pathway to promote polarization of M2 macrophages.

Knockout of HMGA1 Promotes ATRA-Induced Differentiation in MLL-Rearranged Leukemia

Introduction: HMGA1 is a small non-histone chromatin protein characterized by 3 AT-hooks, which modulates transcription by altering the chromatin architecture. It tends to be highly expressed in poorly differentiated cells including hematopoietic stem cells. In various kinds of solid tumors, HMGA1 has been reported to play as an oncogene, and its upregulation was reported to be associated with poorer clinical outcomes. However, although recent study demonstrated that HMGA1 was associated with progression of JAK2V617F positive myeloproliferative neoplasms to myelofibrosis or leukemia, the roles of HMGA1 in myeloid malignancies were still unclear. Therefore, we analyzed its function in myeloid malignancies using HMGA1 knockout (KO) mice. We could not detect clear differences in peripheral blood counts or bone marrow cells, and generated various kinds of models of acute myeloid leukemia (AML). Knockout of HMGA1 could not delay the onset of these leukemia, but we could demonstrate that leukemic cells with MLL-ENL fusion gene which lacked in HMGA1 gene were more susceptible to all-trans retinoic acid (ATRA)-induced expression of CD11b, which suggested that HMGA1 could be associated with the differentiation process induced by ATRA. Method: We generated Vav-iCre HMGA1flox/flox mice, and we first analyzed the fraction of peripheral blood cells and bone marrow cells of these mice using flow cytometry. Next, to evaluate roles of HMGA1 in AML, we made various murine models of AML. We collected c-kit positive fractions of bone marrow cells from the KO mice or Vav-iCre(-) HMGA1flox/flox mice and retrovirally introduced MLL-ENL, MOZ-TIF2, BCL2-MYC fusions. Primary transplantation was performed to sublethally irradiated (5Gy) mice with 4.0×105 infected cells for each fusion gene. Secondary transplantation was also performed for MLL-ENL and BCL2-MYC with 1.0×104 cells for each. Blood samples were collected weekly, and disease-free survivals were compared by Kaplan-Meier method with Vav-iCre(-) HMGA1flox/flox mice. Also, leukemic cells infected with MLL-ENL were collected, and these cells were cultured for four days with different concentrations (1μM, 5μM or 10μM) of ATRA. Flow cytometry analysis was performed to evaluate the differentiation induced by ATRA with antibodies to murine CD11b, c-kit and Gr-1. Result: There was no significant difference in birth rate between Vav-iCre(+) and Vav-iCre(-) mice, which suggested that HMGA1 was not essential for fetal hematopoiesis. Peripheral blood cell counts were measured at week 8 and week 20, and fractions of peripheral blood and bone marrow cells were assessed at week 8 using flow cytometry, but no significant differences were observed. As for AML models, there were no significant differences in survival periods after primary or secondary transplantation. Leukemic cells retrovirally induced with MLL-ENL were collected from primary recipients, and these cells were treated with ATRA. Although growth rates were not show significantly different, expression levels of CD11b after exposure to ATRA were significantly higher in Vav-Cre (+) mice (5μM: 70% vs 44%, respectively; p=0.035. 10μM: 80% vs 46%, respectively; p=0.0006) as shown in the following figure. Discussion: We established HMGA1 KO mice and assessed the function of HMGA1 both in normal and leukemic hemopoiesis. Although HMGA1 might have little effect on normal hematogenesis, it seemed that HMGA1 had inhibitory effect on ATRA-induced differentiation process of MLL-ENL leukemic cells. Though more conditions including AML types and differentiation methods should be tested and the molecular mechanisms should be scrutinized, there is a possibility that inhibition of HMGA1 is effective in combination with differentiation treatments. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

All-trans Retinoic Acid-incorporated Glycol Chitosan Nanoparticles Regulate Macrophage Polarization in Pg-LPS-Induced Inflammation.

