ROS as Regulators of Cellular Processes in Melanoma.

In this review, we examine the multiple roles of ROS in the pathogenesis of melanoma, focusing on signal transduction and regulation of gene expression. In recent years, different studies have analyzed the dual role of ROS in regulating the redox system, with both negative and positive consequences on human health, depending on cell concentration of these agents. High ROS levels can result from an altered balance between oxidant generation and intracellular antioxidant activity and can produce harmful effects. In contrast, low amounts of ROS are considered beneficial, since they trigger signaling pathways involved in physiological activities and programmed cell death, with protective effects against melanoma. Here, we examine these beneficial roles, which could have interesting implications in melanoma treatment.

Genistein reduces proliferation of EP3-expressing melanoma cells through inhibition of PGE2-induced IL-8 expression.

Genistein, a natural isoflavone found in soybean products, is considered as a powerful anti-cancer agent, although the involved mechanisms are not fully understood. There is a growing body of evidence that, among the genes inhibited by genistein and responsible for cell cycle progression, invasion, metastasis, and angiogenesis, IL-8 occupies a relevant place. On the other hand, it is equally well documented that IL-8 is upregulated by prostaglandin E2 (PGE2) in different pathological conditions, particularly in neoplastic disease. Here we investigated whether genistein could affect cell growth in a panel of oral, uveal and cutaneous melanoma cell lines by interfering with basal or PGE2-induced IL-8 production. To this end, experiments were performed to evaluate the effect of PGE2 treatment on IL-8 levels, the expression and the role of PGE2 receptors and whether genistein could be able to interfere with these events. Finally, it was evaluated whether the inhibition of oral, uveal and cutaneous melanoma cell proliferation in the presence of genistein could be related to a reduction of IL-8 levels. We show that PGE2 enhances IL-8 synthesis via the EP3 receptor and that genistein is able to down-regulate the latter, as well as to decrease IL-8 mRNA and protein expression, thereby inhibiting oral, uveal and cutaneous melanoma cell proliferation. Taken together, our data provide new insights into the anti-cancer properties of genistein by showing that this flavonoid may affect the development and growth of melanoma at oral, uveal and cutaneous sites. Moreover, these results provide evidence that genistein may exert its therapeutic activity through its ability to prevent PGE2-mediated IL-8 induction.

Epidrugs in the Immunotherapy of Cutaneous and Uveal Melanoma.

Epigenetic modifications can affect numerous mechanisms used by neoplastic cells to evade immune control. In melanoma epigenetic defects, caused by dysregulations in the expression of genome writers, erasers, or readers, play a significant role in the reduced expression of molecules required for efficient immune recognition as well as antigen presentation and processing. Alterations in gene expression were identified in tumor-associated antigens (TAAs), human leukocyte antigen (HLA) complex, co-stimulatory/accessory molecules, antigen processing machinery (APM), and NKG2D ligands that have shown to be silenced or down-regulated in melanoma. In agreement with the inherent reversibility of epigenetic silencing, epigenetic drugs such as inhibitors of DNA methyltransferases (DNMTs), histone deacetylases (HDACs), histone methyltransferase enhancer of Zeste homolog 2 (EZH2), and modifiers of microRNA (miRNA) dysregulation or antagomirs can restore the expression of these molecules, favouring the recognition of cancer cells by immune responses, reducing the resistance to Natural Killer (NK) and cytotoxic T cells (CTL), and enhancing the functions of antigen presenting cells. Moreover, inhibitors of reader proteins seem to preferentially affect the NF-kB-induced activation of pro-inflammatory cytokine genes. At present an increasing interest is shown toward new combined therapeutic approaches employing epidrugs or new molecular inhibitors and in vivo immunotherapies, such as vaccines and adoptive T-cell transfer (ACT). This review summarizes the current understanding of the role of epidrugs in the modulation of molecules involved in the melanoma immune response and focuses on their future clinical use in new therapeutic combinations for melanoma treatment.

