UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte-derived Cell Lines and in Human Keratinocytes

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC-derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2 and 20 J/cm2) and UVB (8 mJ/cm2 and 16 mJ/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC-derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2 led to a marked decrease in soluble VEGF, whereas doses as high as 32 mJ/cm2 UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC-derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.     

Effects of a Single Exposure to UVB Radiation on the Activities and Protein Levels of Copper-Zinc and Manganese Superoxide Dismutase in Cultured Human Keratinocytes

Abstract— Ultraviolet B irradiation has been believed to decrease or impair the activity of reactive oxygen species (ROS) scavenging enzymes such as superoxide dismutase (SOD) in the skin. It has been recently reported that two isozymes of SOD, namely copper-zinc SOD (Cu-Zn SOD) and manganese SOD (Mn SOD), exist in mammalian cells and that the two enzymes play different roles in living systems. The aim of this study was to investigate changes in SOD activities and protein levels in cultured human keratinocytes after acute UVB irradiation. In addition, the protein levels of Cu-Zn SOD and Mn SOD were quantified separately. A single exposure to UVB irradiation produced an increase in SOD activity and protein level that peaked immediately after UVB irradiation, after which a decline was observed, with subsequent recovery to baseline levels 24 h after irradiation. In individual assays of Mn SOD and Cu-Zn SOD, the amount of Mn SOD protein decreased and then gradually recovered 24 h after irradiation. In contrast, the amount of Cu-Zn SOD protein increased immediately after UVB irradiation, and then gradually declined. To evaluate the mechanisms of these changes, we examined the effects of the cytokines, interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), which can be secreted from keratinocytes after UVB irradiation, on the SOD activity and protein levels in keratinocytes. Interleukin-la and TNF-α enhanced both the SOD activity and protein level of Mn SOD, while these cytokines had no effect on Cu-Zn SOD protein levels in cultured human keratinocytes after incubation for 24 h. Furthermore, when neutralizing antibodies against IL-1α and TNF-α were added separately or together to the culture medium before UVB irradiation, the recovery of total SOD activity and Mn SOD protein level were markedly inhibited 24 h after irradiation. Our results suggest that significant increases in SOD activity and protein level occur as a cutaneous antioxidant defense mechanism that protects against the cytotoxicity as a result of UVB irradiation, and that this increase in SOD is attributed to Cu-Zn SOD. The Cu-Zn SOD and Mn SOD protein levels changed in a different manner after UVB irradiation. The former may participate in an early phase and the latter in a late phase defense mechanism directed against oxidant cytotoxicity through UVB irradiation. In addition, the recovery of Mn SOD to baseline levels 24 h after UVB irradiation seems to be mediated through cytokines such as IL-1α and TNF-α, which are secreted from keratinocytes.     

PARP‐1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light

This study investigated poly(ADP ‐ribose) polymerase‐1 (PARP ‐1) activation in cultured human lens epithelial cells exposed to two levels of UVB light (312 nm peak wavelength), 0.014 and 0.14 J cm⁻² (“low” and “high” dose, respectively). At the low dose, PARP ‐1 and poly(ADP ‐ribose) (PAR ) polymers acted to repair DNA strand breaks rapidly with no subsequent major effects on either cell morphology or viability. However, following the high UVB dose, there was a dramatic second phase of PARP ‐1 activation, 90 min later, which included a sudden reappearance of DNA strand breaks, bursts of reactive oxygen species (ROS ) formation within both the mitochondria and nucleus, a translocation of PAR from the nucleus to the mitochondria and an ultimate 70% loss of cell viability occurring after 24 h. The results provide evidence for an important role for PARP ‐1 in protecting the human lens epithelium against low levels of UVB light, and possibly participating in the triggering of cell death following exposure to toxic levels of radiation.     

The Effect of Chronic Exposure to Artificial UVB Radiation on the Survival and Reproduction of Daphnia magna Across Two Generations

We examined the effects of daily (chronic) exposure to artificial UVB radiation on the survival and reproduction of Daphnia   magna over two generations. Control and experimental animals in each generation (parental and F1) were exposed to 16 h of UVA radiation and photosynthetically active radiation daily. In addition, experimental animals were exposed to 6 h of UVB during the middle of the light period. Survival and reproduction were followed for 12 days for each individual. Survival and production of F1 were significantly lower in the UVB exposed parental generation Daphnia than in controls. F1 exposure to UVB significantly decreased F1 survival and reproduction. Reproduction was lowest in UVB exposed F1 animals whose parents were also exposed to UVB. Adverse effects of UVB on offspring production may be magnified in successive generations suggesting that short-term experiments could underestimate the impact of increased UVB exposure on populations.     

