Dermatology

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with com...

Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2 0-Deoxy-5-fluorouridylyl-(3 0-5 0)-3 0-C-ethynylcytidine [5-FdU(3 0-5 0)ECyd] and 3 0-C-ethynylcytidinylyl-(5 0 fi1-O)-2-O-octadecyl-sn-glycerylyl-(3-Ofi5 0)-2 0-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3 0-5 0)ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 lM. In vivo the duplex drug 5-FdU(3 0-5 0)ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation.

Intravenous application of high-dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate-mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC 50 values were determined on the NCI60 cancer cells. The cell cycle, the influence of cobalt chloride-induced hypoxiainducible factor-1a (HIF-1a) and the glucose transporter 1 (GLUT-1) expression (a pro-survival HIF-1a-downstream-target) were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O 2) globally increased the IC 50 of ascorbate in the 60 cancer cell lines from 4.5 AE 3.6 mM to 10.1 AE 5.9 mM (2.2-fold increase, P < 0.001, Mann-Whitney t-test), thus inducing cellular resistance towards ascorbate. This ascorbate resistance depended on HIF-1a-signalling, but did not correlate with cell line-specific expression of the ascorbate transporter GLUT-1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC 50 reduced the expression of GLUT-1 in the cancer cells. Our data show a ROS-induced, HIF-1a-and O 2-dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.

Sulforaphane (SFN), an isothiocyanate, is part of an important group of naturally occurring small molecules with anti-inflammatory properties. The published reports are best conceivable with an inhibition of T cell function, but the mode of action remains unknown. We therefore analyzed the effect of SFN on T cell-mediated autoimmune disease. Feeding mice with SFN protected from severe experimental autoimmune encephalomyelitis. Disease amelioration was associated with reduced IL-17 and IFN-g expression in draining lymph nodes. In vitro, SFN treatment of T cells did not directly alter T cell cytokine secretion. In contrast, SFN treatment of dendritic cells (DCs) inhibited TLR4-induced IL-12 and IL-23 production, and severely suppressed Th1 and Th17 development of T cells primed by SFN-treated DCs. SFN regulated the activity of the TLR4-induced transcription factor NF-kB, without affecting the degradation of its inhibitor IkB-a. Instead, SFN treatment of DCs resulted in strong expression of the stress response protein heme oxygenase-1 (HO-1), which interacts with and thereby inhibits NF-kB p65. Consistent with these findings, HO-1 bound to p65 and subsequently inhibited the p65 activity at the IL23a and IL12b promoters. Importantly, SFN suppressed Il23a and Il12b expression in vivo and silenced Th17/Th1 responses within the CNS. Thus, our data show that SFN improves Th17/Th1-mediated autoimmune disease by inducing HO-1 and inhibiting NF-kB p65-regulated IL-23 and IL-12 expression.

The RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways are activated through multiple mechanisms and appear to play a major role in melanoma progression. Herein, we examined whether targeting the RAS-RAF-MEK-ERK pathway with the RAF inhibitor sorafenib and/or the PI3K-AKT-mTOR pathway with the mTOR inhibitor rapamycin has therapeutic effects against melanoma. A combination of sorafenib (4 mM) with rapamycin (10 nM) potentiated growth inhibition in all six metastatic melanoma cell lines tested. The absolute enhancement of growth inhibition rates ranged from 13.0-27.8% in different cell lines (Po0.05, combination treatment vs monotreatment). Similar results were obtained with combinations of the MEK inhibitors U0126 (30 mM) or PD98059 (50 mM) with rapamycin (10 nM). The combined treatment of melanoma cells with sorafenib and rapamycin led to an approximately twofold increase of cell death compared with sorafenib monotreatment (Po0.05) as assessed by propidium iodide staining and cell death detection ELISA. Moreover, sorafenib in combination with rapamycin completely suppressed invasive melanoma growth in organotypic culture mimicking the physiological context. These effects were associated with complete downregulation of the antiapoptotic proteins Bcl-2 and Mcl-1. Sorafenib combined with rapamycin appears to be a promising strategy for the effective treatment of melanoma and merits clinical investigation.

