The Protecting Effect of Deoxyschisandrin and Schisandrin B on HaCaT Cells against UVB-Induced Damage

Schisandra chinensis is a traditional Chinese medicine that has multiple biological activities, including antioxidant, anticancer, tonic, and anti-aging effects. Deoxyschisandrin (SA) and schisandrin B (SB), the two major lignans isolated from S. chinensis, exert high antioxidant activities in vitro and in vivo by scavenging free radicals, such as reactive oxygen species (ROS). Ultraviolet B-ray (UVB) radiation induces the production of ROS and DNA damage, which eventually leads to cell death by apoptosis. However, it is unknown whether SA or SB protects cells against UVB-induced cellular DNA damage. Our study showed that both SA and SB effectively protected HaCaT cells from UVB-induced cell death by antagonizing UVB-mediated production of ROS and induction of DNA damage. Our results showed that both SA and SB significantly prevented UVB-induced loss of cell viability using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays showed that the production of ROS following UVB exposure was inhibited by treatment with SA and SB. Moreover, SA and SB decreased the UVB-induced DNA damage in HaCaT cells by comet assays. In addition, SA and SB also prevented UVB-induced cell apoptosis and the cleavage of caspase-3, caspase-8 and caspase-9. In a word, our results imply that the antioxidants SA and SB could protect cells from UVB-induced cell damage via scavenging ROS.     

Protein oxidation,UVA and human DNA repair

Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency – with particular reference to NER and skin cancer risk.     

Lycium barbarum polysaccharide protects human keratinocytes against UVB-induced photo-damage

Ultraviolet B (UVB) irradiation plays a key role in skin damage, which induces oxidative and inflammatory damages, thereby causing photoaging or photocarcinogenesis. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses significant antioxidative and anti-inflammatory effects on multiple tissues. In the present study, the photoprotective effects and potential underlying molecular mechanisms of LBP against UVB-induced photo-damage were investigated in immortalized human keratinocytes (HaCaT cells). The data indicated that pretreatment with LBP significantly attenuated UVB-induced decrease in cell viability, increase in ROS production and DNA damage. LBP also significantly suppressed UVB-induced p38 MAPK activation, and subsequently reversed caspase-3 activation and MMP-9 expression. Notably, LBP was found to induce Nrf2 nuclear translocation and increase the expression of Nrf2-dependent ARE target genes. Furthermore, the protective effects of LBP were abolished by siRNA-mediated Nrf2 silencing. These results showed that the antioxidant LBP could partially protect against UVB irradiation-induced photo-damage through activation of Nrf2/ARE pathway, thereby scavenging ROS and reducing DNA damage, and subsequently suppressing UVB-induced p38 MAP pathway. Thus, LBP can be potentially used for skincare against oxidative damage from environmental insults.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm²). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.     

NADPH oxidase and cyclooxygenase mediate the ultraviolet B-induced generation of reactive oxygen species and activation of nuclear factor-kappaB in HaCaT human keratinocytes

The detrimental effects of ultraviolet B (UVB) irradiation have been connected with the enhanced generation of reactive oxygen species (ROS) by UVB. However, the exact source of ROS produced by UVB has not been clearly revealed yet. In this study, we determined the source of ROS production and its role in the UVB-induced activation of nuclear factor (NF)-kappaB in HaCaT human keratinocytes. UVB irradiation generated ROS in a dose-dependent manner, and this was significantly inhibited by diphenylene iodonium (DPI), apocynin (Apo) and neopterine (Neo), inhibitors of the NADPH oxidase, and indomethacin (Indo), a cyclooxygenase (COX) inhibitor, but not by the mitochondrial electron transport inhibitors and other cytosolic enzyme inhibitors. In addition, these inhibitors of the NADPH oxidase and COX significantly blocked the UVB irradiation-induced nuclear translocation of NF-kappaB. These results suggest that the NADPH oxidase and COX may be major sources for the UVB-induced ROS generation, and play an essential role in the activation of NF-kappaB which is involved in the expression of a variety of genes induced by UVB in HaCaT cells. These results further suggest that these enzymes may be good targets for the preventive strategy of UVB-induced skin injury.     

Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.     

Augmentation of ultraviolet B radiation-induced tumor necrosis factor production by the epidermal platelet-activating factor receptor.

Ultraviolet B radiation (UVB) has been shown to damage human keratinocytes in part by inducing oxidative stress and cytokine production. Indeed, UVB-induced production of the pro-inflammatory and cytotoxic cytokine tumor necrosis factor alpha (TNF-alpha) has been implicated in the epidermal damage seen in response to acute solar radiation. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in UVB-induced epidermal cell cytokine production. These studies examined the role of the PAF system in UVB-induced epidermal cell TNF-alpha biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Treatment of PAF-R-expressing KB cells with the metabolically stable PAF-R agonist carbamoyl-PAF resulted in increased TNF-alpha mRNA and protein, indicating that activation of the epidermal PAF-R was linked to TNF-alpha production. UVB irradiation of PAF-R-expressing KB cells resulted in significant increases in both TNF-alpha mRNA and protein in comparison to UVB-treated control KB cells. However, UVB treatment up-regulated cyclooxygenase-2 mRNA levels to the same extent in both PAF-R-expressing and control KB cells. Pretreatment with the antioxidant vitamin E or the PAF-R antagonists WEB 2086 and A-85783 inhibited UVB-induced TNF-alpha production in the PAF-R-positive but not control KB cells. These studies suggest that the epidermal PAF-R may be a pharmacological target for UVB in skin.     

Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.     

Proteomic analysis of UVB-induced protein expression- and redox-dependent changes in skin fibroblasts using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis

UVB is the most energetic and DNA-damaging to humans in ultraviolet radiation. Previous research has suggested that exposure to UVB causes skin pathologies because of direct DNA damage and the generation of reactive oxygen species (ROS). However, the detailed molecular mechanisms by which UVB leads to skin cancer have yet to be clarified. In the current study, normal skin fibroblast cells (CCD-966SK) were exposed to various doses of UVB, and the changes in protein expression and thiol reactivity were monitored with lysine- and cysteine-labeling 2D-DIGE and MALDI-TOF mass spectrometry. Our proteomic analysis revealed that 89 identified proteins showed significant changes in protein expression, and 37 in thiol reactivity. Many proteins that are known to be involved in protein folding, redox regulation and nucleotide biosynthesis were up-regulated under UVB irradiation. In contrast, proteins responsible for biosynthesis and protein degradation were down-regulated. In addition, the thiol-reactivity of proteins involving cytoskeleton, metabolism, and signal transduction were altered by UVB. In summary, these UVB-modulated cellular proteins and redox-regulated proteins might play important roles in the early stages of skin cancer formation and photoaging induced by UVB-irradiation. Such proteins might provide a potential target for the rational design of drugs to prevent UVB-induced diseases.     

Involvement of UVB-induced reactive oxygen species in TGF-beta biosynthesis and activation in keratinocytes

TGF-beta produced by keratinocytes in response to UVB (290-320 nm) is a potential mediator for effects of acute and chronic solar radiation on skin. This study was designed to determine whether reactive oxygen species (ROS) mediate UVB-induced TGF-beta biosynthesis in keratinocytes and the subsequent activation of the latent TGF-beta complex. UVB irradiation elevated both total (latent plus active) and active TGF-beta in the keratinocyte supernatants, with a greater increase in the active form. UVB irradiation induced up to a 30% increase in ROS, and the ROS were detected up to 90 min after irradiation. NAC and Trolox, cytoplasmic ROS scavengers, abolished the UVB-induced TGF-beta and intracellular ROS, suggesting that UVB-induced ROS are involved in TGF-beta regulation. Inhibitors of NADPH oxidase activity, DPI and apocynin, decreased UVB-induced ROS. The increase in NADPH oxidase activity was mediated by EGFR activation. UVB-induced ROS also activated latent TGF-beta complex by stimulating MMP-2 and -9 activities. In summary, physiological doses of UVB increase intracellular ROS, which upregulate TGF-beta biosynthesis and activation of TGF-beta through increased activity of MMPs.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.     