The occurrence and development of inflammation are closely correlated to the polarization of macrophages. All-trans retinoic acid (ATRA) has been proven to promote the polarization of macrophages from M1 to M2, but this lacks an effective carrier to participate in the biological response. The present study aims to determine whether retinoic acid-incorporated glycol chitosan (RA-GC) nanoparticles can regulate macrophage polarization in Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS)-induced inflammation. Mouse 264.7 cell lines were treated with 1 µg/mL Pg-LPS to induce inflammation. After the effects of ATRA and RA-GC on the activity of macrophages were detected by CCK-8 assay, cells induced with Pg-LPS were assigned to the blank control group (GC) nanoparticles without ATRA, and experimental groups (GC nanoparticles loaded with different concentrations of ATRA: 1, 10 and 100 µg/mL). The effects of RA-GC on inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-10 and IL-12 in macrophages were detected by enzyme-linked immunosorbent assay (ELISA). Subsequently, the effects of GC nanoparticles loaded with/without ATRA on macrophage polarization in an inflammatory environment were detected by RT-PCR and Western blotting. The results revealed that RA-GC had no significant effect on macrophage activity. However, RA-GC could effectively inhibit the Pg-LPS-induced inflammatory factor expression in macrophages. Meanwhile, the experimental results confirmed that RA-GC could downregulate the expression of inducible nitric oxide synthase (iNOS) (a marker of M1 macrophages) and upregulate the expression of mannose receptor and Arginase-1 (a marker of M2 macrophages) in a dose-dependent manner. The present study confirms that RA-GC can promote the M2 polarization of macrophages in an inflammatory environment, and proposes this as a promising target for the clinical treatment of Pg-LPS-related diseases.

Effects of Vitamin A on Yanbian Yellow Cattle and Their Preadipocytes by Activating AKT/mTOR Signaling Pathway and Intestinal Microflora.

Simple SummaryVitamin A is a fat-soluble vitamin that not only plays a role in vision, growth, and development, but also in fat production and metabolism in animals. To improve the production of high-grade beef, it is necessary to explore the molecular mechanism of intramuscular fat deposition in beef cattle through molecular biology techniques. In this study, we selected Yanbian yellow cattle, one of the five major cattle breeds in China, to investigate the effects of vitamin A and its metabolite, all-trans retinoic acid (ATRA), on the proliferation and differentiation of preadipocytes and changes in intestinal microorganisms. It was found that ATRA inhibited adipogenic differentiation of preadipocytes in Yanbian yellow cattle via the AKT/mTOR signaling pathway. This study provides insight into nutritional management and reveals the role of vitamin A in lipid metabolism in Yanbian yellow cattle.In this study, the effects of vitamin A and its metabolite, all-trans retinoic acid (ATRA), on the proliferation and differentiation of preadipocytes and the intestinal microbiome in Yanbian yellow cattle were investigated. Preadipocytes collected from Yanbian yellow cattle treated with different concentrations of ATRA remained in the G1/G0 phase, as determined by flow cytometry. Quantitative reverse-transcription polymerase chain reaction and western blotting analyses showed that the mRNA and protein expression levels of key adipogenic factors, peroxisome proliferator- activated receptor gamma (PPARγ), CCAAT enhancer-binding protein α (C/EBPα), and extracellular signal-regulated kinase 2 (ERK2), decreased. ATRA was found to regulate the mTOR signaling pathway, which is involved in lipid metabolism, by inhibiting the expression of AKT2 and the adipogenic transcription factors SREBP1, ACC, and FAS; the protein and mRNA expression levels showed consistent trends. In addition, 16S rRNA sequencing results showed that a low concentration of vitamin A promoted the growth of intestinal microflora beneficial to lipid metabolism and maintained intestinal health. The results indicated that ATRA inhibited the adipogenic differentiation of preadipocytes from Yanbian yellow cattle through the AKT/mTOR signaling pathway, and that low concentrations of vitamin A may help maintain the intestinal microbes involved in lipid metabolism in cattle.

The effect of all‐trans retinoic acid on the mitochondrial function and survival of cardiomyoblasts exposed to local photodamage

AbstractUsing H9C2 cardiomyoblasts, we have shown that all‐trans retinoic acid (ATRA), the biologically active metabolite of vitamin A, affects mitochondrial dynamics and functions. The low dose (10 nM) ATRA stimulates the expression of nuclear retinoid receptors and induces mechanisms that are protective against severe local damage caused by laser irradiation at the mitochondrial level. These changes include increased density of the mitochondrial network, higher number of mitochondrial junctions, and enhanced mitochondrial velocity. Moreover, the treated cells had lower basal level of reactive oxygen species (ROS) and could maintain mitochondrial potential (ΔΨm) after photodamage. Cells treated with 10 nM ATRA had significantly better survival rate after photodamage in comparison to control cells. Cells treated with pharmacological concentration of ATRA (1 µM) expressed higher mitochondrial connectivity without increased motility, which did not lead to better survival or decreased ROS level as was in the case of low‐dose ATRA. The proteomics analysis showed changes in proteins related to cellular metabolism (glycolysis) and respiration in ATRA‐treated cells. The l‐lactate assay confirmed the shift to anaerobic glycolysis in cells treated with 1 µm ATRA, whereas the 10 nM ATRA decreased the level of lactate in medium. The increased levels of cytochrome c or peroxiredoxins 5 level and also lower expression of retinoid and rexinoid receptors were observed in cells treated with 1 µM ATRA. The effect of ATRA is concentration‐dependent; the increased mitochondrial dynamics and slower metabolism at 10 nM ATRA contributed significantly to the chance of survival of the cells after photodamage whereas the higher concentration of ATRA overrode the protective effect and led to the unfavorable ones.

The involvement of hormone-sensitive lipase in all-trans retinoic acid induced cleft palate.

Abnormally high concentrations of all-trans retinoic acid (atRA) induce cleft palate, which is accompanied by abnormal migration and proliferation of mouse embryonic palatal mesenchyme (MEPM) cells. Hormone-sensitive lipase (HSL) is involved in many embryonic development processes. The current study was designed to elucidate the mechanism of HSL in cleft palate induced by atRA. To establish a cleft palate model in Kunming mice, pregnant mice were administered atRA (70 mg/kg) by gavage at embryonic Day 10.5 (E10.5). Embryonic palates were obtained through the dissection of pregnant mice at E15.5. Hematoxylin and eosin (H&E) staining was used to evaluate growth changes in the palatal shelves. The levels of HSL in MEPM cells were detected by immunohistochemistry, quantitative real-time reverse transcription-polymerase chain reaction (qRT‒PCR) and western blotting. RNAi was applied to construct vectors expressing HSL small interference RNAs (siRNAs). The vectors were transfected into MEPM cells. Cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The palatal shelves in the atRA group had separated at E15.5 without fusing. In MEPM cells, the expression of HSL was reversed after atRA treatment, which caused cleft palate in vivo. In the atRA group, the proliferation of HSL siRNA-transfected cells was remarkably promoted, and the migration rate significantly increased in the HSL siRNA-transfected MEPM cells. These results suggested that HSL may be involved in cleft palate induced by atRA and that atRA enhances HSL levels to inhibit embryonic palate growth.

Effects of ATRA on proliferation, migration and cell cycle of gastric cancer MGC-803 cells-siftdesk

