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.     

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.     

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.     

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.     

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.     

Chafuroside B,an Oolong Tea Polyphenol,Ameliorates UVB-Induced DNA Damage and Generation of Photo-immunosuppression Related Mediators in Human Keratinocytes

Chafuroside B was recently isolated as a new polyphenolic constituent of oolong tea leaves. However, the effects of chafuroside B on skin function have not been examined. In this study, we investigated the protective effects of chafuroside B against UVB-induced DNA damage, apoptosis and generation of photo-immunosuppression related mediators in cultured normal human epidermal keratinocytes (NHEK). Chafuroside B at 1 µM attenuated both UVB-induced apoptosis, evaluated in terms of caspase-3/7 activity, and UVB-induced DNA damage, evaluated in terms of formation of cyclobutane pyrimidine dimers (CPD), in NHEK exposed to UVB (20 mJ/cm²). In addition, chafuroside B at 0.3 or 1 µM suppressed the UVB-induced production of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE₂), as determined by ELISA, and conversely enhanced IL-12 mRNA expression and production, as measured by RT-PCR and ELISA. Further, chafuroside B at 1 µM also suppressed UVB-induced expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA. These results indicate that chafuroside B promotes repair of UVB-induced DNA damage and ameliorates the generation of IL-10, TNF-α, PGE₂, and RANKL, all of which are UVB-induced immunosuppression related mediators. These effects of chafuroside B may be mediated at least in part through induction of IL-12 synthesis in human keratinocytes. Because chafuroside B might have practical value as a photoprotective agent, a further study of the in vivo effects of chafuroside B seems warranted.     

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes

Objective: Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes.

Methods: Human keratinocytes were pretreated with Pter (5 and 10?μM) for 24?h prior to UVB irradiation (300?mJ/cm²). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay.

Results: Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity.

Conclusion: The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.     

Differential role of basal keratinocytes in UV-induced immunosuppression and skin cancer

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced photolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated by UV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmful biological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice to ubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basal keratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basal keratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression of genes associated with repair and recombinational processing of DNA damage, as well as apoptosis and a response to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from the genome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does not affect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basal keratinocytes in these processes, providing novel insights into the skin's acute and chronic responses to UV in a lesion- and cell-type-specific manner.     

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.     

RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling

Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations, and the onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that lead to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB, encoding a small GTPase has been identified as a DNA damage-inducible gene. RhoB is involved in epidermal growth factor (EGF) receptor trafficking, cytoskeletal organization, cell transformation, and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP-bound form of RhoB is increased rapidly within 5 min of exposure to UVB, and then RhoB protein levels increased concomitantly with EGF receptor (EGFR) activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific small interfering RNAs inhibits AKT and glycogen synthase kinase-3beta phosphorylation through inhibition of EGFR expression. Moreover, down-regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicated that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.     

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.     

Silibinin up-regulates DNA-protein kinase-dependent p53 activation to enhance UVB-induced apoptosis in mouse epithelial JB6 cells

In the present study, we employed a well established JB6 mouse epithelial cell model to define the molecular mechanism of efficacy of a naturally occurring flavonoid silibinin against ultraviolet B (UVB)-induced skin tumorigenesis. UVB exposure of cells caused a moderate phosphorylation of ERK1/2 and Akt and a stronger phosphorylation of p53 at Ser(15), which was enhanced markedly by silibinin pretreatment. Kinase activity of ERK1/2 for Elk-1 and Akt for glycogen synthase kinase-3beta was also potently enhanced by silibinin pretreatment. Furthermore, silibinin increased the UVB-induced level of cleaved caspase 3 as well as apoptotic cells. Based on these observations, next we investigated the role of upstream kinases, ATM/ATR and DNA-PK, which act as sensors for UVB-induced DNA damage and transduce signals leading to DNA repair or apoptosis. Whereas UVB strongly activated ATM as observed by Ser(1981) phosphorylation, it was not affected by silibinin pretreatment. However, pretreatment of cells with the DNA-protein kinase (PK) inhibitor LY294002 strongly reversed silibinin-enhanced Akt-Ser(473) and p53-Ser(15) as well as ERK1/2 phosphorylation together with a dose-dependent decrease in cleaved caspase 3 and apoptosis (p < 0.05). In addition, silibinin pretreatment strongly enhanced H2A.X-Ser(139) phosphorylation and DNA-PK-associated kinase activity as well as the physical interaction of p53 with DNA-PK; pretreatment of cells with LY294002 but not caffeine abolished the silibinin-caused increase in both DNA-PK activation and p53-Ser(15) phosphorylations. Together, these findings suggest that silibinin preferentially activates the DNA-PK-p53 pathway for apoptosis in response to UVB-induced DNA damage, and that this could be a predominant mechanism of silibinin efficacy against UVB-induced skin cancer.     

