Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

Heparanase promotes myeloma stemness and in vivo tumorigenesis.

Heparanase is known to enhance the progression of many cancer types and is associated with poor patient prognosis. We recently reported that after patients with multiple myeloma were treated with high dose chemotherapy, the tumor cells that emerged upon relapse expressed a much higher level of heparanase than was present prior to therapy. Because tumor cells having stemness properties are thought to seed tumor relapse, we investigated whether heparanase had a role in promoting myeloma stemness. When plated at low density and grown in serum-free conditions that support survival and expansion of stem-like cells, myeloma cells expressing a low level of heparanase formed tumor spheroids poorly. In contrast, cells expressing a high level of heparanase formed significantly more and larger spheroids than did the heparanase low cells. Importantly, heparanase-low expressing cells exhibited plasticity and were induced to exhibit stemness properties when exposed to recombinant heparanase or to exosomes that contained a high level of heparanase cargo. The spheroid-forming heparanase-high cells had elevated expression of GLI1, SOX2 and ALDH1A1, three genes known to be associated with myeloma stemness. Inhibitors that block the heparan sulfate degrading activity of heparanase significantly diminished spheroid formation and expression of stemness genes implying a direct role of the enzyme in regulating stemness. Blocking the NF-κB pathway inhibited spheroid formation and expression of stemness genes demonstrating a role for NF-κB in heparanase-mediated stemness. Myeloma cells made deficient in heparanase exhibited decreased stemness properties in vitro and when injected into mice they formed tumors poorly compared to the robust tumorigenic capacity of cells expressing higher levels of heparanase. These studies reveal for the first time a role for heparanase in promoting cancer stemness and provide new insight into its function in driving tumor progression and its association with poor prognosis in cancer patients.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.

Immunostimulatory effect on dendritic cells of the adjuvant-active exopolysaccharide from Leuconostoc mesenteroides strain NTM048.

This study evaluated the immunostimulative effect on bone marrow-derived dendritic cells (DCs) of adjuvant-active exopolysaccharide (EPS) produced by Leuconostoc mesenteroides strain NTM048. EPS stimulation increased IL-6, IL-10, IL-12, and retinal dehydrogenase (RALDH) gene expression levels and induced retinoic acid-synthesizing RALDH-active DCs, which play a crucially important role in controlling adaptive immune responses in mucosa.

Antineoplastic effects of clove buds (Syzygium aromaticum L.) in the model of breast carcinoma.

It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF-7 cells was carried out. Dietary administered CLO caused the dose-dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl-2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase-3 and ALDH1 expression increase after high-dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro-apoptotic effects of CLO extract in MCF-7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase-7, Bcl-2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.

Retinaldehyde dehydrogenase 2 as a molecular adjuvant for enhancement of mucosal immunity during DNA vaccination.

In order for vaccines to induce efficacious immune responses against mucosally transmitted pathogens, such as HIV-1, activated lymphocytes must efficiently migrate to and enter targeted mucosal sites. We have previously shown that all-trans retinoic acid (ATRA) can be used as a vaccine adjuvant to enhance mucosal CD8+ T cell responses during vaccination and improve protection against mucosal viral challenge. However, the ATRA formulation is incompatible with most recombinant vaccines, and the teratogenic potential of ATRA at high doses limits its usage in many clinical settings. We hypothesized that increasing in vivo production of retinoic acid (RA) during vaccination with a DNA vector expressing retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA biosynthesis, could similarly provide enhanced programming of mucosal homing to T cell responses while avoiding teratogenic effects. Administration of a RALDH2- expressing plasmid during immunization with a HIVgag DNA vaccine resulted in increased systemic and mucosal CD8+ T cell numbers with an increase in both effector and central memory T cells. Moreover, mice that received RALDH2 plasmid during DNA vaccination were more resistant to intravaginal challenge with a recombinant vaccinia virus expressing the same HIVgag antigen (VACVgag). Thus, RALDH2 can be used as an alternative adjuvant to ATRA during DNA vaccination leading to an increase in both systemic and mucosal T cell immunity and better protection from viral infection at mucosal sites.