DSS1 promoter hypomethylation and overexpression predict poor prognosis in melanoma and squamous cell carcinoma patients.

Previous studies have found a link between high expression levels of the Deleted in Split hand/Split foot 1 (DSS1) gene and cancer progression. The aim of this study was to examine whether overexpression of DSS1 is a feature of melanoma and squamous cell carcinoma (SCC) and if any epigenetic modifications are involved. Evaluation of DSS1 expression profile indicated that the gene is overexpressed in 112 of 130 cutaneous melanomas (86.1%), 41 of 64 uveal melanomas (64.1%), 67 of 82 mucosal melanomas (81.7%), and 61 of 75 SCC samples (81.3%), relative to normal skin. An inverse correlation between DSS1 expression and methylation status of the promoter was found. In vitro studies showed that treatment of DSS1-methylated melanoma and SCC cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine significantly increased DSS1 expression at mRNA and protein levels. Interestingly, a significant association between high DSS1 expression levels and some clinicopathological variables, such as metastasis, ulceration, and reduced overall/disease-free survival was observed. In summary, these data suggest that the extent of promoter methylation plays a role in modulating DSS1 gene expression and highlight that promoter hypomethylation is a frequent event in melanoma and SCC closely linked to poor prognosis.

miR-92a-3p and MYCBP2 are involved in MS-275-induced and c-myc-mediated TRAIL-sensitivity in melanoma cells.

Increasing evidence has demonstrated that in several tumors c-myc acts either as an oncogène or as a proapoptotic agent, depending on binding partner interactions. Recently, we showed that up-regulation of this gene by the histone deacetylase inhibitor MS-275 was responsible for sensitization to TRAIL-induced apoptosis through c-FLIP repression in melanoma. The present study aimed at investigating whether, in addition to inducing H3 hyperacetylation at the c-myc promoter, MS-275 could enhance cell death through the regulation of miRNAs involved in apoptosis, such as the miR-17-92 cluster. Following MS-275 treatment, a decrease in miR-92a-3p was observed either in TRAIL-resistant or TRAIL-sensitive cutaneous and uveal melanoma cells. Prediction tools revealed that miR-92a-3p targeted MYCBP2. Gain- and loss-of-function experiments showed that the 3'-UTR of MYCBP2 mRNA was the target of miR-92a-3p, as ectopic expression of miR-92a-3p resulted in MYCBP2 downregulation whereas miR-92a-3p knockdown markedly increased the expression of MYCBP2. Silencing of MYCBP2 counteracted the pro-apoptotic effects exerted by the down-regulation of miR-92a-3p and prevented c-myc-induced repression of c-FLIP, indicating a pivotal role of MYCBP2 as a mediator of miR-92a-3p and c-myc function. Together, our findings indicate that the MS-275-triggered downregulation of the oncogenic miR-92a-3p- which leads to the overexpression of its target gene MYCBP2 - is an event required for the enhanced susceptibility of melanoma cells to TRAIL-mediated apoptosis. Our data illustrate another epigenetic mechanism activated by MS-275 at the post-transcriptional level in melanoma, in addition to its best-known effects at the transcriptional level.