MicroRNAs are small noncoding RNAs that regulate gene expression and have important roles in various types of cancer. Previously, miR-137 was reported to act as a tumor suppressor in different cancers, including malignant melanoma. In this study, we show that low miR-137 expression is correlated with poor survival in stage IV melanoma patients. We identified and validated two genes (c-Met and YB1) as direct targets of miR-137 and confirmed two previously known targets, namely enhancer of zeste homolog 2 (EZH2) and microphthalmiaassociated transcription factor (MITF). Functional studies showed that miR-137 suppressed melanoma cell invasion through the downregulation of multiple target genes. The decreased invasion caused by miR-137 overexpression could be phenocopied by small interfering RNA knockdown of EZH2, c-Met, or Y box-binding protein 1 (YB1). Furthermore, miR-137 inhibited melanoma cell migration and proliferation. Finally, miR-137 induced apoptosis in melanoma cell lines and decreased BCL2 levels. In summary, our study confirms that miR-137 acts as a tumor suppressor in malignant melanoma and reveals that miR-137 regulates multiple targets including c-Met, YB1, EZH2, and MITF.

In melanoma, the PI3K-AKT-mTOR (AKT) and RAF-MEK-ERK (MAPK) signaling pathways are constitutively activated and appear to play a role in chemoresistance. Herein, we investigated the effects of pharmacological AKT and MAPK pathway inhibitors on chemosensitivity of melanoma cells to cisplatin and temozolomide. Chemosensitivity was tested by examining effects on growth, cell cycle, survival, expression of antiapoptotic proteins, and invasive tumor growth of melanoma cells in monolayer and organotypic culture, respectively. MAPK pathway inhibitors did not significantly increase chemosensitivity. AKT pathway inhibitors consistently enhanced chemosensitivity yielding an absolute increase of cell growth inhibition up to 60% (Po0.05, combination therapy vs monotherapy with inhibitors or chemotherapeutics). Cotreatment of melanoma cells with AKT pathway inhibitors and chemotherapeutics led to a 2-to 3-fold increase of apoptosis (Po0.05, combination therapy vs monotherapy) and completely suppressed invasive tumor growth in organotypic culture. These effects were associated with suppression of the antiapoptotic Bcl-2 family protein Mcl-1. These data suggest that inhibition of the PI3K-AKT-mTOR pathway potently increases sensitivity of melanoma cells to chemotherapy.

Isoforms of the casein kinase 1 (CK1) family have been shown to phosphorylate key regulatory molecules involved in cell cycle, transcription and translation, the structure of the cytoskeleton, cell-cell adhesion and receptor-coupled signal transduction. They regulate key signaling pathways known to be critically involved in tumor progression. Recent results point to an altered expression or activity of different CK1 isoforms in tumor cells. This review summarizes the expression and biological function of CK1 family members in normal and malignant cells and the evidence obtained so far about their role in tumorigenesis.

In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fra...

Highlights d Melanoma cells express a cGMP signaling pathway involving PDE5 d The cGMP pathway promotes MAPK signaling and melanoma cell growth and migration d PDE5 degrades cGMP and thus acts as a brake on the growth-promoting cGMP pathway d The PDE5 blocker sildenafil releases the PDE5 brake, leading to increased tumor growth

Melanoma is a malignant tumor in which UVA (320e400 nm) radiation is considered to be an important risk factor. But the role of UVA in melanoma progression toward an invasive phenotype is still not adequately investigated. For most proliferating tumor cells the preference of aerobic glycolysis has been described as the Warburg effect. Here we investigate the effect of UVA irradiation on changes in the Warburg effect and tumor progression toward invasive potential. On UVA irradiation, melanoma cell lines from initial tumors show an induction of the Warburg effect with increased glucose consumption and lactate production, which is at least partially mediated by reactive oxygen species. Associated with UVA treatment and enhanced lactic acid production, tumor-relevant proteases and in situ invasion is upregulated. Simultaneously, UVA increases intracellular concentrations of progression marker transketolase and activated protein kinase Akt, both involved in metabolic changes that increase with proliferation. Using invasion assays we show that lactic acid, resulting from the UVA enhanced and partially reactive oxygen species-mediated Warburg effect, increases the invasive potential of all melanoma cell lines investigated. Therefore, we demonstrate in melanoma cells that production of lactic acid, induced by UVA irradiation, increases invasiveness of melanoma cells via expression of tumorrelevant proteases.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK-or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly,-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of-catenin in melanoma progression and therapy resistance. We further demonstrate that-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.

