Selective inhibition of human type-5 17ß-hydroxysteroid dehydrogenase (AKR1C3) by baccharin, a component of Brazilian propolis.

The human aldo-keto reductase (AKR) 1C3, also known as type-5 17ß-hydroxysteroid dehydrogenase and prostaglandin F synthase, has been suggested as a therapeutic target in the treatment of prostate and breast cancers. In this study, AKR1C3 inhibition was examined by Brazilian propolis-derived cinnamic acid derivatives that show potential antitumor activity, and it was found that baccharin (1) is a potent competitive inhibitor (K(i) 56 nM) with high selectivity, showing no significant inhibition toward other AKR1C isoforms (AKR1C1, AKR1C2, and AKR1C4). Molecular docking and site-directed mutagenesis studies suggested that the nonconserved residues Ser118, Met120, and Phe311 in AKR1C3 are important for determining the inhibitory potency and selectivity of 1. The AKR1C3-mediated metabolism of 17-ketosteroid and farnesal in cancer cells was inhibited by 1, which was effective from 0.2 µM with an IC(50) value of about 30 µM. Additionally, 1 suppressed the proliferation of PC3 prostatic cancer cells stimulated by AKR1C3 overexpression. This study is the first demonstration that 1 is a highly selective inhibitor of AKR1C3.

Royal jelly facilitates restoration of the cognitive ability in trimethyltin-intoxicated mice.

Trimethyltin (TMT) is a toxic organotin compound that induces acute neuronal death selectively in the hippocampal dentate gyrus (DG) followed by cognition impairment; however the TMT-injured hippocampal DG itself is reported to regenerate the neuronal cell layer through rapid enhancement of neurogenesis. Neural stem/progenitor cells (NS/NPCs) are present in the adult hippocampal DG, and generate neurons that can function for the cognition ability. Therefore, we investigated whether royal jelly (RJ) stimulates the regenerating processes of the TMT-injured hippocampal DG, and found that orally administered RJ significantly increased the number of DG granule cells and simultaneously improved the cognitive impairment. Furthermore, we have already shown that RJ facilitates neurogenesis of cultured NS/NPCs. These present results, taken together with previous observations, suggest that the orally administered RJ may be a promising avenue for ameliorating neuronal function by regenerating hippocampal granule cells that function in the cognition process.

Effects of long-term administration of royal jelly on pituitary weight and gene expression in middle-aged female rats.

To determine the effects of ingested royal jelly (RJ) on the pituitary in middle-aged female rats, we performed a long-term RJ administration test. Several animals showed age-related increases in pituitary weight, and RJ administration compensated for the increase. RJ tended to down-regulate prolactin mRNA and up-regulated thyroid-stimulating hormone beta mRNA in the pituitary. This suggests that RJ compensates for age-associated decline in pituitary functions.

Effects of propolis on cell growth and gene expression in HL-60 cells.

Brazilian propolis obtained from honeybee hives was extracted with water or ethanol. Cell growth-inhibitory activities of these propolis extracts were found in HL-60 human myeloid leukemia cells. The extracts-induced apoptosis in the cells, which was characterized by morphological and nucleosomal DNA fragmentation analysis. The apoptosis was mainly attributed to the induction of granulocytic differentiation, which was evaluated by nitro blue tetrazolium (NBT) reducing assays and cytofluorometric analysis for the expression of cell surface marker CD11b. DNA microarray analysis was performed to examine the gene expression profiles in the propolis-treated HL-60 cells accompanied with granulocytic differentiation, which were compared with those in all-trans retinoic acid-treated cells. Several genes were up- or down-regulated. Two genes encoding S100 calcium binding protein A9 and ferritin, heavy polypeptide 1 were up-regulated, which were also confirmed by semi-quantitative reverse transcriptase-PCR (RT-PCR). Propolis-induced growth inhibition in HL-60 cells was, at least in part, due to differentiation with gene expression profiles, which are similar to those induced by all-trans retinoic acid.

Propolis cinnamic acid derivatives induce apoptosis through both extrinsic and intrinsic apoptosis signaling pathways and modulate of miRNA expression.

Propolis cinnamic acid derivatives have a number of biological activities including anti-oxidant and anti-cancer ones. In this study, we aimed to elucidate the mechanism of the anti-cancer activity of 3 representative propolis cinnamic acid derivatives, i.e., Artepilin C, Baccharin and Drupanin in human colon cancer cell lines. Our study demonstrated that these compounds had a potent apoptosis-inductive effect even on drug-resistant colon cancer cells. Combination treatment of human colon cancer DLD-1 cells with 2 of these compounds, each at its IC20 concentration, induced apoptosis by stimulating both intrinsic and extrinsic apoptosis signaling pathways. Especially, Baccharin plus Drupanin exhibited a synergistic growth-inhibitory effect by strengthening both intrinsic and extrinsic apoptotic signaling transduction through TRAIL/DR4/5 and/or FasL/Fas death-signaling loops and by increasing the expression level of miR-143, resulting in decreased expression levels of the target gene MAPK/Erk5 and its downstream target c-Myc. These data suggest that the supplemental intake of these compounds found in propolis has enormous significance with respect to cancer prevention.

