Timeframes of UVA-induced bystander effects in human keratinocytes

The bystander effect is defined as the induction of cellular damage in unirradiated cells, induced by irradiated cells in the surrounding area. Our laboratory has previously identified that an environmentally relevant dose of UVA is able to induce the effect in human keratinocytes and fibroblasts, seen as reduced clonogenic survival. Here we report on our investigations into the periods over which the bystander signals are released by the irradiated cells and for how long unirradiated cells need to be exposed to them for the effect to be induced. Using a coincubation system we have identified that irradiated cells do not release the signals immediately following irradiation but have a time lag of over 24 h before levels are sufficient to induce the effect, with the signals being released for a minimum of 3 days following irradiation. We have also found that the recipient cells only require at most 24 h of exposure to these signals for induction of the effect. These data indicate that a single exposure to UVA can exert an effect for several days postirradiation, thus amplifying the deleterious effects of exposure.     

Hypericum perforatum L. extract - novel photosensitizer against human bladder cancer cells

The polar methanolic fraction (PMF) of the Hypericum perforatum L. extract has recently been developed and tested as a novel, natural photosensitizer for use in the photodynamic therapy (PDT), and photodynamic diagnosis (PDD). PMF has been tested on HL-60 leukemic cells and cord blood hemopoietic progenitors. In the present study, the efficacy of PMF as a phototoxic agent against urinary bladder carcinoma has been studied using the T24 (high grade metastatic cancer), and RT4 (primary low grade papillary transitional cell carcinoma) human bladder cancer cells. Following cell culture incubation, PMF was excited using 630 nm laser light. The photosensitizer exhibited significant photocytotoxicity in both cell lines at a concentration of 60microg/ml, with 4-8 J/cm(2) light dose, resulting in cell destruction from 80% to 86%. At the concentration of 20microg/ml PMF was not active in either cell line. These results were compared with the results obtained in the same cell lines, under the same conditions with a clinically approved photosensitizer, Photofrin. Photofrin was used in the maximum clinically tolerable dose of 4microg/ml, and it was also excited with 630 nm laser light. In the T24 cell Photofrin exhibited slightly less photocytotocixity, compared with PMF, resulting in 77% cell death with 8J/cm(2) light dose. However, against the RT4 cells Photofrin resulted in minimal cell death (9%) with even 8J/cm(2) light dose. Finally, the type of cell death induced by PMF photoactivation was studied using flow cytometry and DNA laddering. Cell death by PMF photodynamic action in these two bladder cell lines is caused predominently by apoptosis. The reported significant photocytotoxicity, selective localization, natural abundance, easy, and inexpensive preparation, underscore that the PMF extract hold the promise of being a novel, effective PDT photosensitizer.     

On the photobiological properties of chimeras combining quaternary ammonium derivatives of retinoic amides and psoralen. A study with cultured human keratinocytes

The effectiveness of the combination of retinoids with 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light in the treatment of some cutaneous proliferative diseases has motivated the synthesis of new "chimera-type" molecules built from psoralen derivatives and retinoic amides and related molecules. The chimeras result from the combination of 8-(3-bromopropyloxy)-psoralen with amides prepared by reacting 4-amino-pyridine with 13E- and 13Z-retinoic acids or a "retinoid-like" derivative with an alkene chain of only three double bonds. The synthesis of chimeras built with the 8-(3-bromopropyloxy)-psoralen and the amide of cinnamic acid or its 4-methoxy derivative has also been carried out. In contrast to 8-MOP, all the chimeras exhibit strong molar absorptivities in the range 20 000-40 000 M(-1) cm(-1) in the 340-390 nm UV-A region. The "retinoid-like"- and retinoid-psoralen chimeras are characterized by a marked dark toxicity toward proliferating NCTC 2544 keratinocytes (with a lethal dose corresponding to 50% cell survival [LD50] of 1-5 microM) as compared with that of the cinnamic acid derivative-psoralen chimeras (LD50 > or = 50 microM). This toxicity leads to alteration of the mitochondrial membrane potential. At nontoxic concentrations, the chimeras demonstrate effective psoralens + UV-A-induced photocytotoxicity. They are moderate photosensitizers of membrane lipid peroxidation. Cell apoptosis is a major photocytotoxic process as suggested by the fluorescence-activated cell-sorting technique using annexin-fluorescein isothiocyanate and propidium iodide as apoptotic markers.     

