Apoptosis In Human Keratinocytes Research Articles

Protective Effect of Fermented Sea Tangle Extract on Skin Cell Damage Caused by Particulate Matter.

The skin is directly exposed to atmospheric pollutants, especially particulate matter 2.5 (PM2.5) in the air, which poses significant harm to skin health. However, limited research has been performed to identify molecules that can confer resistance to such substances. Herein, we analyzed the effect of fermented sea tangle (FST) extract on PM2.5-induced human HaCaT keratinocyte damage. Results showed that FST extract, at concentrations less than 800 μg/mL, exhibited non-significant toxicity to cells and concentration-dependent inhibition of PM2.5-induced reactive oxygen species (ROS) production. PM2.5 induced oxidative stress by stimulating ROS, resulting in DNA damage, lipid peroxidation, and protein carbonylation, which were inhibited by the FST extract. FST extract significantly suppressed the increase in calcium level and apoptosis caused by PM2.5 treatment and significantly restored the reduced cell viability. Mitochondrial membrane depolarization occurred due to PM2.5 treatment, however, FST extract recovered mitochondrial membrane polarization. PM2.5 inhibited the expression of the anti-apoptotic protein Bcl-2, and induced the expression of pro-apoptotic proteins Bax and Bim, the apoptosis initiator caspase-9, as well as the executor caspase-3, however, FST extract effectively protected the changes in the levels of these proteins caused by PM2.5. Interestingly, pan-caspase inhibitor Z-VAD-FMK treatment enhanced the anti-apoptotic effect of FST extract in PM2.5-treated cells. Our results indicate that FST extract prevents PM2.5-induced cell damage via inhibition of mitochondria-mediated apoptosis in human keratinocytes. Accordingly, FST extract could be included in skin care products to protect cells against the harmful effects of PM2.5.

Research on the resistance of isoviolanthin to hydrogen peroxide-triggered injury of skin keratinocytes based on Transcriptome sequencing and molecular docking.

Apoptosis of skin keratinocytes is closely associated with skin problems in humans and natural flavonoids have shown excellent biological activity. Hence, the study of flavonoids against human keratinocyte apoptosis has aroused the interest of numerous researchers. In this study, methyl thiazolyl tetrazolium (MTT) assay and Western blots were used to investigate the skin-protective effect of isoviolanthin, a di-C-glycoside derived from Dendrobium officinale, on hydrogen peroxide (H2O2)-triggered apoptosis of skin keratinocytes. Transcriptome sequencing (RNA-Seq) was used to detect the altered expression genes between the model and treatment group and qRT-PCR was used to verify the accuracy of transcriptome sequencing results. Finally, molecular docking was used to observe the binding ability of isoviolanthin to the selected differential genes screened by transcriptome sequencing. Our results found isoviolanthin could probably increase skin keratinocyte viability, by resisting against apoptosis of skin keratinocytes through downregulating the level of p53 for the first time. By comparing transcriptome differences between the model and drug administration groups, a total of 2953 differential expression genes (DEGs) were identified. Enrichment analysis showed that isoviolanthin may regulate these pathways, such as DNA replication, Mismatch repair, RNA polymerase, Fanconi anemia pathway, Cell cycle, p53 signaling pathway. Last, our results found isoviolanthin has a strong affinity for binding to KDM6B, CHAC2, ESCO2, and IPO4, which may be the potential target for treating skin injuries induced by reactive oxide species. The current study confirms isoviolanthin potential as a skin protectant. The findings may serve as a starting point for further research into the mechanism of isoviolanthin protection against skin damage caused by reactive oxide species (e.g., hydrogen peroxide).

Deoxynivalenol exposure induces oxidative stress and apoptosis in human keratinocytes via PI3K/Akt and MAPK signaling pathway.

