Inhibitory Effect of Lupeol on MMPs Expression using Aged Fibroblast through Repeated UVA Irradiation

In this study, the aged dermal fibroblast model was constructed by repeated irradiation with UV light and the effect of lupeol, a triterpenoid, on anti‐aging was confirmed. SA‐β‐galactosidase (SA‐β‐gal) stained aged cells increased by about 40% and expression of p‐p53, p21, p16 and MMPs (MMP‐1, ‐2, ‐3) increased in aged fibroblast. As an efficacy result, the treatment of lupeol on aged fibroblast induced by UVA repeated irradiation showed a dose‐dependent reduction of SA‐β‐gal stained aged cells, the expression of p‐p53, p21, p16 and inhibition of MMPs. Interestingly, lupeol increased dephosphorylation of p‐ERK in repeated UV irradiated conditions. Additionally, lupeol compensated MMPs expression when p‐ERK phosphorylation was inhibited by p‐ERK inhibitor PD98059. Thus, these results showed that lupeol has a possible effect on MMPs expression using inhibition of the p‐ERK pathway. Taken together, we confirmed that lupeol inhibits senescence through inhibiting MMP‐1, ‐2, ‐3 as well as p‐p53, p21 and p16 expression and SA‐β‐gal activity in repeated UVA‐irradiated senescent FB models, therefore suggesting that lupeol may be useful as an anti‐aging agent.     

The Protective Effect of Violaxanthin from Nannochloropsis oceanica against Ultraviolet B‐Induced Damage in Normal Human Dermal Fibroblasts

Skin photoaging, which is mainly induced by ultraviolet B (UVB) radiation, is prevented by the application of UV‐protective agents. The microalga Nannochloropsis oceanica (N. oceanica) has been primarily reported as a potential biofuel; however, in this study, we investigated whether N. oceanica extracts exerted photoprotective effects against UVB‐irradiated human dermal fibroblasts (HDFs) and which single component was responsible for the protective effect of the extracts. Two extracts—pigment and nonpigment—were prepared from N. oceanica biomass. WST‐1 assay and expression analysis of interleukin genes showed that the pigment extracts were not significantly cytotoxic to HDFs. Further experiments revealed that treatment with the pigment extract upregulated the expression of collagen genes and significantly blocked UVB‐induced damage such as decreased cell viability and increased ROS production. Next, to investigate the pigment composition of the extracts, HPLC analysis was conducted and violaxanthin was identified as the major pigment. The UVB photoprotective effect of the pigment extracts was confirmed in violaxanthin‐treated HDFs. In addition, violaxanthin significantly attenuated UVB‐induced G1 phase arrest, senescence‐associated β‐galactosidase activation, p16 and p21 upregulation, ERK phosphorylation and the downregulation of ECM molecules in HDFs. Therefore, we concluded that violaxanthin was a potential antiphotoaging agent.     

Inflammation Due to Voriconazole‐induced Photosensitivity Enhanced Skin Phototumorigenesis in Xpa‐knockout Mice

Voriconazole is an antifungal agent and used as a prophylactic measure, especially in immunocompromised patients. However, there have been several reports of its adverse reactions, namely photosensitivity with intense inflammatory rashes and subsequent skin cancer development. To assess the effects of photosensitizing drugs voriconazole and hydrochlorothiazide (HCTZ ) on the enhancement of UV ‐induced inflammatory responses and UV ‐induced tumorigenesis, we utilized Xpa ‐knockout mice, which is DNA repair‐deficient and more susceptible to UV ‐induced inflammation and tumor development than wild‐type mice. Administration of voriconazole prior to broadband UVB exposure significantly upregulated multiple inflammatory cytokines compared with the vehicle‐ or HCTZ ‐administered groups. Voriconazole administration along with chronic UVB exposure produced significantly higher number of skin tumors than HCTZ or vehicle in Xpa ‐knockout mice. Furthermore, the investigation of UVB ‐induced DNA damage using embryonic fibroblasts of Xpa ‐knockout mice revealed a significantly higher 8‐oxo‐7,8‐dihydroguanine level in cells treated with voriconazole N‐oxide, a voriconazole‐metabolite during UV exposure. The data suggest that voriconazole plus UVB ‐induced inflammatory response may be related to voriconazole‐induced skin phototumorigenesis.     

The Autophagy Receptor Adaptor p62 is Up‐regulated by UVA Radiation in Melanocytes and in Melanoma Cells

UVA (315–400 nm) is the most abundant form of UV radiation in sunlight and indoor tanning beds. However, much remains to be understood about the regulation of the UVA damage response in melanocytes and melanoma. Here, we show that UVA , but not the shorter waveband UVB (280–315 nm), up‐regulates adaptor protein p62 in an Nrf2‐ and reactive oxygen species (ROS )‐dependent manner, suggesting a UVA ‐specific effect on p62 regulation. UVA ‐induced p62 up‐regulation was inhibited by a mitochondria‐targeted antioxidant or Nrf2 knockdown. In addition, p62 knockdown inhibited UVA ‐induced ROS production and Nrf2 up‐regulation. We also report here a novel regulatory feedback loop between p62 and PTEN in melanoma cells. PTEN overexpression reduced p62 protein levels, and p62 knockdown increased PTEN protein levels. As compared with normal human skin, p62 was up‐regulated in human nevus, malignant melanoma and metastatic melanoma. Furthermore, p62 was up‐regulated in melanoma cells relative to normal human epidermal melanocytes, independent of their BRAF or NRAS mutation status. Our results demonstrated that UVA up‐regulates p62 and induces a p62‐Nrf2 positive feedback loop to counteract oxidative stress. Additionally, p62 forms a feedback loop with PTEN in melanoma cells, suggesting p62 functions as an oncogene in UVA ‐associated melanoma development and progression.     