Quality and antitumour activity evaluation of extract of Hypericum ascyron

Similarity assessment of complex chromatographic profiles is a potential tool for the identification and quality control of herbal medicinal products to guarantee the expected biological activity. In this paper, a high‐performance liquid chromatography method was established for controlling the quality of extract of Hypericum ascyron for the first time. With this method, the correlation coefficients of similarity of 10 batches extract of H. ascyron were >0.97. The extract of H. ascyron displayed steadily inhibitorty activities on the growth of human cervical cancer Hela cell lines. Therefore, the present study successfully set up a sensitive efficient method which might confirm stable biological activity of the extract of H. ascyron. Copyright © 2014 John Wiley & Sons, Ltd.     

Anti-inflammatory and analgesic effects of human placenta extract

In this study, we investigated the effects of human placenta extract (HPE, Laennec inj.) on pro-inflammatory cytokines and mediators secreted from lipopolysaccharide-stimulated RAW264.7 macrophages. We found that HPE significantly inhibited the production of nitric oxide, tumour necrosis factor-α and cyclooxygenase-2. We studied the anti-inflammatory and analgesic potential of HPE in murine models of inflammation/inflammatory pain. Rats were assigned to six groups and were administered either saline or HPE (0.33, 1, 3 and 6 mL kg?1) intraperitoneally. Diclofenac was used as a positive control. HPE attenuated the swelling of the rat's hind paw. The vascular permeability induced by acetic acid was significantly reduced by HPE. HPE reduced the formation of granuloma in carrageenan air pouch and hind paw oedema in complete Freund's adjuvant-induced chronic arthritis in rats. HPE attenuated writhing episodes. An increase in hot-plate latency was observed in mice receiving HPE. HPE also increased the pain threshold in the Randall-Selitto test. In the tail-flick assay, HPE prolonged the reaction time of rats to radiant heat stimulation. These results suggest that HPE has potent anti-inflammatory and anti-nociceptive activities.     

Protective effects of cyanidin-3-O-beta-glucopyranoside against UVA-induced oxidative stress in human keratinocytes

Ultraviolet-A (UVA) radiation causes significant oxidative stress because it leads to the generation of reactive oxygen species (ROS), leading to extensive cellular damage and eventual cell death either by apoptosis or necrosis. We evaluated the protective effects of cyanidin-3-O-beta-glucopyranoside (C-3-G) against UVA-induced apoptosis and DNA fragmentation in a human keratinocyte cell line (HaCaT). Treatment of HaCaT cells with C-3-G before UVA irradiation inhibited the formation of apoptotic cells (61%) and DNA fragmentation (54%). We also investigated antioxidant properties of C-3-G in HaCaT cells against ROS formation at apoptotic doses of UVA; C-3-G inhibited hydrogen peroxide (H2O2) release (an indicator of cellular ROS formation) after UVA irradiation. Further confirmation of the potential of C-3-G to counteract UVA-induced ROS formation comes from our demonstration of its ability to enhance the resistance of HaCaT cells to the apoptotic effects of both H2O2 and the superoxide anion (O2*-), two ROS involved in UVA-oxidative stress. Furthermore, in terms of Trolox Equivalent Antioxidant Activity, C-3-G treatment led to a greater increase in antioxidant activity in the membrane-enriched fraction than in the cytosol (55% vs 19%). The protective effects against UVA-induced ROS formation can be attributed to the higher membrane levels of C-3-G incorporation. These encouraging in vitro results support further research into C-3-G (and other anthocyanins) as novel agents for skin photoprotection.     