We previously identified CK1α as a novel tumor suppressor in melanoma and reported that the loss of CK1α leads to increased proliferation and invasive growth of melanoma cells by strong activation of the Wnt/β-catenin signaling pathway. In this study we analyzed expression and the functional effects of the dominantly expressed CK1- isoforms α, δ and ε in melanoma cells by quantitative real-time PCR, western blot and immunohistochemistry. We down-regulated CK1 kinase activity with isoform specific siRNAs and small molecule inhibitors. Vice versa we overexpressed the CK1 isoforms α, δ and ε using viral vectors and tested the biological effects on melanoma cell proliferation, migration and invasion. We show that protein expression of all three CK1-isoforms is downregulated in metastatic melanoma cells compared to benign melanocytic cells. Furthermore, the CK1δ and ε isoforms are able to negatively regulate expression of each other, whereas CK1α expression is independently regulated in ...

Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for &quot;brain-specific&quot; therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro. We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases. Buparlisib inhibited AKT activity, decreased proliferation and induced apoptosis in metastatic melanoma cell lines and short-term brain melanoma cells, irrespective of their BRAF and NRAS mutation status. Additionally, buparlisib inhibited hyperactivated AKT and induced apoptosis in melanoma cells that were stimulated with astrocyte-conditioned...

Chronic leg ulcers (CLUs) are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT) has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regula...

Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected. Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant mel-anoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2a and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process. Conclusions: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 23(20); 6203-14. Ó2017 AACR.

The nutritional curcumin (CUR) is beneficial in cell-mediated autoimmune diseases. The molecular mechanisms underlying this food-mediated silencing of inflammatory immune responses are poorly understood. By investigating antigen-specific immune responses we found that dietary CUR impairs the differentiation of Th1/Th17 cells in vivo during encephalomyelitis and instead promoted Th2 cells. In contrast, feeding CUR had no inhibitory effect on ovalbumin-induced airway inflammation. Mechanistically, we found that CUR induces an anti-inflammatory phenotype in dendritic cells (DC) with enhanced STAT3 phosphorylation and suppressed expression of Il12b and Il23a. On the molecular level CUR readily induced NRF2-sensitive heme oxygenase 1 (HO-1) mRNA and protein in LPS-activated DC. HO-1 enhanced STAT3 phosphorylation, which enriched to Il12b and Il23a loci and negatively regulated their transcription. These findings demonstrate the underlying mechanism through which a nutritional can interfere with the immune response. CUR silences IL-23/Th17-mediated pathology by enhancing HO-1/STAT3 interaction in DC. Silencing inflammatory pathways by nutritionals may have sustainable impact on treatment strategies for chronic immune-mediated diseases. Despite the direct role of nutritionals as allergens or inducers of oral tolerance, increasing data suggests that dietary factors or their metabolites can affect the nature of T cell-mediated immune responses 1. This has been intensively studied for dietary fibre metabolites like short-chain fatty acids in experimental models of colitis 2 or allergic airway inflammation 3. Another group of nutritional factors modifying the course of chronic inflammatory disorders consists of phytochemicals. Some phytochemicals seem to be beneficial in the treatment of cancer but also in certain settings of chronic inflammation. Among the well characterized phytochemicals are sulforaphane, resveratrol and curcumin. The isothiocyanate sulforaphane that naturally occurs in cruciferous vegetables has been reported to protect from cancer and from inflammatory autoimmune disease 4,5. Other examples of nutritional compounds with anti-tumoral and anti-inflammatory activities are the natural phenol resveratrol 6,7 , and the polyphenol diferuloylmethane (curcumin; CUR). This component of turmeric isolated from the rhizome of Curcuma longa has been reported to have anti-tumor properties and to dampen inflammatory conditions 8. As a nutritional product, CUR has been tested in several preclinical settings of T cell-dependent organ-specific inflammatory diseases including arthritis 9 , colitis 10 , diabetes 11 or graft-versus-host disease 12. Similarly, some studies reported a beneficial effect of CUR treatment in rodent models of multiple sclerosis 13-16. Different mechanisms have been proposed to explain the beneficial effects of CUR. While its anti-tumoral effects seem to be mediated by the regulation of tumor suppressor genes, cellular apoptosis and oxidative stress, its anti-inflammatory mechanisms are not fully understood. CUR has been suggested to affect the phenotype of immune cells like T cells and dendritic cells (DC) and to interfere with different signaling pathways like NF-κ B, NRF2 and JAK/STAT 10,13,17-19. The effects of CUR on DC and DC-associated signaling pathways are of special interest since DC are the most relevant cell type for initiating inflammatory autoimmune responses and for

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAF V600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both and Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimens...