L-Type Calcium Channel Blocker Enhances Cellular Delivery and Gene Silencing Potency of Cell-Penetrating Asymmetric siRNAs.

The efficient delivery of small interfering RNAs (siRNAs) to the target cells is critical for the pharmaceutical success of RNA interference (RNAi) drugs. One of the possible strategies to improve siRNA delivery is to identify auxiliary molecules that augment their cellular uptake. Herein, we performed a chemical library screening in an effort to discover small molecules that enhance the potency of cholesterol-conjugated, cell-penetrating asymmetric siRNAs (cp-asiRNAs). Interestingly, three compounds identified from the screen share a common dihydropyridine (DHP) core and function as L-type calcium channel blockers (CCBs). Using confocal microscopy and quantitative analysis of small RNAs, we demonstrated that the L-type CCBs increased the endocytic cellular uptake of cp-asiRNAs. Furthermore, these small molecules substantially improved the potency of cp-asiRNAs, not only in vitro but also in vivo on rat skin. Collectively, our study provides an alternative pharmacological approach for the identification of small molecules that potentiate the effects of therapeutic siRNAs.

The natural phytochemical trans-communic acid inhibits cellular senescence and pigmentation through FoxO3a activation.

Ageing is characterized by the accumulation of chronic and irreversible oxidative damage, chronic inflammation and organ dysfunction. To attenuate these ageing-related changes, various natural phytochemicals are often applied. Trans-communic acid (TCA), an active component of brown pine leaf extract, has antimicrobial and cancer chemopreventive activity and inhibits ultraviolet B (UVB)-induced MMP-1 expression. To determine whether the phytochemical TCA could affect the lifespan of an ageing model, Caenorhabditis elegans prevent ageing-related phenotypes of the skin. Caenorhabditis elegans (C. elegans) wild-type N2 and mutant strains were used in this study to explore the lifespan extension effect of TCA and its mechanism. We estimated lipofuscin accumulation and melanin levels, which are closely associated with skin senescence. Moreover, we explored the mechanism of action associated with ageing attenuation. We performed oxidative stress resistance and thermotolerance assays in C. elegans and surface plasmon resonance analysis of TCA binding with the forkhead box-O3a (FoxO3a) protein. TCA, which is the active component in Korean red pine (Pinus densiflora), attenuated ageing-related changes in skin cells. TCA lowered lipofuscin accumulation in fibroblasts and decreased melanin levels in melanocytes. These protective effects were mediated by activation of the representative longevity gene FoxO3a, which was induced by direct binding with TCA. Interestingly, TCA extended the lifespan of C. elegans, although it did not affect stress resistance, oxidative stress or thermotolerance. These results strongly suggest that TCA prevents the senescent phenotype of model organisms and exhibits beneficial effects on ageing-related skin phenotypes through direct FoxO3a activation.

Dehydroabietic Acid Induces Regeneration of Collagen Fibers in Ultraviolet B-Irradiated Human Dermal Fibroblasts and Skin Equivalents.

BACKGROUND/AIMS: Dehydroabietic acid (DAA) is a natural phytochemical found in red pine trees and herbal plants. While DAA and its derivatives are known for improving diabetes and hyperlipidemia, the antiaging effect and its underlying mechanisms of DAA on skin have not been fully examined. Here, we assessed the antiaging effects of DAA on human dermal fibroblasts and skin equivalents. METHODS: We investigated the effect of DAA on the secretion of type I procollagen and matrix metalloproteinase-1 (MMP-1) in ultraviolet B (UVB)-irradiated neonatal normal human dermal fibroblasts (NHDFn). Using nonlinear optical imaging techniques, we visualized quantitative and qualitative changes of collagen fibers by DAA treatment in human skin equivalent models. RESULTS: DAA induces increases in type I procollagen secretion when treated on UVB-irradiated NHDFn. DAA also downregulates secretion of MMP-1 through the inhibition of the JNK signaling pathway. In human skin equivalent models, we successfully visualized the spatial distribution of collagen fibers in the dermis and found that quantity, diameter, and arrangement of collagen fibers in the dermis were significantly improved by DAA treatment. CONCLUSION: Our results suggest that DAA could be a useful agent for improving skin photoaging through the protection and regeneration of collagen fibers in skin.

Dehydroabietic Acid Suppresses Inflammatory Response Via Suppression of Src-, Syk-, and TAK1-Mediated Pathways.