Measurement of UVB-Induced DNA damage and its consequences in models of immunosuppression

Exposure to UVB results in formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These can be quantified by a variety of techniques including alkaline gel electrophoresis, ELISAs, Southwestern blotting, and immunohistochemistry. Damage to DNA results in activation of damage response pathways, as indicated by Western blotting using antibodies specific for p53 and breast cancer-associated gene 1 (BRCA1) phosphorylation. The signal from DNA damage to activation of these response pathways appears to be mediated by FKBP12-rapamycin-associated protein (FRAP), since these phosphorylation events are blocked by rapamycin. UVB-induced DNA damage also leads to induction of immunosuppressive cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10 in skin. Induction of TNF-alpha by UVB is readily detectable in cultured normal human epidermal keratinocytes (NHEKs) using ELISA, while induction of IL-10 is readily detectable in cultured mouse keratinocytes but not in NHEKs. Induction of DNA damage by liposome-encapsulated HindIII results in induction of immunosuppressive responses similar to UVB. Clinical testing shows that liposome-encapsulated T4 endonuclease V or photolyase stimulates repair of CPDs in the skin of human subjects, and prevents UVB-induced immunosuppression. Stimulation of repair and prevention of immunosuppression have been linked to prevention of skin cancer by liposome-encapsulated T4 endonuclease V in repair-deficient xeroderma pigmentosum patients.     

The osmolyte taurine protects against ultraviolet B radiation-induced immunosuppression

Organic osmolytes, such as taurine, are involved in cell volume homeostasis and cell protection. Epidermal keratinocytes possess an osmolyte strategy, i.e., they take up taurine upon hyperosmotic stress and express the corresponding transporter TAUT. UVB irradiation also triggers taurine uptake and TAUT expression in this cell type. We therefore asked whether taurine plays a role in photoprotection. By using a TAUT-deficient mouse model, lack of taurine in the skin was found to cause a significantly higher sensitivity to UVB-induced immunosuppression. This was not due to an increased generation or decreased repair of UVB-induced DNA photoproducts in the skin of these animals. Instead, decreased skin taurine levels were associated with an increased formation of the soluble immunosuppressive molecule platelet-activating factor (PAF) from the membranes of UVB-irradiated epidermal cells. Blocking PAF activity in taut-deficient mice with a PAF receptor antagonist abrogated their increased sensitivity to UVB-induced immunosuppression. Moreover, taut -/- mice were more sensitive to PAF-mediated immunosuppression than taut +/+ mice. These data suggest that taurine uptake by epidermal cells prevents undue PAF formation, and thereby photoimmunosuppression. Thus, similar to nucleotide excision repair, taurine uptake is critically involved in photoprotection of the skin.     

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21^CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence.     

Ligand-activated PPARδ inhibits UVB-induced senescence of human keratinocytes via PTEN-mediated inhibition of superoxide production

UV radiation-mediated photodamage to the skin has been implicated in premature aging and photoaging-related skin cancer and melanoma. Little is known about the cellular events that underlie premature senescence, or how to impede these events. In the present study we demonstrate that PPARδ (peroxisome-proliferator-activated receptor δ) regulates UVB-induced premature senescence of normal keratinocytes. Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly attenuated UVB-mediated generation of ROS (reactive oxygen species) and suppressed senescence of human keratinocytes. Ligand-activated PPARδ up-regulated the expression of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and suppressed the PI3K (phosphatidylinositol 3-kinase)/Akt pathway. Concomitantly, translocation of Rac1 to the plasma membrane, which leads to the activation of NADPH oxidases and generation of ROS, was significantly attenuated. siRNA (small interfering RNA)-mediated knockdown of PTEN abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt/Rac1 signalling and on generation of ROS in keratinocytes exposed to UVB. Finally, when HR-1 hairless mice were treated with GW501516 before exposure to UVB, the number of senescent cells in the skin was significantly reduced. Thus ligand-activated PPARδ confers resistance to UVB-induced cellular senescence by up-regulating PTEN and thereby modulating PI3K/Akt/Rac1 signalling to reduce ROS generation in keratinocytes.