Narrow-band UVB induces apoptosis in human keratinocytes

Narrow-band ultraviolet (NB-UVB) phototherapy emits mostly 311/312 nm light and is commonly used in the treatment of inflammatory skin disorders. As a source of UVB irradiation, NB-UVB causes apoptosis in T lymphocytes but its effects on keratinocytes are unknown. Herein, we have investigated the ability of NB-UVB to induce apoptosis in keratinocytes. Two types of human keratinocytes, primary and immortalized, were exposed to NB-UVB and broad-band UVB (BB-UVB; 315-280 nm) and tested for apoptosis. Both UVB light sources induced apoptosis in keratinocytes as determined by the presence of DNA ladders, although NB-UVB required approximately ten fold higher doses; NB-UVB (1000 mJ/cm2) and BB-UVB (125 mJ/cm2). By comparison, lower doses of NB-UVB (750 mJ/cm2) induced apoptosis in T lymphocytes, suggesting cell type specificity for NB-UVB induced apoptosis. Approximately, 50% or more of the cells underwent apoptosis when exposed to NB-UVB or BB-UVB as revealed by TUNEL assay. Electron micrographs showed that NB-UVB irradiated keratinocytes contained marked chromatin condensation, extensive cytoplasmic vacuolization and fragmentation of the nuclear envelope. Furthermore, Western blot analysis confirmed the presence of activated products of caspase 3 in keratinocytes that received apoptotic doses of NB-UVB. This study defines conditions by which NB-UVB irradiation causes apoptosis in keratinocytes.     

UV-ABC screens of luteolin derivatives compared to edelweiss extract

Pure luteolin is a remarkably heat (200°C/6 days) and UV stable UV-A screen, however, native luteolin enriched to 37% in an edelweiss extract lost its UV-A screen properties upon UV irradiation (～4MJm(-2)). This contrasting behavior led to the examination of a series of purified luteolin derivatives as UV screen candidates. 3',4',5,7-Tetralipoyloxyflavones were synthesized from luteolin (3',4',5,7-tetrahydroxyflavone) and fatty acid chlorides. These acylated semi-biomolecules show a hypsochromic shift in UV-Vis spectra of about Δλ(A→B)=58nm and absorbed in the centre of the harmful UV-B band (λ(max)=295nm). Luteolin was also hydroxyethylated with Br(CH(2))(2)OH. This substitution has no effect on the λ(max)=330nm absorption of luteolin (UV-A band). Finally the natural 4'-O-β-glucosyl-3',5,7-trihydroxyflavone was extracted from edelweiss and used as a purified natural benchmark. Glycosylated and hydroxyethylated luteolin are both UV stable. Fully acylated luteolin derivatives degrade upon UV exposure to a stable UV-C screen with a hypsochroic shift Δλ(B→C)=35nm. All in all, three molecular structures based on luteolin with sunscreen properties were found, distinguishable in: UV-A, UV-B, and UV-C filters. The natural product based UV-absorbers show promise as alternatives to synthetic molecules and nanoparticles in sunscreen products.     

Syntheses of arbutin-alpha-glycosides and a comparison of their inhibitory effects with those of alpha-arbutin and arbutin on human tyrosinase

The effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) and 4-hydroxyphenyl beta-glucopyranoside (arbutin) on the activity of tyrosinase from human malignant melanoma cells were examined. The inhibitory effect of alpha-arbutin on human tyrosinase was stronger than that of arbutin. The K(i) value for alpha-arbutin was calculated to be 1/20 that for arbutin. We then synthesized arbutin-alpha-glycosides by the transglycosylation reaction of cyclomaltodextrin glucanotransferase using arbutin and starch, respectively, as acceptor and donor molecules. The structural analyses using 13C- and 1H-NMR proved that the transglycosylated products were 4-hydroxyphenyl beta-maltoside (beta-Ab-alpha-G1) and 4-hydroxyphenyl beta-maltotrioside (beta-Ab-alpha-G2). These arbutin-alpha-glycosides exhibited competitive type inhibition on human tyrosinase, and their K(i) values were calculated to be 0.7 mM and 0.9 mM, respectively. These arbutin-alpha-glycosides possessed stronger inhibitory activity than arbutin, but less activity than alpha-arbutin. These results suggested that the alpha-glucosidic linkage of hydroquinone-glycosides plays an important role in the inhibitory effect on human tyrosinase.     