Deoxynivalenol (DON) is a mycotoxin frequently occurring in human and animal food worldwide, which raises increasing public health concerns. In the present study, we used human keratinocytes (HaCaT cells) as an in vitro model to explore the cytotoxic effect of DON. The results showed that the cells exhibited varying degrees of damage, including decreased cell number and viability, cell shrinkage and floating, when treated with 0.125, 0.25, and 0.5 μg/mL DON for 6, 12, and 24 h, respectively. Furthermore, exposure to DON for 24 h significantly increased the lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS), and prominently decreased the superoxide dismutase (SOD) and catalase (CAT) activity. Additionally, DON exposure induced mitochondrial damage and cell apoptosis through reducing mitochondrial membrane potential. Then, we performed RNA-sequencing to investigate the molecular changes in HaCaT cells after DON exposure. The RNA-sequencing results revealed that DON exposure altered the gene expression involved in apoptosis, MAPK signaling pathway, and PI3K/Akt signaling pathway. Moreover, DON exposure significantly decreased the mRNA and protein expression of Bcl-2, and increased the mRNA and protein expression of Bax, Caspase 3 and COX-2, the protein expression of PI3K, and the phosphorylation levels of Akt, ERK, p38, and JNK. Taken together, these findings suggest that DON exposure could induce cell damage, oxidative stress, and apoptosis in HaCaT cells through the activation of PI3K/Akt and MAPK pathways.

The Protective Effects of Sodium Pentaborate Tetrahydrate Against UVB-induced Apoptosis in Human Keratinocytes

Ultraviolet radiation (UV) is an environmental carcinogen causing human skin cancer. Exposure of the skin to UV produces apoptotic keratinocytes called sunburn cells within the epidermis. Boron, an essential element for plants, has several biological properties, such as anti-cancer, anti-microbial, and anti-oxidant. In the present study, the possible protective effects of sodium pentaborate pentahydrate (SPT) against UVB-induced apoptosis in human keratinocyte cells, HaCaT, were investigated. They were treated with SPT at different concentrations (7.8-125 μg/mL) for 24h after UVB irradiation (20, 30 and 60mJ/cm2). Cell viability, annexin V assay, cell cycle analysis, and apoptosis-related gene levels were measured using RT-PCR. Treatment with SPT (15.6-31.25μg/mL) after 30 mJ/m2 UVB exposure significantly increased cell survival. Annexin V apoptosis analysis demonstrated a robust protective effect by treatment with SPT at concentrations of 15.6 and 31.25μg/mL after 30mJ/cm2 UVB irradiation. The cell cycle analysis revealed that UVB irradiation elevated the number of cells at the G0/G1 phase while SPT treatment after UVB irradiation increased the number of cells at G2/M phase, suggesting the changes were partially reversed. Furthermore, treatment with 15.6μg/mL SPT after 30 mJ/m2 UV irradiation blocked the activation of caspase 3, caspase 9, Bax, and p53. These results indicate that treatment with SPT exerts protective effects after UVB irradiation. Thus, treatment with SPT led to strong protection against UVB-induced apoptotic cell death in HaCaT cells.

Euphorbia supina Extracts Block NADPH Oxidase-Mediated, Ceramide-Induced Apoptosis Initiated by Diesel Particulate Matter.