Combination of Natural Extracts and Photobiomodulation in Keratinocytes Subjected to UVA Radiation

Natural extracts (NE) with antioxidant properties can minimize the effects of photoaging. Photobiomodulation (PBM) has proven to be a useful tool for the modulation of cell metabolism. Here, we investigate the associations of antioxidants with PBM with the aim of promoting skin rejuvenation. We began with standardization of the experimental protocol. Extracts of chamomile, rosemary, blueberry, green tea, figs, pomegranate and nutwood were tested. A custom irradiation system (366 ± 10 nm) was used to simulate sun exposure. A light emitting diode system (640 ± 12.5 nm) was used for PBM. Viability assessments were performed by the (3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) MTT assay method. Based on the results, radiant exposure to UVA was defined as 9 and 1 J cm^?2 for PBM. Extract concentrations were established on the basis of dark toxicities, which ranged from 0.01% to 0.3%. The data show that PBM is a promising therapy to restore keratinocytes after UVA damage; however, the detailed mechanism and effects require further exploration. Moreover, although the combination of PBM with NE may be a useful strategy, the choice of a NE is challenging, since the working concentration and other properties, such as photosensitivity, may bring about unwanted results.     

Assessment of DNA Damage Induced by Broadband and Narrowband UVB in Cultured Lymphoblasts and Keratinocytes Using the Comet Assay

Phototherapy with broadband UVB is an effective treatment for inflammatory dermatoses. A newly developed fluorescent UVB lamp (Philips TL01) that emits a narrowband UVB around 311 nm was shown to be superior for the phototherapy of psoriasis. In order to contribute to the knowledge about the carcinogenic potential of this UVB source, we measured the DNA damage in lymphoblasts and keratinocytes induced by narrowband UVB and compared it with that by conventional broadband UVB using the single cell gel electrophoresis (comet as-say). At equal doses, broadband UVB produced more DNA damage than narrowband UVB. However, in phototherapy of psoriasis, up to 10-fold higher doses are used with TLO1. When therapeutically equivalent doses were compared (10-fold correction for narrowband UVB), we found only slight differences in the amount of DNA damage produced by broadband and narrowband UVB. This supports the already existing evidence that for phototherapy narrowband UVB is not more carcinogenic than broadband UVB.     

The Role of Protein Kinase C Activity in the Killing of Chinese Hamster Ovary Cells by Ionizing Radiation and Photodynamic Treatment

In several recent studies it has been shown that protein kinase C (PKC) activity may either potentiate or antagonize cell killing by different cytotoxic agents. These apparently conflicting observations suggest that the effects of PKC activity on cell survival may depend on the different properties of different cell types but do not exclude the possibility that the effects may also depend on the nature of the cytotoxic agent. In this context the effects of PKC activation and PKC inhibition or down-regulation on Chinese hamster ovary (CHO) cell survival after photodynamic treatment and ionizing radiation were studied. It appeared that PKC activation by short-term incubation with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) protected CHO cells against ionizing radiation but, in contrast, sensitized the cells to photodynamic treatment. Conversely, inhibition of PKC by H7 and down-regulation of PKC activity by prolonged incubation with TPA sensitized CHO cells to ionizing radiation but protected the cells against photodynamic treatment. These results demonstrate that in one particular cell type PKC activity may have opposite effects on cell survival following cellular damage, depending on the nature of the cytotoxic agent .     

Synthesis of Trifluoromethylated 3-(3-Pyrazolyl)indole-N-glycosides and their Cytotoxic Activity against Human Keratinocytes (HaCaT)

The first 3-(3-pyrazolyl)indole-N-glycosides were prepared starting from indole-N-glycosides by conversion with 4-ethoxy-1,1,1-trifluorobut-3-en-2-one and following cyclization with hydrazine. The cytotoxic activity of the products against human keratinocytes (HaCaT) was studied.     