The 5‐Methylisocytosine β‐D‐Ribopyranosyl (4′ → 2′)‐Oligonucleotide

In the context of Eschenmoser's work on pyranosyl‐RNA (‘p‐RNA’), we investigated the synthesis and base‐pairing properties of the 5‐methylisocytidine derivative. The previously determined clear‐cut restrictions of base‐pairing modes of p‐RNA had led to the expectation that a 5‐methylisocytosine β‐D ‐ribopyranosyl (= D ‐pr(^MeisoC)) based (4′ → 2′)‐oligonucleotide would pair inter alia with D ‐pr(isoG) and L ‐pr(G) based oligonucleotides (D ‐pr and L ‐pr = pyranose form of D ‐ and L ‐ribose, resp.). Remarkably, we could not observe pairing with the D ‐pr(isoG) oligonucleotide but only with the L ‐pr(G) oligonucleotide. Our interpretation concludes that this – at first hand surprising – observation is caused by a change in the nucleosidic torsion angle specific for isoC.     

Plantamajoside Inhibits UVB and Advanced Glycation End Products‐Induced MMP‐1 Expression by Suppressing the MAPK and NF‐κB Pathways in HaCaT Cells

Photoaging and glycation stress are major causes of skin deterioration. Oxidative stress caused by ultraviolet B (UVB) irradiation can upregulate matrix metalloprotease 1 (MMP‐1), a major enzyme responsible for collagen damage in the skin. Advanced glycation end products (AGEs) accumulate via gradual formation from skin proteins, especially from long‐lived proteins such as dermal elastin and collagen. Plantamajoside (PM), isolated from Plantago asiatica, has various biological effects including anti‐inflammatory and antioxidant effects. In this study, we assessed the protective effects of PM on a human keratinocyte cell line (HaCaT) and primary human dermal fibroblasts (HDF) against stress caused by glyceraldehyde‐induced AGEs (glycer‐AGEs) with UVB irradiation. We found that PM attenuated UVB‐ and‐glycer‐AGEs‐induced MMP‐1 expression in HaCaT and HDF cells and proinflammatory cytokines expression by inhibiting the phosphorylation of mitogen‐activated protein kinases (MAPKs) activated by reactive oxygen species. Specific inhibitors of NF‐κB and MAPKs attenuated the induced expression of MMP‐1. PM also inhibited the phosphorylation of IκBα, and reduced nuclear translocation of NF‐κB in these cells. Furthermore, PM attenuated the upregulation of receptor for AGEs (RAGE) by glycer‐AGEs with UVB irradiation. Therefore, our findings strongly suggest that PM is a promising inhibitor of skin photoaging.     

Nitric oxide donor exisulind is an effective inhibitor of murine photocarcinogenesis(†)

NO‐releasing nonsteroidal anti‐inflammatory drugs (NO‐NSAIDs) have been shown to have anti‐inflammatory, antiproliferative and apoptosis‐inducing effects in tumor cells. Herein, we have investigated the effects of NO‐exisulind on the growth of UVB‐induced skin tumor development in a murine model. We found that the topical treatment with NO‐exisulind significantly reduced UVB‐induced tumors in SKH‐1 hairless mice. The tumors/tumor bearing mouse, the number of tumors/mouse and tumor volume/mouse decreased significantly (P < 0.05) as compared with vehicle‐treated and UVB‐irradiated positive controls. Consistently, NO‐exisulind‐treated animals showed reduced expression of proliferation markers, such as PCNA and cyclin D1. These mice also manifested increased expression of proapoptotic Bax and decreased expression of antiapoptotic Bcl2 with an increase in the number of TUNEL‐positive cells in tumors. We also investigated whether NO‐exisulind‐treated tumors are less invasive and progress less efficiently from benign to malignant carcinomas. For this, tumors were stained for various epithelial‐mesenchymal transition (EMT) markers. NO‐exisulind decreased the expression of mesenchymal markers, such as Fibronectin, N‐cadherin, SNAI, Slug and Twist and enhanced the epithelial marker E‐cadherin. Similarly, UVB‐induced phosphorylation of Erk1/2 and p38 was decreased in NO‐exisulind‐treated animals. These data suggest that NO‐exisulind reduces tumor growth and inhibits tumor progression by blocking proliferation, inducing apoptosis and reducing EMT.     