Air pollutants contribute to the development of diseases such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary cancer, cardiovascular problems, and some skin diseases. We recently found that a major air pollutant, diesel particulate matter (DPM), induces apoptosis in human keratinocytes by increasing a proapoptotic lipid mediator, ceramide. DPM activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), which stimulates sphingomyelinase, leading to an increased conversion of sphingomyelin to ceramide. Interestingly, we characterized that although NOX is a reactive oxygen species (ROS) generator, the activation of sphingomyelinases by NOX is an ROS-independent mechanism. A Korean weed, prostrate spurge Euphorbia supina Rafin (ESR), has been used for centuries as a folk medicine to treat bronchitis, hepatitis, hemorrhage, and skin inflammation. Flavonoids, terpenes and tannins are enriched in ESR, and although ESR has proven antioxidative activity, its biological activities are largely unknown. Here, we investigate whether and how ESR protects keratinocytes against DPM-mediated apoptosis. We found that ESR-extracts (ESR-Ex) protect keratinocytes from DPM-induced apoptosis by inhibiting NOX activation in keratinocytes in response to DPM. We also demonstrated that ESR-Ex suppresses NOX activation via a blockage of the aryl hydrocarbon receptor (AhR) activation-mediated transcription of neutrophil cytosolic factor 1 (NCF1)/p47phox, a subunit of NOX. Our study reveals previously uncharacterized biological activity of ESR-Ex; i.e., its inhibition of Ahr and NOX activation. Thus, because the inhibition of NOX has already been developed to treat NOX-mediated diseases, including various types of cardiovascular diseases and cancers, initiated by air pollutants and because AhR activation contributes to the development of chronic inflammatory diseases, our study provides further advantages for the medical use of ESR.

Gadodiamide Induced Autophagy and Apoptosis in Human Keratinocytes.

Gadolinium has been reported to cause liver lobular necrosis and nephrogenic systemic fibrosis. However, its toxicity to the skin remains unknown. This study aimed to investigate the effect of a high dose of gadolinium-based contrast agent gadodiamide on the human keratinocyte HaCaT cell line. Cell viability was assessed using MTT assay, and autophagy was assessed using acridine orange and LysoTracker Red staining. Western blotting was performed to verify the changes in Bcl2 and Bax levels. The viability of HaCaT cells was significantly suppressed after gadodiamide treatment. Interestingly, gadodiamide caused autophagic vacuoles, whereas the autophagy inhibitors 3-methyladenine and chloroquine significantly alleviated autophagic cell death. Simultaneously, gadodiamide induced apoptosis, which was reduced by caspase inhibitors. Gadodiamide also inhibited Bcl-2 expression and promoted Bax expression. Gadodiamide induced both autophagy and apoptosis in HaCaT cells. Physicians should carefully assess the gadodiamide dosage used clinically.

Astaxanthin Protects Ultraviolet B-Induced Oxidative Stress and Apoptosis in Human Keratinocytes via Intrinsic Apoptotic Pathway.

BackgroundUltraviolet radiation causes skin damage due to increased production of reactive oxygen species (ROS) and inflammatory intermediates and direct attack of DNA of skin cells. Astaxanthin is a reddish pigment that belongs to a group of chemicals called carotenoids and has protective effects as an antioxidant.ObjectiveTo determine the beneficial effects of astaxanthin on damaged human skin after exposure to ultraviolet radiation.MethodsNormal human epidermal keratinocytes (NHEKs) were pre-treated with astaxanthin for 24 hours and exposed to ultraviolet B (UVB) irradiation. After 24 hours, the Cell Counting Kit-8 (CCK-8) assay measured cell viability, ROS assay and flow cytometry analysis assessed apoptosis, and western blotting was performed to determine expression of apoptosis-related proteins.ResultsAstaxanthin significantly inhibited UVB-induced NHEKs cytotoxicity. Pretreatment of NHEKs with astaxanthin reduced UVB-induced ROS production. Astaxanthin caused significant inhibition of UVB-induced apoptosis, as evidenced by flow cytometry analysis and western blotting.ConclusionThese results suggest that astaxanthine has a beneficial effect of reducing damage caused by UVB by effectively inhibiting cell death and reducing ROS production in keratinocytes.

Oat (Avena sativa) Extract against Oxidative Stress-Induced Apoptosis in Human Keratinocytes.