Artepillin C, a major ingredient of Brazilian propolis, induces a pungent taste by activating TRPA1 channels.

Brazilian green propolis is a popular health supplement because of its various biological properties. The ethanol extract of Brazilian green propolis (EEBP) is characteristic for its herb-like smell and unique pungent taste. However, the ingredients responsible for its pungency have not yet been identified. This study provides the first evidence that artepillin C is the main pungent ingredient in EEBP and that it potently activates human transient receptor potential ankyrin 1 (TRPA1) channels. EEBP was fractionated using column chromatography with a step gradient elution of an ethanol-water solution, and the fractions having the pungent taste were determined by sensory tests. HPLC analysis revealed that the pungent fraction was composed primarily of artepillin C, a prenylated derivative of cinnamic acid. Artepillin C was also identified as the pungent compound of EEBP by organoleptic examiners. Furthermore, the effects of artepillin C and other cinnamic acids found in EEBP on TRPA1 channels were examined by calcium imaging and plate reader-based assays in human TRPA1-expressing cells to investigate the molecular mechanisms underlying their pungent tastes. Artepillin C and baccharin activated the TRPA1 channel strongly, whereas drupanin caused a slight activation and p-coumaric acid showed no activation. Because the EC(50) values of artepillin C, baccharin, and allyl isothiocyanate were 1.8 µM, 15.5 µM, and 6.2 µM, respectively, artepillin C was more potent than the typical TRPA1 agonist allyl isothiocyanate. These findings strongly indicate that artepillin C is the main pungent ingredient in EEBP and stimulates a pungent taste by activating TRPA1 channels.

Design, synthesis and evaluation of caffeic acid phenethyl ester-based inhibitors targeting a selectivity pocket in the active site of human aldo-keto reductase 1B10.

Inhibitors of a human aldo-keto reductase, AKR1B10, are regarded as promising therapeutics for the treatment of cancer, but those with both high potency and selectivity compared to the structurally similar aldose reductase (AKR1B1) have not been reported. In this study, we have found that, among honeybee propolis products, caffeic acid phenethyl ester (CAPE) inhibited AKR1B10 (IC(50) = 80 nM) with 7-fold selectivity over AKR1B1. Based on a model of docked CAPE in AKR1B10, its derivatives were designed, synthesized and evaluated for inhibitory potency. Among them, 3-(4-hydroxy-2-methoxyphenyl)acrylic acid 3-(3-hydroxyphenyl)propyl ester (10c) was the most potent competitive inhibitor (K(i) = 2.6 nM) with 790-fold selectivity for AKR1B10 over AKR1B1. Molecular docking of 10c and site-directed mutagenesis of AKR1B10 residues suggested that the interactions between the 2-methoxy and 3-hydroxy groups of 10c and the enzyme's Val301 and Gln114, respectively, are important for the inhibitor's selectivity. Additionally, the sub-µM concentration of 10c significantly suppressed the farnesal metabolism and cellular proliferation in AKR1B10-overexpressing cells.

Effects of propolis from different areas on mast cell degranulation and identification of the effective components in propolis.

Propolis is considered to down-regulate type I allergy, but the effective components of propolis remain unknown. In addition, propolis components vary depending on the area from which they are collected due to variations among wild plants in an area. Therefore, we compared the effects of water and ethanol extracts of propolis from Brazil and China on mast cell degranulation and cytokine production, thereby identifying effective components in propolis. The amount of released beta-hexosaminidase via high-affinity IgE receptor I (Fc epsilon RI) from rat basophilic leukemia (RBL-2H3) cells was used as an index of degranulation. All propolis extracts inhibited degranulation from antigen-stimulated RBL-2H3 cells, but the effective doses differed according to collection areas. The ethanol extract of Chinese propolis, which was the strongest inhibitor of mast cell degranulation, was divided into compounds using normal- and reversed-phase liquid chromatography. The isolated anti-allergic components were identified as chrysin, kaempferol and its derivative, and chrysin was revealed to inhibit IL-4 and MCP-1 production from antigen-stimulated RBL-2H3 cells. HPLC quantification also revealed the Brazilian propolis extract to contain only small amounts of these flavonoids, which suggested that variation in propolis components could affect anti-allergic properties.

Estrogenic activities of Fatty acids and a sterol isolated from royal jelly.