DNA methylation-induced E-cadherin silencing is correlated with the clinicopathological features of melanoma.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, has an important role in epithelial cell function, maintenance of tissue architecture and cancer suppression. Loss of E-cadherin promotes tumor metastatic dissemination and predicts poor prognosis. The present study investigated the clinicopathological significance of E-cadherin expression in cutaneous, mucosal and uveal melanoma related to epigenetic mechanisms that may contribute to E-cadherin silencing. E-cadherin expression was reduced in 55/130 cutaneous (42.3%), 49/82 mucosal (59.7%) and 36/64 uveal (56.2%) melanoma samples as compared to normal skin controls and was inversely associated with promoter methylation. Of the 10 different CpG sites studied (nt 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940), two sites (nt 892 and 940) were 90-100% methylated in all the melanoma specimens examined and the other ones were partially methylated (range, 53-86%). In contrast, the methylation rate of the E-cadherin gene was low in normal tissues (range, 5-24%). In all the three types of melanoma studied, a significant correlation was found between reduced levels of E-cadherin and reduced survival, high mitotic index and metastasis, accounting for the predilection of lymph nodal localization. In cutaneous and mucosal melanoma, low E-cadherin expression was positively correlated also with head/neck localization and ulceration. A high frequency of reduced E-cadherin levels occurred in choroid melanomas. In vitro experiments showed that E-cadherin transcription was restored following 5-aza-2'-deoxycytidine (5-aza-dC) treatment or DNMT1 silencing and was negatively correlated with the invasive potential of melanoma cells. The significant relationship between E-cadherin silencing and several poor prognostic factors indicates that this adhesion molecule may play an important role in melanomagenesis. Therefore, the inverse association of E-cadherin expression with promoter methylation raises the intriguing possibility that reactivation of E-cadherin expression through promoter demethylation may represent a potential therapeutic strategy for the treatment of melanoma.

Role of Genetics and Epigenetics in Mucosal, Uveal, and Cutaneous Melanomagenesis.

Melanoma prevalently occurs on parts of the body that have been overexposed to the sun. However, it can also originate in the nervous system, eye and mucous membranes. Melanoma has been thought for a long time to arise through a series of genetic mechanisms involving numerous irreversible changes within the human genome. However, recently, "epimutations" have attracted considerable attention owing to their high prevalence rate and reversible nature. These observations opened up new perspectives in the use of epidrugs with the potential for restoring the "correct" control of neoplastic genomes. Here, we focused on the common consensus on genetics and epigenetics in melanoma. We also discussed the clinical applications of regulators of epigenetic enzymes able to revert the epigenetic and metabolic hallmarks of melanoma cells. Such anti-neoplastic agents affect the expression profile of antioncogenes, proto-oncogenes, and microRNAs resulting in enhanced differentiation, apoptosis, and growth inhibition.

IL-10Rα expression is post-transcriptionally regulated by miR-15a, miR-185, and miR-211 in melanoma.

BACKGROUND: IL-10 is an immunoregulatory cytokine that increases during malignant diseases. The purpose of this study was to: i) determine the mRNA amounts of IL-10, IL-10Rα, and IL-10Rß in cutaneous and uveal melanoma cells and specimens; ii) evaluate their post-transcriptional regulation by miRNAs; iii) ascertain whether miRNA dysregulation may affect IL-10-induced proliferation. METHODS: Genome-wide miRNA expression profiling was performed using a human microarray platform. The reference gene mRNA was measured through qPCR. miRNAs/mRNAs interactions were predicted by TargetScan, microRNA, and PITA. Transfections of specific miRNA mimics/inhibitors were carried out. Cell proliferation was assessed by MTT assay in the presence of IL-10 after transfection with miRNA mimics/inhibitors. RESULTS: There were no differences in IL-10 mRNA levels between any of the 3 melanoma cell lines tested and normal melanocytes. However, lower IL-10Rα expression was found in G361 and OCM-1 cells, and higher levels of IL-10Rß were observed in G361 cells compared with normal melanocytes. GR-M cells did not exhibit any modifications in IL-10Rα and IL-10Rß expression. miR-15a, miR-185, miR-211, and miR-30d were upregulated in G361 and OCM-1 cells, remaining at similar levels in GR-M cells. miR-409-3p and miR-605were down-regulated exclusively in G361 cells. Prediction tools revealed that miR-15a, miR-185, and miR-211 targeted IL-10Rα whereas none of the miRNAs exclusively downregulated in G361 cells targeted IL-10Rß. Luciferase reporter and western blot assays showed that IL-10Rα expression is directly regulated by miR-15a, miR-185, and miR-211, either alone or in combination. An inverse expression pattern between IL-10Rα, on one side, and miR-15a, miR-185, and miR-211 on the other one was also shown in melanoma samples. Ectopic expression of individual miR-15a, miR-185, and miR-211, and even more their co-expression, caused a marked decrease in the proliferation rate of all the cell lines. Likewise, inhibition of any specific miRNA promoted cell growth, an effect that further increased when inhibition concerned all three miRNA. Moreover, specific knockdown of IL-10Rα prevented the proliferative effect of miRNA inhibitors. CONCLUSIONS: Our results support a key role of IL-10Rα in the development and progression of melanoma and suggest that the IL-10/IL-10 receptor system may become a new therapeutic target for melanoma treatment.