Dehydroabietic acid (DAA) is a naturally occurring diterpene resin acid derived from coniferous plants such as Pinus and Picea. Various bioactive effects of DAA have been studied including antibacterial, antifungal, and anticancer activities. However, the anti-inflammatory mechanism of DAA remains unclear. We evaluated the anti-inflammatory effect of DAA in macrophage cell lines. Dehydroabietic acid clearly reduced nitric oxide (NO) production and inflammatory gene expression decreased according to RT-PCR results. Dehydroabietic acid displayed anti-inflammatory activity at the transcriptional level in results from NF-κB- or AP-1-mediated luciferase assays. To identify the DAA target protein, we investigated NF-κB and AP-1 pathways by Western blotting analysis. Dehydroabietic acid suppressed the activity of proto-oncogene tyrosine protein kinase (Src) and spleen tyrosine kinase (Syk) in the NF-κB cascade and transforming growth factor beta-activated kinase 1 (TAK1) in the AP-1 cascade. Using overexpression strategies, we confirmed that DAA targeted these kinases. Our findings demonstrate the anti-inflammatory effects and molecular mechanism of DAA. This suggests that DAA has potential as a drug or supplement to ameliorate inflammation.

In Vitro Effects of Dehydrotrametenolic Acid on Skin Barrier Function.

Dehydrotrametenolic acid (DTA) is a lanostane-type triterpene acid isolated from Poria cocos Wolf (Polyporaceae). Several studies have reported the anti-inflammatory and antidiabetic effects of DTA; however, its effects on the skin are poorly understood. In this study, we investigated the effects of DTA on skin barrier function in vitro and its regulatory mechanism in human keratinocyte cell line HaCaT cells. DTA increased the microRNA (mRNA) expression of natural moisturizing factor-related genes, such as HAS-2, HAS-3, and AQP3 in HaCaT cells. DTA also upregulated the mRNA expression of various keratinocyte differentiation markers, including TGM-1, involucrin, and caspase-14. Moreover, the protein expression of HAS-2, HAS-3, and TGM-2 were significantly increased by DTA. To examine the regulatory mechanisms of DTA, Western blotting, luciferase-reporter assays, and RT-PCR were conducted. The phosphorylation of mitogen-activated protein kinases (MAPKs) and IκBα were increased in DTA-treated HaCaT cells. In addition, AP-1 and NF-κB transcriptional factors were dose-dependently activated by DTA. Taken together, our in vitro mechanism studies indicate that the regulatory effects of DTA on skin hydration and keratinocyte differentiation are mediated by the MAPK/AP-1 and IκBα/NF-κB pathways. In addition, DTA could be a promising ingredient in cosmetics for moisturizing and increased skin barrier function.

The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells.

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.

Transcriptomic variation in proanthocyanidin biosynthesis pathway genes in soybean (Glycine spp.).

BACKGROUND: Proanthocyanidins are oligomeric or polymeric end products of flavonoid metabolic pathways starting with the central phenylpropanoid pathway. Although soybean (Glycine spp.) seeds represent a major source of nutrients for the human diet, as well as components for the cosmetics industry as a result of their high levels of flavonoid metabolites, including isoflavonoids, anthocyanins and proanthocyanidins, the genetic regulatory mechanisms underlying proanthocyanidin biosynthesis in soybean remain unclear. RESULTS: We evaluated interspecific and intraspecific variability in flavonoid components in soybean using 43 cultivars, landraces and wild soybean accessions. We performed transcriptomic profiling of genes encoding enzymes involved in flavonoid biosynthesis using three soybean genotypes, Hwangkeum (elite cultivar), IT109098 (landrace) and IT182932 (wild accession), in seeds. We identified a Glycine max landrace, IT109098, with a proanthocyanidin content as high as that of wild soybean. Different homologous genes for anthocyanidin reductase, which is involved in proanthocyanidin biosynthesis, were detected as differentially expressed genes between IT109098 and IT182932 compared to Hwangkeum. CONCLUSION: We detected major differences in the transcriptional levels of genes involved in the biosynthesis of proanthocyanidin and anthocyanin among genotypes beginning at the early stage of seed development. The results of the present study provide insights into the underlying genetic variation in proanthocyanidin biosynthesis among soybean genotypes. © 2017 Society of Chemical Industry.

Licochalcone A, a polyphenol present in licorice, suppresses UV-induced COX-2 expression by targeting PI3K, MEK1, and B-Raf.