We have previously reported that royal jelly (RJ) from honeybees (Apis mellifera) has weak estrogenic activity mediated by interaction with estrogen receptors that leads to changes in gene expression and cell proliferation. In this study, we isolated four compounds from RJ that exhibit estrogenic activity as evaluated by a ligand-binding assay for the estrogen receptor (ER) beta. These compounds were identified as 10-hydroxy-trans-2-decenoic acid, 10-hydroxydecanoic acid, trans-2-decenoic acid and 24-methylenecholesterol. All these compounds inhibited binding of 17beta-estradiol to ERbeta, although more weakly than diethylstilbestrol or phytoestrogens. However, these compounds had little or no effect on the binding of 17beta-estradiol to ERalpha. Expression assays suggested that these compounds activated ER, as evidenced by enhanced transcription of a reporter gene containing an estrogen-responsive element. Treatment of MCF-7 cells with these compounds enhanced their proliferation, but concomitant treatment with tamoxifen blocked this effect. Exposure of immature rats to these compounds by subcutaneous injection induced mild hypertrophy of the luminal epithelium of the uterus, but was not associated with an increase in uterine weight. These findings provide evidence that these compounds contribute to the estrogenic effect of RJ.

Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines.

Royal jelly (RJ) has diverse physiological and pharmacological functions. We observed its weak estrogenic activity in the previous study. RJ stimulated the proliferation of mouse osteoblast-like MC3T3-E1 cells at 0.1 mg/ml, and the effect was blocked by the specific estrogen receptor antagonist ICI 182,780. The addition of 0.1-1.0 mg/ml RJ enhanced collagen production in culture medium. Oral administration of RJ to normal female mice for 9 weeks increased the ash content of their tibiae. DNA microarray analysis revealed significant changes in gene expression related to extracellular matrix formation when the femurs of mice fed RJ were analyzed. Quantitative reverse transcriptase-PCR (RT-PCR) confirmed up-regulation of procollagen I alpha1 gene expression. These data suggest that RJ as a whole or some of its individual components stimulates production of type I collagen and other activities for bone formation through action on osteoblasts.

Differential expression pattern of C4 bundle sheath expression genes in rice, a C3 plant.

NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) are specifically expressed in bundle sheath cells (BSCs) in NADP-ME-type and PCK-type C4 plants, respectively. Unlike the high activities of these enzymes in the green leaves of C4 plants, their low activities have been detected in the leaves of C3 plants. In order to elucidate the differences in the gene expression system between C3 and C4 plants, we have produced chimeric constructs with the beta-glucuronidase (GUS) reporter gene under the control of the maize NADP-Me (ZmMe) or Zoysia japonica Pck (ZjPck) promoter and introduced these constructs into rice. In leaves of transgenic rice, the ZmMe promoter directed GUS expression not only in mesophyll cells (MCs) but also in BSCs and vascular cells, whereas the ZjPck promoter directed GUS expression only in BSCs and vascular cells. Neither the ZjPck nor ZmMe promoters induced GUS expression due to light. In rice leaves, the endogenous NADP-Me (OsMe1) was expressed in MCs, BSCs and vascular cells, whereas the rice Pck (OsPck1) was expressed only in BSCs and vascular cells. Taken together, the results obtained from transgenic rice demonstrate that the expression pattern of ZmMe or ZjPck in transgenic rice was reflected by that of its counterpart gene in rice.

Identification of caffeoylquinic acid derivatives from Brazilian propolis as constituents involved in induction of granulocytic differentiation of HL-60 cells.

We have previously reported that Brazilian propolis extracts inhibited growth of HL-60 human myeloid leukemia cells, which is partly attributed to the induction of apoptosis associated with granulocytic differentiation. In this study, we isolated three compounds which induce granulocytic differentiation evaluated by nitroblue tetrazolium (NBT)-reducing assays from the water extract of propolis and identified as 4,5-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 3,4-di-O-caffeoylquinic acids by NMR analysis. Cell growth inhibitory activity of these caffeoylquinic acids was found in HL-60 cell, which was mainly attributed to the induction of apoptosis. Furthermore, the potency of caffeoylquinic acid derivatives to induce granulocytic differentiation was examined in HL-60 cells. Caffeic, quinic, and chlorogenic acids had no effects on the NBT-reducing activity, while 3,4,5-tri-O-caffeoylquinic acid induced more than 30% of NBT-positive cells. These results suggest that the number of the caffeoyl groups bound to quinic acid plays an important role in the potency of the caffeoylquinic acid derivatives to induce granulocytic differentiation. This is the first report demonstrating that the caffeoylquinic acid derivatives induce granulocytic differentiation of HL-60 cells.

Expression of cold-tolerant pyruvate, orthophosphate dikinase cDNA, and heterotetramer formation in transgenic maize plants.

Maize is a typical C4 plant of the NADP-malic enzyme type, and its high productivity is supported by the C4 photosynthetic cycle, which concentrates atmospheric CO2 in the leaves. The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced. Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C4 pathway in maize, loses its activity below about 12 degrees C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures. To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveria brownii into maize by Agrobacterium-mediated transformation. We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F. bidentis and F. brownii with a maximum content of I mg/g fresh weight. In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK. Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved. Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 degrees C lower than that of the wild type.

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.).

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.