Oat (Avena sativa) is well known for its various health benefits. The protective effect of oat extract against oxidative stress-induced apoptosis in human keratinocytes HaCaT was determined. First, extracts of two varieties of oat, Daeyang and Choyang, were analyzed for fat-soluble antioxidants such as α-tocotrienol, γ-oryzanols, lutein and zeaxanthin using an UPLC system and for antioxidant activity using a DPPH assay. Specifically, an 80% ethanol extract of Daeyang oat (Avena sativa cv. Daeyang), which had high amounts of antioxidants and potent radical scavenging activity, was further evaluated for protective effect against oxidative stress-induced cell death, intracellular reactive oxygen species levels, the phosphorylation of DNA damage mediating genes such as H2AX, checkpoint kinase 1 and 2, and p53 and the activation of apoptotic genes such as cleaved caspase-3 and 7 and poly (ADP-ribose) polymerase in HaCaT cells. The Daeyang and Choyang oat 80% ethanol extracts had 26.9 and 24.1 mg/100 g γ-oryzanols, 7.69 and 8.38 mg/100 g α-tocotrienol, 1.25 and 0.34 mg/100 g of lutein and 1.20 and 0.17 mg/100 g of zeaxanthin, respectively. The oat 80% ethanol extract treatment (Avena sativa cv. Daeyang) had a protective effect on oxidative stress-induced cell death in HaCaT cells. In addition, the oat 80% ethanol extracts led to a significant decrease in the intracellular ROS level at a concentration of 50–200 μg/mL, the attenuation of DNA damage mediating genes and the inhibition of apoptotic caspase activities in a dose dependent manner (50–200 μg/mL). Thus, the current study indicates that an oat (Avena sativa cv. Daeyang) extract rich in antioxidants, such as polyphenols, avenanthramides, γ-oryzanols, tocotrienols and carotenoids, has a protective role against oxidative stress-induced keratinocyte injuries and that oat may a useful source for oxidative stress-associated skin damage.

Isolation of cabbage exosome-like nanovesicles and investigation of their biological activities in human cells

There are extensive studies on the applications of extracellular vesicles (EVs) produced in cell culture for therapeutic drug development. However, large quantities of EVs are needed for in vivo applications, which requires high production costs and time. Thus, the development of new EV sources is essential to facilitate their use. Accordingly, plant-derived exosome-like nanovesicles are an emerging alternative for culture-derived EVs. Until now, however, few studies have explored their biological functions and uses. Therefore, it is necessary to elucidate biological activities of plant-derived exosome-like nanovesicles and harness vesicles for biomedical applications. Herein, cabbage and red cabbage were used as nanovesicle sources owing to their easy cultivation. First, an efficient method for nanovesicle isolation from cabbage (Cabex) and red cabbage (Rabex) was developed. Furthermore, isolated nanovesicles were characterized, and their biological functions were assessed. Both Cabex and Rabex promoted mammalian cell proliferation and, interestingly, suppressed inflammation in immune cells and apoptosis in human keratinocytes and fibroblasts. Finally, therapeutic drugs were encapsulated in Cabex or Rabex and successfully delivered to human cells, demonstrating the potential of these vesicles as alternative drug delivery vehicles. Overall, the current results provide strong evidence for the wide application of Cabex and Rabex as novel therapeutic biomaterials.

Tetrandrine Enhances H2O2-Induced Apoptotic Cell Death Through Caspase-dependent Pathway in Human Keratinocytes.