Objective：To study the effects of all-trans-retinoic acid (ATRA) on proliferation, migration and cycle of human gastric cancer MGC-803 cells. Methods：The proliferation activity of gastric cancer MGC-803 cells treated with different concentrations of ATRA was detected by CCK-8 assay. The morphological changes of MGC-803 cells in each group were observed under inverted microscope. The migration ability of cells in each group was detected by scratch test. Flow cytometry was used to detect the change of MGC-803 cell cycle. Results：CCK-8 results showed that ATRA could significantly inhibit the proliferation of MGC-803 cells, and the inhibition rate of cell proliferation increased gradually with the increasing of ATRA concentrations and action time, in a dose-time dependent manner(P&lt;0.05). Under inverted microscope, the morphology of MGC-803 cells changed and the number of viable cells decreased significantly after ATRA treatment. The results of scratch assay showed that ATRA could significantly inhibit the migration of gastric cancer MGC-803 cells, and the repair rate of scratch area decreased significantly with the increasing of ATRA concentration(P&lt;0.05).Flow cytometry showed that the proportion of G0/G1 phase cells increased significantly after ATRA treatment, and the proportion of S phase cells decreased (P&lt;0.05).And there was no significant change in G2/M phase cell proportion (P&gt;0.05).

All-trans retinoic acid impairs glucose-stimulated insulin secretion by activating the RXR/SREBP-1c/UCP2 pathway.

Diabetes is often associated with vitamin A disorders. All-trans retinoic acid (ATRA) is the main active constituent of vitamin A. We aimed to investigate whether ATRA influences diabetic progression and its mechanisms using both Goto-Kazizazi (GK) rats and INS-1 cells. Rat experiments demonstrated that ATRA treatment worsened diabetes symptoms, as evidenced by an increase in fasting blood glucose (FBG) levels and impairment of glucose homeostasis. Importantly, ATRA impaired glucose-stimulated insulin secretion (GSIS) and increased the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and uncoupling protein 2 (UCP2) in the rat pancreas. Data from INS-1 cells also showed that ATRA upregulated SREBP-1c and UCP2 expression and impaired GSIS at 23 mM glucose. Srebp-1c or Ucp2 silencing attenuated GSIS impairment by reversing the ATRA-induced increase in UCP2 expression and decrease in ATP content. ATRA and the retinoid X receptor (RXR) agonists 9-cis RA and LG100268 induced the gene expression of Srebp-1c, which was almost completely abolished by the RXR antagonist HX531. RXRα-LBD luciferase reporter plasmid experiments also demonstrated that ATRA concentration-dependently activated RXRα, the EC50 of which was 1.37 μM, which was lower than the ATRA concentration in the pancreas of GK rats treated with a high dose of ATRA (approximately 3 μM), inferring that ATRA can upregulate Srebp-1c expression in the pancreas by activating RXR. In conclusion, ATRA impaired GSIS partly by activating the RXR/SREBP-1c/UCP2 pathway, thus worsening diabetic symptoms. The results highlight the roles of ATRA in diabetic progression and establish new strategies for diabetes treatment.

Role of aryl hydrocarbon receptor (AHR) in overall retinoid metabolism: Response comparisons to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure between wild-type and AHR knockout mice

Young adult wild-type and aryl hydrocarbon receptor knockout (AHRKO) mice of both sexes and the C57BL/6J background were exposed to 10 weekly oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; total dose of 200 μg/kg bw) to further characterize the observed impacts of AHR as well as TCDD on the retinoid system. Unexposed AHRKO mice harboured heavier kidneys, lighter livers and lower serum all-trans retinoic acid (ATRA) and retinol (REOH) concentrations than wild-type mice. Results from the present study also point to a role for the murine AHR in the control of circulating REOH and ATRA concentrations. In wild-type mice, TCDD elevated liver weight and reduced thymus weight, and drastically reduced the hepatic concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid (CORA) and retinyl palmitate (REPA). In female wild-type mice, TCDD increased the hepatic concentration of ATRA as well as the renal and circulating REOH concentrations. Renal CORA concentrations were substantially diminished in wild-type male mice exclusively following TCDD-exposure, with a similar tendency in serum. In contrast, TCDD did not affect any of these toxicity or retinoid system parameters in AHRKO mice. Finally, a distinct sex difference occurred in kidney concentrations of all the analysed retinoid forms. Together, these results strengthen the evidence of a mandatory role of AHR in TCDD-induced retinoid disruption, and suggest that the previously reported accumulation of several retinoid forms in the liver of AHRKO mice is a line-specific phenomenon. Our data further support participation of AHR in the control of liver and kidney development in mice.