Using high-throughput screening data to discriminate compounds with single-target effects from those with side effects

The most desirable compound leads from high-throughput assays are those with novel biological activities resulting from their action on a single biological target. Valuable resources can be wasted on compound leads with significant 'side effects' on additional biological targets; therefore, technical refinements to identify compounds that primarily have effects resulting from a single target are needed. This study explores the use of multiple assays of a chemical library and a statistic based on entropy to identify lead compound classes that have patterns of assay activity resulting primarily from small molecule action on a single target. This statistic, called the coincidence score, discriminates with 88% accuracy compound classes known to act primarily on a single target from compound classes with significant side effects on nonhomologous targets. Furthermore, a significant number of the compound classes predicted to have primarily single-target effects contain known bioactive compounds. We also show that a compound's known biological target or mechanism of action can often be suggested by its pattern of activities in multiple assays.     

Reactions of psoralen radical cations with biological substrates

The reactions of several psoralen and coumarin radical cations with biological substrates such as nucleotides, amino acids and alkenes that serve as models for unsaturated fatty acids have been examined. The radical cations were generated by laser photoionization of the parent psoralen or coumarin in aqueous buffer in most cases. Easily oxidized substrates such as tyrosine, tryptophan and guanosine monophosphate react with the 8-methoxypsoralen and several methoxy-substituted coumarin radical cations with rate constants in excess of 2 x 10(9) M-1 s-1. In each case reaction occurs via electron transfer, as demonstrated by the observation of quencher-derived radical cations or radicals by transient absorption spectroscopy. For other substrates such as histidine, methionine and adenosine monophosphate the measured rate constants are significantly slower and vary with the oxidation potential of both the parent psoralen or coumarin and the quencher, again indicative of electron transfer reactivity. Most of the alkenes studied also react with the psoralen or coumarin radical cations via electron transfer, although there is some evidence for addition for linoleic acid. Product studies carried out using both lamp and laser irradiation in the presence of deoxyguanosine as a radical cation trap lead to the formation of characteristic base-derived Type-I (electron transfer) products. This lends support to our previous hypothesis that photoionization occurs via a monophotonic process and is thus relevant to conditions used in clinical phototherapeutic applications of psoralens. The results demonstrate the relevance of electron transfer chemistry to the use of psoralens and related compounds as photoactivated drugs.     

Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes paragraph sign

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B-induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B-mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10-40 microg/mL) for 24 h before UV-B (40 mJ/cm(2)) exposure dose dependently inhibited UV-B-mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 microg/mL) inhibited UV-B-mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B-mediated activation of nuclear factor kappa B (NF-kappaB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated degradation and phosphorylation of IkappaBalpha and activation of IKKalpha. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated nuclear translocation and phosphorylation of NF-kappaB/p65 at Ser(536). Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B-induced modulations of NF-kappaB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.     

Reproducibility estimates from single-laboratory ruggedness tests compared with those derived from collaborative trials of six microbiological measurement methods

This work investigated the feasibility of single-laboratory ruggedness experiments to estimate between-laboratory reproducibility. Six microbiological measurement methods that had already been subject to multi-laboratory validation were studied. Ruggedness experiments were designed and executed to determine sensitivity coefficients for factors judged likely to vary between laboratories. These were combined with estimates of factor variation to give reproducibility estimates. The single-laboratory estimates of reproducibility were generally not similar to those generated from multi-laboratory work. The experimental plans were difficult to design and execute, and were only partially successful in producing useful sensitivity coefficients. The authors conclude that ruggedness tests do not offer a single-laboratory alternative to multi-laboratory method validation or a practical approach to the evaluation of measurement uncertainty for microbiological methods. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Campden BRI was previously known as the Campden and Chorleywood Food Research Association.     