Tetrandrine, a bis-benzylisoquinoline alkaloid, induces apoptosis of many types of human cancer cell. Hydrogen peroxide (H2O2) is a reactive oxygen species inducer; however, there are no reports to show whether pre-treatment of tetrandrine with H2O2 induces more cell apoptosis than H2O2 alone. Thus, the present study investigated the effects of tetrandrine on H2O2-induced cell apoptosis of human keratinocytes, HaCaT, in vitro. HaCaT cells were pre-treated with and without tetrandrine for 1 h, and then treated with H2O2 for examining cell morphological changes and cell viability using contrast-phase microscopy and propidium iodide (PI) exclusion assay, respectively. Cells were measured apoptotic cell death by using annexin V/PI double staining and further analyzed by flow cytometer. Cells were further assessed for DNA condensation using 2-(4-amidinophenyl)-6-indolecarbamidine staining. Western blotting was used to measure expression of apoptosis-associated proteins and confocal laser microscopy was used to measure the protein expression and nuclear translocation from the cytoplasm to nuclei. Pre-treatment of tetrandrine for 1 h and treatment with H2O2 enhanced H2O2-induced cell morphological changes and reduced cell viability, whilst increasing apoptotic cell death and DNA condensation. Furthermore, tetrandrine significantly increased expression of reactive oxygen species-associated proteins such as superoxide dismutase (Cu/Zn) and superoxide dismutase (Mn) but significantly reduced the level of catalase, which was also confirmed by confocal laser microscopy. It also increased expression of DNA repair-associated proteins ataxia telangiectasia mutated, ataxia-telangectasia and Rad3-related, phospho-P53, P53 and phosphorylated histone H2AX, and of pro-apoptotic proteins BCL2 apoptosis regulator-associated X-protein, caspase-3, caspase-8, caspase-9 and poly ADP ribose polymerase in HaCaT cells. These are the first and novel findings showing tetrandrine enhances H2O2-induced apoptotic cell death of HaCaT cells and may provide a potent approach for the treatment of proliferated malignant keratinocytes.

Beta HPV Type 15 Can Interfere With NF-κB Activity and Apoptosis in Human Keratinocytes

E7 protein from cutaneous as well as mucosal HPV types can alter NF-κB activity. Conflicting literature data show a HPV-induced up- or down-regulation of the NF-κB pathway in different cell lines. In a previous study we detected the expression of E7 gene of HPV15 in a subungual tumor of a patient affected by incontinentia pigmenti (IP). IP is a rare X-linked genodermatosis in which the IKKγ gene is altered. From observations in transgenic IKKγ defective mice, it was suggested that IKK-deficient cells may undergo rapid hyper-proliferation and apoptosis/necrosis, leading to increased pro-inflammatory cytokine production in the neighboring IKK-positive cells. The objective of this study was to ascertain if beta HPV 15 can alter apoptosis and NF-κB pathway in normal and IKKγ-deficient keratinocytes. The human immortalized keratinocyte cell line (HaCaT), and human primary keratinocyte (HPK) cells were transduced with a retrovirus expressing E6–E7 proteins of HPV 15 and IKKγ was successful silenced mimicking the HPV15 infection and IP. HPV15 E6–E7 gene expression improved NF-κB activity in human keratinocytes even when IKKγ was silenced by siRNA. In IKKγ silenced keratinocyte cells, TNF-α-induced apoptosis was strongly reduced by the expression of HPV15 E6–E7 genes. Beta HPV15 exerted this anti-apoptotic activity by decreasing pro-apoptotic BAK and cleaved Caspase 3 proteins. In conclusion, we can speculate that presence of persistent infection by beta papillomavirus might influence the biological fate of IP by altering NF-κB activation and apoptosis in IKKγ mutated cells, favoring their survival and possibly the development of tumors in the late stage of disease. Taken together, our data reinforce the importance of host genetic background in the pathogenesis of HPV-associated skin lesions.

Extract of Cornus officinalis Protects Keratinocytes from Particulate Matter-induced Oxidative Stress.

The skin is one of the large organs in the human body and the most exposed to outdoor contaminants such as particulate matter < 2.5 µm (PM2.5). Recently, we reported that PM2.5 induced cellular macromolecule disruption of lipids, proteins, and DNA, via reactive oxygen species, eventually causing cellular apoptosis of human keratinocytes. In this study, the ethanol extract of Cornus officinalis fruit (EECF) showed anti-oxidant effect against PM2.5-induced cellular oxidative stress. EECF protected cells against PM2.5-induced DNA damage, lipid peroxidation, and protein carbonylation. PM2.5 up-regulated intracellular and mitochondrial Ca2+ levels excessively, which led to mitochondrial depolarization and cellular apoptosis. However, EECF suppressed the PM2.5-induced excessive Ca2+ accumulation and inhibited apoptosis. The data confirmed that EECF greatly protected human HaCaT keratinocytes from PM2.5-induced oxidative stress.