Determination of Myristicin and Linalool in Plants Exposed to Microwave Radiation by High-Performance Liquid Chromatography

The goal of this study was to develop and validate a high-performance liquid chromatography method for the quantification of two essential oil constituents (myristicin and linalool) from extracts of the three aromatic plants exposed to low-intensity microwave radiation. The analysis was performed by high-performance liquid chromatography using a mobile phase consisting of ultrapure water and acetonitrile. The separations were carried out in the following conditions: isocratic conditions for 30 minutes with 100% acetonitrile and then linear gradient conditions from 100% acetonitrile to 25% acetonitrile for 15 minutes. The developed method was validated in terms of linearity, limits of detection, limits of quantification, precision, accuracy, and robustness. The results show that plants react differently under microwave stress and change their essential oil content as a response.     

Differences in Sensitivity to UVC, UVB and UVA Radiation of a Multidrug-Resistant Cell Line Overexpressing P-Glycoprotein

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best-studied mechanism involves the expression of a membrane protein that acts as an energy-dependent efflux pump, known as P-glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non-MDR) cell line.     

Development and Characterization of Curcumin-Silver Nanoparticles as a Promising Formulation to Test on Human Pterygium-Derived Keratinocytes

Pterygium is a progressive disease of the human eye arising from sub-conjunctival tissue and extending onto the cornea. Due to its invasive growth, pterygium can reach the pupil compromising visual function. Currently available medical treatments have limited success in suppressing efficiently the disease. Previous studies have demonstrated that curcumin, polyphenol isolated from the rhizome of Curcuma longa, induces apoptosis of human pterygium fibroblasts in a dose- and time-dependent manner showing promising activity in the treatment of this ophthalmic disease. However, this molecule is not very soluble in water in either neutral or acidic pH and is only slightly more soluble in alkaline conditions, while its dissolving in organic solvents drastically reduces its potential use for biomedical applications. A nanoformulation of curcumin stabilized silver nanoparticles (Cur-AgNPs) seems an effective strategy to increase the bioavailability of curcumin without inducing toxic effects. In fact, silver nitrates have been used safely for the treatment of many ophthalmic conditions and diseases for a long time and the concentration of AgNPs in this formulation is quite low. The synthesis of this new compound was achieved through a modified Bettini’s method adapted to improve the quality of the product intended for human use. Indeed, the pH of the reaction was changed to 9, the temperature of the reaction was increased from 90 °C to 100 °C and after the synthesis the Cur-AgNPs were dispersed in Borax buffer using a dialysis step to improve the biocompatibility of the formulation. This new compound will be able to deliver both components (curcumin and silver) at the same time to the affected tissue, representing an alternative and a more sophisticated strategy for the treatment of human pterygium. Further in vitro and in vivo assays will be required to validate this formulation.     

Recent Advances in Synergistic Antitumor Effects Exploited from the Inhibition of Ataxia Telangiectasia and RAD3-Related Protein Kinase (ATR)

As one of the key phosphatidylinositol 3-kinase-related kinases (PIKKs) family members, ataxia telangiectasia and RAD3-related protein kinase (ATR) is crucial in maintaining mammalian cell genomic integrity in DNA damage response (DDR) and repair pathways. Dysregulation of ATR has been found across different cancer types. In recent years, the inhibition of ATR has been proven to be effective in cancer therapy in preclinical and clinical studies. Importantly, tumor-specific alterations such as ATM loss and Cyclin E1 (CCNE1) amplification are more sensitive to ATR inhibition and are being exploited in synthetic lethality (SL) strategy. Besides SL, synergistic anticancer effects involving ATRi have been reported in an increasing number in recent years. This review focuses on the recent advances in different forms of synergistic antitumor effects, summarizes the pharmacological benefits and ongoing clinical trials behind the biological mechanism, and provides perspectives for future challenges and opportunities. The hope is to draw awareness to the community that targeting ATR should have great potential in developing effective anticancer medicines.     