Kinetic characterization and regulation of the human retinaldehyde dehydrogenase 2 enzyme during production of retinoic acid.

Retinoic acid (RA) is an important regulator of embryogenesis and tissue homoeostasis. Perturbation of RA signalling causes developmental disorders, osteoarthritis, schizophrenia and several types of tumours. RA is produced by oxidation of retinaldehyde from vitamin A. The main enzyme producing RA in the early embryo is retinaldehyde dehydrogenase 2 (RALDH2, ALDH1A2). In the present study we describe in depth the kinetic properties and regulation of the human RALDH2 (hRALDH2) enzyme. We show that this enzyme produces RA using in vivo and in vitro assays. We studied the naturally occurring all-trans-, 9-cis- and 13-cis-retinaldehyde isomers as substrates of hRALDH2. Based on the values measured for the Michaelis-Menten constant Km and the maximal rate Vmax, in vitro hRALDH2 displays the same catalytic efficiency for their oxidation. We characterized two known inhibitors of the vertebrate RALDH2 and determined their kinetic parameters on hRALDH2. In addition, RA was studied as a possible inhibitor of hRALDH2 and a regulator of its activity. We show that hRALDH2 is not inhibited by its oxidation product, all-trans-RA, suggesting the absence of a negative feedback regulatory loop. Expression of the Raldh2 gene is known to be regulated by RA itself, suggesting that the main regulation of the hRALDH2 activity level is transcriptional.

Retinoid Homeostatic Gene Expression in Liver, Lung and Kidney: Ontogeny and Response to Vitamin A-Retinoic Acid (VARA) Supplementation from Birth to Adult Age.

Vitamin A (VA, retinol) metabolism is homeostatically controlled, but little is known of its regulation in the postnatal period. Here, we determined the postnatal trajectory of VA storage and metabolism in major compartments of VA metabolism-plasma, liver, lung, and kidney from postnatal (P) day 1 to adulthood. We also investigated the response to supplementation with VARA, a combination of VA and 10% all-trans-retinoic acid that previously was shown to synergistically increase retinol uptake and storage in lung. Nursling pups of dams fed a VA-marginal diet received an oral dose of oil (placebo) or VARA on each of four neonatal days: P1, P4, P7, and P10; and again as adults. Tissues were collected 6 h after the final dosing on P1, P4, P10, and at adult age. Gene transcripts for Lrat and Rbp4 in liver and Raldh-1 and Raldh-3 in lung, did not differ in the neonatal period but were higher, P<0.05, in adults, while Cyp26B1, Stra6, megalin, and Raldh-2 in lung did not differ from perinatal to adult ages. VARA supplementation increased total retinol in plasma, liver and lung, with a dose-by-dose accumulation in neonatal liver and lung, while transcripts for Lrat in liver, megalin in kidney, Cyp26A1/B1 in liver and lung, respectively, and Stra6 in lung, were all increased, suggesting pathways of VA uptake, storage and RA oxidation were each augmented after VARA. VARA decreased hepatic expression of Rbp4, responsible for VA trafficking from liver to plasma, and, in lung, of Raldh-1 and Raldh-2, which function in RA production. Our results define retinoid homeostatic gene expression from neonatal and adult age and show that while supplementation with VARA acutely alters retinol content and retinoid homeostatic gene expression in neonatal and adult lung, liver and kidney, VARA supplementation of neonates increased adult-age VA content only in the liver.

Thyroid hormone activation of retinoic acid synthesis in hypothalamic tanycytes.