The overriding of TRAIL resistance by the histone deacetylase inhibitor MS-275 involves c-myc up-regulation in cutaneous, uveal, and mucosal melanoma.

Malignant melanoma is a highly aggressive tumor which may occur in the skin, eye, and mucous membranes. The prognosis of melanoma remains poor in spite of therapeutic advances, emphasizing the importance of innovative treatment modalities. Currently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is showing promising clinical responses, however its use is hampered by intrinsic or acquired melanoma resistance to apoptosis. Recently, we showed that the combination of TRAIL with the class I-specific histone deacetylase inhibitor (HDACi) MS-275 was a privileged way to override TRAIL resistance through down-regulation of cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP). Here, we elucidated the underlying mechanism and provided evidence that a crucial step in the c-FLIP downregulation triggered by MS-275 implies the up-regulation of c-myc, a transcriptional repressor of c-FLIP. Notably, MS-275 caused H3 histone acetylation at the promoter of c-myc and increased its binding to the c-FLIP promoter, that in turn led to reduced c-FLIP gene transcription. Knockdown of c-myc prevented the MS-275-mediated downregulation of c-FLIP and hindered TRAIL-plus MS-275-induced apoptosis. Findings reported here provide additional knowledge tools for a more aware and effective molecular therapy of melanoma.

Cellular Mechanisms of Oxidative Stress and Action in Melanoma.

Most melanomas occur on the skin, but a small percentage of these life-threatening cancers affect other parts of the body, such as the eye and mucous membranes, including the mouth. Given that most melanomas are caused by ultraviolet radiation (UV) exposure, close attention has been paid to the impact of oxidative stress on these tumors. The possibility that key epigenetic enzymes cannot act on a DNA altered by oxidative stress has opened new perspectives. Therefore, much attention has been paid to the alteration of DNA methylation by oxidative stress. We review the current evidence about (i) the role of oxidative stress in melanoma initiation and progression; (ii) the mechanisms by which ROS influence the DNA methylation pattern of transformed melanocytes; (iii) the transformative potential of oxidative stress-induced changes in global and/or local gene methylation and expression; (iv) the employment of this epimutation as a biomarker for melanoma diagnosis, prognosis, and drug resistance evaluation; (v) the impact of this new knowledge in clinical practice for melanoma treatment.

Involvement of epimutations in meningioma.

Epimutations are heritable and reversible cell markers, which can influence cell function going beyond the effects of DNA mutations. They result from multiple and coordinated mechanisms able to modulate gene expression. Regarding the significance of epigenetics in meningioma, few and somehow contradictory results are available, although promising information has been obtained. Here we highlight the most recent advances about the impact of DNA methylation, histone modifications, and microRNA regulation on meningioma development as well as the interplay between genetic and epigenetic alterations. Data indicate that epigenetics can help to identify novel candidate genes for the management and treatment of meningioma.

Epigenetic marks responsible for cadmium-induced melanoma cell overgrowth.