All-trans retinoic acid controls differentiation, proliferation, and lipolysis in isolated subcutaneous adipocytes from peripartal Holstein cows

Preadipocyte proliferation and differentiation are critical for normal adipose tissue development, including achieving a mature phenotype, characterized by its ability to accumulate triacylglycerol and release fatty acids. In nonruminants, it is well known that all-trans retinoic acid (ATRA), the most-active form of vitamin A, helps regulate proliferation, differentiation, and apoptosis in several types of cells including adipocytes. The purpose of this study was to evaluate the effect of ATRA on proliferation, apoptosis, differentiation, and lipolysis of primary bovine adipocytes isolated from subcutaneous adipose tissue of 5 healthy Holstein cows at 17 (±4 standard deviations) d postpartum. Cells were stimulated with increasing concentrations of ATRA (0.2, 2, and 20 nM) at the preconfluent (2 d) and postconfluent (8 d) preadipocyte stage or at the mature adipocyte stage (2 d). All concentrations of ATRA inhibited preconfluent preadipocyte proliferation with decreased proportion of S-phase cells and reduced protein abundance of cyclins (CCND1, CCND2, CCND3, CCNE1) and cyclin-dependent kinases (CDK2, CDK4, CDK6). Compared with vehicle, ATRA treatment induced apoptosis in preconfluent preadipocytes. Additionally, ATRA (0.2, 2, and 20 nM) supplementation also inhibited differentiation of postconfluent preadipocytes through downregulation of protein abundance of PPARγ and C/EBPα. After induction of differentiation, basal lipolysis in mature adipocytes increased upon treatment with all concentrations of ATRA. However, data on phosphorylated hormone-sensitive lipase or PLIN1 indicated that ATRA had no effect on epinephrine-stimulated lipolysis in mature adipocytes. Overall, these results demonstrate that ATRA might inhibit lipid accumulation by suppressing preadipocyte proliferation and differentiation, subsequently leading to apoptosis in postconfluent preadipocytes and promoting basal lipolysis in mature adipocytes. Overall, these in vitro responses provide some insights into the potential for nutritional management to modulate adipose tissue lipolysis, particularly in overconditioned cows during the dry period, which are more susceptible to suffer metabolic disorders due to excessive fat mobilization postpartum.

Enhanced Oral Absorption of All-trans Retinoic Acid upon Encapsulation in Solid Lipid Nanoparticles.

All-trans retinoic acid (ATRA) is widely employed in the treatment of various proliferative and inflammatory diseases. However, its therapeutic efficacy is imperiled due to its poor solubility and stability. Latter was surmounted by its incorporation into a solid matrix of lipidic nanoparticles (SLNs). ATRA loaded SLNs (ATRA-SLNs) were prepared using a novel microemulsification technique (USPTO 9907758) and an optimal composition and were characterized in terms of morphology, differential scanning calorimetry (DSC), and powder X-ray diffraction studies (PXRD). In vitro release, oral plasma pharmacokinetics (in rats) and stability studies were also done. Rod-shaped ATRA-SLNs could successfully incorporate 3.7 mg/mL of ATRA, increasing its solubility (from 4.7 μg/mL) by 787 times, having an average particle size of 131.30 ± 5.0 nm and polydispersibility of 0.283. PXRD, DSC, and FTIR studies confirmed the formation of SLNs. Assay/total drug content and entrapment efficiency of ATRA-SLNs was 92.50 ± 2.10% and 84.60 ± 3.20% (n=6), respectively, which was maintained even on storage for one year under refrigerated conditions as an aqueous dispersion. In vitro release in 0.01 M phosphate buffer (pH 7.4) with 3% tween 80 was extended 12 times from 2h for free ATRA to 24 h for ATRA-SLNs depicting Korsmeyer Peppas release. Oral administration in rats showed 35.03 times enhanced bioavailability for ATRA-SLNs. Present work reports preparation and evaluation of bioenhanced ATRA-SLNs containing a high concentration of ATRA (>15 times than that reported by others). Latter is attributed to the novel preparation process and intelligent selection of components. Lay Summary: All-trans retinoic acid (ATRA) shows an array of pharmacological activities but its efficacy is limited due to poor solubility, stability and side effects. In present study its solubility and efficacy is improved by 787 and 35.5 times, respectively upon incorporation into solid lipid nanoparticles (ATRA-SLNs). Latter extended its release by 12 times and provided stability for at least a year under refrigeration. A controlled and sustained release will reduce dose related side effects. ATRA-SLNs reported presently can thus be used in treatment /prophylaxis of disorders like cancers, tuberculosis, age related macular degeneration and acne and as an immune-booster.