Thyroid hormone (TH) is essential for adult brain function and its actions include several key roles in the hypothalamus. Although TH controls gene expression via specific TH receptors of the nuclear receptor class, surprisingly few genes have been demonstrated to be directly regulated by TH in the hypothalamus, or the adult brain as a whole. This study explored the rapid induction by TH of retinaldehyde dehydrogenase 1 (Raldh1), encoding a retinoic acid (RA)-synthesizing enzyme, as a gene specifically expressed in hypothalamic tanycytes, cells that mediate a number of actions of TH in the hypothalamus. The resulting increase in RA may then regulate gene expression via the RA receptors, also of the nuclear receptor class. In vivo exposure of the rat to TH led to a significant and rapid increase in hypothalamic Raldh1 within 4 hours. That this may lead to an in vivo increase in RA is suggested by the later induction by TH of the RA-responsive gene Cyp26b1. To explore the actions of RA in the hypothalamus as a potential mediator of TH control of gene regulation, an ex vivo hypothalamic rat slice culture method was developed in which the Raldh1-expressing tanycytes were maintained. These slice cultures confirmed that TH did not act on genes regulating energy balance but could induce Raldh1. RA has the potential to upregulate expression of genes involved in growth and appetite, Ghrh and Agrp. This regulation is acutely sensitive to epigenetic changes, as has been shown for TH action in vivo. These results indicate that sequential triggering of two nuclear receptor signalling systems has the capability to mediate some of the functions of TH in the hypothalamus.

Retinoic acid signaling biomarkers after treatment with retinoic acid and retinoic acid receptor alpha antagonist (Ro 41-5253) in canine testis: an in vitro organ culture study.

Retinoic acid (RA) is an essential component for development and maintenance of the male genital tract and for spermatogenesis. Aldehyde dehydrogenase (ALDH)1, cytochrome P450 (CYP)26b1, RA receptor (RAR)α, cellular RA-binding protein (CRAB)II, and stimulated by RA gene (STRA)8 are involved in synthesis, metabolism signaling pathways, and as downstream effectors of RA. The objective was to elucidate the effects of exogenous RA and a RARα antagonist on gene expression of ALDH1, CYP26b1, RARα, cellular RA-binding protein II, and STRA8 in an in vitro organ culture model of canine testis. Testicular tissues from medium-sized mixed breed dogs (N = 5; age 8 ± 0.17 mo) were subjected to exogenous all trans-RA (final concentrations of 1, 2, and 10 µM, and DMSO as control) for 24 h. Similarly, testicular tissues were treated with Ro 41-5253 (RARα antagonist), at 1, 10, and 50 µM final concentrations (DMSO as control) for 24 h. Exogenous RA or the RARα antagonist decreased (P < 0.05) mRNA abundance of ALDH1 in a dose-dependent manner compared with control. The CRABII mRNA abundance was greater after RA treatment compared with control (P < 0.01), but only 50 µM Ro 41-5253 effectively decreased CRABII mRNA abundance compared with control (P < 0.01). Although RA did not affect RARα mRNA abundance, the RARα antagonist treatment lowered RARα mRNA abundance compared with control (P < 0.05). Abundance of CYP26b1and STRA8 mRNA were greater (P < 0.05) after RA treatment, but lower (P < 0.05) after RARα antagonist treatment compared with control. In conclusion, exogenous RA decreased mRNA abundance of ALDH1 and increased mRNA abundance of RA signaling molecules and its downstream effectors (CYP26b1, CRABII, and STRA8), whereas treatment with a RARα antagonist effectively decreased RARα and RA metabolism molecules and its downstream effectors in canine testis. Perhaps pharmacological intervention via the RA pathway would enable canine male contraception or treatment of testicular pathology.

Fenretinide treatment prevents diet-induced obesity in association with major alterations in retinoid homeostatic gene expression in adipose, liver, and hypothalamus.