Cadmium (Cd) is a human carcinogen that likely acts via epigenetic mechanisms. However, the precise role of Cd in melanoma remains to be defined. The goals of this study are to: (i) examine the effect of Cd on the proliferation rate of cutaneous and uveal melanoma cells; (ii) identify the genes affected by Cd exposure; (iii) understand whether epigenetic changes are involved in the response to Cd. The cell growth capacity increased at 48 h after Cd treatment at doses ranging from 0.5 to 10 µM. The research on the key genes regulating proliferation has shown that aberrant methylation is responsible for silencing of p16(INK4A) and caspase 8 in uveal and cutaneous melanoma cells, respectively. The methylation and expression patterns of p14(ARF), death receptors 4/5, and E-cadherin remained unmodified after Cd treatment in all the cell lines analyzed. Ectopic expression of p16(INK4A) abolished the overgrowth of uveal melanoma cells in response to Cd and the overexpression of caspase 8 drastically increased the apoptotic rate of Cd-treated cutaneous melanoma cells. In conclusion, our data suggest that hypermethylation of p16(INK4A) and caspase 8 represents the most common event linked to Cd-induced stimulation of cell growth and inhibition of cell death pathway in melanoma.

Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma.

Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2'-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5' CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.

Class I-specific histone deacetylase inhibitor MS-275 overrides TRAIL-resistance in melanoma cells by downregulating c-FLIP.

Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) has selective killing effect toward malignant cells; however some human melanomas are intrinsically resistant. In this study, we have shown that class I-specific histone deacetylase inhibitor (HDACi) MS-275 can synergize with TRAIL to induce apoptosis in TRAIL-resistant cell lines and to enhance susceptibility of sensitive cells. Conversely, class II-selective HDACi MC1575 has shown no effect on the resistance of melanoma cells and was able exclusively to increase TRAIL-induced cell death in responsive cells. Both the HDACis variably increased DR4, DR5, and procaspase 8 expression, regardless whether cells were TRAIL-sensitive or TRAIL-resistant. However, only MS-275 markedly decreased the expression levels of both the long and short c-FLIP isoforms. RNAi-mediated c-FLIP silencing resulted in caspase 8-dependent apoptosis in survivor cells which was comparable to that observed following MS-275 treatment. Accordingly, enforced expression of ectopic c-FLIP has abolished the cooperative induction of apoptosis by the combination of MS-275 and TRAIL. These data indicate that c-FLIP is a critical regulator of death ligand sensitivity in melanoma. Inhibition of class I HDAC isoenzymes 1, 2 and 3 has resulted to be functionally important for c-FLIP downregulation by MS-275. In contrast, knockdown of class II HDACs has had no effect on c-FLIP expression, thus explaining the dual incapacity of MC1575 to inhibit c-FLIP expression and sensitize cells resistant to TRAIL. The data reported here suggest that MS-275 represents a promising therapeutic approach in combination with TRAIL for treatment of cutaneous and uveal melanoma due to its ability to reduce c-FLIP expression.

Identification of microRNA expression patterns in cutaneous and uveal melanoma cell lines.

AIMS AND BACKGROUND: microRNA (miRNA)-mediated epigenetic regulation of tumor suppressor genes and oncogenes has been shown to play a central role in melanomagenesis. Here, we focused on the identification of miRNA signatures in the cutaneous melanoma cell line G361 and the uveal melanoma cell line OCM-1. METHODS AND STUDY DESIGN: We carried out genome-wide miRNA expression profiling using a human miRNA microarray platform (Agilent Sanger miRBase, release 10.1) in both cell lines. RESULTS: Our screening revealed significant alteration of miRNA expression profiles in melanoma cell lines compared with normal human epidermal melanocytes. We defined 208 differentially expressed miRNAs in OCM-1 and 112 in G361. By comparison analysis between the resulting miRNA expression profiles, we identified 96 miRNAs that were modified in both cell models. Among these commonly modified miRNAs, 65 were downregulated, 28 upregulated, and 3 exhibited a different expression trend. CONCLUSIONS: Although preliminary, our analysis identified new melanoma-associated miRNAs providing novel miRNA candidates for the development of anticancer target therapy.