Effect of Hypoxia on Gene Expression in Cell Populations Involved in Wound Healing.

Wound healing is a complex process regulated by multiple signals and consisting of several phases known as haemostasis, inflammation, proliferation, and remodelling. Keratinocytes, endothelial cells, macrophages, and fibroblasts are the major cell populations involved in wound healing process. Hypoxia plays a critical role in this process since cells sense and respond to hypoxic conditions by changing gene expression. This study assessed the in vitro expression of 77 genes involved in angiogenesis, metabolism, cell growth, proliferation and apoptosis in human keratinocytes (HaCaT), microvascular endothelial cells (HMEC-1), differentiated macrophages (THP-1), and dermal fibroblasts (HDF). Results indicated that the gene expression profiles induced by hypoxia were cell-type specific. In HMEC-1 and differentiated THP-1, most of the genes modulated by hypoxia encode proteins involved in angiogenesis or belonging to cytokines and growth factors. In HaCaT and HDF, hypoxia mainly affected the expression of genes encoding proteins involved in cell metabolism. This work can help to enlarge the current knowledge about the mechanisms through which a hypoxic environment influences wound healing processes at the molecular level.

Ginsenoside compound K ameliorates imiquimod-induced psoriasis-like dermatitis through inhibiting REG3A/RegIIIγ expression in keratinocytes

BackgroundPsoriasis is a chronic inflammatory skin disease characterized by keratinocyte hyperproliferation. Ginsenoside compound K (CK), a bioactive metabolite of ginseng, modulates various skin disorders with an impact on keratinocyte biology. However, the effect of Ginsenoside CK in psoriasis has not been explored. ObjectiveOur aim was to investigate whether ginsenoside CK could affect the homeostasis of keratinocytes and their expression of psoriasis-associated antimicrobial protein regenerating islet-derived protein 3-alpha (REG3A) and its murine ortholog RegIIIγ. We further explored the therapeutic potential of ginsenoside CK in imiquimod (IMQ)-induced psoriasis-like dermatitis. MethodsThe effects of ginsenoside CK in cell growth and apoptosis of human keratinocytes were measured by MTT assay and flow cytometry, respectively. Bax levels were evaluated by Western blot in HaCaT cells following ginsenoside CK stimulation. REG3A levels were assessed by RT-PCR and Western blot in human keratinocytes following interleukin (IL)-36γ and ginsenoside CK co-simulation. Utilizing IMQ-induced psoriasis mouse model, the therapeutic effects of 0.1% and 1% ginsenoside CK cream were assessed by skin thicknesses and histological examinations, and RegIIIγ level in the lesional skin was detected by Western blot and immunofluorescence. ResultsGinsenoside CK prohibited human keratinocyte proliferation but did not affect their apoptosis. Moreover, it inhibited IL-36γ-induced REG3A expression in HaCaT cells. Ginsenoside CK alleviated imiquimod-induced psoriasis-like hyperkeratosis and reduced RegIIIγ expression in the keratinocytes from lesional skin. ConclusionGinsenoside CK ameliorated IMQ-induced psoriasis-like dermatitis possibly through inhibiting REG3A/RegIIIγ expression in keratinocytes, which highlighted a therapeutic potential of ginsenoside CK in psoriasis.

Cytoprotective effects of taxifolin against cadmium-induced apoptosis in human keratinocytes.