Licorice is a traditional botanical medicine, and has historically been commonly prescribed in Asia to treat various diseases. Glycyrrhizin (Gc), a triterpene compound, is the most abundant phytochemical constituent of licorice. However, high intake or long-term consumption of Gc has been associated with a number of side effects, including hypertension. However, the presence of alternative bioactive compounds in licorice with anti-carcinogenic effects has long been suspected. Licochalcone A (LicoA) is a prominent member of the chalcone family and can be isolated from licorice root. To date, there have been no reported studies on the suppressive effect of LicoA against solar ultraviolet (sUV)-induced cyclooxygenase (COX)-2 expression and the potential molecular mechanisms involved. Here, we show that LicoA, a major chalcone compound of licorice, effectively inhibits sUV-induced COX-2 expression and prostaglandin E2 PGE2 generation through the inhibition of activator protein 1 AP-1 transcriptional activity, with an effect that is notably more potent than Gc. Western blotting analysis shows that LicoA suppresses sUV-induced phosphorylation of Akt/ mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinases (ERK)1/2/p90 ribosomal protein S6 kinase (RSK) in HaCaT cells. Moreover, LicoA directly suppresses the activity of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK)1, and B-Raf, but not Raf-1 in cell-free assays, indicating that PI3K, MEK1, and B-Raf are direct molecular targets of LicoA. We also found that LicoA binds to PI3K and B-Raf in an ATP-competitive manner, although LicoA does not appear to compete with ATP for binding with MEK1. Collectively, these results provide insight into the biological action of LicoA, which may have potential for development as a skin cancer chemopreventive agent.

Development of Long Asymmetric siRNA Structure for Target Gene Silencing and Immune Stimulation in Mammalian Cells.

Post-transcriptional regulation of transcript abundances by RNA interference (RNAi) is a widely conserved regulatory mechanism to control cellular processes. We previously introduced an alternative siRNA structure called asymmetric siRNA (asiRNA), and showed that asiRNA exhibits comparable gene-silencing efficiency with reduced off-target effects compared with conventional siRNAs. However, to what extent the length of the guide strand affects the gene-silencing efficiency of asiRNAs is still elusive. In this study, we analyzed in detail the gene-silencing ability of asiRNAs along the guide strand length and immunostimulatory capacity of asiRNAs. We generated asiRNAs containing various guide strand lengths ranging from 25 to 29 nt, called long asiRNA (lasiRNA). We found that the gene-silencing activity of lasiRNAs decreased as the length of the guide strand increased. Nonetheless, the 3'-end overhangs that are complementary to the target gene have higher efficiency for gene silencing compared with mismatched overhangs. In addition, we found that the silencing efficiency of lasiRNAs correlates with their Ago2-binding affinity. Finally, replacing the mismatched overhang with a TLR7- or TLR9-associated immune response motif induced a toll-like receptor (TLR)-specific immune response and retained gene-silencing activity. Our findings demonstrate that lasiRNA structures can be tailored to function as bifunctional siRNA, which trigger a specific immune response combined with target gene silencing. Taken together, we anticipate that our findings provide a road map for the subsequent development of immune-stimulating lasiRNA, which bear the potential to be applied for therapeutic benefits.

RIG-I-Mediated Innate Immune Stimulation by Chemically Synthesized Long Double-Stranded RNAs Is Structure and Sequence Dependent.

Double-stranded RNAs (dsRNAs) longer than 30 bp have not been considered desirable RNA interference (RNAi) triggering structures in mammalian cells as they nonspecifically activate innate immune response. However, in earlier studies, not only dsRNA length but also 5'-triphosphate moiety produced by in vitro transcription might have affected the stimulation of innate immune system. Herein, using chemically synthesized long dsRNAs without 5'-triphosphate, we elucidated direct relationship between length of dsRNAs and innate immune stimulation. First, we found that blunt-ended, chemically synthesized 38/40-60 bp-long dsRNAs induced retinoic acid-inducible gene I (RIG-I)-mediated innate immune response, which was suppressed by the introduction of the 2-nt 3' overhang structure. Surprisingly, we discovered that RIG-I activation by these long dsRNAs is also sequence dependent, and the sequence composition at dsRNA termini is important for RIG-I activation. In addition, we identified that long dsRNAs over 38 bp could elicit single- or dual-target gene silencing in a Dicer-independent manner. Taken together, our findings may serve as guidelines to develop an immunostimulatory RNAi trigger to exploit host's innate immune system, as well as a specific dual-gene targeting RNAi therapeutics platform without nonspecific innate immune stimulation by RIG-I.