The synthetic retinoid, Fenretinide (FEN), inhibits obesity and insulin resistance in mice and is in early clinical trials for treatment of insulin resistance in obese humans. We aimed to determine whether alterations in retinoic acid (RA)-responsive genes contribute to the beneficial effects of FEN. We examined the effect of FEN on 3T3-L1 adipocyte differentiation and alterations in gene expression in C57Bl/6 and retinaldehyde dehydrogenase (RALDH) 1 knockout (KO) mice fed a high-fat (HF) diet. FEN completely inhibited adipocyte differentiation by blocking CCAAT/enhancer-binding protein (C/EBP) α/peroxisome proliferator-activated receptor (PPAR) γ-mediated induction of downstream genes and upregulating RA-responsive genes like cellular retinol-binding protein-1. In mice fed an HF diet, RA-responsive genes were markedly increased in adipose, liver, and hypothalamus, with short-term and long-term FEN treatment. In adipose, FEN inhibited the downregulation of PPARγ and improved insulin sensitivity and the levels of adiponectin, resistin, and serum RBP (RBP4). FEN inhibited hyperleptinemia in vivo and leptin expression in adipocytes. Surprisingly, hypothalamic neuropeptide Y expression was completely suppressed, suggesting a central effect of FEN to normalize hyperglycemia. Moreover, FEN induced RA-responsive genes in RALDH1 KO mice, demonstrating that FEN can augment RA signaling when RA synthesis is impaired. We show that FEN-mediated beneficial effects are through alterations in retinoid homeostasis genes, and these are strong candidates as therapeutic targets for the treatment of obesity and insulin resistance.

Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration.

Zebrafish heart regeneration occurs through the activation of cardiomyocyte proliferation in areas of trauma. Here, we show that within 3 hr of ventricular injury, the entire endocardium undergoes morphological changes and induces expression of the retinoic acid (RA)-synthesizing enzyme raldh2. By one day posttrauma, raldh2 expression becomes localized to endocardial cells at the injury site, an area that is supplemented with raldh2-expressing epicardial cells as cardiogenesis begins. Induced transgenic inhibition of RA receptors or expression of an RA-degrading enzyme blocked regenerative cardiomyocyte proliferation. Injured hearts of the ancient fish Polypterus senegalus also induced and maintained robust endocardial and epicardial raldh2 expression coincident with cardiomyocyte proliferation, whereas poorly regenerative infarcted murine hearts did not. Our findings reveal that the endocardium is a dynamic, injury-responsive source of RA in zebrafish, and indicate key roles for endocardial and epicardial cells in targeting RA synthesis to damaged heart tissue and promoting cardiomyocyte proliferation.

Chemotype-dependent metabolic response to methyl jasmonate elicitation in Artemisia annua.

Considerable difference in artemisinin and its direct precursors, artemisinic acid and dihydroartemisinic acid, was detected between two chemotypes within the species Artemisia annua (A. annua). These two chemotypes showed differential metabolic response to methyl jasmonate (MeJA) elicitation. Exogenous application of MeJA resulted in an accumulation of dihydroartemisinic acid and artemisinin in Type I plants. In Type II plants, however, artemisinic acid and artemisinin level decreased dramatically under MeJA elicitation. Squalene and other sesquiterpenes, (e.g., caryophyllene, germacrene D), were stimulated by MeJA in both chemotypes. The effect of MeJA elicitation was also studied at the transcription level. Real time RT-PCR analysis showed a coordinated activation of most artemisinin pathway genes by MeJA in Type I plants. The lack of change in cytochrome P450 reductase (CPR) transcript in Type I plants indicates that the rate-limiting enzymes in artemisinin biosynthesis have yet to be identified. Other chemotype-specific electron donor proteins likely exist in A. annua to meet the demand for P450-mediated reactions in MeJA-mediated cellular processes. In Type II plants, mRNA expression patterns of most pathway genes were consistent with the reduced artemisinin level. Intriguingly, the mRNA transcript of aldehyde dehydrogenase1 (ADHL1), an enzyme which catalyzes the oxidation of artemisinic and dihydroartemisinic aldehydes, was upregulated by MeJA. The differential metabolic response to MeJA suggests a chemotype-dependent metabolic flux control towards artemisinin and sterol production in the species A. annua.