Cadmium (Cd) is a heavy metal widely used in industry, and the skin is an important target of this metal. Taxifolin (Tax), a natural source of bioflavonoids found in various conifers, exerts multiple biologic effects on skin cells. However, the mechanisms by which Tax protects keratinocytes against Cd are currently unclear. We investigated the cytoprotective effects of Tax against Cd-induced apoptosis in the human HaCaT keratinocyte. The water-soluble tetrazolium salt (WST-1) assay and Annexin V/propidium iodide double-staining assay results showed that Cd-induced cell death was lower in cells treated with Tax (0-100 μM) than in cells treated with Cd alone. Additionally, a reduction of Cd-induced DNA fragmentation by Tax was shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay. The levels of reactive oxygen species were also lower in Cd/Tax-treated cells than in Cd-treated cells. We employed a two-dimensional electrophoresis-based proteomic analysis to identify treatment-related alterations in protein expression. Tax downregulated cathepsin B and D and upregulated hsp27, cyclophilin A, and peroxiredowin-1. Western blotting confirmed the downregulation of cathepsin B and D and the upregulation of hsp27. The cytoprotective effects of Tax against Cd-induced apoptosis were also characterized by the changes in the activity of caspase 3, -7, poly ADP-ribose polymerase, the cellular proliferation-related ERK1/2, and AKT. Furthermore, the levels of cell cycle-related proteins, such as SP1 and p21, decreased, whereas p53 level increased. We concluded that Tax reduced Cd cytotoxicity and Cd-induced apoptosis by inhibiting the apoptotic pathway.

Pterostilbene Activates the Nrf2-Dependent Antioxidant Response to Ameliorate Arsenic-Induced Intracellular Damage and Apoptosis in Human Keratinocytes.

The NF-E2 p45-related factor 2 (Nrf2), a transcription factor that regulates the cellular adaptive response to oxidative stress, is a target for limiting tissue damage from exposure to environmental toxins, including arsenic. In the current study, we determine whether Pterostilbene (Pts), as a potent activator of Nrf2, has a protective effect on arsenic-induced cytotoxicity and apoptosis in human keratinocytes. Human keratinocytes (HaCaT) or mouse epidermal cells (JB6) were pretreated with Pts for 24 h prior to arsenic treatment. Harvested cells were analyzed by MTT, DCFH-DA, commercial kits, Flow cytometry assay and western blot analysis. Our results demonstrated that Pts effectively regulated the viability in HaCaT and JB6 cells, decreased the reactive oxygen species (ROS) generation and lipid peroxidation (MDA), and improved the NaAsO2-induced depletion of superoxide dismutase (SOD). Moreover, Pts treatment further dramatically inhibited NaAsO2-induced apoptosis, specifically the mitochondrial mediation of apoptosis, which coincided with the effective recovery of NaAsO2-induced mitochondrial membrane potential (ΔΨm) depolarization and cytochrome c release from the mitochondria. Furthermore, arsenic-induced decrease of anti-apoptotic factor Bcl-2 and Bcl-xl, and increase of pro-apoptotic factor Bax and Bad, as well as survival signal related factor caspase 3 activation were reversed by Pts treatment. Further mechanistic studies confirmed that Pts increased antioxidant enzyme expression in a dose-dependent manner, which was related to Nrf2 nuclear translocation. In addition, the effects of Pts on NaAsO2-induced cell viability were largely weakened when Nrf2 was knocked down. Together, our results provide evidence for the use of Pts to activate the Nrf2 pathway to alleviate arsenic-induced dermal damage.

Potentiation of Psoriasis-Like Inflammation by PCSK9

Psoriasis is a systemic inflammatory disease, associated with metabolic disorders, including high level of low-density lipoprotein. PCSK9, which promotes the degradation of low-density lipoprotein receptors and, therefore, the increased concentration of circulating low-density lipoprotein, is also involved in inflammation. This study aims to examine the role of PCSK9 in psoriasis and to investigate the potential of topically applying small interfering RNA targeting Pcsk9 as a psoriasis treatment. We investigated the expression of PCSK9 in lesions of psoriasis patients and imiquimod-induced psoriatic reactions in Pcsk9-knockout and Pcsk9 small interfering RNA-treated mice, and we also used cultured human keratinocytes to investigate the role of PCSK9 in regulating cell proliferation and apoptosis. We found that PCSK9 is overexpressed in psoriatic lesions and that suppressing Pcsk9 can decrease the inflammatory reaction induced by imiquimod treatment and inhibit hyperproliferation of keratinocytes. We also found that suppressing PCSK9 can significantly alter the cell cycle and induce apoptosis of human keratinocytes. Taken together, our findings indicate that PCSK9 plays an important role in psoriasis and may be a therapeutic target.