Epigenetic effects of cadmium in cancer: focus on melanoma.

Cadmium is a highly toxic heavy metal, which has a destroying impact on organs. Exposure to cadmium causes severe health problems to human beings due to its ubiquitous environmental presence and features of the pathologies associated with pro-longed exposure. Cadmium is a well-established carcinogen, although the underlying mechanisms have not been fully under-stood yet. Recently, there has been considerable interest in the impact of this environmental pollutant on the epigenome. Be-cause of the role of epigenetic alterations in regulating gene expression, there is a potential for the integration of cadmium-induced epigenetic alterations as critical elements in the cancer risk assessment process. Here, after a brief review of the ma-jor diseases related to cadmium exposure, we focus our interest on the carcinogenic potential of this heavy metal. Among the several proposed pathogenetic mechanisms, particular attention is given to epigenetic alterations, including changes in DNA methylation, histone modifications and non-coding RNA expression. We review evidence for a link between cadmium-induced epigenetic changes and cell transformation, with special emphasis on melanoma. DNA methylation, with reduced expression of key genes that regulate cell proliferation and apoptosis, has emerged as a possible cadmium-induced epigenetic mechanism in melanoma. A wider comprehension of mechanisms related to this common environmental contaminant would allow a better cancer risk evaluation.

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma.

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.

NOD2 triggers PGE2 synthesis leading to IL-8 activation in Staphylococcus aureus-infected human conjunctival epithelial cells.

We previously showed that Staphylococcus aureus and Pseudomonas aeruginosa stimulate IL-8 expression in human conjunctival epithelial cells through different signal transduction pathways. As in some cell types both the bacteria may induce the release of prostaglandin E2 (PGE2) and PGE2 may affect the expression of IL-8, we aimed at investigating whether in human conjunctival cells infected with S. aureus or P. aeruginosa the activation of IL-8 transcription was mediated by PGE2 and which were the underlying molecular mechanisms. We found that S. aureus, but not P. aeruginosa, triggered IL-8 activation by increasing COX-2 expression and PGE2 levels in a time-dependent manner. Overexpression of nucleotide-binding oligomerization domain-2 (NOD2) resulted to be essential in the enhancement of IL-8 induced by S. aureus. It dramatically activated c-jun NH2-terminal kinase (JNK) pathway which in turn led to COX2 upregulation and ultimately to IL-8 transcription. The full understanding of the S. aureus-induced biochemical processes in human conjunctival epithelium will bring new insight to the knowledge of the molecular mechanisms involved in conjunctiva bacterial infections and develop novel treatment aiming at phlogosis modulation.

Hepatitis B virus (HBV) induces the expression of interleukin-8 that in turn reduces HBV sensitivity to interferon-alpha.

High levels of serum interleukin-8 (IL-8) have been detected in chronic hepatitis B (CHB) patients during episodes of hepatitis flares. We investigated whether hepatitis B virus (HBV) may directly induce IL-8 production and whether IL-8 may antagonize interferon-alpha (IFN-α) antiviral activity against HBV. We showed that CHB patients had significantly higher IL-8 levels both in serum and in liver tissue than controls. In HBV-replicating HepG2 cells, IL-8 transcription was significantly activated. AP-1, C/EBP and NF-kB transcription factors were concurrently necessary for maximum IL-8 induction. Moreover, HBx viral protein was recruited onto the IL-8 promoter and this was paralleled by IL8-bound histone hyperacetylation and by active recruitment of transcriptional coactivators. Inhibition of IL-8 increases the antiviral activity of IFN-α against HBV. Our results indicate that HBV activates IL-8 gene expression by targeting the epigenetic regulation of the IL-8 promoter and that IL-8 may contribute to reduce HBV sensitivity to IFN-α.