Haplotype-based study of the association of alcohol-metabolizing genes with alcohol dependence in four independent populations.

BACKGROUND: Ethanol is metabolized by 2 rate-limiting reactions: alcohol dehydrogenases (ADH) convert ethanol to acetaldehyde that is subsequently metabolized to acetate by aldehyde dehydrogenases (ALDH). Approximately 50% of East Asians have genetic variants that significantly impair this pathway and influence alcohol dependence (AD) vulnerability. We investigated whether variation in alcohol metabolism genes might alter the AD risk in four non-East Asian populations by performing systematic haplotype association analyses to maximize the chances of capturing functional variation. METHODS: Haplotype-tagging SNPs were genotyped using the Illumina GoldenGate platform. Genotypes were available for 40 SNPs across the ADH genes cluster and 24 SNPs across the two ALDH genes in four diverse samples that included cases (lifetime AD) and controls (no Axis 1 disorders). The case control sample sizes were the following: Finnish Caucasians: 232, 194; African Americans: 267, 422; Plains American Indians: 226, 110; and Southwestern American (SW) Indians: 317, 72. RESULTS: In all four populations, as well as HapMap populations, 5 haplotype blocks were identified across the ADH gene cluster: (i) ADH5-ADH4; (ii) ADH6-ADH1A-ADH1B; (iii) ADH1C; (iv) intergenic; (v) ADH7. The ALDH1A1 gene was defined by 4 blocks and ALDH2 by 1 block. No haplotype or SNP association results were significant after correction for multiple comparisons; however, several results, particularly for ALDH1A1 and ADH4, replicated earlier findings. There was an ALDH1A1 block 1 and 2 (extending from intron 5 to the 3' UTR) yin yang haplotype (haplotypes that have opposite allelic configuration) association with AD in the Finns driven by SNPs rs3764435 and rs2303317, respectively, and an ALDH1A1 block 3 (including the promoter region) yin yang haplotype association in SW Indians driven by 5 SNPs, all in allelic identity. The ADH4 SNP rs3762894 was associated with AD in Plains Indians. CONCLUSIONS: The systematic evaluation of alcohol-metabolizing genes in four non-East Asian populations has shown only modest associations with AD, largely for ALDH1A1 and ADH4. A concentration of signals for AD with ALDH1A1 yin yang haplotypes in several populations warrants further study.

Retinoic acid modulates retinaldehyde dehydrogenase 1 gene expression through the induction of GADD153-C/EBPbeta interaction.

Mammalian class I aldehyde dehydrogenase (ALDH) plays an important role in the biosynthesis of the hormone retinoic acid (RA), which modulates gene expression and cell differentiation. RA has been shown to mediate control of human ALDH1 gene expression through modulation of the retinoic acid receptor alpha (RARalpha) and the CCAAT/enhancer binding protein beta (C/EBPbeta). The positive activation of these transcription factors on the ALDH1 promoter is inhibited by RA through a decrease of C/EBPbeta binding to the ALDH1 CCAAT box response element. However, the mechanism of this effect remains unknown. Here we report that the RARalpha/retinoid X receptor beta (RXRbeta) complex binds to the mouse retinaldehyde dehydrogenase 1 (Raldh1) promoter at a non-consensus RA response element (RARE) with similar affinity to that of the consensus RARE. We found that C/EBPbeta binds to a Raldh1 CCAAT box located at -82/-58bp, adjacent to the RARE. Treatment with RA increases GADD153 and GADD153-C/EBPbeta interaction resulting in a decreased cellular availability of C/EBPbeta for binding to the Raldh1 CCAAT box. These data support a model in which high RA levels inhibit Raldh1 gene expression by sequestering C/EBPbeta through its interaction to GADD153.