Distinct Role of Sesn2 in Response to UVB‐Induced DNA Damage and UVA‐Induced Oxidative Stress in Melanocytes

Ultraviolet (UV) radiation, including both UVB and UVA irradiation, is the major risk factor for causing skin cancer including melanoma. Recently, we have shown that Sesn2, a member of the evolutionarily conserved stress‐inducible protein family Sestrins (Sesn), is upregulated in human melanomas as compared to melanocytes in normal human skin, suggesting an oncogenic role of Sesn2. However, the role of Sesn2 in UVB and UVA response is unknown. Here, we demonstrated that both UVB and UVA induce Sesn2 upregulation in melanocytes and melanoma cells. UVB induces Sesn2 expression through the p53 and AKT3 pathways. Sesn2 negatively regulates UVB‐induced DNA damage repair. In comparison, UVA induces Sesn2 upregulation through mitochondria but not Nrf2. Sesn2 ablation increased UVA‐induced Nrf2 induction and inhibits UVA‐induced ROS production, indicating that Sesn2 acts as an upstream regulator of Nrf2. These findings suggest previously unrecognized mechanisms in melanocyte response to UVB and UVA irradiation and potentially in melanoma formation.     

Silibinin is a potent sensitizer of UVA radiation-induced oxidative stress and apoptosis in human keratinocyte HaCaT cells

UVA radiation (315-400 nm), which constitutes ca 95% of the UV irradiation in natural sunlight reaching earth surface, is a major environmental risk factor associated with human skin cancer pathogenesis. UVA is an oxidizing agent that causes significant damage to cellular components through the release of reactive oxygen species (ROS) and leads to photoaging and photocarcinogenesis. Here we investigate the effect of silibinin, the flavonolignan from Silybum marianum, on UVA-induced ROS and cell death in human keratinocyte cell line HaCaT. In addition, the effect of silibinin on UVA-induced intracellular ROS-mediated endoplasmic reticulum (ER) stress was also analyzed. UVA irradiation resulted in ROS production and apoptosis in HaCaT cells in a dose-dependent manner, and the ROS levels and apoptotic index were found to be elevated significantly when the cells were treated with 75 μmsilibinin for 2 h before UVA exposure. When the cells were pretreated with 10 mmN-acetyl cysteine, the enhancement of UVA-induced apoptosis by silibinin was compromised. Furthermore, we found that silibinin enhances ER stress-mediated apoptosis in HaCaT cells by increasing the expression of CHOP protein. These results suggest that silibinin may be beneficial in the removal of UVA-damaged cells and the prevention of skin cancer.     

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 role of autophagy in cadmium-induced acute toxicity in glomerular mesangial cells and tracking polyubiquitination of cytoplasmic p53 as a biomarker

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development.Subject terms: Macroautophagy, Diagnostic markers     

The Global Response of Nostoc punctiforme ATCC 29133 to UVA Stress,Assessed in a Temporal DNA Microarray Study

Cyanobacteria in nature are exposed not only to the visible spectrum of sunlight but also to its harmful ultraviolet components (UVA and UVB). We used Nostoc punctiforme ATCC 29133 as a model to study the UVA response by analyzing global gene expression patterns using genomic microarrays. UVA exposure resulted in the statistically detectable differential expression of 573 genes of the 6903 that were probed, compared with that of the control cultures. Of those genes, 473 were up‐regulated, while only 100 were down‐regulated. Many of the down‐regulated genes were involved in photosynthetic pigment biosynthesis, indicating a significant shift in this metabolism. As expected, we detected the up‐regulation of genes encoding antioxidant enzymes and the sunscreen, scytonemin. However, a majority of the up‐regulated genes, 47%, were unassignable bioinformatically to known functional categories, suggesting that the UVA stress response is not well understood. Interestingly, the most dramatic up‐regulation involved several contiguous genes of unassigned metabolism on plasmid A. This is the first global UVA stress response analysis of any phototrophic microorganism and the differential expression of 8% of the genes of the Nostoc genome indicates that adaptation to UVA in Nostoc has been an evolutionary force of significance.     