Enhanced Activity and Substrate Specificity by Site-Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole

P450 119 peroxygenase was found to catalyze the sulfoxidation of thioanisole and the sulfonation of sulfoxide in the presence of tert-butyl hydroperoxide (TBHP) for the first time with turnover rates of 1549 min⁻¹ and 196 min⁻¹ respectively. Several mutants were designed to improve the peroxygenation activity and thioanisole specificity by site-directed mutagenesis. The F153G/T213G mutant gave an increase of sulfoxide yield and a decrease of sulfone yield. Moreover the S148P/I161T/K199E/T214V mutant and the K199E mutant with acidic Glu residue contributed to improving the product ratio of sulfoxide to sulfone. Addition of short-alkyl-chain organic acids to the P450 119 peroxygenase-catalyzed sulfur oxidation of thioanisole was investigated. Octanoic acid was found to induce a preferred sulfoxidation of thioanisole catalyzed by the F153G/T213G mutant to give approximately 2.4-fold increase in turnover rate with a k_cat value of 3687 min⁻¹ relative to that of the wild-type, and by the F153G mutant to give the R-sulfoxide up to 30 % ee. The experimental control and the proposed mechanism for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in the presence of octanoic acid suggested that octanoic acid could partially occupy the substrate pocket; meanwhile the F153G mutation could enhance the substrate specificity, which could lead to efficiently regulate the spatial orientation of thioanisole and facilitate the formation of Compound I. This is the most effective catalytic system for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole.     

Diel Cycles of DNA Damage and Repair in Eggs and Larvae of Northern Anchovy, Engraulis mordax, Exposed to Solar Ultraviolet Radiation

Abstract— Damage from UVB radiation (280–320 nm) in the form of cyclobutane pyrimidine dimers (CPD) in DNA and the capacity for their repair were measured in newly spawned eggs and yolk-sac larvae of northern anchovy, Engraulis mordax, exposed to natural diel cycles of sunlight. The CPD were measured by a newly developed chemiluminescent immunoblot assay capable of measuring CPD in samples as small as 50 ng DNA. Eggs and yolk-sac larvae exposed to full irradiance levels died. At lower dose rates, equivalent to deeper more natural locations in the water column, there was a diel cycle of dimer concentration that tracked solar intensity. This diel cycle was due to the interaction of damage and repair processes. Repair of CPD in anchovy eggs and larvae could be attributed to true photodependent repair that could be stopped by moving samples into the dark. The CPD present at sunset remained until the following morning. The diel cycles of damage and repair were maintained over at least 4 days without a long-term upward or downward trend in dimer concentration. This indicates that at the UVB doses used for these experiments, there was no long-term accumulation of CPD nor an induction of increased repair capacity. Unhatched embryos spawned in the dark also exhibited a strong photorepair response, suggesting that photolyase expression was innate and not dependent on previous light exposure. The diel cycle observed here indicates that, at least for northern anchovy, the CPD concentration at the time of sampling is a good indicator of dose rate but a poor indicator of cumulative dose (i.e. late afternoon samples have the highest cumulative dose but relatively low CPD concentrations). The CPD immunoassay described here has the required sensitivity for measuring DNA damage in wild populations of ichthyoplankton exposed to natural sunlight. These results will guide the collection and interpretation of field data on natural levels of CPD in wild larvae collected at different depths and times of the day.     

In Vitro and in Vivo Anticancer Activity of Sophorolipids to Human Cervical Cancer

Six characteristic di-acetylated lactonic sophorolipids with C16:1, C16:0, C18:0, C18:1, C18:2, and C18:3 fatty acid were obtained from Starmerella bombicola CGMCC 1576. In order to confirm their anticancer activity against human cervical cancer cells and reveal the structure-activity relationships, their anti-proliferation effects on HeLa and CaSki cells were estimated. The cytotoxicity of sophorolipid molecules with different degrees of unsaturation was proved to be influenced by carbon chain length of sophorolipids. The longer the carbon chain length, the stronger the cytotoxicity of sophorolipids. The inhibitory mechanism of a di-acetylated lactonic C18:1 sophorolipid on HeLa cells was investigated. The cells developed many features of apoptosis and cell cycle was blocked at G₀ phase and partly at G₂ phase. The expression of CHOP and Bip/GRP78 was induced. Caspase-12 and caspase-3 were both activated. However, mitochondrial membrane potential and concentration of cytosolic cytochrome C did not change. The induced apoptosis of HeLa cells was probably triggered through endoplasmic reticulum signaling pathway without involvement of mitochondria. In vivo, 5, 50, and 500 mg/kg lactonic sophorolipids showed 29.90, 41.24, and 52.06 % of inhibition without significant toxicity to tumor-bearing mice, respectively. Our findings may suggest a potential use of sophorolipids in human cervical cancer treatment.