Specific Binding of a d‐RNA G‐Quadruplex Structure with an l‐RNA Aptamer

G‐quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4‐binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l ‐RNA aptamer that targets a d ‐RNA G‐quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l ‐RNA aptamer, Ap3‐7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA‐rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4‐specific tools for diverse applications.     

Artocarpin‐enriched (Artocarpus altilis) Heartwood Extract Provides Protection Against UVB‐induced Mechanical Damage in Dermal Fibroblasts

This study aimed to evaluate the protective effect of artocarpin‐enriched (Artocarpus altilis) heartwood extract on the mechanical properties of UVB‐irradiated fibroblasts. Human skin fibroblasts were pretreated with 50 μg/mL^?1 extract and later irradiated with UVB (200 mJ/cm^?2). They were then cultured within three‐dimensional of free‐floating and tense collagen lattices. The pretreatment of fibroblasts with the extract prior to UVB radiation showed cells protection against UVB‐induced suppression of α‐SMA expression, fibroblast migration and contraction. These results reveal that the extract prevents mechanical damages induced by UVB irradiation in fibroblast‐embedded collagen lattices, and therefore, has a potential as a natural photo‐protectant.     

Naproxen Inhibits UVB‐induced Basal Cell and Squamous Cell Carcinoma Development in Ptch1+/−/SKH‐1 Hairless Mice

Naproxen possesses anti‐proliferative and pro‐apoptotic effects besides its known anti‐inflammatory functions. Here, we demonstrate the anticancer effects of naproxen against UVB‐induced basal cell carcinoma (BCCs) and squamous cell carcinoma (SCCs) in a highly susceptible murine model of UVB carcinogenesis. Naproxen significantly inhibited UVB‐induced BCCs and SCCs in this model. Tumor number and volume were significantly decreased (P < 0.005 and P < 0.05, respectively). Inhibition in UVB‐induced SCCs and BCCs was 77% and 86%, respectively, which was associated with reduced PCNA and cyclin D1 and increased apoptosis. As expected, inflammation‐related iNOS, COX‐2 and nuclear NFκBp65 were also diminished by naproxen treatment. Residual tumors excised from naproxen‐treated animal were less invasive and showed reduced expression of epithelial‐mesenchymal transition (EMT) markers N‐cadherin, Vimentin, Snail and Twist with increased expression of E‐cadherin. In BCC and SCC cells, naproxen‐induced apoptosis and activated unfolded protein response (UPR) signaling with increased expression of ATF4, p‐eIF2α and CHOP. Employing iRNA‐based approaches, we found that naproxen‐induced apoptosis was regulated by CHOP as sensitivity of these cutaneous neoplastic cells for apoptosis was significantly diminished by ablating CHOP. In summary, these data show that naproxen is a potent inhibitor of UVB‐induced skin carcinogenesis. ER stress pathway protein CHOP may play an important role in inducing apoptosis in cancer cells.     

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra‐S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6‐4 pyrimidine–pyrimidone photoproducts was highest in DNA from UVC‐irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA–UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8‐oxo‐7,8‐dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.     

Fisetin Inhibits UVB‐induced Cutaneous Inflammation and Activation of PI3K/AKT/NFκB Signaling Pathways in SKH‐1 Hairless Mice

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB‐exposed SKH‐1 hairless mouse skin. Mice were exposed to 180 mJ cm^?2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB‐exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX‐2, PGE₂ as well as its receptors (EP1–EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL‐1β and IL‐6 in UVB‐exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB‐induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB‐induced cutaneous inflammation and DNA damage.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Baicalin Protects Keratinocytes from Toll‐like Receptor‐4 Mediated DNA Damage and Inflammation Following Ultraviolet Irradiation

UVB radiation contributes to both direct and indirect damage to the skin including the generation of free radicals and reactive oxygen species (ROS), inflammatory responses, immunosuppression and gene mutations, which can ultimately lead to photocarcinogenesis. A plant‐derived flavonoid, baicalin, has been shown to have antioxidant, anti‐inflammatory and free radical scavenging activities. Previous studies from our laboratory have shown that in murine skin, Toll‐like receptor‐4 (TLR4) enhanced both UVB‐induced DNA damage and inflammation. The aim of this study was to investigate the efficacy of baicalin against TLR4‐mediated processes in the murine keratinocyte PAM 212 cell line. Our results demonstrate that treating keratinocytes with baicalin both before and after UV radiation (100 mJ cm^?2) significantly inhibited the level of intracellular ROS and decreased cyclobutane pyrimidine dimers and 8‐Oxo‐2′‐deoxyguanosine (8‐oxo‐dG)—markers of DNA damage. Furthermore, cells treated with baicalin demonstrated an inhibition of TLR4 and its downstream signaling molecules, MyD88, TRIF, TRAF6 and IRAK4. TLR4 pathway inhibition resulted in NF‐κB inactivation and down‐regulation of iNOS and COX‐2 protein expression. Taken together, baicalin treatment effectively protected keratinocytes from UVB‐induced inflammatory damage through TLR pathway modulation.