Red Light Interferes in UVA‐Induced Photoaging of Human Skin Fibroblast Cells

The possible regulation mechanism of red light was determined to discover how to retard UVA‐induced skin photoaging. Human skin fibroblasts were cultured and irradiated with different doses of UVA, thus creating a photoaging model. Fibroblasts were also exposed to a subtoxic dose of UVA combined with a red light‐emitting diode (LED) for five continuous days. Three groups were examined: control, UVA and UVA plus red light. Cumulative exposure doses of UVA were 25 J cm^?2, and the total doses of red light were 0.18 J cm^?2. Various indicators were measured before and after irradiation, including cell morphology, viability, β‐galactosidase staining, apoptosis, cycle phase, the length of telomeres and the protein levels of photoaging‐related genes. Red light irradiation retarded the cumulative low‐dose UVA irradiation‐induced skin photoaging, decreased the expression of senescence‐associated β‐galactosidase, upregulated SIRT1 expression, decreased matrix metalloproteinase MMP‐1 and the acetylation of p53 expression, reduced the horizon of cell apoptosis and enhanced cell viability. Furthermore, the telomeres in UVA‐treated cells were shortened compared to those of cells in the red light groups. These results suggest that red light plays a key role in the antiphotoaging of human skin fibroblasts by acting on different signaling transduction pathways.     

Ultraviolet A Eye Irradiation Ameliorates Atopic Dermatitis via p53 and Clock Gene Proteins in NC/Nga Mice

Atopic dermatitis (AD ) is a widespread chronic skin condition that severely affects quality of life and can lead to more serious complications. Although ultraviolet (UV )A eye irradiation can exert various effects on the skin, it is unknown whether UVA can affect AD . To investigate potential associations, we used an NC /Nga mouse model of AD to study the effects of UVA eye irradiation. The eyes of mice were irradiated with a UVA dose of 100 kJ m⁻² using a FL 20SBLB ‐A lamp. Our histological data demonstrated that AD symptoms could be ameliorated by UVA eye irradiation. We also observed an increase in the levels of adrenocorticotropic hormone (ACTH ), p53 and retinoid X receptor α (RXR α ) in mice with UVA ‐irradiated eyes. In contrast, the levels of thymic stromal lymphopoietin (TSLP ), period 2 (PER 2) and differentiated embryo chondrocytes 1 (DEC 1) protein were decreased in mice treated with UVA irradiation. Furthermore, UVA eye‐irradiated mice exhibited reduced DEC 1 and RXR α colocalization compared with nonirradiated mice. These results suggested that p53 and various clock gene proteins played important roles in the amelioration of AD symptoms observed after UVA eye irradiation; this technique may have therapeutic applications in AD .     

In vitro and In vivo Antiproliferative Effect of a Combination of Ultraviolet‐A and Alkoxy Furocoumarins Isolated from Umbelliferae Medicinal Plants,in Melanoma Cells

We examined the effects of six furocoumarins with alkoxy groups at the C‐5 or C‐8 position isolated from Umbelliferae medicinal plants on cell proliferation, and their mechanisms of action against B16F10 melanoma cells or in melanin‐possessing hairless mice implanted with B16F10 cells, under UVA irradiation. Three furocoumarins with an alkoxy group at C‐5, isoimperatorin (1), oxypeucedanin (2) and oxypeucedanin hydrate (3), showed antiproliferative activity and caused G₂/M arrest at concentrations of 0.1–10.0 μm . Furthermore, three furocoumarins with an alkoxy group at C‐8, imperatorin (4), heraclenin (5) and heraclenol (6), inhibited the proliferation of melanoma cells and cell cycle at G₂/M at concentrations of 0.1–1.0 μm . UVA plus 1, 2, 3, 4 and 6 reduced tumor growth and final tumor weight in B16F10‐bearing mice at a dose of 0.3, 0.5 or 1.0 mg kg^?1 (intraperitoneal injection). UVA plus 1, 3 and 6 increased Chk1 phosphorylation and reduced cdc2 (Thr 161) phosphorylation in melanoma cells. We suggest that the antitumor actions of UVA plus furocoumarins with an alkoxy group at C‐5 or C‐8 were due to G₂/M arrest of the cell cycle by an increase in phosphor‐Chk1 and decrease in phospho‐cdc2.     