Exploring of bioactive compounds in essential oil acquired from the stem and root derivatives of Hypericum triquetrifolium callus cultures

The chemical profile of the essential oil of callus and cell suspension cultures derivatives from stem and root of Hypericum triquetrifolium were explored by ITEX/GC-MS. The major constituents for stem derivatives were undecane (78.44%) and 2,4,6-trimethyl-octane (9.74%) for fresh calli, 2,4-dimethyl-benzaldehyde (46.94%), 2,3-dimethyl-undecane (28.39%), 2,4-dimethyl-1-hexene (10.17%), 1,2-oxolinalool (3.64%) and limonene (3.55%) for dry calli and undecane (61.24%), octane, 2,4,6-trimethyl- (16.73%), nonane, 3-methyl-(3.74%), 2,5-diphenyl-benzoquinone (3.70%) and limonene (3.60%) for cell suspension. However, for root derivatives, the dominated components were: undecane (49.94%), eucalyptol (12.07%), limonene (9.98%), toluene (9.03%) and 3-methyl-nonane (4.29%) for fresh calli, 2,4-dimethyl-benzaldehyde (29.80%), 1,1-dimethylethyl-cyclohexane (14.99%), 3-methyl-pentanal (14.99%), undecane (10.04%), beta-terpinyl acetate (8.60%), 1,2-oxolinalool (6.27%) and 2-pentyl-furan (4.09%) for dry calli, undecane (52.38%), 2,4,6-trimethyl-octane (13.81%), 3-methyl-nonane (5.73%), toluene (4.82%) and limonene (4.57%) for cell suspension derivative in root. The attained outcomes indicated that the alkane, aldehyde and monoterpene fractions dominated the chemical composition of essential oils.     

Photoreaction of skin-sensitizing trimethyl psoralen with lipid membrane models

The concerted photoreaction between trimethyl psoralen (TMP) and isomeric model compounds of oleic acid methyl ester (OAME) and eadilic acid methyl ester (EAME) are explored using density functional theory. The S(1) surfaces all reveal large barriers (25-31 kcal/mol) to reach the decay channels, whereas the S(2) surfaces differ considerably between the furan and pyrone side adducts. For the pyrone side adducts, a small barrier to reach the intersection between the S(1) and S(2) states is found, followed by a small second barrier on the S(1) surface to the low-lying decay channel. For the furan-side adducts, no such intersection between S(1) and S(2) is seen, which thus prevents these products from being formed in high yields. The photoinduced cycloaddition is more favorable between OAME and the pyrone side double bond of TMP than for any of the other systems, which agrees with the experimental findings that this is formed in the highest yield. The computed UV absorption spectra of TMP and the TMP(pyrone)-OAME cycloadduct agree well with the experimental spectra.     

Neutral liposome-mediated delivery process of fluorescein-modified oligonucleotides in cultured human keratinocytes

We propose a model of the intracellular delivery process in which fluorescein-labeled natural oligonucleotides (F-DNA) are transferred into the nuclei of cultured human keratinocytes. By encapsulation in neutral multilamellar lecithin liposomes, the F-DNA appeared to be protected against intracellular interactions with cellular materials and nuclease attacks in the cytoplasm during the process. The intracellular behavior of F-DNA and fluorescent phospholipid-labeled liposomes was observed by means of fluorescence analysis. Results showed that: F-DNA encapsulated in neutral multilamellar liposomes reached the cellular nuclei more efficiently than either free F-DNA, or F-DNA in unilamellar liposomes; the liposomal membranes appeared to be left in the cytoplasm. The reaction of F-DNA with complementary DNA was suggested by a rapid quenching of the fluorescence in the nucleus. In addition, the fluorescence decrease was evidently suppressed in the cytoplasm, indicating a protective effect of the neutral multilamellar liposomes against the interaction of F-DNA with cytoplasmic materials. The application of these findings to ‘photo’-antisense studies has been discussed, where suppression of a gene expression is attempted by using oligonucleotide-attached fluorescein with the aid of a photo-induced covalent binding property.