In Vitro Anticancer Effects of All-trans Retinoic Acid in Combination with Dacarbazine against CD117+ Melanoma Cells.

Malignant melanoma is a common form of skin cancer that contains different cell types recognized by various cell surface markers. Dacarbazine-based combination chemotherapy is frequently used for the treatment of melanoma. Despite its potent anticancer properties, resistance to dacarbazine develops in malignant melanoma. Here, we aim to improve response to dacarbazine therapy by pretreatment with all-trans retinoic acid (ATRA) in CD117+ melanoma cells. The CD117+ melanoma cells were sorted from A375 malignant melanoma cell line using magnetic-activated cell sorting (MACS). The cell viability was examined by cell proliferation assay (MTT). Apoptosis was determined by acridine orange/ ethidium bromide staining. Indeed, we performed flow cytometry to evaluate the cell cycle arrest. Here, the CD117+ melanoma cells were incubated with various concentrations of ATRA, dacarbazine, and their combination to determine IC50 values. We found that 20 µM ATRA treatment followed by dacarbazine was found to be more effective than dacarbazine alone. There was an indication that the combination of ATRA with dacarbazine (ATRA/dacarbazine) caused more apoptosis and necrosis in the melanoma cells (P<0.05). Furthermore, ATRA/dacarbazine treatment inhibited the cell at the G0/G1 phase, while dacarbazine alone inhibited the cells at S phase. Collectively, combined treatment with ATRA and dacarbazine induced more apoptosis and enhanced the cell cycle arrest of CD117+ melanoma cells. These results suggested that ATRA increased the sensitivity of melanoma cells to the effect of dacarbazine.

All-trans retinoic acid increases ARPE-19 cell apoptosis via activation of reactive oxygen species and endoplasmic reticulum stress pathways

To explore the apoptosis of ARPE-19 cells after the treatment with different doses of all-trans-retinoic acid (ATRA). ARPE-19 cells were used in the in-vitro experiment. Flow cytometry assay was employed to evaluate the level of reactive oxygen species (ROS) and apoptosis. The effects of ATRA (concentrations from 2.5 to 20 µmol/L) on the expression of endoplasmic reticulum stress (ERS) markers in vitro were evaluated by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR) assays. The contribution of ROS and ERS-induced apoptosis in vitro was determined by using N-acetyl-L-cysteine (NAC) and Salubrinal, an antagonist of NAC and ERS, respectively. Flow cytometry showed that ATRA significantly increased ARPE-19 cell apoptosis and ROS levels in each group (F=86.39, P<0.001; F=116.839, P<0.001). Western blot and qRT-PCR revealed that levels of CHOP and BIP were elevated in a concentration-dependent pattern after the cells were incubated with ATRA (2.5-20 µmol/L). The upregulation of VEGF-A and CHOP induced by ATRA could be inhibited by NAC (antioxidant) and Salubrinal (ERS inhibitor) in vitro. ATRA induces the apoptosis of ARPE-19 cells via activated ROS and ERS signaling pathways.

Neurobehavioral effects of cyanobacterial biomass field extracts on zebrafish embryos and potential role of retinoids

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L−1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.