Selenadiazole Derivatives Inhibit Angiogenesis‐Mediated Human Breast Tumor Growth by Suppressing the VEGFR2‐Mediated ERK and AKT Signaling Pathways

Selenadiazole derivatives (SeDs) have been found to show promise in chemo‐/radiotherapy applications by activating various downstream signaling pathways. However, the functional role of SeDs on angiogenesis, which is pivotal for tumor progression and metastasis, has not yet been elucidated. In the present study, we have examined the antiangiogenic activities of SeDs and elucidated their underlying mechanisms. The results showed that the as‐synthesized SeDs not only enhanced their anticancer activities against several human cancer cells but also showed more potent inhibition on human umbilical vein endothelial cells (HUVECs). The in vitro results suggested that SeDs, especially 1 a , dose‐dependently inhibited the vascular endothelial growth factor (VEGF)‐induced cell migration, invasion, and capillary‐like structure formation of HUVECs. Compound 1 a also significantly suppressed VEGF‐induced angiogenesis in a Matrigel plug assay as part of a C57/BL6 mice assay by means of down regulation of VEGF. Furthermore, we found that 1 a significantly inhibited MCF‐7 human breast tumor growth in nude mice without severe systematic cytotoxicity. Compound 1 a was more effective in inhibiting cell proliferation and induced a much more pronounced apoptosis effect in endothelial cells than MCF‐7 cells, which implies that endothelial cells might be the primary target of 1 a . Further mechanistic studies on tumor growth inhibition effects and neovessel formation suppression demonstrated that 1 a inhibited cell viability of MCF‐7 and HUVECs by induction of cell apoptosis, accompanied by poly(adenosine diphosphate ribose)polymerase (PARP) cleavage and caspase activation. Additionally, the 1 a ‐induced antiangiogenesis effect was achieved by abolishing the VEGF‐VEGFR2‐ERK/AKT (ERK=extracellular signal–regulated kinases; AKT=protein kinease B) signal axis and enhanced the apoptosis effect by triggering reactive oxygen species (ROS)‐mediated DNA damage. Taken together, these results clearly demonstrate the antiangiogenic potency of SeDs and the underlying molecular mechanisms.     

PTEN/MMAC1 enhances the growth inhibition by anticancer drugs with downregulation of IGF-II expression in gastric cancer cells

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of advanced and metastatic cancers. Its major function is likely to be the phosphatase activity that regulates the phosphotidylinositol (PI)3-kinase/Akt pathway. On the other hand, IGF system plays an important role in cell proliferation and cell survival via PI3-kinase/Akt and mitogen-activated protein kinase pathways in many cancer cells. To evaluate effect of PTEN on cell growth and IGF system in gastric cancer, human gastric adenocarcinoma cells (SNU-5 & -216) were transfected with human PTEN cDNA. Those PTEN- transfected gastric cancer cells had a lower proliferation rate than the pcDNA3-transfected cells. PTEN overexpression induced a profound decrease in the IGF-II and IGF-IR expression levels, and downregulation of IGF-II expression by PTEN was mediated through the regulation of the IGF-II promoter. In addition, a PI3-kinase inhibitor, LY294002, induced the downregulation of IGF-II expression. The PTEN-overexpressing SUN-5 and -216 cells were more sensitive to death induced by etoposide and adriamycin that induce DNA damage than the pcDNA3-transfected cells. These findings suggest that PTEN suppresses the cell growth through modulation of IGF system and sensitizing cancer cells to cell death by anticancer drugs.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

Ambient UVA‐Induced Expression of p53 and Apoptosis in Human Skin Melanoma A375 Cell Line by Quinine

This study aimed to analyze the phototoxic mechanism and photostability of quinine in human skin cell line A375 under ambient intensities of UVA (320–400 nm). Photosensitized quinine produced a photoproduct 6‐methoxy‐quinoline‐4‐ylmethyl‐oxonium identified through LC‐MS/MS. Generation of ¹O₂, O₂^??, and ^?OH was measured and further substantiated through their respective quenchers. Photosensitized Quinine (Q) caused degradation of 2‐deoxyguanosine, the most sensitive nucleotide to UV radiation. The intracellular ROS was increased in a concentration‐dependent manner. Significant reduction in metabolic status measured in terms of cell viability (54%) at 25 μg mL^?1 was observed through MTT assay. Results of MTT assay accord NRU assay. Single strand DNA breaks and apoptosis were increased significantly (P < 0.01) as observed through comet assay and EB/AO double staining. Photosensitized quinine caused cells to arrest in G₂ phase of cell cycle and induced apoptosis (5.08%) as revealed through FACS. Real‐Time PCR showed upregulation of p21 (4.56 folds) and p53 (2.811 folds) genes expression. Thus, our study suggests that generation of reactive oxygen species by quinine under ambient intensity of UVA may result into deleterious phototoxic effects among human population.     

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.