Prevention of UVB Radiation-induced Epidermal Damage by Expression of Heat Shock Protein 70

Irradiation with UV light, especially UVB, causes epidermal damage via the induction of apoptosis, inflammatory responses, and DNA damage. Various stressors, including UV light, induce heat shock proteins (HSPs) and the induction, particularly that of HSP70, provides cellular resistance to such stressors. The anti-inflammatory activity of HSP70, such as its inhibition of nuclear factor kappa B (NF-κB), was recently revealed. These in vitro results suggest that HSP70 protects against UVB-induced epidermal damage. Here we tested this idea by using transgenic mice expressing HSP70 and cultured keratinocytes. Irradiation of wild-type mice with UVB caused epidermal damage such as induction of apoptosis, which was suppressed in transgenic mice expressing HSP70. UVB-induced apoptosis in cultured keratinocytes was suppressed by overexpression of HSP70. Irradiation of wild-type mice with UVB decreased the cutaneous level of IκB-α (an inhibitor of NF-κB) and increased the infiltration of leukocytes and levels of pro-inflammatory cytokines and chemokines in the epidermis. These inflammatory responses were suppressed in transgenic mice expressing HSP70. In vitro, the overexpression of HSP70 suppressed the expression of pro-inflammatory cytokines and chemokines and increased the level of IκB-α in keratinocytes irradiated with UVB. UVB induced an increase in cutaneous levels of cyclobutane pyrimidine dimers and 8-hydroxy-2′-deoxyguanosine, both of which were suppressed in transgenic mice expressing HSP70. This study provides genetic evidence that HSP70 protects the epidermis from UVB-induced radiation damage. The findings here also suggest that the protective action of HSP70 is mediated by anti-apoptotic, anti-inflammatory, and anti-DNA damage effects.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

Hypoxia-inducible factor-1α regulates the expression of nucleotide excision repair proteins in keratinocytes

The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1α (HIF-1α) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1α increased XPC mRNA expression due to competition between HIF-1α and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1α protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1α also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1α is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1α downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1α in the repair of UVB-induced DNA damage.     

Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans.

We tested the hypothesis that DNA is a target for solar-simulated ultraviolet radiation (ssUVR)-induced suppression of the reactivation of memory immunity in humans. T4N5 liposomes contain the DNA repair enzyme T4 endonuclease V. This cleaves DNA at the site of ultraviolet radiation (UVR)-induced cyclobutane pyrimidine dimers (CPD), initiating DNA repair. It has previously been used to show that CPDs are a key molecular trigger for UVR-induced immunosuppression in mice. To determine whether CPD formation is involved in UVR immunosuppression in humans, nickel-allergic volunteers were irradiated with a range of doses of ssUVR. T4N5 or empty liposomes were then applied after irradiation. Nickel-induced recall immunity was assessed by reflectance spectrometry. T4N5 liposomes inhibited immunosuppression and prevented ssUVR from reducing the number of epidermal dendritic cells. T4N5 liposomes also reduced macrophage infiltration into irradiated epidermis. These studies show that enhanced removal of CPDs from human skin protects from immunosuppression, hence demonstrating that these photolesions are an important molecular event in ssUVR-induced immunosuppression in humans. CPDs also triggered loss of dendritic cells and infiltration by macrophages. It is possible that these changes to antigen presenting cells contribute to ssUVR induced suppression of recall immunity to nickel in humans.