Cytotoxicity measurement of Bisphenol A (BPA) and its substitutes using human keratinocytes

Bisphenol-A (BPA) was first synthesized in the 1890s and has been used in many plastic products. However, BPA is known to act as an endocrine disruptor and has been found to be toxic to human health. Many alternative substances have been developed to replace BPA, but it is still widely used worldwide. In this study, we identified the potential cytotoxicity of BPA by evaluating toxicity using human keratinocytes. Also, we evaluated cytotoxicity of BPA substitutes to determine their suitability as an alternative to BPA. The proliferation assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and western blot analysis showed that BPA significantly affect cell viability, induction of apoptotic fraction and increased activation of DNA-damage marker protein. In addition, through the same experiments, the substitutes of BPA were shown to be significantly less toxic than BPA, and the least toxicity was observed with 1,4-cyclohexanedimethanol (CHDM) and terephthalic acid (TPA). In conclusion, this study suggests that cytotoxicity of BPA induces apoptosis of human keratinocytes, and that CHDM and TPA are the most suitable substitutes for BPA.

Voriconazole-induced photocarcinogenesis is promoted by aryl hydrocarbon receptor-dependent COX-2 upregulation

Voriconazole (VRCZ) induces the development of UV-associated skin cancers. The mechanism underlying the VRCZ-induced carcinogenesis has been largely unknown. Here, we showed that VRCZ metabolites plus UVA generated reactive oxygen species and resultant DNA damage of the epidermis, but did not induce substantial apoptosis in human keratinocytes (KCs). Furthermore, VRCZ per se stimulates aryl hydrocarbon receptor (AhR) and upregulates COX-2, which is a pivotal enzyme for the promotion of UV-associated tumors, in an AhR-ARNT dependent manner of the classical (genomic) pathway. Our findings suggest that the phototoxic moieties of VRCZ metabolites may participate in the initiation phase of VRCZ skin cancer, while VRCZ per se promotes the tumor development. Therefore, during VRCZ therapy, sun exposure protection is essential to prevent photocarcinogenesis caused by VRCZ metabolites plus UV. Chemoprevention with selective COX-2 inhibitors may be helpful to repress the development of skin cancers derived from DNA-damaged KCs.

P 003 - Ambient UV-B exposure attenuate the binding affinity of ofloxacin with bacterial DNA gyrase and induced apoptosis in human keratinocytes via Reactive Oxygen Species mediated pathway

Ofloxacin (OFLX) is a known synthetic broad spectrum antibiotics which inhibit bacterial DNA topoisomerase activity. Previous studies showed reduced antimicrobial activity of photosensitized OFLX and its photo-products. Here, we have addressed the binding affinity of OFLX and its photo-products against DNA gyrase to measure the antimicrobial activity. Further, the study was extended to explore the molecular mechanism of photogenotoxicity on human keratinocyte cells (HaCaT) under environmental UV-B irradiation. Photochemical experiments showed the generation of Reactive Oxygen Species (ROS) such as 1O2, O2•- and •OH by photosensitized OFLX. OFLX shows a concentration dependent decrease in cell viability of HaCaT through MTT and NRU tests. Significant intracellular ROS generation was measured by DCFDA assay. ROS caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. OFLX induced cell cycle arrest in G1 phase with appearance of sub-G1 peak. OFLX triggered apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of photo-products of OFLX with DNA gyrase is mainly responsible for attenuated antimicrobial activity. Thus, study suggests that sunlight exposure should avoid by drug users during peak hours.